
Proceedings of the AACR

Volume 59 | April 2018

Part A: Abstracts 3028-5930

TABLE OF CONTENTS

TUMOR BIOLOGY:

Cancer Imaging: Immunology and Systems Analysis in Vivo

Cancer Stem Cell Characterization

Carcinogenesis 1

Harnessing the Power of Cell Lines for Cancer Research

Immune Cells in the Microenvironment

Metastasis, Invasion, and Migration 1

Pediatrics 2: Preclinical Therapies, Resistance, and Stem Cells

Radiation Studies Using in Vitro and Computational Models

EPIDEMIOLOGY:

Biomarkers of Exogenous and Endogenous Risk Factors in Cancer Epidemiology

PREVENTION RESEARCH:

Prevention, Interception, and Early Detection Research

BIOINFORMATICS AND SYSTEMS BIOLOGY:

Sequence Analysis and Unique Database Resources

Systems and Computational Biology

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

Epigenetic Changes as Molecular Markers of Cancer

Exploring Oncogenic Transcription Factors

Genomic Instability

Genomic Profiling of Tumors 1

Genomic Profiling of Tumors 2

Kinases 2

Metabolomics

Oncogene Growth Factors and Their Receptors

Therapeutic Approaches

Ubiquitylation, Vesicles, and Membranes

CLINICAL RESEARCH:

Adoptive Cell Therapy 3

Biomarker Discovery 4

Immune Checkpoints 3

Liquid Biopsy 3

Molecular Classification of Tumors 1: Epigenetic Therapy, Functional and Molecular Imaging, and Tumor Heterogeneity

CANCER CHEMISTRY:

Drug Delivery

ENDOCRINOLOGY:

Steroid Receptors and Preclinical Studies of Endocrine-Related Cancers

IMMUNOLOGY:

Immunomodulatory Agents and Interventions 1

Innate Immune Responses in Cancer

Therapeutic Antibodies, Including Engineered Antibodies 3

EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

Novel and Canonical Targets

Novel Assay Technology and Delivery Systems

Pharmacogenetics and Pharmacogenomics

Receptor Targeting and the Tumor Microenvironment

Resistance and Biology

Targeting Oncogenes, Tumor Suppressors, or Gene Products

Therapeutic Targeting

TUMOR BIOLOGY:

Carcinogenesis 2

Cell Adhesion and Extracellular Matrix

Determining How the Immune System Drives Tumor Progression

Mechanisms and Models of Gastrointestinal Malignancies

Molecular Imaging: Novel Probes and Preclinical Studies

Pediatrics 3: Signaling, Transcription, and Metastasis

Radiation Studies Using in Vivo and Clinical Models

Therapeutic Approaches to Metastasis

EPIDEMIOLOGY:

Biomarkers of Prognosis and Pharmacoepidemiology

Cancer in Minority Populations, Health Disparities, and Survivorship Research

BIOINFORMATICS AND SYSTEMS BIOLOGY:

Statistical Methods, Mathematical Modeling, and Molecular Modeling

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

Checkpoints and Cell Cycle Progression

Epigenomics

Genomic Profiling of Tumors 3

GTPases and Their Regulators and Effectors

Immunologic and Other Cancer Cell Death

MicroRNA Therapeutics

Noncoding RNAs: From Biology to Therapy

Post-transcriptional and Translational Control of Cell Fate

Tumor Suppressor Genes 1

Tumor-Stroma and Cell-Cell interactions

CLINICAL RESEARCH:

Diagnostic Biomarkers

Immune Checkpoints 4

Immune Mechanisms Invoked by Therapies 2

Liquid Biopsy 4

Molecular Classification of Tumors 2: Molecular Predictors of Response, Tumor Staging, and Correlation of Clinical and Molecular Markers

Novel Preclinical Therapies in Pediatric Solid Tumors

CANCER CHEMISTRY:

Drug Discovery Tools

IMMUNOLOGY:

Adaptive Immunity in Tumors

Immunomodulatory Agents and Interventions 2

New Immunosuppressive Mechanisms in Cancer

REGULATORY SCIENCE AND POLICY:

Regulatory Science and Science Health Policy

EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

Canonical Targets 1

Combination Chemotherapy 1

DNA Damage and Cell Cycle Regulation Experimental Therapeutics

Novel Targets and Inhibitors

Microenvironmental and Cell Nonautonomous Factors in Mediating Therapeutic Resistance

Pharmacokinetics and Pharmacodynamics

CLINICAL RESEARCH:

Liquid Biopsy: Poster Discussion

Emerging Immunotherapy Targets and Combination Strategies to Overcome Treatment Resistance

EPIDEMIOLOGY:

Endogenous and Exogenous Factors in Cancer Risk and Mortality

EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

A Therapeutic "About Face": Reversing Drug Resistance

Early Novel Drug Development

IMMUNOLOGY:

Epigenetic and Metabolic Regulation of Cancer Immunity

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

Metabolism: Emerging Concepts and Therapy

Understanding the Genomic Dark Matter

PREVENTION RESEARCH:

Preclinical Studies of Cancer Prevention

TUMOR BIOLOGY:

Stemness and Cancer

Molecular Mechanisms Driving Metastasis

Expanding the Definition of the Tumor Microenvironment

TUMOR BIOLOGY:

Applications of 3D Models for Tumor Biology and Therapeutic Studies

Carcinogenesis 3

Carcinoma-Associated Fibroblasts in Tumor Progression

Dissecting Tumorigenesis in Vivo Using Genetic Approaches and Spontaneous Tumor Models

Metabolism and the Microbiome: Defining the Greater Microenvironment

Metastasis, Invasion, and Migration 2

Molecular Profiles, Circulating Cancer Cells, and Metastasis

The Systemic Microenvironment in Tumorigenesis

ENDOCRINOLOGY:

Clinical Endocrinology

EPIDEMIOLOGY:

Diet, Alcohol, Tobacco, and Other Lifestyle Risk Factors

PREVENTION RESEARCH:

Population and Behavioral Studies in Cancer

BIOINFORMATICS AND SYSTEMS BIOLOGY:

New Algorithms

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

DNA Methylation

Genomic Profiling of Tumors 4

Genomic Profiling of Tumors 5

MicroRNAs as Biomarkers

Signaling and Hormonal Inputs to Transcription Factor Regulation

Signaling and Therapy

Targets Affecting Metabolism

Tumor Suppressor Genes 2

CLINICAL RESEARCH:

Diagnostic and Prognostic Biomarkers in Clinical Trials

Immunomodulatory Agents and Interventions 3

Liquid Biopsy 5

Liquid Biopsy 6

Therapeutic Antibodies, Including Engineered Antibodies 4

Vaccines 2

CANCER CHEMISTRY:

Emerging Proteomic Technologies for Cancer Research

IMMUNOLOGY:

Emerging Tools and Models in Immuno-oncology Research

Immune Monitoring / Clinical Correlates

Neoantigens in Cancer

Oncogenes, Inflammation, and Cancer

EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

Antibodies, Fusion Proteins, and Related Biologics

Canonical Targets 2

Combination Chemotherapy 2

Epigenetic and Metabolic Pathways in Mediating Therapeutic Resistance

Novel Targets and Therapeutics

Regulation of Gene Expression in Drug Resistance

Therapeutic Approaches Based on Gene Delivery and Vector System

# Tuesday, April 17, 2018

## TUMOR BIOLOGY:

### Cancer Imaging: Immunology and Systems Analysis in Vivo

#3028

**PET imaging with the bispecific** 89 **Zr-antibody ERY974 targeting CD3 and glypican 3 in tumor-bearing mouse models.**

Stijn J. Waaijer,1 Danique Giesen,1 Takahiro Ishiguro,2 Yuji Sano,2 Norihisa Ohishi,2 Athos Gianella-Borradori,3 Carolien P. Schröder,1 Elisabeth G. de Vries,1 Marjolijn N. Lub-de Hooge1. 1 _University Medical Center Groningen, Groningen, Netherlands;_ 2 _Chugai Pharmaceutical Co., Ltd., Japan;_ 3 _Chugai Pharma USA, NJ_.

BACKGROUND ERY974, a modified monoclonal IgG4 bispecific antibody directed against human CD3 on T cells and glypican 3 (GPC3) on tumor cell, is currently in phase I clinical trial. The oncofetal protein GPC3 is overexpressed in several tumor types. Radiolabeling ERY974 with positron emission tomography (PET) isotope zirconium-89 (89Zr) enables non-invasive molecular imaging of tumor targeting and whole-body distribution. We aimed to evaluate 89Zr-ERY974 tumor targeting and effect of T cells on tumor uptake in mouse models, including a humanized mouse model.

METHODS ERY974 and two control molecules namely bispecific CD3xkeyhole limpet hemocyanin (KLH) and KLHxKLH antibodies were radiolabeled with 89Zr. Studies were performed in immunodeficient NOD/Shi-SCID/IL-2Rgnull (NOG) as well as human CD34+ hematopoietic stem cell engrafted NOG mice (huNOG), all subcutaneously inoculated with GPC3 overexpressing human hepatocellular carcinoma HepG2 cells. Mice received 10 µg 89Zr-ERY974, 89Zr-CD3xKLH or 89Zr-KLHxKLH intravenously, with subsequent µPET scanning at 24, 72, 120 and 168 h followed by ex vivo biodistribution. Organs of interest were quantified on µPET scans as mean standardized uptake value (SUVmean) and with ex vivo biodistribution as % injected dose/gram of tissue (%ID/g). Tumor, spleen and lymph nodes were analyzed with autoradiography and immunohistochemical CD3 staining.

RESULTS µPET imaging revealed increased tumor-to-blood ratio (TBR) of 89Zr-ERY974 in NOG over time with maximal TBR of 2.2±0.3 at 168 h post tracer injection (pi). At 168 h, tumor uptake was specific as 89Zr-CD3xKLH and 89Zr-KLHxKLH showed a TBR of only 0.6±0.2 and 0.8±0.3, respectively. In huNOG mice human CD3+ T cells were present in tumor, spleen and lymph nodes. In huNOG mice tumor uptake of 89Zr-ERY974 was higher than in NOG mice as measured on µPET scans (SUVmean at 168 h pi 6.9±2.6 vs 2.9±0.2; P<0.01) and with ex vivo biodistribution (60.9±26.2 %ID/g vs 16.7±2.3 %ID/g; P<0.001), whereas 89Zr-CD3xKLH tumor uptake in both mouse models was lower (P<0.05) but were similar in these mouse models. Autoradiography 168 h following 89Zr-ERY974 administration to huNOG mice showed 89Zr in extensive T cell infiltrate areas in the tumors of huNOG mice, whereas T cell infiltrate was lower in tumors of 89Zr-CD3xKLH and 89Zr-KLHxKLH injected huNOG mice. Spleens of huNOG mice showed CD3+ specific uptake as 89Zr-ERY974 and 89Zr-CD3xKLH uptake were higher than 89Zr-KLHxKLH uptake(P<0.05), whereas spleen uptake in NOG mice of the 3 tracers was similar. Moreover, in huNOG CD3+ mesenteric lymph nodes 89Zr-ERY974 uptake was higher than 89Zr-KLHxKLH uptake (P<0.05)

CONCLUSION 89Zr-ERY974 demonstrates specific tumor uptake in NOG and huNOG mice, while in huNOG mice tumor uptake colocalized with T cell rich infiltrate and also uptake in in spleen and lymph nodes was observed.

#3029

FAP-mediated tumor accumulation of a T-cell agonistic FAP/4-1BB DARPin drug candidate analyzed by SPECT/CT and quantitative biodistribution.

Christian Reichen,1 Ralph Bessey,1 Christine DePasquale,2 Stefan Imobersteg,2 Martin Behe,2 Alain Blanc,2 Roger Schibli,2 Alexander Link,1 Laurent Juglair,1 Joanna Taylor,1 Patricia Schildknecht,1 Julia Hepp,1 Elmar vom Baur,1 Hong Ji,1 Christof Zitt,1 Victor Levitsky,1 Keith M. Dawson,1 Michael T. Stumpp,1 Dan Snell1. 1 _Molecular Partners AG, Zurich, Switzerland;_ 2 _Paul Scherrer Institute, Villigen, Switzerland_.

Immunomodulating agents have revolutionized anti-cancer therapy. However, monotherapy is often not sufficient, and development of combination treatments is hampered by cumulative toxicity. In an attempt to overcome this challenge, a tumor-restricted agonistic 4-1BB/FAP DARPin drug candidate, which induces T-cell co-stimulation only when clustered by binding to fibroblast activation protein alpha (FAP) expressing cells, has been developed. FAP is a type II membrane-bound glycoprotein abundantly expressed in the stroma of many solid tumors by cancer-associated fibroblasts. As shown previously using in vitro and in vivo models (HT-29), co-stimulation induced by a FAP-targeted 4-1BB agonistic DARPin molecule leads to enhanced activation and expansion of CD8+ T-cells. To support clinical development of the drug candidate, tumor localization and accumulation were studied by whole-body SPECT/CT imaging and quantitative biodistribution using Indium-111 labeled DARPin molecules in a human colorectal adenocarcinoma (HT-29) xenograft model in CD1 nude mice. Immunohistochemical staining of tumor stroma confirmed local expression of FAP. Labeled 4-1BB/FAP DARPin molecules specifically accumulated in FAP-expressing tumor in vivo. SPECT/CT imaging and biodistribution revealed a maximum tumor accumulation of around 15% of the injected dose per gram of tissue around 72 h post injection. High tumor/blood ratios were observed one week post injection because the activity in the blood decreased according to the expected serum half-life of 26 h, determined in separate pharmacokinetic studies in BALB/c mice following single dose intravenous bolus injections. Based on the decrease of radioactivity in the tumor, a tumor residence half-life of approximately 4 days was calculated, indicating an extended tumor retention potentially due to FAP binding. No accumulation was observed in the muscle tissue that was choosen as a rather weakly-perfused control tissue. Taken together, FAP-targeting of a 4-1BB agonist DARPin molecule resulted in expected high tumor accumulation and retention compared to an untargeted version of the molecule, both relevant observations for further preclinical and clinical studies. These findings suggest that tumor-targeting via FAP has the potential to induce T-cell activation restricted to the tumor site, and thereby reducing toxicities caused by systemic 4-1BB activation. In conclusion, immunostimulatory drugs with tumor-targeted activity may have the potential to circumvent current limitations of immunotherapy and allow safe and effective use, in particular in combination therapy.

#3030

**Immuno PET imaging of PD-L1 expression in syngeneic and human xenograft tumor mouse models using a site-specific** 89 **Zr labeled PD-L1 antibody.**

Camilla Christensen,1 Lotte K Kristensen,2 Hanna Toftevall,3 Maria Nordgren,3 Brian J. Agnew,4 Carsten H. Nielsen,2 Andreas Kjaer1. 1 _Dept. of Clinical Physiology, Nuclear Medicine & PET and Cluster for Molecular Imaging, Dept. of Biomedical Sciences, Rigshospitalet and University of Copenhagen, Copenhagen Ø, Denmark; _2 _Minerva Imaging, Copenhagen, Denmark;_ 3 _Genovis AB, Lund, Sweden;_ 4 _Thermo Fisher Scientific, Eugene, OR_.

Objectives: One of the major checkpoints probed for therapy is programmed cell death protein 1 (PD-1) and its ligand PD-L1. Despite remarkable clinical efficacy, not all patients benefit from immune checkpoint therapy with PD-1/PD-L1 antibodies, and a cohort of non-responding patients exists. Tumor PD-L1 expression measured by immunohistochemistry has been applied as a biomarker for selecting patients to increase the response rate of PD-1/PD-L1 blockage, but the methods are invasive and prone to sampling error. The objective of the current work was therefore to develop an image-based biomarker for non-invasive Positron-Emission Tomography (PET) imaging of PD-L1 expression

Methods: Anti-PD-L1 (clone 6E11, Genentech) was site-specifically conjugated to dibenzocyclooctyne-Deferoxamine (DIBO-DFO) using GlyclNATOR® (EndoS2) and SiteClickTM technology. Site-specific conjugated 6E11 was subsequently radiolabeled with 89Zr.

The immunoreactivity and KD value were determined by cell binding assays. In vitro stability was assessed by high-performance liquid chromatography (HPLC) and the degree of labeling (DOL) was determined by LC/MS. A dose escalating study was performed with or without co-injection with unlabeled 6E11 to determine the optimal mass dose for PET imaging. Longitudinal PET/CT imaging was performed at various time-points after tracer injection in HCC827 tumor (lung adenocarcinoma) bearing animals and ex vivo biodistribution was performed after the last imaging time-point. Additionally, PET/CT imaging studies were carried out in different human xenograft and syngeneic tumor models with varying degree of PD-L1 expression. The syngeneic tumors received either fractionated external radiation therapy (XRT) or mouse Interferon gamma (IFNγ) treatment 3 days prior to 89Zr-DFO-6E11 PET/CT imaging in order to evaluate treatment induced up regulation of PD-L1 expression.

Results: 89Zr-DFO-6E11 was successfully labeled with a radiochemical purity >99% and the KD value was determined to 0.23 nM. The HCC827 tumors were identified by 89Zr-DFO-6E11 PET imaging (3.7 ± 0.2 %ID/g, mean ± SD), and co-injection of unlabeled 6E11 increased the relative tumor uptake. Ex vivo biodistribution confirmed the in vivo results (5.4 ± 1.7 %ID/g) at 144 hours post injection. Non-invasive PET /CT imaging with 89Zr-DFO-6E11 was able to detect a treatment induced up regulation of PD-L1 expression following treatment with XRT or IFNγ.

Conclusions: Site-specific labeling of antibodies with 89Zr is feasible for immuno-PET imaging and tumor PD-L1 imaging by 89Zr-DFO-6E11. PET/CT is an attractive approach for non-invasive whole body visualization of PD-L1 expression and could potentially aid in therapy guidance. Site-specific labeled antibodies create the opportunity to utilize imaging biomarker-driven approaches to achieve best clinical outcomes in immunotherapy.

#3031

**Positron emission tomography imaging of activated T cells by targeting OX40 reveals spatiotemporal immune dynamics and predicts response to in situ** **tumor vaccination.**

Aaron T. Mayer, Israt S. Alam, Idit Sagiv-Barfi, Kezheng Wang, Ophir Vermesh, Debra K. Czerwinski, Emily M. Johnson, Michelle L. James, Ronald Levy, Sanjiv S. Gambhir. _Stanford University, Stanford, CA_.

Clinical success of cancer immunotherapies has renewed interest in imaging the behavior of immune cells. Due to the spatiotemporally varying signatures of immune response, it has been difficult to monitor and predict patient outcomes using traditional clinical tests. ImmunoPET, defined herein as positron emission tomography utilizing radiolabeled antibodies, has the potential to enable noninvasive, sensitive and longitudinal interrogation of immune cell subset and state. Cell states including activation, anergy, and exhaustion may be more prognostic of disease outcome than the presence of tumor-infiltrating immune cells alone. In particular, T cell activation is thought to be critical to treatment success across many classes of cancer immunotherapy. In this work, we present the first radionuclide imaging of OX40, a novel and specific biomarker of activated antigen-specific T cells. Activation dependent and T cell restricted expression of OX40 was validated in vitro via flow cytometric analysis. Cell uptake studies with radiolabeled 64Cu-DOTA-AbOX40 demonstrated ~11 fold [p < .0001] higher uptake in dyna-bead activated T cells compared to resting. The tracer showed negligible nonspecific uptake in OX40 blocked or OX40-/- T cells and low background levels across a panel of 5 cancer cell lines tested. In vivo, ImmunoPET imaging revealed new insights into response following in situ tumor vaccination with CpG, an adjuvant immunotherapy currently in clinical trials. Balb-C mice bearing dual A20 lymphoma tumors were administered low dose CPG directly in the left tumor (n=7-10), while vehicle control mice received PBS (n=7-10). Early after vaccination, imaging revealed increased OX40 radiotracer uptake in the CPG treated tumor (TT) [~37%; p<0.05] confirmed by immunofluorescent staining. ViSNE, a visualization technique for high-dimensional cytometry data, classified OX40+ single cells in a cluster associated with a nonregulatory, activated CD4 T cell phenotype. CPG treatment led to local expansion of this unique OX40 cell population [~63%; p<0.05]. By late time points, a full systemic response emerged as evidenced by increased Luminex cytokine measurements in the plasma of CPG-treated mice. Unsupervised hierarchical clustering based on radiotracer or cytokine measurements correctly assigned mice into CPG-treated or vehicle cohorts, with few exceptions. More remarkably, a generalized linear regression model indicated early PET signal (mean %ID/g) in the local tumor environment to be highly predictive of response outcomes at late timepoints [r2=0.746]. OX40 ImmunoPET provides a readily translatable approach for monitoring activated T cells with high sensitivity and specificity. In this instance, integration of molecular imaging and computational immunology enabled systems-level interrogation of vaccine response.

#3032

Quantitative three-dimensional imaging cytometry of tumor immune microenvironment.

Steve Seung-Young Lee, Stephen J. Kron, Vytautas P. Bindokas. _The University of Chicago, Chicago, IL_.

The tumor microenvironment is a three-dimensional (3D) system of diverse cellular and non-cellular components whose heterogeneous structure is typically defined by haphazard growth of cancer cells and a disordered microvasculature. Tumor-immune cell interactions occur within this context, providing a challenge for analysis of the infiltrate and anti-tumor immune responses by two-dimensional (2D) methods such as immunohistochemistry (IHC). Toward overcoming the limitations of 2D methods, we introduce Transparent Tissue Tomography (T3) as a tool for quantitative 3D imaging cytometry of the tumor immune microenvironment. For T3 imaging cytometry, tumors were sliced into 400 μm macrosections to facilitate immunofluorescence staining, optical clearing, and confocal microscopic imaging. Macrosections were stained overnight with panels of four to six directly labeled fluorescent primary antibodies then cleared by D-fructose. The full volume of each macrosection was scanned in each channel using a confocal microscope and tumor images were tomographically reconstructed from the macrosection images. The tumor images were segmented to discriminate cell types, map biomarkers, and perform spatial analysis. As an application of T3 imaging cytometry, we examined the distribution of programmed death-ligand 1 (PD-L1) expression in spontaneous Her2+ mammary tumors formed in BALB-NeuT mice. T3 analysis of whole tumors determined PD-L1 expression by tumor cells at the periphery and CD31+ vascular endothelium in the core. For the first time, 3D tomographic projection disclosed vascular PD-L1 expression localized between the endothelium and inner layer of smooth muscle cells. In turn, T3 revealed a strong spatial correlation between CD45+ immune cell distribution and PD-L1 expression. Toward translation, T3 was adapted to analyze whole core needle biopsies. Using T3 to map CD3+CD8+ cytotoxic T cells (CTLs) in BALB-NeuT tumors revealed marked inhomogeneity across single 18 gauge needle cores, confirmed by serial section IHC assays after T3 imaging. Modeling on-treatment biopsy analysis, T3 confirmed a compound effect of radiation and anti-PD-L1 therapy on infiltration of effector cytotoxic lymphocytes such as granzymeB+ NK cells and T cells. Applying T3 imaging cytometry to analyze needle cores from EGFR+ human head and neck cancer tissue, we mapped the distribution of CD3+CD8+ CTLs with respect to the CD31+ microvasculature and tumor cells. By assessing multiple tumor parameters simultaneously at cellular resolution, T3 provides a unique window into the heterogeneity of the tumor immune microenvironment. In particular, spatial, multiparameter T3 analysis may serve as a tool to improve diagnostic, prognostic and predictive testing of patient biopsies as part of evaluation for immune checkpoint blockade therapy.

#3033

Immuno-PET detection of LAG-3 expressing intratumoral lymphocytes using the zirconium-89 radiolabeled fully human anti-LAG-3 antibody REGN3767.

Marcus P. Kelly, Richard Tavare, Jason T. Giurleo, Sosina Makonnen, Carlos Hickey, Makenzie A. Danton, T Cody Arnold, Dangshe Ma, Jie Dai, Jerry Pei, Jessica R. Kirshner, William C. Olson, Gavin. Thurston. _Regeneron Pharmaceuticals, Tarrytown, NY_.

Persistent antigen exposure and inflammatory signals in tumors induce expression of various co-inhibitory or immune checkpoint receptors on T cells, including programmed death protein 1 (PD-1) and Lymphocyte-Activation Gene 3 (LAG-3). Therapeutic antibodies blocking such co-inhibitory receptors have produced durable antitumor responses as single agents and in combinations. In order to monitor LAG-3 expression and potential changes in expression due to therapeutic intervention, we have developed a radionuclide-conjugated antibody to LAG-3 for immuno-PET. The fully human anti-LAG3 antibody REGN3767 was radiolabeled with the positron-emitting radionuclide Zirconium-89 (89Zr) using the bifunctional chelator p-SCN-Bn-Deferoxamine (DFO). 89Zr-REGN3767 demonstrated high radiochemical purity and immunoreactivity in cell binding assays. The ability of 89Zr-REGN3767 to successfully identify LAG-3 expression in vivo was initially assessed using MC38 mouse tumors expressing human LAG-3 (MC38/hLAG-3) implanted into immune-deficient mice. 89Zr-REGN3767 demonstrated higher uptake in MC38/hLAG-3 tumors compared to an 89Zr-isotype control antibody using immuno-PET, and specificity was confirmed by ex vivo biodistribution at day 6 post radiotracer injection (~35 and ~5 %ID/g for 89Zr-REGN3767 and 89Zr-isotype, respectively). Furthermore, a dose titration study of 89Zr-REGN3767 in immune deficient mice co-implanted subcutaneously with Raji lymphoma cells and human peripheral blood mononuclear cells (hPBMCs) demonstrated the ability of 89Zr-REGN3767 to target LAG-3-expressing intratumoral T-cells. 89Zr-REGN3767 immuno-PET and ex vivo biodistribution demonstrated specific localization to Raji/hPBMC co-implanted tumors; this uptake was significantly higher at antibody doses of 0.03 - 0.3 mg/kg than at 5 mg/kg. Doses of 0.03-0.3 mg/kg 89Zr-REGN3767 were also able to detect LAG-3 positive T cells in the spleen. This study shows the ability of 89Zr-REGN3767 to successfully image LAG-3 expressed on intratumoral and splenic T lymphocytes. This work supports the clinical translation of anti-LAG-3 immuno-PET for the assessment of LAG-3 expression, with the goal to investigate its utility for predicting and monitoring response to checkpoint blockade therapy.

#3034

18 **F-FDG-positron emission tomography (PET)/CT enables the identification of checkpoint inhibitor immunotherapy (CIT) responders by determination of CIT-induced metabolic changes in secondary lymphatic organs.**

Johannes Schwenck, Barbara Schörg, Francesco Fiz, Kilian Wistuba-Hamprecht, Andrea Forschner, Thomas Eigentler, Benjamin Weide, Claus Garbe, Martin Röcken, Christina Pfannenberg, Bernd J. Pichler, Christian la Fougere, Manfred Kneilling. _University of Tuebingen, Tuebingen, Germany_.

Although the majority of patients with metastatic melanoma achieve a prolongation of overall survival by using checkpoint inhibitor based immunotherapy (CIT), there is still a larger number of patients who do not benefit from this therapy. As a CIT-induced systemic immune response is required to promote the anti-tumor effect we analyzed the glucose metabolism in secondary lymphoid organs such as the spleen by 18F-FDG-Positron Emission Tomography (PET). In preclinical studies, we were able to distinguish responders from non responders by focusing on the spleen 18F-FDG-uptake of mice with CIT in experimental tumor models. Thus, we aimed to gain deeper insights into impact of CIT on the metabolism in secondary lymphatic organs to identify responders and to stratify patients for differential treatment strategies.

We retrospectively analyzed 18F-FDG-PET/CT scans (baseline and post-therapy) of 38 patients with metastatic melanoma with CTLA-4 or PD-1 Ab treatment as third line therapy (21 responder: 5x nivolumab; 7x pembrolizumab; 9x ipilimumab; 17 non-responder: 2x nivolumab; 11x pembrolizumab; 4x ipilimumab). Spleen regions of interest (ROI) were defined in the CT data, copied to the coregistered PET and analyzed semiquantitatively. Total lesion glycolysis (TLG) was calculated by multiplication of the spleen volume and the SUVmean.

We determined in the baseline 18F-FDG-PET/CT-scans (prior to CIT), no significant differences in spleen volume (221±18 cm3 vs. 209 ±22 cm3) and in the spleen 18F-FDG-uptake (SUVmean: 1,74±0,06vs.1,72±0,05; TLG: 384±37 vs. 359±36) between responders and non-responders. In the follow up 18F-FDG-PET/CT-scans 110±68 days after onset of CIT we measured a similar increase in spleen volume in responders (+8±6%) and non-responders (+7±5%). 15 out of 21 responders revealed an enhanced spleen 18F-FDG uptake when compared to the baseline 18F-FDG-PET/CT-scans. The mean standard uptake values in the spleen of responders increased by +10±9% SUVmean. In sharp contrast, we determined hardly any change in the spleen 18F-FDG uptake of non-responders (SUVmean -1,3±2,6%). Additionally, the total lesion glycolysis (TLG) in the CIT-responders increased stronger (+25±22%) than in non-responders (+6±6%).

Our results suggest that CIT-induced metabolic changes in secondary lymphatic organs are associated with therapy responds. Thus, non invasive 18F-FDG-PET/CT investigations might represent a powerful tool to monitor CIT-induced systemic immune responses in patients. Consequently, preclinical research is prerequisite to uncover the exact mode of action of CIT-induced systemic immune response in secondary lymphatic organs. Moreover, prospective clinical studies are essential to evaluate the prognostic value of our method.

#3035

89 **Zr-labeled anti-PD-L1 Probody therapeutic CX-072 biodistribution in mice bearing human xenograft or murine syngeneic tumors.**

Danique Giesen,1 Linda N. Broer,1 Marjolijn N. Lub-de Hooge,1 Irina Popova,2 Bruce Howng,2 Olga Vasiljeva,2 Elisabeth G. de Vries,1 Martin Pool1. 1 _University Medical Center Groningen, Groningen, Netherlands;_ 2 _CytomX Therapeutics Inc., San Francisco, CA_.

BACKGROUND Immune checkpoint inhibiting antibodies have antitumor activity across several tumor types, but are not effective in all patients and can elicit side effects. CX-072, a fully human Probody™ therapeutic currently in a phase 1/2 clinical trial, is reactive to the murine and human programmed cell death-ligand 1 (PD-L1) immune checkpoint. Probody therapeutics are engineered antibodies with target-binding region blocking masking peptides, which can be preferentially cleaved by tumor-associated proteases, yielding fully active antibodies. CX-072 may thus preserve anti-tumor efficacy, while limiting side effects. We radiolabeled CX-072 with the positron emission tomography (PET) isotope zirconium-89 (89Zr) to reveal its tumor targeting properties and whole body distribution using non-invasive PET imaging.

METHODS CX-072 and a non-specific Probody therapeutic control (PbCtrl) were radiolabeled with 89Zr. For in vivo studies, PD-L1 expressing MDA-MB-231 human breast cancer cells were subcutaneously (sc) engrafted in Balb/c nude mice. To assess tracer protein dose dependency of the tumor uptake, mice received 10 μg 89Zr-CX-072 or 89Zr-PbCtrl (~5 MBq) supplemented with 0, 40 or 240 µg of unlabeled CX-072 or PbCtrl. To evaluate 89Zr-CX-072 biodistribution in an immune-competent setting, C57BL6 mice were implanted sc with low PD-L1 expressing MC38 syngeneic murine colon adenocarcinoma cells. All mice underwent serial in vivo PET imaging 1, 3 and 6 days post injection (pi), quantified by mean standardized uptake value (SUVmean) and followed by ex vivo biodistribution. Activated Probody species in tissues were detected by Western capillary electrophoresis.

RESULTS PET imaging revealed increasing 89Zr-CX-072 tumor accumulation between 1-6 days pi, with the highest SUVmean of 1.5 (± 0.2) observed for 10 µg at 6 days pi. Ex vivo biodistribution analysis showed 8.7 % injected dose per gram (%ID/g) tumor uptake for 10 µg 89Zr-CX-072 versus 3.8 %ID/g for 10 µg 89Zr-PbCtrl (P<0.01) in MDA-MB-231 xenografted mice. In the syngeneic MC38 model biodistribution analysis showed modest tumor uptake for 10 μg 89Zr-CX-072 and 89Zr-PbCtrl (6.5 vs 5.5 %ID/g, P=0.24; tumor-to-blood ratio of 0.61 vs 0.45, P<0.05). 89Zr-CX-072 uptake in lymphoid tissues (spleen, lymph nodes) was similar to 89Zr-PbCtrl. Activated Probody species were predominantly detected in tumor with lesser amounts present in lymphoid tissues.

CONCLUSION 89Zr-CX-072 accumulates more in PD-L1-expressing tumor tissues than in lymphoid tissues. A sub-study of an ongoing clinical study (PROCLAIM-CX-072) is designed to validate study drug distribution in patients using a good manufacturing practice (GMP) quality 89Zr-CX-072 tracer.

#3036

**PEGylated hyaluronidase increases tumor uptake of** 89 **Zr-DFO-HuMab-5B1 (MVT-2163) in a CA19-9 positive hyaluronan-accumulating pancreatic cancer model.**

Jonah Rainey,1 Paul Maffuid,1 Wolfgang W. Scholz,1 Jack Ostrowski,1 H Toni Jun,1 Paul Resnick,1 Xiaoming Li,2 Jesse D. Bahn,2 Susan Zimmerman,2 Kelly Chen,2 Barbara Blouw,2 Curtis B. Thompson,2 Daniel C. Maneval,2 David W. Kang2. 1 _MabVax Therapeutics Holdings Inc., San Diego, CA;_ 2 _Halozyme Therapeutics, Inc., San Diego, CA_.

Hyaluronan (HA) accumulates in the extracellular matrix of many solid tumors, and is associated with poor prognosis in pancreatic ductal adenocarcinoma. In non-clinical models, enzymatic degradation of HA with PEGylated recombinant human hyaluronidase PH20 (PEGPH20) has been shown to remodel the tumor stroma, reduce tumor interstitial fluid pressure, and expand tumor blood vessels, resulting in enhanced delivery of therapeutic and imaging agents, such as monoclonal antibodies. 89Zr-DFO-HuMab-5B1 (MVT-2163) is a targeted ImmuoPET imaging agent for CA19-9 positive malignancies currently in clinical evaluation. CA19-9 plays a role in tumor adhesion and metastasis, and is an independent prognostic indicator of cancer survival. CA19-9 is expressed in pancreatic and other cancers, including small cell lung and colon. In this study, we aimed to show the effects of PEGPH20 on the biodistribution of MVT-2163 in a CA19-9 positive HA-rich human pancreatic tumor xenograft model.

Nude mice were peritibially implanted on the right hind limb with 5x106 human pancreatic tumor cells engineered to overexpress hyaluronan synthase 3 (BxPC3/HAS3). Tumor volumes were measured by MR imaging, and mice were staged when average tumor size reached 320 mm3. The control group received intravenous (IV) vehicle, and the test group received PEGPH20 (IV, 1 mg/kg). Both control and test groups also received MVT-2163 (IV, 3 mg/kg) 24 hours later. PET images were captured from 2 to 120 hours post MVT-2163, and region of interest (ROI) of tumor and liver were analyzed (N=6/group). After the final PET image, tumors and livers were harvested for ex vivo gamma counting.

Ex vivo gamma counting demonstrated improved MVT-2163 accumulation in animals treated with PEGPH20, with a 50.9% increase in counts in the excised tumors. ROI analysis of the tumors showed increased tumor SUV of 8.0, 9.4, and 24.1% at 72, 96, and 120 hours post-injection, respectively, with PEGPH20 compared to vehicle. ROI analysis of liver regions showed an 11.4 to 26.4% reduction in SUV in the PEGPH20 group compared to control. Ex vivo counts of liver tissue confirmed the PET signal reduction with PEGPH20. Analysis of tumor-to-liver ratios showed average increases of 34.1, 35.7, and 58.5% at 72, 96, and 120 hours, respectively, post MVT-2163 injection in PEGPH20 treated mice.

In summary, as measured by SUV, PEGPH20 increased both the tumor uptake and the tumor-to-liver ratios of MVT-2163 in a CA19-9 positive xenograft mouse model of HA accumulating pancreatic cancer. Ex vivo analysis confirmed in vivo results. Taken together, the increased tumor uptake and the decreased liver uptake support further investigation into the potential clinical utility for the combination of PEGPH20 and MVT-2163.

#3037

Comparison of multiplexed imaging mass cytometry in FFPE tissue to monoplex immunohistochemistry.

Navi Mehra,1 Carsten Schnatwinkel,1 Elliott Ergon,1 Joseph Krueger,1 Karl Calara-Nielsen,2 Brad Foulk,2 Kirti Sharma,2 Denis Smirnov,2 Chandra Rao,2 Tatiana Perova,2 Rengasamy Boominathan2. 1 _Flagship Biosciences, Westminster, CO;_ 2 _Janssen Pharmaceuticals, Spring House, PA_.

Introduction: Multiplexed analysis of limited tissue samples can improve our understanding of tumor biology and tumor microenvironment. Chromogenic and fluorescent multiplexed immunohistochemistry (IHC) approaches are available and offer great insights while conserving limited tissue however these approaches have their limitations. Multiplexed chromogenic IHC methods can at best accommodate up to 2-3 distinct markers. Fluorescence-based approaches can support higher degree of multiplexing however spectral overlap issues and differences in labeling efficiency/photostability complicate experimental procedures and data interpretation. Imaging mass cytometry system (IMC) has recently emerged as a novel technology for tissue imaging that enables multiplexed analysis protein expression (up to 40 markers) in a single tissue sample while circumventing the limitations of chromogenic and fluorescent IHC techniques. Metal-conjugated antibodies are used to perform qualitative and quantitative analysis of expression of multiple proteins of interest on a single formalin fixed paraffin embedded (FFPE) tissue slide. Here, we compare the performance of the IMC method with conventional, established IHC techniques using a small panel of markers.

Methods: Serial sections of FFPE tonsil and non-small cell lung carcinoma tissues were assessed by monoplex IHC and multiplex IMC for CD3 (Cell Signaling Technology, D7A6E), CD8 (LS Bio, C8/144B), CD68 (Abcam, KP1), PD-L1 (Spring Bio, SP142) and Histone H3 (D1H2). Digital image analysis using Flagship's image analysis software was used to compare performance characteristics of multiplex IMC platform with standard monoplex chromogenic IHC.

Results: The staining patterns of the corresponding biomarkers were similar between IMC and IHC on sequential sections. Digital image analysis demonstrated concordance in the percentage of biomarker positive cells within analyzed matched IHC and IMC areas. It was also demonstrated that cellular image segmentation can be performed on IMC images thus allowing for utilization of various software packages for high dimensional single cell analysis of IMC data.

Conclusion: Comparative digital image analysis indicates that on FFPE tissues multiplexed IMC platform generates data comparable to that obtained from the monoplex chromogenic IHC platform. We believe that an IMC platform is a new tool capable of dramatically enhancing our ability to study biology of cancer using highly multiplexed analysis of limited tissue samples.

#3038

Intraoperative breast cancer margin detection via polarization-sensitive optical coherence tomography.

Marina Marjanovic,1 Jianfeng Wang,1 Yang Xu,1 Eric J. Chaney,1 Darold R. Spillman,1 Anna M. Higham,2 Natasha N. Luckey,2 Kimberly A. Cradock,2 George Z. Liu,2 Stephen A. Boppart1. 1 _University of Illinois at Urbana-Champaign, Urbana, IL;_ 2 _Carle Foundation Hospital, Urbana, IL_.

Current standard-of-care for breast conserving surgery (BCS) procedures relies on post-operative histopathology to provide a microscopic view and assessment of surgical margins. To avoid high reoperation rates there has been a great interest in developing new imaging solutions to visualize tissue intraoperatively, and provide real-time feedback on the margin status. Optical coherence tomography (OCT) is a high-resolution label-free, imaging technology that is the optical analogue to ultrasound imaging, except images are based on backscattered near-infrared light. Fundamentally, the contrast mechanism of OCT relies on the light scattering properties associated with different tissue structures. Unlike normal tissue with well-organized tissue structure, the structure of cancer tissue is often disorganized and typically characterized by variable cell sizes, abnormal shapes, and enlarged nuclei, resulting in different optical scattering properties, and enabling OCT to reveal differences between normal and cancerous tissues with high spatial resolution. We have previously demonstrated OCT for in vivo label-free intraoperative assessment of the breast conserving surgery (BCS) with a hand-held OCT probe that is integrated with a portable OCT system. Despite the technological advance to achieve in vivo OCT imaging during BCS, there remains a critical need to improve the OCT-based detection, differentiation, and diagnosis of breast tumor tissue.

Polarization-sensitive optical coherence tomography (PS-OCT) is a functional extension of standard OCT that maps changes in the polarization state of light induced by anisotropic tissue properties. By exploiting the interaction between the polarization state of light and tissue, additional structural and functional information can be extracted. Form birefringence is the main optical property present in tissues responsible for altering the polarization state of light. In breast tissue, birefringent structures include the connective tissue or normal stroma, composed largely of collagen fibers. In the presence of breast cancer, the birefringent collagen fibers are degraded, displaced, reorganized, and/or disrupted in ways that dramatically alter the PS-OCT signal, serving as a fundamentally new optical imaging biomarker for the presence of cancer in the breast.

In this study, we report data from 23 cancer surgeries, compared with healthy breast tissue samples obtained from the breast reduction surgeries. Our results show strong correlation of observed tissue birefringence and collagen fiber density, and the lack of the tissue birefringence, due to low collagen fiber density, in the cancerous areas. Our PS-OCT imaging system provides faster and more informative intraoperative tumor margin assessment. It has the potential to determine margin status in real-time during the surgical procedure and reduce reoperation rates.

#3039

Role of IDH mutation status on molecular and spatial heterogeneity in glial tumors across progression and recurrence.

Michael E. Berens,1 Jill S. Barnholtz-Sloan,2 Miribella Rusu,3 John Graf,3 Anup Sood,3 Sanghee Cho,3 Maria Zavodszky,3 Sara Byron,1 Rebecca Halperin,1 Yi Fritz,2 Seungchan Kim,4 Fiona Ginty3. 1 _TGen (The Translational Genomics Research Institute), Phoenix, AZ;_ 2 _Case Western Reserve University Comprehensive Cancer Center, Cleveland, OH;_ 3 _GE Global Research, Niskayuna, NY;_ 4 _Prairie View A &M University, Prairie View, TX_.

We developed and deployed a workflow for generating multi-scale, multiparametric imaging data, feature extraction and/or converting to higher scales which equips multiple analysis approaches to differentiate clinically variable phenotypes of glial tumors. The workflow quantifies spatial heterogeneity (concordance of adjoining cells) and molecular heterogeneity (varied cell states determined by protein abundance) of glial tumors at the genomic, tissue, and medical imaging scales including IDH mutation status and progression/recurrence status. A panel of 24 multiplexed immunofluorescence (MxIF) markers (addressing 9 hallmarks of cancer) was used to profile single cells (in the thousands) in tissue sections from each of 31 glial tumors (ranging from primary grade II to IV, and recurrent grade IV). Pre-resection multi-parameter MR images were feature extracted from discreet habitats (necrosis, enhancing, and edema); whole exome and transcriptome sequencing from bulk viable tumor were analyzed. By MxIF, the various states of individual cells from treatment-naive patient specimens resolved unsupervised into 7 clusters, for which Cluster 2 (including cells from 9 patients) and Cluster 6 (including cells from 8 patients) contained the two larger bundles of patient cases. When separated into IDHmt and IDHwt cases, cells from IDHmt cases frequently contained cell populations dominated by a single cluster (low molecular heterogeneity); cells from cases with IDHwt represented multiple different clusters (high molecular heterogeneity). In grade III astrocytomas, and grade IV recurrent glioblastomas, spatial heterogeneity of the hallmark "inducing angiogenesis" was elevated in the IDHmt tumors compared to IDHwt, while between the same groups, molecular heterogeneity was lower in the IDHmt cases than wild type. Edema from T1w post contrast MR imaging was found to be elevated in IDHwt gliomas relative to IDHmt, while enhancement was reduced in IDHwt compared to IDHmt tumors. The findings demonstrate that IDHmt gliomas, irrespective of grade, show less edema, greater enhancement, and greater spatial heterogeneity of the "inducing angiogenesis" hallmark but lower molecular heterogeneity than IDHwt tumors. Molecular heterogeneity of "cancer invasion" also differed between IDHmt and IDHwt cases. Longer survival duration following diagnosis for patients with IDHmt gliomas may reflect generalized altered molecular and spatial heterogeneity, which is a phenotype evident on medical imaging. [Clinically-annotated specimens originated from the Ohio Brain Tumor Study and the Ivy GBM Clinical Trials Consortium]

#3040

Radiomics discriminates pseudo-progression from true progression in glioblastoma patients: A large-scale multi-institutional study.

Srishti Abrol,1 Aikaterini Kotrotsou,1 Ahmed Hassan,1 Nabil Elshafeey,1 Tagwa Idris,1 Naveen Manohar,1 Anand Agarwal,1 Islam Hassan,1 Kamel Salek,1 Nikdokht Farid,2 Carrie McDonald,2 Shiao-Pei Weathers,1 Naeim Bahrami,2 Samuel Bergamaschi,3 Ahmed Elakkad,1 Kristin Alfaro-Munoz,1 Fanny Moron,4 Jason Huse,1 Jeffrey Weinberg,1 Sherise Ferguson,1 Evangelos Kogias,5 Amy Heimberger,1 Raymond Sawaya,1 Ashok Kumar,1 John de Groot,1 Meng Law,3 Pascal Zinn,4 Rivka R. Colen1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _University of California San Diego, San Diego, CA;_ 3 _University of South California, Los Angeles, CA;_ 4 _Baylor College of Medicine, Houston, TX;_ 5 _University Medical Center Freiburg, Germany_.

BACKGROUND: Treatment-related imaging changes are often difficult to distinguish from true tumor progression. Treatment-related changes or pseudoprogression (PsP) subsequently subside or stabilize without any further treatment, whereas progressive tumor requires a more aggressive approach in patient management. Pseudoprogression can mimic true progression radiographically and may potentially alter the physician's judgment about the recurrent disease. Hence, it can predispose a patient to overtreatment or be categorized as a non-responder and exclude him from the clinical trials. This study aims at assessing the potential of radiomics to discriminate PsP from progressive disease (PD) in glioblastoma (GBM) patients.

METHODS: We retrospectively evaluated 304 GBM patients with new or increased enhancement on conventional MRI after treatment, of which it was uncertain for PsP versus PD. 149 patients had the histopathological evidence of PD and 27 of PsP. Remaining 128 patients were categorized into PD and PsP based on RANO criteria performed by a board-certified radiologist. Volumetrics using 3D slicer 4.3.1 and radiomics texture analysis were performed of the enhancing lesion(s) in question.

RESULTS: Using the MRMR feature selection method, we identified 100 significant features that were used to build a SVM model. Five texture features (E, CS, SA, MP, CP) were found to be most predictive of pseudoprogression. On Leave One Out Cross-Validation (LOOCV), sensitivity, specificity and accuracy were 97%, 72%, and 90%, respectively. Using 70% of the patient data for training and 30% for validation, an AUC of 94% was achieved, with the sensitivity of 97% and specificity of 75%.

CONCLUSION: 3D radiomic texture features of conventional MRI successfully discriminated pseudoprogression from true progression in a large cohort of GBM patients.

#3041

Molecular, histopathologic, MRI and PET findings in syngeneic oncologic mouse models.

Julia Schueler,1 Artem Shatillo,2 Jussi Rytkönen,2 Nina Zanella,1 Antti Nurmi,2 Tuulia Huhtala2. 1 _Charles River, Freiburg, Germany;_ 2 _Charles River, Kuopio, Finland_.

Experimental tumors raised in rodents represent an important preclinical tool to develop innovative anticancer compounds before clinical testing. Amongst others such models include solid tumors raised in syngeneic fully immunocompetent hosts and tumors spontaneously growing in genetically engineered mice (GEM) and derivate thereof. These model platforms have gained additional value since the manipulation of the immune system to fight cancer has led to tangible benefits for cancer patients. Imaging has become important part of the basic and translational research. It enables e.g. metabolic activity, volumetric and biodistribution monitoring within same individual over time. Magnetic resonance imaging (MRI) and positron emission tomography (PET) are widely used for clinical diagnosis and disease follow up. Choosing the most suitable imaging application depends of the prioritization of features. MRI offers unprecedented soft tissue contrast, high spatial resolution and non-invasive nature renders MRI in rodents a perfect tool for preclinical work in oncological applications. Similarly to clinical setting, MRI in rodents is used for detection of the tumors, evaluation of therapeutic response and induced changes, with longitudinal follow-up for early detection of possible tumor recurrence. PET is an excellent tool to study tumor metabolism, location as well biodistribution of novel antibodies. The purpose of this work was to study molecular as well volumetric, metabolic and functional changes in syngeneic oncological mouse model using MRI, MRS and PET imaging. Our panel consists of 32 fully characterized mouse tumor models covering 12 major cancer types. We characterized those models by molecular profiling (whole exome sequencing and RNAseq), histopathological examination (tissue micro array as well as whole slide analysis of Ki67, CD31, SMA, TIL infiltration) and sensitivity towards checkpoint inhibitors. Amongst other findings we saw an enhanced activity of anti-CTLA-4 treatment in combination with anti-PD1 in 4T1 orthotopically implanted into the mammary fat pad in comparison to single agent therapy. In a follow-up study we implanted 14 mice with breast cancer cell line 4T1 orthotopically into the mammary fat pad. When tumors were palpable treatment with the checkpoint inhibitor combination started. During the course of the experiment we determined tumor volume of the primary lesion twice weekly. In addition to molecular markers, MRI, MRS and PET were applied to study changes in tumor over cancer progression. As a conclusion, several syngeneic oncological mouse models have been characterized using molecular profiling, histological techniques and in vivo imaging. These readouts provide a powerful and translational research tool together with oncological disease animal models allowing comprehensive evaluation of disease progression and treatment interventions for in vivo studies.

#3042

Nuclear morphology predicts prostate cancer metastasis at diagnosis.

Fangjin Huang,1 Nathan Ing,1 Eric N. Miller,2 Hootan Salemi,1 Michael S. Lewis,2 Isla P. Garraway,2 Arkadiusz Gertych,1 Beatrice S. Knudsen1. 1 _Cedars-Sinai Medical Center, Los Angeles, CA;_ 2 _David Geffen School of Medicine at UCLA, Los Angeles, CA_.

Background: Prostate needle biopsies (PNBX) that are obtained during the initial diagnostic workup are often the only source of tumor tissue that is available to determine the severity of prostate cancer (PC). Interestingly, tumor grades and pathologic features in PNBXs of men with lethal metastatic disease (M1 stage) are indistinguishable from those of high-grade nonmetastatic tumors of patients who do not progress after treatment (M0 stage). We hypothesize that the morphology of tumor nuclei can be used as a source of biomarker development to distinguish M1 tumors from high-grade localized M0 tumors.

Methods: Our study consists of a cohort of 85 high-grade M0 and 78 M1 cases, within a biorepository of 2150 patients at the Greater Los Angeles Veterans Affairs hospital. PNBX slides were digitized at 40X and pathologists annotated all cancer foci. These annotated regions were divided into smaller image tiles (dimensions) and fed into our digital image analysis pipeline for nuclear segmentation and feature extraction. We applied two distinct feature extraction methods to capture morphologic information within tumor nuclei. The 64 Handcrafted (HC) features describe nuclear properties such as shape, area, and chromatin conformation. The 62 Autoencoder (AE) features are abstract descriptors generated by a deep learning algorithm, which learns to redraw the nuclei. We built 2 machine learning models using AE or/and HC features for classification of M1 versus M0 cases. We divided our patients into 3 groups: training, testing and validation. A 7-fold cross-validation was performed in the training and testing sets and the area under the curve of the receiver operating characteristic (ROC) was calculated in the validation set.

Results: Model 1 utilizes processed features derived from patient-level distributions and generalized linear model. The estimated AUC for predicting M1 stage is 0.79 and 0.74 for HC and AE features, respectively. Model 2 uses the average of each features obtained from dominant group of nuclei within individual tiles and neural network models. Nuclei groups were assigned by unsupervised clustering method. For this model, the AUC of predicting M1 stage is 0.77 and 0.75 for HC and AE features, respectively.

Conclusion: By applying two distinct feature extraction methods and two approaches to summarize features, we obtain similar prediction accuracies. These results demonstrate that quantitative nuclear features contain information to classify M1 and M0 cases, which cannot be classified based on tumor grade or pathologic features.

#3043

Quantitative MRI during neoadjuvant therapy for predicting breast cancer response in the community setting.

Anna Sorace,1 Jack Virostko,1 Chengyue Wu,1 Angela M. Jarrett,1 Stephanie L. Barnes,1 Debra Patt,2 Boone Goodgame,3 Sarah Avery,4 Thomas E. Yankeelov1. 1 _University of Texas at Austin, Austin, TX;_ 2 _Texas Oncology, Austin, TX;_ 3 _Seton Healthcare Family, Austin, TX;_ 4 _Austin Radiological Association, Austin, TX_.

Purpose: Quantification of accurate and early response to neoadjuvant therapy (NAT) provides the opportunity to replace an ineffective treatment with an alternative regimen, thereby potentially avoiding ineffective systemic therapy. Quantitative magnetic resonance imaging (MRI) has been shown to predict breast cancer response to treatment early during the course of NAT within academic medical centers. Integrating quantitative imaging techniques into community-based medical practices has the potential to reach a larger percentage of breast cancer patients, and allow more community center participation in clinical trials that require quantitative imaging. This study evaluated the reproducibility and accuracy of quantitative dynamic contrast-enhanced MRI (DCE-MRI) and diffusion-weighted MRI (DW-MRI) in the community setting and the ability to implement these techniques to predict the eventual response of breast tumors to NAT.

Experiment Procedure: MRI was performed at two community imaging centers and one academic research facility using a 3T Siemens Skyra scanners equipped with a breast coil. To assess reproducibility across sites, normal subjects (N=3) were scanned at three imaging centers. Quantitative T1 (required for pharmacokinetic modeling of DCE-MRI) and apparent diffusion coefficient (ADC) maps were calculated of normal fibroglandular breast tissue. Accuracy was tested through evaluation of T1 and DW-MRI in phantoms. Women undergoing NAT for breast cancer (N=10) were scanned with DCE-MRI and DW-MRI at baseline (prior to beginning therapy) and three longitudinal time points during the course of NAT to evaluate early prediction of response to therapy. Quantitative measures of ADC (evaluating cellularity from DW-MRI) and Ktrans (evaluating vascular perfusion and permeability from DCE-MRI) were calculated for the segmented tumor volume. Imaging was compared to pathology reports at the conclusion of NAT.

Results: Reproducibility scans of normal breast fibroglandular tissue yielded an average difference of 8.4% and 7.0% in T1 and ADC measurements, respectively, across sites. Phantom studies revealed accurate measurements of T1 mapping and ADC, with reproducibility measurements showing a difference of 2.8% and 1.6%, respectively. Patients achieving a pathological complete response (pCR) revealed a 13.8% ± 19.0% increase in the mean ADC values of the tumor from t1 (baseline, prior to beginning NAT) and t2 (following one round of NAT) and a 15.4% ± 40.9% decrease in mean Ktrans. Conversely, patients that did not achieve non-pCR had little change in ADC (-0.9% ± 12.6% change between t1 and t2) and a 15.3% ± 42.8% increase in Ktrans.

Conclusions: Quantitative MRI has been shown to be accurate and reproducible across community medical centers. Furthermore, the preliminary results discussed above parallel those previously reported in the academic research setting.

#3044

Label-free detection and identification of cancer-associated vesicles in human breast cancer.

Sixian You,1 Yi Sun,1 Haohua Tu,1 Ronit Barkalifa,1 Eric J. Chaney,1 Marina Marjanovic,1 Anna M. Higham,2 Natasha N. Luckey,2 Kimberly A. Cradock,2 Z. George Liu,2 Stephen A. Boppart1. 1 _University of Illinois at Urbana-Champaign, Urbana, IL;_ 2 _Carle Foundation Hospital, Urbana, IL_.

Detection and identification of cancer-associated vesicles in their native tissue environment is essential to understand vesicle-mediated communication in cancer progression and potentially facilitate the treatment and management of cancer. Here we develop a label-free optical imaging method that can reliably detect cancer-associated vesicles in vitro, in vivo and in untreated human tissue ex vivo. Preliminary analysis conducted on fresh human breast tissue obtained from healthy and cancer subjects showed that a significant portion of vesicles from the cancer subjects have unique optical signatures in comparison with those from healthy subjects. Additionally, the spatial distribution of these vesicles with unique optical signatures in the tumor micro- and macro-environment appeared correlated with the staging of human breast cancer. These results suggest the potential capability of this methodology to identify and characterize the tumor associated vesicles in the authentic tumor microenvironment, which could open new windows in the explorations of their diagnostic values and physiological roles in cancer progression.

#3045

Pilot study of kernel-based dynamic (KBD) FDG-PET in patients with borderline resectable pancreatic cancer.

John P. Schwerkoske, Guobao Wang, Kit W. Tam, Jasmine C. Huynh, Heather H. Hunt, Michael L. Rusnak, Cameron C. Foster, Michael T. Corwin, Karen E. Matsukuma, Dorina Gui, May T. Cho, Richard J. Bold, Ramsey D. Badawi, Edward J. Kim. _UC Davis, Sacramento, CA_.

Introduction: Patients (pts) with borderline resectable (BR) pancreatic cancer (PC) derive a clear survival benefit when margin-negative resection can be achieved after neoadjuvant treatment (tx). As such, reliable imaging is desperately needed to identify those patients with the best chance of surgical benefit. Current imaging with CT/MRI and standard 18F-fludeoxyglucose (FDG) PET lacks sensitivity and specificity to resolve residual tumor contact with the large blood vessels of the vascular groove. Dynamic PET with kernel-based image reconstruction developed by our group has potential to overcome limits of standard static PET by robust parametric imaging of radiotracer kinetics. KBD FDG-PET provides the FDG kinetic parameter K1 for tumor perfusion and Ki for tumor glucose utilization. A lower Ki/K1 ratio indicates a better perfused, less metabolically active tumor and is derived by our method without need for separate perfusion CT/MRI. Methods: Pts with PC staged as BR as defined by consensus guidelines were eligible. Pts were assessed by CA 19-9 and CT/MRI along with KBD FDG-PET both pre-tx and post-tx. KBD FDG PET was performed by IV bolus of 10 mCi of FDG followed by 60-min PET data acquisition. A 3-compartment model with 5 micro kinetic parameters K1, k2, k3, k4, and fv was used where K1 (mL/min/mL) denotes the rate constant of FDG transport from plasma to tissue, k2 (1/min) the transport rate from tissue to plasma, k3 (1/min) the rate of FDG conversion to FDG 6-phosphate, k4 (1/min) the rate of dephosphorylation, and fv the fractional blood volume. Ki was calculated from micro parameters by the formula K1*k3/(k2+k3). Parametric images of these kinetic parameters were obtained by voxel-wise implementation of kinetic modeling. Results: 4 pts were enrolled, 3 pts had pre- and post-tx KBD FDG-PET, and 2 pts were resected. In the 3 evaluable pts, an overall 62% decrease in tumor glucose utilization was observed (mean pre-tx Ki=.03, [range .013-.045]; mean post-tx Ki=.011 [range .004-.016]. Mean pre-tx K1=.37 (range .26-.50) and mean post-tx K1=.38 (range .31-.46). Delta Ki was -17%, -47%, and -91% and delta Ki/K1 was -9%, -28%, and -95% in pts 1, 2, 3 respectively. Pt 3 had the highest pre-tx Ki and the lowest pre-tx K1, but had a >90% decrease in Ki and Ki/K1 post-tx, indicating a significant decrease in metabolic activity and concomitant increase in perfusion. Despite persistent superior mesenteric vein (SMV) abutment on standard post-tx imaging, margin-negative resection was achieved. In contrast, pt 1 had the lowest pre-tx Ki and highest pre-tx K1, but had a <20% decrease in Ki and Ki/K1 post-tx. Like Pt 3, Pt 1 also had SMV contact on standard post-tx imaging, but in contrast, experienced margin-positive resection. Conclusions: KBD FDG-PET is a promising modification of standard static PET that provides useful kinetic parameters for evaluation of PC resectability.

#3046

Evaluation of pancreatic ductal adenocarcinoma with acidoCEST MRI.

Rachel A. High,1 Edward A. Randtke,1 Kyle M. Jones,1 Mark D. Pagel2. 1 _University of Arizona, Tucson, AZ;_ 2 _UT MD Anderson Cancer Center, Houston, TX_.

Introduction: Our goal is to evaluate the role of using extracelluar pH (pHe) as a predictor of pancreatic ductal adenocarcinoma (PDAC) development using a non-invasive, in vivo imaging technique called acidoCEST MRI, to improve early detection of pancreatic tumors.

Methods: Spontaneous PDAC development was initiated by administering 14 caerulein injections over a 62 hour period to a KrasLSL.G12D/+; PdxCre mouse model. Caerulein induces pancreatitis which drives tumor development in the context of a Kras mutation. Animals were imaged with a Bruker BioSpin 7T MRI scanner with an acidoCEST MRI protocol at pre-injection, 1 hour and 48 hours post final injection of caerulein, and weekly until tumors reached a size of 200mm3. During all MR imaging, mice were anesthetized with 2.0% isofluorane, maintained at a respiration rate of 35-40 bpm, and maintained at a body temperature of 37°C. For acidoCEST MR imaging, mice were injected with 370 mg/mL iopamidol (200 μL IV bolus and 400 μL/hr IV infusion) and scanned with a 6 sec saturation time at 3.5 μT, with a 300 ms acquisition time. Pixel-wise parametric maps of pancreatic pHe values were generated via fitting the CEST spectrum with the Bloch-McConnell equations in MATLAB R2016a. Average pancreatic pHe was recorded.

Results: AcidoCEST MRI was successful in acquiring in vivo pHe of normal and tumor pancreatic tissue, both with sufficient uptake of the iopamidol contrast agent. The pHe values for healthy pancreatic tissue ranged from 6.83 to 7.33 with an average of 6.96 (n=14). 1 hour after the last caerulein injection, during pancreatic inflammation, pHe ranged from 6.78 to 7.00, with an average of 6.92 (n=6). Tumors began to reach a size of 200mm3 at 5 weeks post caerulein injections. In tumor tissue, pHe was 6.59 (n=1), demonstrating tumor acidosis.

Conclusions: We have demonstrated that acidoCEST MRI can be used to quantify pHe in healthy pancreatic tissue, pancreatitis, and pancreatic ductal adenocarcinoma. These pHe values will be used to determine if acidity in pancreatitis correlates with future development of PDAC and/or a more aggressive phenotype. Further pre-clinical studies using acidoCEST MRI may be performed to monitor early therapeutic response to pancreatic tumor treatment.

#3047

Development and initial application of a porcine-specific MRI and MRE protocol for liver imaging in a large animal cancer model.

Faith M. Thomas,1 Bradley P. Sutton,1 Tracey M. Wszalek,1 Megan J. Dailey,1 Kyle M. Schachtschneider,2 Lawrence B. Schook,2 Ron C. Gaba2. 1 _University of Illinois at Urbana-Champaign, Urbana, IL;_ 2 _University of Illinois at Chicago, Chicago, IL_.

While global cancer mortality is generally decreasing, the incidence of hepatocellular carcinoma (HCC) is projected to increase given the growing prevalence of chronic liver diseases that increase the risk for carcinogenesis. The current diagnosis of liver cirrhosis is invasive tissue biopsy, and those at-risk undergo an HCC surveillance program of ultrasound imaging to survey tumor development. Such screening programs center on static radiologic imaging snapshots prescribed at arbitrary intervals based on empiric histologic cirrhosis staging schemes and observational tumor developmental data that neither reflect the transition between normal and diseased states nor biologically relevant disease thresholds. In contrast, magnetic resonance imaging (MRI) and magnetic resonance elastography (MRE) may provide a more foundational portrayal of early stages of liver disease and liver oncogenesis that could result in early diagnosis when curative therapies could be employed. In addition, MRI/MRE-correlated systemic molecular biomarkers indicative of specific disease stages also have potential to serve as important non-invasive diagnostic tools. In this study, we developed a clinically translatable MRI/MRE liver imaging protocol in a clinically relevant large animal platform, the Oncopig Cancer Model (OCM). The OCM is a novel transgenic swine model that recapitulates human HCC through development of site and cell specific tumors after Cre recombinase induced expression of heterozygous KRASG12D and TP53R167H transgenes. A clinical imaging workflow consisting of respiratory gated acquisitions was developed with the Siemens 3 T Prisma MRI scanner to include the following: T1 Flash in-phase and out-of-phase, T2-HASTE/BLADE acquisitions to provide motion-robust imaging, VIBE imaging, diffusion-weighted imaging with IntraVoxel Incoherent Motion (IVIM) for estimating blood flow, and MRE for quantifying liver stiffness. Additional MRI imaging for tumor characterization was also performed, spanning multiphase (arterial, venous, delayed) contrast-enhanced T1-weighted imaging. This novel imaging protocol was successfully applied in normal control Oncopig subjects (n = 2), resulting in high quality, high resolution MRI/MRE images for clinical interpretation. In the future, integration of MRI/MRE, histology, and molecular measures in Oncopigs presenting with liver cirrhosis and HCC may be employed to investigate biomarkers that may define normal versus diseased liver, and may serve as a new approach to identify optimal time points for initiation of disease screening and treatment administration for clinical translation.

#3048

Deep learning to predict survival prognosis for patients with non-small cell lung cancer using images and clinical data.

Edward H. Lee, Mu Zhou, Noah Gamboa, Kevin Brennan, Haruka Itakura, Viswam Nair, Sandy Napel, Simon Wong, Olivier Gevaert. _Stanford University, Stanford, CA_.

Goal

We present a deep learning framework to predict survival prognosis of patients with non-small cell lung cancer using CT images and clinical data developed and validated in multi-institutional cohorts.

Methods

We developed 3D convolutional neural networks (CNNs) using CT images, tumor nodule segmentation, clinical data (i.e. age, sex, histology, and stage) and survival times as input to predict survival of non-small cell lung cancer. Both the CT images and the segmentations were used as inputs for the CNN, the latter to localize the volumes-of-interests (VOIs) in the CT scans. We used three cohorts to evaluate our framework: Moffitt (n=186), Maastro (n=311), and Stanford (n=130). We investigated pre-training with the LIDC-IDRI dataset (n=1010) and compared deep learning performance with 1674 manually-curated radiomics features and clinical data. We used two ways to evaluate the models. First, we split the three cohorts into training and test sets by selecting two of the cohorts for training and the remaining for testing. Secondly, we combined all cohorts and performed a 10-fold stratified cross-validation. We used the Concordance Index (CI) to estimate model performance. Next, we evaluated priming whereby we use 10% of the validation cohort during training and test on the remaining patients in the test cohort. To ensure that the test set was identical for both priming and non-priming experiments, we discarded these samples for the non-priming tests.

Results

Table 1 shows the results of the two evaluation strategies. We observed consistently high CIs for all three cohorts. LIDC pretraining and priming yielded the highest CIs as compared to radiomic and clinical-only pipelines. For the 10-fold cross-validation experiment, our pipeline achieved an average CI=0.64.

Conclusion

Our study highlights the promising results of our pipeline to predict survival prognosis across three cohorts without the need to define radiomics features. | |  | |

|

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

Train on two cohorts, test on 3rd cohort | pretrain on LIDC-IDRI | test

w/o priming | test

w/ priming | test w/o priming

(only radiomics

features / clinical) | test w/ priming

(only radiomics features / clinical)

train on Moffitt + Maastro, test on Stanford | yes | 0.62 | 0.63 | 0.52 (radiomics only) | 0.53 (radiomics only)

train on Moffitt + Maastro, test on Stanford | no | 0.61 | 0.62 | 0.57 (clinical only) | 0.57 (clinical only)

train on Moffitt + Stanford, test on Maastro | yes | 0.6 | 0.6 | 0.55 (radiomics only) | 0.55 (radiomics only)

train on Moffitt + Stanford, test on Maastro | no | 0.58 | 0.6 | 0.56 (clinical only) | 0.56 (clinical only)

train on Stanford + Maastro, test on Moffitt | yes | 0.58 | 0.6 | 0.53 (radiomics only) | 0.53 (radiomics only)

train on Stanford + Maastro, test on Moffitt | no | 0.6 | 0.57 | 0.56 (clinical only) | 0.56 (clinical only)

#3049

Dexamethasone pretreatment impairs the TS inhibition-mediated flare in thymidine salvage pathway activity.

Sharyn I. Katz, Xiao Chen, Yizeng Yang. _Univ. of Pennsylvania, Philadelphia, PA_.

Introduction: Inhibition of thymidylate synthase (TS) by pemetrexed, an inhibitor of thymidylate synthase, results in an early transient burst or "flare" in thymidine salvage pathway activity at 2 hrs of therapy, which, we have demonstrated in prior publications, can be measurable with FLT ([18F]thymidine)-positron emission tomography (PET) in non-small cell lung cancer (NSCLC). Administration of dexamethasone with pemetrexed-based therapy, as typically practiced in the clinic, could potentially confound this imaging approach since dexamethasone is known to inhibit expression of thymidine kinase 1, a key enzyme in the thymidine salvage pathway. Here we examine the potential impact of dexamethasone on the TS inhibition-mediated thymidine salvage pathway "flare" in NSCLC.

Materials and Methods: In order to determine NSCLC cell line sensitivity to dexamethasone and pemetrexed, IC50 studies were performed on NSCLC cell lines H23, H1975, H460, H1299. TS inhibition-mediated "flare" in thymidine salvage pathway activity was then measured at 2hrs of exposure to pemetrexed and cisplatin in NSCLC cells lines following using 3H-thymidine incorporation assays under the following conditions: control (no chemotherapy or dexamethasone), treated with pemetrexed and cisplatin without dexamethasone, with 24 pretreatment of dexamethasone, or with dexamethasone administered together with chemotherapy. These conditions were chosen to model the delivery of pemetrexed-based therapy in the clinic.

Results: The IC50 of H23, H1975, H460, H1299 for dexamethasone and pemetrexed were 40, 5.9, 718, 362 uM and 0.22, 0.73, 0.14 and 0.66 uM, respectively. Significant blunting of the thymidine salvage pathway "flare" is observed at 2hrs of pemetrexed-based therapy when dexamethasone-sensitive cell lines H23 and H1975 were pretreated with dexamethasone but not when dexamethasone was given together with pemetrexed therapy or in the setting of dexamethasone resistance (H460 and H1299).

Conclusion: Imaging of TS inhibition may be impacted by pretreatment with dexamethasone but not administration of dexamethasone on the same day of therapy delivery.

#3050

Imaging tissue extracellular pH for the differential diagnosis of coccidioidomycosis and lung cancer.

Leila Renee Lindeman,1 Edward A. Randtke,2 Lisa F. Shubitz,3 Christine M. Howison,2 Kyle M. Jones,2 Mark D. Pagel4. 1 _Univ. of Arizona Cancer Ctr., Tucson, AZ;_ 2 _University of Arizona, Tucson, AZ;_ 3 _Univeristy of Arizona, Tucson, AZ;_ 4 _MD Anderson Cancer Ceter, Houston, TX_.

Introduction: Our objective was to determine whether acidoCEST MRI—a novel, non-invasive MRI method that measures extracellular pH (pHe)—can differentiate between lung tumors and coccidioidomycosis (valley fever) granulomas within in vivo mouse models of lung cancer and valley fever.

Methods: To develop a spontaneous murine lung tumor model, A/J mice received orthotopic injections of urethane to induce formation of lung adenocarcinomas. The Valley Fever Center for Excellence at the University of Arizona infected SW mice with a BSL 2-compatible mutant Coccidioides strain, Δcps1, to develop our preclinical valley fever model. All scans were performed with a Bruker BioSpin 7T MRI system. For all MRI scans, mice were anesthetized with 2.0% isofluorane, respiration and body temperature were monitoreduring scans, and body temperature was maintained at 37 °C. Respiration-triggering (gating) was used in all imaging sequences to compensate for motion artifacts in the lung. For optimal gating, the mouse's respiration rate was maintained at < 50 breaths per minute. Each mouse was scanned with acidoCEST MRI using 370 mg/mL Iopamidol (200 μL IV bolus, 400 μL/hr IV infusion). AcidoCEST MRI (3.5 μT, 300 ms imaging time, 6000 ms presaturation pulse) was performed according to previously published methods, updated with improved respiration gating. For acidoCEST MRI, the saturation pulse continued until terminated by the gating trigger. Spatial pHe maps of tumor and granuloma ROI were produced using Bloch fitting in Matlab 2014a. Average lesion pHe and iopamidol concentration we also recorded.

Results: AcidoCEST MRI was successfully applied to the in vivo imaging of murine lung tumors and coccidioidomycosis granulomas. Lung tumors demonstrated successful uptake of the iopamidol contrast agent, with an average concentration of approximately 40 mM. pHe values in the tumor ROI ranged from 6.5 to 7.2 with an average value of 6.64. Granulomas demonstrated successful uptake of iopamidol with an average concentration of about 75 mM. pHe values in granulomas ranged from 7.2 to 7.4 with an average value of 7.29.

Conclusion: AcidoCEST MRI may be used to quantify the pHe of murine lung tumors and coccidioidomycosis granulomas in vivo. Our results show that pHe is a promising biomarker for the differential diagnosis of coccidioidomycosis and lung cancer.

#3051

**Prognostic value of** 18 **F-FDG PET/CT parameters in patients with primary pulmonary lymphoepithelioma-like carcinoma.**

Yueh-Fu Fang, Chun-Yu Lin. _Chang Gung Memorial Hospital, Taipei, Taiwan_.

Introduction Pretreatment tumor metabolic burden, which measured by fluorine-18 fluorodeoxyglucose positron emission tomography/computerized tomography (18F-FDG PET/CT) have been shown to predict outcomes in various malignancies. Epstein-Barr virus (EBV) serology titer in pulmonary LELC is correlated to stage. However, the prognostic value of 18F-FDG PET/CT in patients with pulmonary lymphoepithelioma-like carcinoma (LELC) and the relationship between EBV viral load remained unknown. The current study aims to investigate the prognostic value and association between functional parameters of 18F-FDG PET/CT and serum EBV DNA in patients with pulmonary LELC.

Methods This retrospective study comprised 32 patients with pulmonary LELC who underwent pretreatment 18F-FDG PET/CT between January 2008 and December 2016. The EBV viral load and the functional parameters of 18F-FDG PET/CT were used for survival analysis.

Results The value of pre-treatment serum EBV DNA had significantly positive correlation with whole MTV (r = 0.63, P = 0.0337) and whole TLG (r = 0.77, P = 0.0093). Advanced tumor stage (≥III) and high whole MTV (≥72.58) significantly predicted poor PFS. Multivariate survival analysis for PFS showed that there was a trend toward a lower risk of recurrence in patients with early tumor stage (≤II) (HR = 0.112, 95% CI =0.011-1.117, P = 0.062, Table 3), albeit not significantly so. Advanced tumor stage (≥III), high whole MTV (≥72.58) and high whole TLG (≥278.4) were predictors of poor OS. However, in multivariate survival analysis, none of them were independent predictors for OS. EBV viral load was not prognostic factor for both PFS and OS.

Conclusion Greater whole MTV and whole TLG predicted poorer OS in patients with primary pulmonary LELC. Functional parameter of 18F-FDG PET/CT may provide useful prognostic information. Serum EBV DNA was closely related to whole MTV and TLG, but not a prognostic factor. 

### Cancer Stem Cell Characterization

#3052

Identification and characterization of DPPA2 as a potential cancer stem cell marker in hepatocellular carcinoma.

Dandan Yu,1 Qian Yan,1 Yu Zhang,1 Ling Guo,2 Chuanyue Wu,2 Xin-yuan Guan1. 1 _The University of Hong Kong, Hong Kong, Hong Kong;_ 2 _Southern University of Science and Technology, Shenzhen, China_.

Cancer stem cells (CSC) are a small subpopulation of tumor cells that share many properties with embryonic stem cells, including the ability to differentiate and self-renew. The study of stemness-associated genes might give us a hint on the tumorigenesis and tumor progression. By comparing the expression profiles of human embryonic stem cells (ESC), endoderm, liver progenitors, premature hepatocytes as well as two pairs of non-tumor and tumor tissues of hepatocellular carcinoma (HCC), we found that the gene Developmental Pluripotency Associated 2 (DPPA2) was highly expressed in the stage of ESC, and decreased dramatically along with development, suggesting its role as a stemness associated gene. Analysis of HCC clinical samples showed that elevated expression of DPPA2 was significantly associated with differentiation, TNM stage, the number of tumor nodules and thrombus, as well as poorer prognosis. Functional studies demonstrated that DPPA2 - overexpressed cells displayed enhanced stemness feature, increasing the ability of self-renewal, tumorigenicity and chemo-resistance. We also found that overexpression of DPPA2 could increase the expression of some cancer stem cell markers. RNA-sequencing results suggested that Wnt signaling was abnormally activated after DPPA2 overexpression. Our findings point to a new potential cancer stem cell marker which may provide an attractive therapeutic target for HCC patients.

#3053

Characterization of cancer stem cell subpopulations from patient-derived xenografts of advanced prostate cancer.

Sofia Karkampouna,1 Marta De Menna,1 Markus Germann,2 Joel Grosjean,1 Peter Clark Gray,3 George Niklaus Thalmann,1 Marianna Kruithof-de Julio1. 1 _University Bern, Bern, Switzerland;_ 2 _ISREC, EPFL, Lausanne, Switzerland;_ 3 _Clayton Foundation Laboratories for Peptide Laboratories, Salk Institute, La Jolla, CA_.

Androgen-deprivation therapy is the standard treatment for prostate cancer. Despite the initial response, a fraction of cases manifest progression to castration-resistant prostate cancer. PCa recurrence is possibly due to androgen-independent cancer stem cells that reinitiate tumor growth. The properties of cancer stem cells versus highly proliferative progenitor cells remain to be further characterized at the cellular and gene expression level. To address the molecular basis of CSC switch from androgen dependency to independence, we have employed patient-derived xenograft models (LAPC-9 and BM-18) that have different androgen sensitivity properties. We have performed microarray and proteomic analysis of BM-18 tumor tissues prior to and following castration as well as androgen replacement. To characterize the cancer stem cells in these models we have isolated different subpopulations by FACS cytometry, based on combination of selected markers (CD44 and ALDH activity measured by ALDELFUOR assay) aiming to assess the transcriptomic profile of these different populations. To assess response of these CD44+/-/ALDH+/- cells to androgen targeting compounds we have established an androgen-sensitive xenograft model (PNPCa) and tested it by ex vivo tissue culture and organoid viability assays. Proteomic and microarray analysis of bulk BM-18 tumors indicates enrichment of stem cell markers upon castration: CD44, NKX3.1 and ALDH1 isoforms. Readministration of testosterone induces decrease of these markers. Castration induces a rapid tumor volume decrease in the BM-18 as opposed to LAPC-9, reflecting different androgen-dependent cell states. CD44+/-/ALDHhigh/low subpopulations were isolated by flow cytometry and for transcriptomic analysis. Castration in the BM18 model induces an increase in the subpopulations of CD44+/ALDHlow and CD44+/ALDHhigh, while ALDHsingle and ALDHhigh total populations are decreased. Both CD44 expression and ALDH activity is increased in the LAPC-9 model upon castration. Treatment of PNPCa tumor parts ex vivo with enzalutamide inhibited expression of androgen receptor and keratin 8 expression. CD44+ organoids derived from the same tissue did not show altered viability after enzalutamide treatment. Different androgen-independent cancer stem cell populations may be distinguished by ALDH activity status and CD44. Androgen-independent cells in the BM-18 androgen-dependent model are reflected by enrichment of CD44 expression. The least abundant subpopulation CD44+/ALDHhigh is present in the LAPC-9 and expanded upon castration, while in the BM-18 model it is exclusively present after castration, reflecting different androgen sensitivity. Ongoing analysis may elucidate the molecular mechanisms controlling cancer stem cell fates, androgen sensitivity and drug resistance.

#3054

High-throughput microfluidic Labyrinth for the label-free isolation of circulating tumor cells.

Eric Lin. _University of Michigan, Ann Arbor, MI_.

Circulating tumor cells (CTCs) present in the blood are the instruments of metastasis and are of high biologic and clinical relevance. Single-cell technologies are playing an increasing role in profiling CTCs in the peripheral blood for detection and real-time monitoring of cancer metastasis. CTCs also help in identifying its distinct drivers. However, current approaches are limited to manual selection of CTCs, which hinders the unbiased comprehensive study of CTCs on a single-cell level. We present a unique label-free microfluidic "Labyrinth" device to isolate CTCs at a high throughput of 2.5mL/min, offering the first biomarker-independent single-cell isolation and genomic characterization platform to study heterogeneous CTC subpopulations in cancer patients. The Labyrinth takes advantage of inertial forces on the microscale in curved geometries to differentially focus cells based on their size. This novel strategy of multicourse path traversing across inner loops to outer loops yielding highest hydrodynamic path length enables focusing of white blood cells (WBCs), leading to high recoveries (> 90%) and efficient separation of WBCs from CTCs, resulting in high purity (~ 200 WBCs/mL), even in whole blood samples without any preprocessing. CTCs were successfully isolated from 56 breast cancer (9.1 CTCs/mL average, range 2-31/mL) and 20 pancreatic cancer (51.6 CTCs/mL average, range 11-115/mL) patients. We detected not only CTCs typically defined by epithelial markers, but also significant numbers of CTCs (>50%) lacking epithelial markers but expressing mesenchymal and cancer stem cell (CSC) markers. Patient samples were then analyzed using single-cell multiplex gene expression. Seventy single cells were successfully recovered and used to identify different subpopulations of CTCs based on their genetic signature, unlike other methods where a positive or negative selection based on protein expression is used. Interestingly, both inter- and intrapatient molecular heterogeneity at the single-cell level in CTCs were observed with cells expressing genes uniquely related to epithelial, MET, and EMT phenotypes. The Labyrinth platform allows a thorough molecular understanding of the heterogeneity among CTCs. This platform also shows CTCs' potential as a biomarker to noninvasively evaluate tumor progression and response to treatment in cancer patients.

#3055

A model of CSC converted from iPSC in the conditioned medium of HCC paving the way to establish HCC CSC.

Said M Afify,1 Anna Sanchez Calle,2 Kazuki Kumon,1 Hend M Nawara,1 Apriliana C Khairani,1 Hafizah Mahmud,1 Aung Ko Ko Oo,1 Du Juan,1 Maram H Zahara,1 Akimasa Seno,1 Tomonari Kasai,1 Yoshiaki Iwasaki,3 Masaharu Seno1. 1 _Graduate School of Natural Science and Technology, Okayama University, Japan, Okayama, Japan;_ 2 _National Cancer Center Research Institute, Tokyo, Japan;_ 3 _Graduate School of Medicine Okayama University, Okayama, Japan_.

Hepatocellular carcinoma (HCC) represents the major histological subtype, accounting for 70%–85% of cases of primary liver cancer. Liver CSCs sustain their self-renewal remains largely unknown. The self-renewal and pluripotency of liver CSC are maintained by several signaling cascades. However, the mechanism of regulating these signaling is unknown.

In this study, we tried to convert mouse iPSCs into CSCs with the conditioned medium (CM) from human HCC cell line Huh7 cells without any genetic manipulation aiming at establishing liver CSCs.

First of all, CM was collected from confluent culture of Huh7 cells. Then, mouse iPSCs cells without MEF feeder cells were cultured in the presence of 50% CM for 4 weeks. The medium was changed every day with fresh medium containing 50% of CM. Mouse iPSCs cultured in the complete medium with LIF were used as a control.The survived cells (5x105 cells) were suspended in HBSS and injected into the liver of BALB/c nude mice. After 25 days malignant tumor was formed in the liver while benign teratoma was formed by the injection of iPSCs. Tumors were then excised and partly fixed in 10% neutral formalin buffer solution for HE staining and immunohistochemical analysis. The rest of tumors were subjected to rt-qPCR anaylsis and primary culture. Immunohistochemical analysis with GFP antibody showed that malignant tumor sustained GFP expression while teratoma from miPSCs did not. Immunocytochemistry of the primary cells from malignant tumor showed high expression of both glypican-3 (GPC3) and cytokeratin19 (CK19) when compared to that in miPSCs. Expression of stem cell markers (Nanog, Oct3/4, Sox2, Klf4) and CSC markers (CD44, EpCAM) were also confirmed by rt-qPCR in both the CSCs converted from iPSCs (miPS-Huh7cm cells) and the primary culture cells. These results indicate that the primary cells from the malignant tumor are rich in CSCs with high expression of GPC3 and CK19, which paves the way to establish a model of HCC CSC. This model should be very important and useful to assess the significant molecular mechanisms necessary to maintain HCC CSC, which will help develop effective therapy of liver cancer.

#3056

Lung tumor spheres as in vitro platform for testing new therapeutic strategies against cancer stem cells.

Alejandro Herreros Pomares,1 Cristóbal Aguilar-Gallardo,1 Hector Amado,2 Eva Escorihuela,1 Ana Blasco,3 Silvia Calabuig-Fariñas,4 Juan Murga-Clausell,5 Eloísa Jantus-Lewintre,6 Carlos Camps7. 1 _Fundación para la Investigación, Hospital General de Valencia -CIBERONC, Valencia, Spain;_ 2 _Fundación para la Investigación, Hospital General de Valencia, Valencia, Spain;_ 3 _Hospital General Universitario de Valencia-CIBERONC, Valencia, Spain;_ 4 _Fundación para la Investigación, Hospital General de Valencia -CIBERONC; Departament de Patologia, Universitat de València, Valencia, Spain;_ 5 _Dept de Química Inorgànica i Orgànica, Universitat Jaume I, Castellón, Spain;_ 6 _Fundación para la Investigación, Hospital General de Valencia -CIBERONC; Dept Biotecnologia, Universitat Politècnica de València, Valencia, Spain;_ 7 _Fundación para la Investigación, Hospital General de Valencia -CIBERONC; Dept Medicina, Universitat de València; Servicio de Oncología Médica, Hospital General Universitario de Valencia, Valencia, Spain_.

Background: Treatment resistance is related to cancer stem cells (CSCs), a highly tumorigenic subpopulation of cells capable of growing and forming tumor spheres under non-adherent conditions. This study aimed to isolate and characterise CSCs from resected non-small cell lung cancer (NSCLC) patients' tumor-tissue and cell lines like tumor spheres and to use them as an in vitro platform for drug screening.

Methods: The study was performed on tumour cells from 8 resected NSCLC patients and 12 NSCLC cell lines grown in monolayer and as spheres. The expression of 60 genes, including CSC-markers, pluripotency inducers, cell cycle regulators and components of the Notch, Wnt and Hedgehog pathways was analysed by RTqPCR. Protein levels of relevant markers were assessed by WB and IF. Based on CSCs' characterization, two new-synthetized compounds, called compound A and compound B, were tested in the 12 cell lines and on tumour cells from 4 resected NSCLC patients, in 8 different concentrations. Cell viability was measured after 48 hours using MTS assay normalized to the respective mock-treated control cells and presented as a percentage of the control. Statistical analyses were considered significant at p<0.05.

Results: Patients´ median age was 65 years [56-76], 73.3% were male and 45% were adenocarcinomas. Lung tumor spheres had a significantly higher expression of CSC-related genes CD44, ALDH1A1, CD90, CDKN1A, JUNB, NANOG, KLF4 and MDM2 than their paired-adherent cells. Invasion promoters SNAI1, ITGA6, and MMP9 were overexpressed in tumor spheres. Notch pathway ligands, JAG1 and DLL4, and receptors, NOTCH1 and NOTCH3, showed increased expression in spheroids than in differentiated cells. In Wnt, higher expression levels of CTNNB1 and GSK3B were found in spheres; when comparing the expression for both conditions. WB and IF revealed that EpCAM, CD44, ALDH1A1, Sox2 and β-catenin were overexpressed in spheroids whereas E-cadherin was overexpressed in the adherent cells. Drug screening showed that classical anticancer drugs had mild cytotoxic effects on both conditions. Stemness pathways inhibitors IWP2, XAV939, Salinomycin and Vismodegib had stronger cytotoxic effects on spheroids than on the cells grown in monolayers. Cytotoxic effect was stronger in the tumor spheres of the lines and patients treated with compounds A and B. Currently, these compounds are being tested in vivo, in tumors induced in xenografts with cells of patients tested in vitro, to determine the mechanism of action and cytotoxic effect on cancer stem cells in the future.

Conclusions: Our data suggest that lung tumor spheres provide a useful short-term culture platform for the simple and cost-effective characterization of CSCs. We found molecules that could be used as novel therapeutic approach in NSCLC. Supported by grants RD12/0036/0025 from RTICC-FEDER, and PI12-02838 and PI15-00753 from ISCIII.

#3057

ABCG2-positive cells derived from ABCG2-negative pancreatic cancer cells in 3D-culture conditions show high stemness and anti-cancer drug resistance.

Norihiko Sasaki,1 Fumio Hasegawa,1 Masaki Michishita,2 Yoko Matsuda,3 Tomio Arai,3 Naoshi Ishikawa,1 Yoko Itakura,1 Junko Aida,1 Kaiyo Takubo,1 Masashi Toyoda,1 Toshiyuki Ishiwata1. 1 _Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan;_ 2 _Nippon Veterinary and Life Science University, Tokyo, Japan;_ 3 _Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology, Tokyo, Japan_.

ATP-binding cassette subfamily G member 2 (ABCG2), an important multidrug resistance transporter, can mediate the efflux of various chemotherapy drugs and contribute to drug resistance in cancer cells. The correlation between ABCG2 expression and cancer stem cell (CSC) phenotypes has been examined in hepatocellular carcinoma, as well as in glioma, breast, prostate, and colon cancer; however, the results remain controversial. In this study, we compared the characteristics of low- (ABCG2-) and high-expressing (ABCG2+) pancreatic ductal adenocarcinoma (PDAC) cells using a human pancreatic cancerous cell line (PANC-1) because the role of ABCG2 in CSC-related malignant characteristics in PDAC is not well elucidated.

ABCG2- and ABCG2+ PDAC cells were separated using flow cytometric cell sorting. In adherent cell culture conditions, 10% of all PANC-1 cells were ABCG2+. Using transmission electron microscopy, we found that ABCG2+ cells showed more abundant and longer microvilli on the cell surface than ABCG2- cells. Unexpectedly, ABCG2+ cells did not demonstrate significantly greater drug resistance against 5-FU, gemcitabine, and vincristine than ABCG2- cells, as assessed using a WST-8 assay. Furthermore, ABCG2- cells exhibited better sphere formation ability and higher stemness marker expression, including that of Sox2, Oct4, ALDH1, CD44v9, and Nestin, than ABCG2+ cells, as observed using qRT-PCR. Cell growth rates and motilities, examined using the Boyden chamber and scratch assays, were higher in ABCG2- cells than in ABCG2+ cells. However, the epithelial-mesenchymal transition (EMT) ability, assessed by examining the alteration of E-cadherin, N-cadherin, Snail, and Vimentin expression after TGFβ addition, was comparable between ABCG2- and ABCG2+ cells.

In 3D-culture conditions using ultra low-attachment plates, ABCG2- cells formed spheres containing a large number of ABCG2+ cells, and expression of stemness markers in these spheres was higher than that of spheres derived from ABCG2+ cells. Furthermore, spheres derived from ABCG2- cells included large populations of ABCG2+ cells and exhibited high resistance against anti-cancer drugs, presumably depending on ABCG2 expression.

We found that in adherent culture conditions, ABCG2+ PDAC cells do not exhibit stemness and malignant behaviors, but ABCG2+ cells derived from ABCG2- cells after sphere formation have high stemness and anti-cancer drug resistance. This suggests that ABCG2- cells have the capacity to generate ABCG2+ cells, and the malignant potential of ABCG2+ cells in PDAC depends on the environment. The 3D-culture system was expected to mimic in vivo environments. Therefore, ABCG2+ cells in pancreatic cancer tumors may exhibit high stemness and anti-cancer drug resistance. Thus, ABCG2+ cells should be considered as novel therapeutic targets for pancreatic cancer.

#3058

Data and resource sharing: Advancing our understanding of glioblastoma.

Hema A. Luchman,1 Fiona Coutinho,2 Samuel Weiss,1 Stand Up to Cancer Stem Cell Dream Team Consortium, Peter Dirks3. 1 _University of Calgary, Calgary, Alberta, Canada;_ 2 _Hospital for Sick Children, Ontario, Ontario, Canada;_ 3 _Hospital for Sick Children, Toronto, Ontario, Canada_.

Glioblastoma multiforme (GBM), characterized by an aggressive clinical course, therapeutic resistance, and striking molecular heterogeneity, remains incurable. GBM has a median survival of only 12-15 months post-surgery in adults even with standard of care chemotherapy and radiation. Through a collaborative pan-Canadian Stand up to Cancer (SU2C) Stem Cell Dream Team effort, our group is focusing on brain tumour stem cells (BTSCs), a subpopulation of cells within tumours, which are hypothesized to drive tumour growth, relapse, and resistance to conventional chemotherapies. We are completing large-scale phenotypic cell biology, tumourigenicity, drug screening, and multi-platform 'omic studies (genomics, bulk and single cell transcriptomics, epigenomics, metabolomics, proteomics, ATACseq) on a large number of adult GBM patient-derived BTSC cultures. Using both in vitro and in vivo approaches and the above multi-platform approaches, we are investigating the unique growth and stem cell characteristics of the BTSCs. In addition, we are using BTSC cultures to investigate drug response and genetic targeting approaches to identify new therapeutic strategies for clinical translation and understand mechanisms of resistance and recurrence. The wealth of information derived from this project is being integrated using systems biology approaches and will be shared in the public domain via data visualization and big data sharing platforms. Contemporaneously, we are depositing a number of well-characterized BTSC cultures in the American Type Culture Collection repository for unrestricted access to the scientific community. Our cross-Canada multi-disciplinary Cancer Stem Cell Dream Team, comprised of scientists, clinicians and patient advocates supports the ideal of data and resource sharing to help improve patient outcome for this devastating disease. Our goal is to provide the broader community of scientists, clinicians and patient groups an enduring resource and data sharing model to advance our understanding of glioblastoma.

#3059

SOX2 is a novel biomarker for poor long-term outcomes in oral cancer.

Xuefeng Zhang, Hao Xu, Chongkui Sun, Xinhua Liang, Peng Deng, Xin Zeng, Qianming Chen. _West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan, China_.

Purpose: Sex determining region Y-BOX2 (SOX2), a key regulator of the self-renewal ability of stem cells, plays a role in the initiation and development of head and neck squamous cell carcinoma (HNSCC). However, the association between SOX2 expression and the outcomes of patients with oral squamous cell carcinoma (OSCC) has not been investigated. Thus, in this study, we investigated the possibility of predicting the survival outcomes of patients with OSCC by evaluating SOX2 expression.

Experiment design: First, we analyzed SOX2 expression in patients with OSCC in The Cancer Genome Atlas (TCGA) dataset by a bioinformatics approach. In addition, samples of 219 patients with OSCC from two independent Chinese centers were collected. The median follow-up time and 95% confidence interval (CI) for the cohorts were 36 (25.06, 46.93) months and SOX2 expression in these samples was determined by immunostaining using tissue microarray. The Kaplan-Meier analysis, log-rank test, and Cox proportional hazards regression model were used to determine univariate and multivariate analyses, and hazard ratios for overall survival (OS), respectively.

Results: In TCGA database, amplification of SOX2 was negatively correlated with the OS rate for patients who survived more than 300 days after the first diagnosis. Moreover, 39.7% of the samples from the cohorts showed high SOX2 expression. We found a significant difference in the survival rate between patients with high and low SOX2 expression ranging from 13 to 126 months.

Conclusion: High SOX2 expression can predict poor long-term, but not short-term, survival outcomes in patients with OSCC. Our findings not only indicated that SOX2 may be a novel biomarker for the long-term prognosis of OSCC but also strongly supported the cancer stem cell theory that dysregulation of the self-renewal ability of stem cells may contribute to the development of OSCC.

#3060

EGFR-mediated interleukin enhancer-binding factor 3 contributes to formation and survival of cancer stem-like tumorspheres through regulating HER3/ERBB3 expression as a therapeutic target against EGFR-positive non-small cell lung cancer.

Chun-Chia Cheng,1 Kuei-Fang Chou,1 Cheng-Wen Wu,2 Nai-Wen Su,1 Cheng-Liang Peng,3 Ying-Wen Su,1 Jungshan Chang,4 Ai-Sheng Ho,5 Huan-Chau Lin,1 Caleb Gon-Shen Chen,1 Yu-Cheng Chang,1 Ken-Hong Lim,1 Yi-Fang Chang1. 1 _Mackay Memorial Hospital - Tamsui Branch, Tamsui, New Taipei City, Taiwan;_ 2 _National Yang-Ming University, Taipei, Taiwan;_ 3 _Institute of Nuclear Energy Research, Atomic Energy Council, Taoyuan, Taiwan;_ 4 _Taipei Medical University, Taipei, Taiwan;_ 5 _Cheng Hsin General Hospital, Taipei, Taiwan_.

Objectives: YM155, an inhibitor of interleukin enhancer-binding factor 3 (ILF3), significantly suppresses cancer stemness property, implying that ILF3 contributes to cell survival of cancer stem cells. However, the molecular function of ILF3 inhibiting cancer stemness remain unclear. This study aimed to uncover the potential function of ILF3 involving in cell survival of epidermal growth factor receptor (EGFR)-positive lung stem-like cancer, and investigate the potential role to improve the efficacy of anti-EGFR therapeutics.

Materials and Methods: The regulated association of EGFR and ILF3 was investigated at first. ILF3 was knockdowned and RNAseq was utilized to search for the putative genes regulated by ILF3. Meanwhile, HCC827- and A549-derived cancer stem-like cells were used to investigate the role of ILF3 in the formation of cancer stem-like tumorspheres.

Results: We found that EGFR induced ILF3 expression, and YM155 reduced EGFR expression. The knockdown of ILF3 reduced not only EGFR expression in mRNA and protein levels, but also cell proliferation in vitro and in vivo, demonstrating that ILF3 was an oncoprotein contributing to cancer cell survival. Moreover, the knockdown and inhibition of ILF3 by shRNA and YM155, respectively, reduced the formation and survival of HCC827- and A549-derived tumorspheres through inhibiting HER3/ERBB3 expression, and synergized the therapeutic efficacy of afatinib, a tyrosine kinase inhibitor, against EGFR-positive A549 lung cells.

Conclusion: This study demonstrated that ILF3 played an oncogenic role maintaining the EGFR-mediated cellular pathway as a therapeutic target to improve the therapeutic efficacy of afatinib. Therefore, we suggested that YM155, an ILF3 inhibitor, was potential for utilization in cancer therapy against the EGFR-positive cancers.

#3061

The clinicopathologic roles of SOX2 and Oct4 expressions in stage 1 and 2 oral squamous cell carcinoma.

Tetsuya Tamatani, Natsumi Takamaru, Go Ohe, Takayuki Nakagawa, Naito Kurio, Keiko Kudou, Yoshiko Yamamura, Takamitsu Mano, Youji Miyamoto. _Institute of Biomedical Sciences, Tokushim Univ Graduate School, Tokushima, Japan_.

[Background] Cancer stem cells (CSCs) exhibit self-replication, self-differentiation, drug resistance, and immune evasion activities. Recently, CSC has become increasingly important in the treatment of malignant tumors. Cancer stem cells express specific molecules termed CSC marker, including sex determining region Y-box2 (SOX2), and octamer-binding transcription factor 4 (Oct4), and their expression has been reported to be the potential prognostic values. However, the prognostic values of SOX2 and Oct4 expression in patients with stage I and II oral cancer are less understood.

[Purpose] The aims of present study were to evaluate the expression of SOX2 and Oct4 in stage I and II oral squamous cell carcinoma (OSCC) and to elucidate the relationships among the CSC marker expression, clinical stages, histological differentiation, classification of invasion mode, cerebral lymph node metastasis, distant metastasis, and disease-free survival rate.

[Materials and Methods] Tissue specimens were obtains from 83 patients with OSCC after surgery or biopsy. Immunohistochemistry was used to assess SOX2 and Oct4 protein using at least 10% staining-positive cells as the definition of positive staining. [Results] Immunohistochemical analysis of 83 cases showed that 31 cases (37%) expressed SOX2. There was no significant association between SOX2 expression and tumor size, invasion mode or histological differentiation. However, there was significant association between SOX2 expression and distant metastasis or disease-free survival rate at stage 1 and 2 patients. Otherwise, fifty three cases (64%) of 83 OSCC patients expressed Oct4. There was significant association between Oct4 expression and histological differentiation. There was no significant association between Oct4 expression and tumor size, invasion mode, metastasis, or disease-free survival rate.

[Conclusions] These findings suggested that the expression of SOX2 may be good marker indicating survival in patients with stage 1 and 2 OSCC.

#3062

Ablation of parathyroid hormone-related protein in the triple negative human breast cancer line MDA-MB-231 inhibits its cancer stem cell phenotype.

Jiarong Li, Louis Doré-Savard, Karine Sellin, Anne Camirand, Richard Kremer. _McGill Univ. Health Center, Montreal, Quebec, Canada_.

Parathyroid hormone-related protein (PTHrP) is a pleiotropic hormone involved in a wide range of developmental & physiological processes. It is also dysregulated in advanced cancers where it causes malignancy-associated hypercalcemia & plays a major role in the progression of bone metastases. We have shown previously that PTHrP ablation delays tumor initiation & that it is preferentially expressed in triple negative breast cancer (TNBC).Here we investigated how PTHrP can influence the phenotype of the human TNBC cell line MDA-MB231 using CRISPR/cas 9 knockout technology. We used U6G RNA-Cas9-2A-red fluorescence protein (RFP) vector with a human PTHrP specific guide RNA directed to exon 4(knockout/KO) or an empty vector as control. PTHrP KO cells (RFP+) were isolated using AutoMacs Pro Separator & single cells were exp&ed in 96 well plates. Positive clones were confirmed by DNA sequencing. Real time PCR & immunofluorescence staining confirmed that 5/96 KO clones had between 95-99% decrease in PTHrP expression compared to control cells. We then assessed cancer stem cell (CSC) characteristics of both KO & control cell lines. Mammosphere formation was reduced by over 60% in KO cells. Stem cell phenotype assessed by ALDH1 & CD44+/CD24 low using FACS analysis were significantly reduced by over 30% in KO cells. Cell morphology analysis showed a shift from a mesenchymal phenotype towards an epithelial phenotype. Overall our data show that PTHrP regulates TNBC towards a more aggressive phenotype & suggest that it could be targeted therapeutically.

#3063

Stem cell lineage hierarchy by keratin profiling in normal human prostate epithelial cells and prostate cancer.

Wenyang Hu,1 Danping Hu,1 Lishi Xie,1 Hong Hu,1 Ye Li,1 Larisa Nonn,1 Toshi Shioda,2 Gail S. Prins1. 1 _University of Illinois at Chicago, Chicago, IL;_ 2 _Massachusetts General Hospital Center for Cancer Research and Harvard Medical School, Charlestown, MA_.

Background: Both mechanical and nonmechanical functions of keratins have been implicated in stem cells and cancers. Using a sphere-based label retention assay, we recently isolated and characterized prostate stem cells and cancer stem-like cells from mixed progenitor populations (PMID 28651114), identifying keratin 13 (KRT13) as a specific prostate stem cell marker regulating self-renewal. Herein we utilize detailed keratin profiles to further clarify the human prostate epithelial lineage hierarchy and identify prostate cancer stem-like cells.

Methods and Results: Primary prostate epithelial cells were 3D cultured (5 days) to form prostaspheres (PS), followed by PS-based long-term label retention and FACS to separate stem cells from progenitors. In normal prostate tissues from three healthy donors, RNA-seq revealed enrichment of KRT13, 23, 80, 78, 86 and 4 in label-retaining prostate stem cells while KRT6, 17, 14, 5, 8, 18 and P63 were enriched in nonretaining progenitors. We next used Fluidigm C1 captured single-cell RNA-seq and identified three major clusters in the label-retaining stem cell population; Cluster I represents quiescent stem cells (KRT13, 23, 80, 78, 4 enriched) and Clusters II and III contain active stem cells and bipotent progenitors, respectively (KRT16, 17, 6 enriched). GSEA analysis found stem cell and cancer-related pathways enrichment in Cluster I. Three additional clusters were identified in nonretaining progenitor cells, with Cluster IV representing unipotent basal progenitor cells (KRT5, 14, 6, 16 enriched) and Clusters V and VI early- and late-stage unipotent luminal progenitors (KRT8, 18, 10 enriched). Cancer stem-like cells were similarly isolated from three prostate cancer specimens and RNA-seq with MetaCore pathway analysis found enrichment of cytoskeleton remodeling keratin filaments. Interestingly, in addition to normal stem cell keratins (KRT13, 23, 80, 78, 4), other keratins (KRT10, 19, 6, 75, 16, 79, 3, 82) were enriched in cancer stem-like cells. Surprisingly, stem-like cells from patient-matched benign regions revealed a similar keratin profile, suggesting a cancer field effect for stem-like cell populations.

Conclusion: Taken together, using gene profiling with an emphasis on keratin patterns, we have delineated the lineage hierarchy of human prostate stem cells originating from the activation of quiescent stem cells to bipotent progenitors that give rise to unipotent basal and luminal progenitor cells. We have identified common keratins enriched in stem cells from normal prostate and cancer/benign tissues as well as keratins unique in stem-like cells from prostate cancer. This clarification of the stem cell lineage hierarchy and keratin profiling of human prostate stem cells and cancer stem-like cells may provide enhanced opportunities for translational studies that target therapeutic-resistant cancer stem-like cells. (CA-172220)

#3064

**Oral cancer stem cells modulate** Fusobacterium nucleatum **to acquire the capability to induce tumor stemness switch.**

Bidisha Pal,1 Seema Bhuyan,2 Debabrat Baishya,2 Bikul Das1. 1 _The Forsyth Institute, Cambridge, MA;_ 2 _KaviKrishna Laboratory, Guwahati Biotech Park, Guwahati, India_.

Background: Fusobacterium nucleatum is an oral bacteria that contributes to oral carcinogenesis. Its potential role in oral cancer is not known. Fusobacterium nucleatum (henceforth FN) is a gram-negative anaerobe associated with periodontal diseases and also colon cancer. The bacteria modulates the host cell signaling mechanism including the activation of beta-catenin signaling and p38 pathway. The Fap2 is the outer membrane proteins of FN that inhibit lymphocytes and NK cells. The bacteria also directly binds to NKp46 receptor of NK cells. These properties of FN may be exploited by oral CSCs to defend their own niche as a part of niche-defense mechanism against immune system. Hence, it is not surprising that oral CSCs may hijack this specific oral bacterium to enhance tumor stemness. We speculate that oral CSCs may hijack specific bacteria type present in oral mucosa, and enhance their stemness-inducing mechanisms to switch non-CSCs to CSCs.

Methods: We have established an in vitro model of oral cancer cells/FN host/pathogen interaction model. Using this model, we wanted to study potential role of the bacteria in cancer stemness. We also used the saliva of subjects with oral cancer to infect oral cancer stem cells and non-stem cancer cells.

Results: When the SCC-25 oral cancer cell line was treated with the saliva of subjects with oral cancer, the EpCAM+/ABCG2+ oral CSCs population was increased and showed the presence of FN. The FN recovered from CSCs exhibited high expression of FadA and Fap2, two surface proteins present in FN that can modulate signaling pathways of host cells. Importantly, FN recovered from the infected CSCs induced stemness in the non-stem cancer cell population of SCC-25, suggesting that CSCs may modulate the pathogen. Indeed, our preliminary observation indicates that FN recovered from the FN strain ATCC 25586 infected SCC-25 ABCG2+ cells showed increased expression of FADA and Fap2 as compared to the parental strain ATCC 25586.

Conclusion: Our data suggests that CSCs have the capability to reprogram F. nucleatum genome in order to make the bacteria a potent inducer of tumor stemness in non-stem cancer cells.

#3065

Role of noncoding RNA miR 146 alpha and let 7c in epithelial mesenchymal transition, proliferation, and cancer stem cell-like transformation analyzed by gene expression studies in a cohort of prostate cancer patients.

Hirendra N. Banerjee,1 Fazlul Sarkar,2 Myla Worthington,1 Sasha Hodge,1 Mukesh Verma,3 Santosh Mandal4. 1 _Elizabeth City State University, Elizabeth City, NC;_ 2 _Wayne State University, Detroit, MI;_ 3 _National Cancer Institute, Bethesda, MD;_ 4 _Morgan State University, Baltimore, MD_.

Mir 146 alpha and let 7c are considered tumor suppressor miRs and are responsible for downregulating polycomb repressor proteins like EZH2, SUZ12, etc., and thereby prevent epithelial-mesenchymal transition (EMT),proliferation, spheroid formation and metastasis in prostate cancer cells. In this study, we investigated the role of these microRNAs in a cohort of 105 patients, both White (CA) and African American (AA), by gene expression studies using real-time PCR techniques. Our results showed definite downregulation of these noncoding RNAs in the cDNAs obtained from the matched biopsy samples of normal and cancer tissues. We further transfected these miR full-length constructs in PC3 prostate cancer cell lines and validated their effects by downregulation of their target 3'UTR target mRNAs. Our results showed decrease in EMT markers ALDH, increase in EMT marker E-cadherin, decrease in cell proliferation and spheroid formation; fluorescence microscopy studies showed a decrease in CD44-positive cells in the spheroids, signifying a possible hindrance in the growth of cancer stem cells (CSCs). Thus, miR 146 alpha and let7c could be ideal targets for diagnosis, prognosis and therapeutic intervention.

#3066

**Chemotherapy-induced GSTO1 interacts with ryanodine receptor RYR1 to trigger Ca** 2+ **-dependent breast cancer stem cell enrichment.**

Haiquan Lu, Gregg Semenza. _Johns Hopkins University, Baltimore, MD_.

Breast cancer stem cells (BCSCs) play a critical role in tumor recurrence and metastasis. Exposure of breast cancer cells to chemotherapy leads to an enrichment of BCSCs. Here, we demonstrate that chemotherapy induces the expression of glutathione S-transferase omega 1 (GSTO1), which is dependent on hypoxia-inducible factor 1 (HIF-1) and HIF-2. Knockdown of GSTO1 expression abrogates carboplatin-induced BCSC enrichment, decreases tumor initiation and metastatic capacity, and delays tumor recurrence after chemotherapy. GSTO1 interacts with ryanodine receptor RYR1 and promotes calcium release from endoplasmic reticulum. Increased cytosolic calcium levels activate PYK2 → SRC → STAT3 signaling, leading to increased expression of pluripotency factors and BCSC enrichment. HIF inhibition blocks chemotherapy-induced GSTO1 expression and BCSC enrichment. Combining HIF inhibitors with chemotherapy may improve clinical outcome in breast cancer.

#3067

The novel function of keratin 14 (KRT14) in regulating cancer stem cell properties in lung adenocarcinoma.

Isabella Kit-Nam Chin, Goretti Hoi-Yan Cheung, Hazel Yuk-Yan Kwok, Victor Wan-San Ma, Eunice Yuen-Ting Lau, William Chi-Shing Cho. _Queen Elizabeth Hospital, Hong Kong, Hong Kong_.

Lung cancer remains the leading cause of cancer deaths worldwide. Among all classification subtypes, lung adenocarcinoma (LUAD) accounts for the largest proportion of all cases of lung cancer. Although the expanding development of novel targeted therapies has been improving the overall survival of lung cancer patients, recurrence and therapeutic resistance remains as the major reasons of treatment failure. Cancer stem cells (CSCs) have been revealed to be one of the major drivers for tumor recurrence and therapeutic resistance. Understanding the molecular pathways in regulating CSCs may provide insights on designing novel drug combinations in targeting the disease. Various studies showed that there is a contrasting expression of keratins in stem and differentiated cells, implying the functional importance of keratins in maintaining stem cell characteristics. Keratin 14 (KRT14) is a keratin isoform shown to be enriched in breast and bladder CSCs. In LUAD, KRT14 has been reported to be a key player in tumor invasion, yet its role in regulating LUAD CSCs remains largely unknown. In this study, we aim to delineate the clinical significance and the functional role of KRT14in LUAD. From The Cancer Genome Atlas (TCGA) data, the mRNA expression of KRT14 was found to be higher in LUAD tumor when compared with paired normal tissues (p=0.0057).This data was also confirmed in protein level with our in-house cohort, in which LUAD tumor showed an upregulation of KRT14 protein expression. TCGA data also revealed that patients with high KRT14 mRNA expression have a poorer disease-free survival (p=0.0147) and overall survival (p=0.0043). By qPCR and western blot analyses, we found that the expression of KRT14 is upregulated in our established enriched lung CSC population. Using lenti-viral based knockdown approach, KRT14 was found to regulate the traits of CSCs, including tumorigenicity, self-renewal, drug resistance and the expression of lung CSC markers. Interestingly, KRT14 was found to be upregulated in the erlotinib-resistant LUAD cells, and the abrogation of KRT14 expression re-sensitized these cells to erlotinib treatment. The genetic profiles of KRT14 knockdown and control cells will be compared in the future to identify the potential downstream targets of KRT14 in regulating lung CSCs. In summary, our study highlights the crucial role of KRT14 in maintaining lung CSCs and may provide a novel therapeutic target for the disease.

#3068

Chronic alcohol exposure induces malignant transformation of pancreatic ductal epithelial cells and generates stem cell characteristics.

We Yu,1 Yiming Ma,1 Sharmila Shankar,2 Rakesh K. Srivastava2. 1 _Kansas City VA Medical Center, Kansas City, Kansas City, MO;_ 2 _Louisiana State University Health Sciences Center, New Orleans, LA_.

Findings from epidemiologic studies and research with experimental animal models provide insights into alcohol toxicity-related disease pathogeneses. Epidemiologic data indicate that heavy drinking is associated with high risk of pancreatic cancer. However the molecular mechanisms by which chronic ethanol exposure changes pathophysiology of human pancreatic ductal epithelial (HPNE) cells is not well understood. Cancer stem cells (CSCs) play significant role in cellular transformation, and their emergence is responsible for cancer initiation, progression and metastasis. SATB2 is required for normal mammalian development; however, it is not expressed in normal adult cells. Inappropriate activation of SATB2 gene may cause malignant cellular transformation. SATB2 regulates transcription of pluripotency maintaining factors which are required for sustaining the stem cells capacity for self-renewal. The main goal of the paper is to examine the molecular mechanisms by which chronic alcohol consumption induces cellular transformation of HPNE cells. To investigate the role of SATB2 at an early step of cell transformation, we utilized HPNE cells to generate progenitor cells either by chronic ethanol exposure or overexpression with SATB2. Our data demonstrate that chronic exposure of HPNE cells to ethanol induces cellular transformation by upregulating SATB2. Furthermore, during ethanol-induced cell transformation, cells gain the phenotype of progenitor cells / CSCs and express pluripotency maintaining factors and epithelial-mesenchymal transition (EMT) markers. Suppression of SATB2 expression in ethanol-transformed HPNE cells inhibits stemness and EMT. SATB2 is highly expressed in pancreatic cancer tissues derived from human and KrasG12D mice. In conclusion, chronic ethanol exposure may contribute in pancreatic carcinogenesis by inducing the expression of SATB2 gene.

#3069

DSP-0692, a novel and selective stearoyl-CoA desaturase (SCD) inhibitor targeting cancer stem cells.

Eiji Sugaru, Yudai Furuta, Yuichi Fukunaga, Hiroki Umehara, Tsuguteru Otsubo, Manabu Watanabe, Futoshi Hasegawa, Shingo Tojo, Miki Hashizume, Hitoshi Ban. _Sumitomo Dainippon Pharma Co.,Ltd., Osaka, Japan_.

In recent years evidence has accumulated in support of the cancer stem cells (CSCs) model in cancer chemotherapy resistance, highlighting the urgency and necessity of identifying CSC-specific targets for new cancer therapeutics.

SCD, a key enzyme for lipid metabolism, is overexpressed in various types of cancer and plays an important role in cancer cell proliferation. In particular, recent studies have implicated SCD involved also in the maintenance of CSCs.

Here, the identification and the functional characterization of DSP-0692, a novel and selective SCD inhibitor, is reported, using various pharmacological approaches to address the mechanism of SCD inhibition in cancer cells and its efficacy in preclinical tumor models. In these preclinical models, DSP-0692 inhibited human SCD activity with low nanomolar IC50 values. SCD expression was induced in tumor cells cultured in sphere conditions, which were resistant to conventional agents within therapeutic concentration. DSP-0692 showed strong growth inhibition when tumor cells were cultured in sphere conditions, but had little impact on cells cultured under normal two-dimensional conditions. In addition, DSP-0692 inhibited the CSC-related signaling pathway and decreased CSC markers expression in tumor cells as well as in tumor tissues. Treatment of DSP-0692 as single agent suppressed tumor growth in multiple models. Furthermore, this efficacy was enhanced in combination with conventional chemotherapies in those tumor models. Of note, SCD protein expression in tumor tissues was useful as a predictive marker for the susceptibility to DSP-0692 treatment.

Taken together, these data suggest that inhibition of fatty acid desaturation by DSP-0692 could be a new therapeutic strategy in SCD-positive cancer, with potential to overcome resistance to standard chemotherapies.

#3070

Cancer stem cells in field cancerization of oral squamous cell carcinoma.

Simple Mohanta,1 Ravindra DR,2 Vikram Kekatpure,3 Naveen Hedne,3 Vijay Pillai,1 Shubhra Chauhan,3 Naveen BS,3 Athira Ramakrishnan,3 Bichu Jacob,3 Vishak Surendra,3 Leeky Mohanty,4 Anjana Muralidharan,1 Amritha Suresh,1 Moni Abraham Kuriakose3. 1 _Mazumdar Shaw Medical Foundation, Bangalore, India;_ 2 _Narayana Hrudayalaya Foundation, Bangalore, India;_ 3 _Mazumdar Shaw Medical Center, Bangalore, India;_ 4 _Oxford Dental College and Hospital, Bangalore, India_.

Introduction: Field cancerization, the occurrence of transformed cells in the area adjacent to the tumor, has been attributed to the probable reasons of local recurrence of oral squamous cell carcinoma. Cancer stem cells (CSCs) have properties of tumor initiation, migration, and metastasis. The objective of the study was to evaluate their role in field cancerization.

Experimental Procedure: A panel of CSCs and its related markers was established from literature and validated in i) samples from surgical margins, ii) samples from 1 cm and 2 cm distance from margin around the tumor and in additional sides. First-phase validation was done in retrospective surgical margins of tumor (N=23) by qPCR and immunohistochemistry. The best marker subset from qPCR to predict recurrence was identified by ROC curve and logistic regression analysis and validated by immunohistochemistry. The final validation is being carried out in prospective samples from tumor-adjacent normal area as mentioned above. The marker profiles were correlated with the histology of the samples and the clinical outcome of the patients on follow-up.

Summary of Data: Out of 18 markers selected from literature CD44, CD147, SOX2, Snail, ATR, CyclinD1, MMP9 were selected from gene expression of first-phase validation. Among these SOX2, ATR, Cyclin D1, CD44 and CD147 were selected from IHC of first phase for second-phase validation in prospective samples (n=10) collected from 27 patients, which were clinically normal with varied grades of dysplasia; these samples were evaluated by the selected markers from the first set of validation. The correlation of markers expression with disease outcome of patients and with other clinical parameters is currently ongoing.

Conclusion: Our results suggest that CSCs have role in field cancerization and might be predictive of tumor recurrence/reinitiation and/or development of second primary tumor.

#3071

The role of Apela in high-grade serous ovarian cancer.

Laura R. Daily,1 Debolina Ganguly,2 D Neil Hayes,1 Yinan Wang,2 Michelle M. Sims,2 Jinjun Cheng,2 Adam C. ElNaggar,1 Lawrence M. Pfeffer2. 1 _West Cancer Center/University of Tennessee Health Sciences Center, Germantown, TN;_ 2 _University of Tennessee Health Sciences Center, Memphis, TN_.

Objective: To assess the expression of Apela and evaluate its co-expression with cancer stem cell markers and angiogenesis markers in high-grade serous ovarian cancer and further elucidate the role of this poorly characterized gene.

Background: High-grade serous ovarian cancer (HGSOC) presents a major therapeutic challenge due to advanced stage at diagnosis, aggressive metastatic behavior, and therapeutic resistance. A critical barrier to progress in early diagnosis and treatment is the lack of understanding of the mechanisms that underlie ovarian cancer tumorigenesis. Apela (Apelin early ligand A), also called Toddler and Elabela, is a novel ligand for the G-coupled protein receptor Apelin-R, which has been implicated in cancer tumorigenesis and angiogenesis through activation of the PIK3-AKT (phosphatidylinositol-4,5-bisphosphate 3-kinase and protein-kinase B) pathway. This signaling pathway plays an important role in embryonic stem cell growth and self-renewal. Current research indicate that Apela may be involved in cancer stem cells and angiogenesis pathways as well. Markers of angioneogenesis (Angptl4 [angiopoietin-like protein 4]) and cancer cell stemness (Nestin and CD133/PROM1), were selected for study with Apela because of known expression in HGSOC and other studies showing co-expression in other high-grade malignancies with Apela.

Methods: RNA was extracted from established ovarian cancer cell lines (OVCAR3, OVCAR8, CAOV4, OV90, and SKOV3) and expression of Apela, Apelin (another ligand for the Apelin-R), Apelin-R and ANGPTL-4 was determined by qPCR and normalized to actin expression. Formalin-fixed paraffin-embedded (FFPE) slides of HGSOC tissue and normal adjacent ovarian tissue were subjected to RNAscope Multiplex Fluorescent in situ hybridization (ISH) using individual gene probes for Angptl4, Nestin, CD133 and Apela to detect expression at the mRNA level. Immunohistochemistry (IHC) was then performed to confirm Angptl4, Nestin, CD133 and Apela co-expression at the protein level in the FFPE HCSOC tissue. All images were analyzed by confocal microscopy. The TCGA database was analyzed to determine levels of upregulation of Apela in ovarian cancer. An ovarian cancer microarray using 10 paired matched controls was utilized to further validate the presence of the

Results: Apela expression can be detected in FFPE tissue at the RNA level and the same pattern of protein expression is seen with IHC. Apela and Angptl4 co-localize with the cancer stem cell markers nestin and CD133. Apela is upregulated in HGSOC when examining the TCGA database compared to other forms of cancer.

Conclusions: Apela is expressed at relatively low levels in established ovarian cancer cell lines. However, Apela is highly localized in ovarian cancer tissue, and is co-expressed with markers of angiogenesis and stem cells, possibly representing a stem cell niche that promotes tumorigenesis.

#3072

MiRNA-2861 behaves as LCSC biomarker and regulates the HDAC5-ERK signal pathway.

Yimin Zhu, Mengya Zhao, Lin Li. _Suzhou Inst. of Nano-Technology and Nano-Bionics, CAS, Suzhou, China_.

Cancer stem cells (CSCs) are responsible for cancer initiating, recurrence and drug-resistance. Discovery of novel biomarkers for CSCs is helpful for early diagnosis and prognosis. In our study, the important role of miR-2861 in maintaining the stemness of LCSCs was investigated. The LCSCs differentiation model was established through introducing serum into the medium of H460 spheres. MiR-2861 expression was significant higher in LCSCs no matter compared to the differentiation cells or normal cells. HDAC5 expression was positively correlated with miR-2861 in LCSCs and knockdown of miR-2861 decreased the expression of HDAC5, which implied that HDAC5 may be involved in the differentiation of LCSCs mediated by miR-2861.The role of HDAC5 in the regulation of LCSCs differentiation was further verified by the inhibitory effect of LMK-235 on the phosphorylation of ERK1/2, which was recognized as the regulator of CSCs differentiation. Our study provided a better understanding of miR-2861 and HDAC5 axis in maintaining the stemness of LCSCs and laid a foundation for molecular targeted therapy.

#3073

Low copy number of mtDNA is associated with cancer stemness in ESCC.

Takeo Hara, Yasunori Masuike, Koji Tanaka, Tomoki Makino, Makoto Yamasaki, Yasuhiro Miyazaki, Tsuyoshi Takahashi, Yukinori Kurokawa, Hidetoshi Eguchi, Kiyokazu Nakajima, Masaki Mori, Yuichiro Doki. _Osaka university, Suita, Japan_.

Alterations of mitochondrial DNA (mtDNA) copy numbers in various human cancers have been studied. However, those of esophageal squamous cell carcinoma (ESCC) is still unclear. In the present study, we investigated the correlation of mtDNA copy number with clinicopathologic features, prognosis and malignant potential of ESCC. MtDNA copy numbers of resected specimen from 80 patients treated with radical esophagectomy were measured by quantitative real-time PCR analyses. Human ESCC cells, TE8 and TE11, were cultured and depletion of mtDNA content was induced by knockdown of mitochondrial transcription factor A expression or treatment with ethidium bromide. The mRNA expression, proliferation, invasion and cell cycle were investigated. The results showed that mtDNA copy number of cancerous parts was 56.0 (37.4-234.5) % of non-cancerous parts and significantly lower (p<0.05). Low mtDNA copy number in resected cancerous tissues was significantly correlated with pathological depth of tumor invasion and pathological stage (p<0.05). The patients with lower mtDNA copy number had significantly poorer 5-year overall and recurrence-free survival than higher group (p<0.01). MtDNA depleted TE8 and TE11 cells proliferated more slowly than control cells under normoxia, but proliferated at almost the same rate even under hypoxic condition. In mtDNA depleted cells, the mRNA expression of cd44 were increased, and the sphere formation abilities were enhanced and the cell cycle arrest at G0/G1 phase was induced. In conclusion, low copy number of mtDNA is associated with cancer stemness in ESCC. MtDNA copy number may be a novel therapeutic target for esophageal squamous cell carcinoma.

#3074

CD95/Fas increases type I interferon expression through NF-κB activation.

Abdul Qadir Syed, Marcus Peter. _Northwestern University, Chicago, IL_.

CD95/Fas, an apoptosis-inducing death receptor, has also been shown to be required for the proliferation and survival of cancer cells. Chronic stimulation through CD95 enhanced formation of cancer stem cell enriched spheres and it increased expression of cancer stem cell markers and a set of genes consistent with a Type I Interferon (IFN) signature. We reported that CD95 stimulation induces upregulation of Type I IFNs that bind to both Type I receptors IFNAR1 and IFNAR2, resulting in activation of JAK kinases, activation of STAT1, and induction of a number of STAT1-regulated genes. However, the mechanism through which CD95 induces Type I IFNs and STAT1 activation is unknown. In this study we have investigated the mechanism of CD95 stimulation of Type I IFN production in the context of breast cancer. We show that knockout of CD95, FADD or Caspase-8 in MCF7 cells using the CRIPSR/Cas9 gene editing system resulted in a loss of the ability of CD95 to increase IFNα, IFNβ, STAT1, STAT2 expression and the phosphorylation of STAT1 and STAT3. The caspase-8 selective inhibitor zIETD-fmk blocked phosphorylation of STAT1 and STAT3 and blocked Type I IFN expression following CD95 stimulation. This identifies caspase-8 as a critical mediator of the Type I IFN-inducing activity of CD95. On other hand, we also showed that CD95 stimulation induced an antiviral program through RIG-I signaling, which is known to induce NF-κB and IFN signaling. RIG-1, MDA5, IRF7 expression and phosphorylation of IRF3 were increased with prolonged CD95 stimulation. Knocking down IRF3 using a siIRF3 smart pool partially reduced the phosphorylation of STAT1 and STAT3 in response to CD95 stimulation. Finally, we show that NF-κB activation is a key regulator of CD95-mediated Type I IFN expression. A NF-κB inhibitor completely blocked STAT1, STAT2 expression and phosphorylation of STAT1 and STAT3 following CD95 stimulation. Knocking out CD95, FADD or Caspase-8 or knocking down IRF3 also reduced NF-κB activation in response to CD95 stimulation. We conclude that CD95 stimulation induces Type I IFN expression through activation of caspase-8 and NF-κB and identify RIG-1 as a possible mediator of NF-κB activation during CD95 stimulation.

### Carcinogenesis 1

#3075

Type IIB DNA topoisomerase is downregulated by trastuzumab and doxorubicin to synergize cardiotoxicity.

Jiangsong Jiang, Nishant Mohan, Yukinori Endo, Yi Shen, Wen Jin Wu. _FDA, Silver Spring, MD_.

Despite heightened risk of cardiotoxicity associated with combination therapy of anthracyclines and trastuzumab in HER2-positive breast cancer patients, little research effort has been invested in exploring the molecular mechanisms of cardiotoxicity induced by this combination therapy. Using human primary cardiomyocytes as a model, we demonstrate that trastuzumab downregulates both gene and protein expressions of type IIB DNA topoisomerase/DNA topoisomerase IIB (TOP2B), a major intracellular target mediating doxorubicin-induced cardiotoxicity. This downregulation of TOP2B in turn induces DNA damage activity and DNA double strand breaks. However, unlike doxorubicin, trastuzumab alone does not induce apoptosis. Further investigation discovers that concurrent or sequential treatment of doxorubicin and trastuzumab significantly reduces protein levels of TOP2B, enhances apoptosis induced by doxorubicin, and inhibits cell growth. Furthermore, treatment of cardiomyocytes with doxorubicin and trastuzumab in either concurrent or sequential setting also enhances production of reactive oxidative and nitrative species as compared to either trastuzumab or doxorubicin treatment in cardiomyocytes. Our data reveal that doxorubicin treatment increases the cellular level of ErbB2/HER2 protein and enhances downstream signaling activity of this pathway. Consequently, this may render cardiomyocytes more sensitive to trastuzumab treatment after doxorubicin exposure. This study provides molecular basis for significantly increased cardiotoxicity in cancer patients treated with anthracyclines and trastuzumab-based combination regimens.

#3076

Smoking behavior weakens the protective effect of mosaic loss of chromosome Y on lung cancer.

Na Qin, Cheng Wang, Zijian Ma, Jiaping Chen, Hongbing Shen. _Nanjing Medical University, Nanjing, China_.

Mosaic loss of chromosome Y (mLOY) is the most commonly detectable mosaic chromosomal event in cancers, however, the underlying relationship with tumorigenesis is still unclear. We conducted a Mendelian randomization study to systematically investigate the effect of mLOY on lung cancer based on published GWASs study. We identified that genetically defined mLOY was a protective factor for lung cancer in non-smokers but not in smokers (lifelong non-smokers: OR=0.80, CI=0.69-0.93, P=4.03×10-3; ever-smokers: OR=0.96, CI=0.89-1.04, P=2.90×10-1; PHeterogeneity=3.83×10-2). A U-shaped curve between the copy-number level of chromosome Y and lung cancer risk was further fitted (P for Linearity Wald=8.81×10-10) to support that heavy mLOY caused by acquired damaging environmental factors may have different effects on lung cancer from the genetically defined mLOY, while genetically predicted mLOY was linearly associated with decreased lung cancer risk (P for Linearity Wald=0.15). Taken together, we proposed a two "sides" model: the "natural" mLOY reduced the lung cancer risk, while the effect can be abolished by aberrant loss of chromosome Y caused by environmental risk factors. Our results revealed a complex relationship between mLOY and lung cancer, and emphasized the importance of heterogeneity in the mLOY studies.

#3077

Krüppel-like factor 5 and its connection to Sonic Hedgehog signaling in Barrett's esophagus and esophageal adenocarcinoma.

Christopher K. Ng, Yulan Cheng, John M. Abraham, Stephen J. Meltzer. _Johns Hopkins University School of Medicine, Baltimore, MD_.

Aims: We sought to determine whether KLF5 was involved, at either phenotypic or functional levels, in the genesis and progression of Barrett's esophagus (BE) to esophageal adenocarcinoma (EAC), and whether this involvement was related to sonic hedgehog (SHH) signaling. In human adults, KLF5 is expressed in the less differentiated cells of the intestinal crypts, where its activity promotes cell proliferation, contributes to intestinal epithelial homeostasis, and is necessary for villus formation. KLF5 has been found to be highly active in a number of other cancer types, including gastric cancer; moreover, the known role of KLF5 in intestinal epithelial differentiation may contribute to metaplasia and dysplasia in BE and EAC development. We also sought to ascertain whether KLF5 activity was connected to the sonic hedgehog (SHH) pathway, since this pathway is already known to be highly active in BE and EAC.

Methods and results: Quantitative reverse transcriptase PCR (qRT-PCR) performed on RNA obtained from BE cell lines (GhTRT and QhTRT) and EAC cell lines (SKGT4 and OE33) revealed that KLF5 is underexpressed in BE cell lines and overexpressed in EAC cell lines relative to normal primary squamous esophageal epithelial cells (HEEpic). Moreover, qRT-PCR performed on clinical BE and EAC tissue specimens showed the same pattern, with downregulation in BE tissues and upregulation in EAC tissues (relative to matched normal esophageal control tissues). Next, we determined KLF5 functional effects in EAC in vitro. The EAC cell line SKGT4 was transfected with KLF5 siRNA, and WST-1 cell proliferation assays revealed significantly lower cell proliferation at 72 hours (p<0.00005) and 96 hours (p<0.05) after transfection, relative to mock-transfected SKGT4. We then assayed KLF5 downstream effects on esophageal adenocarcinogenesis as well as SHH signaling in vitro. qRT-PCR performed on KLF5-silenced SKGT4 RNA revealed significantly diminished expression (relative to mock-transfected SKGT4) of the following known EAC-related genes: CDX1 (p<0.005), VIL1 (p<0.05), MUC2 (p<0.005), and MUC5ac (p<0.0005); moreover, GLI1, a known SHH pathway target gene, was significantly downregulated by KLF5 inhibition (p<0.005), while PTCH1, a known SHH pathway-inhibitory gene, was significantly upregulated (p<0.05).

Conclusion: These tissue phenotypic and in vitro functional data identify KLF5 as a potential oncogene in esophageal adenocarcinogenesis. KLF5 is a potential therapeutic and/or chemopreventive target that can be evaluated in future studies of EAC or BE, such as with metformin, which is known to inhibit KLF5 activity. We also identify a potential new connection between KLF5 and SHH pathway activity in the development of EAC. This potential connection deserves further exploration in other malignancies, as well as in physiologic functions such as human development or adult stem cell maintenance.

#3078

**The fer/FerT cell survival pathway of the ancestral sponge (** Sycon raphanus **) becomes a mitochondrial proto-oncogene in the human genome.**

Joseph G. Sinkovics. _St Joseph's Cancer Institute, Tampa, FL_.

The Fer/FerT (truncated) genes and their encoded 879 aa / 100 kDa proteins (fujinami poultry sarcoma; feline sarcoma-related kinases) were discovered in the sponge Sycon raphanus [1]. The recognition of other related genes and their protein products (ras/RAS; src/SRC; the human Bruton kinase-related Tec family proteins, etc) in the descendants of ancestral sponges (Suberites; Spongilla) rapidly followed [1]. Primordial cell survival pathways were essential for the life style of early diploblastic multicellular eukaryotes (ctenophores; cnidarians; ciona, etc) in the hostile environment of the ancient Earth [2]. These preserved ancient cell survival pathways evolved into proto-oncogenes in triploblastic hosts (Homo), in order to allow switching back to them under circumstances of life-threatening intrusions, and in chronic infections of the inflammasomes [2]. The Author refers to this inherent fundamental biological principle as "retrograde cellular immortalization" [2]. A common form of this process is recognized in medical clinics as 'cancer' [2]. Constitutively up-regulated Fer/FerT genes and proteins redirect sugar metabolism toward Warburg's aerobic glycolysis with reduced Oxphosph (for ATP production) and tricarboxylic acid cycles. Downregulation of Fer/FerT results in a non-functional ETC. Tumor cells with nonfunctional mitochondrial ETC undergo either mTOR-directed autophagy, and/or necrotic death promoted by poly ADP-ribose polymerase (PARP-1) [3]. The novel inhibitor E260 of enzyme-like activity to Fer/FerT-induced kinases, attacks selectively mitochondrial (or cytoplasmic) Fer/FerT proteins. E260-treated cancer cells lose their ATP content and increase their glycolytic activity (especially in high glucose concentration), thus delaying (but not canceling) their death. These cancer cells operate with low hexokinase II levels and deregulated PARP-1. ATP-deficient cells activate AMPK, de-activate mTOR, and fail to recover from the state of autophagy, resulting in necrosis. So far, all healthy cells remained intact upon exposure to E260 [4]. Recalled ancient cell survival pathways operate as "protooncogenes-to-oncoproteins" in extant triploblastic hosts [2].

References

1. Ćetković H et al Gene 1998;216(1):77-84; Genomics 2004;83(4):743-745.

2. Sinkovics J G RNA/DNA & Cancer. Springer Verlag 2016 ERRATA in Author's CV; Int J Oncol 2015;47:1211-1229 ERRATUM Hydra viridis viridissima DOI: 10.3892/ijo.2016.3762 IJO 2017;50(1):338; Int J Oncol 2012;40:305-349; Inverteb Surviv J 2016;13:68-75 ERRATUM A. japonicus

3. Yaffe E et al Cancer Res 2014;74(22):6474-85.

4. Elkis Y et al Nature Commun 2017;8:940 DOI: 10.1038/s41467-017-00832-W.

#3079

Epstein Barr virus-encoded LMP1 reprograms glucose metabolism to enhance cell motility in nasopharyngeal epithelial cell.

Jun Zhang, Weitao Lin, Chi Man Tsang, Teng Fei Liu, Wing Chong Tang, Yim Ling Yip, Wen Deng, Lin Jia, Sai Wah Tsao. _The University of Hong Kong, Hong Kong, Hong Kong_.

How oncogenic virus rewires glucose flux to regulate cell motility is not well-understood. Epstein-Barr virus (EBV) infection of preinvasive nasopharyngeal epithelium is believed to be an essential initiation event during nasopharyngeal carcinoma pathogenesis. Here we demonstrate that EBV-encoded LMP1 enhances glycolysis thereby accelerating cell mobility of nasopharyngeal epithelial (NPE) cells. Activation of IGF1/mTORC2/AKT/PDHE1α signaling which mediates Histone 3 acetylation is involved in cell motility in EBV-infected NPE cells. Blocking the glucose uptake by pharmacological inhibitors effectively suppress glycolysis as well as the cell motility, linking the glucose metabolism to cell movement. We further show that mTORC2/AKT mediated phosphorylation of pyruvate decarboxylase kinase 1 (PDHK1) and its substrate, pyruvate decarboxylase E1α (PDHE1α) component plays a significant role in cell motility. PDHE1α knockdown effectively blocks LMP1-induced cell motility while overexpression of its phosphomimetic mutant S293A but not S293D inhibits LMP1-induced cell motility, supporting the involvement of PDHE1α in enhanced cell motility is depended on Ser293 phosphorylation. Furthermore, PDHE1α translocation into nucleus enhances Histone 3 acetylation and its binding to the promoter region of snail to enhance cell motility. Finally, we find that LMP1 increases the mRNA expression and the secretion of the ligand IGF1, which promotes phosphorylation of IGF1R as the key regulator for LMP1-induced mTORC2/AKT activation. In summary, this study provides evidences of involvement of EBV infection in metabolic reprogramming of NPE cells to promote cell motility involving activation of mTORC2/AKT/PDHE1α.

Acknowledgment: This project was supported by funding from the Research Grants Council, Hong Kong: General Research Fund (17120814, 17161116, 17111516, 171110315), Area of Excellence (AoE/M-06/08), Theme-Based Research Scheme (T12-401/13-R), Collaborative Research Fund (Project reference: C7027-16G); Health and Medical Research Fund of Hong Kong (12110782, 13120872, 04151726); and CRCG, University of Hong Kong, Hong Kong.

#3080

Chronic exposure to arsenic induces malignant transformation in human kidney epithelial cells.

Yu-Wei Chang, Kamaleshwar Singh. _Texas Tech Univ., Lubbock, TX_.

Arsenic contamination is a significant environmental and public health issue worldwide including the United States. Accumulating evidence suggests that kidney is one of the target organ for arsenic-induced carcinogenesis and toxicological effects. However, the mechanism of arsenic-induced renal carcinogenesis is not well understood. Therefore, the objective of this study was to evaluate the carcinogenicity of chronic exposure to low dose arsenic on kidney epithelial cells, and identify the molecular mechanism underlying this process. HK-2 kidney epithelial cells derived from normal human kidney were treated with arsenic for acute, long-term and chronic durations and cellular response to arsenic exposure at these time points was determined by evaluating the changes in growth, morphology, and expression of genes. The results revealed a significant dose-dependent growth inhibition after acute exposure, whereas increased growth after long-term and chronic exposure to arsenic of HK-2 cells as compared to untreated control cells. The anchorage-independent growth assay for colony formation further confirmed malignant transformation of arsenic exposed cells. Additionally, the morphological changes of EMT and stemness were also observed in long-term arsenic exposed cells. These changes observed above were further confirmed by analysis of gene and protein expression. Our data suggest that cellular response of EMT and stemness act a driving factor and mechanistic basis for increased survival and malignant transformation in arsenic exposed cells. In summary, results of this study suggest that acute exposure to arsenic can cause loss of kidney epithelial cells with potential significance to acute kidney injury, whereas the chronic exposure to arsenic can increase the risk of kidney cancer through enhancing cell proliferation.

#3081

Methylation changes and somatic mutations as early events in Lynch syndrome-associated colorectal cancer.

Satu Mäki-Nevala,1 Satu Valo,1 Ari Ristimäki,2 Laura Renkonen-Sinisalo,2 Anna Lepistö,3 Jukka-Pekka Mecklin,4 Päivi Peltomäki1. 1 _University of Helsinki, Helsinki, Finland;_ 2 _Helsinki University Hospital and University of Helsinki, Helsinki, Finland;_ 3 _Helsinki University Hospital, Helsinki, Finland;_ 4 _Jyväskylä Central Hospital and University of Eastern Finland, Jyväskylä, Finland_.

Lynch syndrome (LS) is a hereditary cancer predisposition syndrome caused by germline mutations in the DNA mismatch repair (MMR) genes resulting in an increased risk of colorectal cancer (CRC) and other malignancies. Those germline defects with other genetic and epigenetic changes accelerate multistep tumorigenesis. However, early events leading to polyp formation and the timing and order of the molecular "hits" remain unknown. Studies on sporadic CRC have indicated that promoter hypermethylation leading to gene silencing can act as an alternative mechanism to mutations at early stages of tumorigenesis, but its importance in hereditary CRC is obscure. In this study we aimed to define 1) methylation changes that occur at different stages of LS-associated colorectal tumor progression, and 2) somatic mutations that occur in adenomas comparing the status of low- and high-grade dysplasia and MMR deficiency (MMR-D) and proficiency (MMR-P). Colorectal biopsies including normal mucosae, adenomas and carcinomas were prospectively collected from 104 LS patients during colonoscopy surveillance, supplemented with retrospective tumor specimens from 56 patients. Promoter methylation was analyzed using the methylation-specific multiplex ligation-dependent probe amplification test (MS-MLPA) including selected tumor suppressor genes (TSGs) associated with early colon oncogenesis. Hypermethylation tendency was evaluated based on the frequency of CpG island methylator phenotype (CIMP) according to the methylation status of eight established CIMP marker genes. LINE-1 methylation was studied as a surrogate marker for global hypomethylation. Immunohistochemistry was used to detect MMR protein expression in neoplastic lesions. As an ongoing study mutation status of adenomas are studied using the Ion AmpliSeqTM Colon and Lung Cancer Panel targeting mutational hot spots in 22 genes. Results indicate that the expression of the MMR protein corresponding to the gene mutated in the germline decreases along with dysplasia but occurs as a relatively late event in the tumor progression, suggesting the presence of other somatic events that drive neoplastic transformation. Taken together, methylation of TSGs and frequency of CIMP increased and LINE-1 methylation decreased in adenomas and carcinomas along with dysplasia. However, a significant increase of methylation in some genes was detected already in adenomas with low-grade dysplasia compared to normal mucosae. In addition, a proportion of low-grade adenomas could already be classified CIMP positive. When comparing MMR-P and MMR-D adenomas, LINE-1 methylation decreased along the loss of MMR protein expression, and interestingly, higher methylation of SFRP1 was observed in MMR-P adenomas compared to MMR-D cases and normal mucosae. These findings emphasize the importance and early appearance of epigenetic changes in LS-associated tumorigenesis.

#3082

Epidermal-specific overexpression of T-cell protein tyrosine phosphatase in mouse attenuates chemically-induced skin carcinogenesis.

Mihwa Kim,1 Liza D. Morales,1 Iksoon Jang,2 Andrew Tsin,1 Dae Joon Kim1. 1 _University of Texas Rio Grande Valley, Edinburg, TX;_ 2 _Korea Basic Science Institute, Daejeon, Republic of Korea_.

Tyrosine phosphorylation signaling, which is regulated by the counter-activities of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), is critical in maintaining cellular homeostasis. The aberrant increase of tyrosine phosphorylation by mutation and/or overexpression of PTKs can contribute to skin carcinogenesis. While PTKs have been extensively studied in skin carcinogenesis, PTPs have not been studied due to their inactivation by environmental toxicants. Our recent studies showed that T-cell protein tyrosine phosphatase (TC-PTP; encoded by Ptpn2) deficiency in mouse epidermis significantly increased skin tumor formation, demonstrating TC-PTP deficiency predisposes mice to skin carcinogenesis. To further examine the tumor suppressive role of TC-PTP in skin carcinogenesis, we generated epidermal-specific TCPTP-overexpressing (K5HA.Ptpn2) transgenic mice. TC-PTP overexpression led to sensitization to tumor initiator 7,12-dimethylbenz[a]anthracene (DMBA)-induced apoptosis both in vivo epidermis and in vitro keratinocytes. TC-PTP overexpression in epidermis significantly reduced epidermal thickness and hyperproliferation compared to wild-type control following treatment with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Furthermore, TC-PTP overexpression significantly induced epidermal differentiation following TPA treatment as evidenced by the increased expression of epidermal differentiation markers loricrin, involucrin, keratin 10, and transglutaminase 5 in comparison to control. Two-stage skin carcinogenesis analysis using a DMBA/TPA regimen clearly revealed that K5HA.Ptpn2 mice exhibited delayed tumor development and significantly reduced tumor numbers compared to control mice. Taken together, our results suggest that TC-PTP is a potential therapeutic target for the prevention of skin cancer given its ability to promote epidermal apoptosis and differentiation and inhibit epidermal proliferation.

#3083

**Ameliorative effect of ethanolic extract of** Annona muricata **against sodium arsenite-induced hepatotoxicty in Wistar rats.**

Adenike M. Adegboyega. _The Polytechnic, Ibadan, Nigeria_.

Ingestion of arsenic in drinking water causes cancer at multiple tissues and there is no cure. Cancer has become a great monster to human race, as it places a significant emotional and economic burden on families and governments all over the world. Research is therefore directed at chemoprevention using medicinal herbs for the management of arsenicosis. In this study, we evaluated the in vitro antioxidant and protection offered by Annona muricata L. (AM) against sodium arsenite induced hepatotoxicity in rats. Antioxidant and radical scavenging activities of AM were compared to vitamin C and butylated hydroxytoluene (BHT). Proximate and phytochemical analyses were also carried out. Hepatoprotective study was investigated with six groups of rats that received distilled water (Control), 5.0 mg/kg bwt of NaAsO2, 250 mg/kg bwt AM, 500 mg/kg bwt AM, NaAsO2 plus 250 mg/kg AM, NaAsO2 and 500 mg/kg AM. The NaAsO2 was given once on days 7, 14 and 21, while AM was administered orally daily for 21 days. Serum transaminases and alkaline phosphatase activities were determined and liver histopathology carried out. AM contained 2.00% ash, 1.94% crude fat, 25.65% crude fibre, 2.88% protein and 58.62% carbohydrate. Phytochemical analysis indicated the presence of alkaloids, flavonoids and cardiac glycosides. The reducing power and metal chelating ability was in the order Vit C > BHT > AM, while for DPPH scavenging ability AM > Vit C > BHT. The NaAsO2 significantly (p < 0.05) increased the liver function enzymes relative to control. However, AM treatment markedly reduced the marker enzymes and restores the severe vaculation of hepatocytes in the NaAsO2 group to near normal. Our findings suggest that AM may constitute a remedy against arsenic induced hepatic injuries.

#3084

Epstein Barr virus-encoded LMP1 activates the mTORC2 signaling pathway to reprogram glucose metabolism in nasopharyngeal epithelial cell.

Jun Zhang, Weitao Lin, Chi-Man Tsang, Teng Fei Liu, W C. Tang, Tim Ling Yip, Wen Deng, Lin Jia, Sai-Wah Tsao. _Univ. of Hong Kong, Hong Kong, China_.

Epstein-Barr virus (EBV) infection of preinvasive nasopharyngeal epithelium is believed to be an essential initiation event during nasopharyngeal carcinoma pathogenesis, but the underlying mechanisms are largely undefined. Here we demonstrate that EBV-encoded LMP1 reprograms the glucose metabolism from oxidative phosphorylation to glycolysis of nasopharyngeal epithelial (NPE) is a major process for NPC pathogenesis. RNA-seq analysis indicates that EBV infection reprograms the glucose metabolism from glycolysis and oxidative phosphorylation and enhances the metabolic associated pathways. Our study show EBV encoded LMP1 is important for this event, EBV infection and LMP1 expression enhance the extracellular acidification rate and decrease the oxygen consumption rate in NPE cells. We further show that activation of mTORC2/AKT signaling is involved in this metabolic reprogramming in EBV-infected NPE cells. Genetic or pharmacologic inhibition of the mTORC2/AKT signaling blocks the LMP1-induced glycolysis. In summary, this study provides evidences of involvement of EBV infection in metabolic reprogramming of NPE cells. Acknowledgment: This project was supported by funding from the Research Grants Council, Hong Kong: General Research Fund (17120814, 17161116, 17111516, 171110315), Area of Excellence (AoE/M-06/08), Theme-Based Research Scheme (T12-401/13-R), Collaborative Research Fund (Project reference: C7027-16G); Health and Medical Research Fund of Hong Kong (12110782, 13120872, 04151726); and CRCG, University of Hong Kong, Hong Kong.

#3085

Epithelial S100A2 reduced upper aerodigestive track carcinogenesis by immunomodulation.

Li-Wha Wu. _National Cheng Kung Univ., Tainan, Taiwan_.

Upper aerodigestive track cancer (UATC) including oral and esophageal cancer is the 3rd leading cause of cancer death in the world. Most of these patients demonstrate compromised immunity systems. Myeloid-derived suppressor cells (MDSCs), a population of cells exerting immunosuppressive effects, can sustain cancer progression by providing a pro-tumor microenvironment through the enhancement of T-cell suppressive factors like arginase 1 (ARG1). MDSCs can subsequently facilitate the development of immunosuppressive regulatory T cells (Tregs). Tumor-associated macrophages (TAMs) produce multiple inflammation modulators and are other key players in tumor microenvironment and divided into M1 and M2 phenotypes with specific expression of different cytokines in each type. Several S100 proteins are recently shown to participate in tumor promotion or suppression through both autocrine and least known receptor-mediated paracrine release. RAGE and TLR4, frequently expressed on immune cells, are two known receptors for S100 proteins. Among many S100 proteins, we previously showed a tumor suppressor role of S100A2 in oral cancer. Whether S100A2 functions as a tumor suppressor in mice and plays any role in regulating these immune cell populations during upper aerodigestive track carcinogenesis remains elusive. Due to lack of S100A2 expression in mice, epithelial-specific human S100A2 transgenic mice and their wildtype littermates were used for tumor induction in upper areodigestive track by the co-treatment of 4NQO and arecoline. Epithelial S100A2 significantly repressed tumor formation in esophagus. To understand the involvement of immune cell recruitment in these mice, flow cytometry and qRT-PCR analyses were used. Among the key molecules associated with MDSCs, Tregs and TAMs, S100A2 preferentially induced tumor macrophage polarization to M1 phenotype with a marginal decrease of Tregs recruitment at 7 month post tumor induction. Together, epithelial S100A2 exerts anti-tumor effect in part through differential impact on immune cell recruitment although more studies are needed to underscore the action mechanism.

#3086

CPAP enhances diethylnitrosamine-induced hepatocarcinogenesis via STAT3 pathway.

Liang-Yi Hung. _National Cheng Kung Univ., Tainan, Taiwan_.

Chronic and persistent inflammation is a well-known carcinogenesis promoter. Hepatocellular carcinoma (HCC) is one of the most common inflammation-associated cancers; most HCC arises in the context of chronic inflammation and hepatic injury. Both NF-κB and STAT3 are important regulators for inflammation. Our previous studies indicated that CPAP (centrosomal P4.1-associated protein), a centrosomal protein that majorly participates in centrosome functions, is overexpressed in HCC and can increase TNFα-mediated NF-κB activation and IL-6-induced STAT3 activation. A hepatocyte-specific CPAP-expressing transgenic mouse was generated to investigate the physiological role of CPAP in hepatocarcinogenesis. An obvious inflammatory cell accumulation and fatty change were shown in the CPAP transgenic (Tg) mouse liver. The ALT level as well as the expression of inflammatory genes, such as IL-6, IL1β and TNF-α in CPAP Tg mouse are higher than in the wild type (WT) mouse. By high-dose and short-term diethylnitrosamine (DEN) treatment, the ALT level, pro-inflammatory genes and STAT3 activation are increased in CPAP Tg mouse; low-dose and long-term DEN treatment induces more severe liver tumor formation in CPAP Tg mouse than in WT mouse. In summary, these results suggest that CPAP may promote hepatocarcinogenesis through enhancing the inflammation pathway via activating the STAT3 pathway.

#3087

Identification of EBV infection and its life cycle in pan-cancer samples.

Hyojin Song,1 Hogune Im,2 Sung-Soo Yoon,3 Youngil Koh3. 1 _Cancer Research Institute, Seoul National University, Seoul, Republic of Korea;_ 2 _Genome Opinion, Seoul, Republic of Korea;_ 3 _Seoul National University Hospital, Seoul, Republic of Korea_.

Viral infection is associated with 10-15 percent of human cancers and EBV (human herpesvirus 4 (HHV-4)) contributes to cancer development in diverse cancer types. The life cycle of EBV is divided into latent and lytic cycles. Lytic replication cycle begins when early transcription factors are activated, and initiation complex is formed. Subsequently, lytic antigens are expressed once infected cells enter the lytic cycle. But the contribution of each cycle in cancer development is not well known yet. Hence we explored EBV integration and gene expression pattern by analyzing TCGA whole transcriptome sequencing (WTS) data.We investigated the integration of EBV by using FASTQ-formatted TCGA tumor WTS data. We designed a three-step EBV detection workflow. 1) Overall viral integration into human genome sequence was detected by VirusSeq. 2) "Spliced Transcripts Alignment to a Reference" algorithm was used for mapping against a hybrid reference genome of human and EBV. 3) To quantify the expression of EBV-related genes, we applied "RNA-Seq by Expectation Maximization" algorithm to obtain Transcripts Per Million values. Furthermore, we validated this detection workflow in EBV-positive cell lines from the public data repository and analyzed mutation profiles by using Mutation Annotation Format files.We investigated 851 WTS samples over 23 cancer types and scanned viral presentations by mapping against four types of EBV strains (Human HHV-4 complete wild-type, GD1, AG876, and artificial join). Consequently, we detected 88 samples mapped against the hybrid genome, and identified 46 of them were EBV-infected. By performing gene expression analysis, we recognized that 39/46 samples were expressing EBV lytic genes. Three types of cancers (Colorectal adenocarcinoma (COAD), Rectum adenocarcinoma (READ), and Stomach adenocarcinoma (STAD)) showed EBV lytic gene expression. Along with STAD, both BZLF1 and BALF2 were highly expressed in COAD; BZLF1 encodes an early transcription factor, and BALF2 is one of the viral genes constructs the initiation complex. Using our method, we also identified the expression of EBV lytic and latent genes in EBV-positive cell lines, hence validating our results. In addition, we found out an association between mutational events and EBV infection in COAD by profiling mutational signatures: increased C to T transitions and TP53 disruptions in EBV-infected samples. Overall, we could identify biologically relevant EBV-infected cancer samples considering EBV life cycle. We hereby drew two major conclusions based our findings. First, we established an analysis workflow to find out biologically meaningful EBV infection in cancer by quantifying gene expression of composing the EBV life cycle, beyond a simple viral integration. Second, we discovered EBV expression pattern in small number of colon cancer patients. Our integrative data will provide clues for comprehensively understanding of EBV-associated cancers.

#3088

Deciphering components of mutational signatures arising from carcinogen co-exposures: A genome-scale experimental approach.

Manuraj Pandey,1 Maria Zhivagui,1 Mona I. Churchwell,2 Alvin W. Ng,3 Liacine Bouaoun,1 Vincent Cahais,1 Martha R. Stampfer,4 Magali Olivier,1 Zdenko Herceg,1 Ewy Mathé,5 Steven G. Rozen,3 Frederick A. Beland,2 Michael Korenjak,1 Jiri Zavadil1. 1 _Int. Agency for Research on Cancer, Lyon, France;_ 2 _National Center for Toxicological Research, Jefferson, AR;_ 3 _Duke-NUS Medical School, Singapore, Singapore;_ 4 _Lawrence Berkeley National Laboratory, Berkeley, CA;_ 5 _Ohio State University, Columbus, OH_.

Pan-cancer analyses of tumor genomes reveal mutational signatures characteristic of particular etiologic factors. The signatures are extracted mathematically from mixed patterns typically observed by tumor sequencing. However, the components of signatures originating from complex carcinogen mixtures have not been studied in detail. Lung, head and neck and liver tumors of tobacco smokers exhibit COSMIC signature 4 marked by predominant G>N mutations, involving mainly G:C>T:A transversions with transcription strand bias, consistent with the mutagenic effects of benzo[a]pyrene (B[a]P). Additionally, A>N mutations (strand-biased A:T>T:A transversions and A:T>G:C transitions) are also prominently present, yet their origins are less understood. By using exposure-coupled clonal immortalization of human and mouse primary cells and deep sequencing, we were able to dissect 'clean' mutational signatures of tobacco smoke carcinogens B[a]P and glycidamide (GA), a key reactive metabolite of acrylamide (ACR). Whole-genome sequencing of multiple clones derived from primary B[a]P-treated human mammary epithelial cells identified a robust mutational signature marked by strand-biased G>N mutations and increased GG>TT dinucleotides, while no apparent enrichment of A:T>T:A mutations was observed. Next, in ACR and GA-treated primary mouse embryonic fibroblasts, we established by the LC-MS/MS DNA adduct analysis that ACR exerts its mutagenic effects exclusively via GA. We then extracted from 15 treated clones the exome-scale mutational signature of GA, marked by predominant A:T>T:A transversions followed by A:T>G:C transitions and G:C>T:A transversions, all showing transcription strand bias. Similarity analysis involving known primary-cancer and experimental mutational signatures indicated that the GA mutational signature was novel. A more in-depth comparison with mutation patterns from lung adenocarcinomas of heavy smokers revealed that the GA signature, including its strand bias features, matched closely with and may thus account for the A>N mutation component of the tobacco smoking-derived signature 4. Thus, mutational signatures generated in controlled experimental settings may explain particular sub-features of cancer signatures arising from co-exposures to multiple carcinogens. Furthermore, the use of innovative in vitro systems, characterized by biological barrier bypass to mimic early steps of cell transformation, can provide revealing insights into the molecular links between mutagenesis and carcinogenesis. Funding: INCa-INSERM Plan Cancer 2015; NIH/NIEHS 1R03ES025023-01A1

#3089

YAP and TAZ induce MCM protein expression to facilitate tumor-supporting properties in liver cancer.

Maria Knaub, Sofia Weiler, Stefan Thomann, Peter Schirmacher, Kai Breuhahn. _University Hospital Heidelberg, Heidelberg, Germany_.

Overexpression and nuclear enrichment of the Hippo pathway-regulated transcriptional co-activator Yes-Associated Protein (YAP) is frequently detected in human hepatocellular carcinoma (HCC) and correlates with poor clinical outcome of cancer patients1. In mice, inducible overexpression of the constitutively active isoform YAPS127A induces hepatocellular proliferation leading to hepatomegaly followed by tumor formation within 12-15 weeks. How oncogenic YAP and its paralogue TAZ (syn. WWTR1, WW Domain Containing Transcription Regulator 1) induce uncontrolled proliferation in liver cells is not known, yet. Analysis of expression profiling data, derived from human liver cancer cells after siRNA-mediated inhibition of endogenous YAP, revealed that the Minichromosome Maintenance (MCMs) family members MCM2-7 were positively regulated. MCMs are key components of the pre-replication complex and involved in the formation of the replication fork, which is essential for efficient DNA duplication followed by mitosis2. We confirmed that silencing of YAP and TAZ by independent siRNAs reduced the mRNA and protein expression of MCM2-7 in liver cancer cell lines. In vitro analyses revealed that transcription factors of the TEAD family were the most relevant YAP binding partners needed for the transcriptional regulation of MCMs. Using chromatin immunoprecipitation and Dual Luciferase Reporter Assay, we showed that YAP, TAZ and TEAD4 directly bound the MCM promoter. First in vitro data suggested that viability of liver cancer cells, subjected to drug induced replication stress, was exclusively reduced in cells with reduced YAP, TAZ or MCM protein expression. Administration of the YAP/TAZ/TEAD inhibitor Verteporfin reduced the expression of all MCMs in liver cancer cells. Vice versa, the overexpression of YAPS127A in transgenic mice showed significantly increased levels of the proliferation marker Ki67 and MCM2-7. Expression data from 242 HCC patients illustrated an association between YAP and MCM mRNA levels, with elevated MCMs significantly correlating with worse overall survival and early cancer recurrence3. Immunohistochemical stains of HCC tissue micro-arrays revealed a highly significant correlation between Ki67 expression, nuclear YAP overexpression and nuclear MCM2-7 enrichment. In summary, our results strongly suggest that YAP and TAZ facilitate their tumor-supporting properties through the regulation of MCM2-6, a complete protein complex. We conclude that combined inhibition of YAP and TAZ or perturbation of MCM activity might represent an efficient therapeutic approach for the treatment of a subgroup of HCC patients. 1Tschaharganeh D, et al., Gastroenterol. 2013; 2Deegan TD, et al., Curr Opin Struct Biol. 2016; 3Roessler S, et al., Cancer Res. 2010.

#3090

Phenol modifying influence on genotoxic effect in organism during peroral combined administration with benzo[a]pyrene.

Olga Ostash,1 Igor Chernichenko,1 Nina Balenko,1 Elena Vinarska,1 Lyudmila Grigorenko,1 Svetlana Lukyanchuk,1 Oksana Shvager2. 1 _O.M. Marzeyev Inst. for Public Health of The National Academy of Medicine, Kyiv, Ukraine;_ 2 _Bogomolets National Medical University, Kyiv, Ukraine_.

In the modern world in different spheres of life, people experience a load of the complex of numerous chemical compounds of different types, which have a wide range of biological effects - from toxic ones to mutagenic and carcinogenic. For instance, people suffer from the growth of cancer, which is one of the direct consequences of the impact of environmental pollution by chemical compounds. It has now been proved that the development of cancer is determined not only by the initiating effect of chemical carcinogens, but also by the modifying effect of the concomitant toxic compounds. The aim of this work was experimental study of the role of toxic substances (phenol) in the modification of the evidence of genotoxic and immunological changes in organism in the early stages of carcinogenic activity (benzo[a]pyrene).The studies were conducted with random-bred white male mice. Peroral administration was applied to animals taken benzo[a]pyrene (day-dose 0.1mg), benzo[a]pyrene - phenol (day-dose 0.1; 0.002 mg). Genotoxic changes in forestomach were assessed with the help of micronuclear test. The carcinogenic effect (forestomach papillomas) and common regularities of genotoxical and immunological changes in relation to carcinogenesis and their dose and treatment duration dependence under benzo[a]pyrene (day-dose 0,1mg), benzo[a]pyrene and phenol (day-dose 0,1; 0,002 mg) complexes oral administration in random-bred white mice were established. The noted regularities conducted in interrelation and identical directivity of mutagenic (increasing of micronucleus incidence) and immunological (reducing the relative number of T-lymphocytes) indexes changes in relation to carcinogenesis, presence of reliable cross-correlation connections between them in the end of the first months. Besides, the modifying effect of phenol on the carcinogenesis that appeared by increasing of micronucleus incidence, immunosuppression and index of forestomach papillomas multiplicity was determined. The findings detect the presence of cancer risk from the action of carcinogenic and toxic substances, the formation of which begins in the early period (at the end of the first month) and manifests itself as a genotoxic effect in combination with immunosuppression, which must be taken into account in the hygienic assessment of the state of the environment.

#3091

Functional screening of genes involved in carcinogenesis using a cDNA expression library of ovarian clear cell carcinoma cells.

Tsutomu Miyamoto, Yasushi Yamada, Koichi Ida, Ryoichi Asaka, Hisanori Kobara, Hirofumi Ando, Hiroyasu Kashima, Akihisa Suzuki, Tanri Shiozawa. _Shinshu Univ. School of Medicine, Matsumoto, Japan_.

Background: Ovarian clear cell carcinoma (OCCC) is one of the 4 major histological types of ovarian carcinoma (OVC) and is frequently arising from ovarian endometriotic cysts. OCCC is rare in the United States and western countries, but it is more frequent in Japan and may be seen in almost 25% of OVC. OCCC commonly shows chemo-resistance which makes difficult to treat OCCC. In order to develop the new therapeutics for OCCC, genetic abnormalities that lead to the development of OCCC need to be elucidated in detail. The comprehensive analysis using next-generation sequencing has rapidly revealed the accumulation of genetic abnormalities in many cancers. However, the significance of each abnormality has not been elucidated. Therefore, functional screening using a cDNA expression library was performed in order to identify genes involved in the pathogenesis of OCCC.

Methods: The mRNA was extracted from an OCCC cell line, RMG-1, and a cDNA library was established using a retroviral vector. This cDNA library was transfected into mouse NIH3T3 cells, and then the transformed foci which formed multilayered growth were picked up and the inserted DNAs of those foci were detected by sequencing. The mRNA and protein expression and the transforming ability of the identified genes were confirmed by immunohistochemistry and focus-formation assay.

Result: Seven genes were isolated; Dishevelled 1(DVL1), beta subunit of Sec61 (SEC61B), K-specific demethylase 4A (KDM4A), chromosome 1 open reading frame 38 (C1ORF38), splicing factor 1 (SF1), ribosomal protein S2 (RPS2), and ribosomal protein S3A (RPS3A). Of these genes, RT-PCR and immunostaining revealed that the expression of SEC61B and C1ORF38 was stronger than that in other histological types of ovarian cancer. A mutational analysis of the DVL1 c-terminal region detected 3 point-mutations in RMG1 cells, whereas no mutation was found in 24 cases of OCCC. Focus formation was confirmed by the transfection of SEC61B, C1ORF38, and DVL1 into NIH3T3 cells.

Conclusion: These results suggest that SEC61B, C1ORF38, and DVL1 may be involved in the pathogenesis of OCCC.

#3092

Redirection of human HER2-positive breast cancer cells.

Anastasia Frank-Kamenetskii, Julia M. Mook, Brian W. Booth. _Clemson Univ., Clemson, SC_.

Breast cancer is the most common form of cancer among women, affecting one eigth of the female population. While advances in clinical management and therapeutic modalities have been achieved, serious limitations in the improvement of outcomes after the diagnosis remain, largely due to the complexity of the underlying biology. Studying the mechanism that makes cancer cells tumorigenic and how they can lose the ability to cause malignant formations is the first step towards understanding complex mechanisms of this disease. The normal mammary microenvironment can redirect cells to assume a normal mammary epithelial phenotype. This phenomenon also applies to tumorigenic cells; cancer cells lose their tumor-forming capacity and differentiate, assuming a normal, non-tumorigenic phenotype. This phenomenon has been termed "cancer cell redirection." Comparing cells that went through this transformation with original cancer cells, features that identify tumorigenic activity can be acknowledged. We have established an in vitro model of cancer cell redirection, in which HER2+ breast cancer cells are grown alone or in co-culture with normal human breast epithelial cells. The ratios of cells used in co-cultures, 1:1 and 1:50 (cancer: normal) were previously established through in vivo and in vitro studies. Redirection was confirmed by immunostaining. The metric used to determine cancer cell redirection was the absence of receptor tyrosine kinase phosphorylation without a change in surface expression of the receptor. HER2 and P-HER2 expression were both present in the HER2\+ cancer cells and in the: MCF10A and 1:1 co-cultures. However, HER2 expression was observed in 1:50 co-cultures, but an absence of P-HER2 was noted. Based on attenuation of receptor phosphorylation in the 1:50 co-cultures we can confirm the breast cancer cell redirection, which matches our previous results. Furthermore, alteration of HER2 dimerization patterns in redirected cells were investigated. The capacity of cancer and redirected cells to form tumors was examined with use of mammosphere assay. Formation of mammospheres has correlated to stem cell function and tumor-initiation capacity. The HER2+ breast cancer cell lines formed significantly higher numbers of spheres compared to the non-tumorigenic breast epithelial cells. Mammosphere formation is decreased in redirected cancer cells. Breast cancer stem cells are HER2+ regardless of pathological or genetic classification. This suggests that HER2 expression is not only a target in HER2+ breast cancer but should be targeted in all breast cancers to prevent breast cancer stem cells from recapitulating tumors following treatment. Our data demonstrates that by attenuating activity of HER2 theses cancer cells lose the capacity to form tumors in vivo and lose their cancer phenotype in vitro.

#3093

Genomic characteristics of myeloproliferative neoplasms in patients exposed to ionizing radiation following the Chernobyl nuclear accident.

Larysa Poluben,1 Maneka Puligandla,2 Donna Neuberg,2 Christine R. Bryke,1 Nancy Hsu,1 Sergiy Klymenko,3 Olga Mishcheniuk,3 Oleksandr Shumeiko,3 Steven Balk,1 Xin Yuan,1 Olga Voznesensky,1 German Pihan,1 Miriam Adam,4 Ernest Fraenkel,4 Paula G. Fraenkel1. 1 _Beth Israel Deaconess Medical Center, Boston, MA;_ 2 _Dana-Farber/Harvard Cancer Center, Boston, MA;_ 3 _National Research Center for Radiation Medicine, Kyiv, Ukraine;_ 4 _Massachusetts Institute of Technology, Cambridge, MA_.

Philadelphia-chromosome negative chronic myeloproliferative neoplasms (MPNs) are a unique group of hematological malignancies including Polycytemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) characterized by impaired function and structure of bone marrow. MPN driver mutations are usually found in Janus Kinase 2 (JAK2), Thrombopoietin Receptor (MPL) and Calreticulin (CALR) genes, however, 10-15% of MPN cases are triple negative (TN) for these mutations. A previous study showed a lower rate of JAK2 V617F mutations in PMF patients exposed to sublethal doses of ionizing radiation (IR) from the Chernobyl accident in Ukraine. We hypothesized a lower rate of the usual driver mutations in IR-exposed MPN patients. To examine whether there are distinct driver mutations, 281 Ukrainian IR-exposed and unexposed MPN patients, were enrolled in the study. Their records were reviewed for classification by the WHO 2016 MPN criteria. Genomic DNA was obtained from the peripheral blood leukocytes of 281 MPN patients to identify JAK2 V617F, MPL W515, and type 1- and 2-like CALR mutations by allele-specific PCR, Sanger Sequencing and RT-PCR, respectively. Copy number alterations and copy-neutral loss of heterozygosity (cnLOH) were assessed in 30 PMF patients by high-density Affymetrix CytoScan HD oligo-SNP microarray platform. Whole exome sequencing was used to identify additional genetic variants in these 30 PMF patients. Statistical significance for categorical variables and continuous variables were evaluated by Fisher's exact test and Wilcoxon's rank sum test using Statistical Analysis R, version 3.4.2. Clinical features of exposed and unexposed MPN patients were similar. More PMF IR-exposed patients were transfusion dependent (32.4%) than PMF unexposed patients (14.1%) (p = 0.04). JAK2 V617F was detected in 58% of IR-exposed and in 74% of unexposed MPN patients (p = 0.007). JAK2 V617F was also less frequent in IR-exposed PV patients, but not statistically significant. Type 1-like CALR mutation was detected in 12% of exposed and 3% of unexposed patients (p = 0.033). Overall, exposed patients were TN in 28% of IR-patients versus 16% of unexposed patients (p = 0.027). Among other genetic variants, ATM S1691R mutation with cnLOH at 11q22.3 was identified in one TN IR-exposed PMF patient. Previously the mutation was reported, but not in MPN patients. Missense mutations in EZH2 at 7q36.1 and SUZ12 at 17q11.2 with copy number loss were also identified in TN IR-exposed PMF patients. Our results confirm a lower rate of JAK2 V617F and higher rate of TN cases among IR-exposed MPN patients versus unexposed. We also demonstrated a higher frequency of type 1-like CALR mutation in IR-exposed MPN patients and new potential MPN driver mutations. Thus, IR-exposed MPN patients represent a disease group with distinct genomic characteristics worthy of further study.

#3094

Effects of aryl hydrocarbon receptor and estrogen receptor beta crosstalk on benzo(a)pyrene induced AhR target genes.

Kenneth J. Harris,1 Kelly L. Harris,1 Mary K. Washington,2 James Amos-Landgraf,3 Aramandla Ramesh1. 1 _Meharry Medical College, Nashville, TN;_ 2 _Vanderbilt University, Nashville, TN;_ 3 _University of Missouri, Columbia, MO_.

Colorectal cancer (CRC) is the third most common diagnosed cancer and the third leading cause of cancer-related deaths in the United States. Epidemiological evidence show estrogen might influence the incidence of CRC in women by acting in a protective role via estrogen receptor beta (ERβ) but the mechanism of action is not known. Benzo(a)pyrene [B(a)P], a member of the polycyclic aromatic hydrocarbon (PAH) family of compounds is a well-characterized environmental toxicant that has been proven to be a major contributor to the development of sporadic colon cancer. Literature provides evidence of crosstalk between Aryl Hydrocarbon Receptor (AhR), a receptor for B(a)P, and estrogen receptors (ERs) which negatively affect ER-mediated transcription. This study aims to elucidate the effect of AhR/ERB crosstalk on B(a)P-induced expression of AHR target genes in adult Polyposis In the Rat Colon (PIRC) model. We hypothesize that estrogen inhibits B(a)P-induced expression of AHR target genes and attenuates the formation of colon polyps in female PIRC rats. Groups of female and male PIRC rats (n = 8) received sub-chronic exposure to 25, 50 and 100 µg B(a)P/kg body wt. via oral gavage for 60 days. Female PIRC rats that received 25, 50 and 100 µg B(a)P/kg body wt. showed significant decrease in total polyp count when compared to males with respective treatments. Polyp sizes of female PIRC rats receiving 25, 50 and 100 µg B(a)P/kg body wt. were increased when compared to males respectively. Histopathological analysis of colon polyps revealed that female animals exhibited low-grade to no dysplasia while high-grade dysplasia was recorded in male animals treated with corresponding doses. As expected B(a)P increased expression of Cytochrome P450 isoform 1A1 (CYP1A1), CYP1B1, Sulfotransferase Family 1A Member 1 (SULT1A1), and Glutathione S-transferases (GST) in the colon of PIRC rats. Female PIRC rats show increase in CYP1B1, SULT1A1 and GST when compared to males in various treatment groups. It is possible that the increase in Phase II enzymes may provide clearance of B(a)P, preventing polyp development in female PIRC rats. In future studies, by measuring the expression of other phase 1 and phase 2 drug metabolizing enzymes (DME), along with measuring circulating estrogen levels, analyzing [B(a)P] metabolite profile, and probing B(a)P-DNA interactions, we will provide insight into how estrogen protects females from developing colon cancer. This research was funded by NIH grants 5RO1CA142845-04, 5R25GM059994-3, and G12MD007586-29.

#3095

Promotion of pancreatic cancer by perfluorooctanoic acid (PFOA).

Barbara A. Hocevar, Lisa M. Kamendulis. _Indiana University, Bloomington, IN_.

Pancreatic cancer is the fourth leading cause of cancer deaths and one of the most lethal forms of cancer diagnosed in the United States. While the exact cause of pancreatic cancer is unknown, established risk factors for pancreatic cancer include smoking, alcohol consumption and pancreatitis; all of which share the ability to generate oxidative stress - a condition known to promote cancer progression. Perfluorooctanoic acid (PFOA), a chemical widely used in consumer and industrial applications, has been shown to induce pancreatic acinar cell tumors in rodents through a yet to be determined mechanism. In humans, epidemiologic studies have linked PFOA exposure to adverse chronic health effects including several types of cancer. PFOA has been detected in essentially all of the American population, with mean serum levels close to 4 ng/ml and a predicted half-life of ~ 4 yrs. We have previously shown that exposure of mice to PFOA for 7 days triggered oxidative stress in the pancreas, which was associated with focal ductal hyperplasia and inflammation. The purpose of this study was to determine if PFOA exposure promotes the progression of pancreatic cancer in a well-characterized mouse model of pancreatic cancer, the LSL-KRasG12D;Pdx-1 Cre model. For our studies, LSL-KRasG12D;Pdx-1Cre mice received either tap water or tap water supplemented with 5 ppm PFOA, starting at 2 months of age. Mice were sacrificed at 6 and 9 months of age, corresponding to 4 and 7 months of PFOA treatment, at which time serum and tissues were collected. Pancreata were processed for histologic examination (H&E stain), immunohistochemistry for CK19, used as a marker to identify neoplastic lesion area, and RNA isolation for gene expression. Serum was utilized for quantitation of amylase, lipase, PFOA and inflammatory cytokine levels. Our preliminary results show that PFOA accumulates in the serum and pancreas of treated LSL-KRasG12D;Pdx-1 Cre mice. In addition, exposure to PFOA increased the CK19+ lesion area in LSL-KRasG12D;Pdx-1 Cre mice indicating an acceleration of pancreatic cancer progression. qPCR analysis of pancreata verified increased expression of both CK19 and the early lesion marker Sox9 in PFOA-treated mice. Analysis of mRNA expression in the pancreas revealed an increase in oxidative stress, evidenced by elevated Sod1 expression, and an increase in inflammatory markers (Tnfα and IL1α). Together, these results demonstrate that PFOA leads to expansion of the pancreatic lesion area in the LSL-KRasG12D;Pdx-1 Cre mouse model which is accompanied by oxidative stress and an inflammatory response. These results, coupled with the widespread human exposure to PFOA and its biological persistence, suggest that it could be a prominent agent promoting pancreatic cancer progession.

#3096

LINE-1 retrotransposition L1-FGGY promotes the occurrence and progression of lung squamous cell carcinoma by inhibiting the tumor-suppressor gene FGGY.

Rui Zhang, Jinpu Yu. _Tianjin Medical University Cancer Institute & Hospital, Tianjin, China_.

Retrotransposition is a kind of chromatin rearrangement containing the non-coding region, which makes up about half of the human genome. Long interspersed element-1 (LINE-1) retrotransposition is the only currently known active autonomous transposon in humans and might cause genetic instability, which was reported to occur with high frequency in a variety of tumor tissues, including colon cancer, prostate cancer, ovarian cancer, hepatocellular carcinoma, etc. However, the relationship between LINE-1 and lung squamous cell carcinoma (LUSC) is unclear. In this study, we analyzed 504 cases of LUSC samples based on the RNA-seq database in The Cancer Genome Atlas (TCGA), and found that 1/3 of tumor samples possessed LINE-1 inserted retrotransposons. We screened out 14 LINE-1 retrotransposons with the highest occurrence frequency and detected the expression of LINE-1 retrotransposons in 110 clinical LUSC samples and 52 matched adjacent normal tissue samples. The results showed that among 14 screened retrotransposon genes, the expression of 13 retrotransposon genes in LUSC was significantly higher than that in the corresponding para-carcinoma tissues, indicating that the presence of LINE-1 retrotransposon was related to the occurrence of LUSC. Furthermore, by analyzing the correlationship between the expression levels of LINE-1 retrotransposons and overall survival time of patients, we also found that the survival time of patients with low retrotransposon gene expression was long, while the survival time of patients with high retrotransposon gene expression was short. Among them, L1-FGGY and L1-ATP8B1 showed significant differences. In this study, we focused on one LINE-1 retrotransposon, i.e. L1-FGGY. Then we further investigated the mechanism of the LINE-1 retrotransposition in regulating the occurrence and progression of LUSC and discovered that the expression of L1-FGGY and FGGY was negatively correlated in LUSC patients (coefficient of association=-0.232, p<0.05). In order to further explore the function of FGGY in cancer development, we constructed 3 different siRNAs which could specifically interfere with the expression of FGGY and discovered that knockdown of FGGY could promote cell proliferation, inhibit cell apoptosis, as well as facilitate cell migration and invasion in vitro. The further study showed that inhibition of FGGY could promote tumorigenesis and tumor metastasis in vivo, which collectively indicated that FGGY might function as a tumor-suppressor gene. In conclusion, we not only uncovered that the LINE-1 retrotransposition L1-FGGY promotes the development of LUSC by inhibiting the expression and function of the tumor-suppressor gene FGGY, but also revealed that LINE-1 retrotransposon might be a new biomarker for early diagnosis, prognosis evaluation, and targeted therapy in the future clinical translation.

#3097

Chemo prevention effect of Withaferin A, a natural compound, on mouse models of colorectal carcinogenesis.

Deeksha Pal, Balaji Chandrasekaran, Becca Von Baby, Houda Alatassi, Murali Ankem, Chendil Damodaran. _Univ. of Louisville, Louisville, KY_.

Introduction: Colorectal cancer (CRC) is the most common cancer and one of the leading causes of cancer-related deaths in United States as well as worldwide. Development of sporadic CRC takes an average of 15-20 years, so establishing preventive measures are possible. We are interested in a dietary compound, Withaferin-A (WA), which possesses both chemotherapeutic and chemopreventive properties. WA's effect was studied on both colitis-mediated colon and spontaneous-intestinal carcinogenesis mouse models.

Methods: In the present study, two animal models of colon cancer were used to study the chemo- preventive effect of WA. In intestinal tumorigenesis model C57BL/6-ApcMin/+ , mice at six weeks of age were orally fed with the vehicle or WA (4mg/kg body weight of mice) for five days per week for 12-14 weeks. The other model used was AOM/DSS (Azoxymethane/dextran sulfate sodium), which induces gut inflammation and tumorigenesis. Intraperitoneal injection of AOM (8mg/kg) was followed by three cycles of DSS. WA (3mg/kg body weight of mice) was administered five times per week for 8-10 weeks. After completion of the studies, the tumor tissues were subjected to histopathological and molecular analysis. An unpaired Student's t-test was used for statistical analysis.

Results: Oral administration of WA to ApcMin/+ mice leads to a significant decrease in the number of intestinal polyps and colon tumors (colon: 42%, p=0.025), proximal: 53%, p=0.0063, middle: 27.84%, p= 0.28, distal: 32.8, p= 0.001) when compared to vehicle-treated mice. Similarly, in AOM/DSS, oral administration of WA reduced polyp multiplicity by 40% (p= 0.02) compared to vehicle-treated controls. Moreover, the treatment group of both colon cancer models showed inhibition of pro-survival signaling markers (Notch1, pAKT and NFκB) as well as a decrease in proliferative markers.

Conclusion: Our results suggest that WA effectively suppresses intestinal polyp development and colitis-mediated colon carcinogenesis, suggesting a preventive and therapeutic role in colon cancer models. These findings warrant further investigation to develop novel strategies to achieve maximum benefit of dietary agents for cancer chemoprevention.

#3098

Promotion of in vivo growth of Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma by miR-BARTs.

Anna Chi Man Tsang,1 Shaina Chor Mei Huang,1 Ming Han Tsai,2 Henri-Jacques Delecluse,2 Honglin Chen,1 George Sai Wah Tsao1. 1 _University of Hong Kong, Hong Kong, Hong Kong;_ 2 _German Cancer Research Centre (DKFZ), Heidelberg, Germany_.

Epstein-Barr virus (EBV) infection is nearly 100% associated with undifferentiated nasopharyngeal carcinoma (NPC). However, EBV episomes are rapidly lost under in vitro culturing if there is no selective advantage to their retention. The persistence of the viral episomes within the in vivo tumors therefore implies that the virus must be contributing to the growth and/or survival of the tumors. Since most of the growth-promoting EBV latent proteins are immunogenic, their expressions are usually low and heterogeneous within a tumor. Hence, the abundant expression of the non-immunogenic microRNAs derived from EBV-encoded BART transcripts, i.e. miR-BARTs, strongly implicates their pathogenic roles in this cancer. We found that EBV infected NPC cells could develop into bigger tumors in nude mice than the non-infected counterparts. Importantly, the miR-BARTs were all upregulated after the in vivo growth conditions. Nevertheless, EBV-infected and non-infected cells had comparative growth in in vitro culturing, suggesting EBV infection could provide growth advantage to NPC in vivo, but not in vitro. We have recently established a new and representative NPC cell line (NPC43) to study the pathogenesis of EBV in the development of NPC. In consistent will other previous publications, the EBV genomes were lost gradually when growing in vitro, resulting in a cell line with only half of the population is EBV-positive (at PD 50). Interestingly, after this mixture of EBV-positive and EBV-negative cells were grown as tumor in nude mice, the tumor cells were detected to be nearly 100% positive for EBV-infection. This suggests the in vivo growth conditions could select for the EBV-positive NPC cells. miR-BARTs are known to have a predominant role in suppressing cellular apoptosis. We hypothesized that miR-BARTs may help to resist apoptosis under metabolic stress in vivo, when tumor growth outstrips the nutrient supply. In our in vitro experiments, we observed that EBV infection protected the cells from apoptosis if they were grown in culture medium deprived of glucose and amino acids. Moreover, the expression levels of miR-BARTs were also upregulated in this metabolically stressed condition. To further investigate the roles of miR-BARTs, we infected NPC cells (NPC43) and immortalized nasopharyngeal epithelial cells (NP460) with either wild-type M81 EBV or mutant M81 EBV (with BART deleted). We found that NP460-M81WT showed a general trend of higher resistance to starvation-induced cell death than NP460-M81∆BART. Besides in the spheroid cultures of the pair of NPC43-M81WT and NPC43-M81∆BART, the WT cells could grow into bigger spheroids, reflecting they could sustain their growth under a gradient of nutrient deficiency which is present in the 3D structure of a spheroid. Taken together, all these suggest miR-BARTs could promote in vivo growth by enhancing survival of the EBV-infected cells.

#3099

Hydrocarbon carcinogenicity reexamined: An extremly potent direct-acting mutagen is common to smoke, soot and ash from a variety of combustion sources, including cigarette tobacco.

Gary R. Blackburn. _PetroLabs, Inc., Ivyland, PA_.

Percivall Pott (1714-1788), an English surgeon, is generally credited with being the first to suggest that cancer may be induced by exposure to environmental carcinogens -- in his case, cancer of the scrotum in London chimney sweeps occupationally exposed to soot. It is perhaps not surprising, therefore, that combustion products became a primary focus of later research into the mechanisms of chemical carcinogenesis. Early on, those studies pointed toward a particular class of compounds as being primarily responsible for cancer induction by such materials, a group of environmentally ubiquitous compounds called polycyclic aromatic hydrocarbons, or PAHs. One such PAH, benzo[a]pyrene (BaP), is perhaps the most studied compound in all of cancer research. While there is little doubt that BaP and its congeners are the primary carcinogens in incomplete combustion products like coal tar, their concentrations in other hydrocarbon mixtures are too low to fully account for their overall carcinogenic potency. It is possible that other factors such as tumor promotion contribute to the additional potency, but it might also be expected that there are significant contributions from nitro-PAH derivatives, in particular the isomers of dinitropyrene (DNP) found in carbon black, diesel exhaust and other combustion products. One such isomer, 1,6-dinitropyrene was shown by Iwagawa et al. to have three-times the carcinogenic potency of BaP when injected directly into the lungs of rats. In order to indirectly test nitroaromatic contribution to combustion product carcinogenicity, extracts of smoke soot, ash and food products exposed to smoke during processing were fractionated by HPLC, and the fractions tested in the Ames Test without metabolic activation. All showed a common peak of extremely potent direct-acting mutagenicity at a retention time similar to that of dinitropyrene. Efforts are currently underway to fully characterize the mutagen by a combination of standard analytical means. 

### Harnessing the Power of Cell Lines for Cancer Research

#3100

Replacing fetal calf serum by human platelet lysate in cancer research and toxicology.

Oliver H. Krämer. _Institut für Toxikologie Universitätsmedizin der Johannes Gutenberg-Universität Mainz, Mainz, Germany_.

Experiments with cultured mammalian cells represent a common in vitro alternative to animal experiments. Fetal calf serum (FCS) is the most commonly used medium supplement. FCS contains a mixture of largely undefined growth factors and cytokines. Since FCS is received from unborn calves older than three months, it represents a massive burden for the pregnant cows and their fetuses. A needle is inserted into the heart of the fetus to collect blood. Since the animal is not under anesthesia, it may suffer pain and discomfort. Furthermore, the undefined nature of FCS is a source of experimental variation, undesired immune responses, and possible contaminations. Thus, alternative, defined, valid, and reliable medium supplements should be identified. We follow the principles of the 3Rs (Replacement, Reduction, Refinement). We established a method to receive human platelet lysate (hPL) from human blood platelets. We investigated if hPL represents potential true alternative for FCS in cell culture experiments and thereby the cruel slaughtering of unborn fetuses. hPL is received by freeze and thawing cycles and centrifugation steps. We successfully adapted three human leukemia cell lines (K562, NB4 and MV4-11) and HCT116 colon cancer cells to culture medium containing 1-5% hPL. We analyzed cell morphology by microscopy. Cell growth kinetics were examined by counting and cell cycle distributions were investigated by flow cytometry. Protein expression patterns were determined globally by global scale proteomics. We additionally determined possible FCS- or hPL-dependent reactions of cells to the well-established anti-cancer drugs Irinotecan, Hydroxyurea and Imatinib, and to epigenetic modulators of the HDAC inhibitor family (FK228 and the new, highly selective HDAC6 inhibitor Marbostat-100) by flow cytometry and Western blot. In these tests, we particularly focused on the expression of the pan-leukemic marker and transcription factor Wilms tumor 1 (WT1) and on the tumor suppressor p53. All tested cell lines grow with 5% FCS and 5% hPL. Cell morphology, cell cycle distribution, and protein expression patterns were not affected by such different culture conditions in resting and treated cells. We further show that the class I HDACs HDAC1-3, but not HDAC6, regulate WT1 and p53. Our findings demonstrate that hPL is a promising replacement for animal serum.

#3101

Influence of targeted knockout of the BRCA1 gene on the pharmacologic profile of the mouse breast cancer cell line EMT6 in vitro and in vivo.

Anya Avrutskaya,1 Cordula Tschuch,2 William Durham,1 Gerhard Kelter,2 Astrid Jensen,3 Armin Maier,2 Aidan Synnott,1 Anne-Marie Zuurmond,3 Julia Schüler2. 1 _Discovery Services Charles River, Morrisville, NC;_ 2 _Charles River Research Services Germany GmbH, Freiburg, Germany;_ 3 _Charles River, Leiden, Netherlands_.

Around 10% of breast cancer cases are attributed to genetic disorders like mutations in BRCA-1/2 genes. Targeted therapy of BRCA-deficient cancers has been achieved using poly(ADP-ribose) polymerase (PARP) inhibitors, which block BRCA-independent DNA repair. To study the effect of this common mutation on sensitivity towards innovative therapies in more detail, we knocked out the BRCA1 gene in the murine EMT6 breast cancer cell line. Subsequently, we analyzed the sensitivity towards PARP as well as checkpoint (CP) inhibitors in the mutated as well as the parental line in vitro and in vivo. The EMT6/BRCA1-/- cell line was created by a CRISPR/CAS9 based removal of Exon2 of the BRCA1 gene. The homozygous clone of the modified cell line was used for drug testing in comparison to the wildtype (wt) cell line. PARP inhibitors Niraparib, Rucaparib, Talazoparib and Olaparib were investigated in EMT6/BRCA1wt as well as EMT6/BRCA1-/- in vitro in 2D as well as 3D cell culture using different cell concentrations. The latter showed higher sensitivity towards Talazoparib and Olaparib in 2D assays: The respective IC50 values were 2-4 times lower in the EMT6/BRCA1-/- as compared to EMT6/BRCA1wt. In 3D assays Niraparib, Rucaparib and Talazoparib showed pronounced activity in the EMT6/BRCA1-/- : IC50 values depicted a 2-3fold difference between the two investigated lines. To further characterize the two lines, tumor growth and sensitivity towards PARP as well as CP inhibitors was determined in vivo. Tumor growth behavior of subcutaneously implanted breast cancer cells was similar in both lines: mean doubling times were 1.9 (± 0.38) days for EMT6/BRCA1-/- and 1.62 (± 0.37) days for EMT6/BRCA1wt . Both lines showed a distinct sensitivity profile towards CP inhibitors: anti-CTLA4 was more active in EMT6/BRCA1-/- : optimal T/C (test/control) value of 22% (KO line) was accompanied by 34.9% in the parental line. In contrast, anti-PD1 treatment led to optimal T/C values of 30.9% in the wt line and 54.8% in the KO line. The evaluation of Niraparib as well as Olaparib in mono-and combined therapy with anti-CTLA4 and anti-PD1 in vivo is currently under investigation. Weekly cytokine analysis in the serum of tumor bearing mice will elucidate possible interactions between PARP and CP inhibitors and give guidance to optimal combination and schedules. We expect that further evaluation of PARP inhibitors in combination with different immune modulatory interventions in our mouse breast cancer model will increase the clinical utility of this strategy for the treatment of patients with BRCA1 mutated cancers. In general, the possibility to compare a mutated vs a parental line in vivo will help to identify novel promising combination as well as patient stratification strategies for immuno-oncology.

#3102

Identification of actionable cancer genes and treatment options for metastatic ovarian carcinomas using patient-derived xenografts and PDX-derived tumor cells.

Martina Olivero,1 Jessica Erriquez,2 Maddalena Arigoni,3 Sonia Capellero,1 Concetta D'Ambrosio,1 Gloria Mittica,1 Fulvio Borella,4 Dionyssios Katsaros,4 Silvana Privitera,4 Enrico Berrino,2 Tiziana Venesio,2 Giorgio Valabrega,1 Raffaele Calogero,3 Maria Flavia Di Renzo1. 1 _Candiolo Cancer Institute FPO-IRCCS, Univ. of Torino, Candiolo (Torino), Italy;_ 2 _Candiolo Cancer Institute FPO-IRCCS, Candiolo (Torino), Italy;_ 3 _University of Torino, Torino, Italy;_ 4 _AOU Città della Salute, S.Anna Hospital, University of Torino, Torino, Italy_.

Patients with advanced ovarian cancers have experienced little improvement in overall survival with standard treatments even after the incorporation of anti-angiogenic therapies. Besides anti-PARP inhibitors, matching individual critical genomic alterations with the best available drugs has not advanced as in other cancers, likely because a handful of cancer-related genes are mutated at high frequency, while many more are found mutated at much lower frequencies. This so called "mutation tail" is not only long but also mostly unexplored.

We used Patient Derived Xenografts (PDXs) to identify actionable cancer genes and PDX Derived Tumor Cells (PDTCs) to accelerate the discovery of treatment options. We envisioned that the alleged weakness of PDX models, i.e. lack of human stromal and immune cells, might be instrumental to identify mutations in cancer and to test approved or experimental targeted drugs as monotherapy or in different combinations to link biomarkers to treatments.

Fourty-nine PDX lines from metastatic epithelial ovarian carcinomas have been propagated and fully characterized as far as histology, immunohistochemistry of epithelial and high-grade serous-specific markers and presence of TP53 and BRCA1/2 mutations.

Copy number variations (CNV) analysis and Whole Exome Sequencing (WES) were carried out of 12 PDX lines derived from naïve metastatic high-grade serous epithelial ovarian carcinomas. We studied non-synonymous mutations with allele frequencies ≥0.1. Only mutations in cancer genes listed in databases were further analyzed. SNPdb allowed ruling out polymorphisms. SIFT and PROVEAN softwares predicted deleterious or damaging effects onto the protein sequences. DGIdb helped selecting actionable genes.

We identified mutations in 1-4 cancer genes in 8/12 PDX lines. In one PDX line, a possibly loss-of-function mutation of the PIK3R1 gene (encoding the p85alpha regulatory subunit of PI3K) had an allele frequency=0.9 in early and late passages. Moreover, in two micro-dissected FFPE samples of the source tumor this mutation had an allele frequency nearly identical to that of the mutated TP53. Hence, PIK3R1W624R could be a trunk mutation in the PDX line and possibly in the human counterpart.

Treatment options were assayed ex-vivo, on short-term cultures of PDTCs of the PIK3R1W624R PDX line. Buparlisib, a pan-class I PI3K inhibitor, showed the ability to block proliferation of PDTCs and the growth in vivo of PDXs in regression preclinical trial. These data proofed-the-concept that a PDX-based pipeline is able to unveil actionable pathways for the treatment of advanced/metastatic ovarian cancer.

#3103

Analysis of breast cancer lines and PDXs using a blood brain niche (µm-BBN) microfluidic device and algorithms to aid diagnosis of brain metastatic potential.

Christopher Ryan Oliver,1 Megan Altemus,1 Trisha Westerhof,1 Aki Morikawa,1 Xu Cheng,1 Jason Heth,1 Shuichi Takayama,2 Maria Castro,1 Sofia Merajver1. 1 _Univ. of Michigan, Ann Arbor, MI;_ 2 _Georgia Institute of Technology, Atlanta, GA_.

Metastasis from the primary tumor site to the brain is the most lethal complication of advanced breast cancer. There is no translational approach to detect if a primary tumor has brain metastatic potential. This is due to a lack of blood brain barrier (BBB) models that can classify a cells metastatic potential. Moreover, the mechanisms by which circulating cancer cells extravasate through the BBB are unknown. Currently used in vivo murine models are slow to manifest metastasis therefore, an alternative approach is an in vitro microfluidic model that re-capitulates the BBB niche micro-environment. Our goal is to develop and use this model to identify the metastatic potential of cancer cell populations. Therefore, we have developed a blood brain niche (µm-BBN) on-a-chip and studied the phenotypic differences between cancer cells in the µm-BBN. The device has two chambers separated by a 5µm porous membrane coated with Matrigel and a BB human endothelial cell (hCMEC/D3) mono-layer. The bottom chamber contains a brain stromal ECM and Normal Human Astrocytes (NHA), while the top chamber acts as the blood vessel. Cancer cells are introduced into the top chamber and allowed to extravasate into the brain like stroma. We measured the ability of the endothelial layer to prevent fluorescent small molecules from diffusing into the brain stromal space. The barrier measured 8.3X lower max fluorescent values than when no barrier was present and were confirmed by TEER. Using this model, we characterized the MDA-231 breast cancer cell line against a brain-seeking subclone (MDA-231-BR), normal-like cell lines (MCF10A) and brain met patient derived xenografts (PDXs) in terms of their ability to extravasate, migrate and survive in the niche for 24-48 hrs. Phenotypic and migratory behavior was recorded using confocal tomography to measure cancer cell properties (volume, shape, position) relative to the endothelial layer. Brain-seeking subclones cluster around the endothelial layer, the MCF10A cell line has no preferred location and the parent line (MDA-231) extravasates deeper into the brain stromal space than the other two cell lines. We also found significant variation in the shape of each cell line before and during extravasation suggesting differences in plasticity. The effects of chemoattractants within the µm-BBN on extravasation have been explored by omitting astrocytes in the collagen-infused brain niche and replacing with astrocyte conditioned media. These findings confirm that the system is capable of measuring both variations in cancer cell populations and individual cells. This approach may enable classification of subclone populations with higher metastatic potential, meeting a major need in Oncology. Future work will employ this emerging tool to study the mechanisms by which the cancer cells extravasate and survive in the niche.

#3104

Establishment of an oral tongue squamous cell carcinoma cell line from a never-smoking patient.

Steven J. Wang, Saurabh Asthana, Annemieke van Zante, Adam B. Olshen, Frank McCormick, Osamu Tetsu. _UCSF, San Francisco, CA_.

(Objective) The rising incidence of oral tongue squamous cell carcinoma (OTSCC) in patients who have never smoked and the paucity of knowledge of its biological behavior prompted us to develop a new cell line originating from a never-smoker. (Materials and methods) Fresh tumor tissue of keratinizing OTSCC was collected from a 44-year-old woman who had never smoked. Serum-free media with a low calcium concentration were used in cell culture, and a multifaceted approach was taken to verify and characterize the cell line, designated UCSF-OT-1109.(Results) UCSF-OT-1109 was authenticated by STR DNA fingerprint analysis, presence of an epithelial marker EpCAM, absence of human papilloma virus (HPV) DNA, and SCC-specific microscopic appearance. Sphere-forming assays and mouse xenografts supported its tumorigenic potential. Spectral karyotype (SKY) analysis revealed numerical and structural chromosomal abnormalities. Whole-exome sequencing (WES) identified 46 non-synonymous and 13 synonymous somatic single-nucleotide polymorphisms (SNPs) and one frameshift deletion in the coding regions. Specifically, mutations of CDKN2A, TP53, SPTBN5, NOTCH2, and FAM136A were found in the databases. Copy number aberration (CNA) analysis revealed that the cell line loses chromosome 3p and 9p, but lacks amplification of 3q and 11q (as does HPV-negative, smoking-unrelated OTSCC). It also exhibits four distinctive focal amplifications in chromosome 19p, containing 131 genes without SNPs. Particularly, 52 genes showed >3- to 4-fold amplification and could be potential oncogenic drivers.(Conclusion) We have successfully established a novel OTSCC cell line from a never-smoking patient. UCSF-OT-1109 is potentially a robust experimental model of OTSCC in never-smokers.

#3105

Oncopig carcinoma cell lines: A foundation for co-clinical trials.

Laurie A. Rund,1 Kyle M. Schachtschneider,2 Ron C. Gaba,2 Daniel R. Principe,2 Paul Grippo,2 Regina M. Schwind,2 Howard Ozer,2 Lawrence B. Schook1. 1 _Univ. of Illinos at Urbana-Champaign, Urbana, IL;_ 2 _Univ. of Illinos at Chicago, Chicago, IL_.

The Oncopig cancer model (OCM) was developed to support a broad range of solid and liquid tumor malignancies while providing the appropriate anatomical and physiological models for preclinical and co-clinical trials to expedite human clinical trials. The OCM develops tumors following Cre recombinase induced expression of KRASG12D and TP53R167H transgenes in a temporally and spatially controlled manner. To date, in vitro transformation and in vivo tumor formation of soft tissue sarcomas (STS), hepatocellular carcinoma (HCC), and pancreatic ductal adenocarcinoma (PDAC) have been demonstrated. However, the OCM was designed to generate cancers of all tissue origins. To demonstrate the potential importance of the OCM, we have successfully isolated and transformed multiple OCM cell types, each a progenitor of clinically relevant human disease. Cell type specific cell isolations were performed, and cells cultured under appropriate conditions. Within the first 48 hours, isolated cells were treated with adenoviral vector encoding Cre recombinase (AdCre) at 200-400 MOI for 5 hours in low serum (5%) medium. Both control and transformed (AdCre treated) cells underwent basic histopathologic screening (H&E staining, immunohistochemical staining for vimentin and cytokeratin), and verification of transgene expression (RT-PCR) and tumorigenicity (xenograft into SCID mice). Cell types isolated include fibroblasts, hepatocytes, pancreatic ductal cells, dermal epithelial cells, splenocytes, ovarian surface epithelial cells, fallopian tube secretory epithelial cells, renal proximal tubule epithelial cells, urinary bladder epithelial cells, bone marrow (without specific cell isolation), and testis (no specific cell isolation). Each cell type was successfully transformed, as shown by in vitro phenotypic changes and tumor formation in vivo. Additional gene expression analysis (RNA-Seq) of the STS and HCC cell lines verified the OCM transformed cells recapitulate transcriptional hallmarks of human cancers. Consistent with human STS, OCM STS cell lines display altered TP53 signaling, Wnt signaling activation, and evidence of epigenetic reprogramming. OCM HCC cell lines show transcriptional evidence of TERT reactivation, apoptosis evasion, angiogenesis activation, and Wnt signaling activation, all similar to human HCC. While not an exhaustive list of Oncopig cell types, we have not encountered cells resistant to malignant transformation through Cre recombinase exposure. These results support the unprecedented potential for modeling of additional cancer types, including colorectal, ovarian, fallopian tube, uterine, renal, bladder, skin, and hematological cancers to support and expand preclinical, translational, and co-clinical investigations.

#3106

**Generation and characterization of IDH1** R132H **and IDH2** R140Q **in vitro models for drug discovery and development.**

Diana Douglas,1 Lysa-Anne Volpe,2 Luping Chen,2 Metewo S. Enuameh,1 Fang Tian,2 Weiguo Shu1. 1 _ATCC, Gaithersburg, MD;_ 2 _ATCC, Manassas, VA_.

Isocitrate dehydrogenase (IDH) is a metabolic enzyme that converts isocitrate to α-ketoglutarate. This process leads to NADPH production, and is involved in the control of oxidative cellular damage. Mutations in this enzyme have been linked to human cancers such as glioma and acute myeloid leukemia (AML). While there are three isoforms of IDH, mutations that lead to cancer have only been identified in IDH1 and IDH2, which result in simultaneous loss of normal catalytic activity, the production of α-ketoglutarate (α-KG), and gain of a new function, the production of 2-hydroxyglutarate (2-HG). 2-HG is structurally similar to α-KG, and acts as an α-KG antagonist to competitively inhibit multiple α-KG-dependent dioxygenases, including lysine histone demethylases (KDM) and DNA hydroxylases (TET), causing widespread changes in histone and DNA methylation and potentially promoting tumorigenesis. A number of pre-clinical and clinical studies from the mutant IDH inhibitors indicate that IDH is a valid target for a new class of cancer therapeutics. However, there is a lack of well-established and characterized in vitro models containing IDH mutants.

The most prominent IDH1 mutation takes place at residue R132H and plays a role in the development of gliomas, while the majority of IDH2 mutations take place at residue R140Q, linked to AML. Given the prevalence of these mutations, we sought to use CRISPR/Cas9 gene editing technology to create two in vitro disease models harboring either the IDH1 or IDH2 mutations. To develop the most clinically relevant models we selected highly appropriate parental cell lines from our extensive portfolio. An IDH1R132H mutation was introduced in the malignant glioblastoma U-87 MG cell line, and an IDH2R140Q mutation was introduced in the TF-1 erythroblast cell line derived from an AML patient. The introduction of the IDH mutations in the respective cell lines was then confirmed via sequencing at the genomic and transcriptional levels. Furthermore, to validate the isogenic IDH mutations confer gain-of-function in vitro, we tested the intracellular and extracellular levels of 2-hydroxyglutarate (2-HG). Bio-functional evaluation data indicated that IDH1R132H U-87 MG cells showed an increase in cellular 2-HG and elevated level of histone methylation. In IDH2R140Q TF-1 cells, an increase in cellular 2-HG was also observed. In response to IDH2 specific inhibitors, AG-221 and AGI-6780, we demonstrated that IDH2R140Q TF-1 cells exhibited decreases in both cellular 2-HG and histone methylation levels. Taken together, these isogenic in vitro models are valuable tools for elucidating mechanisms involved in cancer-associated tumorigenesis and use in screening anti-cancer compounds for drug discovery and development.

#3107

Modeling DICER1 syndrome in cells.

Mona K. Wu. _McGill University, Montreal, Quebec, Canada_.

DICER1 is an endoribonuclease central to generating microRNAs (miRNAs), small RNA molecules that downregulate the expression of approximately 30% of protein-coding genes. Germ-line mutations in DICER1 have been identified in patients afflicted with a pleiotropic tumor predisposition syndrome, usually referred to as DICER1 syndrome (OMIM 606241). DICER1 syndrome is thought to originate from a mesenchymal cell bearing a frameshift or nonsense mutation on one allele and a missense mutation affecting the RNase IIIb domain on the other allele. While interest in the role of DICER1 in normal development and pathogenesis has been explored in both the knockout and overexpression context, neither situation is relevant for understanding DICER1 syndrome. We hypothesize that defective DICER1 protein production in a mesenchymal stem cell at a critical time in development leads to altered miRNA populations that, in turn, initiate or prime cells for tumorigenesis. The goal of this study was to develop and characterize a model of the putative tumor-initiating cell namely a mesenchymal stromal cell (MSC) bearing a DICER1 RNase IIIb mutation. SV-40 immortalized mouse mesenchymal stromal cells (MSCs) containing a homozygous floxed exon for DICER1 were ex vivo cre inactivated to produce Dicer-/- MSCs. Into these Dicer-/- MSCs, a FLAG-tagged human DICER1, a FLAG-tagged RNase IIIa mutant DICER1, and a FLAG-tagged RNase IIIb mutant DICER1 were stably introduced. Cells were characterized by Western blot in response to serum starvation and stimulation. Their ability to form anchorage-independent colonies was assessed by soft agar assay pre- and post-oncogene introduction. Transcriptomes were interrogated by gene expression microarray.

Results: Alterations in protein expression were observed in cells in response to starvation and stimulation. Certain cell lines were able to produce anchorage-independent colonies.

Conclusions: We have created MSCs bearing RNase IIIb-mutated DICER1 to model DICER1 syndrome. We suggest that the ability for an MSC to produce only 3p miRNAs in the presence of an oncogene can allow for anchorage-independent growth.

#3108

Modeling translocation driven tumors with human induced pluripotent stem cells (hiPSCs) using CRISPR-Cas9: Pseudomyogenic hemangioendothelioma as a proof of principle.

David G. van IJzendoorn, Francijna van den Hil, Karoly Szuhai, Judith V. Bovée, Valeria V. Orlova. _Leiden University Medical Center, Leiden, Netherlands_.

Within the group of vascular tumors balanced chromosomal translocations often drive tumorigenesis. For pseudomyogenic hemangioendothelioma (PHE) no cell line is available, therefore to model this tumor of endothelial origin, we induced a t(7;19)(q22;q13) chromosomal translocation leading to a SERPINE1-FOSB gene fusion in "healthy" human induced Pluripotent Stem Cells (hiPSCs). The hiPSC line was derived using Sendai virus (SeV)-based reprogramming and has been fully characterized. To create a model for PHE we used CRISPR-Cas9 to induce double-stranded breaks in SERPINE1 and FOSB. Using a repair template we introduced a removable neomycin resistance cassette to facilitate selection of cells with translocation. Droplet digital PCR (ddPCR) showed that the wild-type allele of both SERPINE1 and FOSB was mutated in 50% of the cells, and 12% of cells had SERPINE1-FOSB translocations. Screening of single cell-derived clones resulted in 2 clones harboring the correct chromosomal translocation out of 73 screened colonies. Fluorescence in situ hybridization (FISH) using dual-fusion probes confirmed presence of translocation in hiPSCs. Next, isogenic "healthy" and SERPINE1-FOSB targeted hiPSC clones were used to derive endothelial cells (ECs). SERPINE1-FOSB fusion had no effect on differentiation efficiency of ECs from hiPSCs. Fluorescence-activated cell sorting (FACS) confirmed comparable expression of endothelial-specific markers, such as Ve-cadherin, PECAM1, and VEGFR2. Interestingly, expression of CD105 and VEGFR3 that were shown to be elevated in tumor associated endothelial cells were increased in ECs derived from SERPINE1-FOSB targeted hiPSCs compared with ECs derived from "healthy" hiPSCs. Functional assessment of hiPSC-derived ECs showed increased proliferation, tube formation and a decrease in endothelial barrier function in the presence of a SERPINE1-FOSB fusion. Real-time PCR confirmed elevated expression of FOSB (5.9 log2 fold), CD105 (1.9 log2 fold), as well as known pro-angiogenic and pro-inflammatory cytokines, such as VEGF (2.2 log2 fold), IL6 (1.2 log2 fold) and IL8 (2.9 log2 fold). In summary, we demonstrate SERPINE1-FOSB translocation can be introduced into hiPSCs. Importantly, hiPSC-derived ECs that harbor the SERPINE1-FOSB translocation display abnormal tumor endothelium-like phenotype and therefore could serve as an excellent model to study the pathogenesis of PHE. This method could also be potentially interesting to introduce other translocation-driven tumors where no cell lines are available for the discovery of novel targeted therapy and deeper understanding of tumorigenesis.

#3109

Investigating the role of wild-type TFE3 in renal cell carcinoma cells harboring TFE3 fusions with spliceosome machinery associated genes.

Nur P. Damayanti, Khunsha Ahmed, May Elbanna, Chinghai Kao, Roberto Pili. _Indiana University, indianapolis, IN_.

Background: Translocation Renal Cell Carcinoma (tRCC) represents an aggressive subtype of kidney cancer associated with various gene fusions involving translocation of one of two members of micropthalmia transcription factor (MiT) family, TFE3 or TFEB. Despite the identification of multiple TFE3 gene fusions in tRCC, heterogeneous phenotype and various dysregulated signaling pathways resulting from variety of gene fusion partners pose a challenge to establish effective treatments for these patients. In this work, we assessed the role of wild type TFE3 (wt-TFE3) in RCC cell bearing TFE3 fusion with genes associated with pre-mRNA splicing factor machinery (NONO, SFPQ and PRCC).Methods: Endogenous SFPQ-TFE3 expressing cells, RP-RO7, were generated from patient derived xenograft established in NSG mice. UOK-109 and UOK-146 (kindly provided by Dr. Marston Lenehan, NCI) were used as endogenous NONO-TFE3 and PRCC-TFE3 expressing cells, respectively. RNA interference (RNAi) mediated knockdown with differential exon targeting strategy was employed to study wt-TFE3 loss of function in RP-R07, UOK-109 and UOK-146, respectively. Real time monitoring of cell viability assay with multiplex readout were developed to investigate the effect of wt-TFE3 loss of function in 2D and 3D culture system. Two different 3D models were used;1) Tumor growth model, in which cancer cell interaction with extracellular matrix and stromal cells were represented in a multiculture-spheroid system; 2) Invasion model, in which cell ability to invade basement membrane barrier was modeled with matrix restricted spheroid, and the invasion trajectories were observed and quantified. Exogenous expression of wt-TFE3-EGFP was utilized to study the protein subcellular localization in RP-R07, UOK-109 and UOK-146 2D cultures and 3D culture system.Results: Consistent with the expression of chimeric TFE3, wt-TFE3-EGFP ectopic expression demonstrated strong nuclear localization in RP-R07, UOK-109 and UOK-146. Multiplex readout of cells viability assay showed that transient loss of wt-TFE3 in RP-R07, UOK-109 and UOK-146 curtails the proliferation rate of these cells in 2D and 3D models in fusion partner dependent manner (P<0.05). 3D invasion assay coupled with RNAi strategy in RP-R07, UOK-109 and UOK-146 demonstrated the role wt-TFE3 in invasion potential of these cell in fusion partner dependent fashion (P<0.05). Downstream analysis suggested that the wt-TFE3 transient silencing affected cell proliferation and invasion ability via inhibition of the IRS-PI3K-mTOR axis.Conclusion: Our results suggest the potential role of wt-TFE3 in tRCC oncogenesis irrespective of its different fusion partner.

#3110

Heterokaryon formation and nuclear fusion of hybridizing cancer cells visualized in real time by color-coded imaging.

Atsushi Suetsugu,1 Takuro Matsumoto,1 Kousuke Hasegawa,1 Miki Nakamura,1 Takahiro Kunisada,1 Masahito Shimizu,1 Shigetoyo Saji,1 Hisataka Moriwaki,1 Michael Bouvet,2 Robert M. Hoffman3. 1 _Gifu University, Gifu, Japan;_ 2 _University of California, San Diego, CA;_ 3 _AntiCancer, Inc., San Diego, CA_.

Background: Fusion of cancer cells has been studied for over half a century. However, the steps involved after initial fusion between cells, such as heterokaryon formation and nuclear fusion, have been difficult to observe in real time. In order to be able to visualize these steps, we have established cancer-cell sublines from the human HT-1080 fibrosarcoma, one expressing green fluorescent protein (GFP) linked to histone H2B in the nucleus and a red fluorescent protein (RFP) in the cytoplasm and the other subline expressing RFP in the nucleus (mCherry) linked to histone H2B and GFP in the cytoplasm.

Materials and Methods: The two reciprocal color-coded sublines of HT-1080 cells were fused using the Sendai virus. The fused cells were cultured on plastic and observed using an Olympus FV1000 confocal microscope.

Results: Multi-nucleate (heterokaryotic) cancer cells, in addition to hybrid cancer cells with a single fused nucleus undergoing mitosis, were observed by confocal microcopy. Red, green, orange and yellow nuclei were observed in cells. The orange and yellow nuclei indicate nuclear fusion. Red and green nuclei remained unfused.

Conclusion: Cell fusion with subsequent heterokaryon formation followed by nuclear hybridization may be an important natural phenomenon between cancer cells that may make them more malignant. The ability to image the complex processes following cell fusion using reciprocal color-coded cancer cells will allow greater understanding of the genetic basis of malignancy.

#3111

Expansion of mouse prostate epithelial stem cells in serum-free ProstaCult Organoid Growth Medium.

John Stingl, David Rowbotham, Terry E. Thomas, Allen C. Eaves, Sharon A. Louis. _STEMCELL Technologies, Vancouver, British Columbia, Canada_.

Growing prostate epithelial cells as organoids in a three-dimensional (3D) cell culture environment represents a more physiological model system than conventional 2D adherent cell culture systems for studying many different aspects of prostate epithelial cell biology. Two types of prostate epithelial organoids have been previously described: large hollow cystic organoids, and smaller solid organoids (Karthaus et al, Cell 2014). Both types of organoids are composed of androgen receptor (AR)-expressing luminal cells and keratin (K) 5-expressing basal cells, but only vary in the proportion of each cell type, with the cystic organoids enriched for luminal cells and solid organoids enriched for basal cells. We are currently developing ProstaCult™ Organoid Growth Medium, a serum-free medium for the long-term propagation of both types of organoids from mouse prostate tissue. To initiate the cultures, mouse prostates are enzymatically dissociated sequentially in collagenase type II, trypsin and dispase to generate a single cell suspension, and 5x103 the liberated cells are then embedded in Corning® Matrigel® and cultured in ProstaCult™ Organoid Growth Medium. Approximately 10±2% (mean±sem; n=6) of the seeded cells will proliferate and generate approximately equivalent numbers of cystic and solid organoids, which can be dissociated and passaged every 7 days as a single cell suspension. Typical total epithelial cell expansion observed during media development ranges from 21-240-fold per passage for a minimum of 4 passages. Experiments are ongoing to determine the long-term passageability of organoids maintained in ProstaCult™ Organoid Growth Medium. Organoids that are generated at multiple passages are composed of a polarized epithelium with AR+K18+ luminal cells and K5+ basal cells. These results demonstrate that ProstaCult™ Organoid Growth Medium efficiently generates and expands prostate epithelial organoids.

#3112

Distinct oncogenic events induce different DNA methylation and copy number changes in human mammary epithelial cells.

Stan P. du manoir,1 Claire Fonti,1 Anne Saumet,1 Amanda Abi-Khalil,1 Béatrice Orsetti,1 Cleroux Elouan,2 Ambre Bender,2 Michael Dumas,2 Jacques Colinge,1 Michael Weber,2 Charles Theillet3. 1 _IRCM,INSERM U1194, University of Montpellier, Montpellier, France;_ 2 _CNRS, University of Strasbourg, UMR 7242, Illkirch, France;_ 3 _IRCM,ICM,INSERM U1194, Montpellier University, Montpellier, France_.

Gene expression differences, combined with distinct patterns of genomic rearrangements and epigenetic modifications constitute the bases of molecular classification of breast cancer. Molecular subtypes may originate from different cell lineages in the mammary gland, but also from the early activation of oncogenes that may drive the establishment of these molecular subtypes. However, in the natural history of human cancer, it is difficult to discriminate between these two factors : cell lineage and initial oncogenic alterations. In this work, we designed an experimental strategy aiming at determining whether activation of distinct oncogenic pathways in human mammary epithelial cells (HMEC) could lead to different patterns of genetic and epigenetic changes. We show that initial activation of CCNE1, WNT1 and RASv12 in shp53 immortalized HMECs results in different and reproducible profiles of mRNA and miR expression, copy number alterations (CNA) and DNA methylation modifications. Interestingly, first the extend of CNA measured as fraction of the genome altered was lower in HMECs transformed by RAS than CCNE1 and WNT1 transformed HMECs revealing a lower genetic instability. This was confirmed by less numerous γH2Ax and 53BP1 nuclear foci in HMECs transformed by RAS. Second, HMECs transformed by RAS bore specific profiles of CNAs and DNA methylation, clearly distinct of those shown by CCNE1 and WNT1 transformed HMECs. Genes differentially expressed in the RAS and the CCNE1/WNT1 clusters and included in CNAs or with variable CpG methylation were mostly different, depicting the activation of distinct signaling pathways in these two clusters. These data indicate that early activation of distinct oncogenic pathways may leads to adaptive events resulting in specific pattern of CNAs and DNA methylation changes. We postulated that the early activation of oncogenes may be an important determinant of the establishment of breast cancer molecular subtypes along with cell lineage origin.

#3113

Expression of autophagy- and mitophagy-related genes in clear cell renal cell carcinoma.

Adrian G. Simon, Joerg Ellinger, Yuri Tolkach, Stefan C. Mueller, Glen Kristiansen, Marieta I. Toma. _University Hospital, Bonn, Germany_.

Objectives: First objective of this study was to establish primary cell cultures from renal cell carcinoma (RCC). Second objective was to analyze mRNA and protein expression of PINK1, PARK2, PACRG and a-Synuclein, genes involved in cell survival, apoptosis and mitophagy.

Methods: Tumor and non-neoplastic tissue from 15 patients undergoing surgery for clear cell renal cell carcinoma (ccRCC, n=9), papillary renal cell carcinoma (pRCC, n=3) as well as three retroperitoneal metastases (two clear cell, one papillary type) were obtained. After tissue digestion and filtration malignant and non-malignant cells were cultivated in two different media in order to establish an optimized cultivation procedure. DNA and RNA were extracted and immunohistochemical analyses (IHC) and qPCR from cultivated cells were performed for VHL and CAIX to verify tumor histotype. RNA was isolated from fresh-frozen primary ccRCC samples and corresponding non-malignant tissues from 50 patients (30 male, 20 female). Expression of PINK1, PARK2, PACRG and a-synuclein was measured by qPCR in the cultivated ccRCC cell lines as well as in the patient samples.

Results: All tumor specimens were successfully cultivated to first confluence. Six out of nine ccRCC cell lines and all other tumor specimens could be passaged more than two times. Cultivated RCC cells showed protein expression matching to their tumor of origin in IHC analyses, confirming tumor identity. QPCR demonstrated VHL expression downregulated or lost in all ccRCC cell lines and CA9 highly upregulated in most cases, which is both typical for ccRCC. The mRNA expression of PINK1, PARK2 and PACRG was downregulated in ccRCC cultures as well as in the 50 patient tumors in comparison to normal tissue. A-synuclein expression was highly variable.

Conclusions: We successfully established primary cell cultures from various renal tumor entities suitable for further investigation. Loss of PACRG and PARK2 are common events in ccRCC. PACRG and PARK2 might function as tumor suppressors in ccRCC and their loss may be involved in tumor progression as reported before for other tumor entities. Further investigation of PINK1, PARK2 and PACRG might allow new therapeutic approaches.

#3114

**In vitro** **culture system for circulating tumor cell.**

Chen Yin-Ju,1 Long-Sheng Lu,1 Hsin-Lun Lee,2 Lai-Lei Ting,2 Jeng-Fong Chiou2. 1 _Taipei Medical University, Taipei, Taiwan;_ 2 _Taipei Medical University Hospital, Taipei, Taiwan_.

Circulating tumor cells (CTCs) detection could be considered a real-time "liquid biopsy" approach and contains several advantages such as being minimally invasive, easy and safe to perform, multiple samples can be taken over time, better prognosis to indicate an elevated risk of metastases, improved therapy monitoring, and providing live disease status information. CTC analysis is a promising new diagnostic field for estimating the risk for metastatic relapse and metastatic progression in patients with cancer. However, the number of CTCs is very low so the establishment of cell culture from CTCs becomes the most challenging over the past year. In this study, we development a cost-effective and reproducible in vitro system for primary CTC cultures or established cell lines from CTCs. 8 CTC cell lines were generated from patients including breast cancer, small cell lung cancer, pancreatic cancer and expanded for more than one year. These cell lines exhibit the tissue-specific markers and also correlated with clinical treatment response from patients. This system provides an opportunity for broad applications of CTCs for personalized oncology and advancing precision medicine.

#3115

Physiologic comparisons of five bladder cancer cell lines.

Bonnie L. Richmond, Kyle Damen, Ryan Moore, Alex White, Julia H. Carter. _Wood Hudson Cancer Research Lab., Newport, KY_.

The use of tumor-derived cultured cells continues to be a major resource for understanding the biological activities of tumors and the effects of environmental factors and pharmacological agents on tumors. With comparison of data from the International Cancer Genome Consortium and the Cancer Genome Atlas comes the realization that some frequently used cells lines may not closely share genomic similarity with patient tumors, whereas less frequently used cell lines may share high similarity. For example, the bladder cancer cell line T24 (HTB-4) is more frequently used than other cell lines. We selected five commercially available bladder cancer cell lines, all derived from patient primary tumors, for a comparison of selected proteins, with the intent of determining how similar or dissimilar the cultured cells are. In selection of cultured cell lines, we considered reported grade of tumor from which the cell line was derived and the sex and age of the patient. We evaluated protein expression in newly confluent cultures and in sub-confluent cultures. The following cell lines were selected: RT4 (transitional cell papilloma), 5637 (grade II carcinoma), T24 (grade III carcinoma), HT 1376 (grade III carcinoma) and TCCSUP (grade IV carcinoma). We hypothesized that variations exist between bladder cancer cell lines isolated from low and high-grade carcinomas. Further, we identified physiological characteristics of the different bladder cancer cell lines at both newly confluent and sub confluent culture states as additional parameters for a bladder cancer model. Selection of an appropriate tissue culture cell line(s) and state of confluency are critical considerations in cellular research designed to understand the biological activities of tumors. Growth rates of the five cell lines were determined. Next, the basal-luminal status of the cell lines was determined using molecular subtyping markers (transcription factors GATA3 and p63, Cytokeratin 6, and Estrogen Receptor alpha). The presence of Cathepsin L, a cysteine protease, was evaluated. Further, we evaluated the expression of key proteins in the MAPK pathway (p38 MAPK, SAPK/JNK, and ERK1/2) as indicators of cellular stress. Last, the receptor status of Alpha 7 receptor, the putative receptor for nicotine, was determined. Our results indicate there is a difference in growth rates between the cell lines, with line 5637 having a delayed growth rate. Three cell lines were determined to be basal, and two cell lines were determined as mixed baso/luminal. The RT4 cell line had greater expression of cathepsin L. We observed no significant changes in the protein expression levels of the MAPK pathway nor of the alpha-7 receptor. Our results suggest that there are differences between the five bladder cancer cell lines suggesting that more than one cell line should be used and evaluated prior to beginning experiments to evaluate the potential effects of environmental factors and pharmacological agents.

#3116

Developing engineered and primary cancer cell lines for oncology drug development.

Feng Hao, Wenna Zhang, Hao Peng, Feng He, Zhaoshuai Bai, Changpeng Liu, Guoqian Wang, Juan Xu, Yang Qu, Jinying Ning. _KYinno Biotechnology Co., Ltd, Beijing, China_.

Cancer cell lines are effective in vitro systems in oncology drug development. The success of many targeted cancer drugs benefited from the utilization of cancer cell lines such as ones from American Type Culture Collection (ATCC) and Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). Recent multiplatform omics research revealed complex diversity of cancers, many of which were classified into subtypes that have distinct genomic signatures, show different prognosis patterns, and may respond differently to same drug treatment. For example, colorectal cancers were separated into four consensus molecular subtypes, and subtype 1 is marked by high microsatellite instability and has worse survival after relapse1. Cancer subtypes may not be well represented in existing cell line libraries. Immortal cell lines for some cancers, such as soft-tissue sarcoma2, may also be rare. It is therefore pertinent to expand current cell line collection, a common approach being to establish primary cell lines directly from cancer patients, preferably with complete clinical information. In addition, current library may also lack of cell lines with rare mutations that exist at low frequency in naïve patient tumors and frequently occur under drug treatment. For example, Erlotinib—a first generation of EGFR inhibitor—induces the EGFR T790M mutation, and Tagrisso—a third generation of EGFR inhibitor—induces the EGFR C797S mutation. It is desirable to create engineered lung cancer cell line with both mutations. An engineered cell line with both mutations can be more efficient in developing EGFR inhibitors overcome the resistance of both mutations. In this study, we report the establishment and characterization of over 120 engineered cell lines and several dozens of primary cell lines across multiple cancers. For the engineered cell lines, we used CRIPSR technology to create cell lines bearing specific mutation for well-studied target proteins such as EGFR, FLT3, ELM4-ALK. In addition, we also created cell lines that may be used for immunotherapy development targeting PD-1, PD-L1, TIM3, OX40, etc. We used Sanger sequencing, flow cytometry, cell viability assay, in vitro and in vivo efficacy studies to validate and characterize the cell lines. To make the resources easily accessible to researchers, we created a searchable database named InnopediaTM that presents detailed information of the engineered and primary cell lines. Access is open with registration. The database also provides comprehensive genomic and efficacy data, retrieved from published research, for over 1000 conventional cell lines.

References 1. Justin, G., Rodrigo, D., et al. (2015). The consensus molecular subtypes of colorectal cancer:. Nature Medicine, 21(11), 1350-6. 2. Salawu, A., Fernando, M., et al. (2016). Establishment and molecular characterisation of seven novel soft-tissue sarcoma cell lines:. British Journal of Cancer, 115(9), 1058-1068.

#3117

Full spectrum of in vitro disease models with optimized CRISPR-Cas9 gene editing in difficult-to-transfect blood-lineage cells.

Huanyu Jin, Zhongsheng Yu, Vladimir Pak, Yin Zhang, Padmaja Tummala, Pavithra Rajeswaran, Diana Nguyen, Monika Maleszewska, Jinling Li, Charles Cao, Ruby Yanru Chen-Tsai. _Applied StemCell, Inc., Milpitas, CA_.

CRISPR/Cas9 technology is a powerful tool for gene editing in mammalian cells. However, the efficiency of gene editing in certain types of cells, especially suspension cells such as several blood-lineage cell lines, is extremely low. This is due to a variety of factors including low transfection efficiency of CRISPR/Cas9 reagents, cytotoxicity, and low or undesired gRNA and Cas9 activities in the cell lines. By optimizing the protocol and transfection conditions, we were able to successfully generate a spectrum of gene modifications in blood-lineage cells. Here, we describe techniques optimized for genome editing in some difficult-to-transfect blood-lineage cell lines and the types of modifications achieved: (1) For leukemia cell lines such as KG-1 and MOLM-13 cells, we compared DNA-based versus RNP-based Cas9 delivery efficiency, and found that plasmid-based Cas9 transfection resulted in cytotoxicity. In contrast, the RNP-based route demonstrated significantly lower cytotoxicity and higher efficiency of gene editing. Furthermore, we found that the application of a DNA-dependent protein kinase inhibitor dramatically enhanced the homology directed repair (HDR) in these cell lines; (2) For T lymphocyte cell lines such as Jurkat cells, we could efficiently delete a targeted 5kb regulatory region of a critical gene. We were also able to sequentially knockout (KO) two genes (double KO) in Jurkat cells using a Cas9-plasmid protocol; (3) In the case of the lymphoblastic cell line T2, we successfully created a large fragment knock-in, as well as a gene knockout cell line model, through DNA-based CRISPR/Cas9 delivery. Our studies demonstrate that for CRISPR/Cas9 genome editing in blood-lineage cells, different cell lines may require different approaches or modified protocols to deliver the CRISPR/Cas9 components for efficient and successful modifications of the targeted genes.

#3118

Oncogenesis modeling in malignant pleural mesothelioma.

Nathanael D. Pruett, Anand Singh, Nisan Bhattacharyya, Chuong D. Hoang. _National Institutes of Health/National Cancer Institute, Bethesda, MD_.

Malignant pleural mesothelioma (MPM) is causally associated with exposure to asbestos and accounts for roughly 75% of mesothelioma cancers. It is an aggressive tumor with a median survival time of approximately 12 months. Treatment options are limited, largely palliative, and still based on cisplatin and pemetrexed, a regimen that remains unchanged since 2003. Despite widespread study, the complexities of MPM biology remain poorly described, especially at the point of tumor initiation where development of novel clinical interventional strategies would be expected to yield the most effect. The extended latency period in humans consisting of decades (over 20-30 years) remains a prohibitive barrier to gain accurate, in vivo insights about MPM oncogenic processes. As such, all in vitro MPM models, which are designed to accelerate tumor development as a practical endpoint of study, to date have not yielded clinically translatable molecular targets. Here, we present a novel in vitro model system for exploring the earliest molecular mechanisms contributing to the development of MPM using normal, human mesothelial cell lines (MeT-5a and LP9-hTERT) engineered to harbor constitutively enhanced NFkB signaling activity. Both 3D-sphere and soft agar assays were used to assess cellular transformation status in these induced cells compared to their wild-type counterparts. Heterotopic xenograft implantation studies were performed in NOD scid-gamma (NSG) immunocompromised mice (sub-cutaneous dorsal flank injection) for tumorigenic potential. In all cases, these induced cells (despite the complete absence of asbestos exposure) exhibited a significant malignant-like phenotype. Tumor growth in mouse xenograft models occurred very rapidly, and was marked by extensive vascular recruitment with invasion into surrounding tissues. In addition, these transformed cells expressed typical mesothelioma markers such as Wilms tumor protein (WT1 gene product) as revealed by immunohistofluorescence. This new MPM model system could represent a powerful construct to explore the early molecular events involved in transformation of normal mesothelial cells leading to malignancy.

### Immune Cells in the Microenvironment

#3119

Treatment prediction by subset analysis of tumor infiltrating lymphocytes (TILs) in combination therapy with trastuzumab, pertuzumab, and docetaxel for advanced HER2-positive breast cancer.

Koji Takada, Shinichiro Kashiwagi, Yuka Asano, Goto Wataru, Tamami Morisaki, Satoru Noda, Tsutomu Takashima, Naoyoshi Onoda, Kosei Hirakawa, Masaichi Ohira. _Osaka City University Graduate School of Medicine, Osaka, Japan_.

Background: The trastuzumab, pertuzumab, and docetaxel (TPD) regimen is strongly recommended as a treatment option for first-line therapy for advanced human epidermal growth factor receptor (HER) 2-positive breast cancer. The immune microenvironment in cancer is involved in many antitumor treatment effects, and tumor-infiltrating lymphocytes (TILs) is being established as a biomarker for therapeutic effect and prognosis. Recently, subset analysis of TILs is also under way. We have previously reported the clinical validity and benefits of evaluating TILs for neoadjuvant chemotherapy (NAC). Chemotherapy with a TPD regimen is garnering attention for its clinical outcomes and impact on cancer microenvironments. In this study, we evaluated the impact of chemotherapy with a TPD regimen, on immune micro environments in HER2-positive breast cancer using immune related proteins as indicators.

Methods: The subjects consisted of 30 patients who received the TPD regimen. The expression levels of estrogen receptor (ER), progesterone receptor (PgR), Ki67, CD8, forkhead box protein (FOXP) 3, programmed death (PD) 1, and programmed death ligand (PD-L) 1 were evaluated in biopsy specimens, by immunostaining. We also examined the ratio of CD8 and FOXP3 (CFR).

Results: The objective response rate (ORR) in the high CFR group was higher than in the low CFR group (p=0.013). The CD8 positive, high CFR and PD-L1 negative group had significantly longer PFS than the CD8 negative, low CFR and PDL1 positive group (p=0.045, log-rank) (p=0.007, log-rank) (p=0.040, log-rank), respectively. The high CFR group had significantly better OS than the low CFR group (p=0.034, log-rank). Receiver operating characteristic (ROC) analyses showed that, for advanced HER2-positive breast cancer patients, the CFR results [area under the curve (AUC): 0.708] were better than those for the other factors (AUC: CD8=0.681, FOXP3=0.639, PD1=0.528, PD-L1=0.681).

Conclusions: This study shows with the TPD regimen, a high CFR leads to a high ORR and long PFS in HER2-positive breast cancer. CFR, therefore, may be one of the important prognostic factors for this disease.

#3120

Tumor-infiltrating lymphocyte profiling in hepatitis B virus pre-S2 mutant-positive hepatocellular carcinoma.

Chiao-Fang Teng,1 Woei-Cherng Shyu,1 Long-Bin Jeng2. 1 _China Medical University, Taichung, Taiwan;_ 2 _China Medical University Hospital, Taichung, Taiwan_.

Chronic hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC), the leading cause of cancer-related deaths worldwide. Pre-S2 mutant is well demonstrated as a HBV oncoprotein that disturbs multiple cellular signaling pathways, leading to HCC development. HCC patients with pre-S2 mutant are recognized as a high-risk population for postoperative recurrence. Plentiful evidences reveal that tumor infiltrating lymphocytes (TILs) play an essential role in the stepwise progression of HCC development, and are closely associated with clinical outcomes. However, to date, the role of pre-S2 mutant in regulation of TILs distribution in HCC remains totally unknown. In this study, we collected plasma and paraffin-embedded liver samples from HBV-related HCC patients. The plasma samples were used for detection of the pre-S2 mutant with polymerase chain reaction-based method. As a result, the patients were divided into two groups: the pre-S2 mutant-positive and -negative groups. The paraffin-embedded liver samples were used for examination of the distribution of specific population of TILs with fluorescent immunohistochemistry staining. We analyzed 6 cases of HBV-related HCC samples, half of which were detected as pre-S2 mutant-positive. The distribution level of the cytotoxic CD3+CD8+ TILs in HCCs was examined. We found that patients with pre-S2 mutant had a considerably lower density of cytotoxic CD3+CD8+ TILs in HCC tissues than patients without pre-S2 mutant. The mean ± SEM of cytotoxic CD3+CD8+ TILs density was 3.74 ± 0.60 and 6.66 ± 0.66 in patients with and without pre-S2 mutant, respectively (p value=0.031). Our result suggests that pre-S2 mutant may have a role in regulation of TILs distribution in HBV-related HCCs. Systematic evaluation of specific population of TILs may help guide development of novel strategies for prognosis and/or therapies of HCC, especially for the HBV-related pre-S2 mutant-positive high-risk patient population.

#3121

Characterization of immune profiling of pancreatic intraductal papillary mucinous neoplasm using multiplex immunofluorescence and image analysis approaches.

Luisa M. Solis,1 Naohiro Uraoka,1 Edwin Roger Parra,1 Yu Shen,1 Wei Wei,1 Mei Jiang,1 Barbara Mino,1 Kajsa Affolter,2 Courtney L. Scaife,2 Michele T. Yip-Schneider,3 C. Max Schmidt,3 Matthew Firpo,2 Sean Mulvihill,2 Eugene J. Koay,,1 Huamin Wang,1 Ignacio I. Wistuba,1 Anirban Maitra,1 Subrata Sen1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _University of Utah, Salt Lake City, UT;_ 3 _Indiana University School of Medicine, Indianapolis, IN_.

Pancreatic intraductal papillary mucinous neoplasms (IPMN) are cystic mucin-secreting tumors that arise within the pancreatic ductal system and are considered precursor lesions of pancreatic ductal adenocarcinoma. Profiling the immune infiltrate of IPMNs is essential to understand the immune mechanisms associated with the progression of IPMN. The aim of this study was to characterize the immune profiling of IPMN. We examined 31 formalin-fixed and paraffin-embedded tumor samples from surgically resected IPMN. The diagnostic slides were histologically reviewed by 2 expert pathologists in pancreas and classified as low-grade IPMN (low to intermediate grade dysplasia, N=11), high-grade IPMN (high grade dysplasia, N=18) and IPMN with invasive adenocarcinoma (N=2). Multiplex immunofluorescence (mIF) was used to characterize the immune profiling using two panels: Panel 1: PD-L1, PD1, CD3 (pan T cells), CD8 (T-cytotoxic), CD68 (macrophages), CK (AE1/AE3), DAPI. Panel 2: CD20 (B cells), CD45RO (T-memory), CD57 (NK), Granzyme B (NK and cytotoxic), FOXP3 (T reg), CK (AE1/AE3), DAPI. The slides were scanned using the Vectra multispectral microscope and analyzed by the InForm software (PerkinElmer). We quantified and co-localized immune phenotypes (epithelial cells PD-L1+; Total T-cell lymphocytes; T-cells antigen-experienced; Cytotoxic T-cells; Total macrophages; Macrophages PD-L1+; Total B-cell lymphocytes; Memory T cells; Granzyme B+ cells; Regulatory T cells; NK cells) in low grade dysplasia (LGD), high grade dysplasia (HGD), and invasive carcinoma areas in epithelial and stromal compartments. The Wilcoxon rank sum test or the Wilcoxon singed-rank test was used to test the equality of medians between two independent groups or paired samples, respectively. In all patients, HGD areas showed significantly higher density of macrophages (P= 0.041) and lower density of B-cell lymphocytes (P= 0.028) compared with LGD areas in the epithelial/stromal and epithelial compartments, respectively. Similar differences were observed when patients with high-grade IPMN (n=18) were analyzed for immune infiltrates in the HGD areas compared with LGD areas in the same tumor samples: HGD areas showed significantly higher density of macrophages (P= 0.035) and lower density of B-cell lymphocytes (P= 0.031). Patients with high-grade IPMN had significantly lower densities of B-cell lymphocytes (P= 0.011), T-cell lymphocytes (P= 0.034), and memory T-cells (P= 0.031) in the HGD areas when compared with LGD areas from patients with low-grade IPMN, in the epithelial compartment. No significant differences were found between LGD areas from patients who had high-grade IPMN and LGD areas from patients with low-grade IPMN. In conclusion, we characterized the immune landscape of IPMN and identified immune cell biomarkers associated with progression of IPMN.

#3122

Density of CD3+ and CD8+ tumor-infiltrating T lymphocytes as predictor of survival in colon cancer stage I-III.

Kjersti E. Hestetun,1 Luka Stanisavljević,2 Mette P. Myklebust,2 Olav Dahl1. 1 _University of Bergen, Bergen, Norway;_ 2 _Haukeland University Hospital, Bergen, Norway_.

Introduction: The risk of recurrence after surgery for colon cancer is not only determined by cancer stage and molecular characteristics, but also by the tumor microenvironment. This study aims to assess the prognostic value of CD3+ and CD8+ T lymphocyte infiltration and the relation to mismatch repair (MMR) deficiency.

Material and methods: The study cohort includes 289 patients with surgically treated colon cancer stage I-III (no adjuvant chemotherapy) with clinical- and follow-up data. Immunohistochemistry for CD3+ and CD8+ T lymphocytes and MMR proteins MLH1, MSH2, MSH 6 and PMS2 was performed on Tissue Micro Arrays. (Results were available from 242/258 patients, respectively). Tumors were characterized as MMR deficient/proficient according to expression pattern. Densities of CD3+ and CD8+ lymphocytes were assessed within the tumor margins (tumor infiltrating lymphocytes, TILs) and stroma <100 μm from tumor margin (stromal lymphocytes) and results were combined into semi-quantitative composite scores. In cases of intratumoral heterogeneity, the cylinder with the highest density of TILs was included in the analyses. Results were dichotomized at the median value into low and high density.

Results: MMR deficiency (60 patients, 24.8%) was significantly associated with high density of CD3+ and CD8+ TILs and stromal lymphocytes (p<0.001, p=0.001 Chi square test), but no independent prognostic value of MMR deficiency was demonstrated in our multivariate models. CD3+ and CD8+ TILs and stromal CD3+ and CD8+ lymphocytes were analyzed in separate multivariate cox regression models adjusting for TNM-stage, sex, age, MMR status, histology and tumor differentiation. Low density of CD3+ and CD8+ TILs was an adverse independent prognostic marker of recurrence in colon cancer stages I-III (HR 4.63, CI 1.72-12.49, p=0.002) and the colon cancer stage III subgroup (HR 4.83, CI 1.06-21.97, p=0.042). TNM-stage and differentiation grade were other statistically significant variables in the colon cancer stage I-III model. The stromal lymphocyte score did not reach statistical significance in our multivariate models. Kaplan Meier plots illustrate marked differences in survival between groups of various TIL densities. The low TIL density group had a mean recurrence-free survival of 50.5 months (SE 1.89) versus 63.5 months (SE 1.11) for patients with high TIL density (p<0.001) in colon cancer stage I-III.

Conclusions: Our results support the prognostic impact of tumor infiltrating CD3+ and CD8+ lymphocyte density in colon cancer stage I-III and indicate that a high TIL density is a stronger predictor of survival than MMR deficiency.

#3123

**Peripheral T cell lymphoma-associated fibroblasts promote tumor growth in an** in vivo **model.**

Martina Magni,1 Sara Rizzitano,1 Alessio Pellegrinelli,1 Paolo Corradini,2 Cristiana Carniti1. 1 _Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy;_ 2 _Fondazione IRCCS Istituto Nazionale dei Tumori - Università degli Studi di Milano, Milan, Italy_.

Peripheral T-cell lymphomas (PTCLs) represent a rare and heterogeneous group of aggressive non-Hodgkin's lymphomas comprising different entities. Anthracycline-based regimens (usually CHOP, cyclophosphamide/doxorubicin/vincristine/prednisone) followed by stem cell transplantation are considered the standard of care in the front-line setting. However, long-term disease control can be achieved only in 30-40% of young patients. Increasing evidences highlight the role of tumor microenvironment in sustaining tumor progression and aggressiveness. Among microenvironment components, a central role is played by cancer associated fibroblast (CAFs). Recent studies demonstrated that this is applicable not only to solid tumors but also to haematological malignancies. Here, we report for the first time the isolation and characterization of PTCL-CAFs and their role in promoting cell proliferation.

We kept in culture a skin biopsy of a cutaneous localization of a peripheral T cell lymphoma. Fibroblast-like cells grown out from the biopsy were isolated, cultivated for few passages and then characterized. Flow cytometric analyses revealed that the cell population entirely expressed cell-surface antigens specific for fibroblasts, such as CD140b and CD90, while they did not express CD45 and CD31, thus excluding hematopoietic or endothelial origin. Biochemical analyses confirmed that all cells were positive for vimentin, while αSMA expression highlighted the activated status of the cells isolated. We then assessed the in vitro effect of CAFs on PTCL cell growth by co-colture experiments. For this purpose, we coltured for 8 days the PTCL cell line OCI-Ly12 in fresh medium or in medium previously conditioned by CAFs or normal skin fibroblasts (NFs). PTCL cell growth was not affected by co-colture either with CAFs or with NFs. Moreover, the presence of conditioned medium did not alter the response of OCI-Ly12 cells to CHOP treatments. We then assessed the in vivo effect of PTCL-CAFs. We subcutaneously injected CAFs alone, OCI-Ly12 cells alone and OCI-Ly12 together with CAFs (ratio 1:1) in NOD/SCID mice and monitored tumor growth for 20 days. In contrast to the effect observed in vitro, co-injection of CAFs with cancer cells substantially promoted tumor growth (tumor volume OCI-Ly12 0,87±0,47 cm3; OCI-Ly12+CAFs 2,30±0,26 cm3). As expected, CAFs alone did not develop a measurable mass. Subsequent immunohistochemical staining with CD31 revealed the presence of more vascular structures in tumors originated by OCI-Ly12+CAFs co-injection than by OCI-Ly12 alone, suggesting that CAFs presence sustains tumor growth by promoting angiogenesis.

In summary, we show that PTCL-derived CAFs promote cancer cell growth in vivo, while leaving unaffected cell growth in vitro and the response to chemotherapy. This is the first evidence of the role played by the microenvironment in promoting malignant cell proliferation in PTCLs.

#3124

Cytotoxic effects of tumor-specific T lymphocytes on autologous cancer initiator stem-like glioblastoma cells.

Yin-Cheng Huang,1 Kuo-Chen Wei,1 Chen-Nen Chang,1 Wen K. Yang2. 1 _Chang Gung Memorial Hospital, Taoyuan, Taiwan;_ 2 _Den-Mei Brain Tumor Educational FoundationDen-Mei Brain Tumor Educational Foundation, Taichung, Taiwan_.

[Purpose] In clinical trial of autologous dendritic cell/autologous tumor cell antigens (ADCTA) adjuvant immunotherapy of glioblastoma multiforme (GBM), we studied if and how GBM CD133(+) stem-like initiator cells are susceptible to immune cytotoxic eradication

[Methods] Tumor-infiltrating T lymphocytes (TILs) were isolated from GBM patients after the ADCTA vaccination and tested for their effects on CD133(+) and CD133(-) glioma cells separated from the same patients.

[Results] TILs isolated from GBM patients after ADCTA vaccination were cytotoxic to both CD133(+) and CD133(-) autologous glioma cells. However, the cell lysis was detectable in CD133(-) in 4 hours but did not occur until more than 18 hours later in the CD133(+) cancer stem-like initiator cells. Surprisingly, we found that, upon exposure to the TILs, the GBM tumor cells showed phenotype shift from CD133(+) to CD133 (-). Similar CD133(+) to CD133(-) phenotype shift was also observed by exposure to culture condition medium of the TILs. The responsible factors secreted by the TILs that caused the phenotype shift was determined to be INFγ and TNFα.

[Conclusion] ADCTA vaccination in GBM patients would generate TILs that are not only specifically cytotoxic to autologous CD133(-) glioma cells but also secret INFγ and TNFα etc to induce phenotypic shift of the resistant CD133(+) stem-like GBM to susceptible CD133(-) glioma cells.

#3125

Functional validation of respective gene from a nineteen gene-based risk score classifier, TCA19 as a prognostic indicator and response to immune system of colorectal cancer patients.

Jong Lyul Lee, Jin Cheon Kim, Chan Wook Kim, Seon Ae Roh. _Asan Medical Center, Seoul, Republic of Korea_.

Background: Colorectal cancer (CRC) is a leading cause of cancer death worldwide. Pathologic staging is the gold standard for prognosis, concurrently selecting patients for adjuvant chemotherapy. A recent study reports that the risk score based 19 gene regulated by TREM1 or CTGF activation, TCA19, was an independent risk factor for disease-free survival.

Objective: The aim of this study was to validate TCA19 as a prognostic indicator of colorectal cancer patients and to select candidate genes related to clinicopathologic variables and immune system. Patients and Method: A retrospective review was conducted on patients who underwent surgery followed by adjuvant chemotherapy for CRC at the Asan Medical Center, Seoul, Korea, between December 2008 and December 2014. To validate TCA19, the medical records of 60 patients who met inclusion criteria were reviewed. According to the previous report, RNA sequencing using reverse transcription-quantitative polymerase chain reaction (RT-PCR) was done and then TCA19 risk score using 2ΔΔCt values was checked. After checking TCA19 genes, we selected key candidate genes according to the correlation with clinicopathologic variables and PFS. Immunohistochemistry (IHC), Western blot, and immunofluorescence staining (IFS) were done for the selected candidate genes, TREM1, CKS2, SLAMF7.

Results: Among 60 study cohort, 59 patients were in stage IV and 1 patient was in stage IIIC. Among 30 patients with high TCA risk score, the PFS rates between 4 patients who received 5-FU regimen and 8 patients received the target regimens (bevacizumab or cetuximab) were significantly different. On multivariate analyses for PFS, lymphovascular invasion (LVI), circumferential resection margin (CRM), SLAMF7, CKS2, and TREM1 were risk factors associated with PFS. Based on the association with clinicopathologic variables and the survival results, the SLAMF7, the CKS2, and the TREM1 can become a key gene among the TCA 19 genes in terms of prognostic factor and previous results. Primary and metastatic tumor IHC of the SLAMF7, the CKS2, and the TREM1 was significantly overexpressed, compared with normal tissue IHC of the three genes. Like IHC results, 5 days' IFS imaging of SLAMF7 after co-culture showed significant hypoexpression compared with 0 day imaging of SLAMF7.

Conclusion: TCA 19 risk score might be one of prognostic predictors related to using the target regimens (bevacizumab or cetuximab). TREM1, CKS2, and SLAMF7 were key candidate genes among the TCA19 genes in terms of clinicopathologic variables and survival outcomes. TREM1 was overexpressed in primary or metastatic tumor on the contrary; downregulation of SLAMF7 was shown in the primary or metastatic tumor. Further functional study using a large cohort will be needed.

#3126

Calnexin induces impairment of anti-tumor immunity of CD4+ and CD8+T cells.

Zhi Wang, Yichen Chen, Da Ma, Juan Fang, Xi Wang. _School of Stomatology, Sun Yat-sen Univ., Guangzhou, China_.

Aim: Elucidation of molecular mechanism underlying the inhibited infiltration effector function of T cells induced is essential for improvement of anti-tumor immunotherapy. This study is to elucidate the importance of chaperon protein Calnexin as a new anti-tumor immunotherapy target.

Methods: OSCC tissue microarray was used to correlate Calnexin with TIL infiltrates and OSCC prognosis. Construction of Calnexin-specific knock-out and overexpressed tumor cell lines, combined with Calnexin fusion protein were used to elucidate the function of Calnexin on T cell response and its anti-tumor immunity in vitro and in humanized OSCC mice model and melanoma models. Bisulfite pyrosequencing of PD-1 promoter was used to identify the demethylation statuses of PDCD1 promoter.

Results: we found that Calnexin is significantly up-regulated in oral squamous cell carcinoma (OSCC) tumor cells and multipleother tumors. Up-regulation of Calnexin in tumor cell membrane links with limited infiltration of T cells in OSCC tumor core and poorer survival of OSCC patients. Importantly, we found that Calnexin mediates inhibition of proliferation of CD4+ and CD8+T cells and effector functions of expression of IFN-γ, TNF-α, IL-2 by T cells. While knock-down of Calnexin enhanced the infiltration and effector functions of T cells in tumor cores and conferred better control of tumor growth, treatment of recombinant Calnexin induced impairment of infiltration and effector functions of T cells in tumor cores and promotes tumor growth in mice. We also found that Calnexin expressed in tumor cells or its recombinant protein enhanced expression of PD-1 on CD4+ and CD8+T cells in tumor core via sustaining DNA demethylation statuses in CFG island of PD-1 promoter, and then Calnexin recombinant protein and treatment of anti-PD-1 mAb can synergistically enhance infiltration and effector functions of T cells in tumor cores and limits tumor growth in mice.

Conclusion: This work uncovers a previously unknown mechanism in which infiltration of T cells and effector functions of T cells are inhibited by Calnexin expressed in tumor cells, and implicates that Calnexin may serve as an important target for improvement anti-tumor immunotherapy.

#3127

Peripherally induced Tregs or pTregs are the potent tolerance inducer for the growth and metastasis of cancer.

Upendra P. Hegde, Nitya G. Chakraborty. _Univ. of Connecticut School of Medicine, Farmington, CT_.

Regulatory T cells act as the most important tolerance inducer for the growth and metastasis of cancer. It remains a wonder how Tregs induce tolerance for the development of cancer. Previously we have shown with melanoma patients that, increase in pTreg cell number in blood are related to the poor prognosis of the disease. pTreg and iTreg are remarkably similar and significantly different in functionality from tTreg. Here, we worked with four melanoma patients two HLA A2 + and two HLA A2 negative. PBL and tumor cells were obtained from the patients with informed consent. Treg cells were generated in four different culture conditions: isolated from tumor +PBL IVC or peptides, Mart-1 A2 or Flu A2 pulsed DC + PBL IVC, with purified CD4 cells stimulated with anti CD3 and antiCD28 plus IL-2 with or without TGF-b. Here we show some phenotypic and functional characteristics of the induced Treg cells in cultures from the patients' PBL derived CD4+CD5- cells and analyzed those in separate CTL generation assays. We observed that induced Treg cells under different conditions do not uniformly express CD25, FoxP3, PDL-1 or CTLA 4 as the known surface markers. When analyzed for their functionality, with adjusted number of cells, in suppressing the anti tumor CTL response, a significant difference was observed. The most effective Tregs cells were found to be those isolated from autologous tumor +PBL IVC or from Mart-1 peptide pulsed IVC. Those cells completely blocked the CTL induction and secreted huge amount of IL-10 upon re-stimulation. Further analysis with these different types of induced Treg cells in terms of various gene expressions will be useful to find a target molecule to block such expansion of pTregs cells for better therapeutic outcome.

#3128

Immune microenvironment of colorectal tumors and matched liver metastases.

Vegar J. Dagenborg,1 Serena Elizabeth Marshall,1 Krzysztof Grzyb,1 Åsmund A. Fretland,1 Marius Lund-Iversen,1 Gunhild M. Mælandsmo,1 Anne-Lise Børresen-Dale,1 Anne H. Ree,2 Bjørn Edwin,1 Sheraz Yaqub,1 Kjersti Flatmark1. 1 _Oslo University Hospital, Oslo, Norway;_ 2 _Akershus University Hospital, Lørenskog, Norway_.

Introduction: The presence of tumor-infiltrating lymphocytes, in particular T-cells in the tumor and its microenvironment, has been shown to be associated with prognosis in primary colorectal cancer (CRC) and CRC liver metastases (CLM). In this study we examined T-cell composition in the immune microenvironment in surgical specimens from 64 cases with CRC and resectable CLM.

Methods: Patients were enrolled in the Oslo-CoMet randomized trial (NCT 01516710). CRC and corresponding CLM were examined by immunohistochemistry to quantify all T-cells (Ttot) and subpopulations of cytotoxic (CTL), helper (TH), and regulatory T-cells (Treg). Densities of T-cells and subtypes (cells/mm2) were calculated at T-cell hotspots in the invasive margin (IM) and intra-tumoral (IT) locations. Median densities with interquartile ranges were calculated and compared for each region in CRC and CLM.

Results (Table 1): The highest density of Ttot was observed in the IM for both primary CRC and CLM, with the density in CLM being two-fold higher than CRC (CLMIM median 2853 cells/mm2 vs CRCIM 1257 cells/mm2; p<0.001). For IT regions, the density of Ttot was much lower than in IM, and slightly higher in CRC compared to CLM (CRCIT median 486 vs CLMIT 332; p=0.02). Examining T-cell subtypes, the differences in CTL and TH mirrored Ttot, with the highest densities in the IM (CLMIM>CRCIM) and lower densities in IT regions (CLMIT<CRCIT). For the Treg, the IM densities were similar, while for IT, again, the density was lower in CLM than CRC.

Conclusion: In this study, we analyzed the immune microenvironment in matched CRC and CLM surgical specimens. We are currently analyzing associations with long-term outcome and results will be presented at the conference.

Table1:Median T-cell density (interquartile range).

---

|

CRCIM | CLMIM | CRCIT | CLMIT

Ttot | 1257 (931 - 1735) | 2853 (2305 - 3813) | 486 (285 - 704) | 332 (189 - 564)

CTL | 497 (315 - 722) | 1083 (762 - 1426) | 106 (51 - 209) | 99 (49 - 219)ns

TH | 759 (584 - 983) | 1888 (1449 - 2745) | 348 (192 - 480) | 198 (118 - 383)

Treg | 222 (155 - 375) | 233 (126 - 375)ns | 144 (75 - 225) | 43 (22 - 82)

CRC and CLM densities at each location (IM and IT) were compared using the Mann-Whitney U test. P-values were <0.05 for all comparisons unless indicated by ns.

#3129

PD-L1 expression differs across cancer metastatic sites from breast tumors.

Mariaelena Pierobon,1 K Alex Hodge,1 Elisa Baldelli,1 Steven Anthony,2 Nicholas J. Robert,3 Donald W. Northfelt,4 Mohammad J. Jahanzeb,5 Linda Vocila,6 Lance A. Liotta,1 Bryant Dunetz,7 Emanuel F. Petricoin1. 1 _George Mason University, Manassas, VA;_ 2 _Evergreen Hamtology & Oncology, Spokane, WA; _3 _Virginia Cancer Specialists, Fairfax, VA;_ 4 _Mayo Clinic Arizona, Scottsdale, AZ;_ 5 _University of Miami, Miami, FL;_ 6 _TD2 Translational Drug Development, Scottsdale, AZ;_ 7 _Side Out Foundation, Fairfax, VA_.

Background: Although the efficacy of different immunotherapeutic agents varies greatly across tumor types, little is known about the role of different metastatic tumor microenvironments in modulating the expression of immune-checkpoints like PD-L1. Using metastatic breast cancer as a model, this work explored whether metastatic lesions derived from different patients, but colonizing the same host target organ, showed organ-specific immune signatures.

Methods: Snap frozen metastatic lesions collected from 30 breast cancer patients enrolled in a prospective phase II multi-omic precision medicine clinical trial ("Side-Out II") were used for this analysis. Sites of metastasis were: liver (LI) (n=10), skin/chest wall (SC) (n=10), and other lesions (OL) (n=10) [including lymph nodes (LN) (n=4), lung (LU) (n=3), and intra-abdominal lesions (IA) (n=3)]. Pure tumor epithelia were isolated from each specimen via Laser Capture Microdissection (LCM). PD-L1 expression (Antibody Clone E1L3N) was quantitatively measured using the Reverse Phase Protein Microarray (RPPA), a high-throughput, multiplex immunoassay that provides operator-independent quantitative data starting from a small amount of biological material. Proportion of patients with PD-L1 expression above the median and the 75th percentile of the population was compared across metastatic sites.

Results: PD-L1 expression had a >5 fold dynamic range across breast cancer-derived metastatic lesions with different distribution across metastatic sites. Specifically, PD-L1 expression of 6/10 LIs, 5/10 OLs (including 2 LU and 3 LN positive lesions), and 4/10 SC metastases was greater than the population median of all metastases combined. LI metastases were the most represented group above the 75th percentile of the population (4/10), followed by OS (2/10) (including 1 LU and 1 LN positive lesions), and SC (1/10). Of the 15 lesions above the population median, 6 were LI (40%), 5 were OS (33%), and 4 were SC (27%); of the 7 metastases above the 75th percentile, 4 were LI (57%), 2 were OS (29%), and 1 was SC (14%).

Conclusions: The LCM-RPPA workflow can capture PD-L1 protein expression on a continuous quantitative scale and provide a broader understanding of the dynamic range of expression. Our data suggest that organ specific microenvironments in which metastatic lesions develop may strongly influence overall PD-L1 tumor cell expression. If different metastatic host organs can modulate PD-L1 expression, location of the metastatic lesion should be kept in consideration when selecting patients that are candidates for immunotherapy. Such hypotheses should be further validated in prospective clinical trial where quantitative PD-L1 expression of different metastatic lesions is evaluated along with response to targeted immuno-oncology compounds.

#3130

Immune profiling in uterine papillary serous carcinoma cancer patients reveals distinct subpopulations and clinical outcomes.

Angela Rynne Vidal, Karen H. Lu, Samuel C. Mok. _MD Anderson Cancer Center, Houston, TX_.

Uterine papillary serous carcinoma (UPSC) is a rare but aggressive type of endometrial cancer (EC) that represents 10% of cases of EC but accounts for 40% of EC deaths. Patients with UPSC have poor survival rates, a high risk of recurrence and frequently metastasize to the lymph nodes and the omentum. The mechanisms that drive UPSC progression have not been elucidated yet and immune profiling can be a valuable tool to understand the different patient outcomes. In this work, we used formalin fixed paraffin embedded (FFPE) tumor samples from a cohort of 140 patients with UPSC, the largest used to date, to study a panel of 7 immune markers by multiplex immunofluorescence, using the OpalTM system (Perkin Elmer) in the same tissue section. Samples were imaged with Vectra 3.0 multispectral microscope (Perkin Elmer) and images were analyzed using Visiopharm software. Tumor samples from primary, metastatic and recurrent sites were used to study the tumor microenvironment. We observed significant differences in the tumor-infiltrating immune cells between patients that recurred and those that did not. Similarly, immune subpopulations distinguished patients with metastasis compared to those with a less aggressive form of UPSC. Spatial distribution of cytotoxic T cells (CD8+ GranzymeB+) and cancer cells was also found to significantly correlate with overall patient survival. Collectively, our work shows the immune populations play a role in patient outcomes and should be taken into account for future therapeutic strategies.

#3131

Acute myeloid leukemia derived SDF1 repels tumor infiltrating lymphocytes in the bone marrow microenvironment.

Manar S. Shafat,1 Amina A. Abdul-Aziz,2 Christopher Marlien,2 Rachel Piddock,2 Kristian M. Bowles,3 Stuart A. Rushworth2. 1 _University College London, London, United Kingdom;_ 2 _University of East Anglia, Norwich, United Kingdom;_ 3 _Norwich and Norfolk University Hospital, Norwich, United Kingdom_.

Introduction: Acute myeloid leukaemia (AML) is an age-related disease with a median age at diagnosis of 71. Combined with co-morbidities, survival of patients with AML above this age bracket is poor with 90% of older adults dying of their disease. Even in younger patients who achieve remission with chemotherapy, relapse is common and occurs from minimal residual disease sequestered in protective niches in the bone marrow microenvironment (BMM). AML as a disease is highly dependent on the bone marrow microenvironment and accordingly it is envisaged that improved outcomes will come from novel treatment strategies derived from an improved understanding of the biology of AML within the bone marrow and their interaction with the immune cells therein.

Objective: Here, we investigate a possible immune evasive mechanism imparted by AML blasts attributed to their localization within the BMM.

Methods and Results: Using RNA-seq data generated from bone marrow derived AML (BM-AML) compared to peripheral blood derived AML (PB-AML) (GEO ID: GSE49642, GSE48846) we identified SDF-1 as the most differentially expressed in favour of BM-AML. Further investigation using matched primary clinical samples (BM verses PB) by real-time PCR analysis and ELISA confirmed the in-silico findings. In addition, primary AML engrafted into NSG mice demonstrated high SDF-1 in the BM derived AML blasts compared to AML blasts derived from the spleen. Lentiviral knock-down of SDF-1 in the AML blasts impaired blast engraftment and prolonged survival of the animals. SDF1-mediated migration of T-cell subsets have been previously identified using varying concentrations of SDF1. We therefore, investigated the migration of CD8+ T cells when co-cultured with AML. We show that CD8+ T-cells migrate away from AML when in culture and this fugetaxis is reduced by the knock-down of SDF1 in the blasts.

Conclusion: We conclude that SDF-1 is secreted by the AML blasts within the BMM contributing to fugetaxis of CD8+ cells away from the blasts. Implications of these finding suggest a possible immune evasive mechanism by the blasts that can be manipulated to potentiate CD8+ infiltration into the AML bone marrow.

#3132

Therapeutic blockade of Foxp3 in experimental breast cancer: Immune stimulation and direct antitumor effects.

Alejandro J. Nicola,1 Mariela A. Moreno Ayala,1 Maria F. Gottardo,1 Antonela S. Asad,1 Camila F. Zuccato,1 ELISA Bal de Kier Joffé,1 Adriana Seilicovich,1 Flavia Zanetti,2 Marianela Candolfi1. 1 _University of Buenos Aires School of Medicine, Buenos Aires, Argentina;_ 2 _Instituto Cesar Milstein (CONICET), Buenos Aires, Argentina_.

Our previous results indicate that systemic administration of a cell penetrating peptide (P60) that inhibits Foxp3, a transcription factor required for Treg function, improves the efficacy of antitumor vaccines in experimental breast cancer. We also found that P60 exerts direct antitumor effects, inhibiting tumor progression in Foxp3+ breast tumors. Although there is controversy over the role of Foxp3 in tumor cells, we found that P60 inhibits survival, proliferation and release of IL-10 in Foxp3+ breast tumor cells. Here we aimed to evaluate the pathways that control Foxp3 expression in murine LM3 breast tumor cells and to develop gene therapy vectors encoding P60 in order to improve the availability of this peptide in vivo. We assessed the role of TGF-β, mTOR and prostaglandins on the expression of Foxp3 in breast tumor cells, as these pathways have been shown to modulate Foxp3 expression in Tregs. Stimulation of LM3 cells with TGF-β and mTOR inhibitor rapamicyn upregulated Foxp3 expression, as assessed by flow cytometry, whereas indomethacin, an inhibitor of prostaglandin synthase cyclooxygenase, inhibited Foxp3 expression. These observations suggest that the regulatory mechanisms of Foxp3 expression are similar in breast tumor cells and Tregs. We next developed a plasmid that encodes P60 linked through an IRES sequence to Td.Tomato as a reporter gene (pCMV.P60.dTomato), as well a control plasmid. Transduction efficiency of these plasmids was evaluated in murine 4T1 breast tumor cells, which exhibit low expression of Foxp3 and thus are not affected by P60. Expression of Td.Tomato was readily detected by fluorescence microscopy. Conditioned media from pCMV.P60.dTomato-transfected 4T1 cells reduced the proliferation and the secretion of IL-10 in Foxp3\+ LM3 cells when compared to control plasmid-transfected cells, which suggests that functional P60 is released from pCMV.P60.dTomato-transfected cells. In order to develop a gene therapeutic strategy to deliver P60 in vivo, we developed an adenoviral vector (Ad.P60.dTomato) encoding the therapeutic cassette, as well as a control vector (Ad.dTomato). These vectors successfully transduced breast tumor cells in vitro and in vivo, as evaluated by TdTomato expression. Our findings indicate that P60 could be delivered using gene therapy vectors, which could be useful for the treatment of breast cancer.

#3133

Reprogramming of tumor-associated macrophages by a short synthetic peptide eradicates ovarian cancer.

Reshma Bhowmick,1 Elena Vinokour,2 Michael Paul Plebanek,2 Marisol Villanueva,2 Victor Shifrin,3 Jack Henkin,4 Ignacio Melgar-Asensio,4 James Petrik,5 Raghu Kallurie,1 Olga V. Volpert1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Northwestern University, Chicago, IL;_ 3 _Pamdeca LLC, Auburndale, MA;_ 4 _Northwestern University, Evanston, IL;_ 5 _Ontario Veterinary Collage, Guelph, Ontario, Canada_.

Purpose: Ovarian cancer is the deadliest gynecologic malignancy with limited treatment options and novel therapies urgently needed. Immunosuppressive microenvironment is critical for tumor progression and immune checkpoint inhibitors, which enable T-cell anticancer immunity revolutionized the outcomes in multiple cancer types. However, this approach had limited success in ovarian cancer. Our small therapeutic peptides, derived from an endogenous type 2 tumor suppressor, Pigment Epithelium-Derived Factor (PEDF), act through an alternative immune mechanism, repolarization of tumor-associated macrophages (TAMs) to the tumor-suppressive phenotype.Experimental Design: Short peptides based on the PEDF's active domain were modified for improved stability and efficacy. Two peptides (PMD-427, PMD-336) were tested in preclinical ovarian cancer models using the human chemoresistant cell line, OvCar-3, and transformed mouse cell line ID8. We also performed mechanistic analysis of the peptides' anti-tumor action, including effects on macrophages cytotoxic, cytokine secretion and migratory activity in vitro and in vivo. Results: PEDF peptide PMD-427 caused > 20-fold reduction in tumor burden. PMD-427 induced selective apoptosis in ovarian cancer cells but not in normal ovarian epithelium. This selectivity was based on context-specific modulation of extrinsic death cascades, Fas and FasL. More importantly, PMD-427 peptides also stimulated macrophage polarization from M2 to M1 phenotype as was evidenced by the shift in cytokine profile (decreased IL-10 and increased IL-12 expression), altered morphology (increased number of dendrite-like-processes) and other changes in M2 markers (attenuated PD-L1 expression). M2/M1 macrophage polarization was also evident by tumor immunostaining. Critically, PMD peptides ovarian cancer cell killing by macrophages as was determined in co-culture studies; this fratricidal activity was reliant on the expression of TRAIL by the macrophages and of its cognate receptor, DR5 by ovarian cancer cells, respectively. Combined with enhanced macrophage motility as observed by time-lapse micropscopy, these changes resulted in increased macrophage recruitment to the tumors and enhance killing of the cancer cells in vivo. The key role of macrophages in the anti-cancer effects of PMD peptides was confirmed by depletion of macrophages in ovarian tumor bearing mice using clodronate liposomes. Conclusions: We have generated a first-in-class multi-targeted peptide drug, which promotes macrophage polarization that results in eradication of ovarian tumors in mice.

#3134

Pilot study of immune status of GEP-NETs in tumor microenvironment.

Yasushi Ichikawa,1 Noritoshi Kobayashi,1 Ayumu Goto,1 Motohiko Tokuhisa,1 Yukihiko Hiroshima,1 Takashi Ishikawa,2 Shoko Takano,1 Tomio Inoue,1 Itaru Endo1. 1 _Yokohama City Univ. Graduate School of Medicine, Yokohama, Japan;_ 2 _Tokyo Medical University, Tokyo, Japan_.

Introduction: Neuroendocrine tumor (NET) is a rare cancer, however, morbidity rate is increasing every year. Recently, effective molecular targeting drugs for NET were developed and leading acceptable results, however, it is still not enough. Immune checkpoint inhibitor is becoming a very effective drug in malignant melanoma, lung cancer, and some kind of cancer showing microsatellite instability (MSI).

Aim: Aim of this study is to investigate NET about MSI status and immune status of microenvironment using NET patients' operative or biopsy samples.

Method: Patients were 20 NETs, male: 11, female: 9, G1: 6, G2: 8, G3: 6, pancreas: 11, duodenum: 4, colon: 2, liver: 1, unknown: 2. Using patients' samples by operative resection or biopsy, examinations by immunohistochemistry were performed to confirm MSI status using MSH2, MSH6, PMS2 and MLH1 antibody and count the cell number expressing PD-L1, PD-1, CD8 or Foxp3. Expression of MSH2, MSH6, PMS2, MLH1 and PD-L1 was evaluated on tumor cells. Specimens were categorized as IHC negative or positive if < 1% or > 1% of cells were stained by each monoclonal antibody. Expression of PD-1, CD8, Foxp3 was evaluated on the infiltrating lymphocyte of intratumor and peritumor respectively. Positive cells absolute number of each staining were counted in the 3 hot spot field (X400) and then the average of the 3 field of each specimen was utilized.

Results: MSI: Expression of the 4 mismatch repair protein could be detected in the all 20 NETs tumor, then there was no case showing MSI. PD-L1 was detected in the 15 cases (75%). PD-L1 was expressed in the surface of tumors. PD-1 was also detected 15 cases (75%). PD-1 was expressed in the surface of infiltrative intratumoral and peritumoral lymphocyte. Both of PD-L1 in the tumor and PD-1 in the lymphocyte were detected in the 12 cases (60%). PD-L1 was detected in 72.7% of pancreatic NET, 72% of gastroenteral NET. PD-L1 was detected in 100% of NET-G1, 75% of G2 and 50% of G3. The absolute number of intratumoral PD-1+ lymphocyte, CD8+ lymphocyte or Foxp3+ lymphocyte was higher in the PD-L1+ tumor than in the PD-L1- tumor. In peritumor, there was not a similar tendency. In the PD-L1 positive NET, the absolute number of intratumoral PD-1+ lymphocyte or CD8+ lymphocyte increased according to NET grade.

Conclusion: Seventy-five% of GEP-NET expressed PD-L1. It might be higher rate of PD-L1 expression compared with other cancers. The absolute number of Intratumoral infiltrating lymphocyte expressing PD-1 or CD8 was also high. There might be a fraction of a good target for immune check point therapy in NETs.

#3135

Single-cell RNA sequencing identifies macrophage-specific expression signatures associated with phagocytosis of multiple myeloma after treatment with cIAP antagonist.

Nicholas Banovich,1 Martin Boateng,1 Marta Chesi,2 Leif Bergsagel,2 Jonathan Keats1. 1 _Translational Genomics Research Institute, Phoenix, AZ;_ 2 _Mayo Clinic, Scottsdale, AZ_.

Multiple myeloma (MM) is the second most common hematologic cancer, accounting for roughly 2% of all cancer deaths. Unfortunately, MM remains incurable and nearly all patients experience relapse. Recently, we have identified a novel cIAP1 antagonist, LCL161, with remarkable anti-MM effects. Interestingly, unlike in other tumor types, our previous work found that LCL161 does not directly induce apoptosis of MM cells, but rather drives activation and infiltration of macrophages to the tumor bed and phagocytosis of tumor cells. Furthermore, in vitro experiments demonstrate that the anti-MM activity of the agent relies on the presence of the tumor, with no observed increase in MM phagocytosis when macrophages are treated in isolation. However, LCL161 is a strong and indiscriminate transcriptional activator and thus, the molecular mechanisms underlying this immune response in MM remain poorly understood. In an effort to characterize molecular signatures associated with this response, we collected whole spleens from seven WT mice and eleven mice with VK12598 transplanted myeloma for single cell RNA-seq. Of the eleven tumor-bearing mice, three remained untreated, three were treated with LCL161, three with bortezomib, and three with cyclophosphamide. Additionally, one of the WT mice was treated with LCL161. Using the 10X genomics chromium platform, we measured mRNA levels in over 90,000 individual cells. We identified groups of cells with shared expression profiles, corresponding to known cell types, using graph-based clustering. Consistent with our previous work, we observed a marked reduction in tumor cells and activation of the noncanonical NFkB pathway in mice treated with LCL161. Turning our attention to cells identified as monocytes, with a particular focus on putative macrophages, we sought to identify molecular signatures associated with LCL161 treatment. Indeed, using a binomial model we identified over 500 upregulated genes in LCL161-treated macrophages. However, our previous data suggested that the efficacy of LCL161 was dependent on the presence of tumor cells. Thus, we refined these results by comparing the expression profile of macrophages treated with LCL161 in WT and tumor-bearing mice. These contrasts identified dozens of genes that were significantly upregulated in macrophages from LCL161 treated, tumor-bearing mice. Of note, we identified a small number of genes (including Spp1, Irg1, and Lipg) that were active only in macrophages from mice treated with LCL161 and bearing a transplanted tumor and unexpressed in all other cells. We believe these genes—and the pathways in which they are active—hold promise for the development of novel targeted therapies to better treat MM.

#3136

Comprehensive profiling the immune-status of a broad range of in vivo syngeneic models.

Jingqi Huang, Shipzong Hu, Wentao Li. _Pharmaron, Beijing, China_.

Latterly with approval of PD-1, PD-L1 antibodies in clinical oncology, immunotherapy has gain recognition for cancer treatment for changing the way by joining chemotherapy, radiation and surgery. This type of cancer treatment aims to produce greater effectiveness with less toxicity than cancer chemotherapy and mainly acts by boosting the body's natural defenses to fight the cancer. It uses substances made by the body or laboratory agent to improve or restore the function of immune system. Therefore, understood the nature immune status and micro environment of in vivo tumor models is very important to assist exploring immunotherapy. As a results, nearly 40 tumor cell lines over a broad range of tumor type as well as corresponding in vivo syngeneic models were intensively studied on its immune status under two conditions when tumor volume were 100 mm3 and 500 to 600mm3 . The effort has been focus on the immune status including T cell status and levels of immune-suppression via FACS analysis of the population of CD4+, CD8+, Treg, MDSC, macrophage and DC cells. Furthermore, not only population of immune cells but also the expressions of immune related gene were analyzed by RE-PCR which are the genes related regulatory of T cells i.e., CD4, Foxp3, GITR; gene of tumor associated macrophage CSF1R, CD68, CX3CR1, ITGAM, CD163, MARC1; gene of co-stimulator receptors of T cells ICOS, CD137(Tnfrsf9), OX40(Tnfrsf4); co-inhibitory receptors of T cell BTLA, HAVCR2, CTLA4, LAG3, CD28, PDCD1; and gene of co-inhibitory ligands of tumor cells CD274, CD137L(Tnfsf9), PDCD1LG2, HVEM(Tnfsf14), OX40L(Tnfsf4). The profiling data illustrated, indeed, the expression of PD-L1 is different on different tumor cells; the immune status certainly pending on the tumor type but also related to the tumor progression. The bigger tumor size the higher level of expression of immune suppression which deterring the immune cell ability to conquer cancer. We believe that this profiling data will help many scientists to properly select correct model to support R&D and to better understand how immune therapeutically agent acts in the immune system. With this comprehensive research data, we're now at a point where this information has started to become translated into treatment possibilities for cancer patients.

#3137

**Towards analysis of the immune microenvironment in ductal carcinoma** in situ **.**

Mathilde M. Almekinders, Lindy L. Visser, Bram Thijssen, Petra Kristel, Rianne van der Linden, Annegien Broeks, Erik Hooijberg, Karin de Visser, Esther H. Lips, Jelle Wesseling. _Netherlands Cancer Institute, Amsterdam, Netherlands_.

Introduction

Since the introduction of population-based mammographic screening, the incidence of ductal carcinoma in situ (DCIS) increased manifold. DCIS lesions are non-obligate precursors to invasive breast cancer, because only a minority of DCIS patients later develops invasive breast cancer. DCIS patients are treated intensively with surgery, frequently supplemented by radiotherapy and/or endocrine treatment. However, treatment of DCIS lesions did not result in a decreased incidence of advanced stages of breast cancer, suggesting overdiagnosis and hence overtreatment exists. Because the immune microenvironment plays an important role in cancer progression, we performed a pilot study to assess the amount, composition and spatial distribution of immune cells aiming at the identification of biomarkers that distinguish aggressive from indolent DCIS.

Methods

A representative series of 32 paraffin-embedded DCIS lesions was studied with multispectral immunohistochemical imaging, providing simultaneous detection and quantification of CD20+ B-cells, CD8+ T-cells, CD4+ T-cells, CD4+Foxp3+ regulatory T-cells, CD68+ macrophages and pankeratin. Cellular density of immune cell subsets per tissue compartment and spatial distribution was analyzed by Inform software, SPSS and R. The number of CD4+FoxP3+ T-cells within 30µm of a CD8+ T-cell was assessed and expressed in a CD4+FoxP3+ T-cell per CD8+ T-cell ratio. Immune cell density and composition were correlated to grade and immunohistochemical ER, Her2 and p53 status.

Results

Multispectral immunohistochemical quantification showed a range of 30 to 2100 lymphocytes/mm2 in the stroma of DCIS lesions. High grade positively correlated with higher number of stromal lymphocytes/mm2 (p<0.01). Negative ER status, positive Her2 status and aberrantly expressed p53 was significantly associated with higher number of stromal lymphocytes/mm2, CD8+ T-cells/mm2, CD4+FoxP3+ regulatory T-cells/mm2 and CD20+ B-cells/mm2 (p<0.05). Within the DCIS-epithelium, the number of CD4+FoxP3+ regulatory T-cells positively correlated with negative ER-status (p=0.02) and positive Her2 status (p=0.03). The spatial distribution of the number of CD4+Foxp3+ T-cells within 30 μm of a CD8+ T-cell (expressed in a Treg per CD8+T-cell ratio) varied from 0 to 0.23 in the stromal compartment and from 0 to 0.60 in the DCIS compartment.

Conclusions

Within the immune microenvironment, CD20+ B-cells, CD8+ T-cells, CD4+ T-cells, CD4+Foxp3+ regulatory T-cells and CD68+ macrophages were successfully and simultaneously detected. Stromal lymphocyte density and CD8+ T-cell, CD4+ T-cell, CD4+FoxP3+ regulatory T-cell and CD20+ B-cell density positively correlated with negative ER status, positive Her2 status and aberrant expression of p53. The next step will be to analyze this multiplex panel in our nationwide DCIS cohort (1989-2005, median follow-up 12.0 years) for correlation with clinical outcome.

#3138

Key role of secreted kinase FAM20C on tumor associated macrophage (TAM) leading to pancreatic cancer progression.

Jieun Im,1 Yu-Sun Lee,2 Sun Il Choi,2 Beom-Kyu Choi,2 A-Ra Jeon,2 Sang Hyun Park,2 Min-Kyeong Lee,1 Joon-Ki Kim,2 Yun-Hee Kim2. 1 _National Cancer Center Graduate School of Cancer Science and Policy, Goyang-si, Gyeonggi-do, Republic of Korea;_ 2 _National Cancer Center, Goyang-si, Gyeonggi-do, Republic of Korea_.

FAM20C reported as a novel secreted kinase has the potential of phosphorylation on consensus motif, S-x-E/pS, of secretory proteins or ectodomain of membrane proteins. Numerous substrate candidates through prediction implied FAM20C has the function on tumor microenvironment, however, function and regulatory mechanism of cancer progression by FAM20C has not been defined yet. As tumor associated macrophage (TAM) changes to have the tumor supporting phenotype in response to various environmental stimuli, TAM is the potent regulatory target of FAM20C in tumor microenvironment. In this study, we hypothesized that the secreted kinase FAM20C in tumor microenvironment can support pancreatic cancer progression by regulating TAM contents or polarization. In pancreatic orthotopic xenograft model of FAM20C-overexpressing tumor cells, the tumor growth rate was enhanced and TAM contents (F4/80+/CD11b+/MHCII+) were significantly increased compared to control group, while total macrophage population between two groups had no difference. Moreover, the high level of TAM contents was sustained in the presence of FAM20C till the late stage of tumor progression. In addition, infiltrated tissue macrophages were polarized into TAM by FAM20C treatment Furthermore, increased TAM population by FAM20C suppresses the CD8+ cytotoxic T cell proliferation with anti-tumor function. Collectively, FAM20C might be a key regulatory factor in pancreatic cancer progression by promoting polarization of TAM.

#3139

Innate immune cells expressing myeloperoxidase directly inhibit tumor NF-κB transcriptional activation during tumor initiation.

Tracy W. Liu, Seth T. Gammon, David Piwnica-Worms. _UT MD Anderson Cancer Center, Houston, TX_.

Neutrophils are considered the primary defenders of the innate immune system. However, in the context of cancer, the role of neutrophils is complex where they can be host protecting or pro-tumorigenic. For example, myeloperoxidase (MPO), the major protein in neutrophils, produces reactive oxygen species (ROS); ROS can lead to tumor regression from cell cytotoxicity or cause DNA-damage resulting in genetic mutations triggering tumor initiation/progression. Using the melanoma cell lines, B16F10, stably expressing a NF-κB-driven Firefly luciferase (FLuc) reporter or an IκBα-FLuc fusion reporter gene, co-culture studies in vitro with bone marrow-isolated-neutrophils from C57BL/6 animals demonstrated inhibition of NF-κB transcriptional activation in a MPO activity-dependent manner. Using bioluminescence imaging with luminol, neutrophils pre-stimulated with 50uM phorbol myristate acetate (PMA) demonstrated an increase in MPO activity by 20.3 ± 2.4 fold over unstimulated neutrophils. When B16F10 reporter cells were co-cultured with PMA pre-stimulated neutrophils, we observed inhibition of IκBα degradation by 33.0% ± 11.9%, resulting in a decrease in NF-κB transcriptional activation compared to co-culture studies with unstimulated neutrophils. The role of neutrophils during B16F10 tumor initiation and growth was compared in C57BL/6 and syngeneic MPO-null mice. Utilizing intravital imaging with skinfold window chamber animal models and imaging reporters, we evaluated in real-time, the recruitment of MPO-expressing cells and tumor NF-κB activation in vivo. Mean NF-κB transcriptional activation within the tumor compartment in MPO-null mice was 42.2% ± 1.6% greater compared to wild type animals. Tumor growth and survival curves demonstrated that B16F10 tumors grew slower in wild-type animals (mean survival of 26.8 days) compared to MPO-null animals (mean survival time of 23.4 days). Using an MPO inhibitor, 4-aminobenzoic acid hydrazide (4-ABAH), we pharmacologically mimicked the MPO-null phenotype in C57BL/6 animals (40mg/kg intraperitoneal injection twice daily). B16F10 tumors grew slower in C57BL/6 animals treated with 4-ABAH (mean survival of 23.8 days) compared to wild type animals. Taken together, these data demonstrated that MPO-expressing cells contribute to host protection during tumor initiation in a NF-κB-dependent manner.

#3140

Infiltrating macrophage-derived TNF-α promotes PD-L1 expression, leading to poor prognosis of patients with pancreatic cancer.

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

Background: Pancreatic cancer is the eighth leading cause of cancer death in men and the ninth leading cause of cancer death in women worldwide. Solid tumors comprise not only malignant cells but also many other non-malignant cell types. Many leukocytes, including macrophages, are known to exist in tumor tissues, and together with fibroblasts and vascular endothelial cells, constitute the tumor microenvironment. Cancer immunotherapy using immune checkpoint blockades, such as anti-PD-1/PD-L1 antibody drugs has received considerable attention in recent decades. Importantly, previous studies reported that patients with pancreatic ductal adenocarcinoma (PDAC) harboring PD-L1-positive tumor cells showed significantly poorer prognosis than those harboring PD-L1-negative tumor cells. However, the molecular mechanism underlying PD-L1 expression in PDAC cells has not been clearly elucidated. We examined whether infiltrating macrophages in tumor stroma could affect PD-L1 expression in PDAC cells.

Methods: PDAC cells were treated with several cytokines, including tumor necrosis factor (TNF)-α, and then subjected to real-time PCR, Western blot analysis and flow cytometry analyses. On the other hand, PDAC cells were co-cultured with human monocyte-derived macrophages, and then subjected to real-time PCR. Subsequently, immunohistochemistry analyses were performed on consecutive paraffin sections from 122 patients with PDAC using anti-PD-L1 and anti-CD163 antibodies. In addition, double immunostaining was performed on the same paraffin sections from patients with PDAC using anti-PD-L1 and anti-Iba-1 antibodies. Finally, survival analysis was conducted.

Results: Among various cytokines tested (interleukin (IL)-1a, IL-1b, IL-4, IL-6, IL-7, IL-13, IL-24, TNF-α, IFN-γ or LPS), TNF-α promoted strong PD-L1 expression in PDAC cell lines (S2-013 and MIAPaCa2). Western blot analysis showed that TNF-α up-regulated the expression level of PD-L1 via the NF-κB pathway in PDAC cells. In addition, PD-L1 expression was increased in PDAC cells co-cultured with activated macrophages derived from human monocytes, and this up-regulation was inhibited by the anti-TNF-α antibody. Furthermore, a significant correlation between PD-L1 expression in cancer cells and the number of infiltrating macrophages expressing CD163 in PDAC tissues was observed by immunohistochemistry analysis. Finally, survival analysis revealed that PD-L1 expression was significantly associated with poor prognosis in patients with PDAC.

Conclusion: TNF-α derived from tumor-infiltrating macrophages enhances PD-L1 expression in PDAC cells, which may lead to poor prognosis in patients with PDAC.

#3141

Immune gene expression and prognosis in localized clear cell (cc) renal cell carcinoma (RCC).

Pooja Ghatalia,1 Karthik Devarajan,1 Jennifer Gordetsky,2 Essel Dulaimi,1 Sejong Bae,2 Gurudatta Naik,2 Guru Sonpavde,3 Elizabeth Plimack1. 1 _Fox Chase Cancer Center, Philadelphia, PA;_ 2 _University of Alabama at Birmingham, Birmingham, AL;_ 3 _Dana-Farber Cancer Institute, Boston, MA_.

Background: We previously reported that increased infiltration of immune cells (lymphocytes, plasma cells, macrophages, neutrophils) in localized ccRCC is associated with recurrence. In these tumors we now study the immune gene expression to identify a signature to predict recurrence.

Methods: We identified pts with ccRCC with >T1b tumor who underwent nephrectomy for whom we had annotation for objective tumor recurrence and a minimum follow-up of 2 years. All histologic slides were examined and tumor section with the highest amount of intratumoral immune infiltration were selected from each tumor for gene expression profiling. Total mRNA isolated from the macrodissected FFPE tumor underwent gene expression profiling for 770 genes in the Immune Profile Panel by NanoString Technologies. Digital raw counts of mRNA abundance were normalized using positive controls as well as 16 housekeeping genes. The mean signals were used to calculate fold change in gene expression between recurrers and non-recurrers, and a p-value <0.05 using empirical Bayes methods was considered significant. Cox regression was also conducted to correlate gene expression with time to recurrence.

Results: Of 132 patients (pts), 24 (18%) had recurrence and 108 did not. The median age of pts was 59 years and 57 were female. The median time to recurrence was 25.7 months. Fifty pts had low immune infiltration score and 82 pts had high infiltration based on previously conducted morphologic assessment. Only 4 genes (IL8, NCAM1, ARG2, PPBP) had >1.5 times increased mean expression in recurrers vs non-recurrers (p<0.05). Only 5 genes (CX3CL1, HLA-G, VCAM1, IL17RB, CXCL14) had >1.5 times decreased mean expression in recurrers vs non-recurrers (p<0.05). Overexpression of the following immunoregulatory genes was associated with a shorter time to recurrence: TGFB1, STAT3, NFATC4, B7-H3, ADORA2A, OX-40L, IL10, RUNX1, LILRA4, MARCO, CR1, FCER2, IL2RA (HR 1.5-3.6, p<0.05). When compared to pts with low lymphocyte infiltration, pts with high lymphocyte infiltration overexpressed T cell (CD3E, CD5), cytotoxic T cell (CD8B, SLAMF6, IL2RB, GZMK, TCF7, CTSW, EBI3), Th1 (CD38, CXCR3, CXCR6, IL16), Th2 (ITK) and Tregs (IL2RB, IRF4, TIGIT) genes (FDR p<0.01).

Conclusions: Specific immune related somatic genes were differentially expressed based on recurrence and time to recurrence in patients with localized ccRCC undergoing surgery. With further validation, these genes warrant functional validation since they may represent therapeutic targets in the perioperative setting. The results also highlight the heterogeneity of immune cell infiltration.

#3142

Tumor-infiltrating lymphocytes and CD4/FOXP3 ratios reliably predict survival using digital image analysis.

Stephanie G. Craig, Pete Bankhead, Victoria Bingham, Stephen McQuaid, Maria Tumelty, Simon S. McDade, Manuel Salto-Tellez, Jacqueline A. James. _Queens University Belfast, Belfast, United Kingdom_.

Background:Tumour infiltrating lymphocytes (TILs) have been associated with improved prognosis in cancer, including oropharyngeal squamous cell carcinoma (OPSCC); however, the survival benefit appears modest and favours HPV positive OPSCC. The current literature presents a mixed view on the best method to score TILs and if it would be beneficial to score all cancers of the head and neck. Use of immunotherapies to stimulate immune cell activation is increasing in the clinic and as such a robust method of assessment is needed if we want to apply this routinely to OPSCC. This study investigates the prognostic value of assessing TILs by H&E in HNSCC using digital pathology solutions against human eye visual assessment and by comparing TIL estimates in H&Es against markers of adaptive immunity (CD3, CD4,CD8 and FOXP3) using digital image analysis.

Methods:This study utilised 190 HNSCC arising from the oropharynx. TMAs constructed, in triplicate, from these samples were stained with either H&E or IHC for CD3, CD4, CD8 and FOXP3. Visual estimates of TIL counts by human eye from H&E stained slides were assessed on the whole core at x10 magnification based on published thresholds by two observers to form a consensus score. Image analysis was performed on whole cores using image analysis software QuPath on H&E and IHC stained slides. HPV status was determined by p16 IHC and high risk HPV RNAScope. Statistical analyses were performed using R.

Results:Scoring of TILs in H&Es by visual estimates was prone to misestimation and reduced survival estimates when using predetermined thresholds compared to machine scoring by image analysis. Comparison of digital pathology solutions assessing the TIL population in H&E and IHC stained slides found the percentage of TILs present to be significantly higher in the HPV positive's compared to the HPV negative's in the H&E, CD3 and CD8 immunostained slides but not CD4 or FOXP3. When dichotomised into high and low TILs only H&E based TIL counts and CD4/FOXP3 ratios were found to be independently predictive of survival when adjusted for age and smoking history.

Conclusion:Image analysis creates continuous variables from which patient TIL counts can be reliably assessed. The adaptive immune landscape of HPV negative OPSCC is significantly different to HPV positive OPSCC of which only total TIL counts (H&E) and CD4/FOXP3 ratios appears to be predictive of five-year overall survival.

#3143

A novel approach to deplete Treg cells using non-IL-2 blocking anti-CD25-targeting antibodies leads to complete rejection of established tumors.

Isabelle Solomon,1 Frederick Arce Vargas,1 Dimitrios Zervas,1 Chen Qing,1 Josephine Salimu,2 Mark Brown,2 Pascal Merchiers,2 Aghiles Boughetane,2 Karl S. Peggs,1 Anne Goubier,2 Sergio A. Quezada1. 1 _UCL Cancer Institute, London, United Kingdom;_ 2 _TUSK Therapeutics, Stevenage, United Kingdom_.

The accumulation of regulatory T cells (Tregs) in the tumor hampers effector anti-tumor activity and correlates with a bad prognosis in several human cancers. Increasing the effector T cell (Teff) to regulatory T cell (Treg) ratio is known to result in improved control of established tumors. Studies demonstrating high levels of CD25 expression on Tregs but not Teff in human tumors have underscored its relevance as a target for Treg depletion. This supported the development of anti-CD25 depleting monoclonal antibodies (mAbs) as a promising monotherapy as well as combination partner in cancer immunotherapies. To date, anti-CD25 antibodies for clinical use have been designed to deplete CD25 positive cells but also to block IL-2 binding and signaling. Since IL-2 is a critical cytokine involved in T cell activation and proliferation, we hypothesized that a depleting antibody targeting CD25 on Treg cells but not blocking IL-2 signaling on Teff cells would induce a more potent anti-tumor response by depleting Tregs whilst still allowing IL-2 to stimulate effector T cells. We therefore compared the functional activity of anti-mouse CD25 depleting mAbs with and without IL-2 blocking activity. After having confirmed their differential impact on IL-2 signaling, we evaluated the therapeutic activity of these mAbs in various syngeneic tumor models. While both the IL-2 blocking and non-IL-2 blocking mAbs showed equivalent Treg-depleting activity, the antibody sparing IL-2 signaling promoted stronger anti-tumor effect than the IL-2 blocking mAb, with complete tumor regression observed in 70-100% of the mice after a single administration of the antibody. Our data demonstrates that targeting CD25 with ADCC enabled antibodies preserving IL-2 signaling is a novel and powerful strategy for rejection of established tumors via depletion of Treg cells and enhanced, cell intrinsic, IL-2-driven effector T cell activation.

#3144

Active CD11b integrin signaling in tumor associated macrophages inhibits tumor growth in mice.

Anugraha Rajagopalan,1 Terese Geraghty,1 Samia Khan,1 Roberto Vazquez-Padron,2 Vineet Gupta1. 1 _Rush University Medical Center, Chicago, IL;_ 2 _University of Miami, Chicago, FL_.

Tumor associated macrophages (TAMs) are polarized to a tumor promoting state (M2 TAMs) in the tumor microenvironment. These M2 TAMs can effectively suppress the anti-tumor immune response and promote angiogenesis. Increased abundance of M2 TAMs in solid tumors are associated with poor prognosis. Activation of CD11b, an integrin molecule expressed by the M2 TAMs converts them from a tumor promoting state to a tumor inhibiting state. A single point mutation that changes an invariable Isoleucine at position 332 to Glycine (CD11bI332G) leads to constitutive activation, by maintaining the integrin in the active conformation. Expression of CD11bI332G in bone marrow derived macrophages from CD11b-/- mice by lentiviral transduction reprogrammed these macrophages from a pro-tumor to an anti-tumor state. This reprogramming of TAMs led to significant reduction in tumor growth in mice. This study highlights the role of CD11b integrin signaling in regulating immune responses against cancer and provides a rationale to use CD11b agonists to elicit a potent anti-tumor immune response.

#3145

Characterization of novel immune checkpoint receptors within the breast cancer tumor microenvironment.

Austin D. Williams,1 Jean S. Campbell,2 Lauri D. Aicher,2 Kyle K. Payne,3 Jose R. Conejo-Garcia,4 Robert H. Pierce,2 Julia Tchou1. 1 _University of Pennsylvania, Perlman School of Med, Philadelphia, PA;_ 2 _Fred Hutch Cancer Research Center, Seattle, WA;_ 3 _Wistar Institute, Philadelphia, PA;_ 4 _Moffit Cancer Center, Tampa, FL_.

Introduction. The interruption of immune checkpoint receptor (ICR)/ligand interaction within the tumor microenvironment has become an important immunotherapy strategy. In breast cancer (BC), neither the ICR phenotype of immune cells of each BC subtype nor the associated immune response is fully characterized. We hypothesize that there are differences in ICR phenotypes between BC subtypes. Methods. To analyze the expression of ICRs (i.e. PDCD1 (PD-1), TIGIT, HAVCR2 (Tim-3), and LAG3 in the tumor microenvironment (TME) of BC, the 956- patient BC cohort of The Cancer Genome Atlas (TCGA) gene expression data was analyzed and normalized against CD3E, a T-cell specific transcript. As validation, we examined various ICR by flow cytometry on 14 matched dissociated breast tumor and peripheral blood mononuclear cells (PBMC) and compared differences across BC subtypes with t-test and ANOVA. In addition, differential gene expression and multispectral analysis were investigated using FFPE specimens and the NanoString and Vectra platforms. Results. TCGA data revealed a higher expression of TIGIT, LAG3 and PD-1 in triple-negative breast cancer (TNBC) when compared to other subtypes (p <0.001, Table 1). PD-L1 (CD274) expression was higher in TNBC than ER/PR+/Her2- cancer (p=-0.008). On flow cytometry, there was a higher co-expression of TIGIT and Tim-3 on CD4 T cells in TNBC than all other subtypes (p=0.003) while ER/PR+Her2- cancers had the highest CD4 expression of TIGIT alone (p=0.04). Compared to ER/PR+Her2- cancers, TNBC had increased expression of transcripts associated with myeloid derived suppressor cells, and decreased expression of transcription factors that regulate lineage specification for CD4 helper T cells. Conclusions. In addition to PD-1, we demonstrated that other ICRs may play significant roles in TME of Her2+ and TNBC BC subtypes. Our results support the development of novel anti-ICR therapy which may synergize with current treatment strategies against these unfavorable BC subtypes.

#3146

Versican proteolysis: A novel immunostimulatory component of CD8+ T cell responses to colorectal cancer.

Philip B. Emmerich, Susan N. Payne, Connor J. Maloney, Rosabella T. Pitera, Hanna Rainiero, Gioia Sha, Athanasios T. Papadas, Adam C. Pagenkopf, Michael F. Bassetti, Kristina A. Matkowskyj, Fotis Asimakopoulos, Dustin A. Deming. _Univ. of Wisconsin Carbone Comp. Cancer Ctr., WI_.

Background: Infiltration of CD8+ T cells has shown important prognostic implications in colorectal cancers (CRC). Our group has recently correlated proteolysis of an extracellular matrix proteoglycan, versican (VCAN), with infiltration of CRCs by CD8+ T cells. Cleavage by ADAMTS proteases generates a bioactive fragment of versican, versikine (VKINE). VKINE stimulates Batf3-driven dendritic cell activation and maturation, which are critical for immunotherapy efficacy.

Methods: CT26 murine CRC cells were transfected to constitutively express VKINE. BALB/c mice between 50 and 70 days of age were orthotopically injected with either 105 empty vector (CT26-EV) or VKINE-expressing (CT26-VKINE) cells. Tumor growth was tracked weekly with colonoscopy and growth rate assessed using percent lumen occlusion. Mice were euthanized and dissected once moribund. Tissues were formalin-fixed and paraffin-embedded (FFPE). Immunohistochemical (IHC) staining was performed using standard procedures. Additionally, FFPE samples from human CRC liver metastases (n=9) were obtained under the University of Wisconsin Translational Science Biocore Institutional Review Board-approved protocol and IHC performed for VCAN, VKINE and CD8. The number of CD8+ tumor-infiltrating lymphocytes (TILs) per high-powered field (HPF) within the malignant epithelium was calculated using four separate 400x magnification fields of view.

Results: CT26-EV and CT26-VKINE injected mice both had a ~70% tumor initiation rate and these tumors averaged ~50% lumen occlusion by 14 days post injection. Survival data for each group were compared using a log-rank test. A trend towards improved survival was observed in those mice injected with CT26-VKINE (average survival of 27 days for CT26-VKINE compared to 22 days for CT26-EV, p=0.41, n=13). The average number of CD8+ TILs for each tumor were compared. For mice with CT26-VKINE, the average number of CD8+ TILs per HPF was 7.6, compared to 4.7 for mice with CT26-EV (p=0.39).

Human CRC liver metastases were analyzed for VCAN, VKINE, and CD8+ T cells. Several lesions had little to no VCAN staining, whereas others had intense pockets or peripheral staining. VKINE staining was more abundant in metastatic CRC lesions than in primary lesions. CD8+ T cells were excluded in the setting of increased VCAN staining and infiltration was noted specifically in areas of VCAN proteolysis (low VCAN and high VKINE staining). These areas of proteolysis were more often present at the tumor margin and correlated with ADAMTS1 expression.

Conclusions: Immunotherapies offer a unique treatment option for cancer patients, but their use is limited in CRC. VCAN proteolysis and the generation of bioactive VKINE has potential to influence the ability of CD8+ T cells to infiltrate the tumor in both primary and metastatic CRC, indicating the potential to utilize VCAN and VKINE as immune biomarkers or therapeutic targets.

#3147

Adipocytes affect castration-resistant prostate cancer cells in resistance to cytotoxic action of NK cells through alterations of pd-l1/nkg2d ligand levels.

Lijun Xu,1 Mingjing Shen,1 Xiaodong Chen,2 Rongying Zhu,2 Dong-rong Yang,1 Ying Tsai,2 Peter Keng,2 Yuhchyau Chen,2 Soo Ok Lee2. 1 _Soochow Hospital, Jiangsu Sheng, China;_ 2 _Univ. of Rochester Medical Ctr., Rochester, NY_.

Background: One-third of US population is obese. The mortality of obese prostate cancer (PCa) patients is higher than non-obese patients, thus studying molecular mechanism of how obesity affects PCa progression has clinical relevance. The periprostatic adipose tissue adjacent to the prostate is considered a driving force of disease progression and adipocytes are the main cell population in adipose tissues. The paracrine role of adipocytes in PCa progression has been emphasized, but its implication in modulating immune reactions remains unknown. We investigated the role of adipocytes in the surveillance of castration-resistant PCa (CRPC) cells to immune actions. Especially we investigated the role of adipocytes in affecting tumor cell susceptibility to cytotoxic action of natural killer (NK) cells, as the strategy of improving the NK cell-mediated immunity is emerging as an immunotherapeutic option.

Methods: Using primary NK cells as the NK cell source, NK cell cytotoxicities to CRPC cells, either control media treated or adipocyte-conditioned media (CM) treated, were tested in lactate dehydrogenase (LDH) release-based assays. The levels of programmed death receptor ligand (PD-L1) and NK group 2D (NKG2D) ligands in adipocyte CM-treated CRPC cells were analyzed in qPCR analyses. Effects of blocking adipocyte action on altering PD-L1/NKG2D ligand levels and the susceptibility of CRPC cells to NK cell cytotoxicity were investigated.

Results: We found NK cell cytotoxicity to CRPC cells decreased when tumor cells were treated with adipocyte CM, and was associated with PD-L1 (up-regulation) and NKG2D ligand level (down-regulation) alterations. Further, we discovered that the JAK/Stat3 signaling pathway was activated by adipocyte CM. Two adipokine molecules, IL-6 and leptin, were shown to be important in the activation of the JAK/Stat3 signaling to modulate the PD-L1/NKG2D ligand level alterations in CRPC cells. Consequently, adding inhibitors of JAK/Stat3 signaling or neutralizing antibodies of IL-6 or leptin increased the susceptibility of CRPC cells to NK cell cytotoxic action.

Conclusions and Impact: Results from our study showed the influence of adipocytes on tumor cells and resulted in reducing the susceptibility to NK cell-mediated antitumor immune function. This may be one of ways that adipocytes trigger CRPC cells to be more aggressive and out of immune surveillance control. Our study results showed that blocking the adipocyte effect by inhibiting the IL-6/leptin-JAK/Stat3 signaling axis enhanced the NK cell mediated immunity to CRPC cells. We suggest that this strategy can be applied to the development of future immune therapeutics to treat obese PCa patients.

#3148

p140Cap affects breast cancer features through the regulation of the tumor microenvironment composition.

Vincenzo Salemme. _University of Turin, Turin, Italy_.

Increasing data indicate the key role of the tumor microenvironment in cancer progression, metastasis formation and treatment sensitivity, where the tumor stroma and the immune infiltrate strongly influence tumor features. Our recent studies indicate that the adaptor protein p140Cap is highly expressed in about 50% of ERBB2-amplified breast cancer patients. Its expression correlates with increased patient survival and decreased probability to develop metastasis. Causally, p140Cap negatively regulates breast tumor properties both in vitro and in the NeuT preclinical mouse model. In this study, we observed an indirect influence of p140Cap-expression on tumor immune infiltrate composition of a syngeneic ERBB2+ breast cancer model. p140Cap tumor cells and tumors show reduced level of G-CSF mRNA and protein. Accordingly, we observed a reduced mobilization of G-CSF-dependent myeloid-derived suppressor cells (MDSCs- CD11b+Ly6G+Ly6Clo) from bone marrow and a decreased level of MDSCs in tumor, blood and spleen of mice orthotopically injected with p140Cap tumor cells, compared to controls. This "less" immune suppressive microenvironment associated to p140Cap-expression, was also confirmed by an increase in CD8+, M1 macrophages and Natural Killer lymphocytes concomitant with a decrease in M2 macrophages and FoxP3 regulatory T cells. Interestingly, the expression of p140Cap in cells and tumors significantly reduce the fraction of cancer stem cells and their sphere and tumor initiation frequency, by modulating their proliferative potential. Since that the tumor initiating cell exhibit an enhanced G-CSF production, these data suggest that p140Cap, by regulating the cancer stem cell compartment, limits ERBB2 tumor aggressiveness by creating a tumor microenvironment less favorable to tumor progression. 

### Metastasis, Invasion, and Migration 1

#3149

Collagen XIII induces β1-integrin activation and triple-negative breast cancer metastasis.

Hui Zhang, Tricia Fredericks, Ren Xu. _University of Kentucky, Lexington, KY_.

Increased collagen expression and deposition is a risk factor for breast cancer development. Collagen XIII is a type II transmembrane protein within the collagen superfamily. Experiments in tissue culture and knockout mouse model show that collagen XIII is involved in cell adhesion and differentiation of certain cell types. However, function of collagen XIII in cancer development and progression largely remains to be determined. We found that collagen XIII expression is significantly induced during breast cancer development, especially in triple negative breast cancer. Increased collagen XIII mRNA level is associated with poor prognosis. Importantly, silencing collagen XIII expression in MDA MB 231 cells slow down xenograft growth and significantly reduced cancer metastasis in mice. Using gain- and loss-of function approaches, we showed that collagen XIII enhanced cell migration, invasion, and mammosphere/tumorsphere formation. We also found that collagen XIII induced integrin-β1 activation, and subsequently enhanced TGF-β signaling. Blocking integrin-β1 activation with AIIB2 antibody significantly reduced collagen XIII-induced cell migration and mammosphere formation. These findings demonstrate a novel function of collagen XIII in promoting cancer metastasis through enhancing β1 integrin-dependent cell migration and invasion

#3150

Serglycin promotes cell migration mediated through the interaction of its GAG moiety with CD44.

Joanne Jeou-Yuan Chen, Jing-You Guo, Chu-Hsuan Chiu, Mei-June Wang. _Academia Sinica - Institute of Biomedical Sciences, Taipei, Taiwan_.

Tumor microenvironment (TME) plays an important role in cancer progression. We have recently shown that serglycin (SRGN), a chondroitin sulfate proteoglycan (CSPG), is overexpressed in primary non-small cell lung cancers (NSCLCs), by carcinoma as well as stromal cells, and that the secreted SRGN can bind to CD44 and promotes NSCLC aggressiveness (Oncogene 36:2457, 2007). In this study, we further examined the molecular mechanisms underlying SRGN-elicited cell migration. By gain- and loss-of-function studies, we first showed that SRGN promoted cell migration in a CD44-dependent manner. SRGN promoted SRC activation, leading to increased phosphorylation of paxillin (PAX) at Y118 as well as reduced PAX/FAK complex formation, suggesting that SRGN promotes cell migration through facilitating focal adhesion turnover. In support, knockdown of SRC or treatment of cells with PP2, a SRC family inhibitor, abolished SRGN-elicited migratory activity. In addition, we showed that SRGN induced actin cytoskeleton reorganization and promoted lamellipodia and filopodia formation at the leading edge, facilitating a directional movement during wound closure in NSCLC cells. In consistence, increased levels of activated RAC1, which is required for lamellipodia formation, and CDC42, which is required for filopodia formation, were detected. We further examined the role of CS modification in SRGN-mediated function. We generated the SRGN(S/A) mutant in which the eight serine residues involved in GAG modification were converted to alanine, and showed that SRGN(S/A) mutant not only failed to induce the phosphorylation of SRC and paxillin, but also failed to promote cell migration. In consistence, pre-treatment of SRGN-containing conditional medium with Chondroitinase ABC also suppressed SRGN-elicited cell migration. Taken together, our data demonstrated that SRGN interacts with CD44 through its GAG modifications, leading to increased cell migration by promoting FA turnover and cytoskeleton reorganization.

#3151

Novel regulation of cancer cell migration by ABCG2-enhanced autophagy.

Kalkal Anil Trivedi, Rui Ding, Kathleen W. Scotto. _Rutgers University, New Brunswick, NJ_.

ABCG2 is an ABC multidrug transporter best known for its ability to efflux a wide variety of chemotherapeutic agents, resulting in tumor drug resistance. More recently, our laboratory has shown that ABCG2 plays an additional role in tumor cell survival by enhancing the rate of autophagy, a conserved, catabolic recycling process that breaks down damaged proteins and cellular organelles to form cellular building blocks (Ding et al., Autophagy, 2016).

Sporadic studies over the decades have led to somewhat conflicting reports about the impact of ABCG2 expression on migration. Likewise, autophagy can play either a pro-metastatic or anti-metastatic role depending on the context. In the present study, we confirm that ABCG2 enhances tumor cell migration in vitro, and provide novel data to suggest that this enhancement is due to the ability of ABCG2 to augment autophagy flux.

Two cell line pairs were used for these studies: the NSCLC line H460 and its drug-selected ABCG2-overexpressing subline H460/MX20, and the embryonic kidney HEK293 cells stably-transfected either with ABCG2 or an empty vector. Transwell assays were used to study in vitro migration and invasion. ABCG2-overexpressing cells exhibited a marked increase in both cell migration and invasion relative to their parental counterparts. Using gelatin zymography, we also showed that ABCG2 over-expressing cells had enhanced MMP-2 activity, consistent with increased invasion. Both siRNA-mediated knockdown of ABCG2 expression and pharmacological inhibition of ABCG2 transport reduced the migration of ABCG2-overexpressing cells. Moreover, blocking autophagy using either a pharmacological or genetic approach also inhibited migration. Further supporting the hypothesis that ABCG2-enhanced autophagy influenced tumor cell migration, wild-type (WT) and MEFs in which the autophagy essential gene ATG5 was knocked out (ATG5-/-) were transiently transfected with either an empty vector or ABCG2. The ABCG2-transfected WT MEFs showed significantly enhanced migration compared to the empty vector transfected WT MEFs. The autophagy compromised ATG5-/- MEFs, however, did not display a statistically significant difference in the migration of the ABCG2 and empty-vector transfected cells, supporting the hypothesis that ABCG2 requires functional autophagy to enhance cell migration. Taken together, our results demonstrate a novel role for ABCG2 in enhancing cell migration by augmenting autophagy flux. Studies to determine whether ABCG2-regulated autophagy enhances migration and metastasis in vivo are underway.

#3152

RhoA-myosinII axis protects circulating tumor cells from fluid shear stress-induced damage.

Devon L. Moose, Ben L. Krog, Gretchen Burke, Lei Zhao, Lillian Rhodes, Michael D. Henry. _University of Iowa, Iowa City, IA_.

Circulating tumor cells (CTCs) are exposed to hemodynamic forces, which have long been thought to be mechanically destructive to CTCs. However, recent studies show that cancer cell lines from diverse histologies exhibit resistance to brief (millisecond) pulses of high-level (750-6400 dyn/cm2) fluid shear stress (FSS), whereas nontransformed epithelial cells are sensitive to this mechanical insult (PMID: 23226552, 26447202). Moreover, exposure of cancer cells to FSS results in cortical stiffening (PMID: 25908902). Herein, we elucidate the mechanism of FSS resistance in cancer cells, and extend these findings to experimental models of CTCs. We show that although some cancer cell lines exhibit elevated levels of membrane repair relative to nontransformed counterparts, intrinsic resistance to plasma membrane damage is a more consistent feature distinguishing cancer cells. FSS resistance is detectable in cancer cells acutely isolated from primary mouse TP53/PTEN mutant prostate tumors, not just a feature of cultured cancer cell lines. Our findings indicate that cancer cells respond to FSS by activation of RhoA-myosinII contractility, which protects them from nanometer-scale damage to the plasma membrane. Moreover, we present evidence that the RhoA myosinII axis protects CTCs from mechanical damage in animal models. Treatment of PC-3 cancer cells with a nontoxic dose of the myosin II inhibitor blebbistatin (20μM; 3h) reduced the number of intact cells arrested in the lung microvasculature immediately following tail vein injection. Additionally, treatment of mice bearing orthotopically implanted, metastatic PC-3 derived prostate tumors with blebbistatin (2.5mg/kg; 3h) acutely reduced steady-state CTC levels by approximately 10-fold. Taken together, our data indicate that viable CTCs actively resist destruction by hemodynamic forces and are likely to be more mechanically robust than is commonly thought.

#3154

Harnessing the adaptive potential of mechanoresponsive proteins to overwhelm pancreatic cancer dissemination and invasion.

Alexandra Surcel,1 Eric S. Schiffhauer,1 Dustin G. Thomas,1 Qingfeng Zhu,1 Kathleen DiNapoli,1 Maik Herbig,2 Oliver Otto,2 Jochen Guck,2 Elizabeth Jaffee,1 Pablo A. Iglesias,3 Robert A. Anders,1 Douglas N. Robinson1. 1 _Johns Hopkins Univ. School of Medicine, Baltimore, MD;_ 2 _TU Dresden, Dresden, Germany;_ 3 _Johns Hopkins University Whiting School of Engineering, Baltimore, MD_.

Metastatic disease is often characterized by altered cellular contractility and deformability, lending cells and groups of cells the flexibility to navigate through different microenvironments. This ability to change cell shape is driven in large part by the structural elements of the mechanobiome, which includes cytoskeletal proteins that sense and respond to mechanical stimuli. Here, we demonstrate that key mechanoresponsive proteins (those that accumulate in response to mechanical stress), specifically nonmuscle myosin IIA and IIC, α- actinin 4, and filamin B, are highly upregulated in pancreatic ductal adenocarcinoma cancer (PDAC) and in patient-derived pancreatic cancer cell lines. Their less responsive sister paralogs (myosin IIB, α-actinin 1, and filamin A) show a smaller dynamic range or disappear with PDAC progression. We demonstrate that these mechanoresponsive proteins directly impact cell mechanics using knockdown and overexpression cell lines. We further quantify the nonmuscle myosin II family members in patient-derived cell lines and identify a role for myosin IIC in the formation of transverse actin arcs in single cells and cortical actin belts in tissue spheroids. We harness the upregulation of myosin IIC and its impact of cytoskeletal architecture through the use of the mechanical modulator 4-hydroxyacetophenone (4-HAP), which increases myosin IIC assembly and stiffens cells. Here, 4-HAP decreases dissemination, induces cortical actin belts, and slows retrograde actin flow in spheroids. Finally, mice having undergone hemisplenectomies with PDAC cells and then treated with 4-HAP have a reduction in liver metastases. Thus, increasing the activity of these mechanoresponsive proteins (in this case, by increasing myosin IIC assembly) to overwhelm the ability of cells to polarize and invade may be an effective strategy to improve the five-year survival rate of pancreatic cancer patients, currently hovering around 6%.

#3155

Loss of YAP1 dysregulates RhoA signaling and promotes cell invasion.

Natalie M. Hendrick, Marcin P. Iwanicki, Carman Man-Chung Li, Duyen Amy Bui, Laura M. Selfors, Joan S. Brugge. _Harvard Medical School, Boston, MA_.

While Hippo pathway signaling undoubtedly plays an important role in cancer, the role of its effector YAP1 as an oncogene or tumor suppressor is context dependent. The precise conditions that support the tumor-promoting or tumor-suppressing roles of YAP1 remain largely undefined. While YAP1 can drive oncogenic programs through its nuclear function as a transcriptional coactivator of TEAD and other transcription factors, previous findings from our lab and others suggest that loss of YAP1 function can also have tumor promoting consequences. One well-characterized role of YAP1 is that of a nuclear relay of signals triggered by mechanical forces that lead to activation of Rho GTPases. Our new studies using a combination of genetic, biochemical, and microscopy techniques provide evidence that YAP1 is not only activated by Rho, but can also regulate Rho activation and the actin cytoskeleton. Knockdown of YAP1 in MCF10A immortalized mammary epithelial cells resulted in increased lamellipodia formation in culture as seen by time-lapse microscopy, and increased protrusive, invasive activity in 3D Matrigel culture. YAP1 knockdown also induced a scattering phenotype, characterized by cell dissociation from the leading edge of a wounded monolayer. While E-cadherin protein levels were unaffected by YAP1 knockdown, adherens junction maturation at cell-cell junctions was compromised. These morphological alterations were associated with decreased levels of active RhoA, as well as a decrease in downstream myosin light chain phosphorylation. Our results indicate loss of YAP1 decreases active RhoA, leading to reduced cell-cell adhesion and increased protrusive cellular behavior. Our findings raise the possibility that YAP1 is not only a Rho-sensor, but also mediates a feedforward regulatory loop to maintain Rho-regulated cytoskeletal structures, and that loss of YAP1 through genetic deletion in tumor cells could promote invasive behavior.

#3156

C1orf116, an uncharacterized gene with unknown function, restricts movement capacity of would-be metastatic cells through modulation of tight junctions.

Sarah R. Amend, Kenneth J. Pienta. _Johns Hopkins Univ., Baltimore, MD_.

Lethal metastases are the end-result of a cancer cell that escapes the primary tumor and eventually invades a secondary site. Thus, in order for a cancer cell to metastasize from the primary tumor, it must move. In a critical early event of metastasis, a stationary epithelial cell undergoes a dramatic transmogrification event to gain mesenchymal-like features, most notably, ability to move. The full repertoire of genes regulating this switching event and inducing potential metastatic cell movement in the primary tumor remains incomplete.

Using a multi-study discovery analysis (doi: 10.1186/s12885-017-3413-3), we identified C1orf116 as a novel candidate driver of an epithelial phenotype. C1orf116 is a highly conserved unnamed gene of unknown function. It is highly expressed in proliferative tissues (e.g. skin, lung, esophagus; GTeX) and is associated with an epithelial gene expression signature in cancer cell lines (CCLE).

To determine if C1orf116 is a driver of epithelial phenotype, we overexpressed C1orf116 in C1orf116low PC3 cells (PC3-oe). PC3-oe demonstrated distinctive cobblestone epithelial cellular morphology and increased epithelial marker expression (e.g. OVOL1, CDH1, ESRP). Knockdown of C1orf116 expression in C1orf116high C42b cells (C42b-kd) resulted in decreased expression of classical epithelial markers. This data indicates that C1orf116 was sufficient to induce an epithelial transition.

The essential characteristic of a lethal emigrant cell that may give rise to a clinical metastasis is gaining the capacity to move. We found that PC3-oe cultures had significantly reduced motility compared to control, suggesting that C1orf116 restricts motility. Cellular motility is influenced by many factors, including cell-cell adhesion. We observed that PC-oe established cell-cell contacts more rapidly than control cells and hypothesized that C1orf116 promoted cell-cell adhesion. PC3-oe cells had dramatically increased expression of tight junction complex proteins (e.g. ZO-1, ZO2, CLDN1). In contrast, C42b-kd cells had reduced expression of these tight junction components.

These data demonstrate that C1orf116 promotes tight junctions and inhibits cell movement, thereby restricting the active emigrant phenotype of potentially metastatic cells. The putative metastasis suppressor role of C1orf116 is further substantiated by clinical data. C1orf116 expression is reduced in radical prostatectomy specimens from patients who progress with a biochemical recurrence compared to those who do not recur. Similarly, C1orf116 expression is decreased in prostate cancer metastatic lesions compared to primary tumor.

This work shows the critical role of the overlooked gene and protein C1orf116. Further knowledge of how movement is restricted in the primary tumor provides valuable insight into the lethal metastatic process.

#3157

p53 inactivation and STAT2 cooperate to enhance migration and metastasis of colon tumor cells.

Ana M. Gamero, Kevin Kotredes, Aliza Abezis, Sruthi R. Gohimukkula, Alexandra Afanassiev, Tess Cremers, John J. Swain. _Temple University, Philadelphia, PA_.

STAT2 is a pivotal signaling component in the antiviral and antiproliferative effects of type I and type III interferon (IFN), but its role in cancer remains understudied. To date, only IFNs are known to activate STAT2. Work by us and others has demonstrated that STAT2 deficiency impairs the antitumor effects of type I IFN, supporting its role in suppressing tumor growth. With the availability of Stat2-/- mice, we interrogated the tumor-suppressive role of STAT2 by employing the AOM/DSS model of colitis-associated cancer and the spontaneous ApcMin/+ model of colorectal cancer (CRC). In both cancer models, loss of STAT2 reduced the number of adenomas, showing that under specific conditions STAT2 can be tumorigenic. Next, immunohistochemical staining of human normal colon and CRC tumors revealed elevated STAT2 protein in both adenomas and adenocarcinomas, suggesting that STAT2 is implicated in human CRC. Conversion of adenomas to adenocarcinomas is linked to loss of p53 function, which occurs in >50% of colorectal cancers. Therefore, we evaluated whether STAT2 altered the aggressive phenotype of p53-deficient colon carcinoma cells. We silenced STAT2 in isogenic p53 null and p53 wild-type tumor cells. STAT2 knockdown in p53 null colon carcinoma cells reduced migration of 3D tumor spheroids and tumor colony formation while no effect was seen in p53 wild-type tumor cells. We also observed that STAT2 knockdown in p53 null tumor cells increased protein levels of the marker of epithelial-mesenchymal transition, E-cadherin. Conversely, overexpression of STAT2 in p53 null cells reduced E-cadherin protein. In vivo, STAT2 knockdown delayed subcutaneous tumor growth of p53 null tumor cells. Furthermore, using an experimental liver metastasis tumor model, STAT2 knockdown abrogated the formation of tumor nodules in the liver. RNA-seq profiling of subcutaneous p53 null tumors revealed a subset of genes that were differentially expressed in a STAT2-dependent manner. Clinically, analysis of the TCGA database showed a STAT2 transcriptional signature in colorectal tumors that we are presently correlating with p53 alterations. Overall, we conclude that STAT2 enhances tumor growth and can contribute to disease progression by promoting migration and invasion when normal p53 function is lost.

#3158

DLL3 regulates migration and invasion of small cell lung cancer.

Megumi Furuta,1 Jun Konishi Sakakibara,1 Tetsuaki Shoji,1 Yuta Takashima,1 Hajime Kikuchi,1 Eiki Kikuchi,1 Junko Kikuchi,1 Ichiro Kinoshita,2 Hirotoshi Dosaka Akita,2 Masaharu Nishimura1. 1 _First Department of Medicine, Hokkaido University School of Medicine, Sapporo, Japan;_ 2 _Department of Medical Oncology, Hokkaido University School of Medicine, Sapporo, Japan_.

Background: Delta-like protein 3 (DLL3) is a ligand of Notch signaling, which is reported to be a tumor suppressor in small cell lung cancer (SCLC). Previous studies suggest that DLL3 might be associated with neuroendocrine tumorigenesis through inhibition of Notch signaling unlike other activating ligands. Moreover, DLL3 was highly expressed in SCLC, but not in normal lung tissue. However, little is known about function of DLL3 in SCLC. In this study, we examine the effect of DLL3 in tumorigenesis of SCLC.

Methods: The mRNA expression levels and proteins of DLL3, Notch receptors (Notch1, Notch2, Notch3 and Notch4), Hes1 and EMT markers (E-cadherin, Snail and Vimentin) were examined using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot in 9 SCLC cell lines (SBC-3, SBC-5, MS-1, RERF-LC-MA, H69, H82, H209, H529 and H1688). We used small interfering RNA (siRNA) to down-regulate the expression of DLL3 in SCLC cell lines. Anchorage-dependent and anchorage-independent cell growth was measured by MTT assay and migration and invasion were assessed by transwell assay.

Results: The mRNA of DLL3 was expressed in all of 9 SCLC cell lines. The expression of DLL3 was especially higher in H82, H69, H209, H529 and H1688 cells. Notch1 protein was expressed in SBC-3, SBC-5, MS-1 and H82. Based on DLL3 expression analysis data, we used H82 and H69 in the following experiments. The suppression of DLL3 by siRNA resulted in the moderate inhibition of cell growth of H82 cells in both anchorage-dependent and anchorage-independent cell proliferation. The depletion of DLL3 prevented migration and invasion in the two cell lines. The expressions of Notch1 and Notch target gene, Hes1 were downregulated by DLL3 knockdown in both SCLC cells. Because Notch pathway was reported to regulate EMT, we evaluated the EMT markers when DLL3 was inhibited in SCLC cells. Snail was downregulated in DLL3 knockdown SCLC cells, while protein expression levels of E-cadherin and Vimentin were not changed in these DLL3 knockdown cells compared to control cells.

Conclusions: DLL3 promotes the migration and invasion in SCLC cells by modulating Notch1 and Snail.

#3159

Rac1 and Cdc42 as drivers in ovarian cancer metastasis.

Melanie Rivera, Dayna Dominguez, Christine Pauken, Elsa Romero, S. Ray Kenney, Yang Shi, Ji-Hyun Lee, Jennifer Gillette, Laurie G. Hudson, Angela Wandinger-Ness. _University of New Mexico, Albuquerque, NM_.

Five-year ovarian cancer patient survival has not improved for decades, largely due to disease recurrence. Therefore, drivers of relapse need to be identified. We first reported that Rac1 and Cdc42 GTPases are highly overexpressed in serous ovarian cancer using immunohistochemistry. To correlate GTPase expression with survival outcomes, we analyzed gene expression data in The Cancer Genome Atlas, revealing that ovarian cancer patients with the highest RAC1 RNA levels had significantly worse age-adjusted survival than those with low RAC1. Quantitative analyses of Rac1b, a constitutively active Rac1 splice variant, show dramatic overexpression in tumor epithelia relative to stroma and normal epithelia. Since high Rac1 and Cdc42 levels drive cancer cell adhesion, invasion and metastasis, we used multiple assays to assess the dependence of ovarian cancer cell behaviors on Rac1 and Cdc42. Using Matrigel invasion chambers, treatment of multiple ovarian cancer cell lines with specific Rac1 and Cdc42 inhibitors resulted in significantly decreased cellular invasion. Conversely, with overexpression of Rac1, ovarian cancer cells demonstrated increased invasion. Furthermore, IP injection of ovarian cancer cells overexpressing Rac1 resulted in increased adhesion, invasion and metastasis in the omentum and peritoneum in vivo. These data support Rac1 and Cdc42 as important drivers of invasive and metastatic ovarian cancer and as novel high-value therapeutic targets with potential impact in reducing disease metastasis.

#3160

The actin-binding protein Girdin/GIV regulates collective cancer cell migration by controlling cell adhesion and cytoskeletal organization.

Xiaoze Wang,1 Atsushi Enomoto,1 Liang Weng,1 Hisashi Haga,2 Sumire Ishida,2 Masahide Takahashi1. 1 _Medicine School, Nagoya University, Japan;_ 2 _Faculty of Advanced Life Science, Hokkaido University, Japan_.

Pathological observations show that cancer cells frequently invade the surrounding stroma in collective groups rather than through single cell migration. The identification of genes and proteins that are specifically involved in the collective behavior of cancer cells has thus far been limited. Here, we studied the role of the actin-binding protein Girdin in collective cancer cell migration. This protein is a specific regulator of collective migration of neuroblasts born in the subventricular zone of the postnatal and adult brains and participates in the progression of cancer. We found that Girdin was essential for the collective migration of the skin cancer cell line A431 on collagen gels as well as their fibroblast-led collective invasion in an organotypic culture model. We provide evidence that Girdin binds to β-catenin that plays important roles in the Wnt signaling pathway and in E-cadherin-mediated cell-cell adhesion. Girdin-depleted cells displayed scattering and impaired E-cadherin-specific cell-cell adhesion. Importantly, Girdin depletion led to impaired cytoskeletal association of the β-catenin complex, which was accompanied by changes in the supracellular actin cytoskeletal organization of cancer cell cohorts on collagen gels. Although the underlying mechanism is unclear, this observation is consistent with the established role of the actin cytoskeletal system and cell-cell adhesion in the collective behavior of cells.

#3161

The macrolide toxin mycalolide B disrupts actin-driven invasion and metastasis of HER2-positive cancers.

Sarah Nersesian, Rodette Williams, Dr. Andrew Evans, Dr. John Allingham, Dr. Andrew Craig. _Queens University, Kingston, Ontario, Canada_.

HER2 is a driver and clinical target in cancers afflicting women. HER2-positive (HER2+) cancers have high rates of metastasis and lower overall survival rates compared to other cancer subtypes. The goal of this study is to test the vulnerability of HER2+ cancer cells and tumors to disruption of their actin cytoskeleton using the marine macrolide toxin mycalolide B (Myc B), as these cells are highly dependent on rapid actin polymerization and remodeling for migration and invasion into new sites of tumor growth. The effects of Myc B treatment on HER2+ breast (HCC1954) and ovarian (SKOV3) cancer cell lines were profiled in assays of cell viability, motility, and invasion. Treatments of Myc B alone or in combination with Trastuzumab were also performed in HER2+ tumor xenograft assays. Myc B showed potent growth suppressive and cytotoxic effects on HER2+ cancer cells at doses in the 70-100 nM range. At sub-lethal doses, Myc B caused a rapid loss of leading edge protrusions, and sustained defects in HER2+ cancer cell motility and invasion. HER2 internalization and killing of HER2+ cancer cells by Trastuzumab-emtansine was not compromised with Myc B treatment. In a HER2+ tumor xenograft model, Myc B treatment alone, or in combination with Trastuzumab, led to significant reductions in tumor growth and metastasis. Together, these findings identify a major vulnerability in metastasis-initiating HER2+ cancer cells to the actin toxin Myc B, and provide a rationale to exploit this vulnerability with the development of new therapeutics targeting actin.

#3162

Endothelin-1 receptor/beta-arrestin-1 pathway promotes invadopodia and metastatic process by integration with hMENA in human serous ovarian cancer.

Valentina Caprara,1 Francesca Di Modugno,1 Lidia Chellini,1 Piera Tocci,1 Francesca Spadaro,2 Andrea Sacconi,1 Giovanni Blandino,1 Paola Nisticò,1 Anna Bagnato,1 Laura Rosanò1. 1 _Regina Elena National Cancer Institute, Rome, Italy;_ 2 _Istituto Superiore di Sanità, Rome, Italy_.

Aberrant activation of the endothelin-1 receptor (ET-1R) elicits pleiotropic effects relevant for serous ovarian cancer (SOC) cell invasion. The network activated by this receptor might be finely, spatially and temporary orchestrated by β-arrestin1 (β-arr1)-driven interactome. Recently, we uncovered a novel role for ET-1R/β-arr1 as regulator of cytoskeletal remodelling and invasive protrusions, invadopodia. Emerging evidence demonstrated that hMENA protein, an Ena/Vasp family member, is a key invadopodia component. In this study, we set out to molecularly dissect whether hMENA might represent a novel interacting partner of β-arr1 necessary for invadopodial function downstream of ET-1R in SOC cells. ENAH mRNA is significantly upregulated in OC tissues, and in particular in high-grade (HG)-SOC tumors, compared with normal tissues. In a panel of SOC cells, the expression hMENA, along with the spliced isoform hMENAΔv6, is upregulated by ET-1, at mRNA and protein levels, through β-arr1, restricted to mesenchymal phenotype. This effect is inhibited by treatment with the dual ETAR/ETBR antagonist macitentan. As shown by biochemical and imaging assays, ET-1 promotes a physical association between hMENA and β-arr1 as well PDZ-RhoGEF, which in turn activate RhoC. Most importantly, ET-1 induces localization of hMENA in F-actin-containing puncta, which co-localize with cortactin, and extracellular matrix degradation sites, thus promoting invadopodia maturation. Silencing of hMENA, as well as of β-arr1, or treatment with macitentan, impairs ET-1-dependent invadopodia activity, MMP secretion and activation, invasion, transendothelial migration and cell plasticity. In vivo, macitentan is able to inhibit SOC metastatic dissemination and hMENA expression along with other invadopodia markers. Finally, high ETAR/ARRB1/ENAH gene expression is associated with a poor prognosis in SOC patients. Collectively, these data define a pivotal function of hMENA/ hMENAΔv6, which is required for ET-1/β-arr1-induced invadopodial function and metastatic spreading of SOC.

#3163

Wnt7a promotes bladder cancer cell invasion via canonical Wnt/β-Catenin signaling.

Ruimin Huang,1 Xiaojing Huang,2 Hongwen Zhu,1 Junlong Zhuang,3 Yu Dong,4 Hu Zhou,1 Hongqian Guo,3 Jun Yan2. 1 _Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China;_ 2 _Nanjing University, Nanjing, China;_ 3 _Nanjing University Medical School, Nanjing, China;_ 4 _Shanghai University, Shanghai, China_.

The mortality rate of urinary bladder cancer (UBC) increases dramatically, once low grade non-muscle invasive bladder cancer develops into muscle invasive bladder cancer. Hence, understanding the mechanisms underlying UBC invasion/metastasis and identification of biomarkers for invasion/metastasis will help clinicians to determine how to treat patients. Herein we generated two sublines from parental 5637 UBC cells with low invasive and high invasive capacities, which were designated as 5637 NMI and 5637 HMI, respectively. Mass spectrometry analysis was performed to identify the invasion-associated proteins. 5,987 proteins were quantified across all six samples with the coefficients among different channels all nearly 1.00, suggesting good biological repeatability and minor differences between 5637 HMI and NMI samples. Fold change > 1.2 and p value <0.05 were set as the cut-off standard, and 16 upregulated proteins and 26 downregulated proteins were identified in 5637 HMI cells. Among them, Wnt7a overexpression was confirmed in 5637 HMI cells compared to 5637 NMI cells, which was also associated with metastasis potential and worse clinical outcome in UBC patients.

Depletion of Wnt7a in 5637 HMI and T24 cells reduced UBC cell invasion, with the reduction of active β-Catenin and its downstream target genes involved in epithelial-to-mesenchymal transition (EMT) and extracellular matrix (ECM) degradation. Consistently, introduction recombinant Wnt7a protein to 5637 NMI and J82 cells induced cell invasion, EMT, and the expression of ECM degradation-associated genes. Using luciferase reporter driven by the wildtype or mutants of matrix metalloproteinase 10 (MMP10) promoter, a canonical Wnt/β-Catenin regulation pattern was demonstrated.

Four miRNAs downregulated in UBCs were predicted directly targeting Wnt7a using bioinformatics analysis. miRNA mimic could decrease Wnt7a expression, reduce the activation of MMP 10 reporter and suppress the invasion of 5637 HMI cells. Treatment of UBC cells with Wnt7a partially abrogated such miRNAs-induced invasion suppression.

Taken together, our data identify a new miRNA/Wnt7a axis to mediate cell invasion and metastasis of UBC through canonical Wnt/β-Catenin pathway, which may offer prognostic and therapeutic opportunity.

#3164

Impact of cN-II and CD73 inhibition on cancer cell migration.

Octavia Cadassou,1 Muhammad Zawwad Raza,1 Emeline Cros-Perrial,1 Célia Armanet,1 Laura Gudefin,1 Kamel Chettab,1 Serge Manié,2 Charles Dumontet,1 Lars P. Jordheim1. 1 _Anticancer Antibodies Team, CRCL, INSERM U1052, CNRS UMR 5286, CLB, UCBL, Lyon, France, LYON, France;_ 2 _ER stress and tumorogenesis group, CRCL, INSERM U1052, CNRS UMR 5286, CLB, UCBL, Lyon, France, LYON, France, LYON, France_.

Deregulation of nucleotide metabolism can lead to biological disturbances such as genetic instability, energetic homeostasis deregulation and pro-proliferative signaling, that are among the "Hallmarks of Cancer" described by Hanahan and Weinberg. Thus, this complex process has become in hotspot in cancer research. To fully apprehend to what extent nucleotide metabolism can be targeted for new anti-cancer therapies, the involved molecular actors and their function in cancer cell biology need to be better understood. Purine metabolism involves various intracellular and extracellular enzymes including cN-II (cytosolic nucleotidase-II) and CD73 that are two 5'-nucleotidases respectively able to dephosphorylate intracellular and extracellular nucleoside monophosphates into corresponding nucleosides. Considering nucleotide/nucleoside trafficking and their roles in cell biology, it is a possible that cN-II and CD73 are involved together in processes that increase cancer cells aggressiveness. We abolished cN-II and/or CD73 expressions in two human carcinoma cell lines (MDA-MB-231 and NCI-H292), using the CRISPR/Cas9 technique, and evaluated the impact of cN-II and CD73 on cell migration, under an extracellular nucleotide stress (by performing wound healing assays on the Incucyte device). The obtained results showed that cN-II alone or together with CD73, was able to modify cell migration, in absence and in presence of an extracellular nucleotide stress. Indeed, cN-II deficiency was associated with accelerated cell migration, in control conditions. Moreover, exposure to adenosine decreased migration for all the models, with a stronger effect on cN-II deficient cells. We further investigated on the expression or activity of migration regulators, and found that 5'-nucleotidases deficiency was associated with altered transcriptional expression of gelatinases (MMP-2 and MMP9) and their negative regulator (TIMP2). The study of signaling pathways that are known to be related to cell migration are currently studied. These in vitro observations justify upcoming in vivo studies that aim to correlate these results with metastasis occurrence after xenografts in immunodeficient mice. For the first time, our results define cN-II as involved in cell migration, confirming that this 5'-nucleotidase could represent an interesting target to reduce pro metastatic behaviors in cancer cells. Nevertheless, it remains to be defined whether the relation between 5'-nucleotidases and cell migration involves their catalytic activity (and thus nucleotide pools balance) or if it is due to the physical interaction with complexes that are already known to regulate this process.

#3165

PI 3-kinase-beta regulates invadopodia maturation and beta-1 integrin signaling.

Zahra Erami, Anne R. Bresnick, Jonathan M. Backer. _Albert Einstein College of Medicine, Bronx, NY_.

The invasion of tumors cells during metastasis is mediated by invadopodia, actin rich protrusive organelles that secrete matrix metalloproteases and mediate matrix degradation. The PI3K-beta isoform of PI 3-kinase, which is regulated by both receptor tyrosine kinases and GPCRs, is an important regulator of invadopodia formation. We have previously reported that in MDA-MB-231 breast cancer cells, replacement of endogenous PI3K-beta with kinase dead (p110-betaK799R; KD) or GPCR-uncoupled (p110-beta526KK-DD; KK-DD) mutants leads to defects in gelatin degradation. We have now studied the mechanism by which loss of PI3K-beta signaling affects invadopodia formation and matrix degradation. While the number of invadopodia precursors (cortactin-Tks5 positive, degradation negative) is unaffected by mutation of PI3K-beta, formation of mature invadopodia (cortactin-Tks5 positive, degradation positive) is reduced in cells expressing KK-DD and KD PI3K-beta. Previous studies have demonstrated a role for beta-1 integrins in invadopodia maturation, and PI3K-beta has been implicated in integrin signaling in platelets. We therefore tested whether mutation of PI3K-beta would affect integrin signaling in breast cancer cells. In a haptotaxis assay, MDA-MB-231 cells expressing KD or KK-DD PI3K-beta showed a significant decrease in migration. Consistent with these data, cell spreading on collagen I was significantly impaired in cells expressing KD or KK-DD PI3K-beta, and was inhibited by TGX221 (PI3K-beta inhibitor) and LY294002 (pan-PI3K inhibitor), but not by specific inhibitors of other Class I PI3Ks (alpha, delta and gamma). Spreading was also impaired in cells treated with pertussis toxin, which blocks GPCR activation of PI3K-beta. To distinguish whether PI3K-beta was acting on integrins by inside-out versus outside-in mechanisms, we treated cells with the activating beta-1 integrin antibody TS2/16, which bypasses inside-out regulation of integrin binding. TS2/16 increased cell spreading on collagen I, but TS2/16-stimulated spreading was blocked by treatment of cells with TGX221, suggesting that PI3K-beta acts downstream of beta-1 integrins. Finally, to determine whether PI3K-beta was involved in beta-1 integrin signaling in invadopodia, we plated cells on high density fibrillar collagen (HDFC), which enhances invadopodia formation through beta-1 integrin activation. Invadopodia formation on HDFC was blocked by treatment of cells with TGX221. In summary, beta-1 integrin signaling in the context of invadopodia requires GPCR signaling to PI3K-beta. We propose that GPCR activation of PI3K-beta is a critical downstream component of beta-1 integrin signaling in invadopodia.

#3166

The HU177 cryptic collagen epitope selectively regulates melanoma cell behavior by a CDK5 and PTPN12 associated mechanism.

Xianghua Han, Jennifer M. Caron, Peter C. Brooks. _Maine Medical Center, Scarborough, ME_.

Previous studies indicate that selectively inhibiting cellular interactions with the HU177 cryptic collagen epitope, which can be generated in vivo during structural remodeling of the extracellular matrix (ECM), inhibits tumor growth and metastasis. However, a detailed understanding of the mechanisms by which antagonists of the HU177 collagen epitope inhibit these pathological processes is not fully understood. Uncovering the molecular mechanisms by which cellular interactions with this collagen epitope regulate tumor cell behavior may allow the development of more effective clinical strategies targeting tumor cell-ECM interactions. Here we provide evidence for the first time, that specific targeting of the HU177 cryptic collagen epitope reduces phosphorylation of cyclin-dependent kinase (CDK5) and in addition, reduces the expression of the phosphatase PTPN12 in melanoma cells attached to denatured collagen. Importantly, both CDK5 < PTPN12 play a functional role in the ability of the HU177 collagen epitope to regulate migration of melanoma cells as reducing expression of these molecules inhibited the anti-migratory activity of the anti-HU177 antibody. Surprisingly, reduction in either CDK5 or PTPN12 also inhibited nuclear accumulation of YAP. These novel findings help define a previously unknown signaling mechanism by which both CDK5 < PTPN12 may play a selective role in controlling nuclear accumulations of the transcriptional co-activator YAP following melanoma cell interactions with the HU177 cryptic collagen epitope. Taken together, these studies provide new molecular insight into the role of the HU177 collagen epitope in controlling melanoma cell behavior. Given that direct targeting of integrin receptors that mediate tumor cellular interactions with the ECM have demonstrated only limited anti-tumor activity in human clinical trials, a more detailed molecular understanding of how generation of biologically relevant cryptic elements of the ECM control melanoma cell behavior will likely facilitate the development of more effective new clinical strategies to control tumor progression.

#3167

Loss of HP1α alters the integrity of the nuclear lamina, thereby promoting invasion.

Tracy K. Hale, Raoul Solomon. _Massey University, Palmerston North, New Zealand_.

Malignant cell invasion is accompanied by complex changes in nuclear morphology and chromatin patterning that have been utilized by pathologists to determine malignancy. Members of the HP1 family modulate chromatin plasticity, with the HP1α paralog required to maintain the condensation of transcriptionally silent heterochromatin and to sequester it at the nuclear periphery. While increased expression of HP1α is proposed to aid the onset of malignancy, the acquisition of an invasive phenotype correlates with the loss of this architectural protein in many solid tumors including those of the thyroid, kidney, colon and breast. We have shown in a Drosophila model of epithelial cell invasion that loss of HP1α synergistically enhances invasion in cells with an activated oncogenic pathway. In the poorly invasive MCF7 breast cells, loss of HP1α increases their invasive potential and promotes epithelial plasticity. These phenotypic changes are associated altered nuclear morphology including increased nuclear grooves and indentations, diffuse chromatin patterning and a loss of heterochromatin at the nuclear periphery. Since during metastatic invasion, the nucleus, the largest and most rigid cellular of organelle, becomes malleable enough to squeeze through interstitial spaces in the surrounding tissue. This lead us to propose that loss of HP1α causes a remodelling of nuclear periphery thereby increasing nuclear malleability. Here we demonstrate that HP1α knockdown in MCF7 cells decreases mechanical stability of the nuclei and altering the integrity of nuclear lamina. While total levels of Lamin A/C, Lamin B1 and Lamin B2 are unchanged within the cell, their solubility and thus localization within the nucleus is altered. We also demonstrate how this change in the lamina effects other proteins that function at the nuclear periphery. Together, these findings suggest that when loss of HP1α increases invasive potential there is a corresponding disruption to the nuclear periphery, creating a weakness in these cells that could be exploited for therapeutic benefit.

#3168

Serglycin promotes migration and invasion of esophageal cancer cells.

Yun ZHU,1 Bin Li,1 Wenwen Xu,1 Aky Lam,2 Simon Law,1 ALM Cheung1. 1 _University of Hong Kong, Hong Kong, Hong Kong;_ 2 _Griffith Medical School, Medicine and Oral Health Centre, Queensland, Australia_.

Esophageal cancer is the eighth most common cancer in the world and it ranks sixth in mortality rate among cancer diseases. While patients with early stage esophageal cancer without metastasis have a better chance of being cured by surgery, most patients present with locally advanced or metastatic disease. Unfortunately, some common cancer biomarkers such as carcinoembryonic antigen (CEA) and CA19-9 have low sensitivity and specificity in esophageal cancer. A better understanding of the molecular mechanisms underlying esophageal cancer progression is needed to identify novel diagnostic markers and therapeutic targets for intervention. We previously identified serglycin (SRGN) as the top upregulated gene in a highly invasive esophageal squamous cell carcinoma (ESCC) cell subline. Serglycin is a proteoglycan with a core protein and glycosaminoglycan (GAG) chains. Its significance in tumorigenesis is not well understood. The GAG chains have the ability of binding specific growth factors and ligands, which may play an important role in facilitating cancer cell migration and invasion. Immunohistochemical analysis showed that serglycin expression was higher in ESCC compared with normal esophageal epithelium, and in lymph node metastases compared with primary ESCC. Ectopic overexpression of serglycin in ESCC cells enhanced their migration and invasion potential in vitro. Knockdown of serglycin reduced ESCC cell migration and invasion. In invasion chamber assays, conditioned medium (CM) from SRGN-overexpressing ESCC cells stimulated the migration and invasion ability of less invasive ESCC cells. Conditioned medium from ESCC cells expressing serglycin that lacks the GAG attachment site had no such effects. Chemokine antibody array analysis showed that midkine (MDK), a heparin-binding growth factor, was the top upregulated protein in the CM of SRGN-overexpressing cells. Analysis of TCGA and GTEx datasets showed that MDK expression was higher in many cancer types such as breast cancer, colon adenocarcinoma, glioblastoma and lung cancer, than in corresponding normal tissues. Western blot analysis showed that forced expression of serglycin, but not serglycin without the GAG binding domain, led to increase of MDK in the CM of multiple ESCC cell lines. Forced expression of serglycin also increased the expression level of phosphorylated extracellular signal-regulated kinase (p-ERK) in ESCC cells. Taken together, serglycin may play an important role in the progression ESCC by promoting migration and invasion of cancer cells. These effects may be mediated through activation of the ERK signaling pathway. Whether MDK is involved in this regulatory mechanism warrants further investigation. [This study was supported by Research Grants Council of the Hong Kong SAR, China, GRF Project No. 17111016]

#3168A

S100A4 mediates paracrine interactions with breast tumor cells.

Natalya G. Dulyaninova, Adriana Levine, Jonathan M. Backer, Anne R. Bresnick. _Albert Einstein College of Medicine, Bronx, NY_.

S100A4, a member of the S100 family of Ca2+-binding proteins, has a direct and causative role in tumor metastasis. In murine models of breast cancer, S100A4 overexpression in tumor cells promotes an aggressive metastatic phenotype, while inhibition of S100A4 expression significantly reduces metastatic burden. We have now evaluated the contribution of stromal S100A4 to mammary tumor progression. The growth of E0771 orthotopic mammary tumors was reduced in S100A4-/- mice as compared to wild type mice. In addition, in a 3D collagen assay that recapitulates macrophage-tumor cell paracrine interactions that drive metastasis in vivo, S100A4-/- bone marrow-derived macrophages failed to stimulate breast tumor cell invasion. Our cell biological studies on bone marrow-derived macrophages from wild type and S100A4-/- mice show that loss of S100A4 produces two mechanistically distinct phenotypes in macrophages: a myosin-IIA-dependent defect in matrix degradation, due to an inhibition of podosome rosettes (actin-rich degradative structures that allow macrophages to penetrate through endothelial layers and their associated basement membranes), and a defect in invasion, due to a myosin-IIA-independent increase in microtubule acetylation and stability. These studies suggest that S100A4, which has well described pro-metastatic functions in tumor cells, also plays a critical role in immune cells in the tumor microenvironment.

### Pediatrics 2: Preclinical Therapies, Resistance, and Stem Cells

#3169

Mechanisms of novel cytokine therapy for Ph-like B-cell acute lymphoblastic leukemia with overexpression of CRLF2.

Cornelia Stoian,1 Jacqueline S. Coats,1 Hossam Alkashgari,1 Veriah Vidales,1 Ineavely Baez,1 Juliette Personius,1 Hannah Choi,1 WayAnne Watson,1 Brandon Ng,1 Benjamin Becerra,1 Rishikesh Chavan,1 Muhammad Kamal,1 Shadi Farzin Gohar,1 Sinisa Dovat,2 Kimberly J. Payne1. 1 _Loma Linda University, Loma Linda, CA;_ 2 _Penn State Hershey College of Medicine, Hershey, PA_.

B-cell acute lymphoblastic leukemia with overexpression of CRLF2 (CRLF2 B-ALL) comprises ~50% of Ph-like B-ALL, a leukemia that is associated with poor outcomes, high relapse rates and leukemia health disparities in Hispanic children. CRLF2, together with the IL-7 receptor alpha (IL-7Ra), comprises a receptor complex that is activated by the cytokine, TSLP. Receptor activation induces JAK2/STAT5 and PI3/AKT/mTOR signals that are believed to contribute to survival and proliferation of leukemia cells. To study the role of TSLP in CRLF2 B-ALL, we developed a novel patient-derived xenograft (PDX) model of CRLF2 B-ALL that allows us to vary circulating levels of human TSLP (hTSLP). Primary CRLF2 B-ALL cells injected into PDX mice without hTSLP or with circulating hTSLP levels similar to pediatric leukemia patients (~4-10 pg/ml) showed engraftment and expansion of leukemia cells. In contrast, CRLF2 B-ALL cells were essentially eliminated in PDX with elevated physiologic levels of hTSLP (40-140 pg/mL). We observed these results in 5 independent experiments produced using primary CRLF2 B-ALL cells from two different Hispanic pediatric patients with CRLF2 B-ALL (N= 40 PDX). We hypothesize that the observed antileukemia effects are mediated via TSLP-induced upregulation of the Suppressor of Cytokine Signaling (SOCS) genes. SOCS genes encode a family of proteins (SOCS1-7 and CISH) that regulate cytokine signaling via negative feedback through multiple mechanisms including ubiquitin-mediated cytokine receptor degradation. Consistent with our hypothesis, SOCS1, SOCS2, SOCS3 and CISH mRNA were upregulated in primary CRLF2 B-ALL cells cultured with high-dose hTSLP. To gain mechanistic insights we evaluated the CRLF2 B-ALL cell lines, MUTZ5 and CALL4, following culture with and without hTSLP. Flow cytometry analysis showed that high-dose hTSLP upregulated SOCS1 and SOCS3 proteins in both CRLF2 B-ALL cell lines. We found that CRLF2 B-ALL cells cultured with hTSLP for 3 days showed a dose-dependent loss in the ability to induce STAT5 and S6 phosphorylation following hTSLP stimulation. This loss was correlated with the loss of IL-7Ra, and maintained for 24-48 hours following a pulse of high-dose (but not low-dose) hTSLP. The loss of signaling and surface IL-7Ra could be prolonged if high-dose hTSLP levels were maintained. These data provide evidence that TSLP exerts its antileukemia effects by shutting down CRLF2-mediated signals and suggest that these effects are at least partially mediated by the loss of the IL-7Ra component, and potentially through SOCS family proteins. These studies identify the human TSLP cytokine as a potential biologic therapy to treat CRLF2 B-ALL and reduce cancer health disparities for Hispanic children with CRLF2 B-ALL. (Supported by 1R01CA209829.)

#3170

AKT/mTOR pharmacologic inhibition sensitizes RAS/MAPK-activated neuroblastoma cells to MEK1/2 inhibitors.

Marielle Yohe, Norris Lam, Joshua Kowalczyk, Carol J. Thiele. _National Cancer Inst., Bethesda, MD_.

Relapsed neuroblastoma tumors have an increased incidence of activating mutations in the RAS/MAPK pathway. Pre-clinical studies indicate these tumors have an increased sensitivity to inhibitors of the RAS/MAPK pathway but single-agent therapeutic approaches have rarely led to durable responses. For this reason, we evaluated MEK1/2 inhibitors, Selumetinib(SEL) and Trametinib(TRAM), for their efficacy in controlling the growth of NB cells alone or in combination with inhibitors of the AKT/mTORi pathways. MYCNamplified [(MYCNamp) KCNRAKLF1174L, KellyALKF1174L,NGPNF1del] or MYCNwild-type[(WT) ASNF1del,NRASQ61K, SY5YF1174L] NB cell lines were used in the study. For tested NB cells, the TRAM IC50 was 10-80fold lower (4-43nM) compared to SEL IC50 (325-525nM). TRAM induced growth inhibition (10nM in AS at 24hrs) was accompanied by an increase in cells in G1. In other tumor types, MEK inhibition is associated with feedback activation of the PI3K/AKT pathway. While we did not see MEKi induced activation of the AKT/mTOR pathway at 1-3 days by Western analysis, combination dose responses (TRAM or SEL +/- MK2206 or Rapamycin) were initiated for each cell line and continued until control-treated cells reached stationary phase using a 384-well format. In SY5Y cells, treatment with MK2206 (allosteric Akt1/2/3 inhibitor) weakly inhibited cell proliferation (IC50 ~ 250nM). Using the Bliss Independence model, we computationally elucidated doses of MEK1/2i and Akt/mTORi that resulted in the greatest synergy in suppressing cell proliferation. MK2206(200nM) increased the sensitivity of SY5Y cells to TRAM some 2.5fold as the TRAM IC50 decreased from 35 to 13nM with MK2206. Using the Bliss independence model, combination of 200nM MK2206 and 0.01µM TRAM showed the greatest synergy in inhibiting cell numbers. MK2206(200nM) increased the sensitivity of SY5Y cells to SEL ~37-fold as the SEL IC50 decreased from 4.3 to 13µM with MK2206. MK2206(200nM) with 0.37µM SEL showed the greatest synergy in inhibiting cell growth. Similar results were also seen in MYCNamp Kelly NB cells. Next, we determined whether similar sensitization to MEK1/2i could be seen with treatment with the mTORi rapamycin. As a single agent SY5Y and Kelly NB cells are insensitive to rapamycin (1.6nM) but in combination with1.6nM rapamycin in combination with 40nM TRAM synergistically inhibited cell growth. In Kelly cells, 1.6nM rapamycin (insensitive as a single agent) and 330nM TRAM synergistically inhibited cell growth. In conclusion, using the Bliss independence model for synergy, we found that Akt or mTOR inhibition synergized with MEK inhibitors in neuroblastoma cell lines with activating RAS or ALK mutations.

#3171

Vangl2 regulates cancer stem cell self-renewal and growth in rhabdomyosarcoma.

Madeline Hayes,1 Karin McCarthy,1 Alexander Jin,1 Sowmya Iyer,1 Sara Garcia,1 Mariana L. Oliveira,2 Sivasish Sindiri,3 Berkley Gryder,3 Zainab Motala,4 G Petur Nielsen,5 Jean-Paul Borg,6 Matt van de Rijn,7 David Malkin,4 Javed Khan,3 Myron Ignatius,8 David M. Langenau1. 1 _Massachusetts General Hospital, Charlestown, MA;_ 2 _Instituto de Medicina Molecular, Faculdade de Medicina, 3Instituto de Medicina Molecular, Faculdade de Medicina, Lisbon, Portugal;_ 3 _Oncogenomics Section, Center for Cancer Research, National Cancer Institute, Bethesda, MD;_ 4 _Hospital for Sick Children, Toronto, Ontario, Canada;_ 5 _Massachusetts General Hospital, Boston, MA;_ 6 _Centre de Recherche en Cancérologie de Marseille, Marseille, France;_ 7 _Department of Pathology, Stanford University Medical Center, Stanford, CA;_ 8 _Greehey Children's Cancer Research Institute, University of Texas, San Antonio, TX_.

Growth and relapse are driven by cancer stem cells (CSCs) in a subset of tumors, yet mechanisms driving cancer cell fate choices, maintenance and self-renewal are not fully understood. Here, we show that Van Gogh-like 2 (Vangl2), a core regulator of the non-canonical Wnt/planar cell polarity pathway (Wnt/PCP), regulates CSCs self-renewal in human rhabdomyosarcoma (RMS) – a common pediatric cancer of muscle. Wnt/PCP signaling is essential during development and recent work has linked this pathway to cancer growth, invasion and metastasis. However, roles for Vangl2 in regulating tumor self-renewal have not been previously described. Here, we show that VANGL2 is expressed in a majority of human RMS, specifically within early mononuclear progenitor-like cells. VANGL2 depletion inhibited proliferation, reduced self-renewal, and induced differentiation of human RMS. VANGL2 was also required for continued tumor growth and maintenance following engraftment of human RMS using mouse xenografts. Using a zebrafish model of embryonal rhabdomyosarcoma (ERMS) and limiting dilution cell transplantation approaches, we identified that Vangl2 expression enriches for CSCs in vivo and when transgenically expressed, at high levels elevates cancer stem cell number by 9-fold. Mechanistic studies revealed a role for RhoA downstream of Vangl2 in regulating maintenance of stem cell programs in human RMS. Our studies offer novel opportunities to isolate and characterize RMS cancer stem cells in vivo, and identify potential therapeutic targets for patient treatment.

#3172

Targeting genomic instability in embryonal tumors with multilayered rosettes (ETMR).

Sander Lambo,1 Andrey Korshunov,2 Christin Schmidt,1 Carolina Romero,3 Aparna Gorthi,3 Sonja Krausert,1 Tobias Rausch,4 Susanne Gröbner,1 Sebastian Brabetz,1 Sebastian Waszak,4 Alexander J. Bishop,3 Stefan Pfister,1 Marcel Kool1. 1 _Hopp Children's Cancer Center at NCT (KiTZ) & German Cancer Research Center (DKFZ), Heidelberg, Germany; _2 _German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 3 _University of Texas Health Science Center at San Antonio, San Antonio, TX;_ 4 _European Molecular Biology Laboratory (EMBL), Heidelberg, Germany_.

Embryonal Tumors with Multilayered Rosettes (ETMRs) are pediatric brain tumors mainly occurring in infants. Characteristic to ETMRs is the highly recurrent (~90%) amplification of the C19MC miRNA cluster fused to TTYH1 that drives the expression of this cluster. As the overall survival of these patients is very poor, there is an urgent need for a better understanding of these tumors that may lead to other treatment strategies. Whole genome and panel sequencing data have been generated for 60 ETMRs and matching germline when available. Data have been complemented with DNA methylation profiling and m(i)RNA sequencing data. Our results show that ETMR is a single disease entity without molecular subgroups. ETMRs lacking the C19MC amplification (~10%) are highly similar to tumors with C19MC amplification, based on methylation and m(i)RNA profiling, indicating that they do not represent a distinct subgroup. Germline sequencing revealed mutations in genes involved in DNA repair or miRNA processing, while tumor specific mutations included genes involved in the TP53-, SHH-, WNT-, or miRNA processing pathways. These pathways are also highly upregulated compared to other pediatric brain tumors. Mutations in DNA repair, miRNA processing, structural variations (SVs) and mutations in close proximity of SVs occur at high allele frequencies and are conserved in recurrent tumors while many other SNVs are lost. These data suggest that C19MC amplification/fusion, miRNA processing and DNA repair defects are the early (driving) events in tumor formation while aberrations involving for instance the SHH and WNT pathways are later (passenger) events. Aside from frequent and recurrent copy number changes, ETMRs show pluriploidy, complex rearrangements and strong presence of R-loops suggesting that ETMR genomes are highly unstable. We identified a high number of R-loops in the region forming the C19MC aberration and an enrichment of breakpoints in other R-loop forming regions. This may suggest a role for R-loops in both tumor progression and initiation. Finally, we tested whether further inducing the number of R-loops in these tumors may increase replication stress and cell death. Indeed, topoisomerase inhibition coupled to PARP inhibition increased the amount of R-loops and acted synergistically in killing ETMR cells. These data show that targeting the genomic instability in ETMRs could be a viable treatment option for treating ETMR patients.

#3173

Lymphocyte-specific chromatin accessibility predetermines glucocorticoid resistance in acute lymphoblastic leukemia.

Duohui Jing,1 Yizhou Huang,2 Xiaoyun Liu,1 Keith Sia,1 Rebecca C. Poulos,3 Miriam Span,1 Chao Zhang,4 Jianqing Mi,5 Jason WH Wong,3 Dominik Beck,2 John E. Pimanda,3 Richard B. Lock1. 1 _Children's Cancer Institute, Sydney, Australia;_ 2 _Lowy Cancer Research Centre and the Prince of Wales Clinical School, UNSW, Sydney, Australia; Centre for Health Technologies, School of Biomedical Engineering and the School of Software, University of Technology, Sydney, Australia;_ 3 _Lowy Cancer Research Centre and the Prince of Wales Clinical School, UNSW, Sydney, Australia;_ 4 _School of Life Sciences and Technology, Tongji University, Shanghai, China;_ 5 _State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui Jin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China_.

Glucocorticoids are well known for their immunosuppressive activity, and play a critical role in the treatment of lymphoid malignancies. However, the development of resistance remains a significant barrier to cure and the mechanisms are poorly defined. Moreover, glucocorticoids are rarely efficacious in treating myeloid and other non-lymphoid malignancies, and the mechanisms of lymphocyte-specific efficacy are unclear. To address these issues, we first carried out a global analysis of DNase I hypersensitive sites in 18 lymphoid and 64 non-lymphoid cell types to map lymphocyte-specific open chromatin domains (LSOs). We then integrated these domains with genome-wide glucocorticoid-induced gene transcription and epigenetic modulation in an in vivo patient-derived xenograft (PDX) model of acute lymphoblastic leukemia (ALL). Performing chromatin immunoprecipitation and assays for transposase-accessible chromatin (ATAC), we determined a strong correlation between glucocorticoid receptor (GR) binding and chromatin accessibility, acetylated histone marks and binding of a DNA structural protein (CTCF), and identified 1,536 GR bound LSOs. We next analyzed RNA-seq data in glucocorticoid sensitive and resistant ALL PDXs for expression changes in genes located within 100 kb of the LSOs after glucocorticoid treatment in vivo, and identified four groups comprising 389 genes that were significantly differentially expressed. Of the 198 up-regulated genes, 143 showed increased H3K27Ac enrichments at 177 LSOs. Forty-two LSOs showed the increase only in glucocorticoid sensitive but not resistant ALLs. Applying this to an extended panel of ALL PDXs, basal DNA methylation at the 42 LSOs was significantly higher in resistant ALLs despite no difference within the gene bodies, whereas the basal chromatin accessibility indicated by ATAC abundance was diminished. Pathway analysis indicated that the LSO regulated genes were involved in repressing B- and T- cell receptor signaling pathways and activating the apoptotic pathway. One such LSO was at the pro-apoptotic BIM gene locus, where CTCF binding was found only in lymphocytes but not in other cell types. The GR cooperated with CTCF to mediate interactions between the promoter and the BIM LSO to direct DNA looping, thus triggering BIM transcription. Importantly, this LSO was heavily methylated in resistant ALLs as well as non-lymphoid cells. Azacitidine, a DNA demethylating drug that is routinely used in the clinic, could partially reverse these changes and restore glucocorticoid sensitivity. Taken together, this study demonstrated for the first time that lymphocyte-specific epigenetic modifications pre-determine glucocorticoid resistance in ALL and may account for the lack of glucocorticoid sensitivity in other cell type. Reversal of these epigenetic changes may lead to improvements in the use of glucocorticoids in the clinic.

#3174

PLK4 is a rational target for very high-risk medulloblastoma.

Carolina Nor,1 Mark Bray,2 Tak Mak,2 Michael Taylor1. 1 _The Hospital for Sick Children, Toronto, Ontario, Canada;_ 2 _The Campbell Family Institute for Breast Cancer Research, Toronto, Ontario, Canada_.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Despite aggressive therapy (maximal safe surgical resection, craniospinal irradiation and/or chemotherapy) only 65% of patients survive, and survivors are left with severe neuro-cognitive sequelae. MB is comprised of at least four distinct molecular subgroups (SHH, Wnt, Group 3 and Group 4). Within the Sonic hedgehog (SHH) subgroup, a subset harboring inactivating mutations within TP53 represent a very high-risk group with almost universally fatal outcome. TP53 mutated SHH tumors have a high degree of genomic instability with frequent chromothripsis and are resistant to the current generation of SHH pathway inhibitors. The protein PLK4 has a key role in the maintenance of chromosome numeral integrity and as such dysregulation of PLK4 expression causes genomic instability. The PLK4 inhibitor (CFI-400945, produced by the Campbell Family Institute for Breast Cancer Research) was shown to increase genomic instability in aneuploid cells from adult solid tumors and induce these cells to die through mitotic catastrophy. Here we leverage a sporadic transposon based metastatic mouse model (Ptch+/-/Math1-SB11/T2Onc) that faithfully mimics very high-risk MB in children. In order to mimic treatment in children, tumors are microsurgically subtotally resected, followed with the oral administration of CFI-400945 and cranial MRI to monitor response. Blood brain barrier penetration of CFI-400945 was confirmed using mass spectroscopy, specifically pharmacokinetics study revealed an expected availability of CFI-400945 in the circulating blood and concentrated compound in the tumor compared to the normal brain. The administration of CFI-400945 confers an advantage of survival to the mice in this model, median of 18.5 days for surgery group and 49 days for surgery plus CFI-400945 group (p=0.005). In human tumor datasets PLK4 is highly expressed across a variety of malignancies, including MB. Specifically, across two independent, non-overlapping datasets higher PLK4 levels correlate with a trend toward worse outcome, however, when restricting this analysis only to SHH tumors, there is a significant survival difference between high and low PLK4 expression (p=0.012). In paired primary-metastatic MB PLK4 expression is retained at the time of recurrence, specifically in metastatic recurrences, suggesting PLK4 inhibition is a suitable treatment option at the time of recurrence. Taken together, the data reveals PLK4 inhibition as a novel and rational therapeutic target for a subset of very high risk MB and a broad range of other pediatric cancers.

#3175

Sulfated glycopeptide nanostructures activate and extend bone morphogenetic protein 4 ability to increase pediatric malignant glioma chemotherapeutic efficacy.

Guifa Xi,1 Benjamin Best,1 Sonali Nayak,1 Mark McClendon,2 Barbara Mania-Farnell,3 John Kessler,4 Charles David James,4 Samuel Stupp,2 Tadanori Tomita1. 1 _Ann & Robert H. Lurie Children's Hosp. of Chicago, Chicago, IL; _2 _Northwestern University, Chicago, IL;_ 3 _Purdue University Northwest, Hammond, IN;_ 4 _Feinberg School of Medicine, Northwestern University, Chicago, IL_.

Pediatric gliomas, particularly high-grade gliomas, are among the most formidable and devastating cancers in children. These tumors have remained incurable, regardless of the many treatment approaches attempted. We recently identified a small population of glioma cells with stem-like features in pediatric gliomas (glioma stem cells: GSCs), that may be responsible, for therapeutic resistance in pediatric gliomas. Signaling by bone morphogenetic protein 4 (BMP4), an essential molecule for central nervous system (CNS) development, increases GSC therapeutic sensitivity, and whose activation is a promising adjuvant for glioma treatment. Mechanisms through which BMP4 increases therapeutic sensitivity need to be elucidated as this can lead to the identification of additional targets for treating malignant gliomas in children. Delivery systems for administering BMP4 in a clinical setting also need to be developed. Here, we show that BMP4 increases chemosensitivity by decreasing H3 lysine 4 trimethylation (H3K4me3) at the promoter of multidrug resistant gene 1 (MDR1) that, in turn, results in increased BMP4 expression. This appears to be the result of BMP4 decreasing levels of hSETD1A, a critical methyltransferase for H3K4me3. Our work also demonstrates the first use of a novel sulfated glycopeptide (glycol-PA) nanostructure as a vector for delivery of BMP4. Glycol-PA markedly extended and enhanced BMP4 function, and increased chemotherapeutic anti-tumor activity against pediatric malignant glioma cells in culture. Overall, our results expand understanding of how BMP4 brings about glioma therapeutic sensitization through epigenetic mechanisms, and show that highly bioactive glyco-PA nanostructures have potential as a novel delivery mechanism for treating pediatric malignant glioma, as well as other tumors.

#3176

CD99 regulates ERK1/2-MSK1/2-CREB axis in Ewing sarcoma.

Haydar Celik, Erin J. Conn, David V. Allegakoen, Jeff R. Petro, Jeffrey A. Toretsky, Aykut Uren. _Georgetown University Medical Center, Washington, DC_.

Ewing sarcoma (ES) is an aggressive malignancy of bone and soft tissue that affects predominantly children and young adults with a high propensity to metastasize and poor prognosis. ES cells express high levels of a cell surface protein CD99, which is routinely used as a marker antigen in the histological diagnosis of ES, and contributes to its pathogenesis. We performed a phospho-kinase array to uncover specific cellular responses evoked by CD99 inhibition in ES cells. The treatment of ES cells with CD99 antibody or a small molecule CD99 inhibitor (clofarabine) resulted in strong activation of mitogen- and stress-activated kinases 1 and 2 (MSK1/2) as well as its substrate CREB and its upstream kinase ERK1/2. These findings were further confirmed with western blot analysis in ES cells treated with either different CD99 antibodies or clofarabine. The treatment of osteosarcoma (OS) cells with clofarabine or the forced expression of CD99 in OS cells did not change their phosphorylation levels, suggesting that CD99-mediated ERK1/2-MSK1/2-CREB signaling cascade is specific to ES cells. Interestingly, knock-down of the CD99 protein in ES cells led to a significant decrease in ERK1/2 phosphorylation without any effect on the downstream phosphorylation of MSK1/2 and CREB. Cytarabine, a structurally similar pyrimidine analog that has been identified as an inhibitor of EWS/FLI1 transcriptional activity but failed in clinical trials on ES patients, did not activate ERK1/2-MSK1/2-CREB axis unlike clofarabine in ES cells. These findings suggest that clofarabine functions through alternative mechanisms on ES cells that are different than cytarabine. Clofarabine induced a significant increase in phosphorylation levels of MSK1/2 in a small cohort of TC-71 xenografts, supporting its use as a potential pharmacodynamic marker of CD99 inhibitor activity. In conclusion, our findings suggest that CD99 regulates ERK1/2-MSK1/2-CREB axis in ES, which represents an intracellular pathway that may be responsible for CD99's biological activities in ES.

#3177

mTOR inhibitor augments the antitumor effect of p53-expressing oncolytic adenovirus in human neuroblastoma cells.

Terutaka Tanimoto,1 Hiroshi Tazawa,1 Hiroshi Noso,1 Morimichi Tani,1 Takanori Oyama,1 Yasuo Urata,2 Shunsuke Kagawa,1 Takuo Noda,1 Toshiyoshi Fujiwara1. 1 _Okayama UNIV., Okayama, Japan;_ 2 _Oncolys Biopharma,Inc., Okayama, Japan_.

Background: Neuroblastoma (NB) is a primary malignant tumor of the peripheral sympathetic nervous system. High-risk NB patients show an unfavorable clinical outcome, even after treatment with intensive chemotherapy and aggressive surgical resection. Therefore, a novel therapeutic strategy is needed for the treatment of high-risk NB patients. We developed a tumor-specific replication-competent oncolytic adenovirus OBP-702, in which the hTERT promoter drives the expression of the viral E1A and E1B genes for tumor-specific viral replication and the Egr-1 promoter induces p53 expression. Preclinical studies demonstrated that OBP-702 induces autophagy-related cell death in human NB cell lines. To enhance OBP-702-induced autophagy-related cell death, we hypothesized that a mTOR inhibitor temsirolimus, which is well-known autophagy inducer, may promote autophagy-related cell death by OBP-702 infection. In this study, we investigated the antitumor effect of OBP-702 in combination with temsirolimus in human NB cell lines.

Methods: We used 3 human NB cell lines, including IMR-32, CHP-134 and SK-N-SH. The antitumor effect of OBP-702 and temsirolimus was evaluated using XTT assay. In vitro synergistic effect was calculated with CalcuSyn software (BioSoft, Inc.). Virus-mediated cell death and p53 expression were analyzed by Western blot analysis. Ad-p53, a replication-defective adenovirus expressing p53 gene, was used to assess the effect of p53 in combination with temsirolimus. The combined effect of OBP-702 and temsirolimus was further evaluated using a subcutaneous CHP-134 xenograft tumor model.

Results: Combination with OBP-702 and temsirolimus exhibited a synergistic antitumor effect in all NB cell lines in vitro. We confirmed that combination with these drugs enhanced autophagy. In addition, temsirolimus showed an effect to stabilize p53 protein induced by OBP-702. Ad-p53 also showed synergistic effect in combination with temsirolimus. The combination treatment significantly inhibited tumor growth, as compared to monotherapy, in NB xenograft tumor model.

Conclusions: These results suggest that p53-expressing oncolytic adenovirus and mTOR inhibitor synergistically induce autophagic cell death and may be a promising strategy in NB patients.

#3178

Up-regulation of GLI1 expression in drug resistant rhabdomyosarcoma (RMS) and Ewing sarcoma (EWS) cell lines.

Joon Won Yoon, Iris Smith, Sumeed Manzoor, Marilyn Lamm, Philip Iannaccone, David Walterhouse. _Northwestern University Feinberg School of Medicine and Stanley Manne Children's Research Institute of Ann & Robert H. Lurie Children's Hospital of Chicago, Chicago, IL_.

Outcome for pediatric patients with recurrent sarcomas remains poor. Therefore, understanding mechanisms that lead to drug resistance and ways to overcome drug resistance therapeutically are essential. Recent reports have suggested a role for the Hedgehog signaling pathway and its downstream mediator GLI1 in acquisition of a multidrug resistance phenotype in esophageal adenocarcinoma, glioblastoma, and myeloid leukemia. Since GLI1 is expressed in the majority of RMS and EWS cases, we hypothesized that GLI1 up-regulation occurs as cells become drug resistant in vitro and in vivo. We established vincristine (VCR) resistant RMS cell lines (Embryonal RMS: RD, Ruch-2; Alveolar RMS: Rh30, Rh41) by serial exposure to increasing concentrations of VCR. Drug-resistance was defined as an IC50 of ≥30 fold of the baseline that was established using parental cells. In addition, we obtained EWS cell lines, representing untreated cases (CHLA-9 and TC-32) and cases following recurrence (CHLA-258 and TC-71). We used an 86-gene cancer drug resistance PCR array (Qiagen), to characterize gene expression differences between the parental vs. VCR-resistant RMS cells lines and between the untreated and recurrent EWS cell lines. GLI1 expression was significantly increased (p≤0.05) in VCR-resistant alveolar RMS cell lines (Rh30 [2.3 fold], and Rh41 [10.3 fold]) compared with parental cells. Expression of 2 additional genes (MDR1 [13,307 fold in Rh30 and 1,755 fold in Rh41 cells] and MVP [2.4 fold in Rh30 and 8.8 fold in Rh41]) was also significantly up-regulated in these cells. Gel mobility shifts showed interaction of GLI1 with the MVP promoter, and cotransfection assays showed that GLI1 up-regulates reporter gene expression through the MVP promoter. GLI1 expression was significantly up-regulated in Ruch-2 embryonal RMS cells (2 fold). We also showed higher GLI1 expression in recurrent EWS cell lines (TC-71 [3.4 fold] and CHLA-258 [4.8 fold]) compared with untreated CHLA-9 cells. Expression of 1 additional gene (androgen receptor [11.1 fold in CHLA-258 cells and 274.6 fold in TC-71 cells]) was also significantly up-regulated in CHLA-258 and TC-71 cells. Treatment of VCR-resistant UKF Rhb-1 RMS cells with VCR together with either GANT61 or Cpd#33 (GLI1 inhibitors) significantly decreased cell viability by MTT assay at doses that did not reduce cell viability individually. Our results suggest that GLI1 up-regulation contributes to development of drug-resistance in RMS and EWS and that GLI1-inhibitors may reduce multidrug resistance.

#3179

TORC1/2 inhibition sensitizes MYC-driven medulloblastoma cells to carboplatin chemotherapy.

Rachael E. Maynard. _Johns Hopkins School of Medicine, Baltimore, MD_.

Medulloblastoma is the most common malignant pediatric brain tumor. Tumors having high levels of c-MYC have the worst clinical prognosis, with only a minority of patients surviving. To address this unmet clinical need, we generated a human neural stem cell model of high medulloblastoma that recapitulated the most aggressive subtype phenotypically and by mRNA expression profiling. We performed an in silico analysis of these cells, which predicted that mTOR inhibitors would be potential therapeutic agents. The dual TORC1/2 inhibitor TAK228 (also known as MLN128 and INK128) is orally bioavailable and penetrates the brain. TAK228 is currently in early clinical trials for adults with glioblastoma. We hypothesized that TAK228 would have activity against MYC-driven medulloblastoma. We tested this hypothesis using the MYC amplified medulloblastoma cell lines D425 and D283 as well as our human neural stem cell models. We determined that TAK228 was effective in inhibiting both mTORC1/2 as measured by western blots showing decreased p-S6 and p-AKT473 expression. P-S6 expression is decreased by 90% and p-AKT expression is decreased by 70% in D425 cell lines treated with 20nM TAK228. We found that treatment with TAK228 decreases cell growth in D425 and D283 high-MYC medulloblatoma cell lines at concentrations of 20nM (D425 p<0.005 vs vehicle, D283 p<0.0005 vs vehicle). We also found that TAK228 induces apoptosis as measured by cleaved caspase 3 staining and an increased expression of cleaved PARP (D425 p<0.05 vs vehicle, D283 p<0.0005 vs vehicle). Because mTOR inhibition can suppress glutathione production, and glutathione is required to detoxify platinum containing chemotherapy, we hypothesized that TAK228 would cooperate with carboplatin, which is part of the current standard treatment for high-risk medulloblastoma. TAK228 in combination with carboplatin inhibits cell growth as measured by MTS assay (D425 p<0.05 vs vehicle, D283 p<0.05 vs vehicle) and induces apoptosis as measured by cleaved PARP expression. Combination therapy of TAK228 and carboplatin treatment leads to a 7-fold increase in cleaved PARP compared to cells treated with the vehicle. In vivo testing in orthotopic xenografts is currently underway to determine the survival benefit of TAK228 in combination with carboplatin. TAK228 is effective at inhibiting growth and inducing apoptosis in high-MYC medulloblastoma cell lines and shows strong combinatorial efficacy with carboplatin.

#3180

Preclinical validation of EZH2 as a therapeutic target in pediatric Ewing's sarcoma.

Pankita H. Pandya,1 Barbara Bailey,1 Adily E. Elmi,1 Heather R. Bates,1 Courtney N. Hemenway,1 Anthony L. Sinn,1 Khadijeh Bijangi-Vishehsaraei,1 M. Reza Saadatzadeh,1 Harlan E. Shannon,1 Jixin Ding,1 Mark S. Marshall,1 Michael J. Ferguson,2 Lijun Cheng,1 Lang Li,3 Mary E. Murray,2 Jamie L. Renbarger,2 Karen E. Pollok1. 1 _Indiana University School of Medicine, Indianapolis, IN;_ 2 _Indiana University School of Medicine-Riley Hospital for Children, Indianapolis, IN;_ 3 _Indiana University School of Medicine and Ohio State University College of Medicine, Indianapolis, IN_.

Disease-free survival in relapsed Ewing's Sarcoma Family of Tumors (ESFT) has not improved over the past 25 years. Current standard-of-care (SOC) agents result in 70% survival in patients with localized ESFT; however, for relapsed patients the survival rates remain between 15-20%. Approximately 85% of ESFTs have the chromosomal translocation t(11;22)(q24;q12) which encodes for the oncogenic EWS/FL1 fusion protein. The EWS/FL1 functions as a potent transcription factor leading to the dysregulated expression of genes that promote and maintain tumorigenesis. A major epigenetic regulator that is a downstream target of EWS/FL1 is the enhancer of Zeste Homolog 2 (EZH2). EZH2 is the catalytic component of the polycomb repressor complex 2 (PRC2). Notably, it is overexpressed in ESFT and maintains tumor oncogenicity by tri-methylating histone 3 lysine 27 (H3K27me3) to modulate gene expression. Genome and transcriptome data obtained by the Pediatric Precision Genomics Program at Riley Hospital for Children at Indiana University Health (IUH) indicate that EZH2 is expressed at high levels in ESFT biopsies. Additionally, other groups have reported that high levels of EZH2 protein in ESFT and other cancers correlate with increased chemoresistance to SOC therapy. We are testing tested the hypothesis that EZH2 contributes to chemoresistance in ESFT by regulating critical growth and survival genes. In addition, we are investigating if pharmacological inhibition of EZH2 will enhance sensitivity to the cytotoxic effects of SOC agents. Pediatric primary and relapsed ESFT cell lines and ESFT xenografts were validated for the EWS/FLI, EZH2, and H3K27me3 signatures. In vitro- and in vivo-pharmacodynamic studies of EZH2 inhibition via tazemetostat were conducted to optimize dosing effect. In ESFT cell lines (TC71, A673, CHLA-9, and CHLA-10), tazemetostat dose-response experiments indicated a significant reduction of H3K27me3 by one day post-treatment which was either sustained or completely blocked by 7-days post-treatment compared to vehicle treated (p<0.001). EZH2-mediated gene regulation was confirmed via qPCR. Inhibition via siRNA or tazemetostat resulted in transcriptional up-regulation of p21 (p<0.05) and down-regulation of C-MYC (p<0.01) in all ESFT cell lines. In TC71 sarcoma xenografts, 200-400 mg/kg BID was well tolerated, and decreased H3K27me3 was evident by 5 and 10 days post-treatment (n=4-5 per cohort, vehicle vs treated, p<0.001). While decreased tumor growth was evident at day 5 post-treatment, growth had recovered by day 10. In vitro assays indicate that tazemetostat potentiates SOC agents, etoposide and irinotecan (SN-38), in mediating ESFT growth inhibition (p<0.001). These data provide rationale for systematic investigation of EZH2 inhibition in combination with SOC. New information will be obtained on how to reprogram the epigenome to mitigate therapeutic resistance in ESFT that overexpress EZH2.

#3181

Class I histone deacetylases (HDAC) critically contribute to Ewing sarcoma pathogenesis.

Oxana Schmidt, Nadja Nehls, Kristina von Heyking, Tim Hensel, Stefan Burdach, Günther H. Richter. _Technical Univ. of Munich, München, Germany_.

Introduction: Recent evidence demonstrated that a low mutation rate seems a general feature of pediatric cancers. Ewing sarcoma (EwS) is defined by balanced chromosomal EWS/ETS translocations, which give rise to oncogenic chimeric proteins (EWS-ETS). Previously, we identified the histone methyl-transferase enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), the enzymatic subunit of the polycomb PRC2 complex, to be over-expressed in EwS. RNA interference of EZH2 suppressed tumor development and metastasis in vivo and microarray analysis of EZH2 knock down revealed an EZH2-maintained, undifferentiated, reversible phenotype in EwS. Microarray analysis further revealed that treatment with MS-275 or TSA resulted in the induction of a similar pattern of differentiation genes as was previously observed after EZH2 blockade and seems to be dependent on histone deacetylase (HDAC) activity.

Experimental procedures: The role of class I histone deacetylases (HDACs) was determined using different potent inhibitors like Trichostatin A (TSA), Romidepsin (FK228), Entinostat (MS-275) and PCI-34051 as well as a CRISPR/Cas9 approach to knock out class I HDACs. To analyze resulting changes microarray analysis, gene set enrichment analysis (GSEA), qRT-PCR, western blotting, proliferation, apoptosis and invasion assays as well as immunofluorescence experiments were deployed.

Results: Interestingly, treatment with enzymatic inhibitors blocking EZH2 activity could not mimic the results observed after EZH2 RNA interference indicating that EZH2-containing PRC2 complexes may serve as a building block of class I HDAC activity in ES. Consequently, in proliferation assays EwS cells were more susceptible to treatment with HDAC inhibitors than other small round blue cell tumors such as neuroblastoma or pediatric cALL cells. Microarray and GSEA analysis demonstrated that treatment with Romidepsin resulted in an inhibition of an EwS specific expression profile while Entinostat suppressed metastasis relevant genes. In addition, EwS cells demonstrated increased susceptibility to treatment with chemotherapeutics such as Doxorubicin and Vincristine in the presence of HDACi. Consequently, CRISPR/Cas9 knock outs of HDAC1 to HDAC3 in EwS cell lines resulted in a reduction of their proliferative ability and greater sensitivity to treatment with chemotherapeutics.

Conclusion: Class I HDAC proteins seem to be important mediators of the pathognomonic EWS-ETS-mediated transcription program in EwS and thus interesting new treatment opportunities for this malignant disease.

#3182

A human neural stem cell glial brain tumor model identifies the relative contribution of different oncogenic elements to malignant transformation.

Youngran Park, Ping An, Dacheng Ding, Charles G. Eberhart, Eric H. Raabe. _Johns Hopkins University, Baltimore, MD_.

Diffuse intrinsic pontine glioma (DIPG) is an invasive malignancy of the brainstem that accounts for greater than 80% of pediatric brainstem gliomas. In the past 40 years, there have been no significant advances in DIPG treatments and survival, so it remains a leading cause of death from pediatric brain tumors. Nearly 80% of DIPG harbor a point mutation in H3F3A or HIST1H3B, and the presence of this H3.3K27M mutation is inversely correlated with reduced OS, suggesting that epigenetic dysregulation is a key driver to cause the pathogenesis of DIPG. However, it is unclear how the H3.3K27M mutation and the other common alterations in DIPG contribute to tumorigenicity in human neural stem cells. To address the relative contributions of different oncogenic elements to DIPG, we sought to add common DIPG elements in a stepwise fashion to human neural stem cells derived from the developing hindbrain. We chose oncogenic elements that are known to present in DIPG, including the stem cell factor BMI1, mutant (R248W) p53, H3.3K27M, constitutively active AKT (representing the activation of mTOR detected in the vast majority of DIPG) and hTERT. We found that the combination of the stem cell factor BMI1 along with mutant (R248W) p53 and hTERT could immortalize neural stem cells, but was insufficient to form orthotopic xenograft tumors when placed in the pons of immunodeficient mice. Addition of activated AKT led to aggressive tumor formation, with a glial phenotype as evidenced by robust GFAP expression and absence of synaptophysin expression. All mice bearing these four genetic alterations succumbed to their tumors within 100 days of implantation. We found that introduction of H3.3K27M mutation reduces the level of tri-methylation of H3.3KK27 to that seen in patient-derived DIPG cell lines. Furthermore, we found that H3.3KK27 mutation introduction increases expression of LIN28B, a stem cell factor, which is also found in patient-derived DIPGs. Our previous study showing that LIN28A, another family member of LIN28 proteins, regulates invasion and tumorigenicity in adult high-grade gliomas suggests that H3.3K27M mutation might facilitate invasiveness of DIPG through LIN28B and its downstream effectors HMGA2, SNAI1, and SLUG. In summary, we have developed a human hindbrain neural stem cell DIPG model that has both accurate cell of origin and genetic defects including H3.3K27M mutation. Our models allow for assessment of the relative contribution to transformation of each genetic element in a stepwise fashion in the likely cell of origin of this deadly tumor.

#3183

Exosomes secreted under hypoxia enhance aggressiveness in Ewing's sarcoma.

Matthew Kling, Donald Coulter, Nagendra Chaturvedi, Timothy McGuire, John Sharp, Shantaram Joshi. _University of Nebraska Medical Center, Omaha, NE_.

Ewing's sarcoma (EWS) is a malignant tumor that primarily develops in children and adolescents. The main driver in EWS tumor progression is the result of a chromosomal translocation, t(11;22)(q24;q12), leading to the EWS-FLI1 fusion protein that functions as an oncogenic transcription factor. EWS is curable if diagnosed early, however, patients presenting with metastasis upon diagnosis have the worst prognosis of all the bone tumors. There is emerging evidence that a low oxygen (hypoxic) microenvironment contributes to increased EWS-FLI1 expression in EWS tumors and induces cellular changes observed in many cancers leading to the selection of clones with tumor initiating cell (TIC) properties. TICs are widely considered to be responsible for relapse, metastasis, and therapeutic resistance in many tumors. The effects of hypoxia are mediated primarily by hypoxia inducible factors HIF-1α and HIF-2α and have been demonstrated to upregulate EWS-FLI1 and stem cell-related factors, but the mechanism by which hypoxia promotes aggressiveness of this normally treatable cancer is unclear. In our study, we hypothesize that exosomes secreted under hypoxic conditions enhance the aggressiveness in Ewing's sarcoma by promoting tumor initiating cells in EWS cells and in stromal cells of the tumor microenvironment. This hypothesis is based on evidence in the literature showing that small vesicles (exosomes) released from cancer cells can reprogram metabolic and cellular function of both tumor cells and cells that compose the tumor microenvironment. In our study, RNAseq and pathway analysis of exosomes secreted from hypoxic EWS cells revealed high levels of the EWS-FLI1 fusion transcripts and stem cell related factors CD133, SOX2, OCT4, NANOG and MYC. Moreover, various co-culturing assays demonstrated that hypoxic EWS exosomes can increase cell motility, anchorage independent growth, chemoresistance and TIC formation in EWS cells, and transform microenvironment cells. Our in vivo model revealed that EWS exosomes injected into developing EWS tumors in mice significantly increased tumor formation. EWS tumors injected with hypoxic exosomes had increased expression of the CD133 TIC marker when analyzed by flow cytometry. These findings suggest that hypoxic EWS exosomes can enhance aggressiveness in EWS by promoting TIC formation, therapy resistance, invasiveness and motility in their target cells. Here, we describe a mechanism where EWS-FLI1 containing exosomes enhance TIC formation and introduce a new pathway that must be considered to effectively treat aggressive EWS tumors.

#3184

**A secondary mutation in** BRAF **confers resistance to RAF inhibition in a** BRAF **V600E-mutant brain tumor.**

Jiawan Wang,1 Zhan Yao,2 Philip Jonsson,2 Amy Allen,1 Alice Can Ran Qin,1 Sharmeen Uddin,2 Ira J. Dunkel,2 Mary Petriccione,2 Katia Manova,2 Sofia Haque,2 Marc Rosenblum,2 David J. Pisapia,3 Neal Rosen,2 Barry S. Taylor,2 Christine A. Pratilas1. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 3 _Weill Cornell Medical College, New York, NY_.

BRAF V600E hyperactivates ERK and signals as an RAF inhibitor-sensitive monomer. While RAF inhibitors can produce impressive clinical responses in patients with mutant BRAF tumors, the mechanisms of resistance to these drugs are incompletely characterized. Here, we report a complete response followed by clinical progression in a patient with a BRAF V600E-mutant brain tumor treated with dabrafenib. Whole-exome sequencing revealed a secondary BRAF L514V mutation at progression that was not present in the pretreatment tumor. Expressing BRAF V600E L514V induces ERK signaling, promotes RAF dimer formation, and is sufficient to confer resistance to dabrafenib. A novel class of RAF dimer inhibitors and an ERK inhibitor are effective against BRAF L514V-mediated resistance. Collectively, our results validate a novel biochemical mechanism of RAF inhibitor resistance mediated by a secondary mutation, emphasizing that like driver mutations in cancer, the spectrum of mutations that drive resistance to targeted therapy are heterogeneous and perhaps emerge with a lineage-specific prevalence.

#3185

Whole genome sequencing for precision medicine in high-risk neuroblastoma patients: Translation from research to clinical practice in Sweden.

Susanne M. Fransson,1 Angela Martinez-Monleon,1 Susanne Reinsbach,1 Per Sikora,1 Rosa-Marie Sjoberg,1 Per Kogner,2 Tommy Martinsson1. 1 _Univ. of Gothenburg, Gothenburg, Sweden;_ 2 _Karolinska Institutet, Stockholm, Sweden_.

The overall survival of children presenting high-risk neuroblastoma is about 40 %, implying an urgent need for rapid improvement in treatment options and strategies for this particular patient group. Clinical use of next-generation sequencing provides a comprehensive approach for rapid determination of genomic biomarkers for diagnosis/prognosis, and of foremost importance, to achieve precision treatment based on the patients' specific tumor targets. Whole genome sequencing (WGS) was performed on tumor DNA and constitutional DNA for 30 neuroblastoma patients, both retrospective samples and a subset of patients with progressive disease despite ongoing treatment. Sequencing was performed using Illumina instrumentation for an average coverage of at least 60X and 30X for tumor and normal tissue respectively. Read trimming, mapping to hg19 and variant calling were performed using the CLC Genomics Workbench software while copy number profiles were prepared through the CANVAS software. Systematic filtering was performed using the Ingenuity variant analysis tool. Variants with allele frequency above 3% in common population based cohorts were discarded as well as excluding all synonymous variants or variants in non-coding regions except those affecting canonical splice sites. Remaining variants were assessed manually through the Integrative Genomics Viewer. Somatic variants and CNVs were evaluated through Qiagen Clinical Insight in relation to clinical significance and action-ability. In our present setting the approximate time for handling is 2,5-3 weeks, counting from arrival of material to analyzed data. The analysis includes copy number variations, structural aberrations and mutational screening. These alterations have then been thoroughly evaluated with regards to biological relevance and druggability, looking for drugs either used in existing cancer treatment or in clinical trials. Among the 30 patients, potentially actionable targets were detected in 14 (47%) of the cases; including aberrations in ALK, CDK4/MDM2, CDKN2A, CCND1, ROCK2 or FLT1. Inconclusive results were seen for 16 patients. This included seven patients where no actionable target were detected and nine patients with weak support for targeted treatment. The latter group includes patients with either TERT-rearrangements or alterations in different mitogen-associated kinases. Our results demonstrate the feasibility to incorporate WGS in clinical practice. A logistic and analytic pipeline has been established in order to present the results in a clinically meaningful time frame. Actionable targets that could be relevant in therapy were detected in 14 out of 30 neuroblastoma patients. The result from this study show promising results and we predict that integration of WGS in clinical routine will lead to improved management of the therapeutically most challenged group.

#3186

Tegavivint suppresses progression and metastasis of osteosarcoma via blockade of Wnt signaling/ALDH1 axis: Preclinical study of a novel Wnt/β-catenin pathway inhibitor.

Motonari Nomura,1 Nino C. Rainusso,1 Ruolan Han,2 Jeff Larson,2 Ryan L. Shuck,1 Lyazat Kurenbekova,1 Jason T. Yustein1. 1 _Texas Children's Cancer and Hematology Center, Baylor College of Medicine, Houston, TX;_ 2 _Beta Cat Pharmaceuticals, Houston, TX_.

Osteosarcoma (OS) is the most common bone cancer in children and adolescents, and patients with metastatic disease still have extremely poor prognosis and no effective targeted therapy. It has been reported that the activation of Wnt/β-catenin pathway is closely associated with OS development and metastatic progression. Tegavivint (BC2059), a novel small molecule inhibitor of the Wnt/β-catenin pathway, has been reported to be active against multiple types of cancer cells in vitro, and anti-tumor efficacy has been published in animal models of acute myeloid leukemia and multiple myeloma. In this study, we investigated the antitumor activity of Tegavivint against metastatic OS both in vitro and in vivo using established human OS cell lines (143B, SaOS-2, LM7) and patient-derived xenograft (PDX) models. In vitro, Tegavivint effectively inhibited tumor cell survival in a dose-dependent manner in all established OS cell lines and OS PDX-derived cells. Subsequent in vivo studies using an orthotopic model of LM7 cells engrafted in the tibia of NSG mice demonstrated complete regression of the primary tumors in all treated mice as well as a significant reduction in lung metastasis by the treatment with Tegavivint. We further examined the activity of Tegavivint using a pair of PDX models derived from an inherently chemo-resistant OS patient at different disease stages: PDX63 was derived from the pre-treatment biopsy of the primary tumor, and PDX84 was derived from the relapsed metastatic lung lesion of the same patient after chemotherapy. The growth of subcutaneously engrafted PDX63 tumors was significantly suppressed by the treatment with Tegavivint alone and Tegavivint enhanced the antitumor activity of doxorubicin. Analysis of mRNA expression by qPCR in the tumor tissue demonstrated significant downregulation of critical genes by Tegavivint, including c-Myc and ALDH1, which is a potential marker for cancer stem cells. Furthermore, in an innovative lung metastasis model using PDX84 tumor-dissociated cells intravenously (IV) injected in NSG mice, we demonstrated that Tegavivint treatment markedly reduced the number of lung metastases. Finally, PDX84-derived cells were sorted into ALDH1-high and ALDH1-low populations. ALDH1-high cells showed higher expression of β-catenin and higher sensitivity to Tegavivint than the ALDH1-low cells in vitro and subsequent IV injection of ALDH1-high PDX84 or ALDH1-low PDX84 cells demonstrated significantly more lung lesions in the ALDH1-high group than the ALDH1-low group. The number of ALDH1-high derived lung metastases was significantly suppressed by Tegavivint. Taken together, our preclinical findings demonstrate that Tegavivint has promising therapeutic potential for primary and metastatic OS through the blockade of Wnt signaling /ALDH1 axis.

#3187

Pediatric Preclinical Testing Consortium evaluation of the menin inhibitor, VTP-50469, against xenograft models of MLL-rearranged infant acute lymphoblastic leukemia.

Richard B. Lock,1 Kathryn Evans,1 Tara Pritchard,1 Stephen W. Erickson,2 Yuelong Guo,2 David A. Claremon,3 Gerard M. McGeehan,3 Beverly A. Teicher,4 Malcolm A. Smith4. 1 _Children's Cancer Institute, Sydney, Australia;_ 2 _RTI International, Research Triangle Park, NC;_ 3 _Vitae Pharmaceuticals, Fort Washington, PA;_ 4 _National Cancer Institute, Bethesda, MD_.

Introduction: Rearrangements involving the MLL (mixed lineage leukemia, KMT2A) gene (MLLr) occur broadly in acute leukemia, in ~80% of infant acute lymphoblastic leukemia (ALL) cases, and are associated with poor outcome. Menin is a ubiquitously expressed nuclear protein, and the MLLr-menin interaction is the key impetus for transformation of MLLr-expressing cells. VTP-50469 is a potent, orally available, small molecule inhibitor of the MLL-menin interaction with pM binding affinity for menin. Therefore, it was of interest to test the in vivo efficacy of VTP-50469 against preclinical models of infant MLLr-ALL.

Methods: Infant MLLr-ALL patient-derived xenografts (PDXs) grew in an orthotopic manner in NSG mice. Engraftment and response to treatment were assessed by enumeration of the % human leukemic blasts in the murine peripheral blood (%huCD45+). Treatment commenced when the median %huCD45+ exceeded 1%, and mice received VTP-50469 (120 mg/kg by oral gavage twice daily x 28) or vehicle. An event was defined as the %huCD45+ >25% or leukemia-related morbidity. The Kaplan-Meier method was used to compare event-free survival (EFS) between treated (T) and control (C) groups. Stringent objective response measures were assigned to each mouse and reported as group medians (Houghton et al, Pediatr. Blood Cancer, 2007;49:928-40). Leukemia infiltration into the femoral bone marrow was also assessed at Day 28 following treatment initiation. VTP-50469 was provided by Vitae Pharmaceuticals.

Results: VTP-50469 was well tolerated, with maximum average weight losses of 1.6-6.4% across treatment groups compared to 0-2.0% in vehicle control treated groups. VTP-50469 induced significant differences in EFS distribution compared to control in 6 of 6 (100%) of the evaluable MLLr-ALL PDXs. VTP-50469 T-C values in MLLr-ALL PDXs ranged from 1.2 to 100 days (T/C 1.3-21.1), and Maintained Complete Responses (MCRs) were observed in 5 of 6 PDXs. Two of 8 mice engrafted with an MLLr-ALL harboring the MLL-AFF1 (t4;11) translocation had not reached event >230 days following treatment initiation. A significant reduction (P<0.001) in bone marrow infiltration at Day 28 was observed in 2 of 4 evaluable MLLr-ALL PDXs. VTP-50469 at 30 mg/kg (4-fold lower than its maximum tolerated dose) also elicited MCRs in 8 of 8 mice engrafted with an MLL-AFF1 PDX. The on-target activity of VTP-50469 was verified by its lack of efficacy against an ALL PDX harboring the BCR-ABL1 translocation.

Conclusions: VTP-50469 exerted profound in vivo efficacy against ALL PDXs derived from infants harboring MLL-AFF1, MLL-GAS7, and MLL-ENL translocations, and significantly reduced leukemia infiltration in the bone marrow. VTP-50469 was also effective across a broad dose range, indicating that it may represent a novel treatment for MLLr leukemia. (Supported by NCI Grants CA199222 & CA199000)

#3188

Combination treatment strategies to enhance the effect of standard chemotherapeutic agents in Ewing sarcoma cells.

Anish Ray,1 Sagar Shelake,2 Umesh Sankpal,2 Lina Albeer,2 Holly Lout,2 Abigail Hunter,2 Kathryn Dunlap,2 Rajasekhar Maram,2 W. Paul Bowman,2 Riyaz M. Basha2. 1 _Cook Children's Medical Center, Fort Worth, TX;_ 2 _UNT Health Science Center., Fort Worth, TX_.

EWS/FLI1 is a unique chimeric fusion protein found in approximately 85% of Ewing sarcoma (ES) tumors. Blocking this fusion protein in conjunction with administration of anticancer agents could be an effective strategy for treating ES. Baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5), also known as survivin, is overexpressed in several malignancies, including ES. Survivin is associated with aggressive growth of cancer and resistance to standard chemotherapy and thus serves as a prognostic marker for ES and other cancers. EWS/FLI1 and Specificity protein1 (Sp1) jointly mediate the expression of cyclins to facilitate cell growth. Mithramycin-A (Mit-A) has been identified as a potent inhibitor of EWS-FLI1 and its downstream targets. Preclinical research from our laboratory demonstrated that clotam, a small molecule, exhibits anticancer activity by targeting Sp1 and survivin. The objective of our investigation is to test the efficacy of Mit-A or clotam to induce the antiproliferative activity of standard chemotherapeutic agents, vincristine (Vin) or etoposide (Eto), in ES cell lines. TC71, TC32, CHLA32, CHLA10 and TC205 cells were treated with increasing concentrations of Mit-A/clotam/Vin/Eto. Cellular growth inhibition was evaluated at 48 h post-treatment using CellTiter-Glo kit (Promega). In order to determine the combination response, cell growth inhibition of each chemotherapeutic drug was assessed in the presence or absence of Mit-A or clotam. Further studies were performed to investigate the effect of individual agents and combinations on apoptosis and cell cycle phase distribution. Levels of cleaved poly (ADP-ribose) polymerase (c-PARP) were determined by Western blot analysis and the apoptotic cell population quantitated by flow cytometry analysis of Annexin-V stained cells. Cell cycle phase distribution was also measured by flow cytometry using propidium iodide. Results showed a dose-dependent antiproliferative effect with all agents. The combination of Mit-A or clotam with Vin or Eto showed greater effect on apoptosis and cell cycle arrest compared to the effects of Vin or Eto alone. Overall, these results indicate the potential clinical advantage of combination treatment involving Mit-A or clotam for inhibiting the growth of ES cells. Molecular profiling analysis is under way to elucidate the candidate pathways associated with beneficial effect of our combination treatments and with other chemotherapeutic agents.

#3189

Targeting NPM1 for controlling growth of Ewing sarcoma.

Geri Traver,1 Konjeti R. Sekhar,1 Narsimha R. Renthala,2 Peter A. Crooks,2 Maxwell Ofori,1 Michael L. Freeman1. 1 _Vanderbilt Univ. School of Medicine, Nashville, TN;_ 2 _University of Arkansas for Medical Sciences, Little Rock, AR_.

Ewing sarcoma is a rare cancer found in children and young adults. Long term survival rates are approximately 70% for patients who present without clinically overt metastases. However, 5 year survival is less than 20% for patients with recurrent tumors or metastasis. Thus, there is a need to improve local tumor control and to treat metastatic disease. Eighty five percent of these cancers are driven by the reciprocal chromosomal translocation t (11;22) (q24;q12), a consequence of a fusion between the 5' portion of Ewing sarcoma breakpoint region 1 on EWSR1 (chromosome 22) and the 3' portion of Friend leukemia virus integration site 1 on FLI1 (chromosome 11). Nucleophosmin1 (NPM1) is a chaperone protein involved in many cellular functions, including DNA repair. NPM1 has been shown to be a novel prognostic biomarker for patients with localized Ewing's sarcoma. Overall survival is significantly lower for patients whose tumors express high levels of NPM1. Besides promoting repair of DNA double strand breaks NPM1 directly interacts with and induces c-Myc-induced hyperproliferation and transformation. Consistent with this knowledge is the observation that relapsed Ewing's patients exhibit high levels of c-Myc expression compared to disease-free patients. Therefore, we wanted to ascertain whether NPM1 represented a molecular target for controlling growth of Ewing's sarcoma. CRISPR/Cas9-mediated targeting of NPM1 exons 3 and 4, as well as lentivirus-mediated shRNA targeting of NPM1 mRNA revealed that targeting of NPM1 expression reduced survival (colony formation) of TC32 and A673 cells (P<0.05). NPM shRNA also reduced TC32 sphere formation under non-adherent conditions. YTR107 and NSC348884 are small molecule inhibitors of NPM1 oligomerization, which is required for NPM1 activity. Both drugs reduced survival of TC32 and A673 Ewing's sarcoma cells in a dose-dependent fashion (P<0.05). We interpret these data to indicate that NPM1 represents a potential molecular target for controlling growth of Ewing's sarcoma. Supported in part by RO1CA140409.

#3190

DFMO preferentially drives cancer stem cells in neuroblastoma towards senescence.

Tracey Avequin, Austin Goodyke, Joseph Zagorski, Tyler Maser, Giselle L. Saulnier Sholler. _Helen DeVos Children's Hospital, Grand Rapids, MI_.

Background: High-risk neuroblastoma (HRNB) is an aggressive form of the most common extracranial solid tumor cancer in children, often characterized by amplified levels of MYCN. HRNB patients in remission have a high relapse rate, resulting in poor survival. Recent studies have shown that cancer stem cells (CSCs) play a role in the progression, chemoresistance, and relapse of many cancers. Difluoromethylornithine (DFMO) is being evaluated to prevent relapse in clinical trials. Our current study examines the in vitro and in vivo effects of DFMO on tumor-initiating cells through evaluating CSC viability, CSC markers and senescence.

Methods: BE(2)-C and SMS-KCNR established HRNB cell lines were injected subcutaneously at limiting cell dilutions into nude mice. DFMO was administered in the drinking water (2%, by volume) for 50 and 75 days for BE(2)-C and SMS-KCNR, respectively. Tumor formation was monitored, and LDA was performed. In a second xenograft model BE(2)-C cells were injected into nude mice, once tumors were palpable, the mice were given either normal drinking water (vehicle) or DFMO (2%)-containing water. Following 7 days of treatment, the tumors were resected and measured for volumes/weights as well as the harvested for Western blot analysis of CSC markers (OCT4, SOX2, NANOG, KLF4). BE(2)-C cells were cultured for 48 and 72h with or without 5mM DFMO and stained for SA-βGal activity as a marker of senescence. NB cell lines treated for 48 and 72h with a range of DFMO concentrations (0, 10μM, 50μM, 100μM, 1mM, 2.5mM, and 5mM) were harvested and labeled with cell surface antibodies for CSCs (CD133, CD114), senescence (CD148), differentiation (CD24), and NB (CD56) for 30min in the dark, and cell viability was determined by 7AAD staining immediately before flow cytometric analysis.

Results: Limiting dilution analysis (LDA) on xenograft mice receiving DFMO showed both prevention of tumor formation (p value<0.001) and a decrease in frequency of tumor-initiating cells (p value p<0.04). Tumors harvested from mice treated with DFMO for 7 days showed decrease in volume and weight relative to control (p value <0.001) as well as a decrease in the presence of CSC markers shown by Western blot. Galactosidase (SA-βGal) staining of cultured HRNB cells showed the induction of senescence in HRNB cells when treated with DFMO by 72h (p value <0.001). Flow cytometric analysis determined that DFMO specifically targeted the CSC subpopulation at as low as 50uM concentrations (100uM p value<0.05); CSCs had decreased viability and higher levels of senescence-associated protein expression than the overall NB population (p value<0.05).

Conclusions: Our results show the preferential induction of senescence and overall decrease in the CSC population at very low doses of DFMO. This may expose a novel treatment mechanism to inhibit CSC function and prevent relapse in HRNB patients.

#3191

The MDM2 inhibitor CGM097 synergizes with the BET inhibitor OTX015 to induce cell death in neuroblastoma cells.

Tyler P. Maser, Joseph W. Zagorski, Austin J. Goodyke, Elizabeth A. VanSickle, Jeffrey P. Bond, Giselle L. Saulnier Sholler. _Helen DeVos Children's Hospital, Grand Rapids, MI_.

Background: Neuroblastoma (NB) is the most common extracranial solid tumor in infants and children, with amplification of the oncogene MYCN being a hallmark of high-risk disease and poor prognosis. Most NB tumors initially respond to chemotherapy; however, over half of these cases relapse, resulting in chemoresistant disease. In this stage of NB there is evidence of p53 inactivation, which suggests a role for p53 in chemo-sensitivity. Although highly mutated in other cancer types, p53 mutations in NB are rare. Previous data have shown that amplification or deregulation of MDM2, a negative regulator of p53, may be the more common mechanism of p53 suppression and chemoresistance in NB. MYCN and MDM2 have been shown to interact and contribute to NB growth and disease progression. In vitro treatment of NB cells with MDM2 inhibitors has shown promise in increasing the expression of p53, leading to a decrease in proliferation, and increasing apoptosis. BET (Bromodomain and Extra-Terminal domain) inhibitors have also been shown to be effective in treating NB cells in vitro, decreasing MYCN expression, and resulting in increased apoptosis and differentiation. We hypothesize that the combination treatment of a MDM2 inhibitor (CGM097) with a BET inhibitor (OTX015) will result in greater p53 activation and a subsequent synergistic increase in NB cell death.

Methods: Single-agent IC50 values for both CGM097 and OTX015 were obtained for the established NB cell lines SMS-KCNR, SH-SY5Y, BE(2)-C, and CHLA-90 using Calcein AM and Cell Titer-Glo cell viability assays after 48 hours of treatment. Drug combination studies were performed to evaluate synergism between CGM097 and OTX015 in the treatment of NB cells. IncuCyte ZOOM live cell imaging was used for kinetic monitoring of apoptosis with single-agent and combination drug treatment in NB cells. Western blot analysis was used to determine protein levels of apoptosis (Caspase-3) and MYCN/p53 pathway targets (MYCN, MDM2 and p53).

Results: IC50 values for the four established NB cell lines ranged from 0.384 µM-13.5 µM and 7.25 µM-42.79 µM for CGM097 and OTX015, respectively. The combination treatment exhibited synergistic effects in wild-type p53 NB cells, with supporting data obtained from IncuCyte imaging tracking real-time cell death. IncuCyte showed induction of caspase-mediated apoptosis confirmed by Western blot analysis of Cleaved Caspase 3. Pathway analysis shows the importance of wild-type p53 for treatment effect.

Conclusion: This study indicates that the combination of CGM097 and OTX015 synergistically decreases viability in NB cells with wild-type p53 expression. Further in vitro and in vivo work will be necessary to elucidate the mechanisms behind this interaction and in vivo efficacy.

#3192

Aptamers, antibodies and radiotherapy for the treatment of DIPG.

Naiara Martinez-Velez,1 Miguel Marigil,2 Javier Aristu,3 Luis Ramos,1 Fernando Pastor,4 Ana Patiño-García,1 Marc García-Moure,1 Juan Fueyo,5 Candelaria Gomez-Manzano,5 Ricardo Diez-Valle,1 Sonia Tejada,1 Marta M. Alonso1. 1 _FIMA - University Clinic of Navarra, Pamplona (Navarra), Spain;_ 2 _Toronto Western Hospital, Pamplona (Navarra), Ontario, Canada;_ 3 _University Clinic of Navarra, Pamplona (Navarra), Spain;_ 4 _FIMA, Pamplona (Navarra), Spain;_ 5 _UT MD Anderson Cancer Center, Houston, TX_.

Diffuse intrinsic pontine gliomas (DIPGs) is one of the most aggressive pediatric brain tumors. Radiotherapy (RT) constitute the standard of care of these tumors being its therapeutic effect just palliative. There is plenty of evidence that RT is able to activate immune responses through the induction of immunogenic cell death. However, it also elicits immunosuppressive effects and enforces immunological tolerance. The result is that in DIPGs the RT effect is just transitory. In this context, immune costimulatory or inhibitory molecules with the capability to boost the immune effect of RT could be amenable agents to use in combination to treat DIPGs. Agonist antibodies has been widely used in immunotherapy. Aptamers are high-affinity single-stranded nucleic acid ligands that exhibit remarkable affinity and specificity to their targets, comparable or exceeding that of antibodies. 4-1BB is a major costimulatory receptor promoting the survival and expansion of activated T cells. TIM-3 is a negative regulator of lymphocyte function that is involved in T-cell exhaustion. To this end, we examined the effect of RT in combination with either an agonist 4-1BB antibody or an aptamer against TIM-3. Importantly, both combined treatments showed a safe profile. Moreover, combination treatment of 4-1BB agonist antibody or TIM-3 aptamer with RT resulted in a significant improvement in the median survival of mice bearing DIPG orthotopic tumors when compared with single treatment in around 20 days (P=0.001 and P=0.04, for the combination of RT and 4-1BB or TIM-3,respectively). In addition, both combination led to long-term survivors (90 days). Rechallenge experiments in these animals showed the generation of memory against the tumors in both combined treatment. Mechanistic studies performed on day 16 showed an increase in CD8 effector cells, a decrease in T-regulators Foxp3+ cells and an increase in INF-gamma expression suggesting the triggering of an antitumor-immune response. Our data underscore that combination of RT with immune-boosting strategies for DIPGs are worth exploring.

#3193

Combination treatment with selinexor and bortezomib for management of highly aggressive neuroblastoma.

Basia Galinski,1 Marcus Luxemburg,1 Yosef Landesman,2 Daniel Weiser1. 1 _Albert Einstein College of Medicine, Bronx, NY;_ 2 _Karyopharm Therapeutics, Boston, MA_.

Background: Neuroblastoma is an embryonic tumor of the peripheral nervous system. Comparative proteomics of high-risk neuroblastoma patients with poor prognosis outcomes demonstrated increased expression of Exportin-1 (XPO1), a nuclear transport protein with over 200 recognized cargo, in those who do not survive. Inhibition of nuclear export with the selective inhibitor of nuclear export, orally bioavailable drug molecule selinexor (Karyopharm Therapeutics) has shown anticancer activity. Treatment decreased tumor burden and increased survival in animal models. Advance-phase clinical trials using selinexor in hematologic and solid cancers are ongoing. In neuroblastoma cell lines, we have found correlation between decreased steady state protein levels of IkB, the inhibitor of NF-kB, and increased XPO1 protein expression. Likewise, proteasome inhibition with bortezomib leads to decreased NF-kB levels and decreased proliferation in neuroblastoma cells. We hypothesize that combination treatment of selinexor and bortezomib will have a greater than additive effect on the inhibition of neuroblastoma cellular proliferation. Such effect would be due in part to decreased NF-kB transcription activity through stabilization of IkB as a result of proteasome activity inhibition and forced localization of IkB into the nucleus where it may be protected from degradation.

Methods: Neuroblastoma cell lines were used for all experiments. MTT assays were used to generate dose-response curves for single-agent selinexor and bortezomib, as well as sequential treatment combination (bortezomib then selinexor six hours later). Apoptosis and cell cycle regulation were assessed for all treatment conditions. NF-kB activity was measured by ELISA in cell lysates with decreased XPO1 expression through siRNA knockdown of XPO1. Western analysis and immunofluorescence were used to quantify NF-kB and IkB protein expression in treated and knockdown conditions.

Results: Sequential combination treatment showed decreased cell viability compared to treatment with a single agent. Western analysis of NF-kB in treated single- and double-agent conditions saw decreased protein expression of NF-kB active and inactive forms. We expect flow cytometry experiments to demonstrate increased levels of apoptosis and cell cycle arrest. Future ELISA analysis of siRNA and combination treated cells will assess NF-kB transcription activity.

Conclusions: Regulation of IkB protein expression through nuclear localization as well as decreased proteasome degradation can halt NF-kB-driven cellular proliferation. Combination therapy with selinexor and bortezomib has potential to be effective against highly aggressive neuroblastoma that is, in part, driven by NF-kB. Future work will define additional pathways altered with combination treatment and guide in vivo testing and clinical development of this therapeutic strategy.

#3194

**Development of an** in vitro **drug profiling platform with primary Rhabdomyosarcoma cells for tailoring patient-specific treatments.**

Marco Wachtel, Gabriele Manzella, Michaela Römmele, Luduo Zhang, Joelle Tchinda, Felix Niggli, Beat Schäfer. _University Children's Hospital Zurich, Zurich, Switzerland_.

The paradigm of cancer therapy currently shifts from broadly used cytotoxic drug cocktails to patient specific precision therapies. These novel therapies are normally directed towards tumor specific driver oncogenes, identified by genomic analysis of individual tumors. In case of tumors with a very low mutational burden and/or currently undruggable driver oncogenes this approach is less suitable. There, direct drug profiling of in vitro cultured tumor cells represents a promising alternative for identification of patient specific therapies. Conditions to maintain primary tumor cells in culture for this purpose however are largely unknown for most tumor entities. Here, we aimed to establish an in vitro drug profiling platform for Rhabdomyosarcoma (RMS), a childhood cancer with above described characteristics. Towards this aim, we isolated cells from 6 alveolar and 8 embryonal RMS patient derived xenografts and systematically tested 18 different culture conditions in order to find suitable parameters for in vitro propagation of primary RMS cells. This approach revealed that cells from most tumors survive and proliferate only in serum-free medium, whereas in serum containing medium tumor cells are progressively lost over time. Importantly, cells preserve the clonal composition and phenotypic characteristics of the primary PDX under these conditions, as assessed by genomic and copy number analysis. Drug profiles established with a library of 204 drugs revealed, beside patient-specific vulnerabilities, a yet unrecognized sensitivity of a subgroup of alveolar RMS towards AKT inhibitors. Furthermore, screening with the same drugs for resensitization of resistant relapse samples identified the BCL-2 family inhibitor ABT-263 as most potent resensitizer towards standard-of-care chemotherapy. Detailed molecular analysis revealed that this effect is based on blockade of the BCL-XL-MCL-1 axis. Overall, our proof of concept study highlights the feasibility of in vitro drug profiling of primary RMS cells for patient-specific treatment selection in a co-clinical setting.

### Radiation Studies Using in Vitro and Computational Models

#3195

The molecular mechanism of action and cellular targets of TTFields.

Aarat P. Kalra,1 Jack Xiao,1 Cameron Hough,1 Piyush Kar,1 Vahid Rezania,2 John D. Lewis,1 Karthik Shankar,1 Jack A. Tuszynski1. 1 _University of Alberta, Edmonton, Alberta, Canada;_ 2 _MacEwan University, Edmonton, Alberta, Canada_.

Tumor Treating Fields (TTFields) are AC electric fields of intensity 1-2 V/cm in the frequency range 100-300 kHz, which have been shown to be an effective FDA-approved adjuvant therapy for Glioblastoma Multiforme. However, the mechanism of action for TTFields is not well understood. It is known that microtubules (MTs) and actin filaments re-orient themselves and behave abnormally when subjected to external electric fields indicating that they may be cellular targets of TTFields. The purpose of our study was to determine the molecular mechanism of action of TTFields. Previously, using impedance spectroscopy we observed the effect of unpolymerized tubulin and microtubules on ionic conductivity of buffer solution, and found that unlike tubulin, microtubules increased electrical conductance, which peaked at TTField-like frequencies1. Microtubules have been modeled as conductive cables that attract and guide counterions to increase the solution's conductance. protonic transport through the lumen of the microtubule has also been modeled. To investigate the additional possibility of electronic transport along α, β- tubulin dimers we are performing spectroscopic characterization of MTs. Using Dynamic Light Scattering (DLS) we have characterized the thickness of the solvation layer around the tubulin dimer, which elucidates the mechanism of ionic conduction. We have found that the thickness of the solvation layer increases as the concentration of a highly polar molecule such as DMSO is increased. We have found that increasing the solution temperature leads to a reduction of the solvation layer's thickness and have also characterized response to changes in pH of solution. We have polymerized various morphologies of tubulin assemblies, e.g. planar zinc sheets and (300-500 nm diameter) macrotubes to gain insight into associated conductivity processes. We have imaged these interesting morphologies using Transmission Electron Microscopy (TEM) and epifluorescence microscopy. Impedance spectroscopy of microtubules and actin filaments decorated with ligands such as microtubule associated proteins and drugs and anesthetics is under way. Numerical estimates of the magnitudes of the currents, energy and power generated in a cancer cell using TTFields have been computed. This research provides physical insights into the subcellular mechanisms involved in TTField therapy and can assist in its' optimization.

1.Santelices, Iara B., et al. "Response to Alternating Electric Fields of Tubulin Dimers and Microtubule Ensembles in Electrolytic Solutions." Scientific Reports 7.1 (2017): 9594.

#3196

Investigation of mechanism on photoimmunotherapy focused on cell membrane damage.

Kanta Ando, Kohei Nakajima, Hideo Takakura, Mikako Ogawa. _Hokkaido University, Sapporo, Japan_.

Objectives: Photoimmunotherapy (PIT) is a new type of cancer treatment using IR700, a hydrophilic near-infrared (NIR) photosensitizer, conjugated to monoclonal antibodies. However, the mechanism of the phototoxicity has not been clarified well. Previous study showed that the major cytotoxic mechanism of PIT is different from conventional photo-therapies which require singlet oxygen, and the cells were swelled in the process of cell death induced by PIT. We hypothesized that cell plasma membrane damage should be an important trigger to cause cell death in PIT. In order to verify the hypothesis, we investigated the plasma membrane permeability during PIT and photoresponse property of PIT agent.

Methods: To evaluate the permeability of the plasma membrane during PIT, inflow or outflow of various sized compounds, such as Na+ (diameter 0.1 nm), 111In3+ (0.09 nm), 111In-DTPA (1.0 nm), calcein (1.4 nm), or EthD-1 (2.6 nm) was investigated after NIR light irradiation to NIH3T3/HER2 cells treated with IR700 conjugated to trastuzumab, an anti-HER2 antibody. We also investigated the permeability under high osmotic pressure conditions by small or large molecules such as NaCl or dextran (3.7 nm), respectively. To evaluate the photoresponse property of PIT agent under various conditions, IR700 was irradiated by the NIR light in PBS in the presence of electron donors or accepters with or without oxygen. The irradiated solution was analyzed by HPLC and the photolytes were characterized.

Results: Na+ flowed into cells immediately after NIR light irradiation although intracellular calcein did not flow out from the cells. Calcein was flowed out within 3 min after the irradiation and EthD-1 gradually flowed into cells at 30 min. The uptake of 111In3+ was almost the same at 3 min and 60 min after the irradiation although that of 111In-DTPA increased at 60 min compared to 3 min. The uptakes of these compounds were not observed under the high osmotic pressure condition by dextran. HPLC analyses of the irradiated solution with electron donors showed that the photolytes were lipophilic. In high concentration of IR700, hydrophobic precipitations were observed after NIR light irradiation. IR700 was photodegradable in the presence of electron donors, and the degradation was promoted under oxygen depletion condition.

Discussion: We found that the size of initial damage on the cell membrane immediately after NIR irradiation was tiny that only small ions flowed into cells. Then, the size gradually increased with time and finally was larger than macromolecules, indicating that the tiny cell membrane damage should be the initial step of cell death induced by PIT. We also found that the hydrophilicity/hydrophobicity of IR700 was drastically changed. We considered the change of physiochemical property must be related to cell membrane damage.

#3197

Beta-Apopicropodophyllin, a derivative of podophyllotoxin, has a function as anticancer drug and radiosenstizer against non-small cell lung cancer cells.

Ju Yeon Kim,1 Jeong Hyun Cho,1 Sang-Gu Hwang,1 Jie-Young Song,1 EunAh Lee,2 Hong-Duck Um,1 Jong Kuk Park1. 1 _Korea Inst. of Radiological & Medical Sci., Seoul, Republic of Korea; _2 _Kyung Hee University, Seoul, Republic of Korea_.

We have synthesized new 32 derivatives of podophyllotoxin and screened candidate showed anti-cancer effect. Among these derivatives, we found beta-Apopicropodophyllin (APP) is candidate derivative inducing cell death against non-small cell lung cancer (NSCLC) cell lines: A549, NCI-1299 and NCI-460 (IC50 values =16.9, 13.1 and 17.1 nM, respectively). We also analyzed MOA (mode of action) of APP and found APP induced cell death via various mechanisms. Firstly, APP inhibits the polymerization of microtubule and induces G2/M phase arrest as well as DNA damage. APP treatment induces expressions of mitosis-related proteins, phospho-ATM, γ-H2AX and p21. Next, the intrinsic and extrinsic apoptotic pathways by APP are activated as shown in the activations of caspase-3, -8 and -9. APP also induces pro-apoptotic ER stress signaling pathway as indicated by elevated expression levels of BiP, phospo-eIF2α, CHOP and ATF4. Additionally, APP also showed radiosensitizer function. Combination of APP and gamma-ionizing radiation (IR) enhanced apoptotic cell death of NSCLC cell via amplification of ER stress. Taken together, these results implied APP might be a potent anti-cancer drug candidate inducing microtubule disruption, DNA damage, cell cycle arrest and ER stress.

#3198

Comprehensive transcriptome analysis identifies integrated mRNA-lncRNA signature of triple-negative breast cancer treated with radiotherapy.

Xingxing CHEN, Zhaozhi YANG, Yizhou JIANG, Jinli MA, Xiaoli YU, Zhimin SHAO, Xiaomao GUO. _Fudan University Shanghai Cancer Center, Shanghai, China_.

Triple-negative breast cancer (TNBC), with a lack expression of ER, PR, and HER-2 as potential treatment targets, is insensitive to the hormonal and trastuzumab therapies. Thus, local and regional treatment, such as radiotherapy is placed with more emphasis when treating TNBC patients. In the current study, expression profiles of mRNAs and long noncoding RNAs (lncRNAs) in TNBC patients, who received radiotherapy in our institute, was investigated to develop radiation-related gene signatures to facilitate individualized TNBC treatment. Specifically, 44 TNBC samples including 14 recurrences and 30 non-recurrences after radiotherapy were analyzed by using transcriptome microarrays. A total of 626 mRNAs were identified as differentially expressed (DEGs) between recurrent patients and non-recurrent patients (|fold change| > 1.5, p < 0.05). GeneOntology analysis showed that these DEGs were enriched in immune response, mitotic cell cycle, inflammatory response, cell adhesion as well as extracellular matrix organization, which indicated that these biological processes may exert significant impacts on the therapeutic effects of radiation. In addition, a total of 150 lncRNAs were identified as differentially expressed between recurrent patients and non-recurrent patients (|fold change| > 1.5, p < 0.05). The differentially expressed mRNAs and lncRNAs were confirmed by quantitative real-time PCR. Cox regression model was further applied to explore potential lncRNAs involved in certain signaling pathways such as JAK/STAT, EGRF/ATK/PI3K are highly co-expressed with mRNAs in these signaling pathways. Overall, our results revealed a panel of mRNAs and lncRNAs with significant impacts on evaluating the TNBC radio-therapeutic effects as well predicting the possibility of recurrence.

#3199

Computational studies show that Tumor Treating Fields can be delivered to the infratentorial brain at therapeutic levels.

Shay Levy,1 Ariel Naveh,1 Ze'ev Bomzon,1 Eilon Kirson,1 Uri Weinberg2. 1 _Novocure, Haifa, Israel;_ 2 _Novocure Gmbh, Lucerne, Switzerland_.

TTFields is an antimitotic cancer treatment that utilizes low intensity (1-3 V/cm) alternating electric fields in the intermediate frequency (100-300 kHz), approved by the Food and Drug Administration (FDA) for the treatment of Glioblastoma Multiforme (GBM) located in the supratentorial regions of the brain. A clinical trial testing the efficacy of TTFields for treating brain metastases is currently underway (METIS, NCT02831959). TTFields are delivered using two pairs of transducer arrays placed on the patient's skin in the proximity of the tumor. The electric field generated by the arrays is localized to a region located roughly between the arrays. The standard array layouts used to deliver TTFields involve placement of the arrays on the supratentorial regions of the head. With these array layouts,therapeutic field intensities above 1 V/cm are only achieved within the supratentorial brain. Therefore, treatment with TTFields has been limited to tumors located in the supratentorium. Although Tumors located in the infratentorial brain are rare in adult patients, they are common in the pediatric population. In addition, brain metastases commonly occur in the infratentorium. Hence, there is a need to identify array layouts that can effectively deliver TTFields to the infratentorial brain. Here we present computer simulations designed to identify new array layouts that deliver high field intensities to the infratentorial brain. To simulate delivery of TTFields to the brain, we used realistic computerized head models of an adult human female and an adult human male. Virtual transducer arrays were placed on the models, and boundary conditions were set to simulate delivery of TTFields at 200 kHz. Various array layouts were tested, and field distributions resulting from the layouts were evaluated. In both head models, the highest field intensities were delivered to the infratentorial brain by a layout, in which each array of one pair was laterally placed superficially to the lower region of the occipital lobe, and the two arrays of the second pair were placed on the calvarium and the superior aspect of the neck. In both models, the pair of arrays in which one array was placed on the calvarium and one array on the superior aspect of the neck delivered field intensities above 1.1 V/cm to over 95% of the volume of the infratentorial brain, and the pair of arrays placed on the lateral aspects of the occipital lobe delivered field intensities above 1 V/cm to over 95% of the infratentorial brain. Both pairs of arrays delivered field intensities above 1 V/cm to significant portions of the supratentorial brain. This work shows that TTFields at therapeutic intensities can be delivered to the cerebellum, stem and surrounding regions which are located inferior to the tentorium, suggesting that treatment of tumors within these with TTFields is feasible.

#3201

A theranostic approach using photodynamic therapy to efficiently kill neuroblastoma cancer cells through induction of apoptosis.

Lester M. Davids,1 Anja Schwar,2 Dirk Lang2. 1 _University of the Western Cape, Cape Town, South Africa;_ 2 _University of Cape Town, Cape Town, South Africa_.

Neuroblastoma is an extra-cranial tumour of childhood and arises from the sympatho-adrenal lineage of the neural crest tissue. Despite advances in treatment strategies, significant complications in curing this cancer remain. Furthermore, severe consequential effects on patient health have been associated with chemotherapy and/or radiation. Photodynamic therapy (PDT) with the photosensitiser hypericin (HYP) presents an attractive and efficient adjunctive therapy alternative for this aggressive cancer. HYP, an extract from Hypericum perforatum (St John's Wort) species, has a high quantum yield following photo-activation and a low dark toxicity and genotoxiciy. This study tests the anti-cancer efficacy of HYP-PDT in SK-N-SH neuroblastoma cells using an in vitro tissue culture model. Results show through detailed uptake kinetics that HYP is absorbed effectively by SK-N-SH cells after 6 hours of incubation and live fluorescent confocal microscopy revealed primary localisation of HYP to lysosomes and mitochondria. Through cell viability assays it was determined that 2, 3 and 4μM HYP-PDT resulted in 45.6±5.1% cell death within 24 hours of treatment. Additionally, 24 hours post 0.25μM and 2μM HYP-PDT, we observed distinct morphological changes in SK-N-SH cells using phase contrast microscopy, and determined a high apoptotic mode of cell death using Fluorescent Activated Cell Sorting (FACS). These results demonstrate that HYP-PDT is effective in killing neuroblastoma cells and that the mode of cell death is apoptotic in nature. This study is ongoing with further investigation into the signalling underlying this photo-toxicity with particular emphasis on cell death resistance mechanisms in neuroblastoma cells.

#3202

Radiation increases invasive activity of breast cancer cells via altering lysosome exocytosis.

Ping-Hsiu Wu,1 Yasuhito Onodera,1 Amato J. Giaccia,2 Quynh-Thu Le,2 Hiroki Shirato,1 Jin-Min Nam1. 1 _Hokkaido University, Sapporo City, Japan;_ 2 _Stanford University School of Medicine, Stanford, CA_.

Radiotherapy is a standard treatment for many localized solid cancers. However, previous studies have shown that radiation may increase the invasive activity of cancer cells and potentially distant metastasis. Recently, lysosome exocytosis has been linked to cancer cell invasiveness and progression. In this study, we evaluate the role of lysosome exocytosis on invasive activity of breast cancer cells upon radiation.We used both human and murine breast cancer cell lines (MDA-MB-231 and 4T1). The cells were treated with a single radiation dose of 4 Gy. Cell invasive activity was measured by matrigel chemoinvasion assay. Lysosome exocytosis was quantified by fluorescein isothiocyanate conjugated dextran (FITC-dextran) intake assay, cell-surface lysosomal associated membrane protein 1 (LAMP1) expression and cellular lysosome distribution assay. To validate the lysosome function, lysosome inhibitors, bafilomycin A1 and chloroquine were used. Short hairpin RNA (shRNA) was used to knockdown ARL8B, which is a small GTPase protein that regulates lysosome distribution and exocytosis. The invasive activity and lysosome exocytosis of tested breast cancer cell lines were increased after radiation treatment. Treatment with lysosome inhibitor bafilomycin A1 or chloroquine decreased the invasive activity of cancer cells, with or without radiation treatment. The protein level of ARL8B increased in the lysosome fraction upon radiation. Down-regulation of ARL8B with shRNA led to a decrease in lysosome exocytosis with a concomitant inhibition radiation induced invasive activity of the breast cancer cells without affecting the basal invasiveness. In addition, overexpression of ARL8B increased the invasive activity of breast cancer cells, which was similar to the result obtained after radiation.In summary, radiation enhances lysosome exocytosis in breast cancer cells that can lead to their increased invasive activity. Our findings provide a novel mechanism to understand cancer invasion after radiotherapy and suggest novel approaches to counteract this undesirable effect of radiotherapy in the future.

#3203

Modulation of cellular phospholipids correlates with tumor regression grade and radio resistance in rectal cancer.

Liam Robert Poynter,1 Reza Mirnezami,1 Renata Soares,1 James McKenzie,1 Pranav Patel,1 Nadia Peppa,2 Alexander Mirnezami,2 James Kinross,1 Ara Darzi,1 Zoltan Takats1. 1 _Imperial College London, London, United Kingdom;_ 2 _University of Southampton, United Kingdom_.

Objectives

Progression through the adenoma-adenocarcinoma sequence has been shown to demonstrate hallmark shifts in the tissue lipidome. The incorporation of certain phospholipid subclasses into cellular membranes has previously been found to correlate with susceptibility to external stressors through alteration of membrane biodynamics. On this basis, it is hypothesized that modulation of cellular lipid modulation may influence radioresistance in rectal cancer. A desorption electrospray ionization mass spectrometry imaging (DESI-MSI) platform was utilized to demonstrate lipidomic shifts reflective of tumor heterogeneity and modulation of cellular lipids as a radioresistance mechanism.

Methods

26 specimens of fresh, human rectal cancer tissue were harvested at surgical resection from matched cohorts; 13 of each from those who had received RT, and those who proceeded straight to surgery (STS). DESI-MSI was performed using an Orbitrap Fourier transform mass spectrometer in negative ion mode (mass range 150-2000m/z). Lipid spectra acquired were spatially aligned to subsequent H&E staining of the tissue section in a chemometric toolbox in MatLab, annotating morphologically distinct regions of the tumour microenvironment. Statistical modeling was performed incorporating clinicopathological metadata. ANOVA identified spectral peaks with discriminating power between both RT and STS groups, and between clusters of patients across the range of Mandard tumor regression grades (TRG). Lipid classes and putative structures were determined using an existing MS/MS data reference library (MetLin).

Results

Significant clustering of lipid species was demonstrated in both RT and STS groups, for both adenocarcinoma and stroma. Leave-One-Out, all-pixels cross-validation (LOOCV) yielded high predictive values (>97.9%) for RT vs. STS adenocarcinoma pixels. Cancers subjected to RT demonstrated significantly increased abundance of phospholipids compared to the STS group, notably phosphatidylglycerols (PG), phosphatidylethanolamines (PE) and phosphatidylserines (PS) on univariate analysis of all spectra, with a defined p/q value of 0.05 and negative log2 fold change of 0.1. Three-class PCA and discriminant analysis demonstrated clustering of samples according to Mandard TRG 1-2, TRG 3, and TRG 4-5. Clear separation of spectra was observed on LOOCV between all three classes. Compounds in the range 240-450m/z were implicated in this shift, in addition to the phospholipids as described.

Conclusion

This work has demonstrated a putative role for phospholipids and fatty acids in radioresistance, in a human rectal cancer cohort. DESI-MSI provides a unique foundation from which to continue 'bottom-up' pathway analysis - utilizing both immunohistochemistry and tissue genotyping – which will assist in elucidating the possible mechanisms of response to radiotherapy in rectal cancer.

#3204

Transducer array configuration optimization for treatment of pancreatic cancer using Tumor Treating Fields (TTFields).

Ariel Naveh,1 Ze'ev Bomzon,1 Ori Farber,1 Noa Urman,1 Ofir Yesharim,1 Eilon Kirson,1 Uri Weinberg2. 1 _Novocure, Haifa, Israel;_ 2 _Novocure Gmbh, Lucerne, Switzerland_.

Introduction: TTFields is an antimitotic cancer treatment that utilizes low intensity (1-3 V/cm) alternating electric fields in the intermediate frequency (100-300 kHz) that are delivered in two orthogonal directions using 2 pairs of transducer arrays. A phase II clinical trial (PANOVA, NCT01971281) showed that TTFields in combination with chemotherapy was safe in patients with locally advanced pancreatic cancer. Preclinical studies show that the effect of TTFields is intensity-dependent with a therapeutic threshold of 1 V/cm. The field distribution within the body is known to changes with array placement. Therefore, there is a need to develop principles for personalizing array placement to optimize TTFields delivery when treating pancreatic cancer. The aim of such guidelines should be to ensure delivery of maximal field intensities to the region of disease, whilst minimizing the size of the arrays placed on the body. Minimizing array size is important to enable periodic shifting of the arrays for potentially reducing skin irritation associated with TTFields therapy, as well as potentially improving overall patient comfort. Here we present a systematic study investigating how the location and size of the arrays on the abdomen influences the field distribution.

Methods: To simulate delivery of TTFields to the abdomen, we used 3 realistic computerized models (from ZMT_Zurich) of: a male (DUKE 3.0); b) , a female (ELLA 3.0); and an obese male (FATS 3.0). For each model, 6-8 different layouts utilizing combinations of arrays with either 13 or 20 disks per-array were tested. The arrays were placed over the upper 6 standard abdominopelvic, and field intensity distributions within these regions were evaluated. In order to generate TTFields, an alternating voltage at a frequency of 150 KHz was imposed on the outer surfaces of the disks of each pair of arrays. The voltage was set to deliver a current of 200 mA peak to peak per-disk (total current 2.6 A for 13-disk arrays, 4.0 A for 20-disk arrays). The simulations were performed using ZMT's Sim4Life V3.0 electro-quasi-static solver.

Results: In all simulations, the large arrays generated higher field intensities than the small arrays. However, On ELLA and DUKE, the large arrays covered most of the skin around the abdomen, leaving little to no room for shifting the arrays. FATs body surface is large enough so that even when large arrays are used, there is still ample space for shifting the arrays. On DUKE and ELLA, when using small arrays, the average field intensity in the abdominopelvic region over which the arrays were placed was above the therapeutic threshold of 1 V/cm.

Conclusion: This work shows that TTFields array placement can be optimized to deliver therapeutic field intensities to specific abdominopelvic regions. Tailoring the size and position of the arrays based on disease site and patient size may help to improve overall treatment outcome.

#3205

Histone tyrosine phosphorylation determines glioblastoma cell survival.

Mohammad Belayat Hossain,1 Rehnuma Shifat,1 Debora K. Kim,1 Yiesel Rivera-Molina,1 Francisco Puerta Martinez,1 Hong Jiang,1 Teresa Nguyen,1 David G. Johnson,2 Mark T. Bedford,2 Mien-Chie Hung,1 Erik P. Sulman,1 Frederick Lang,1 Raymond Sawaya,1 Juan Fueyo,1 Candelaria Gomez-Manzano1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _UT MD Anderson Cancer Ctr., Smithville, TX_.

Histone modification is crucial for cell signaling pathways and regulates predominantly chromatin functions related with DNA damage response (DDR) that lead cancer cells to survive upon different therapies. Receptor tyrosine kinases (RTKs) have been extensively studied in cancer progression and DDR and, recently, several reports described the role of RTKs in histone tyrosine modification. We have been focusing on the role of the receptor tyrosine kinase, TIE2, in gliomas. Our study identified the expression of TIE2 in human surgical glioma specimens in relation to malignancy, specifically in brain tumor stem cells. We have revealed that the unexpected membrane-to-nuclear trafficking of TIE2 is related with radioresistance of brain tumor stem cells, functioning as a genotoxic stress sensor to activate DNA repair machinery. Interestingly, TIE2 binds, upon IR stress, to DNA/protein complexes and phosphorylates several core histones. The tandem mass spectrum of H4 and H2B demonstrates that TIE2 directly phosphorylates not previously described H4pY51 and H2BY40 respectively, which reinvigorates TIE2's role in histone code modification. We screened for pY-readers using a SH2 domain array and found that H4pY51 and H2pY40 modifications are read by the proto-oncogenes ABL1 and CRK, respectively. Our data also showed that TIE2/H4pY54/ABL1 complex binds to DNA repair proteins, activating an NHEJ DNA repair mechanism. We summarize that upon IR stress TIE2 localizes to the nucleus where it is involved in key cellular functions by directly phosphorylating core histones, and recruiting SH2 domain proteins to the DNA damage sites, complexing to the DNA repair machinery, which in turns results in radioresistance. Our data highlight the role of epigenetics in modulating the DNA damage response of glioma radiation therapy and open new avenues to find novel targetable pathways for treatment of patients with glioblastomas.

#3206

Synthetic lethal interactions with APOBEC3A identified by functional genomic screening.

Abby M. Green, Katharina E. Hayer, Julia H. Szeto, Ophir Shalem, Matthew D. Weitzman. _Children's Hospital of Philadelphia, Philadelphia, PA_.

The APOBEC3 (A3) family of cytosine deaminase enzymes is integral to innate defenses by editing viral genomes to limit infectivity. However, several members of the A3 enzyme family (A3A-A3H) have also been implicated in mutational signatures observed in human cancers. The A3 mutational signature has emerged as the most prevalent across all cancers, implicating A3 enzymes as powerful mutators of the human genome. While few prospective studies have addressed the potential for A3 enzymes to induce specific mutational patterns in human genomes, the possibility of exploiting the mutagenic activity of A3 for cancer therapy is beginning to be explored. We recently showed that A3A is highly expressed in human acute myeloid leukemia (AML), and that deamination activates the replication checkpoint via ATR kinase signaling. In an AML model, we showed that inhibition of ATR or downstream Chk1 resulted in accumulated DNA damage by A3A and ultimately death of leukemia cells. In search of additional cellular pathways that may be manipulated to take advantage of A3 activity for therapeutic effect, we performed a functional genomics screen using a genome-wide CRISPR knock-out approach in a model of human leukemia with inducible A3A expression. We introduced Cas9 and the Brunello guide RNA lentivirus library into the human AML cell line, THP1, which we engineered to express doxycycline-inducible A3A. The Brunello library consists of 4 guide RNAs targeting each human gene. Transduction was performed to enable one lentivirus integration per cell, thus resulting in a single gene disruption per cell. Cells were treated with doxycycline and genomic DNA was harvested from treated and untreated cells every week. Guide RNAs were amplified by PCR from genomic DNA and identified by next-generation sequencing. We applied the MaGECK algorithm to identify the quantity of each guide RNA remaining in treated samples and compared to untreated samples. In THP1 cells induced to express A3A, knockout of genes in cell cycle and DNA repair pathways resulted in synthetic lethality. Specifically, expression of A3A synergized with genes essential for cell cycle checkpoints and DNA damage responses to cause leukemia cell death. Clinical-grade inhibitors are available for protein products of many of the genes identified; thus these represent feasible targets for therapeutic intervention in AML that has elevated A3A activity. In addition to leukemia, A3A and the closely related enzyme A3B have been implicated in several malignant processes including breast, bladder, and ovarian cancers. We show that functional genomic screening is a powerful tool for identification of synthetic lethal interactions, which can provide therapeutic opportunities for additional cancers in which A3 enzymes are active.

#3207

CXCR4 endows radioresistance in non-small cell lung cancer cells through STAT3-mediated Slug expression.

Dae-Hee Lee,1 Jeong-Yub Kim,2 Hee-Jin Kim,2 Myung-Jin Park2. 1 _Korea Univ. Medical Center, Seoul, Republic of Korea;_ 2 _Korea Institute of Radiological and Medical Sciences, Seoul, Republic of Korea_.

Lung Cancer is one of the most common causes of cancer-related mortality worldwide, despite of significant advance of treatment strategies including chemotherapy and radiotherapy. Although chemotherapy is the first choice for treatment of non-small cell lung cancer cells (NSCLCs) except surgery, radiotherapy is primary treatment modality for patients with unresectable NSCLC on its own or with another treatment like chemotherapy. CXCR4 is chemokine (C-X-C motif) receptor 4 whose ligand is Stromal-derived-factor-1 (SDF1a), so-called CXCL12. According to the current reports, CXCR4 is widely expressed by malignant tumor and crucial for tumor growth, vascularization, metastasis, cell migration and related to poor prognosis. Here, we show that CXCR4 signaling confers resistance to ionizing radiation (IR) in NSCLCs. Drug-resistant A549/GR cells with enhanced CXCR4 expression exhibited high colony forming ability after IR compared with A549 cells. Transfection of siRNA against CXCR4 or treatment of pharmacological inhibitor (AMD3100) sensitized these cells to IR. Reversely, overexpression of CXCR4 in A549 and H460 enhanced clonogenic survival after IR. IR stimulated CXCR4 expression and CXCR4-mediated STAT3 phosphorylation in NSCLCs. Inhibition of STAT3 by WP1066 or siSTAT3 significantly suppressed the colony forming ability, indicating that CXCR4-mediated STAT3 signaling is important for IR resistance in NSCLC cells. CXCR4/STAT3 signaling upregulated the expression of Slug and down-regulation of Slug with siRNA increased IR sensitivity in NSCLCs. Taken together, our data suggest that CXCR4/STAT3/Slug axis is crucial for IR resistance of NSCLCs and is able to be considered as a therapeutic target for enhancing IR sensitivity of this devastating cancer.

#3208

Optimal array layouts for tumor treating fields therapy in glioblastoma: Oblique array layouts are superior to standard LR and AP positions.

Nikola Mikic, Anders Korshøj. _Aarhus University Hospital, Aarhus, Denmark_.

Introduction: Tumor treating fields (TTFields) is a new and effective treatment for glioblastoma. Two pairs of transducer arrays are placed on the scalp of the patient to deliver low intensity, alternating electric fields, which inhibit mitosis and cancer growth. In the present study, we investigated systematic variations in electrode array positions to identify optimal layouts, which induce the highest electric fields in the tumor tissue.

Method: The electric field distribution was calculated using finite element methods applied to a realistic computational head model. Array positions were rotated systematically in fifteen-degree intervals around a central cranio-caudal axis of the head to obtain thirteen different layouts in the same plane. Tumors were introduced in the computational mesh at nineteen different fronto-parietal positions in the same plane as defined by the electrode positions.

Results: Electrode array layout greatly affected the field distribution. For the vast majority of tumors, two optimal and oblique array orientations were identified. At these positions, field intensities were approximately 30-40% higher compared to the standard anterior/posterior and left/right positions. In addition, the two optimal layouts were oriented at ninety-degree interval to each other. Correspondingly, a single optimum layout was identified when analyzing the combination of two pairs of arrays. Each tumor position was associated with only one optimum (combined) layout, which was generally oblique relative to the sagittal plane (15-45 degrees). An oblique layout at forty-five degrees to the sagittal plane was effective for most tumor positions and in all cases it was superior to the default layout combination of anterior-posterior and left-right positions.

Conclusion: Our results provide guiding principles for optimal electrode placement, which will be helpful, especially in situations where the placement must be changed due to concurring clinical conditions such as shunts, scars, tissue rash etc. Patient specific models should be for more accurate TTF distributions. Additional research is needed to confirm these current results.

#3209

Numerical modeling of intracellular mechanisms in tumor-treating fields.

Kristen W. Carlson,1 Jack A. Tuszynski,2 Socrates Dokos3. 1 _BIDMC/Harvard Medical School, Cambridge, MA;_ 2 _University of Alberta, Edmonton, Alberta, Canada;_ 3 _University of New South Wales, Sydney, Australia_.

Tumor Treating Fields (TTFields) are 100-500 kHz electric fields with intensities of about 1-4 V/cm. They are known to exert an antimitotic effect on cancer cells, most likely by exerting forces on highly polar tubulin dimers, thereby disrupting spindle formation. Calculations show that TTFields-tubulin interaction energy is negligible compared to the thermal energy in the cell (1). Therefore, this interaction is unlikely to disrupt cellular function. Conductivity of polymerized tubulin, microtubules (MTs), was measured to be 20 S/m, which is 400 times higher than that of the ambient cytosol (0.05 S/m) (2). Thus when TTFields penetrate the cytosol, they may induce electric currents along MTs that are strong enough to disrupt key cellular functions. In particular, if the power (energy per unit time) deposited by these currents is on par with that the power consumed by the molecular motor kinesin, then TTFields may disrupt the forces needed for cell division, thereby disrupting mitosis. To test this hypothesis, we performed numerical simulations evaluating the magnitude of the electric current along MTs exposed to TTFields at 200 kHz. Based on studies of MTs and their microenvironment, we model the MT as a layered cylindrical structure (1): Innermost is the lumen (15 nm in thickness), surrounded by 13 strands of alpha-beta tubulin dimers linked in a helix (4.5 nm). C-termini extend out from the helix with a thickness of 3.5 nm. MTs carry net negative charge; thus they are surrounded by a counter-ion layer (2 nm), and an outer nonconductive Bjerrum layer (3 nm). We built a finite element model in COMSOL Multiphysics (tm) incorporating these layers and examined the current density induced in each layer by TTFields for MTs varying in length from 1-10 µm within an ambient 200 kHz AC electric field of 1-4 V/cm. Our model shows that MTs act as electrical "shunts" that conduct electric current within them. The highest current flows through the counter-ion layer surrounding the C-termini. The current density in this layer exceeds the level likely to disrupt the motor protein kinesin "walk" along the C-termini. The current density is highest when both the field and the MTs are aligned with the cell axis, in accord with in vitro experiments (3). Our model is consistent with the hypothesis that when cells are exposed to TTFields, MTs act as cables carrying high-density electric currents strong enough to disrupt the function of molecular motors, ultimately disrupting mitosis.

References:

1. Tuszynski JA et al. An overview of sub-cellular mechanisms involved in the action of TTFields. Int J Environ Res Public Health 2016.

2. Santelices IB et al. Response to alternating electric fields of tubulin dimers and microtubule ensembles in electrolytic solutions. Sci Rep 2017.

3. Kirson ED et al. Alternating electric fields arrest cell proliferation in animal tumor models and human brain tumors. Proc Natl Acad Sci U S A 2007.

#3210

**Photothermal therapy of malignant mesothelioma with delaminated MXene Ti** 3 **C** 2 **.**

Lanlan Zhou,1 Fayan Meng,2 Babak Anasori,2 Yury Gogotsi,2 Wafik S. El-Deiry1. 1 _Fox Chase Cancer Center, Philadelphia, PA;_ 2 _Drexel University, Philadelphia, PA_.

Malignant mesothelioma is an aggressive and deadly form of cancer that develops in the thin layer of tissue surrounding the majority of internal organs (mesothelium). Malignant mesothelioma has no known cure and has a very poor prognosis because of late diagnosis and limited usefulness of standard treatments. We are exploring newer treatments to tackle this deadly disease. Photothermal therapy is an emerging noninvasive spatiotemporal selective therapeutic strategy that employs near-infrared photoabsorbers to generate heat for thermal ablation of cancer cells. The therapeutic efficacy of photothermal therapy significantly depends on the transformation of light to sufficient heat with photothermal agents. MXene Ti3C2 is an effective two-dimensional light-to-heat conversion material. The internal light-to-heat conversion efficiency of MXene Ti3C2 was measured to be 100%, indicating a perfect energy conversion. In this study we report for the first time using the MXene Ti3C2 nanoplatform as a photothermal agent to efficiently ablate malignant mesothelioma. First, we prepared two-dimensional MXene Ti3C2 and UV-vis-NIR spectrum showed Ti3C2 has a strong absorbance at the region of 790 nm, which showed a high light extinction coefficient of 26.081 L g-1cm-1 and exhibited a good photothermal conversion efficiency of 22%. Photothermal heating curves show temperatures of different concentration of MXene Ti3C2 increase steadily with irradiation of 808nm laser. Recycling heating profiles demonstrate that MXene Ti3C2 can be repeatedly heated stably. Next, the effects of MXene Ti3C2 on normal and mesothelioma cell lines were tested with CellTiter-Glo® luminescent cell viability assays. MXene Ti3C2 has no toxicity to cells at the tested concentration range. Light microscopy revealed that MXene Ti3C2 can accumulate inside of mesothelioma cells but not in normal cells. There was no red blood cell hemolysis within the tested concentration range and the hemolysis ratio was 0.5% even at ten times higher than the test concentration. Then, normal and mesothelioma cells were treated with and without MXene Ti3C2 for 24~48 hours followed by laser irradiation. CellTiter-Glo® luminescent cell viability assays and colony formation assays validated that MXene Ti3C2-treated mesothelioma cells were eliminated by laser irradiation while normal cells and nontreated mesothelioma were spared. Flow cytometry and Western blot analysis were used for determination of apoptosis. Lastly, we established a mesothelioma mouse xenograft model to evaluate the therapeutic effect of MXene Ti3C2 on mesothelioma in vivo. We are also pursuing the combination of MXene Ti3C2-based photothermal therapy and current therapies for mesothelioma. Our results indicate that MXene Ti3C2 is a promising photothermal agent in therapy of mesothelioma.

#3211

**Therapeutic effects of radiotherapy on cancer cell lines using** RadioGx **computational platform.**

Venkata S. Manem,1 Meghan Lambie,1 Petr Smirnov,1 Mark Freeman,1 Victor Kofia,1 Mohamed E. Abazeed,2 Scott V. Bratman,1 Benjamin Haibe-Kains1. 1 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada;_ 2 _Cleveland Clinic, Cleveland, OH_.

Purpose: Radiotherapy (RT) is the foundation of curative treatment regimens for many cancer types and is often delivered with drugs to produce synergistic effects. The "precision medicine" paradigm in which genomic biomarkers guide therapeutic decisions has eluded RT. To support preclinical discovery of biomarker-directed RT dose and drug combinations, we built a computational platform for integrating and interrogating radiogenomics data sets.

Methods: RadioGx integrates in vitro cancer cell line survival data with multimodal molecular profiles from the NCI-60 and Cancer Cell Line Encyclopedia (CCLE) cell line panels. We fit published radiation response data from both clonogenic and high-throughput viability assays to established radiobiologic models of clonogenic survival. We assessed the concordance of radiation response profiles across different survival endpoints (i.e., surviving fraction at 2Gy [SF2] and Area Under the Curve of the radiobiologic model [AUC]). By comparing radiation response with transcriptomic data, we examined indicators of radiosensitivity at the pathway level. We integrated drug response data from Cancer Therapeutics Response Portal (CTRP v2) using an interface between RadioGx and our previously published pharmacogenomics platform, PharmacoGx. Using the oxygen modification factor in established radiobiologic models, we identified pathways that are enriched under hypoxic conditions.

Results: RadioGx includes 600 radiation dose response data, 511 gene expression data sets, and 504 drug dose response data for chemotherapeutic compounds. Goodness-of-fit (given by R2 value) of the linear-quadratic radiobiologic model ranged from 0.927 to 0.99 (median=0.998, n=57) for the clonogenic assay and from 0.043 to 0.99 (median=0.952, n=535) for the viability assay. The concordance between SF2 and AUC using the clonogenic assay and the viability assay was 0.79 (n=12) and 0.82 (n=15), respectively. Among cell lines with survival data from both assays (n=28) across two different studies, the concordance of SF2 and AUC was 0.68 and 0.62, respectively. By interrogating the 504 cell lines that were profiled using the viability assay, we found drugs that behaved similar to RT were enriched in three pharmacologic classes, namely, drugs that impact mitosis, DNA replication, or cytoskeleton. Pathway analysis of radiation response under hypoxia revealed two key DNA repair pathways, namely, DNA double-strand break repair by NHEJ and DNA damage-induced 14-3-3, to be enriched compared to normoxic conditions.

Conclusions: Our work is a major step towards analyzing preclinical models of radiation response using in vitro survival and transcriptomic data. RadioGx coupled with PharmacoGx lays the foundation for the identification of synergistic drug-RT combinations, which can further push the boundaries of translational research towards precision radiation medicine.

#3212

Nuclear-targeted gold nanoparticles as radiosensitizers.

Sunil Krishnan, Maureen Aliru. _UT MD Anderson Cancer Ctr., Houston, TX_.

Purpose

Intracellular localization of gold nanoparticles has been shown to enhance the effects of radiation. However, few studies have investigated the effect of subcellular localization of these particles on enhancing the radiosensitization effect in cells treated with radiation of varying energies and linear energy transfer (LET) levels. Herein, we evaluate the radiosensitization potential of nuclear-targeted gold nanoparticles across a spectrum of energies and LETs.

Methods

Gold nanospheres (AuNSs) were functionalized with a nuclear localization sequence (NLS) peptide to enhance the nuclear accumulation of these particles. Internalization of the particles into the cells was achieved using a cell penetrating peptide (CPP). Cellular and nuclear internalization were assessed in Panc-1 human pancreatic adenocarcinoma cancer cells using western blot (WB), transmission electron microscopy, dark field microscopy and inductively-coupled plasma mass spectrometry (ICP-MS). DNA damage due to radiation was evaluated through γH2AX and 53BP1 foci staining. Clonogenic cell survival assay was performed to evaluate the radiosensitization effect after irradiation with 250-kVp X-rays, Cs-137, Co-60, 6 MV and 100 MeV protons (LET 2.5 and 7.7 keV/µm).

Results

Nuclear-targeted particles (nAuNS) and non-targeted particles (PEGylated, pAuNS) demonstrated similar cellular internalization. However, enhanced uptake of nuclear-targeted particles was observed in the nucleus when compared to non-targeted particles. This was confirmed using dark field microscopy, WB and ICP-MS analysis. DNA double strand breaks through γH2AX and 53BP1 foci staining revealed significantly higher numbers of foci at early (1 hour) and late (24 hour) time points following radiation and nAuNS compared to radiation and pAuNS treatment. Clonogenic cell survival assay revealed significantly higher radiosensitization enhancement factor (REF) at 10% survival fraction for nAuNS compared to pAuNS for all beams assessed. Protons with higher LETs resulted in greater clonogenic cell death and nAuNS further increased radiosensitization whereas pAuNS did not.

Conclusion

Our data demonstrates that, across a spectrum of radiation energies and LETs, nuclear-targeted gold nanoparticles enhance radiosensitization beyond that achievable with strictly intracytoplasmic gold nanoparticles.

#3213

Evaluation of porphyrin biodistribution for use in photodynamic therapy of retinoblastoma in transgenic mice.

Florent Poyer,1 Samuel Hugeut,2 Stéphanie Lemaitre,1 Olivier Madar,2 Carole D Thomas,1 Philippe Maillard,1 Marie-Paule Teulade-Fichou,1 Nathalie Cassoux,3 François Doz,3 Keyvan Rezai2. 1 _Inst. Curie Research Center, Orsay, France;_ 2 _Inst. Curie, Saint Cloud, France;_ 3 _Institut Curie, Paris, France_.

Background: Porphyrins are a group of heterocyclic macrocycle organic compounds. They have shown good efficiency in the photodynamic treatment of tumors in the last twenty years. The photodynamic therapy could be an alternative treatment for retinoblastoma using a photosensitizer with low dark toxicity and low mutagenic properties unlike the conventional chemotherapies. The objectives of this study were to develop a photosensitizer in our lab which is a diethylene glycol mannoconjugated tetraphenyl porphyrin (TPP, MW: 1413.6) and to determine the optimal interval time for photodynamic therapy using the concentration of TPP in eye and blood samples of mice. Materials and method: Transgenic mice developed bilateral retinoblastoma from the retina since 4 weeks of age to 16 weeks of age (tumors fill the ocular globe and the mice will be sacrificed for ethic purpose). The evaluation of the biodistribution of our TPP was realized to know the best drug-light interval to perform the photodynamic therapy. For this purpose, an intraperitoneal injection of 200 µl at 5.25 mg/ml in PEG 400/physiological serum (1:1, v/v) was performed on the LHbetaTag transgenic mice. At different time intervals (8h, 24h, 40h, 48h, and 72h) following injection, the mice were sacrificed and the eyes and blood were collected. TPP in eye samples were extracted in methanol and the level of TPP was measured by ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) analysis (0.5-100 ng/mL). The level of TTP in blood samples was quantified after protein precipitation by UPLC-MS/MS method (5.00-1000 ng/mL).

Results: We observed a time dependency accumulation of TPP in eyes with observed Tmax=48 hours. TPP serum concentrations decreased by the same manner up to 48 h. The determination of drug-light interval allowed us to perform the light delivery when the TPP level is highest (48 h) in the ocular tumor tissue (retinoblastoma).

Conclusions: Our TPP had a good in vitro photocytotoxicity at very low doses and showed a good efficacy in vivo with subcutaneous patient derived retinoblastoma xenografts in immunodeficient mice. The knowledge of Tmax and this interval is essential to realize the photodynamic therapy with the better efficiency, the final goal being to eradicate the retinoblastoma from the retina.

#3214

FePt nanoparticles overcoming radiotherapy resistance in hCtr-1 high expression cells via mitochondria targeting.

Tsung-Lin Tsai,1 Macus Tien Kuo,2 Jen-Ya Wang,1 Wan-Li Shin,1 Helen H. W. Chen,1 Wu-Chou Su1. 1 _National Cheng Kung University Hospital, Tainan, Taiwan;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

The glutathione (GSH) overexpression small-cell-lung-cancer cell lines, SR3A-13 and SR3A-14, transfected with recombinant DNA encoding γ-glutamylcysteine synthetase (γ-GCSh), are radiation resistant. However, they are more sensitive to cisplatin treatment, compared with parental cell (SR3A)1. In addition to be a copper transporter, the hCtr-1 is also a transporter for platinum (Pt)-based agents. Therefore, the cellular uptake of cisplatin is enhanced by the upregulation of human copper transporter 1 (hCtr-1) at the cell membrane. We have synthesized an alloy FePt nanoparticles (FePt NPs) that generated excellent contrast effects in both CT and MRI images2. Interestingly, the uptake of FePt NPs by SR3A-13 and -14 cells was 3-3.5 fold higher than that of SR3A cells, measured with inductively coupled plasma optical emission spectrometry (ICP-OES) and transmission electron microscopy (TEM). Because intracellular NPs have been shown to generate reactive oxygen species (ROS) and overproduction of ROS may enhance the efficacy of radiotherapy, we sought to explore whether treatment with the alloy FePt NPs can reverse the radiation resistance in SR3A-13 and -14 cells. We found co-treatment with the alloy FePt NPs and radiation effectively reduce the survivals of these 2 cells. Furthermore, we found an alternation in mitochondrial appearance accompanied with low signal of Tetramethylrhodamine, methyl ester (TMRM), indicating a malfunction is induced in mitochondria. We conclude that FePt NPs are able to overcome radiation resistance in hCtr-1 high expression cells via mitochondria targeting.

#3215

Combination BB2r targeted radiotherapy with PARP inhibitors in human prostate cancer cells: An in vitro evaluation.

Tammy Rold, Reneise White, Nkemakonam C. Okoye, Timothy J. Hoffman. _Department of Veterans Affairs, Columbia, MO_.

Introduction: Although recent studies employing 177Lu-PSMA beta emitting targeted radiotherapy (TRT) have demonstrated efficacy in treating patients with CRPC, there remains significant opportunities for improvement. Since the TRT mode of action involves DNA damage, inhibiting DNA damage repair with a PARP inhibitor (PARPi) may lead to increased cell death. This pilot in vitro study aims to evaluate the potential synergy of two FDA approved PARPi's, Olaparib and Rucaparib, when used in combination with 177Lu-RM2 TRT to prevent repair of DNA damage caused by TRT in PC3 cells. 177Lu-RM2 TRT, a beta emitting TRT agent targeting the BB2 receptor frequently expressed in CRPC.

Methods: 177Lu-RM2 was prepared via automated radiosynthesis with HPLC QC validation of product purity >90%. PC3 cells cultured overnight were treated with media or 177Lu-RM2 TRT (5uCi to 40uCi/160uL). Cells were incubated with TRT for 4 h prior to addition of fresh media with or without the PARP inhibitors, Olaparib (0.25, 0.5, 1.0, 1.5uM) or Rucaparib (0.5, 1.0, 1.5, 2.0uM). DNA damage was assessed at 24 hrs after TRT addition by enumeration of 53BP1 and gamma H2AX foci. Clonogenic survival was evaluated in cells treated with TRT and Olaparib at 0.25, 0.5, 1.0, 1.5uM as single and combination treatments with media refreshed every 4-7 days. CalcuSyn software (Biosoft™) was used to evaluate synergism.

Results: At 24 hrs after TRT, the addition of either Olaparib or Rucaparib to the cell culture media showed significant increases in DNA damage markers. Combined with TRT dose levels of 10-20uCi/160uL, the addition of Olaparib significantly increased the number of both markers at 0.5-1.25uM as compared to TRT alone. Treatment with Rucaparib resulted in extreme significance (P < 0.0001) being observed in the number of 53BP1 foci at 13/16 drug combinations as compared to TRT alone. In 7/16 drug combinations gamma H2AX foci were noted as extremely significant (P < 0.0001), and the single combination of 40uCi + 1.5uM was noted as very significant (P < 0.005) as compared to TRT alone. Synergy assessment of TRT plus Olaparib showed increasing levels of synergy (synergism to very strong synergism) with increasing dose levels of TRT.

Conclusions: This in vitro investigation supports the use of either Olaparib or Rucaparib in combination with beta emitting TRT to increase PC cell death resulting from beta particle induced DNA damage. Additional in vitro and in vivo preclinical therapeutic evaluation is warranted to support the clinical translation of TRT/PARPi combination therapy.

Acknowledgements: US Veterans Administration (VA) BX001699 and USVA Research Career Scientist (TJH)

#3216

H2AX is a novel prognostic marker of breast cancer.

Eriko Katsuta, Mutsuko Ouchi, Toru Ouchi, Kazuaki Takabe. _Roswell Park Cancer Institute, Buffalo, NY_.

INTRODUCTION: Phosphorylation of histone variant H2AX, termed gamma-H2AX, is well known for its role in DNA damage repair. On the other hand, clinical relevance of expression of H2AX in breast cancer remains to be elucidated. Previously we found that H2AX determines clonogenic cell survival under metabolic oxidative stress and cellular levels of Reactive Oxygen Species (ROS). In this study, we investigated the impact of H2AX expression on breast cancer patients' survival, as well as underlying mechanisms.

METHODS: Breast cancer patients in The Cancer Genome Atlas (TCGA) dataset were classified as either high or low expression of H2AX. Overall Survival (OS) and Disease-Free Survival (DFS) as well as Gene Set Enrichment were compared between these two groups. Gene expression of each cell lines was analyzed by RNA-seq. Radiation sensitivity was quantified using cells from patient-derived xenograft (PDX). Gene expression level after neoadjuvant chemo-radio therapy was compared using publically available database (GSE25113).

RESULTS: H2AX expression level is higher in triple negative breast cancers (TNBC) than non-TNBC (p<0.001) and in HER2 positive tumors than negative tumors (p=0.048) as well as advanced Stage tumors (Stage I vs Stage II/III/IV, p=0.029). H2AX high expressing tumors showed significantly worse OS (p=0.007) as well as DFS (p=0.001) and this was the case only in advanced Stage tumors (Stage I vs Stage II/III/IV, p=0.029). There were more H2AX high tumors with advancement of Stage (Stage I: 2.8%, Stage II: 5.5%, Stage III/IV 7.4%). In agreement, breast cancer cell line clones that develop metastasis expressed higher H2AX compared from their parental cells (4T1 vs 4T1.2; p<0.001, MDA-MB-231 vs LM2-4; p=0.042). Gene Set Enrichment Analysis (GSEA) demonstrated that not only DNA repair (p<0.001) and ROS related gene set (p=0.011), but also Myc target related gene sets (p=0.002) as well as UV response related gene set (p<0.001) were significantly enriched in H2AX high patients. We also demonstrated that H2AX high expressing cells from PDX showed significantly higher sensitivity to radiation therapy with higher ROS production. Indeed, tumors that demonstrated pathological completely response (pCR) after neoadjuvant chemo-radiation therapy had a trend to have higher H2AX levels compared to non-pCR tumors (p=0.093).

CONCLUSIONS: Advanced Stage breast cancers that express high levels of H2AX associated with worse OS and DFS, but showed higher sensitivity to radiation. H2AX can be a prognostic biomarker for recurrent tumor that may be sensitive to radiation therapy.

#3217

Newly identified role of tumor treating fields in DNA damage repair and replication stress pathways.

Narasimha Kumar Karanam, Lianghao Ding, Brock Sishc, Debabrata Saha, Michael D. Story. _UT Southwestern Medical Center, Dallas, TX_.

Tumor treating fields (TTFields) are low-intensity, intermediate frequency, alternating electric fields non-invasively applied to the region of the tumor. TTFields have revolutionized the treatment of recurrent and newly diagnosed glioblastoma and Phase III clinical trials are ongoing for brain metastasis. Phase II trials are ongoing in non-small cell lung, ovarian and pancreatic cancers. The primary mechanism of TTFields function is thought to be the disruption of mitosis; however, other mechanisms are under investigation. Using a panel of 5 non-small cell lung cancer cell lines (NSCLC), we found that cell proliferation and killing as a result of TTFields exposure was variable. To understand the molecular mechanisms underlying the biological effects of TTFields exposure we examined temporal gene expression changes in these NSCLC cell lines after TTFields treatment. Interestingly we found that the expression of the BRCA1 DNA damage repair pathway genes were significantly downregulated (P < 0.05) upon TTFields treatment which was confirmed at the protein level by western blot. Furthermore, TTFields treatment slowed the repair of ionizing radiation-induced DNA damage compared to radiation alone which was evident by an increased number of DNA double strand break repair foci at any given time. Moreover, we found that TTFields treatment alone increased the number of γH2AX foci and the incidence of chromatid aberrations. Given the role of BRCA1/FANC gene downregulation, we examined the status of replication fork integrity as a result of TTFields exposure and determined that the length of newly replicated DNA, slowed as a function of TTFields exposure time. Furthermore, we showed that TTFields increased R-loop formation (DNA:RNA hybrid structures) which was quantified using a DNA-RNA hybrid specific antibody. These results clearly indicate that TTFields induce DNA damage and increase replication stress. Based on newly identified mechanisms of TTFields action we hypothesized that by applying TTFields first, a conditional lethality environment would develop, rendering cells more susceptible to agents such as radiation or in the case of BRCA1 downregulation PARP inhibition. Indeed, by applying TTFields before, rather than after radiation, all cell lines were more susceptible to death. Studies of PARP inhibition in combination with TTFields and TTFields plus radiation are ongoing. Finally, we propose that future use of TTFields in a clinical setting may be more durable if provided post-surgery but prior to or concomitantly with chemotherapy or radiation therapy.

#3218

Maternal embryonic leucine zipper kinase (MELK) confers radioresistance in triple-negative breast cancers (TNBC) through a nonhomologous end joining (NHEJ)-mediated pathway.

Shyam Nyati,1 Ben Chandler,1 Eric Olsen,1 Leah Moubadder,1 Meilan Liu,1 Meleah Cameron,1 Kari Wilder-Romans,1 Theodore S. Lawrence,1 Powel H. Brown,2 Fellix Y. Fang,3 Lori J. Pierce,1 Corey Speers1. 1 _Univ. of Michigan Medical School, Ann Arbor, MI;_ 2 _University of Texas, Austin, TX;_ 3 _University of California at San Francisco, San Francisco, CA_.

Background: Increased rates of locoregional recurrence have been observed in TNBC despite the use of radiation (RT); therefore, approaches that result in radiosensitizaton in TNBC may improve clinical outcomes. Despite some controversy as to the role of MELK kinase as an oncogene, we previously established its clear role in conferring radioresistance in TNBC. Here we extend those studies to understand the mechanism of conferred radioresistance.

Methods: Clonogenic survival assays were used to quantify the degree of radiosensitivity after MELK inhibition. Mass spectrometry was utilized to identify proteins associated with MELK. NHEJ assays were performed using linearized pEYFP vector and quantitated by FACS. qRT-PCR was used to determine MELK RNA expression. Rad51 and γH2AX foci counting were performed to evaluate homologous recombination (HR). Neutral comet assay was used to quantitate dsDNA break repair. Laser microirradiation studies were performed to determine the location and kinetics of protein shuttle to and from sites of dsDNA breaks, including MELK, Ku70 and Ku80 proteins. Fluorescent microscopy was performed for localization of endogenous MELK protein post RT. To study the effect of MELK kinase activity on Ku70-Ku80 complex formation and recruitment at the DNA damage site, cells were lysed and immunoprecipitation was performed using MELK, Ku70 or Ku80-specific antibodies.

Results: We previously demonstrated that genomic or pharmacologic inhibition of MELK confers significant radiosensitization. Mechanistic studies were undertaken to understand the pathways critical to MELK-mediated radioresistance. When MELK is genomically or pharmacologically inhibited, NHEJ and HR reporter assays demonstrate that this radiosensitization is driven through impaired NHEJ at 4, 16, and 24 hrs after RT and not mediated by HR in multiple TNBC cell lines. Tandem mass spectrometry studies of tagged MELK protein identify Ku70 and Ku80 as direct interactors with MELK protein, which is confirmed by Co-IP. Laser microirradiation studies confirm that MELK, Ku70 and Ku80 co-localize to sites of dsDNA breaks, and that MELK kinase function is required to stabilize the Ku70/Ku80 complex at these sites. Comet assay confirms that rapid dissolution of the Ku70/Ku80 complex when MELK is inhibited or functionally dead is not a result of accelerated dsDNA break repair.

Conclusion: MELK kinase function is critical in conferring radioresistance in TNBC and inhibition of function confers radiosensitivity through an NHEJ-mediated pathway. MELK also stabilizes Ku70/Ku80 proteins at sites of dsDNA breaks and allows for more efficient repair of breaks induced by ionizing RT. These results further support the rationale for developing clinical strategies to inhibit MELK in combination with RT treatment as a novel radiosensitizing strategy in TNBC.

#3219

Sensitizing hypoxic small cell lung cancer cells to radiation and hydrogen peroxide-producing agents using CuATSM.

Sebastian Sciegienka, Samuel Rodman, Ann Tomanek-Chalkley, Kelly C. Falls, Douglas R. Spitz, Melissa A. Fath. _Univ. of Iowa, Iowa City, IA_.

Cancer cells have increased steady state levels of reactive oxygen species (ROS; O2•- and H2O2) compared to normal cells. It has been proposed that using redox active agents that further increase ROS levels will result is selective cancer cell death. This metabolic frailty can be targeted using drugs deemed safe for human use, ascorbate (ASC) and disulfiram (DSF), via a mechanism of H2O2 production. CuATSM is a drug being used in clinical trials to treat ALS disease. Imaging studies have shown that CuATSM preferentially concentrates in hypoxic tissues, releasing its Cu after entering cells. Copper (Cu) can participate in oxidation reactions that result in highly toxic hydroxyl radicals. Tumors often have areas of hypoxic tissue that exhibit resistance to ionizing radiation (IR). Our hypothesis is that CuATSM can be used to sensitize hypoxic regions of tumors to IR, ASC and/or DSF.

Method: H2O2 flux after addition of ASC or DSF was measured in small cell lung cancer (SCLC) cells DMS53 using the 3-aminotriazole method. The Cu uptake of these cells was measured in normoxia and hypoxia after treatment with CuATSM. DMS53 and DMS273 (SCLC lines) were treated with ASC, CuATSM, and/or DSF+CuSO4 at varying oxygen tensions with and without IR and clonogenic assays were performed.

Results: Pharmacologically relevant dosing of DSF and ASC resulted in significantly higher fluxes of H2O2 compared to control. Both DSF and ASC enhanced IR clonogenic cell death in SCLC lines. Treatment with CuATSM resulted in increased intracellular Cu and enhanced cell death from ASC and IR in hypoxic conditions.

Conclusion: These observations support the hypothesis that the differences in steady-state level of ROS in small cell lung cancer cells can be exploited to develop effective therapies using ASC and DSF. Furthermore CuATSM can enhance responses in hypoxic tumor tissues to both IR and ASC. (Supported by P50 CA174521, T32 CA078586, P30 CA086862 and Carver Research Program of Excellence in Redox Biology and Medicine.)

#3220

**Preliminary evaluation of** 212 **Pb BB2r targeted radiotherapy in prostate cancer.**

Tammy L. Rold, Nkemakonam C. Okoye, Hoffman Timothy. _Department of Veterans Affairs, Columbia, MO_.

Introduction: Treating castration resistant prostate cancer (CRPC) continues to be a challenge for clinicians. We have recently shown that gamma emitting 203Pb-RM2 (DOTA-4-amino-1-carboxymethyl-piperidine-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) efficiently targets the BB2r expressed on CRPC cells in vivo. We are now exploring the potential of using the alpha-emitting radioisotope 212Pb to generate the radiotherapeutic 212Pb-RM2. Using 212Pb-RM2, we have performed in vitro cell studies to examine treatment effects in terms of DNA damage, clonogenic potential, and cell viability and preliminary in vivo xenograft targeting proof of principle studies.

Methods: 212Pb-RM2 was prepared using an automated radiosynthesizer using 212Pb eluted from a 212Ra/212Pb generator. DNA damage, colony formation, and cell viability were evaluated in PC-3 cells following treatment with 212Pb-RM2 at 0.51, 1, 2, and 4uCi/160uL. DNA damage was assessed using 53BP1 foci following a 4hr incubation with 212Pb-RM2 (T0) and at 24hrs and 48hrs. Colony formation assays were performed on cells treated with TRT for 4hrs, and analyzed at 12-14 days. The MTT assay was conducted up to 144hrs. All cell experiments were performed in triplicate on different occurrences. PC-3 xenografted SCID mice were administered 212Pb-RM2 and organs/tissues were harvested and counted at 15 min, 1hr, 4hr, and 24hrs post injection.

Results: DNA damage was found to be sustained over time with 53BP1 foci/cell at 48hrs of 14.8+1.6, 33.8+2. 6, 29.6+1.1, and 40.6+1.6 (avg+SEM) at 0.51, 1, 2, and 4uCi of 212Pb-RM2/160uL, respectively. Colony formation followed a dose dependent response with surviving fraction decreasing as TRT increased with 17.7+3.2, 4.4+2.9, 0.5+0.5, and 0.0+0.0 (avg+SEM) at 0.51, 1, 2, and 4uCi of 212Pb-RM2/160uL, respectively. 212Pb-RM2 tumor uptake in PC-3 xenografts was found to be 5.2+0.7, 5.0+0.9, 3.9+0.7, and 2.6+0.3 %ID/g at 15min, 1hr, 4hr, and 24hrs respectively. In vivo BB2r blocking studies confirmed 212Pb-RM2 targeting of the BB2r with > 72% blocking of normal pancreas uptake measured at 4hr post injection.

Conclusions: Treatment with alpha emitting 212Pb-RM2 TRT showed sustained levels of DNA damage which was confirmed by impaired colony formation at all dose levels of 212Pb-RM2 TRT studied. Preliminary in vivo tumor targeting and retention of 212Pb-RM2 was observed out to 24hrs post injection warranting further evaluation of 212Pb-RM2 in multiple CRPC cell line models assessing potential therapeutic efficacy.

Acknowledgements: US Veterans Administration (VA) BX001699 and USVA Research Career Scientist (TJH)

## EPIDEMIOLOGY:

### Biomarkers of Exogenous and Endogenous Risk Factors in Cancer Epidemiology

#3221

Plasma B-vitamin and one-carbon metabolites and risk of breast cancer before and after folic acid fortification in the US.

Serena C. Houghton,1 A. Heather Eliassen,2 Shumin M. Zhang,2 Jacob Selhub,3 Bernard A. Rosner,2 Walter C. Willett,4 Susan E. Hankinson1. 1 _University of Massachusetts Amherst, Amherst, MA;_ 2 _Brigham and Women's Hospital and Harvard Medical School, Boston, MA;_ 3 _Tufts University, Boston, MA;_ 4 _Harvard T.H. Chan School of Public Health, Boston, MA_.

Background: Prior epidemiologic findings for plasma folate and B-vitamins and breast cancer risk are inconsistent and have not assessed the possible influence of folic acid fortification mandated in 1998. Excess folate, through the one-carbon cycle, might promote neoplastic lesions through DNA synthesis and methylation and inactivation of tumor suppressor genes. However, deficiencies in folate and other B-vitamins may also stimulate carcinogenesis through this same cycle. With concern for carcinogenesis at both low and high folate levels, folic acid fortification has been controversial. Therefore, we examined the associations between plasma folate, B12, B6, homocysteine, cysteine, and cysteinylglycine and breast cancer risk, before and after fortification.

Methods: We conducted a nested case-control study within the prospective Nurses' Health Study. In 1989-1990 (pre-fortification), 32,826 women donated a first blood sample and 18,743 donated an additional blood sample in 2000-2001 (post-fortification). From 1989 to 2006, 1874 incident breast cancer cases with at least one blood sample and 367 with two blood samples were matched 1:1 to controls on age, date/ time of blood draw, fasting status, postmenopausal hormones use, and menopausal status. Conditional logistic regression was used to estimate relative risks (RR) and 95% confidence intervals (CI) adjusting for age at menarche, parity/age at first birth, age at menopause, family history of breast cancer, history of benign breast disease, height, body mass index at 18, weight change since 18, and alcohol intake. Additionally to assess the effect of fortification, we cross-classified plasma levels at the median for women with two bloods and used unconditional logistic regression adjusting for breast cancer risk factors using low (<median) in both 1990 and 2000 as the referent group.

Results: Overall, higher plasma folate, B12, B6, homocysteine, cysteine, and cysteinylglycine levels were not associated with breast cancer risk. For example, comparing women in the highest versus lowest quintile of 1990 plasma folate, adjusted RR (95% CI) was 0.95 (0.77-1.17) for breast cancer. Associations did not vary by in situ /invasive, hormone receptor status, or molecular subtype of the tumor. Additionally, associations did not vary before/after fortification. For example, compared to those with consistently low plasma folate levels, RRs (95% CI) were 0.90 (0.58-1.40) among those that were low 1990/ high 2000, 0.93 (0.59-1.45) among those high 1990/ low 2000, and 1.10 (0.75-1.63) among those that were consistently high.

Conclusion: Plasma folate, B12, B6, homocysteine, cysteine, and cysteinylglycine were not significantly associated with breast cancer overall, before and after fortification, or with specific molecular subtypes. These results do not support concerns about folic acid fortification and breast cancer risk.

#3222

Evaluation of associations between circulating proteins and breast cancer risk using genetic variants.

Xiang Shu,1 Jiandong Bao,1 Lang Wu,1 Jirong Long,1 Xingyi Guo,1 Kyriaki Michailidou,2 Manjeet K. Bolla,2 Qin Wang,2 Joe Dennis,2 Jacques Simard,3 Douglas F. Easton2. 1 _Vanderbilt University Medical Center, Nashville, TN;_ 2 _University of Cambridge, United Kingdom;_ 3 _Laval University, Quebec, Canada_.

Breast cancer is the most commonly diagnosed cancer in women in many countries. Several circulating protein biomarkers have been identified in relation to breast cancer risk. However, previous studies either had small sample sizes or employed low-throughput techniques. To search for novel biomarkers, we utilized genetic variants as instruments and evaluated over 1,400 proteins in relation to breast cancer risk using data from the Breast Cancer Association Consortium (BCAC).

We extracted beta coefficients from reported protein quantitative trait loci (pQTL) derived from genome-wide association studies of circulating proteins. Summary statistics of these pQTL variants associated with breast cancer risk were obtained from 122,977 cases and 105,974 controls of European descent in the BCAC. Associations of genetically predicted protein levels with breast cancer risk were evaluated using the inverse-variance weighted method. For proteins with a significant association, expression levels of the corresponding gene were predicted using genotyping and transcriptomic data from the Genotype-Tissue Expression project and then evaluated for their associations with breast cancer risk.

We identified 56 protein biomarkers showing a significant association with breast cancer risk after accounting for multiple comparisons (false discovery rate < 0.05). Among them, levels of 32 proteins were influenced by variants close to a newly reported breast cancer susceptibility locus (9q34.2, ABO). Inverse associations of breast cancer risk were found with membrane proteins such as insulin receptor, insulin-like growth factor receptor 1, hepatocyte growth factor receptor, neurogenic locus notch homolog protein 1, and vascular endothelial growth factor receptor 2, with odds ratios ranging from 0.82 to 0.97 per unit increase in genetic risk scores (p-values ranging from 6.53x10-4 to 3.28x10-8). Genetically predicted expression of five genes, CPNE1, CTSF, TFPI, SCG3, and QSOX2, was found to be associated with breast cancer risk at p <0.05 in the same direction as the associations observed for the corresponding proteins. Results from our study suggest that multiple membrane proteins related to insulin resistance may be linked to breast cancer risk through genetic variants at 9q34.2.

#3223

Validation of a blood biomarker for identification of individuals at high risk for gastric cancer.

Meira Epplein,1 Julia Butt,2 Yang Zhang,3 Laura Hendrix,1 Christian Abnet,4 Youlin Qiao,5 Gwen Murphy,4 Philip R. Taylor,4 Wei Zheng,6 Xiao-Ou Shu,6 Taichi Shimazu,7 Shoichiro Tsugane,7 Keun-Young Yoo,8 Sue Park,8 Jeongseon Kim,9 Sun Ha Jee,10 Tim Waterboer,2 Michael Pawlita,2 Wei-cheng You,3 Kai-feng Pan3. 1 _Duke University, Durham, NC;_ 2 _German Cancer Research Center, Heidelberg, Germany;_ 3 _Beijing Institute for Cancer Research, Beijing, China;_ 4 _National Cancer Institute, Bethesda, MD;_ 5 _Chinese Academy of Medical Sciences, China;_ 6 _Vanderbilt University, Nashville, TN;_ 7 _National Cancer Center of Japan, Japan;_ 8 _Seoul National University, Republic of Korea;_ 9 _National Cancer Center of Korea, Republic of Korea;_ 10 _Yonsei University, Republic of Korea_.

BACKGROUND AND AIMS: Infection with Helicobacter pylori is the leading cause of gastric cancer, the fifth most common cancer worldwide, and yet mass eradication is not recommended as the majority of infected individuals will not develop neoplasia. Previously we developed and replicated serologic H. pylori biomarkers for gastric cancer risk among prospective cohorts in East Asia, and now seek to validate these biomarkers in a high-risk area of China.

METHODS: This cross-sectional study included 1,402 individuals from Linqu County who were screened by upper endoscopy from 2002-2004. Blood samples were assessed for H. pylori protein antibody levels using multiplex serology. Multivariable-adjusted logistic regression models were used for calculation of odds ratios (ORs) and 95% confidence intervals (CIs) for prevalent intestinal metaplasia, indefinite dysplasia, or dysplasia, compared to superficial or mild atrophic gastritis (controls).

RESULTS: Compared to individuals sero-negative to Omp and HP 0305, individuals sero-positive to both were seven times more likely to have prevalent precancerous lesions (OR, 7.43; 95% CI, 5.59-9.88). A risk prediction model for precancerous lesions that includes age, smoking, H. pylori sero-positivity, and individual sero-positivity to Omp and HP 0305 resulted in an area under the curve (AUC) of 0.7510 (95% CI, 0.7245-0.7774), which is significantly better than the same model including the previously established gastric cancer risk factor CagA (AUC, 0.7184; 95% CI, 0.6908 -0.7461, p=0.0002).

CONCLUSIONS: These findings validate that biomarkers Omp and HP0305 are strongly associated with pre-malignant gastric lesions in East Asia. Furthermore, the evidence presented here suggests that these two markers can significantly contribute to a risk prediction model in East Asia, as they predict risk beyond the established, and highly prevalent in East Asia, known virulence marker of CagA.

#3224

Prospective serum metabolomic profiling of fatal prostate cancer.

Jiaqi Huang,1 Alison M. Mondul,2 Stephanie J. Weinstein,1 Andriy Derkach,1 Steven C. Moore,1 Joshua N. Sampson,1 Demetrius Albanes1. 1 _NCI-DCEG, Rockville, MD;_ 2 _University of Michigan School of Public Health, Ann Arbor, MI_.

Metabolic dysregulation may play a role in the etiology of fatal prostate cancer, yet the association with pre-diagnostic serum metabolites has not been elucidated. We conducted a prospective serum metabolomic analysis of fatal prostate cancer risk in 523 cases and 523 matched controls nested within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study. Median time from baseline fasting serum collection to prostate cancer death was 18 years (interdecile range 10-26 years). We profiled 860 known metabolites through an ultrahigh-performance LC-MS/MS platform, and conditional logistic regression models were used to estimate odds ratios (OR) and 95% confidence intervals of risk associated with 1-standard deviation increases in the log-metabolite signals. Based on a false discovery rate (FDR) of 0.15 or lower, 34 metabolites are identified as being associated with fatal prostate cancer. Notably, increased serum thioproline and the three cysteine-related amino acids combined (thioproline, cysteine and cystine) are associated with lower odds of fatal disease (OR=0.75 and 0.71, respectively; P≤8.2×10-5). In contrast, the serum dipeptide leucylglycine (OR=1.36, P=8.2×10-5), and three gamma-glutamyl amino acids, including gamma-glutamylvaline, gamma-glutamylglycine and gamma-glutamylleucine (OR=1.28-1.30, P≤4.6×10-4), are associated with increased fatal disease risk. Several other dipeptides including histidylalanine, valylglycine and leucylglutamine, as well as the nucleotides dihydroorotate, pseudouridine, 2'-O-methyluridine, 5,6-dihydrouridine and 5-methyluridine (ribothymidine), also have significant positive associations with fatal disease risk. Findings are not materially altered by adjustment for body mass index, smoking, serum total and HDL cholesterol, serum alpha-tocopherol and retinol, or the ATBC trial supplementation. Our results are also similar across strata of time from blood collection to prostate cancer death and age at blood collection. Gene-set analysis (GSA) reveals a significant peptide super-metabolic pathway association with lethal disease (P<0.0001), and the sub-pathways of dipeptides, pyrimidines (uracil containing), gamma-glutamyl amino acids, glycine/serine/threonine, n3 and n6 polyunsaturated fatty acids, amino-sugars, androgenic steroids, dicarboxylate fatty acids, and endocannabinoids are similarly associated with fatal prostate cancer (FDRs ≤ 0.1). Our study demonstrates a serum risk profile up to two decades prior to death of prostate cancer that is characterized by multiple dysregulated biochemical metabolites of redox status and dipeptide, pyrimidine (uracil), and gamma-glutamyl amino acid metabolism. The findings represent novel leads related to the molecular basis of the etiology or subclinical course of fatal prostate cancer that warrant examination in other populations.

#3225

Cutaneous nevi and cancer risk among US women.

Xin Li,1 Wenting Wu,1 Edward Giovannucci,2 Meir J. Stampfer,2 Xiang Gao,3 Jiali Han1. 1 _Fairbanks School of Public Health, Indiana University, Indianapolis, IN;_ 2 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 3 _College of Health and Human Development, Pennsylvania State University, State College, PA_.

Background: Longer telomeres have been linked to elevated cutaneous nevus number. Recently, a large Mendelian randomization study identified a positive association between telomere length and risk of cancer. We hypothesized that higher nevus count, as a phenotypic marker of longer telomere length, may be associated with increased risk of cancer development.

Methods: We prospectively examined the association between nevus count and total as well as site-specific cancer risk among participants in the Nurses' Health Study (NHS, 1986-2012) and the Nurses' Health Study 2 (NHS2, 1989-2013).

Results: During 3,941,477 person-years of follow-up, we documented a total of 24,428 cancer cases (16,297 in the NHS and 8,131 in the NHS2). Compared to participants who had no nevi, the multivariate HRs were 1.08 (95%, 1.05-1.11) for women with 1-5 nevi, 1.15 (95%, 1.09-1.21) for those who had 6-14 nevi, and 1.26 (95%, 1.18-1.34) for those with 15 or more nevi (p trend < 0.0001). We did not observe significant interactions between the number of nevi and other covariates in the multivariable-adjusted model. We also conducted secondary analyses by excluding 1) only melanoma and 2) both melanoma and breast cancer from the outcomes of interest, respectively. The results were very similar to those of our primary analysis using all cancer sites. For individual cancer sites, most of the associations with mole counts were positive but not statistically significant.

Conclusion: We identified the number of cutaneous nevi as a phenotypic marker associated with cancer risk, which may possibly be explained by telomere biology.

#3226

Overweight and obese women with high volumetric breast density at high breast cancer risk.

Natalie J. Engmann,1 Christopher G. Scott,2 Matthew R. Jensen,2 Stacey J. Winham,2 Lin Ma,1 Kathleen R. Brandt,3 Amir Mahmoudzadeh,1 Serghei Malkov,1 Dana Whaley,2 Carrie Hruska,2 Fang Fang Wu,2 Diana L. Miglioretti,4 Aaron D. Norman,2 John Heine,5 John Shepherd,1 Vernon S. Pankratz,6 Celine M. Vachon,2 Karla Kerlikowske1. 1 _UCSF, San Francisco, CA;_ 2 _Mayo Clinic, Rochester, MN;_ 3 _Mayo Clinic, San Francisco, CA;_ 4 _University of California Davis, Davis, CA;_ 5 _Moffitt Cancer Center, Tampa, FL;_ 6 _University of New Mexico Health Sciences Center, Albuquerque, NM_.

Purpose: Volumetric breast density and body mass index (BMI) are increasingly used for breast cancer risk stratification. We examine if the effect of volumetric breast density on breast cancer risk increases with increasing BMI.

Methods: Participants were from two case-control studies within mammography practices, the San Francisco Mammography Registry and the Mayo Clinic Rochester, Breast Screening Practice. Breast cancers were included if diagnosed between January 2007 and 2015 and with a screening full-field digital mammogram at least 6 months prior to diagnosis; the earliest mammogram within 5 years of diagnosis was selected. Up to three controls were matched to each case on age, race, date, mammography machine, and state of residence. Volumetric percent density (VPD), dense volume (DV) and non-dense volume (NDV) were measured using VolparaTM software. Breast cancer risk was assessed using logistic regression stratified by menopause status and adjusted for matching factors, family history of breast cancer, parity/age at first birth, and postmenopausal hormone therapy. Models for DV were additionally adjusted for NDV, and NDV models for DV. Multiplicative interactions were fit between BMI categories [<25 (underweight), 25-30 (normal weight) and >30 (overweight/obese) kg/m2] and VPD, DV, and NDV, and trend tests assessed for increasing odds ratios (OR) with increasing BMI.

Results: A total of 781 premenopausal and 1850 postmenopausal breast cancers and 3535 controls were included in the analysis. Median age of premenopausal women was 45 years (IQR: 6.1) and 63.3 years (IQR: 14) for postmenopausal women. Cases vs. controls had greater VPD and DV for both premenopausal (VPD: 14.9% vs. 12.0%, DV: 74.1 cm3 vs. 64.4 cm3) and postmenopausal women (VPD: 6.8% vs. 6.1%, DV: 53.4 cm3 vs. 48.0 cm3)(all p's<0.001). Trends between increasing BMI and VPD were evident for both pre (ptrend=0.0007) and postmenopausal (ptrend=0.0005) women. Among premenopausal women, the odds ratio (OR) for breast cancer associated with a 10% increase in VPD was of 1.39, 2.19 and 2.88 for BMI <25, 25-30 and >30 kg/m2 (p-trend=0.0007), respectively. For DV, OR's were 1.39, 1.33 and 1.51 for a 1 SD increase in DV, respectively, though the interaction with DV was not significant (ptrend=0.68). Among postmenopausal women, a 10% increase in VPD was associated with OR's of 1.35, 2.03, 3.6 for BMI <25, 25-29, >30-kg/m2 (ptrend =0.0001), respectively, and 1.31, 1.34 and 1.65 for a 1 SD increase in DV (ptrend =0.01), respectively. Associations between NDV and breast cancer risk did not differ by BMI category for premenopausal (ptrend =0.52) or postmenopausal (ptrend =0.07) women.

Conclusions: The effect of VPD on breast cancer risk is strongest in overweight/obese women. As volumetric breast density and BMI are commonly used in clinical risk stratification, these differences in risk have high clinical relevance for informing prevention decisions.

#3227

Getting rid of obesity in middle-to-late adulthood relates to lower prostate cancer risk: Another piece of evidence supporting the "rapid testosterone reduction theory".

Kai Wang,1 Xinguang Chen,1 Travis A. Gerke,2 Victoria Y. Bird,1 Mattia Prosperi1. 1 _University of Florida, Gainesville, FL;_ 2 _Moffitt Cancer Center, Tampa, FL_.

Background: Previously we demonstrated that a smoothed age-related decline in testosterone was associated with decreased risk of prostate cancer (PCa) ("rapid testosterone reduction theory"). Obesity relates to low testosterone, so body weight pattern reflecting smoothed testosterone decline should be related to lower PCa risk. This study aimed to validate the "rapid testosterone reduction theory" from the perspective of body mass index (BMI) pattern and risk of PCa. Methods: We conducted a nested case-control study in a retrospective hospital cohort with median follow-up of 5.0 years from a tertiary-level hospital in the Southeastern US. Electronic medical records of 18168 patients with prostate-related disease diagnosed between November 4, 1995 and January 10, 2016 were screened. The outcome was newly diagnosed PCa. Only patients with BMI measured at ≥3 differing calendar years before PCa diagnosis or the latest follow-up (for non-PCa patients) were included, resulting in a sample of 1571, among which 128 were PCa patients. BMI was analyzed in cross-section value, change, and developmental trajectories in relation to PCa risk. Results: Participant ages spanned a range of 40 to 90 years, with mean age at baseline was 57 years (standard deviation = 9.6). Baseline BMI was inversely associated with PCa risk (adjusted odds ratio [AOR], 0.76; 95% confidence interval [CI], 0.63-0.91 for every 5-unite increase in BMI). When analyzed in change between baseline and the latest follow-up, BMI increment was associated with increased PCa risk (AOR, 1.26; 95% CI, 1.05-1.51 for every 5-unite increase in BMI change). When analyzed in latent-class trajectory, a downward BMI trajectory of being obese in the 4th, 5th, and 6th decade then progressing to be non-obese since the 7th decade of age was associated with decreased PCa risk (AOR, 0.66; 95% CI, 0.45-0.98), compared to those with consistently normal BMI. When analyzed by strata of PCa diagnosis age, the same associations were more evident for PCa diagnosed after age 65. Conclusions: Weight loss and getting rid of obesity during middle-to-late adulthood is related to a decreased PCa risk, supporting the "rapid testosterone reduction theory" in explaining PCa risk. If this finding can be verified with more rigorous study, provision of testosterone replacement therapy as a weight control measure for men in middle-to-later adulthood has potentials in PCa prevention.

#3228

Tobacco smoking and circulating immune-related biomarkers in monozygotic twins.

Jun Wang,1 David Conti,1 Marta Epeldegui,2 Miina Ollikainen,3 Amie E. Hwang,1 Ann S. Hamilton,1 Larry Magpantay,2 Rachel Tyndale,4 Thomas M. Mack,1 Otoniel Martinez-Maza,2 Jaakko Kaprio,3 Wendy Cozen1. 1 _University of Southern California, Los Angeles, CA;_ 2 _University of California, Los Angeles, Los Angeles, CA;_ 3 _University of Helsinki, Helsinki, Finland;_ 4 _University of Toronto, Toronto, Ontario, Canada_.

Background: Tobacco smoking is a cause of a variety of cancers by various mechanisms. Paradoxically, smoking also increases the risk of atopy and asthma, which are inversely associated with some cancers such as glioma, colorectal cancer and non-Hodgkin lymphoma, but positively associated with others such as lung cancer. Tobacco smoking may affect the immune system, which may explain some of these associations. We assessed the association between smoking and levels of 27 serum immune/inflammatory markers and DNA methylation in healthy monozygotic (MZ) twins.

Methods: 67 MZ twin pairs were identified from the Finnish Twin Cohort Study. Cotinine and immune related biomarkers were measured from fasting serum samples using LC-MS/MS and Luminex multiples assays. Current smoking status was defined by cotinine >3.08 ng/mL. Current smokers were further categorized into low vs high smoking level by the median cotinine (78.17 ng/mL) among smokers. Questionnaire reports of current and former smoking included duration, amount (cigarettes per day [CPD]) and years since quitting. Linear mixed models were used to assess the association between smoking variables and each individual biomarker. For each smoking variable P values were adjusted for multiple comparisons using Pact, taking into account correlations among biomarkers. For biomarkers significantly associated with smoking, we assessed whether blood DNA methylation of biomarker-related genes mediated the smoking-biomarker association.

Results: The median age of the study population was 24.8 years (range 21.0-68.9 years) and 56.7% were female twins. 32.1% of the twins were current smokers according to cotinine levels. Current smoking, defined by either cotinine or self-reports, was significantly associated with CCL17, B-cell activating factor (BAFF) and haptoglobin (Hp) levels respectively, after adjusting for multiple comparisons. For instance, serum cotinine was associated with increasing CCL17 levels (Pact for trend test = 0.002): the geometric mean CCL17, adjusted for age and sex among current high-level smokers was approximately 8.2% higher than noncurrent smokers. Similar positive dose-response relationships were observed for self-reported smoking variables with the 3 biomarkers. However, we found no associations between former smoking and any of the 27 biomarkers. We also found that smoking-associated DNA methylation alterations in 3 CpG sites of BAFF affected circulating CCL17 (CpG site: cg11726530) and Hp (cg09158314 and cg21784254) levels, respectively.

Conclusion: Current but not former smoking may be associated with alterations in circulating levels of CCL17, BAFF and Hp, suggesting that smoking may promote B-cell activation and affect Th2 immune response, which may play a role in carcinogenesis. Further, preliminary mediation analysis suggests that smoking-induced alterations in these biomarkers may partially mediate through DNA methylation.

#3229

Prediagnostic biomarkers of metabolic dysregulation and cancer mortality.

Tomi Akinyemiju. _University of Kentucky, Lexington, KY_.

INTRODUCTION: The obesogenic milieu is a pro-tumorigenic environment that promotes tumor initiation, angiogenesis and metastasis. In this prospective cohort, we examined the association between pre-diagnostic metabolic biomarkers, plasma adiponectin, resistin, leptin and lipoprotein (a), and the risk of cancer mortality.

METHODS: Prospective data was obtained from the REasons for Geographic and Racial Differences in Stroke (REGARDS) cohort of Blacks and Whites followed from 2003 through 2012 for cancer mortality. We determined the association between metabolism biomarkers (log-transformed and tertiles) and risk of cancer mortality using Cox Proportional Hazards models with robust sandwich estimators to calculate the 95% confidence intervals (CIs), and adjusted for baseline covariates, including age, gender, income, education, physical activity, BMI, smoking status, alcohol use, and comorbidity score.

RESULTS: Among 1764 participants with available biomarker data, each SD higher log-leptin was associated with a 54% reduced risk of total cancer mortality (HR: 0.46, 95% CI: 0.23 – 0.92) and obesity-related cancer mortality (HR: 0.55, 95% CI: 0.39-0.79). Among Blacks only, each SD higher log-resistin was associated with a nearly 7-fold increased risk of cancer mortality (adjusted HR: 6.68, 95% CI: 2.10 – 21.21). There were no significant associations of adiponectin or Lp(a) and cancer mortality.

CONCLUSIONS: Leptin is involved in long-term regulation of energy balance, while resistin is involved in chronic inflammation and LDL production. These findings highlight the biological mechanisms linking metabolic dysregulation with cancer mortality, and the influence of resistin on cancer mortality only among Blacks suggests that this hormone may be a useful biomarker of racial differences in cancer mortality that deserves further study.

#3230

Genome-wide blood DNA methylation and breast cancer risk: A meta-analysis of four prospective studies.

Clara Bodelon,1 Srikant Ambatipudi,2 Pierre-Antoine Dugué,3 Annelie Johansson,4 Joshua N. Sampson,1 Melissa C. Southey,5 Graham G. Giles,3 Silvia Polidoro,6 Zdenko Herceg,2 James M. Flanagan,4 Roger L. Milne,3 Montserrat Garcia-Closas1. 1 _Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD;_ 2 _International Agency for Research on Cancer, Lyon, France;_ 3 _Cancer Council Victoria, Victoria, Australia;_ 4 _Imperial College London, London, United Kingdom;_ 5 _The University of Melbourne, Melbourne, Australia;_ 6 _Italian Institute for Genomic Medicine, Torino, Italy_.

Background. Environmental and genetic factors play an important role in the etiology of breast cancer; however the potential epigenetic component to the risk remains largely unexplored. DNA methylation in blood cells could serve as a biomarker for exposure and breast cancer risk. Several small blood-based DNA methylation studies have reported risk associations with individual CpGs and average methylation levels; however, these findings require validation in larger prospective cohort studies. To investigate the role of blood DNA methylation on breast cancer risk, we conducted a meta-analysis of four prospective cohort studies, including a total of 1,941 cases and 1,952 controls, the largest study of blood DNA methylation and breast cancer risk to date.

Methods. We assessed associations with methylation at 365,589 CpGs present in the HumanMethylation450 Beadchip, after excluding CpGs that did not pass quality controls in all studies. Each of the four cohorts (the Melbourne Collaborative Cohort Study, the Italian and IARC cohorts of the European Prospective Investigation into Cancer and Nutrition and the Prostate, Lung, Colorectal and Ovarian screening trial) estimated odds ratios (ORs) and 95% confidence intervals (CI) for the association between each individual CpG and breast cancer risk. In addition, each study assessed the association between average methylation measures and breast cancer risk, adjusted and unadjusted for cell-type composition. Study-specific ORs were combined using fixed-effect meta-analysis with inverse variance weights. The false discovery rate (q-value) was used to account for multiple testing.

Results. Methylation measured at individual CpGs in blood DNA was not associated with breast cancer risk (q-value>0.6). In addition, higher average methylation level was not associated with risk of breast cancer (OR=0.94, 95% CI=0.85, 1.04; P=0.25; P for study heterogeneity=0.83). We found no evidence of modification of this association by age at diagnosis, time since blood collection or CpG location (P-heterogeneity>0.05).

Conclusions. Overall, our data indicates that DNA methylation measured in blood prior to breast cancer diagnosis is unlikely to be associated with substantial breast cancer risk. Larger studies are needed to determine if modest to weak associations exist between blood DNA methylation and breast cancer risk.

#3231

Prospective study of genomic 5-methylcytosine and LINE-1 methylation levels of leukocyte DNA and colorectal cancer risk.

Wen-Yi Huang,1 Mark P. Purdue,1 L. Joseph Su,2 Hormuzd A. Katki,1 Lee E. Moore,1 Srinivasan Yegnasubramanian,3 Sonja I. Berndt1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _University of Arkansas for Medical Sciences, Little Rock, AR;_ 3 _Johns Hopkins University, Baltimore, MD_.

Global hypomethylation in peripheral blood leukocyte DNA, measured by the reduction of genome-wide methylated cytosine (5-mC) or the long interspersed nuclear element-1 (LINE-1) level, has been associated with an increased risk of colorectal cancer, mainly in retrospective studies. Little is known as to how these global methylation markers relate in individuals and their associations with colorectal cancer risk in a prospective study setting.

We studied leukocyte 5-mC and LINE-1 methylation levels in prospectively collected blood specimens from 404 cases and 528 controls who were free of colorectal cancer at blood collection from the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. Leukocyte 5-mC level was determined by an HPLC/Tandem Mass Spectrometry method and expressed as the relative amount of methyl- to total cytosine residues (%5-mC). LINE-1 methylation level was measured by McrBC digestion followed by real-time PCR of the undigested/unmethylated LINE1 promoter sequences. In our analysis, we excluded cases diagnosed within one year of blood draw to minimize the potential for reverse causation from prevalent disease. We estimated odds ratios (ORs) and 95% confidence intervals (CIs) for tertiles of methylation using multivariable logistic regression, adjusting for age, sex, and other potential confounders.

We observed a significant association between low LINE-1 methylation and colorectal cancer risk (1st vs. 3rd tertile: OR=1.63, 95% CI=1.10-2.41; P trend=0.01) and a near-significant association with low %5-mC methylation (OR=1.37, 95% CI=0.92-2.03; P trend=0.15). The two markers were not correlated (Spearman's r=0.01). Comparing subjects with low methylation levels (1st tertiles) at both markers with those having the highest methylation level (3rd tertile) at either marker, we observed a stronger significant association (OR=2.49, 95% CI=1.62-3.81; P trend=0.0001). Interestingly, we also found that the association between natural folate intake and colorectal cancer risk was modified by %5-mC or two markers combined (P interaction= 0.0003 and 0.02, respectively). Among individuals with the highest methylation level at either marker, high natural folate intake (3rd tertile) was associated with a reduced risk of colorectal cancer compared to low (1st tertile) folate intake (OR=0.49, 95% CI=0.25-0.96); no association was observed among individuals with lower levels of methylation. No significant risk modification was found for other suspected risk factors, such as smoking, obesity, and aspirin/ibuprofen use.

This prospective investigation showed that reduced LINE-1 methylation in leukocyte DNA, especially in conjunction with reduced 5-mC methylation, was associated with increased colorectal cancer risk. Folate may protect against colorectal carcinogenesis through mechanisms involving adequate DNA methylation in the genome.

#3232

Circulating sCD27 and sCD30 in samples collected fifteen years apart and non-Hodgkin lymphoma risk: Findings from the CLUE-I and CLUE-II cohorts.

Mark P. Purdue,1 Qing Lan,1 Judith Hoffman-Bolton,2 Allan Hildesheim,1 Paul Strickland,2 Kala Visvanathan,2 Nathaniel Rothman1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Johns Hopkins Bloomberg School of Public Health, Baltimore, MD_.

Elevated serum levels of the immune activation markers sCD27 and sCD30 have been consistently associated with increased future risk of non-Hodgkin lymphoma (NHL). However, past studies could not clarify patterns of these markers over time preceding diagnosis, given their use of a single banked sample per participant. To address this question, we conducted a nested case-control study within the CLUE-I and CLUE-II cohorts including 102 case-control matched pairs with two banked samples per participant, collected fifteen years apart. The CLUE-II sCD27 concentrations among cases were higher than in the earlier CLUE-I samples (P<0.0001), whereas no systematic difference was observed for controls (P=0.89). The sCD27 increase was greater for cases diagnosed less than 10 years vs. 10+ years after CLUE-II enrollment (P = 0.0007). For sCD30, an increase between samples was observed for both cases (P = 0.0002) and controls (P=0.02), and the magnitude of increase among cases did not significantly differ across the two follow-up periods (P = 0.12). In comparison to having low (below-median) sCD27 levels in both CLUE-I and CLUE-II samples, associations with NHL were observed for having low CLUE-I sCD27 and high CLUE-II sCD27 ("low / high"; odds ratio 2.4, 95% confidence interval 1.0-6.2) as well as having high sCD27 levels in both samples ("high / high"; 2.1, 1.0-4.4). For sCD30, only the "high-high" category was associated with NHL (2.1, 0.9-5.0). The "low-high" sCD27 association was apparent only for cases diagnosed less than 10 years after CLUE-II (4.1, 1.3-13.0), while the "high-high" associations were consistent across follow-up periods. Our findings suggest that multiple factors may underlie NHL associations with these markers; rising sCD27 closer to diagnosis is likely driven by occult disease, whereas associations with stable elevated analyte levels may reflect etiologically relevant effects.

#3233

The effects of electronic cigarette exposure on genome-wide expression in human bronchial epithelium.

Daniel Y. Weng,1 Min-Ae Song,1 Theodore M. Brasky,1 Joseph P. McElroy,1 Ewy Mathe,1 Jo L. Freudenheim,2 Mark D. Wewers,1 Peter G. Shields1. 1 _The Ohio State University, Columbus, OH;_ 2 _University at Buffalo, Buffalo, NY_.

Background: Although smoking rates continue to decline in US adults, the number of electronic cigarette (e-cig) users is rapidly growing, especially among youth. Exposure to cigarette smoke is known to result in lung cancer and chronic obstructive pulmonary disease, is associated with inflammatory and immune responses generally, and effects gene expression related these and other pathways. However, the effects of e-cigs on the gene expression and inflammation in the bronchial epithelium are largely unknown. We conducted a cross-sectional study to evaluate the effects of e-cig use on gene expression in the bronchial epithelium, in comparison to effects on never smokers and cigarette smokers.

Methods: Epithelia brushings were obtained by bronchoscopy from never smokers (n=42), e-cig users (n=14), and current smokers (n=16) (overall, age 21-30, 78% European-American, 54% male). RNA was extracted and profiled for the whole genome transcriptome (Affymetrix Human Transcriptome Array 2.0). Between group differences were determined with the t-test. False Discovery Rate (FDR) < 0.05 was considered significant. Functional and network analyses were performed using Ingenuity Pathway Analysis (IPA) software.

Results: The average number of cigarettes smoked per day in the smoker group was 16, ranging from 10 to 20 cigarettes/day. In the e-cig user group, the average number of puffs inhaled per day was 173, ranging from 20 to 600, and the average volume of e-liquid per day was 8 ml, ranging from 2 to 20 ml. Of the 14 e-cig users, eleven identified themselves as former smokers (average 24 months since last cigarette), while three were never-smokers. In microarray analysis, comparing never smoker and smoker groups, there were 2,536 differentially expressed genes (DEGs; 1,235 up-regulated genes, 1,301 down-regulated genes in smokers) and 69 DEGs comparing never smoker and e-cig user groups (59 up-regulated genes, 10 down-regulated genes in e-cig users). In addition, there were 108 DEGs between e-cig user and smoker groups (83 up-regulated genes, 25 down-regulated genes in smokers). Several well-known smoking-related genes such as CYP1B1, AKR1B10, ALDH3A1, CYP2A13, and CX3CL1 were significantly decreased in their expression for e-cig users compared to smokers. Unsupervised hierarchical clustering revealed that the expression profiles of never smokers and e-cig users are more similar to each other compared to profiles of smokers. Pathway analysis of smoking-related genes revealed that NRF2-mediated oxidative stress was the most significant canonical pathway (P = 7.88E-11).

Conclusions: Consistent with the hypothesis that e-cigs are less harmful than smoking, bronchial epithelium gene expression profiles of never smokers and e-cig users are more similar, while smokers exhibit distinct profiles. Associations of different nicotine products are apparent in gene expression profiles.

#3234

Identification of novel risk factors for ovarian cancer in the Nurses' Health Studies using metabolomics.

Oana A. Zeleznik,1 Elizabeth M. Poole,1 Clary Clish,2 Sarah Jeanfavre,2 Amy Deik,2 Kevin Bullock,2 Daniel Hitchcock,2 Julian Avila-Pancheco,2 Peter Kraft,3 A Heather Eliassen,1 Shelley S. Tworoger4. 1 _Harvard Medical School and Brigham and Women's Hospital, Boston, MA;_ 2 _Broad Institute of MIT and Harvard, Boston, MA;_ 3 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 4 _Harvard T.H. Chan School of Public Health and Moffitt Cancer Center, Tampa, FL_.

Background: No reliable early detection modalities are available for ovarian cancer (OC), thus most cases are diagnosed at an advanced stage when treatment is less effective. Bilateral salpingo-oophorectomy can prevent disease, but because of negative health consequences, it is indicated only for high-risk women. Beside high-penetrance mutations, absolute OC risk prediction is poor, emphasizing the need to identify new risk factors that increase prediction to a clinically meaningful level. Thus, we conducted the first-ever prospective assessment of the association of pre-diagnosis plasma metabolites with risk of OC and its histotypes.

Methods: Targeted metabolomics were measured on 300 cases and 300 matched controls. We used logistic regression and network analysis to identify individual and groups of metabolites associated with risk. Fisher's test was used to determine over-represented metabolite classes within the risk-associated metabolites.

Results: At nominal p≤0.05, 24 metabolites were associated with risk of overall OC, 13 metabolites were associated with serous OC, 71 metabolites were associated with endometrioid/clear cell OC, and 5 metabolites were associated with mucinous OC (Table 1). The metabolites for endometrioid/clear cell and mucinous OC were significant after accounting for multiple comparisons (FDR-corrected p≤0.3).

Table 1: OR (95%CI) of top metabolites for an increase from the median of the bottom to top quintile of levels with risk of ovarian cancer overall and by histotype.

---

Overall

OR (95% CI)1 | Serous

OR (95% CI)1 | Endometrioid/Clear Cell

OR (95% CI)2 | Mucinous

OR (95% CI)2

Pseudouridine

2.12 (1.30, 3.47) | Pseudouridine

2.26 (1.33, 3.91) | C36:2 PC plasmalogen

0.17 (0.06, 0.46) | Trihydroxyecdysone

42.3 (7.24,342)

Acetylcytidine

1.82 (1.17, 2.81) | C54:6 TAG

1.92 (1.18, 3.17) | C56:8 TAG

4.85 (1.76, 14.22) | Laserpitin*

23.3 (4.82, 150)

C18:0 Sphingomyelin

1.82 (1.15, 2.88) | Dimethyllysine

1.92 (1.18, 3.16) | C22:0 LPE

0.21 (0.07, 0.57) | Pregnenolone sulfate*

24.0 (4.13, 182)

1nominally significant (p≤0.5), 2significant after adjusting for multiple comparisons (FDR-corrected p≤0.3), PC=phosphatidylcholine, LPE=lysophosphatidylethanolamines, TAG = triglyceride, *isomer

Network analysis identified the metabolite class Organic Acids as nominally associated with overall and serous OC. Several metabolite groups were associated with endometrioid/clear cell tumors after correcting for multiple comparisons, including Carnitines, Fatty Acyls, and PE/PC Plasmalogens. No metabolite groups were associated with mucinous OC.

Conclusion: We identified several metabolites, notably pseudouridine, a component of transfer RNA, as potential novel risk factors for OC. OC histotypes were associated with different individual metabolites and metabolic classes. Validation analyses are on-going.

#3235

Pre-diagnosis neutrophil-to-lymphocyte ratio and lung cancer risk in heavy smokers.

Laurie Grieshober,1 Stefan Graw,2 Matt J. Barnett,3 Mark D. Thornquist,3 Gary E. Goodman,3 Chu Chen,3 Devin C. Koestler,2 Carmen J. Marsit,4 Jennifer A. Doherty1. 1 _Huntsman Cancer Institute, University of Utah, Salt Lake City, UT;_ 2 _University of Kansas Medical Center, Kansas City, KS;_ 3 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 4 _Emory University, Rollins School of Public Health, Atlanta, GA_.

The neutrophil-to-lymphocyte ratio (NLR) is a marker of systemic inflammation that is inversely associated with survival for many chronic diseases, including lung cancer. We hypothesize that the inflammatory profile reflected by DNA methylation-derived NLR (mdNLR) may also be associated with lung cancer risk. We assessed the relationship between pre-diagnosis mdNLR and lung cancer risk in a nested case-control study of the β-Carotene and Retinol Efficacy Trial (CARET), a population at high risk for lung cancer due to heavy smoking (≥20 pack years; current smoker or quit ≤6 years before enrollment) or substantial occupational asbestos exposure (current smoker or quit ≤15 years before enrollment). We matched 319 incident lung cancer cases to controls based on time at risk, age at blood draw, smoking status, sex, race, asbestos exposure, and enrollment year. We computed mdNLR using the ratio of predicted neutrophil and lymphocyte proportions derived from DNA methylation signatures in whole blood samples collected prior to diagnosis (mean 4.31 years in cases). Conditional logistic regression models were adjusted for potential confounding factors: age, pack years of smoking, cigarettes per day, and body mass index. Mean mdNLR was higher in cases at 2.06 than in controls at 1.86 (p=0.04). Each unit increase in mdNLR was associated with a 21% increased risk of lung cancer (Odds Ratio (OR) 1.21, 95% Confidence Interval (CI) 1.01-1.45). There was a 30% increased risk of non-small cell lung cancer (NSCLC; n=240 pairs; 1.30, 1.03-1.63); estimates for adenocarcinoma and squamous cell NSCLC were similar. mdNLR was not associated with small cell lung cancer (n=68 pairs; 1.06, 0.77-1.47). The association between mdNLR and NSCLC risk was most pronounced in those with asbestos exposure (3.39, 1.32-8.67; all men). Estimates for NSCLC cases without asbestos exposure were similar for men (1.15, 0.88-1.51) and women (1.22, 0.75-1.98). We assessed whether mdNLR and NSCLC risk associations varied by tertiles of time at risk among cases: 0-2.7 years, 2.8-5.4 years, and 5.5-9.8 years. Though NSCLC risk was suggestively elevated in each time period stratum, the magnitude of the association was largest for the time period closest to diagnosis (1.49, 0.92-2.41; 1.26, 0.86-1.84; and 1.30, 0.88-1.91, respectively). This is the first study to evaluate whether pre-diagnosis mdNLR is associated with lung cancer risk, and we assessed this association in a sample of heavy smokers. Our findings suggest that the inflammatory response produced by both smoking and asbestos may be reflected by mdNLR. A better understanding of the role of mdNLR in NSCLC etiology may improve detection of nascent lung cancer, thereby improving treatment strategies in NSCLC patients.

#3236

Lung epithelium DNA methylation: Electronic cigarette users, smokers, and never-smokers.

Min-Ae Song,1 Dominic J. Smiraglia,2 Theodore M. Brasky,1 Daniel Y. Weng,1 Joseph P. McElroy,1 Sarah A. Reisinger,1 Kevin L. Ying,1 Quentin A. Nickerson,1 Mark D. Wewers,1 Peter G. Shields,1 Jo L. Freudenheim3. 1 _The Ohio State University, Columbus, OH;_ 2 _Roswell Park Cancer Institute, Buffalo, NY;_ 3 _University at Buffalo, Buffalo, NY_.

Background: The use of electronic cigarettes (e-cigs) among smokers and never-smokers is increasing, and is considered to be less harmful than cigarettes. However, this has not been studied in target organs for smoking-related diseases, especially the lung. Altered DNA methylation is seen in smokers and contributes to lung carcinogenesis. However, it is not known how e-cigs affect methylation in the lung or whether smoking-induced DNA methylation changes can be reversed in former smokers who switch to e-cigs.

Methods: We conducted a cross-sectional bronchoscopy study of e-cig users (n=12), cigarette smokers (n=10), and never-smokers (n=10) (age 21-30, 84% European-American, 66% male). Bisulfite-converted DNA extracted from lung epithelium collected by bronchial brushing was analyzed for 865,859 CpGs with the llumina Infinium Methylation EPIC Chip in a single batch. Probes were filtered out if on sex chromosomes, cross-reactive, or SNP associated, leaving 735,317 CpGs for analysis. One-way analysis of covariance using M-values was used to identify CpGs differentially methylated for the three groups. False Discovery Rate (FDR) of q<0.1 was considered significant. Ingenuity pathway analysis (IPA) was used for potential biologic implications of identified CpGs.

Results: Smokers averaged 18 cigarettes/day (SD: 4.2) and 6 years of smoking (SD: 4.5). E-cig users vaped an average e-liquid nicotine content of 10 mg/mL (SD: 11) and 8 mL/day (SD: 4), for an average of three years (SD: 1). Mean time since smoking among the e-cig users was 31 months (SD: 15); 3 were never-smokers. We identified 517 differentially methylated CpGs among the three groups (FDR q<0.1). Of them, 128 (25%) were in enhancers, 165 (47%) in promoters, and 162 (31%) in CpG islands/surroundings. For e-cig users, methylation of most differentially methylated CpGs (n=505) was between those for smokers and never-smokers. For smoking- and/or lung cancer-related genes: AHRR, ALDH3A1, ALPK3, CYP1B1, and OXCT1, CpGs were less methylated in smokers than in never-smokers; e-cig users were intermediate. There were 330 unique genes with differentially methylated CpGs; 319 were included in an IPA analysis. The most significantly associated disease was cancer (n=283) and biologic function was drug metabolism (n=10). The top canonical pathways included xenobiotic metabolism signaling (n=13) and wnt/β-catenin signaling (n=9).

Discussion: DNA methylation differed for never-smokers and smokers, with e-cig users' methylation intermediate, consistent with the hypothesis that e-cigs are less harmful than smoking; whether e-cig use is more harmful than never-smoking needs to be studied. Because this study is cross-sectional, association, and not causation, is indicated. Additional observational studies and randomized trials are warranted to understand biologic changes in the lung for smokers and never-smokers using e-cigs.

#3237

Electronic cigarettes and inflammation in the human lung.

Min-Ae Song,1 Theodore M. Brasky,1 Jo L. Freudenheim,2 Joseph P. McElroy,1 Daniel Y. Weng,1 Kevin L. Ying,1 Quentin A. Nickerson,1 Sarah A. Reisinger,1 Mark D. Wewers,1 Peter G. Shields1. 1 _The Ohio State University, Columbus, OH;_ 2 _University at Buffalo, Buffalo, NY_.

Background: The use of electronic cigarettes (e-cigs) is increasing rapidly among both adults and youth, including among both smokers and never-smokers, and is thought to be less harmful than cigarettes. However, biological changes including inflammation have not been studied in target organs such as the lung, particularly for e-cigs; further, it is not known if changes are reversible in smokers who switch to e-cigs.

Methods: We conducted a cross-sectional bronchoscopy study of e-cig users (n=13), cigarette smokers (n=16), and never-smokers (n=40), age 21-30, to assess inflammatory cell counts and cytokines in bronchoalveolar lavages (BAL) fluids. BAL total cell counts were measured using the Countess Automated Cell Counter. Differential cells were performed on stained cytospins by a blinded clinical histopathologist. Smoking-associated inflammatory cytokines were measured using a V-PLEX Plus Pro-inflammatory Panel. Non-parametric Mann-Whitney and Kruskal-Wallis tests were used for cell counts. Log10 transformed cytokines were used for 1-way ANOVA.

Results: Smokers averaged 16 cigarettes/day (SD: 4.7) for 7 years (SD: 4.1). E-cig users vaped an average e-liquid nicotine content of 12 mg/ml (SD: 7.6) and 8 ml/day (SD: 3.9), for an average of 3 years (SD: 0.9); mean time since smoking among e-cig users was 24 months (SD: 16) (2 were never-smokers). Smokers compared to never-smokers had significantly increased total cell counts, macrophages and neutrophils, and decreased lymphocytes (7.15E-05<P<0.05). Total cell concentration for the e-cig users were 35% lower than the smokers and 30% higher than the never smokers (overall P=0.0005). Total and differential cell counts were borderline or significantly different for the smokers compared to e-cig users, and followed the same trends for never-smokers compared to e-cig users. Inflammatory cytokines IL-1β, IL-6, and IL-8 were significantly higher for smokers compared to never-smokers, and were lower for IL-2 and IFN-γ (all P's <0.002). Levels of these cytokines in e-cig users were corroborated by patterns of differences for the cell counts, where overall differences were highly significantly different; e-cig users were intermediate. IL-4, IL-10, IL-12p70, IL-13, and TNF-α did not differ across the groups. Time since last cigarette and cigarettes/day among e-cig users was not significantly correlated with any of the cell counts or cytokine levels.

Discussion: Inflammation differed for never-smokers and smokers, with markers of inflammation intermediate for e-cig users, consistent with the hypothesis that e-cigs are less harmful. Study of whether e-cig use is more harmful than never-use and never-smoking is warranted. Because this cross-sectional study provides only indirect evidence of causation, additional observation studies and randomized trials are needed to understand changes in inflammation for e-cig users, both former- never-smokers.

#3238

Annual coal use and stove improvement in relation to cause-specific mortality in a 36-year cohort analysis among anthracite coal users in Xuanwei, China.

Bryan Bassig,1 Bingshu E. Chen,2 Wei Jie Seow,3 Roel Vermeulen,4 Jinming Zhang,1 Wei Hu,1 Jason Wong,1 Bofu Ning,5 H.Dean Hosgood,6 Yunchao Huang,7 Kaiyun Yang,7 George S. Downward,4 Lützen Portengen,4 Nathaniel Rothman,1 Qing Lan1. 1 _NCI, Bethesda, MD;_ 2 _Queen's Cancer Research Institute, Kingston, Ontario, Canada;_ 3 _Saw Swee Hock School of Public Health, Singapore;_ 4 _Utrecht University, Utrecht, Netherlands;_ 5 _Xuanwei Center for Disease Control and Prevention, Xuanwei, China;_ 6 _Albert Einstein Medical Center, New York, NY;_ 7 _Third Affiliated Hospital of Kunming Medical University, China_.

Emissions from burning solid fuels contribute substantially to the global disease burden. Xuanwei (XW), China, has among the highest lung cancer rates in the world, which has been attributed to the use of bituminous ("smoky") coal for cooking and heating. Anthracite ("smokeless") coal is also used in XW and is considered a "cleaner" fuel source compared to bituminous coal, but it emits high-levels of certain indoor pollutants (e.g. nitrogen dioxide and sulfur dioxide) and the disease burden associated with its use has not been well characterized. To evaluate the impact of anthracite coal use and stove improvement on all- and cause-specific mortality, we conducted a cohort study in which 42,400 XW residents were followed from 1976-2011. We ascertained information on fuel use, stove improvement, and lifestyle factors using questionnaires, and mortality data were obtained from hospital records and death certificates. Sex-specific multivariable Cox regression models were used to estimate the hazard ratio (HR) of all- and cause-specific mortality in relation to time-dependent annual coal tonnage use (median: 2 tons/year; 25th and 75th percentile = 1 and 3 tons/year, respectively) and stove improvement among 4,486 lifetime anthracite coal users. Compared to subjects who used <2 tons of anthracite coal/year, higher annual tonnage was associated with increased total mortality (HR=1.16, 95% CI =1.03-1.30 in men; HR=1.18, 95% CI =1.02-1.37 in women), total respiratory disease mortality (HR=1.52, 95% CI =1.16-1.99 in men; HR=1.74, 95% CI=1.28-2.36 in women), which was driven by pneumonia, and total cancer mortality in men (HR=1.66, 95% CI=1.09-2.51) but not women. Lung cancer mortality was non-significantly increased and liver cancer mortality was significantly increased in both men and women for use of ≥2 tons of anthracite coal/year. Suggestive positive associations in relation to higher tonnage were also observed for ischemic heart disease. Changing from the traditional use of an unvented firepit to a portable or vented stove was associated with a reduction in overall mortality (HR=0.70, 95% CI=0.61-0.80 in men; HR=0.72, 95% CI=0.61-0.85 in women). Consistent sex-specific reductions in risk were observed for pneumonia and ischemic heart disease mortality, and a non-significant reduction in total cancer mortality was also observed for those who changed to using a portable or vented stove. Our findings suggest that anthracite coal use contributes to the burden of mortality in XW. This study emphasizes the importance of reducing the use of anthracite coal, which is still widely used in China for indoor cooking and heating, in favor of alternative fuel sources such as gas/electricity where possible.

#3239

Molecular biomarkers and indoor household air pollution in a rural area of China.

Wei Hu,1 Richard M. Cawthon,2 George Downward,3 Boris Reiss,3 Jihua Li,4 Jun He,4 Jun Xu,5 Wei Jie Seow,6 Bryan A. Bassig,1 Jason Y. Wong,1 Dean Hosgood,1 Lützen Portengen,3 Nathaniel Rothman,1 Roel Vermeulen,3 Qing Lan1. 1 _National Cancer Institute, Rockville, MD;_ 2 _University of Utach, Salt Lake City, UT;_ 3 _Utrecht University, Utrecht, Netherlands;_ 4 _Qujing Center for Disease Control and Prevention, Qujing, China;_ 5 _The University of Hong Kong, Hong Kong, Hong Kong;_ 6 _National University of Singapore and National University Health System, Singapore, Singapore_.

To explore the influence of fuel source used for home heating and cooking on biomarkers of potential genomic instability, we analyzed biological samples obtained from a cross-sectional study of 104 female never-smokers enrolled from 30 villages in Xuanwei and Fuyuan in Yunnan Province, a rural region with a high incidence of lung cancer in China. Study subjects who used primarily smoky (i.e., bituminous) coal (n=80), smokeless coal (i.e., anthracite) (n=14) and wood (n=10) were analyzed. Personal 24-h air PM2.5 and polycyclic aromatic hydrocarbon concentrations were measured over two consecutive days in each household. DNA was extracted from peripheral white blood cells. We assayed molecular biomarkers of potential genomic instability including the Alu copy number to albumin gene (ALB) ratio (Alu/ALB), 5.8S ribosomal DNA to ALB ratio (5.8S rDNA/ALB) and 18S ribosomal DNA to ALB ratio (18S rDNA/ALB), which were weakly to highly correlated with each other. Multiple linear regression was performed to analyze the association between fuel type and these biomarkers, adjusted for age, as well as the influence of benzo[a]pyrene (BaP) and PM2.5 concentration. There was a significant difference (p = 0.041) in levels of 18S rDNA/ALB among the three exposure groups [mean (standard deviation [sd]) = 1.30 (0.42), 1.58 (0.72), and 1.65 (0.62) among smoky, smokeless coal and wood users, respectively]. There were substantial differences in personal PM2.5 exposure among smoky coal, smokeless coal, and wood users [mean (sd) =184 (127), 227 (318), and 433 (284) µg/m3, respectively, p = 0.0004]. PM2.5 exposure was significantly and positively associated with 18S rDNA/ALB (p = 0.001). Further, after adjustment for PM2.5, the association between fuel type and 18S rDNA/ALB became non-significant (p = 0.24). Similar positive associations were observed between PM2.5 and 5.8S rDNA/ALB (p=0.009) and Alu/ALB (p=0.0003). There were no associations identified between BaP and these biomarkers after adjustment for PM2.5. Our findings suggest that exposure to PM2.5, which has been associated with increased risks of lung cancer and cardiovascular disease, may alter potential molecular markers of genomic instability. Additional studies are needed to confirm these findings and further understand the role of these biomarkers in predicting human health effects.

#3240

Radon exposure, lung cancer, and respiratory outcomes in a cohort of former and current smokers: An ecologic analysis.

Shaina L. Stacy, Linda Robertson, Renwei Wang, David O. Wilson, Jian-Min Yuan. _University of Pittsburgh, Pittsburgh, PA_.

Background and Objective: Residential radon-222 decay product exposure is the second leading cause of lung cancer overall and the leading cause among nonsmokers. The objective of our study was to assess the potential contribution of radon exposure to lung cancer risk and risk of other respiratory conditions, including chronic obstructive pulmonary disease (COPD), that predispose subjects to lung cancer risk.

Methods: Participants were 50- to 79-year-old current and former smokers recruited through the Pittsburgh Lung Screening Study (PLuSS) between January 2002 and April 2005. For the current analysis, we considered a subset of 3,070 (out of 3,654) PLuSS participants living in a six-county area of southwestern Pennsylvania at the time of enrollment. We obtained estimates of residential (basement) radon exposure from the PA Department of Environmental Protection at the zip code level for each of the six counties. These estimates were mean radon concentrations calculated from measurements made at individual residences between 1990 and 2016. We linked each study participant to the mean radon concentration in their zip code and conducted adjusted logistic regressions to explore associations with tertiles of increasing radon exposure and risk of lung cancer, COPD, and emphysema. Models were adjusted for age, sex, individual-level smoking status (former or current), the number of cigarettes smoked per day, and the number of years participants smoked.

Results: Mean radon levels for the six-county area ranged from 2 to 16 pCi/L. Comparing the third to the first tertile of radon exposure, the odds ratio for lung cancer was 1.10 (95% CI: 0.77-1.57) and not statistically significant. However, higher radon levels were positively associated with increased odds of COPD diagnosis determined at baseline (odds ratio: 1.19, 95% CI: 0.98-1.43, p: 0.07). In linear regression models using baseline spirometry measurements (specifically, the FEV1/FVC ratio) as the outcome, increasing mean radon levels were associated with decreased lung function (β: -139, p: 0.003). In the PLuSS cohort, lung cancer risk decreases as FEV1/FVC ratios increase (i.e., as lung function improves), after accounting for smoking history (odds ratio: 0.78, 95% CI: 0.68-0.89, p: 0.0002).

Conclusions: As COPD is an important predisposing factor for respiratory health, lung cancer development, and overall survival, this potential association between residential radon level and COPD risk warrants additional follow-up. Future work will include using more granular radon measurements to characterize exposure as well as to capture potential changes in exposure status over time.

#3241

Children with acute lymphoblastic leukemia have increased arginase 2 at birth, implicating immunosuppression in leukemogenesis.

Amalie B. Nielsen,1 Mi Zhou,1 Adam de Smith,1 Lucie McCoy,1 Helen Hansen,1 Paige Bracci,1 Libby Morimoto,2 Scott Kogan,1 Catherine Metayer,2 Xiaomei Ma,3 Joseph Wiemels1. 1 _University of California San Francisco, San Francisco, CA;_ 2 _University of California Berkeley, Berkeley, CA;_ 3 _Yale University, New Haven, CT_.

Background: Epidemiologic evidence points to a role for infection and immune development in childhood acute lymphoblastic leukemia (ALL). Emerging evidence suggests that in utero immunosuppressive processes, along with immaturity at birth, cause immune hyporesponsiveness to commensal microbes and susceptibility to early childhood infections. A key regulator of active immunosuppression within the neonatal environment is arginase 2, which is produced by CD71+ erythroid cells and myeloid-derived suppressor cells. Arginase-dependent arginine depletion is suspected to aggravate both infections and tumor growth (the latter in adult settings) due to suppressed T-cell function by downregulation of TCR-CD3ζ. Studies have shown that immune vulnerability of the preterm neonate is critically related to arginase-dependent arginine depletion, but none have investigated this neonatal immunosuppression in relation to ALL.

Methods: Children were identified via California Cancer and Vital Statistics Registries, and their neonatal dried bloodspots derived from the California Genetic Disease Screening Program. Children were born in California between 1999 and 2009, with ALL cases being 0-15 years of age at diagnosis. 137 ALL cases and 500 controls (frequency matched on birthdate, gender, and ethnicity) were analyzed. Arginase 2 levels were measured using enzyme-linked immunosorbent assay (ELISA), with total serum-protein levels measured using the Pierce BCA protein assay. Arginase 2 levels in the analyses were adjusted for total serum protein and analyzed both as a continuous variable and as quartiles based on the distribution among controls. Adjusted unconditional logistic regression was used to estimate odds ratios (OR) for the association between neonatal arginase 2 and ALL with assay plate variance structure accounted for using cluster methods.

Results: Arginase 2 levels in neonatal dried blood spots were significantly higher among cases with ALL than controls (OR = 1.36, 95% CI: 1.02-1.80, p = 0.03). The highest quartile of arginase 2 levels differed significantly from the lowest (OR = 1.81, 95% CI: 1.04-3.41, p = 0.03), while the intermediate quartiles had a trend towards higher arginase 2 levels (p for trend: 0.02).

Conclusions: Our findings suggest that children with ALL have significantly higher immunosuppression at birth due to increased arginase 2. A high neonatal level of this enzyme, an important downregulator of T-cell activity, may lead to altered immune responses to antigenic stimulation and infection during early childhood and potentially impact the immune system's capability to react against nascent cancer. In addition to previously discovered prenatal mutations, this new finding of an immunosuppressed environment at birth contributes to our understanding of early events in the development of ALL.

#3242

Differences in stromal tumor-infiltrating lymphocytes in breast cancer from women of African and European ancestry after accounting for tumor characteristics.

Ting-Yuan D. Cheng,1 Rochelle Payne Ondracek,2 Song Yao,2 Warren Davis,2 Angela Omilian,2 Mateusz Opyrchal,2 Elisa V. Bandera,3 Michael J. Higgins,2 Christine B. Ambrosone,2 Thaer Khoury2. 1 _University of Florida, Gainesville, FL;_ 2 _Roswell Park Cancer Institute, Buffalo, NY;_ 3 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ_.

Background: Women of African ancestry (AA) have higher breast cancer mortality than other US racial/ethnic groups, attributed to a number of social and economic factors. However, breast tumors in AA women are more likely to present with higher grade and lacking estrogen receptor (ER) expression compared to those from women of European ancestry (EA), suggesting the importance of tumor factors in breast cancer disparities. Emerging evidence suggests that stromal tumor-infiltrating lymphocytes (TIL-str) have a role in immune response to therapy and prognosis of breast cancer. The objective of this study was to examine associations between risk factors for breast cancer and TIL-str, with consideration of tumor characteristics.

Methods: We reviewed the hematoxylin and eosin slides from surgical or biopsy tumor blocks of invasive breast cancer from 1,385 cases (986 AA and 399 EA women, aged 20-75 years) in the Women's Circle of Health Study, a multicenter case-control study in New York and New Jersey. In-person interviews were conducted at diagnosis to obtain data on demographics, family histories, hormone use, and reproductive and menstrual histories. Anthropometric measurements were taken by trained staff. TIL-str were scored as 0%, 1%, and 10% to 100% in increments of 10%. Linear regressions were performed to estimate differences in TIL-str scores (β).

Results: In univariate analysis, TIL-str was higher in tumors from AA than those from EA women overall (mean score = 21.5% vs. 12.5; P<0.001). TIL-str score was positively associated with higher grade, larger tumor size, and ER and progesterone receptor (PR) negativity in both AA and EA women (all P<0.05). In EA women, TIL-str score was also higher in more advanced than early stage tumors and human epidermal factor receptor 2 (HER2) positive than negative tumors (both P<0.05), but not in AA women. In multivariable analysis, AA compared to EA race was significantly associated with a higher TIL-str score (β = 5.7%, P<0.001), adjusting for age at diagnosis, body mass index, menopausal status, tumor grade, size, stage, and molecular subtypes defined by ER, PR, and HER2. There was no apparent association between TIL-str and history of breast cancer in first-degree relatives, history of benign breast disease, hormone use, reproductive factors, or body size.

Conclusion: Self-reported African compared to European ancestry is independently associated with higher TIL-str scores in breast tumors. Our findings suggest that immune response may play a role in the differences in breast cancer biology and outcomes between AA and EA women. (Funding: The Breast Cancer Research Foundation; NIH P01CA151135, R01CA100598, R01CA185623, P30CA072720, K07CA201334; US Army Medical Research and Material Command DAMD-17-01-1-0334)

#3243

Associations of polyomavirus seroreactivity with AIDS-related non-Hodgkin's lymphoma.

Minkyo Song,1 Noemi Bender,2 James J. Goedert,1 Cheryl A. Winkler,1 Nicole Brenner,2 Tim Waterboer,2 Charles S. Rabkin1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _German Cancer Research Center, Heidelberg, Germany_.

Background: Human polyomaviruses have a suspected role in carcinogenesis. In the setting of human immunodeficiency virus (HIV) infection, coinfection with MCV polyomavirus is associated with high risk of Merkel cell carcinoma. A role in lymphomagenesis has also been suggested by studies in the general population, but there are no prior studies in individuals with the acquired immunodeficiency syndrome (AIDS).

Methods: We measured antibodies against 11 common polyomaviruses in archived serum and plasma samples from two prospective cohort studies of HIV infection. Patients with incident (n=28) and prevalent (n=38) AIDS-related non-Hodgkin lymphoma (NHL) were matched by age, sex, and CD4 count to 67 HIV-positive AIDS-free controls. Seroreactivity was measured by fluorescent bead-based multiplex serology, quantified as median fluorescence intensity (MFI). Logistic regression models were used to estimate adjusted odds ratios (ORs) and 95% confidence intervals (95% CIs) for NHL risk. Multinomial logistic regression compared incident and prevalent NHL cases to lymphoma-free controls and heterogeneity between case groups was evaluated by Wald tests. Differences in MFI were assessed by Kruskal-Wallis tests. Based on log-transformed MFI values for 15 masked duplicate samples, estimated coefficients of variation (CV) were <20% and intraclass correlation coefficients (ICC) were >90% for all antibodies except LPV (CV 29%, ICC 67%).

Results: Cases had significantly lower prevalence of antibodies to WU polyomavirus than lymphoma-free controls (64% vs. 85%), with OR 0.28 (95% CI 0.12-0.68). Cases had non-significantly lower prevalence of antibodies to JC, TSV, MCV and KI polyomaviruses (ORs 0.42-0.71); similar prevalence of antibodies to BK, HPyV6 and HPyV7 polyomaviruses (ORs 0.98-1.18); and non-significantly higher prevalence of antibodies to HPyV10, LPV and HPyV9 polyomaviruses (ORs 1.35-1.77). MFIs for anti-WU antibodies were also lower for cases (median 552.6, interquartile range [IQR] 140.3-1298.3) than controls (median 1096.3, IQR 410.3-2044.3; p-value 0.047). The association of anti-WU antibody with NHL was stronger in samples obtained post-diagnosis (OR 0.18, 95% CI 0.07-0.48) than pre-diagnosis (OR 0.66, 95% CI 0.19-2.31; p-heterogeneity=0.037).

Conclusion: Our data do not directly implicate known polyomaviruses as lymphomagenic in the setting of HIV-associated immunodeficiency. Nevertheless, impaired antibody response to WU may be a harbinger of AIDS-related lymphoma.

#3244

**Enrichment of** Helicobacter pylori **mutant strains after eradication therapy analyzed by gastric wash-based quantitative pyrosequencing.**

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

The eradication of Helicobacter pylori reduces the risk of gastric cancer. A clear understanding of the factors underlying mixed infection with multiple clarithromycin susceptible and clarithromycin resistant H. pylori strains is necessary to design more effective therapies against H. pylori. We aimed to assess how the abundance and prevalence of H. pylori strains vary after clarithromycin based eradication therapy. Using gastric wash samples, which represent the entire stomach, we sequentially analyzed the abundance and prevalence of H. pylori DNA by 23S ribosomal RNA pyrosequencing before and 1, 2, and 3 years after eradication therapy. Low levels of H. pylori DNA were still detectable at the first year follow up in all samples with negative post treatment urea breath test results. The abundance of H. pylori DNA decreased significantly until the 2 year follow up, but it switched to an increase at the 3 year follow up. Importantly, the ratio of the prevalence of mutant strains to the prevalence of wild type strains had already increased at the first year follow up and continued to increase, suggesting the selection and growth of clarithromycin resistant strains during the follow up periods. Being sensitive and representative, our assay will be useful in effectively addressing gastric cancer development by enhancing the long-term success of intervention strategies and consecutive surveillance for H. pylori eradication.

#3245

The interplay between patient´s features and mutational processes in tobacco related cancers.

João Paulo da Silveira Nogueira Lima,1 Rodrigo Couto Duarte Drummond,1 Monize Nakamoto Provisor Santos,1 Renan Valieris,1 Rafael Andres Rosales Mitrowsky,2 Vladmir Cordeiro Lima,1 Diana Noronha Nunes,1 Emmanuel Dias-Neto,1 Israel Tojal da Silva1. 1 _A.C. Camargo Cancer Center, Sao Paulo, Brazil;_ 2 _Universidade de Sao Paulo, Sao Paulo, Brazil_.

Background: There is a complex interplay between smoking and patient demographics in the emergence and development of cancer. We vetted the possible relationship between gender, race and age with mutational processes operating on aerodigestive tract cancers, known to be highly associated to tobacco. Methods: We used an empirical Bayesian approach to identify genomic patterns that lead to mutational processes in head and neck, esophageal, and lung cancers from TCGA database followed by confirmation in independent cohorts (cBioPortal). Results: After confronting clinical information and smoking-habits to molecular findings in those cancers, we identified imprints of different mutational processes in a disease-, gender- and race-specific fashion. Black and female patients - despite their lower tobacco consumption in comparison to whites or males - had tumors with the highest enrichment for tobacco-induced DNA damage. The relevance of mutational signatures related to aging varied across race and gender and were independent from chronological age. The activity of smoking signature was different according to disease histotype, and stage. tumors of never-smoker lung cancer patients showed tobacco-specific mutational signatures, which could suggest the influence of second-hand smoking. Patients with tumors enriched for the smoking mutational signature presented longer survival, whereas those with ageing-enriched signature had poorer prognosis. Conclusions: Mutational processes may unravel connections between patient features and tumorigenesis, particular to race, gender, and tumor type. Therefore, it is paramount to assess those details with greater granularity in every effort of personalized medicine either in preventive or therapeutic approaches.

#3246

Association between colorectal cancer and red or processed meat among Korean diet: A molecular approach.

Mihi Yang,1 Jeongseon Kim,2 Jong Y. Park3. 1 _Sookmyung Women's University College of Pharmacy, Seoul, Republic of Korea;_ 2 _National Cancer Center, Goyang-si, Republic of Korea;_ 3 _Moffitt Cancer Center and Research Institute, Tampa, FL_.

To study whether red or processed meat cause colorectal cancer in non- western population, we performed a pilot study in Korean people (N=30, case, 15 vs. control 15, mean of age=59.7 yrs) with biological monitoring of exposure and response biomarkers, i.e. metabolites of polycyclic aromatic hydrocarbons and heterocyclic amine (1-OHP, PhIP, and MeIQx), their DNA-adducts (dG-C8-MeIQx), and homocysteine, malondialdehyde (MDA), C-reactive protein and epigenetic alteration (5mC). The subjects also filled out daily consumption of food before diagnoses. As results, they averagely consumed 59.89 g/day and 2.47 g/day of red and processed meat, respectively. There were positive associations among consumption of Kimch(g), folate (ug) and fruits(g). The consumption of red meat was not associated with the cancer and the control even showed higher processed meat (g). From biomonitoring, the average levels of urinary 1-OHP, PhIP, and MeIQx, blood-dG-C8-MeIQx and 5mC were 0.13 ppb, 9.9 ppt, 14.63 ppt, 5.23 ppt/1.8 ug of DNA, and 5.3%, respectively. We found younger people more consumed processed meat or beverage and showed higher urinary levels of MeIQx than elderly people. The urinary levels of PhIP were negatively associated with folate consumption. The cases showed higher levels of LDL-cholesterol, homocysteine, urinary 1-OHP and dG-C8-MeIQx than controls. There were positive associations among levels of MeIQx, MDA, and 1-OHP. In addition, levels of dG-C8-MeIQx were positively associated with those of 1-OHP and total cholesterol. The levels of global methylation of 5mC (%) were higher in well done meat consumers than others. In conclusion, the Korean subjects might consume less volume of red or processed meat than western people. From questionnaire (FFQ), we could not find association between red or processed meat and colorectal cancer. However, biological monitoring showed the Korean colorectal cancer could be affected by red or processed meat, due to association with exposure or response biomarkers, particularly long term exposure biomarkers, such as dG-C8-MeIQx. This molecular epidemiological approach provides more reliable evidences to overcome limitation of case-control studies.

## PREVENTION RESEARCH:

### Prevention, Interception, and Early Detection Research

#3247

Genome-wide cell-free DNA mutation integration for sensitive cancer detection.

Asaf Zviran,1 Steven T. Hill,2 Rafael Schulman,1 Minita Shah,1 Sunil Deochand,3 Gavin Ha,4 Sarah Reed,4 Denisse Rotem,4 Greg Gydush,4 Justin Rhoades,4 Kevin Huang,1 Will Liao,1 Dillon Maloney,1 Nathan Omans,2 Murtaza Malbari,2 Cathy F. Spinelli,2 Selena Kazancioglu,1 Nicolas Robine,1 Viktor Adalsteinsson,4 Brian Houck-Loomis,1 Nasser Altorki,2 Dan A. Landau2. 1 _New York Genome Center, New York, NY;_ 2 _Weill Cornell Medicine, New York, NY;_ 3 _New York University, New York, NY;_ 4 _Broad Institute, Cambridge, MA_.

Solid malignancies are often diagnosed at a late stage with dismal prognosis. Even after cancer is diagnosed, we lack sensitive tools to guide difficult therapeutic decisions such as adjuvant therapy. Sensitive cancer detection by blood biopsy can therefore transform care by enabling early detection and residual disease monitoring.

Cell free DNA mutation detection has shown significant promise in its ability to survey the somatic genome and enable detection of cancer mutations in the peripheral blood. However, the combination of low tumor fraction and limiting number of DNA fragments in a typical plasma sample, restrict the probability of detecting early stage cancer in cfDNA through current deep targeted sequencing methods.

Focusing on non-small cell lung cancer (NSCLC), we reasoned that we would need to supplant depth of sequencing with breadth of sequencing to overcome the fundamental limitation of low input of cfDNA. To do so, we apply whole genome sequencing (WGS) that allows us to base sensitive detection on the cumulative signal provided by 10,000-30,000 somatic mutations observed in a substantial proportion of NSCLC. We developed an analytic method that integrates genome-wide mutation signal to obtain a tumor fraction (TF) estimate, and thus allow sensitive detection of residual disease and quantitative dynamic monitoring of disease burden. Benchmarking on artificial plasma showed TF detection sensitivity as low as 1:100,000, two orders of magnitude more sensitive than currently available methods.

To test this method, we performed WGS on resected NSCLC and matched germline samples of 8 NSCLC patients, as well as on matched pre- and post-surgery cfDNA. Patient-specific somatic mutations were identified in the tumor/normal pairs and used for the estimation of TF in the matched plasma samples. We detect pre-surgery circulating tumor DNA (ctDNA) in all of the early-stage pre-operative samples and in ~40% post-operative patients, correlated with post-operative disease progression.

In early cancer detection, tumor DNA is not available, requiring de-novo mutation detection in cfDNA. To do so, we first trained a convolutional neuronal network to distinguish between cancer altered sequencing reads and reads affected by sequencing errors. This was followed by genome-wide pattern matching to a specific genomic signature that mark lung cancer mutations (Tobacco signature) indicating the presence of ctDNA in the patient plasma. Applying this method to the pre-operative early stage lung cancer samples and plasma samples from 5 patients with benign nodules (CT-detected) showed an accurate discrimination between malignant and benign nodules, suggesting a potential role in improving the positive predictive value of lung cancer screening in at-risk populations.

These results show that genome-wide mutation integration is a promising novel approach for ultra-sensitive early detection and residual disease monitoring.

#3248

Genomic characterization of premalignant lung squamous cell carcinoma lesions.

Joshua D. Campbell,1 Xijun Zhang,2 Catalina Perdomo,1 Sarah Mazzilli,1 Yaron Geshalter,1 Samjot S. Dhillon,3 Gang Liu,1 Sherry Zhang,1 Hanqiao Liu,1 Jessica Vick,1 Christopher Moy,4 Stefano Monti,1 Evan Johnson,1 Matthew Meyerson,5 Matthew Wilkerson,2 Clifton Dalgard,2 Suso Platero,4 Chris Stevenson,4 Marc Lenburg,1 Mary Reid,3 Jennifer Beane,1 Avrum Spira1. 1 _Boston University School of Medicine, Boston, MA;_ 2 _Uniformed Services University, Bethesda, MD;_ 3 _Roswell Park Cancer Institute, Buffalo, NY;_ 4 _Janssen Pharmaceuticals, Spring House, PA;_ 5 _Dana Farber Cancer Institute, Boston, MA_.

Background: Lung squamous cell carcinoma (SqCC) arises in the epithelial layer of the bronchial airway and is often preceded by the development of premalignant lesions. However, not all premalignant lesions progress to lung SqCC and many will regress spontaneously. Understanding the somatic alterations and molecular subtypes associated with progression will allow us to identify biomarkers for early detection and develop therapeutic strategies for disease prevention and interception. Methods: Biopsies were obtained from high-risk smokers undergoing lung cancer screening by auto-fluorescence bronchoscopy and CT at the Roswell Park Cancer Institute. For each subject, multiple sites were sampled repeatedly over time. One biopsy from each region was sent for pathological review while another biopsy was taken for molecular studies. Whole-exome sequencing (WES) was performed at Uniform Services University to 120x coverage and RNA-seq was performed at Boston University School of Medicine. Results: The median number of somatic mutations across all premalignant lesions that underwent DNA-seq (150 biopsies from 20 subjects) was 0.45 per megabase and displayed a modest association with histological grade (p=0.05). The most frequently mutated known lung cancer genes included NOTCH1 (14%), TP53 (6%), FAT1 (3%), PIK3CA (2%), KRAS (<1%), and CDKN2A (<1%). One patient had a moderate dysplastic lesion without any detectable arm-level copy number changes or known cancer mutations. Six months later, this lesion had progressed to severe dysplasia and obtained many genomic alterations commonly observed in squamous cell carcinoma including 3q gain, 3p loss, and mutations in TP53, NOTCH1, and CDKN2A. Using RNA-seq, we identified 4 distinct molecular subtypes using 197 biopsies from 29 subjects. One subtype was enriched for samples with dysplasia histology, high basal cell content, and the "Classical" SqCC tumor gene expression subtype (p<0.001). These associations replicated in an independent set of 111 biopsies from 20 subjects. Genes associated with IFN-gamma signaling and T cell mediated immunity were down-regulated among lesions that persisted or progressed vs. those that regressed within the high-grade subtype. Staining of adjacent biopsies revealed that decreased expression of these immune pathways was associated with decreased numbers of CD4+ and CD8+ T cells within the lesions and surrounding tissue. Conclusions: The somatic alterations observed in known cancer genes may be among the earliest events in lung SqCC development and may be useful as biomarkers for early detection. Molecular classification of these lesions into molecular subtypes may lead to biomarkers of disease progression that could be used to identify at-risk patients for aggressive surveillance or for prevention trials.

#3249

Detection of early-stage malignant lung cancer using methylation signatures in circulating tumor DNA.

Ruijun Liu,1 Xiaojie Li,2 Athurva Gore,3 Zi Qin,2 Jeff Gole,3 Qiye He,2 Rui Liu,2 Shun Lu1. 1 _Shanghai Chest Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China;_ 2 _Singlera Genomics Inc., Shanghai, China;_ 3 _Singlera Genomics Inc., San Diego, CA_.

Low-dose computed tomography (LDCT) is recommended for early lung cancer screening in high-risk populations. Small lung nodules are identified by these CT scans in up to 50% of high-risk patients. While over 50% of lung nodules less than 1 cm are benign, discriminating benign nodules from malignant nodules remains extremely challenging without an invasive lung biopsy. Taking advantage of methylation signatures in circulating tumor DNA (ctDNA), we developed a non-invasive screening assay to identify patients with malignant lung nodules from those with benign lung nodules.

Methods: We first adopted Singlera's MONOD+ assay as a marker screening technology to interrogate the methylation state of over 4,000,000 CpG sites across more than 200,000 methylation haplotype blocks in the human genome from as little as 10 ng of cell-free or tissue DNA. We first processed 129 fresh frozen and FFPE tissue samples from healthy lung, benign lung nodules, and lung cancer at various stages of malignancy. We identified a preliminary set of methylation markers capable of segregating lung nodules by malignancy and invasiveness. To examine the reproducibility of these markers in plasma, we applied the same screening assay to plasma cell-free DNA from 46 healthy patients and 175 lung nodule patients with either benign or malignant nodules (most at an early stage of Ia1/Ia2). We refined the marker set by removing methylation markers that were unstable in plasma samples. We next applied Singlera's MethylTitan assay to deeply interrogate approximately 1,000 methylation haplotype blocks enriched in malignant lung nodules in plasma samples, as MethylTitan's higher conversion rate allowed us to better handle the limited amount of DNA in plasma samples. We ran the MethylTitan assay on both healthy and lung nodule plasma samples; using this method, we confirmed that tissue-derived methylation signatures could be rapidly screened in plasma samples in a high-throughput and highly sensitive manner.

Results: Using the MONOD+ marker screening assay on plasma samples, we were able to utilize cell-free DNA methylation to identify early-stage malignant lung nodule patients with a sensitivity of 80.25% and a specificity of 83.08%. We were able to further improve upon these results using the MethylTitan assay, as we could more efficiently and more accurately screen the methylation markers in a large number of plasma samples.

Conclusion: In summary, we have applied Singlera's MONOD+ and MethylTitan methylation assays to non-invasively identify early stage lung cancer in patients with small lung nodules. We are currently expanding this study to a much larger cohort of lung nodule patients in order to clinically validate our early lung cancer screening assay. The approach of using MONOD+ to identify markers and MethylTitan to analyze plasma samples will allow us to noninvasively screen for additional cancer types in a high-throughput clinical setting.

#3250

Evaluation of a blood test for cancer screening.

Rita Tanos, Amaëlle Otandault, Caroline Mollevi, Safia El Messaoudi, Alain R. Thierry. _Institut de Recherche en Cancerologie de Montpellier, Montpellier, France_.

Circulating DNA (cfDNA) has emerged as a potential biomarker, particularly in cancer, and is the subject of extensive studies in translational and clinical research. It presents a great potential in diagnosis, detection of residual disease, monitoring of recurrence and control of therapeutic response, solely through a non-invasive blood draw. Several efforts are underway to evaluate its potential for the early detection of cancer.

We set a multiparametric screening test on various ccfDNA parameters, to investigate their potential for cancer screening. We targeted, using a specific q-PCR based method, nuclear and/or mitochondrial sequences and determined an index called MNR (Multi Normalized Ratio) in the supernatant of normal (n=6) and cancer cell lines (n=14), and in the plasma of healthy individuals (n=212) and patients with colorectal cancer (CRC) (n=148), or lung cancer (n=51).

When applied to the supernatant of cell culture, the MNR had a discriminative potential of 100% between normal and cancer cell lines. In plasma samples, the MNR showed a high potential with an AUC of 0.85, a 76% sensitivity and 76% specificity. When combined to the total nuclear cfDNA concentration, these two parameters together showed an AUC increase up to 0.88, a 73% sensitivity and 80% specificity. For individuals at risk and patients with early stages of CRC (stage 0/I), the MNR showed high AUCs of 0.9 and 0.88 respectively. We identified tumor biology specific phenomena underlying the MNR performance.

No other biomarker alone has shown an equivalent performance. Our results clearly suggest that the MNR could be a potent biomarker for early tumor detection and could potentially be used to screen for asymptomatic or undiagnosed individuals. An evaluation in a much larger cohort is warranted.

#3251

TaqMan Advanced miRNA cDNA Synthesis Kit to simultaneously study expression of miRNA and mRNA and to detect somatic mutations by qPCR.

Kathleen Hayashibara,1 Harita Veereshlingam,1 Malte Buchholz2. 1 _Thermo Fisher Scientific, South San Francisco, CA;_ 2 _Philipps-Universitaet Marburg, Marburg, Germany_.

MicroRNAs (miRNA) are a class of small non-coding RNAs (approximately 21 nt long) that bind complementary sequences in target mRNAs to specifically regulate gene expression. Aberrant regulation of miRNAs and their targets has been associated with several diseases including cancer. The relationship between miRNA and mRNA has been found to be important in cancer development and progression. Simultaneous expression studies of miRNA and mRNA and detection of mutations in mRNA transcripts can be valuable in understanding molecular mechanisms that have an underlying role in various diseases. We demonstrate the technical verification of a novel method to reverse-transcribe and pre-amplify miRNA and mRNA from sample-limiting serum research samples using the TaqMan® Advanced miRNA cDNA Synthesis Kit. Based on results from previous studies, a signature of 49 mRNA and 37 miRNA targets has been identified that may help distinguish between benign and malignant pancreatic tissues. In this study, these targets and an additional set of transcript mutations were analyzed in serum from normal and test samples. TaqMan assays for miRNA and mRNA targets and custom TaqMan Mutation Detection Assays were placed on TaqMan Array Cards to facilitate investigation of several samples in a single experiment. Results demonstrate that transcript mutations can be detected and miRNA and mRNA targets can be reliably quantified from a single reverse transcription reaction. (For research use only. Not for use in diagnostic purposes.)

#3252

Circulating tumor DNA levels as a prospective marker for survival and prognosis.

Sai Balaji Andugulapati,1 Arun K. Hariharan,1 Ashwini Shanmugam,1 Kumar Prabhash,2 Srikant Sridharan,1 Anuradha Chougule,2 Divyashree Kushnoor,1 Urvashi Bahadur,1 Satish Sankaran,1 Ramesh Hariharan,1 Vamsi Veeramachaneni,1 Vaijayanti Gupta1. 1 _Strand Life Sciences Pvt. Ltd., Bangalore, India;_ 2 _Tata Memorial Hospital, Mumbai, India_.

Background:

Liquid biopsy is increasingly gaining traction as an alternative to invasive solid tumor biopsies for treatment decisions and disease monitoring. Tumor-plasma concordance studies across multiple cancer types and stages are therefore needed to validate emerging technologies and their clinical utility. However, there is no consensus on the prognostic value of pre-surgical/at biopsy (baseline) ctDNA levels.

Experimental Design:

Matched tumor-plasma samples were collected from 141 patients with varied cancers of different stages. Tumors were profiled using next-generation sequencing (NGS) or quantitative PCR (qPCR), and the mutation status was queried in the matched plasma using digital platforms such as droplet digital PCR (ddCPR) or NGS for concordance. Patient survival outcomes were correlated to pre-surgical ctDNA levels. Wherever follow up samples were available, ctDNA was monitored and matched to disease status.

Results:

Tumor-plasma concordance of 76% and 32% was observed in advanced (Stage IIB and above) and early (Stage I to Stage IIA) stage samples respectively. Baseline ctDNA can stratify patients into 3 categories: high ctDNA (>211 copies per ml plasma) correlated with poor survival outcome, (b) undetectable ctDNA with good outcome and (c) low ctDNA whose outcome was ambiguous. Serial monitoring was most apt in the low ctDNA category where the persistence or spike in tumor DNA in the follow up plasma samples correlated with poor outcome.

Conclusions:

ctDNA can be a powerful tool for therapy decisions and patient management in a large number of cancers across a variety of stages. Its levels may help stratify patients into prognostic groups and identify subsets who may require serial monitoring or aggressive treatment. Our results indicate that patients with high baseline ctDNA levels may have poorer survival outcome while those with low ctDNA levels need to be monitored closely for disease progression, and the undetectable ctDNA group had good outcome. Levels of ctDNA can thus impact treatment decisions and patient management.

#3253

Droplet digital PCR is a sensitive method for detecting refractory acute myeloid leukemia (AML) clones in peripheral blood and saliva.

Kimberly E. Hawkins,1 Cesia Salan,1 Madeleine Turcotte,1 Lauren T. Vaughn,1 Mei Zhang,1 Yanping Zhang,1 Barry Sawicki,1 Glenda G. Anderson,2 Nosha Farhadfar,1 Hemant S. Murthy,1 Biljana N. Horn,1 Helen L. Leather,1 Paul Castillo,1 Maxim Norkin,1 John W. Hiemenz,1 Randy A. Brown,1 William Slayton,1 Jack W. Hsu,1 John R. Wingard,1 Christopher R. Cogle,1 Leylah M. Drusbosky1. 1 _University of Florida, Gainesville, FL;_ 2 _Farsight Genome Systems, Inc, Sunnyvale, CA_.

Droplet digital PCR (ddPCR) is a highly sensitive and rapid method for detecting mutant allele frequency (MAF). In preliminary work, our lower limit of detection for common myeloid gene mutations was 0.001% in peripheral blood and bone marrow compared to 0.1% with flow cytometry and 0.01% with real-time quantitative PCR, and turnaround time is 1 day. Furthermore, we detected leukemic mutant alleles in peripheral blood (PB), introducing the possibility of sparing painful bone marrow biopsy procedures to determine treatment response. Thus, we hypothesized that ddPCR is a feasible and accurate method for monitoring leukemic disease burden in PB for the prospective care of patients (pts) with AML. Eighteen patients (pts) with de novo, relapse/refractory, and secondary AML were recruited to an IRB-approved study (NCT02435550) and bone marrow (BM), peripheral blood (PB), and saliva were collected at standard clinical visits. Gene mutations were identified by whole-exome sequencing (WES) of BM specimens at study entry. For ddPCR interrogation, genomic DNA was isolated (Qiagen), and select primers and probes (Bio-Rad/IDT) were developed based on variants identified in WES data. Case-specific primers and probes were validated on archived specimens obtained at study entry. 12/18 pt mutanomes met criteria for primer/probe design. 8 pts are in the primer/probe design and validation stage and 4 have completed validation and serial analyses. WES identified, and ddPCR confirmed, at least 1 mutation per patient at the study entry timepoint. The mutations included NRAS G13R, NRAS G12A, CSF3R T618I, and IDH2 R172K. In 2 cases, we observed a reduction in both PB and saliva MAF that were consistent with the reduction in both BM and PB blasts after treatment, resulting in complete remissions. Although PB blasts were reduced in a third pt receiving ruxolitinib, the persistence of their CSF3R MAF in PB indicated a resistant AML clone. WES revealed the presence of NRAS G13R variant in a secondary AML pt; however, WES did not detect this NRAS G13R variant in a cryopreserved BM specimen obtained at the pts MDS diagnosis. Interestingly, ddPCR was able to detect NRAS G13R variant at 0.1% MAF in a PB sample obtained at MDS diagnosis, demonstrating the ultrasensitive detection of rare variants within a sample, and highlighting the subclonal evolution of this pt's malignancy. Rapid detection of myeloid-related somatic mutations in a variety of tissue sources (i.e., saliva, PB) will allow for noninvasive monitoring of AML tumor burden. ddPCR may be used to observe molecular response to treatment and to detect molecular residual disease and relapse prior to clinically indicated BM biopsies.

#3254

Dissecting genomic determinants of response to platinum-based chemotherapy in advanced NSCLC and colorectal cancer.

Shirley S. Mo,1 David Liu,1 Stacy W. Gray,2 Steven Joffe,3 Nikhil Wagle,1 Judy Garber,1 Levi A. Garraway,4 Lynette Sholl,5 Pasi A. Janne,1 Eliezer M. Van Allen1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _City of Hope, Duarte, CA;_ 3 _University of Pennsylvania, Philadelphia, PA;_ 4 _Eli Lilly, Indianapolis, IN;_ 5 _Brigham and Women's Hospital, Boston, MA_.

Background: Despite the advent of new targeted- and immuno-therapies, chemotherapy continues to be a mainstay in most tumor types. In lung and colorectal cancer, platinum-based regimens are the standard of care as part of front-line systemic, neoadjuvant, or adjuvant chemotherapy regimens. Response to these agents varies widely among patients, and currently there are no mature or reliable biomarkers of clinical response to platinum therapies for these cancer types. We hypothesized that a systematic integrated genomic analysis of pre-treatment tumors from a large cohort of platinum-treated cancer patients would reveal genomic determinants of resistance and response, which may guide a precision medicine-based approach to choosing effective treatments. Methods: 238 metastatic lung and colorectal adenocarcinoma patients were enrolled in the CanSeq study from February 2013 to July 2015. We performed whole-exome sequencing (WES) on pre-treatment tumors, and called variants including single nucleotide variants (SNVs), small insertions and deletions, copy-number alterations, purity and ploidy of tumors, and inferred clonality and subclonality of individual mutations. Clinical records were reviewed to determine response to therapy, progression-free (PFS), and overall survival. Patients were characterized as having clinical benefit (CB), intermediate benefit (IB), and no clinical benefit (NCB) based on the best radiologic response to therapy and PFS. CB included all patients with complete response or partial response + a long PFS in the top quartile of responders, while NCB experienced progressive disease. IB included all other patients. We evaluated associations between genomic features and response using standard statistical methods. Results: 119 patients received platinum-based systemic chemotherapy and had pre-treatment tumors. Our initial analysis comparing CB vs. NCB suggests that mutations in PTPRD, a receptor protein tyrosine phosphatase previously associated with platinum resistance in cancer cell lines, may be significantly associated with chemoresistance (3/9 NCB, 0/23 CB, p < 0.05, Fisher's Exact) and shorter progression-free survival (p < 0.001, Log-Rank). Further, we found that lower mutational burden is associated with response to platinum chemotherapy (p < 0.05, Wilcoxon Rank Sum). Conclusion: Our approach demonstrates the power and potential of harmonizing genomic and clinical data in an unbiased way to identify biomarkers of platinum chemosensitivity. Our findings suggest that lower mutational burden correlates with CB from platinum therapy, while specific gene alterations potentially offer additional predictive value.

#3255

The roles of ALDH2, ADH1B and ADH1C gene polymorphism in predicting upper digestive tract multiple primary malignancies in head and neck cancer patients.

Shiang-Fu Huang,1 Huei-Tzu Chien,2 Sou-De Cheng,2 Chun-Ta Liao,1 Hung-Ming Wang1. 1 _Chang Gung Memorial Hospital, Taoyuan, Taiwan;_ 2 _Chang Gung University, Taoyuan, Taiwan_.

Head and neck cancer was closely related with habitual use of cigarette and alcohol. The genes involving in the metabolism of alcohol are reported to play roles in increasing risks of head and neck cancer. In this study, we investigated the single nucleotide polymorphisms (SNPs) of alcohol metabolizing enzymes (ALDH2, ADH1B and ADH1C) in their distributions among different tumor subsites and their risks of developing multiple primary cancers. We recruited 659 male head and neck cancer patients between Mar 1996 and Feb 2017. Four hundred and twenty-seven normal controls were also recruited. The SNPs in ALDH2 (rs671), ADH1B (rs1229984) and ADH1C (rs698) were analyzed by Taqman PCR primers and TaqMan probes synthesized by Applied Biosystems (Foster City, CA). Genotyping was performed using the Taqman-based allelic discrimination method on an Applied Biosystems 7500 Real-Time Polymerase Chain Reaction System (Applied Biosystems, Foster City, CA). The A allele carriers than G/G homozygotes in ALDH2 is significantly higher in oral cavity cancer (p = 0.016), oropharynx (p = 0.018) and hypopharynx cancer (p = 0.051) than normal population but not in tonsillar cancer (p = 0.099). In ADH1B, the G allele carriers is significantly higher than A/A homozygotes in oropharynx (p = 0.012), tonsillar cancer (p = 0.050) and hypopharynx cancer (p < 0.001) than normal population but not in oral cavity cancer (p = 0.406). We did not observe ADH1C (rs698) SNP significantly related with HNSCC tumor subsites (p > 0.05). For ALDH2 (A allele carriers vs G/G homozygotes) and ADH1B (G allele carriers vs A/A homozygotes), they significantly increased risks to develop multiple primary cancers in upper digestive tract (p < 0.001, OR [95% confidence interval, C.I.]: 5.358 [2.565-11.195]. and p < 0.001, OR [95% C.I.]: 2.907 [1.598 - 5.290]). Conclusion:ALDH2 (rs671) A allele carriers and ADH1B (rs1229984) G allele carriers had higher risks of developing multiple primary cancers in upper digestive tract. For those with high risk allele carriers, routine screening of upper digestive tract including laryngoscope and esophagoscope is suggested to detect new primaries early.

#3256

The prevalence of non-chronic lymphocytic leukemia (CLL) phenotype monoclonal B-cell lymphocytosis (MBL) in a population-based cohort of U.S. adults.

Sameer A. Parikh,1 Sara J. Achenbach,1 Kari G. Chaffee,1 Neil E. Kay,1 Connie E. Lesnick,1 James R. Cerhan,1 Curtis A. Hanson,1 Tait D. Shanafelt,2 Susan L. Slager1. 1 _Mayo Clinic, Rochester, MN;_ 2 _Stanford Medicine, Palo Alto, CA_.

Introduction: CLL phenotype (CD5+CD23+CD20dim) MBL is present in 5-10% of the general population. The prevalence and outcomes of adults with non-CLL like MBL (including CD5+CD23-, CD5+CD20bright, and CD5-) are not known. We screened for MBL using highly sensitive flow-cytometry in a clinic-based cohort of adults from the Mayo Clinic Biobank.

Methods: The Mayo Clinic Biobank is a large scale bio-repository of 50,000 adults seen in primary care-based clinics. Herein, we focused on those adults ≥40 years who had stored peripheral blood mononuclear cells available (10% of the overall cohort). We screened for MBL using an 8 color (CD38, CD45, Kappa, Lambda, CD19, CD23, CD5 and CD20) flow cytometry assay validated to detect clonal B-cell events to the 0.005% level (1/20,000 events). Chi-square tests were used to assess associations with demographic characteristics. Cox regression models were used to estimate hazard ratios (HR) and 95% confidence intervals (95% CI) for death.

Results: A total of 2594 adults were screened; their median age was 63 years (range, 40-96) and 1111 (43%) were male. 2445/2594 (94%) individuals had interpretable flow cytometry results. A total of 336 (14%) individuals had MBL identified, of whom 276 (82%) had CLL-like MBL and 60 (18%) had non-CLL like MBL (including 39 adults with CD5- and 21 with CD5+CD23- or CD5+CD20 bright phenotype). A strong association between age and the prevalence of non-CLL like MBL was observed, with 0/278 (0%) in 40-49 years, 6/613 (1%) in 50-59 years, 20/674 (3%) in 60-69 years, 21/490 (4%) in 70-79 years, and 13/114 (11%) ≥80 years demonstrating non-CLL like MBL (p<0.001). Non-CLL like MBL was more common in men compared to women (5% vs. 1%; p<0.001). Compared to adults with no MBL, a higher proportion of non-CLL like MBL had a first-degree relative with a lymphoproliferative disorder (8% vs. 17%; p=0.03), and prior non-hematologic cancer (22% vs. 38%, p=0.006). The remainder of the present analysis is focused on adults with non-CLL like MBL (n=60). After a median follow-up of 5 years (range 0-8 years), 3/60 (5%) adults with non-CLL like MBL developed a hematologic malignancy (one myelodysplastic syndrome, two mantle cell lymphoma) compared to 5/2109 (0.2%) adults with no MBL (two large cell lymphoma, one multiple myeloma, one acute myeloid leukemia, one myelofibrosis). Among adults with non-CLL like MBL, the estimated 7-year overall survival (OS) was 79% compared to 94% in those with no MBL (p<0.001, HR for death 3.8, 95% CI 1.98-7.3). After adjusting for age, sex, and self-reported prior non-hematologic cancer, the HR for death was 1.50 (95% CI 0.74-3.05, p=0.26).

Conclusion: In this large study, ~1 of 40 adults seen in a primary care setting had non-CLL like MBL. The prevalence of the non-CLL phenotype MBL increased with age. With current follow-up, the risk of progression to an overt lymphoproliferative neoplasm appears to be low.

#3257

Polygenic risk score, stage, and mode of detection in relation to breast cancer risk.

Amy Trentham-Dietz,1 John M. Hampton,1 Irene M. Ong,1 C David Page,1 Michael N. Gould,1 Jill D. Haag,1 Polly A. Newcomb,2 James D. Shull,1 Elizabeth S. Burnside1. 1 _University of Wisconsin-Madison, Madison, WI;_ 2 _Fred Hutchinson Cancer Research Center, Seattle, WA_.

Identification of increasing numbers of breast cancer risk variants holds promise to improve risk prediction and identify population subgroups that could benefit from targeted prevention and early detection. We aimed to examine whether women with increased polygenic risk are more likely to be diagnosed with symptomatic breast cancer. A case-control study including data collected from interviews, DNA from saliva, and cancer registry data collected between 2001-2007 (4,315 cases, 3,919 controls) was used to construct a polygenic risk score (PRS). Single nucleotide polymorphisms (SNPs, N=98) were selected from recently published genome-wide association studies of breast cancer and breast density, and comparative rat genome studies. Logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CI) associated with the SNPs. SNPs with p-values ≤0.10 (N=24) were used to construct quintiles of the PRS by multiplying the log-odds of the SNPs by the number of risk alleles; multivariable logistic regression models evaluated all women and strata of women defined by method of detection and stage at diagnosis. Odds ratios were adjusted for age, family history of breast cancer, age at menopause/menopausal status, body mass index, parity, and age at first birth. Overall, PRS quintile categories were associated with breast cancer risk in a dose response manner (Q1: OR=1, reference; Q2 OR=1.36, 95% CI 1.16-1.58; Q3 OR=1.66, 95% CI 1.43-1.93; Q4 OR=1.83, 95% CI 1.58-2.12; Q5 OR=2.58, 95% CI 2.23-2.98). Evaluated on a continuous scale, the PRS was associated with an OR=1.38 for a 1-unit change in the standard deviation (SD), 95% CI 1.32-1.45. Odds ratios were essentially unchanged when stratified by method of detection (p-value=0.40). The odds ratio of breast cancer associated with the continuous PRS was elevated among women diagnosed with advanced breast cancer: DCIS, OR=1.36, 95% CI 1.26-1.47 per 1 SD; localized OR=1.37, 95% CI 1.30-1.45 per 1 SD; regional OR=1.38, 95% CI 1.28-1.48 per 1 SD; and distant OR=1.82, 95% CI 1.39-2.40. These results suggest that polygenic risk scores are strongly related to breast cancer risk, that the association does not vary by method of breast cancer detection, but that the association is strongest for metastatic breast cancer.

#3258

**Preclinical evaluation of enhanced irofulven antitumor activity in an** ERCC3 **mutant background by** in vitro **and** in vivo **tumor models.**

Sabine Topka, Sara Khalil, Elisa De Stanchina, Joseph Vijai, Kenneth Offit. _Mem. Sloan Kettering Cancer Ctr., New York, NY_.

Background: We recently described a recurrent founder mutation (p.R109X) in the ATP dependent helicase ERCC3 in BRCA1/2 negative, breast cancer-affected individuals of Ashkenazi Jewish ancestry. Modeling of the mutation in ERCC3 deficient or CRISPR/Cas9 edited cell lines showed a consistent pattern of reduced expression of the protein and concomitant hypomorphic functionality when treated with the DNA alkylating agent IlludinS. A derivative of IlludinS- Irofulven- has previously been evaluated as an antitumor agent in multiple clinical trials and has shown high antitumor activity. Higher effective doses of this drug have been shown to give rise to side effects, including retinal toxicity. Based on the observation that ERCC3 mutant cell lines are sensitive to lower doses of IlludinS we aimed to explore the IlludinS-derivative Irofulven as a potential therapeutic agent to treat patients with hypomorphic ERCC3 mutations. Experimental procedures and Results: We tested the efficacy of Irofulven in isogenic WT and ERCC3 mutant mammary epithelial cell lines in vitro and in xenograft models. In vitro cells were treated with various doses of Irofulven and cell viability was assessed at 72h following treatment. For generation of xenograft models hRAS-V12 expressing genetically engineered ERCC3 mutant as well as isogenic ERCC3 wild-type cell lines were injected into flanks of nude mice and treated with different doses of Irofulven. Tumor volumes were measured over the course of treatment and tumors excised after termination of treatment for final weighing and for histological and subsequent molecular-genetic analysis. In vitro studies showed an increased sensitivity of the ERCC3 mutant cells towards the drug Irofulven, with a significant difference in sensitivity observed in the lower dose range. An initial xenograft experiment determining the effective in vivo dose range and schedule demonstrates enhanced sensitivity in the ERCC3 mutant background. Additional xenograft experiments under the determined optimal treatment dose and schedule are in progress as are further experiments including RNA sequencing of tumor tissue and exploration of combinatorial therapies. Conclusions: A functionally significant recurrent ERCC3 mutation has been shown to increase the risk for breast cancer in a genetic isolate. Similar to tumors arising in the background of homologous repair defects, mutations in nucleotide excision repair genes such as ERCC3 could constitute potential therapeutic targets in a subset of hereditary breast cancers. Mutated cell lines showed lower survival after in vitro and in vivo exposure to the DNA alkylating agent Irofulven. The present study provides a potential pre-clinical approach to a subset of breast cancer cases with an inherited mutation of a nucleotide excision repair pathway gene, demonstrating sensitivity to the DNA alkylating agent Irofulven.

#3259

When is cancer not really cancer: The PREvent Ductal Carcinoma In Situ Invasive Overtreatment Now (PRECISION)* initiative.

Jelle Wesseling,1 Alastair M. Thompson,2 Serena Nik-Zainal,3 Andrew Futreal,2 Shelley Hwang,4 Jos Jonkers,1 Esther H. Lips,1 Daniel Rea5. 1 _Netherlands Cancer Institute, Amsterdam, Netherlands;_ 2 _MD Anderson Cancer Center, Houston, TX;_ 3 _Welcome Trust Sanger Institute, Hinxton, United Kingdom;_ 4 _Duke University School of Medicine, Durham, NC;_ 5 _University of Birmingham, Birmingham, United Kingdom_.

Background. Ductal carcinoma in situ (DCIS) represents 25% of all breast neoplasia due to population-based breast cancer screening. As a result, thousands of women are confronted with DCIS each year. Conventional management includes surgery, supplemented by radiotherapy and/or endocrine therapy, but overtreats the majority of DCIS. Uncertainty as to which DCIS lesions will progress to invasive cancer drives this overtreatment. This urges us to learn how to distinguish DCIS that may progress to invasive breast cancer from the majority of indolent DCIS. Such distinction is best achieved by synergistic collaboration between leading global experts from various disciplines, driven by the essential input from patient voices.

Aim. PRECISION (PREvent ductal Carcinoma In Situ Invasive Overtreatment Now) aims to save thousands of women with low risk DCIS the burden of intensive inappropriate treatment of DCIS (surgery, radiation therapy, hormonal therapies) through the discovery of new data and development of novel tests that promote informed and shared decision-making between patients and clinicians, without compromising the excellent outcomes for DCIS management presently achieved.

Methods. First, three large DCIS cohorts and supplementary resources will be collected enabling in depth molecular studies. Second, extensive genomic characterization, immune profiling and imaging analysis will be performed. In vivo and in vitro modeling will be performed to study the biology of DCIS in detail. Finally, all clinical, immune, and molecular data will be incorporated into a clinical risk prediction model. This risk prediction model will be validated in three prospective randomized DCIS trials in the US (COMET trial), UK (LORIS trial), and mainland Europe (LORD trial).

How the results of this research will be used. The discoveries from our laboratory studies, including a risk stratification model, will be cross-validated in three prospective trials of DCIS active surveillance versus conventional treatment (the COMET, LORIS, and LORD trials). As such, the main result of this study will be that we can identify a group of women for which active surveillance for DCIS could be a safer alternative to intensive treatment. Ultimately, this may also contribute to a more reassuring perception of risk regarding non-life threatening precancerous lesions in general, reducing anxiety and preserving quality of life.

* The PRECISION Team is a Cancer Research UK Grand Challenge Award 2017 winning team and will be jointly funded by Cancer Research UK and the Dutch Cancer Society

#3260

Utilizing digital pathology to understand breast epithelial characteristics of benign breast disease among women undergoing diagnostic image-guided breast biopsy.

Maeve Mullooly,1 Samantha Puvanesarajah,2 Shaoqi Fan,3 Ruth M. Pfeiffer,3 Linnea Olsson,2 Manila Hada,3 Erin L. Kirk,2 Pamela M. Vacek,4 Donald L. Weaver,4 John A. Shepherd,5 Amir P. Mahmoudzadeh,5 Jeff Wang,6 Stephen M. Hewitt,7 Sally D. Herschorn,4 Mark E. Sherman,8 Melissa A. Troester,2 Gretchen L. Gierach3. 1 _Royal College of Surgeons in Ireland, Dublin, Ireland;_ 2 _University of North Carolina at Chapel Hill, NC;_ 3 _National Cancer Institute, Bethesda, MD;_ 4 _University of Vermont, Burlington, VT;_ 5 _University of California, San Francisco, San Francisco, CA;_ 6 _MD Anderson Cancer Center, Houston, TX;_ 7 _National Cancer Institute, Betehsda, MD;_ 8 _Mayo Clinic, Jacksonville, FL_.

Introduction: Defining the histologic correlates of mammographic breast density (MD) may provide insights into why elevated MD is related to increased breast cancer risk. Studies suggest that reduced terminal ductal lobular (TDLU) involution is associated with elevated MD, and both are independent breast cancer risk factors among women who have undergone a biopsy for benign breast disease (BBD). Prior digital histologic analyses of normal breast tissues revealed epithelial nuclear density (END) and TDLU involution are correlated. Accordingly, we examined associations of END with TDLU involution and MD in clinical biopsies. Methods: We analyzed 262 image-guided breast biopsies diagnosed as BBD from 224 women. TDLU involution was visually assessed as TDLU count/mm2 and TDLU span (inversely related to level of involution) in background normal tissue, evaluated using digitized images. The Genie Classifier (Aperio) was applied to images to estimate nuclei count per unit epithelial area, (END). Single X-ray Absorptiometry of pre-biopsy craniocaudal digital mammograms was applied to measure global MD (percent fibroglandular volume (%FGV)). Analysis of covariance, adjusted for age and body mass index, examined mean END differences across tertiles of TDLU/MD measures. Analyses were conducted at the biopsy level using SAS PROC GENMOD to account for within-woman correlations. All tests were two-tailed. Results: Overall, 67% of BBD biopsies were proliferative. Higher END was observed among proliferative than non-proliferative BBD (median END: 10,187 vs. 9,953 respectively; p=0.04). Among all women, END significantly increased with increasing tertiles of TDLU measures (p-trends: TDLU count/100mm2=0.0001, TDLU span=0.046). Whereas TDLU metrics were positively associated with %FGV, no relationship was observed between END and %FGV. In analyses stratified by BBD severity, however, END and %FGV were positively associated among women with non-proliferative disease (p-trend=0.04), findings not observed with proliferative disease. Conclusions: Automated END and visually assessed TDLU involution metrics were positively associated with each other and with MD. However, associations were diluted for proliferative lesions, suggesting that applying automated digital pathology tools to unsegmented digital images of whole sections of BBD biopsies does not demonstrate the same associations with MD as visual assessment of TDLU involution.

#3261

Pilot study of the combination of bazedoxifene and conjugated estrogen to modulate risk biomarkers in women with hot flashes at increased risk for breast cancer.

Carol J. Fabian,1 Kandy R. Powers,1 Jennifer L. Nydegger,1 Amy L. Kreutzjans,1 Trina Metheny,1 Teresa A. Phillips,1 Lauren Nye,1 Carola M. Zalles,2 Bruce F. Kimler1. 1 _University of Kansas Medical Center, Kansas City, KS;_ 2 _JFK Medical Center, Atlantis, FL_.

Uptake of anti-hormonal agents for primary prevention of breast cancer is poor due to concern about side effects, especially induction of menopausal symptoms. A combination of 20 mg bazedoxifene plus 0.045 mg conjugated estrogen is FDA approved (as Duavee®) for treatment of hot flashes and prevention of osteoporosis in postmenopausal women with a uterus. We undertook a pilot study to assess the feasibility of using this formulation as a breast cancer prevention agent in women at increased risk for development of breast cancer. Feasibility was to be assessed by accrual, retention, and documentation of a lack of enhanced proliferation in benign breast tissue acquired by random periareolar fine needle aspiration (RPFNA). Eligibility criteria included risk >2X that of average risk woman for age group, postmenopausal having hot flashes or night sweats and not on systemic hormone replacement, and at least 500 cells on a baseline RPFNA. Women were ineligible if they had LCIS or DCIS, a BRCA1/2 germline mutation, had had a hysterectomy, or had >4% Ki-67 positive cells by immunocytochemistry. Fasting blood draw, digital mammography with Volpara software, and DXA scan for body composition was performed at baseline along with QOL questionnaires. Women then received Duavee® daily for 6 months, followed by repeat of baseline tests. We accrued the first 20 subjects in 14 months. Many of the women followed in our cohort and interested in the trial were not eligible due to prior hysterectomy, prior LCIS or a high penetrance gene mutation. Thus accrual was slower than anticipated. All women have reported improvement in hot flash frequency and intensity. None have discontinued prematurely or had a study related serious adverse event. Fourteen women have completed the 6-month intervention and are evaluable for modulation of biomarkers. There have been no protocol-defined increases in proliferation (to >2% Ki-67 for baseline Ki-67 <1% or doubling if baseline Ki-67 ≥1%), with 10 of 14 paired specimens exhibiting a decrease. Ten women had Volpara fibroglandular assessments pre- and post-study with a median relative decrease of 11% (8 decreased and 2 increased). For the first ten subjects where serum hormones and growth factors were assessed in paired assays, favorable modulation was observed for estradiol, testosterone, SHBG, bioavailable testosterone, IGF-1, and the molar ratio of IGF1:IGFBP3. A primary prevention trial in symptomatic women appears feasible given the favorable initial results. The current pilot will continue to accrue so as to inform the design of a randomized, placebo-controlled Phase II trial of Duavee® in women at risk for breast cancer. Financial support provided by grants from the Breast Cancer Research Foundation (BCRF-16-049, BCRF-17-049). Duavee® provided by Pfizer, Inc. which was not involved in design, conduct, or analysis of the study.

#3262

Biomarkers of response to deslorelin in women at high risk of breast cancer.

Jeffrey N. Weitzel,1 Michael F. Press,2 Sharon R. Sand,1 Lei Zhang,1 Catherine Klifa,3 Malcolm Pike,4 Giske Ursin,5 Darcy V. Spicer,6 AnnaMarie Daniels,7 Josef Herzog,1 Yunan Miao,1 Gerald Wuenschell,1 Chun Li,1 Robert Hickey1. 1 _City of Hope, Duarte, CA;_ 2 _University of Southern California, Los Angeles, CA;_ 3 _Median Technologies, Valbonne, France;_ 4 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 5 _University of Oslo, Oslo, Norway;_ 6 _University of Southern California, CA;_ 7 _BioPharmX, Menlo Park, CA_.

Women with BRCA1 or BRCA2 mutations have a high breast cancer (BC) risk. Breast density is an independent biomarker of BC risk. We demonstrated that deslorelin (D), a gonadotropin-releasing hormone agonist (GnRHA), reduced mammographic breast density among BRCA1 carriers (Weitzel, CCR 2007). We report here on correlative studies in a Phase II chemoprevention study of intranasal D plus low-dose add-back estradiol and testosterone for 10 months in 10 premenopausal high-risk women. Pre- and post-study measures included mammographic and MRI breast density; serum hormone levels; breast tissue gene expression by Affymetrix GeneChip Human Genome U133 Plus 2.0 Array; Q-PCR to validate the top genes; analysis of sera by 2D-PAGE and LC-MS/MS Mass Spectrometry, validated by a marker specific ELISA. Nine of 10 participants showed a mean reduction in MRI volumetric breast density of 17.3% (p=0.045), a mean reduction in MRI fibroglandular volume of 15.1% (p=0.041) and a mean reduction in mammographic density of 31.9% (p=0.0033). The top five differentially expressed genes in breast tissue were: FOS, EGR1, FABP3, COL3A1 and COL1A1. From seven protein spots up-regulated following treatment, 65 sera proteins were initially identified as response biomarkers, with two of these, adiponectin and Vitamin D-binding protein, predicted by Ingenuity Pathway Analyses (IPA) to have a direct role in reducing breast density by possibly altering stromal and epithelial tissue viability and fat metabolism. FOS was identified through IPA in the Vitamin D-binding protein network and both COL3A1 and COL1A1 were identified in the adiponectin network. Of the nine subjects two had an increase of ≥ 5% BMI and two had a decrease of ≥ 5% BMI. There was no significant correlation between adiponectin level changes and BMI changes.Tissue micro-array gene expression and proteomic analyses of sera identified changes in biomarkers that correlated with reduced breast density among young high-risk women receiving a GnRHA-based chemoprevention regimen.

Pre/Post Human Serum Adiponectin Levels

---

|

A_Pre: 0 M | B_ Post: 10 M | Fold (Post/Pre) | Log A (0M) | Log B (10M) | T-Test (p) paired

S1 | 0.463 | 0.386 | 0.835 | -0.335 | -0.413 | 0.014

S2 | 0.376 | 0.413 | 1.097 | -0.425 | -0.385

|

S3 | 0.262 | 0.367 | 1.403 | -0.583 | -0.435

|

S4 | 0.664 | 0.802 | 1.209 | -0.178 | -0.096

|

S5 | 0.245 | 0.608 | 2.487 | -0.612 | -0.216

|

S6 | 0.315 | 0.755 | 2.399 | -0.502 | -0.122

|

S7 | 0.413 | 0.799 | 1.936 | -0.385 | -0.098

|

S8 | 0.575 | 0.646 | 1.123 | -0.240 | -0.190

|

S9 | 0.436 | 0.733 | 1.680 | -0.361 | -0.135

|

Average | |  | 1.574224 | |

|

#3264

Targeting bone marrow-resident dormant CTCs to overcome metastasis.

Debasish Boral,1 Haowen N. Liu,1 Wei Yin,1 Monika Vishnoi,1 Antonio T. Scamardo,2 David S. Hong,2 Dario Marchetti1. 1 _Houston Methodist Research Institute, Houston, TX;_ 2 _The University of Texas M.D. Anderson Cancer Center, Houston, TX_.

Metastatic "seeding" is an early event initiated when cancer cells are shed from the primary tumor, enter into the blood stream as circulating tumor cells (CTCs), and travel to distant organs where they can remain dormant as disseminated cancer cells (DTCs) for indefinite lengths of time. The organ serving as the foremost reservoir for DTCs is the bone marrow (BM). Despite widespread knowledge that bone marrow-resident breast cancer cells (BMRCs) affect tumor progression, signaling mechanisms of BMRCs implicated in maintaining long-term dormancy have not been characterized. This is caused by the lack of biomarkers that can identify and isolate viable but dormant BMRCs not expressing neoplastic and/or proliferation markers.

To overcome these hurdles, we developed a patient-derived CTC model of clinical dormancy. We hypothesized that a significant distinction exists between signaling pathways of BM-homing vs metastasis-competent CTCs upon transplantation in xenografts. The model was established by three sequential steps: (a) isolation of CTC-enriched cell populations from the peripheral blood of patients with metastatic breast cancer, followed by CTC implantation in NOD-SCID-gamma mice; (b) in vivo depletion of "normal" human cell lineages over 4-8 months (before onset of overt metastasis), and (c) capture and characterization of viable human CTCs from blood and BM of mice via multi-parametric flow cytometry. This strategy was implemented on a set of breast cancer patients stratified according to their primary tumor subtype (ER+/PR+, HER2+, and ER-/PR-/HER2-), and gene-signatures of putative ex vivo CTCs and BMRCs were obtained by whole genome transcriptomic arrays.

First, comparative analyses of ex vivo vs de novo CTC-gene signatures identified increased mTORC2 along with decreased mTORC1 signaling activity as the most significant characteristic of human BM-resident CTCs. mTORC2/mTORC1 represent the two complementary arms of mTOR signaling - a critical pathway frequently dysregulated in breast cancer and implicated in cell survival and dormancy. Second, heightened mTORC2 downstream targets augmented quiescent CTC populations (Ki67-/RBL2+ cells) in paired metastatic vs primary breast cancer tissues. Third, IHC analyses of breast cancer CTC xenograft tissues showed that solitary BM and tissue-resident breast cancer CTCs had high mTORC2 activity. Finally, shRNA knockdown of Rictor-the essential component of mTORC2 actions, increased Ki67/PCNA expression and proliferation.

Collectively, these findings suggest that the balance between mTORC1 vs mTORC2 signaling pathways regulate CTC-associated mitotic and/or dormancy characteristics. Further elucidation of mTOR-mediated CTC dormancy will provide novel strategies for therapeutic interventions in breast cancer patients having metastatic "seeding" but yet to develop overt metastasis.

#3265

Healthy eating index 2005 and the mucosa associated gut microbiome in healthy individuals.

Yanhong Liu, Nadim J. Ajami, Diane Hutchinson, David Graham, Sarah Plew, Ashley Johnson, Preksha Shah, Liang Chen, Kathryn Royse, Donna L. White, Jennifer Kramer, Matthew C. Wong, Rhonda Cole, Clark Hair, Jason Hou, Nisreen Husain, Maria Jarbrink-Sehgal, Fasiha Kanwal, Gyanprakash Ketwaroo, Rajesh Shah, Maria Velez, Melissa L. Bondy, Hashem B. El-Serag, Joseph F. Petrosino, Li Jiao. _Baylor College of Medicine, Houston, TX_.

Background: Diet is a modifiable risk factor for multiple cancers. It is also known to modulate gut microbial composition and metabolic activity, and plays an important role in maintaining gut homeostasis. However, our understanding of dietary quality and mucosa-associated microbiota in the large intestine in humans is limited. We examined the association between diet quality and gut microbiota in adults underwent routine colonoscopy in a cross-sectional study. Methods: We enrolled 21 men (50-75 years old, 71% White) who were found to have grossly normal colons at colonoscopy completed between July 2013 and April 2016. We obtained 98 colonic mucosa biopsies, with microbial DNA extracted from snap frozen biopsy. The 16S rRNA V4 region was amplified and sequenced on the Illumina MiSeq platform. The UPARSE and SILVA were used for operational taxonomic unit (OTU) classification. A self-administered BLOCK Food Frequency Questionnaire was used to assess dietary intake in the past year. Dietary quality was defined using the Healthy Eating Index (HEI)-2005, and further categorized as low or high using the median of total HEI or 12 individual component scores. We compared alpha-diversity (OTU and Shannon index), beta-diversity (Weighted UniFrac principal coordinates analysis), and relative abundance of bacterial phylum and genus by total HEI and all 12 individual HEI components using Mann-Whitney test. Reported P values were adjusted for multiple testing using false discovery rate. Results: The most abundant bacterial phyla observed were Firmicutes, Bacteroidetes, Proteobacteria, Verrucomicrobia, and Fusobacteria. High-quality diet (total HEI score ≥ 63) was not associated with higher richness and evenness of gut microbiome (P = 0.12), but was significantly associated with bacterial composition (P = 0.046). Compared to men who had low-quality diet (total HEI score < 63), those with high-quality diet had a higher abundance of Proteobacteria (12.2% vs. 8.1%, P = 0.006), and lower abundance of Fusobacteria (0.13% vs. 3.37%, P = 0.004). At the genus level, those with high-quality diet had significantly lower abundance of Fusobacterium than those with low-quality diet (0.11% vs. 3.77%, P = 0.004). At the HEI component level, individuals with a higher consumption of solid fruits and milk had higher richness and evenness of gut microbiome (P < 0.05) as well as higher abundance of phylum Verrucomicrobia (P < 0.05) and genus Akkermansia (P < 0.001). Consuming more solid and saturated fat, alcohol, and added sugar were associated with a higher abundance of phylum Fusobacteria (P < 0.0001) and genus Fusobacterium (P = 0.0001). Conclusions: The colonic microbiome of men consuming a low-quality diet exhibited increased levels of Fusobacteria, which has been associated with colorectal cancer risk. If replicated in prospective research, our findings suggest low quality diet may contribute to colorectal cancer by modulating gut microbiome.

#3266

Measurement and comparison of the gut microbial communities in fecal, rectal swab, and mucosal samples.

Roshonda B Jones,1 Xiangzhu Zhu,2 Emili Moan,3 Harvey J. Murff,2 Qi Dai,2 Martha J. Shrubsole,2 Anthony A. Fodor,1 M. Andrea Azcarate-Peril,4 Reid M. Ness,5 Douglas L. Seidner5. 1 _University of North Carolina at Charlotte, Charlotte, NC;_ 2 _Vanderbilt-Ingram Cancer Ctr., Nashville, TN;_ 3 _North Carolina State University, Raleigh, NC;_ 4 _University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 5 _Vanderbilt University Medical Center, Nashville, TN_.

Background: Studies evaluating the gut microbiome frequently use stool. However, stool, which is predominantly composed of luminal bacteria, may not adequately reflect mucosally adherent bacteria. The purpose of this study is to evaluate similarities and differences in gut bacterial measurements and stability in the microbial communities of three different types of samples that could be used to assess different niches of the gut microbiome: rectal swab, stool, and normal rectal mucosa samples.

Design: Sixty-eight participants were selected from a personalized chemoprevention trial population of individuals with previous colorectal adenomas who had donated stool, swab, and mucosal samples at baseline and three months later. 16S rRNA amplicon sequencing was conducted for 60 participants at two time points (n=120 stool and 120 swab). Whole-genome shotgun metagenomics DNA sequencing was conducted for 50 participants at two time points (n=100 stool, 28 swab, and 16 mucosal).

Results: In swab-stool comparisons, there were substantial taxa differences with some taxa varying largely by sample type (e.g., Thermaceae) while other taxa were predominantly associated with interindividual subject variation (e.g., Desulfovibrionaceae) or by both sample type and participant (e.g., Enterobacteriaceae). At species-level resolution (with WGS sequences) we observed that bacteria associated with colorectal tumors (Escherichia coli and Fusobacterium nucleatum) were of higher relative abundance in swab than stool. There were also statistically significant differences in other bacteria according to the sample type (e.g., Bifidobacterium longum, Bacteroides fragilis). Comparing all three sample types with whole-genome metagenome shotgun sequencing, swab samples were much closer to stool samples than mucosa samples, although all KEGG functional Level 1 and Level 2 pathways were significantly different across all sample types (e.g., transcription and environmental adaptation). However, the individual signature of participants was also observed and was largely stable between two time points. Thus, we found that while the distribution of some taxa was associated with these different sampling techniques, other taxa largely reflected individual differences in the microbial community that were insensitive to sampling technique.

Conclusion: There is substantial variability in the assessment of the gut microbial community according to the type of sample.

#3267

Cervical microbiota and the urinary metabolome in patients with high-risk and low-risk HPV infections.

Gilmary Ortiz,1 Natalyia Chorna,2 Josefina Romaguera-Agrait,2 Magaly Martinez-Ferrer,3 Maria Sanchez,3 Ana P. Ortiz,3 Rafael Guerrero-Preston,2 James R. White,4 Filipa Godoy-Vitorino5. 1 _Inter American University of Puerto Rico, CAGUAS, PR;_ 2 _University of Puerto Rico, School of Medicine, San Juan, PR;_ 3 _Comprehensive Cancer Center, San Juan, PR;_ 4 _Resphera Biosciences, Baltimore, MD;_ 5 _Inter American University of Puerto Rico, San Juan, PR_.

Genital human papillomavirus (HPV) is the world's most commonly diagnosed sexually transmitted infection, and high-risk HPV types are strongly linked to cervical dysplasia and carcinoma. The microbiota of the human cervico-vaginal tract is the interface between the host and environment, and its constituents may act as determinants of susceptibility for HPV infection. There exists the potential for reliable diagnostic biomarkers for cervical cancer in the cervical microbiome as well as in urine, which would be an important biospecimen due to its abundance and non-invasive collection strategy. In this study, we performed comprehensive metabolomic analysis on urine samples from 65 women (54 HPV+, 38 of whom have cervical intraepithelial neoplasia (CIN), and 11 HPV-) and discuss the potential diagnostic implication and metabolic description of high-risk and low-risk HPV infected patients.

The experimental procedure employed gas chromatography mass spectrometry (GC-MS) on the urine-derived products and HPV genotyping was determined by reverse hybridization with the HPV SPF10-LiPA25 kit. We further characterized resident cervical bacteria in these patients using Illumina sequencing of the V4 region of the 16S ribosomal RNA with a high-resolution bioinformatics methodology (Resphera Insight v2.2) for species-level taxonomic assignment.

The hybridization assay detected 11 low-risk and 10 high-risk HPV types many of them co-occurring in the cervical samples. Lactobacillus iners OTUs were shared by all samples but were significantly more dominant in women with low-risk HPV strains (p-value=0.05), while G. vaginalis, S. sanguinegens and L. jensenii were associated with high-risk strains. CIN1 and CIN3 dysplasic biopsies, detected significant enrichments of Gardnerella vaginalis, L.crispatus and Prevotella amnii (p-values <0.05). Preliminary GC-MS analysis identified elevated levels of palmitic (C16:0) and stearic fatty acids (C18:0) as well as lactic acid, abundant in the HPV+ samples while 5-oxo-proline and Neu5Ac (N-Acetylneuraminic acid) were abundant in the HPV- samples.

Our preliminary data identifies possible microbial and metabolic biomarkers associated with oncogenic HPV and cervical intraepithelial neoplasia in women living in Puerto Rico.

#3268

Associations of plasma trimethylamine N-oxide (TMAO), choline, and betaine with the gut microbiome and biomarkers of inflammation and cardiometabolic risk in the Multiethnic Cohort.

Benjamin C. Fu,1 Meredith AJ Hullar,2 Timothy W. Randolph,2 Adrian A. Franke,3 Kristine R. Monroe,4 Iona Cheng,5 Lynne R. Wilkens,3 John Shepherd,6 Loïc Le Marchand,3 Unhee Lim,3 Johanna W. Lampe2. 1 _University of Washington, Seattle, WA;_ 2 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 3 _University of Hawaii Cancer Center, HI;_ 4 _University of Southern California, CA;_ 5 _Cancer Prevention Institute of California, CA;_ 6 _University of California, San Francisco, CA_.

Background: TMAO is produced by gut microbial metabolism of dietary quaternary amines and has been linked to several chronic diseases, including colorectal cancer and cardiovascular disease. However, the microbes and mechanisms involved are still unclear. Here, we aimed to 1) identify microbial taxa associated with TMAO and 2) evaluate associations of plasma TMAO, along with its precursors choline and betaine, with inflammatory and cardiometabolic biomarkers.

Methods: We conducted a cross-sectional analysis in 1,716 participants (men and women, 60-77, and of 5 racial/ethnic groups) of the Adiposity Phenotype Study within the Multiethnic Cohort in Hawaii and California. Plasma TMAO, choline, and betaine were measured by LC-MS/MS. The gut microbiome was analyzed by Illumina MiSeq paired-end sequencing of the 16S rRNA gene V1-V3 region using DNA extracted from stool. Fasting blood was analyzed for lipopolysaccharide-binding protein (LBP) and insulin by ELISA; C-reactive protein (CRP), cholesterol, glucose, and triglycerides using a Cobas autoanalyzer; and HOMA-IR for insulin resistance by derivation. Associations between microbial genera and TMAO were assessed with negative binomial and zero-inflated negative binomial (ZINB) regression models, with control of the false discovery rate (FDR) using the Benjamini-Hochberg procedure. Associations of TMAO, choline, and betaine with biomarkers were determined using multivariable linear regression and a ZINB model for CRP, adjusted for age, sex, race/ethnicity, physical activity, aspirin use, and % body fat.

Results: Of 105 genera examined, 7 were associated with TMAO at an FDR of 0.01: Streptoccoccus, Blautia, Clostridium, a genus from the Ruminococcaceae family, Butyricimonas, Lactobacillus, and an uncultured Verrucomicrobia of the order vadinHA64; all showed a positive relationship. Plasma choline (β±SE: 0.23±0.10; p=0.03) and betaine (0.19±0.08; p=0.02), but not TMAO, were positively associated with CRP, whereas only choline was associated with LBP (1.97±0.80; p=0.01). Choline was inversely associated with HDL cholesterol (-14.86±2.29; p<0.001) and positively associated with LDL cholesterol (9.86±5.01; p=0.049). Betaine (-0.16±0.03; p<0.001) and choline (0.14±0.05; p=0.002) were associated with triglycerides, while TMAO was not (0.01±0.02; p=0.46). All 3 plasma compounds were associated with HOMA-IR, with only betaine having an inverse association (-0.32±0.06; p=0.004).

Conclusion: We found several genera of gut microbes associated with plasma TMAO, including Clostridium, several species of which can metabolize choline to trimethylamine. We also identified associations between plasma TMAO, choline, and betaine with inflammatory and cardiometabolic markers, particularly choline, which indicated risk to adverse health outcomes.

#3269

Metformin to prevent metabolic syndrome associated with androgen deprivation therapy (ADT): Metabolic analysis from a placebo-controlled study of metformin in non-diabetic men initiating ADT for advanced prostate cancer (PCa).

Devalingam Mahalingam,1 Salih Hanni,2 Christos Fountzilas,3 Joel Michalek,2 John Sarantopoulos,2 Sureshkumar Mulampurath Achuthan Pillai,2 John Kuhn,2 Michael Pollak,4 Ian Thompson5. 1 _Northwestern University, Chicago, IL;_ 2 _University of Texas Health Science Center San Antonio, San Antonio, TX;_ 3 _Roswell Park Cancer Institute, Buffalo, NY;_ 4 _McGill University, Montreal, Quebec, Canada;_ 5 _Christus Health, San Antonio, TX_.

Background: ADT results in metabolic syndrome, characterized by hyperinsulinemia, insulin resistance and obesity. The hyperinsulinemia may result in ADT resistance; therefore preventing metabolic syndrome could have a therapeutic impact on PCa. Metformin decreases glucose & insulin by inhibiting hepatic gluconeogenesis. There is preclinical evidence for additional antineoplastic activity due to mTOR inhibition secondary to AMPK activation. Methods: We analyzed serum and PBMC from a recently completed clinical study of men with advanced PCa on ADT that were randomized 1:1 to metformin at 500mg TID or color matched placebo. Subjects serum insulin/glucose, metformin levels, weight and waist circumference (WC) was assessed at baseline, week 12 and 28. The primary endpoint of study was the metabolic consequences of metformin vs placebo cohort. Secondary endpoints were PSA response and PBMC analysis of downstream target of mTOR, phospho-S6 kinase. Results: There were 36 evaluable men. The mean age on study was 68.4. Mean weight, WC and insulin at baseline in metformin cohort was 187 lbs, 41.14 cm and 10.03 mIU/L respectively, and 177.65 lbs, 40.52 cm and 8.02 mIU/L in placebo cohort. An increase in mean weight, WC and insulin levels was seen in both cohorts. At week 12 and 28, no statistical difference in weight, WC and insulin was observe in either cohort. Four men randomized to metformin had undetectable serum drug levels despite drug-diary suggesting compliance; excluding them did not result in significant metabolic change. Assessing efficacy, 50% in metformin and 53.3% in placebo cohort achieved undetectable PSA at week 28; difference not statistically significant. PBMC analysis demonstrated variable down-regulation of phospho-S6 kinase in the metformin cohort. Conclusion: This study detected no impact of MET addition to ADT on the risk of metabolic syndrome and no additional anti-tumor effects. Control of hyperinsulinemia related to diabetes by MET does not necessarily imply MET has a similar action on hyperinsulinemia due to ADT.

#3269A

2-Hydroxyflavanone: A novel estrogen receptor alpha downregulator with potent antitumor effect in breast cancer.

Sharad S. Singhal,1 Shireen Chikara,1 Lokesh Nagaprashantha,1 Jyotsana Singhal,1 David Horne,1 Sanjay Awasthi2. 1 _City of Hope, Duarte, CA;_ 2 _Texas Tech University Health Sciences Center, Lubbock, TX_.

Breast cancer (BC) remains one of the major causes of cancer deaths in women. 2Hydroxyflavanone (2HF), a constituent of citrus fruits, inhibits growth of human breast cancer cell lines in culture. The present study was undertaken to determine in vivo efficacy of 2HF against MDAMB231 human breast cancer xenografts. The 2HF administration retarded growth of MDAMB231 cells subcutaneously implanted in female nude mice without causing weight loss or any other side effects. The 2HF mediated suppression of MDAMB231 xenograft growth correlated with reduced cell proliferation as revealed by immunohistochemical analysis for Ki67 expression. Analysis of the vasculature in the tumors from 2HF treated mice indicated smaller vessel area compared with control tumors based on immunohistochemistry for angiogenesis marker CD31. The 2HF mediated inhibition of angiogenesis in vivo correlated with downregulation of vascular endothelial growth factor (VEGF) level in the tumor. Consistent with these results, 2HF treatment suppressed cell cycle regulators levels in cultured MDAMB231 cells. Moreover, the 2HF treated MDAMB231 cells exhibited reduced capacity for migration compared with vehicle-treated control cells. In addition, 2HF administration elicits apoptotic response as judged by annexin V and PI staining and TUNEL assay. 2HF treatment reduced the levels of ER alpha in ER positive MCF7 breast cancer cells. In conclusion, the present study demonstrates in vivo anticancer efficacy of 2HF against MDAMB231 xenografts in association with reduced cell proliferation and suppression of neovascularization. Our results indicate that 2HF is a promising anticancer agent for breast cancer due to its ability to collectively inhibit major regulators of breast cancer including RLIP76, ER alpha, and HER2. Interestingly, a spontaneously immortalized non-tumorigenic normal human mammary epithelial cell line (MCF10A) is significantly more resistant to 2HF mediated apoptosis compared with breast cancer cells, demonstrating selectivity of 2HF towards malignant breast cells. These preclinical observations merit clinical investigation to determine efficacy of 2HF against human breast cancers. (This work was supported in part by the Department of Defense grant W81XWH 16 1 0641. Funding from the Beckman Research Institute of City of Hope is also acknowledged). 

## BIOINFORMATICS AND SYSTEMS BIOLOGY:

### Sequence Analysis and Unique Database Resources

#3270

Pan-cancer analysis of 1,962 whole-genomes reveals significantly mutated CTCF/cohesin insulators.

Eric Minwei Liu, Alexander Martinez-Fundichely, Tawny Cuykendall, Jason G. Dumelie, Matthew MacKay, Priyanka Dhingra, Samie R. Jaffrey, Ekta Khurana. _Weill Cornell Medical College, New York, NY_.

Recent studies have shown that mutations at non-coding elements, such as the TERT promoter and ESR1 enhancer, can act as cancer drivers. However, an important class of non-coding elements, namely CTCF/cohesin insulators, has been overlooked in the previous driver analyses. It is known that promoter and enhancer interactions are facilitated by partitioning of the human genome into DNA loops. These loops act as insulated neighborhoods preventing the interactions of enhancers and promoters across loops. They are held together by the binding of two CCCTCF binding factors (CTCF) and the cohesin complex, which consists of four core transcription factor subunits (SMC1, SMC3, RAD21 and either STAG2 or STAG1) at the loop ends. Disruption of the loop anchor regions, called CTCF/cohesin insulators, can lead to de novo enhancer-promoter interactions and subsequent dysregulation of associated genes. We used insulator annotations from cohesin ChIA-PET assays and analyzed somatic mutations in 1,962 whole-genomes from 21 cancer types. We find that the mutations predicted to disrupt DNA binding motifs of CTCF, RAD21 and SMC3 are enriched in many cancer types due to the sequence contexts of neutral mutational processes. Using a novel computational approach, CNCDriver (Cornell Non-Coding Driver), that balances the interplay between neutral mutational rate covariates of CTCF/RAD21/SMC3 motif loss and the stronger functional impact of motif-disrupting mutations, we identify the insulators that show signals of positive selection during tumor evolution. We find that the mutations in significantly mutated insulators are associated with differential expression of genes involved in multiple cancer pathways: TGF-β, Hedgehog and Wnt signaling. In particular, we find 16% of melanoma samples show mutations in an insulator that are associated with up-regulation of TGFB1, which is known to promote angiogenesis and tumor cell migration in melanoma. Thus, our study reveals insulators as a novel class of non-coding cancer drivers. Identification of significantly mutated insulators can complement the identification of other types of non-coding cancer drivers (promoters, enhancers and ncRNAs) to fully understand the role of non-coding alterations in tumorigenesis.

#3271

A machine learning approach for somatic mutation discovery.

Derrick Wood,1 James White,1 Andrew Georgiadis,1 Beth Van Emburgh,1 Sonya Parpart-Li,1 Jason Mitchell,1 Valsamo Anagnostou,2 Noushin Niknafs,2 Rachel Karchin,2 Eniko Papp,1 Christine McCord,1 Peter Loverso,1 David Riley,1 Luis A. Diaz,3 Sian Jones,1 Mark Sausen,1 Victor E. Velculescu,2 Samuel Angiuoli1. 1 _Personal Genome Diagnostics, Baltimore, MD;_ 2 _Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD;_ 3 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Variability in the accuracy for somatic mutation detection may affect discovery of alterations and therapeutic management of cancer patients. To address this issue, we developed a somatic mutation discovery method based on machine learning approaches that outperformed other methods using tumor alterations that were experimentally validated (sensitivity of 97% vs 90-99%; positive predictive value of 98% vs 34-92%). Analysis of tumor-normal exome data from 1,376 TCGA samples using this method revealed concordance for 74% of mutation calls but also identified likely false positive and negative changes in TCGA data, including in clinically actionable genes. For melanoma and lung cancer patients treated with immune checkpoint inhibitors, determination of high-quality somatic mutation calls improved mutation load-based predictions of clinical outcome. Integration of high-quality mutation detection in clinical NGS analyses improved the accuracy of test results compared to other clinical sequencing analyses not using these approaches (sensitivity of 100% vs 50-97%; positive predictive value of 100% vs 9-66%). These analyses provide an approach for improved identification of tumor-specific mutations and have important implications for research and clinical management of patients with cancer.

#3272

**A graph remapping framework for** in silico **adjudication of SNVs, INDELs, and structural variants from genetic sequencing data.**

Dillon Lee, Yi Qiao, Gabor Marth. _University of Utah Department of Human Genetics, Salt Lake City, UT_.

Several state-of-the-art, easy to use tools are available both for short-variant detection (e.g. GATK, FREEBAYES), and structural variant (SV) detection (e.g. LUMPY, MANTRA, DELLY), but these tools often produce divergent variant calls, especially INDELs, and it is very difficult to reconcile such variants into a single, accurate set. Furthermore, while it would be highly desirable to also detect larger, structural variants (SV), existing SV detector packages are typically difficult to integrate, highly resource-intensive to run, and result in call sets that require expert manual review to reduce false positive detection rate.

Our algorithm, GRAPHITE (https://github.com/dillonl/graphite) requires as input a collection of variant calls, made by one or more short-variant or SV detection tools. Typically, this starting set is high sensitivity (i.e. inclusive), but low specificity (i.e. have a high false discovery rate). We then apply a novel "variant adjudication" procedure to discard false positives, while keeping true positive calls. This is accomplished by constructing a graph from these variants (the Variant Graph) representing allelic variants as graph branches, in addition to the branches formed by the current, linear genome reference sequence. Using a graph mapping algorithm (GSSW, a graph extension of the Smith-Waterman alignment algorithm) we developed earlier, we re-map all reads from each of the samples contributing to the candidate calls. We retain candidate variants confirmed by mappings to those branches in the graph that represent the corresponding variant allele, and discard those candidates that were not confirmed by such mappings. This procedure results in a highly specific callset that also maintains the high sensitivity of the inclusive starting callset constructed by multiple primary variant calling methods. Because the graph construction and mapping approach works for most types of SVs in addition to all short variants, variants of all different types can be integrated in a single step.

Here we present the application of this method for cross-validating structural variants calls from Pacific Biosciences data by remapping deep Illumina WGS read sets to Variant Graphs constructed using the candidate Pacific Biosciences variants, as part of the Human Genome Structural Variation Consortium (HGSVC) data analysis project. We also present GRAPHITE's application to improving the accuracy of allele frequency measurement in tumor sequencing data, which is essential for the accurate reconstruction of subclonal evolution in longitudinal tumor samples.

#3273

rkmh: A MinHash toolbox for analyzing HPV coinfections.

Eric T. Dawson,1 Sarah Wagner,2 David Roberson,2 Meredith Yeager,2 Joseph Boland,2 Erik Garrison,3 Mark Schiffman,1 Tina Raine-Bennet,4 Thomas Lorey,4 Phillip Castle,5 Stephen Chanock,1 Lisa Mirabello,1 Richard Durbin3. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Leidos Biomedical Research Inc, Frederick, MD;_ 3 _University of Cambridge, Cambridge, United Kingdom;_ 4 _Kaiser Permanente Northern California, Oakland, CA;_ 5 _Albert Einstein College of Medicine, Bronx, NY_.

Human papillomaviruses are common infections transmitted by direct physical contact. Two sexually transmitted types, HPV16 and HPV18, are responsible for almost 75% of cervical cancer cases worldwide; another 10 types of >200 known types are considered carcinogenic. Among the four HPV16 lineages (A, B, C, and D), 100-fold differences in odds ratios for adenocarcinoma are observed. It is therefore informative to distinguish between infection with HPV16 lineages, even though they differ by as little as 2% of their genome. We present a computational toolkit, rkmh, for characterizing viral coinfections. rkmh uses kmer matching strategies to rapidly determine the most similar type or lineage reference genome for a given read. The proportion of reads matching each reference genome can then be calculated and the ratios of the infecting viruses can be estimated. To assess the performance of rkmh we first simulated 100bp paired end Illumina read sets from the PAVE database of HPV reference genomes, then also evaluated performance on a real HPV16 sample sequenced on the Ion Torrent Proton platform (typical read length 250bp), and a set of 3,660 Oxford Nanopore minION reads generated from two HPV16 reference strains (typical read length over 6500bp). We demonstrate that rkmh can adequately classify HPV infections at the type and lineage level. We discuss further applications of the tool in metagenomics. rkmh is freely available at https://github.com/edawson/rkmh.

#3274

Pan cancer analysis of fusion genes in TCGA using ChimeRScope, an alignment free algorithm.

Neetha Nanoth Vellichirammal,1 Jasjit Banwait,1 Abrar Albahrani,1 You Li,2 Chittibabu Guda1. 1 _University of Nebraska Medical Center, Omaha, NE;_ 2 _HitGen, Chengdu, China_.

Fusion transcripts, frequent in cancer formed from the concatenation of two unrelated genes results primarily from the structural rearrangements of the genome. Fusion transcripts that are unique to a cancer type can be exploited to understand the underlying mechanisms for malignancy and can serve as diagnostic or prognostic markers. We used ChimeRScope, a novel alignment-free algorithm developed recently by our group. This program is specifically designed to identify fusions in cancer transcriptomes that contain frequent chromosomal aberrations and structural variations. We explored the fusion landscape of 31 cancers from TCGA with a focus to identify transcriptionally induced fusions along with recurrent fusions across cancer types. Level 1 paired-end RNA sequencing data from The Cancer Genome Atlas (TCGA) GDC data portal was downloaded and unmapped reads extracted from both tumor and tumor adjacent normal tissue. Unmapped fastq files were then analyzed with the ChimeRScope pipeline and fusions identified in tumor adjacent normal tissue was removed from further analysis. Fusions identified by ChimeRScope Examiner module were further screened to remove those with low confidence using custom python scripts. We classified fusions based on the distance of break point from the participating exon boundaries as E-E (both break points near exon-intron junctions), E-M (one break point near junction), and M-M (both break points away from the junction). Further, samples with somatic whole-genome sequence data were downloaded from TCGA database and analyzed for structural variants using BreakDancer to identify fusions supported by genomic events. A total of 8,409 primary tumor samples and 730 normal samples spanning 31 cancer types from TCGA were analyzed in this study. Of the identified fusions across different cancer types, Breast Invasive Carcinoma had the maximum number of fusions. Thymoma, Melanoma, Breast Invasive Carcinoma, Testicular Germ Cell Tumor, and Acute Myeloid Leukemia had the highest number of recurrent fusions. Uterine carcinoma, Lung Squamous cell carcinoma, and Sarcoma had the maximum number of samples with at least one fusion. Approximately 30% of the fusions identified in each tumor was in-frame. We identified a number of fusions that are recurrent across cancer types involving kinases, oncogenes or tumor suppressors. This is a comprehensive analysis of fusions across all of the major class of cancers from TCGA. Data from this study indicate the presence of a vast pool of unexplored fusion events that need to be evaluated for functionality.

#3275

Comparison of somatic variant interpretation results between human experts and automated classification using AMP/ASCO/CAP guidelines.

Niroshan Nadarajah, Manja Meggendorfer, Claudia Haferlach, Wolfgang Kern, Torsten Haferlach. _Munich Leukemia Laboratory, Munich, Germany_.

Introduction: Evaluating the pathogenicity of a variant is challenging given the plethora of types of genetic evidence that laboratories have to consider. Deciding how to weigh each type of evidence is difficult, and standards are needed. In 2017, AMP/ASCO/CAP released a joint consensus recommendation proposing a four-tiered system to categorize somatic sequence variations based on their clinical significance in cancer diagnosis, prognosis, and/or therapeutic.

Aim: Evaluate how the AMP/ASCO/CAP guidelines compare to an accredited laboratory approach to variant classification and explore the variance in the use and interpretation of the pathogenicity criteria. Identifying disease-contributory variants for various human genetic diseases will greatly improve diagnosis and facilitate development of therapies.

Patients and Methods: 50 cases with myeloid malignancies were selected, analyzed either with a 26 genes myeloid panel (ThunderStorm Target Enrichment library; Raindance, Billerica, MA) or a 63 genes panel (TruSeq Custom Amplicon; Illumina, San Diego, CA). Alignment and Variant calling was performed with JSI SeqPilot (JSI Medisys, Ettenheim, Germany). Molecular geneticists in the lab annotated each variant manually in a 3-tier system (pathogenic, uncertain significance, benign) given the lab's SOP for variant classification. Each variant was checked against the following databases: COSMIC (v76), ClinVar, dbSNP (v147) and IARC TP53 (r17). Population frequency information was extracted from ExAC. Mutation impact prediction was performed using PolyPhen-2, SIFT and VEP.

Results: Among the 50 cases 681 variants were classified during routine workup according to SOPs accredited by EN ISO15189, subsequent to the elimination of sequencing artefacts. 405 were classified as benign, 52 with variant of uncertain significance (VUS) and 224 as pathogenic. Using the computed classification yielded 377 Tier IV (Benign), 184 Tier III (Unknown clinical significance), 93 Tier II (Potential clinical significance) and 27 Tier I (Strong clinical significance). To be able to compare, Tier I and II were binned. In 80% (542/681) of instances both approaches are concordant. 4 variants classified diagnostically discrepant (3 VUS to Tier I/II, 1 benign variant to Tier II) (Table 1). Manual interrogation revealed these were difficult variants with scarce public data and poor concordance of prediction tools.

Conclusion: Systematic evaluation of an automated classification based on AMP/ASCO/CAP with manual curated data found a concordance rate of 80%. The automated approach seems to be more cautious, thus the bias towards more VUS calls, which is preferable to miscalls. The guidelines seem to yield results sufficiently good for clinical use, especially for labs with little experience in variant classification and a big step forward regarding standardization.

#3276

Whole exome sequencing identifies significantly linked regions on multiple chromosomes in families with a history of lung cancer.

Anthony M. Musolf,1 Haiming Sun,2 Bilal A. Moiz,1 Diptasri Mandal,3 Mariza de Andrade,4 Colette Gaba,5 Ping Yang,4 Yafang Li,6 Ming You,7 Elena Y. Kupert,7 Marshall W. Anderson,7 Ann G. Schwartz,8 Susan M. Pinney,9 Christopher I. Amos,10 Joan E. Bailey-Wilson1. 1 _Nih Nhgri, Baltimore, MD;_ 2 _Harbin Medical University, Harbin, China;_ 3 _Louisiana State University Health Sciences Center, New Orleans, LA;_ 4 _Mayo Clinic, Rochester, MN;_ 5 _University of Toledo Dana Cancer Center, Toledo, OH;_ 6 _Dartmouth College, Lebanon, NH;_ 7 _Medical College of Wisconsin, Milwaukee, WI;_ 8 _Karmanos Cancer Institute, Wayne State University, Detroit, MI;_ 9 _University of Cincinnati College of Medicine, Cincinnati, OH;_ 10 _Baylor College of Medicine, Houston, TX_.

Lung cancer is the deadliest cancer in the United States, contributing approximately 25% of all cancer deaths. Lung cancer risk is well-documented to increase in response to environmental factors, particularly tobacco smoking. As one might expect for a complex trait, there is also a significant genetic risk component in lung cancer, though it is not very well studied. We obtained whole exome sequencing (WES) genotype data from Washington University on 204 subjects from 25 extended families. These individuals were recruited from families with a history of lung cancer and previous analyses showed these 25 families be informative. The purpose of this study is to identify potential risk variants for lung cancer by performing genetic linkage analysis. Quality control was performed on the sequence data, filtering on parameters such as depth (less than 10), genotype quality (less than 10), missingness, and Mendelian inconsistencies. Identity-by-descent (IBD) values were also calculated to verify correct familial relationships. Quality control procedures left approximately 500,000 SNVs and indels for analysis.

We performed two-point parametric linkage analysis assuming an autosomal dominant mode of inheritance with a disease allele frequency of 1%, a 10% penetrance for carriers and a 1% penetrance for non-carriers. Two discrete sets of linkage analyses were performed. One was a variant-based analysis, which evaluated linkage between the phenotype and individual SNVs or indels. The second was gene-based analysis, which created a multi-allelic pseudomarker corresponding to a gene from haplotypes of rare variants (minor allele frequency <= 0.01) located within that particular gene. Two-point linkage analysis was then performed on the pseudomarkers.

While the variant-based analysis did not identify any genome-wide significant results, several were identified by the gene-based analysis. The highest HLOD scores were both greater than the genome-wide significance level of HLOD = 3.3 and were located on two regions of chromosome 1q: 1q42.13-43 and 1q21.2-21.1. In both regions, the significant HLOD scores clustered around known cancer genes. At the 1q42.13-43 region, signals centered on the cancer-implicated genes OBSCN and RYR2 (also a known mesothelioma gene), while at 1q21.2-21.1 the signals centered on five genes in the neuroblastoma breakpoint family (NBPF) genes, a cluster of recently duplicated genes. NBPF genes have previously been implicated in a variety of different cancers including lung cancer. We are currently performing additional analyses to corroborate the significant results, as well as examining the individual families to determine which families are driving the significant signals.

#3277

**Identification of internal tandem duplication within the** FLT3 **gene from AML patient next-generation sequence data.**

Yevhen Akimov, Samuli Eldfors, Disha Malani, Tero Aittokallio, Caroline Heckman. _Institute for Molecular Medicine Finland (FIMM), Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland_.

Introduction

The presence of the Fms Related Tyrosine Kinase 3 (FLT3) internal tandem duplication (FLT3-ITD) is associated with a poor prognosis and an increased rate of relapse in acute myeloid leukemia (AML). Identification of FLT3-ITD from next-generation sequencing (NGS) data is currently impeded by lack of reliable algorithms to identify large insertions. Therefore, despite the wide adoption of NGS in recent years, FLT3-ITD mutations are still being detected with PCR/electrophoresis assays which are expensive and time consuming. Algorithms that reliably identify FLT3-ITDs from NGS data are urgently required.

Methods

We developed a computational algorithm for FLT3-ITD calling from next-generation sequencing data. The method is based on iterative realignment of unaligned sequence read fragments. The algorithm uses sequence reads mapped to FLT3 exon 14 and unmapped sequence reads sharing similarity (at least 25bp aligned with the Smith-Waterman algorithm) with FLT3 exon 14. The procedure has two steps: (1) Smith-Waterman local pairwise alignment of each read to the reference sequence, and (2) trimming unaligned parts of each read. If a read has an unaligned part(s) identified at step 2, then these are returned to the step 1, and the whole procedure is repeated. The procedure is iterated until each read is fully-aligned to the reference sequence. A sample is considered to be ITD negative if all reads are fully-aligned after the first iteration. Otherwise, the algorithm returns fragmentized reads aligned to the reference sequence. This output can be used to discriminate between ITD positive and negative samples based on the pattern produced by aligned fragments of the reads. Reads coming from FLT3-ITD have an intrinsic property to cluster at the boundaries of the duplicated region; therefore, samples with reads ends clustering at a shared genomic position are considered ITD positive. We calculate the p-value of reads ends clustering at the same genomic position using generalized birthday-problem theory.

Results

We analyzed a total of 273 AML samples from 125 patients. The algorithm assigned 221 samples (~81%) as FLT3-ITD negative, forty-seven (~17%) samples as FLT3-ITD positive and 5 samples (~1.8%) were assigned as undefined (with a p-value between 0.005 and 0.1). We visually inspected the patterns produced by realigned fragments to assign a FLT3-ITD status for each of the undefined samples.

We compared these results with FLT3-ITD status determined by a clinical PCR/electrophoresis assay. Our algorithm was able to recall all the 13 samples where ITD was detected by the clinical assay. Notably, FLT3-ITD mutation in two samples were detected with our method but not by the clinical assay.

Conclusions

1. Our novel algorithm enables accurate identification of FLT3-ITD mutations from NGS sequence data.

2. The method was able to detect FLT3-ITD positive samples that were missed by the clinical assay.

#3278

Performance assessment of de novo assembly-based structural variation detection in the human genome.

Chunlin Xiao, SEQC2 Working Group #3. _NIH, Bethesda, MD_.

Structural variations (SVs) contribute to genetic diversity of human populations, affect biologic functions, and cause various human disorders. However, accurately identifying SVs with correct sizes and locations in the human genome remains challenging due to the complexity of the human genome, limitations of sequencing technologies, and drawbacks of analysis methods. The advancement of next-generation sequencing technologies has dramatically decreased the sequencing cost, while substantially increased the lengths of the sequencing reads. Thus, using de novo assembly-based approaches for discovering a full spectrum of SVs in human genome becomes appealing. While various assembly methods have been developed and proposed for general use by the community, the relative efficiency and predictive accuracy of SVs calling based on these assembly methods have not been fully evaluated. In this study, we applied several popular de novo assembly tools to the sequencing read data that were generated using multiple sequencing technologies with technical replicates for NA12878/HG001, a well-studied individual from NIST-led Genome-in-a-Bottle (GIAB) project: a HapMap Caucasian trio and a Chinese Quartet from FDA-led Sequencing Quality Control Phase II (SEQC2) project. Assemblies and SVs callsets were generated for each of the eight samples, and repeatability in the SVs of the technical replicates and reproducibility across sequencing sites were evaluated. The assembly-based SVs callsets have been compared with alignment-based SVs callsets. These results allow better understanding of the impacts of de novo assembly methods on SVs calling, thus providing a better insight to precision medicine.

#3279

Comprehensive analysis of small nucleolar RNAs across in human cancers.

Jing Gong,1 Yajuan Li,2 Chunjie Liu,3 Yu Xiang,1 An-yuan Guo,3 Chunru Lin,2 Leng Han1. 1 _University of Texas Health Science Center at Houston, Houston, TX;_ 2 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _Huazhong University of Science and Technology, China_.

Recent studies demonstrated that small nucleolar RNAs (snoRNAs) play important roles in tumorigenesis. We systematically investigated the expression landscape and clinical relevance of snoRNAs in >10,000 samples across 31 cancer types. We observed overall elevated expression of snoRNAs and their ribonucleoproteins in multiple cancer types. We showed complex regulation of snoRNA expression by their host genes, copy number variation, and DNA methylation. Unsupervised clustering revealed that the snoRNA expression subtype is highly concordant with other molecular/clinical subtypes. We further identified 46 clinically relevant snoRNAs and experimentally demonstrated functional roles of SNORD46 in promoting cell proliferation, migration, and invasion. We developed a user-friendly data portal, SNORic, to benefit the research community. Our study highlights the significant roles of snoRNAs in the development and implementation of biomarkers or therapeutic targets for cancer and provides a valuable resource for cancer research.

#3280

Utah somatic variant calling pipeline featuring multi-sample joint calling, variant-graph based accurate allele frequency estimation and subclone analysis.

Yi Qiao, Xiaomeng Huang, Dillon Lee, Andrew Farrell, Thomas Nicholas, Brent Pederson, Aaron Quinlan, Gabor Marth. _University of Utah, Salt Lake City, UT_.

Current tumor variant detection software tools are focused on the identification of somatic mutations in a single tumor sample (e.g. the primary tumor) compared to normal control tissue, and are not adequate for studies where multiple samples from the same patient, either at consecutive time points or at different metastatic sites are collected. Here we present a software framework that facilitates simultaneous analysis of such multi-sample datasets.

Our framework utilizes our FreeBayes variant caller for jointly calling all tumor samples and normal controls from the patient. This ensures that even very low frequency mutations are called because read evidence is aggregated across all samples; and that allele frequency information for a called somatic variant is provided uniformly for all samples. Short and medium-length INDELs, and structural variants are identified by our RUFUS software, a new reference-free mutation calling algorithm for both germline and somatic mutation detection, with extremely high sensitivity and specificity. We use our Graphite tool, a graph-genome based read realignment algorithm, to eliminate false positive somatic calls i.e. inherited variants masquerading as somatic mutations, and to substantially increase accuracy of allele frequency estimation of the true somatic calls, a feature critical for downstream subclone analysis. In addition, we use the FACETS package for CNV and LOH analysis, also critical for downstream subclone analysis.

Finally, we utilize SubcloneSeeker, our algorithm to reconstruct the evolution of the cancer at the subclonal level from the somatic variant allele frequencies, to understand how the tumor evolved over time, in response to multiple courses of treatment, or over space, across multiple metastatic sites. We demonstrate the use of this pipeline in published and ongoing studies involving longitudinal and multi-site tumor sample datasets.

#3281

PDX Finder: An open and global catalogue of patient tumor-derived xenograft models.

Nathalie A. Conte,1 Terrence F. Meehan,1 Dale A. Begley,2 Debbie M. Krupke,2 Csaba Halmagyi,1 Jeremy C. Mason,1 Abayomi Mosaku,1 Steven B. Neuhauser,1 Helen Parkinson,1 Carol J. Bult2. 1 _European Bioinformatics Institute (EMBL-EBI), Hinxton, United Kingdom;_ 2 _The Jackson Laboratories, Bar Harbor, ME_.

Patient-derived tumor xenograft (PDX) mouse models have emerged as an important oncology research platform to study tumor evolution, drug response and for tailoring chemotherapeutic approaches to individual patients. PDX models are produced and made available in repositories managed by small academic labs, large research consortia and contract research organizations. Because of the distributed and heterogeneous nature of PDX repositories, finding relevant models of interest to investigators is a challenge. To address this issue, The Jackson Laboratory and EMBL-EBI have co-developed the PDX Finder, a comprehensive open global catalogue of PDX models and their associated data across resources. In support this initiative, we coordinated the community initiative to develop the PDX models Minimal Information standard (PDX-MI) that defines the minimal information necessary for describing key elements of a PDX model including the clinical attributes of a patient's tumor, methods of implantation, host strain, and quality assurance methods used for model validation†. PDX-MI serves as the basis for PDX Finder's comprehensive search and attribute filtering options (e.g., tumor histology, molecular variant, drug response). Within PDX Finder, model attributes are harmonized and integrated into a cohesive ontological data model that supports consistent searching across the originating resources. From PDX Finder, direct links to these resources are provided to allow users to contact the relevant institution for model acquisition and further collaboration. PDX Finder is formally collaborating with several worldwide consortia including PDXnet and EurOPDX to increase "findability" of PDX models and to advance cancer research and drug discovery. PDX Finder is currently displaying over 1200 PDX models for a wide variety of cancers and is actively recruiting more models. The community is invited to explore and provide feedback on our portal as we build this rich resource at : www.pdxfinder.org.

† Meehan et al, 2017. PDX-MI: Minimal Information for Patient-Derived Tumor Xenograft Models. Cancer Res. 2017 Nov 1;77(21):e62-e66.

#3282

Standardization and coordination of variant interpretation knowledgebases improves clinical genome actionability.

Alex H. Wagner,1 Brian Walsh,2 Dmitriy Sonkin,3 Rodrigo Dienstmann,4 Xuan S. Li,5 Jacques Beckmann,6 Georgia Mayfield,2 David Tamborero,7 Núria López-Bigas,7 Jeremy Goecks,2 Adam Margolin,2 Malachi Griffith,1 Obi Griffith1. 1 _Washington University in Saint Louis, Saint Louis, MO;_ 2 _Oregon Health & Science University, Portland, OR; _3 _National Cancer Institute, Rockville, MD;_ 4 _Vall D'Hebron, Barcelona, Spain;_ 5 _MolecularMatch, Houston, TX;_ 6 _University of Lausanne, Lausanne, Switzerland;_ 7 _Institute for Research in Biomedicine, Barcelona, Spain_.

The promise of precision medicine in which a cancer patient's treatment is tailored to their specific cancer variants requires concise, standardized, and searchable clinical interpretations--biomarker-disease associations that provide therapeutic or prognostic value. While many institutions curate interpretations for clinically relevant cancer variants, these efforts are siloed and non-interoperable. Through the Global Alliance for Genomics and Health (GA4GH), we formed a Variant Interpretation for Cancer Consortium (VICC, cancervariants.org). We have evaluated six established sources of cancer variant interpretations hosted by members of the consortium: the Clinical Interpretations of Variants in Cancer (CIViC) knowledgebase, OncoKB, the Jackson Laboratories Clinical Knowledgebase (JAX-CKB), Precision Medicine Knowledgebase (PMKB), MolecularMatch, and the Cancer Genome Interpreter (CGI). We compared the coverage of the biomedical literature across them, and found that from 6,034 publications across the 6 resources, 86% of them were unique (by PubMed ID) across the collective. This illustrates both the value to be gained from aggregating the knowledge of these resources and the enormity of the biomedical literature describing cancer variants that is not yet queryable. To create a framework and resource for sharing these interpretations, we have integrated guidelines and ontologies describing genes, variants, diseases, drugs, and evidence to harmonize and describe clinical interpretations of cancer variants. From this, the VICC has collectively established a set of minimum elements required for describing a cancer variant interpretation, and used this to guide harvesting and harmonizing knowledge from these founding resources. Our results demonstrate that the knowledge from the constituent sources of the VICC are highly heterogeneous across every element defining these interpretations (genes/variants, drugs, diseases, evidence). We have developed a harmonized database and an alpha-stage web interface for exploring and querying this breadth of knowledge (http://g2p-ohsu.ddns.net). We next evaluated the 110,320 somatic mutations of 17,005 patient donors in the AACR Project GENIE cohort against our VICC aggregate interpretation database. When separated out by evidence level (as described by the joint consensus recommendation of the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists), the interpretations within the VICC aggregate knowledgebase provided 2-4x breadth of coverage across each level of evidence when compared to the median coverage from the constituent resources. Finally, we demonstrate how the clinically actionable interpretation of a patient mutation may change dramatically when considering the knowledge from the VICC resources in aggregate instead of individually.

#3283

NDEx, the Network Data Exchange: Collaboration, publication and data sharing for cancer pathways.

Dexter R. Pratt. _UCSD, La Jolla, CA_.

Computable network models of pathway mechanisms, molecular interactions, and cellular function are widely developed and used in cancer genome analysis. Dissemination and reuse of networks, however, needs common data standards, facile access by visualization and analysis applications, mechanisms for pre-publication collaboration, and integration with the publishing process. We present NDEx, the Network Data Exchange, an online resource (www.ndexbio.org) that addresses these needs by providing a framework in which users can store, share, access, and disseminate networks. These networks can range from small pathways to large interaction data or to hierarchical cell models. NDEx is user-driven, where every researcher can be an author, easily creating publicly available network resources with full control of the data content and organization. Networks can range from standard resources disseminated by organizations to novel, up-to-date content from individual scientists. Collaborators can privately share their networks for review and reference or for integration in analysis workflows across multiple scripts and applications. Access to NDEx by an extensive API and client libraries supports network-enabled application development. Published networks can go beyond diagrams and static supplemental data, becoming immediately accessible and interactive. Integration with academic publishing is underway, facilitated by features such as anonymous access by reviewers and minting of DOIs for networks. NDEx breaks down barriers to scientific discourse involving networks, fostering collaboration within and between communities.

#3284

COSMIC: Integrating and interpreting the world's knowledge of somatic mutations in cancer.

Zbyslaw Sondka, Sally Bamford, Charlotte G. Cole, Elisabeth Dawson, Laura Ponting, Raymund Stefancsik, Sari Ward, Harry C. Jubb, Sam Thompson, Dave Beare, Nidhi Bindal, Charambulos Boutselakis, Peter Fish, Bhavana Harsha, Chai Yin Kok, Chris Ramshaw, Claire Rye, John Tate, Shicai Wang, Peter J. Campbell, Simon A. Forbes. _Wellcome Trust Sanger Institute, Cambridge, United Kingdom_.

COSMIC, the Catalogue Of Somatic Mutations In Cancer (http://cancer.sanger.ac.uk/cosmic) is a long-term sustainable effort to collect, standardise and integrate information on somatic mutations and other molecular alterations that cause human cancer. Being the world's largest and most comprehensive database of somatic mutations in cancer, it also provides web-based tools for exploration and interpretation of collected data. The content of the database is obtained primarily by careful curation of the scientific literature by a team of experienced post-doctoral curators, allowing mutations to be described across every form of human cancer at high resolution (covering 1,706 cancer phenotypes). This is then combined with data from a number of on-line sources, including the TCGA and ICGC web portals. During exhaustive manual curation, all the available information about mutations and samples (e.g. disease type, demographic data, treatments) are collected, standardised and integrated to allow for both creation of wide virtual cohorts and large-scale studies, as well as precise analysis at the level of a single sample, gene or mutation.

To support investigations into how genes dysfunction to drive cancer, the 83rd release of COSMIC (Nov 2017) encompasses 5,366,273 coding mutations and 18,845 gene fusions in 1,343,214 cancer samples, hand-curated from 25,501 scientific publications, including 32,514 whole cancer genomes. Further curations support examination of gene dysregulation in cancer, including 16,961,605 non-coding variants, 1,180,789 Copy Number Variants, 9,176,464 gene expression variants, and 7,879,142 differentially methylated CpGs.

Specialised COSMIC projects highlight specific knowledge of cancer in order to characterise events with a higher impact in disease aetiology. The Cancer Gene Census (http://cancer.sanger.ac.uk/census), currently defines and describes 699 genes, how their dysfunctions drive oncogenesis, and characterises their impact on hallmarks of cancer. COSMIC-3D (http://cancer.sanger.ac.uk/cosmic3d) provides an interactive view of cancer mutations in the context of 3D protein structures, and predicts potential drug-binding sites. Mutations causing drug resistance are now described as a new resource (http://cancer.sanger.ac.uk/cosmic/drug_resistance). To allow for the incorporation of new data exploration tools, the COSMIC web page layouts have been updated and their usability has been improved, giving more options to filter webpage content.

COSMIC is significantly updated 4 times a year, and is available free-of-charge for academic and non-profit users via COSMIC webpage (http://cancer.sanger.ac.uk/cosmic) or to download through COSMIC downloads (http://cancer.sanger.ac.uk/cosmic/download)

#3285

COSMIC-3D: Impacts of cancer mutations on protein structure, function, and druggability.

Harry C. Jubb,1 Harpreet K. Saini,2 Marcel L. Verdonk,2 Simon A. Forbes1. 1 _Wellcome Trust Sanger Institute, Cambridge, United Kingdom;_ 2 _Astex Pharmaceuticals, Cambridge, United Kingdom_.

In recent years there has been an explosion in cancer genomic data that has enabled understanding of the incidence and patterns of mutations in cancer patients. The challenge is to translate this vast resource of genomic research and data into precision medicines that target specific mutations in patient populations and improve clinical outcomes. Delivering personalised cancer medicines requires greater understanding of how cancer mutations impact the structure, function, and druggability of known and potential protein drug targets.

In this study we annotate each missense mutation in COSMIC, via COSMIC-3D (http://cancer.sanger.ac.uk/cosmic3d), with protein structural features derived from the wwPDB, and rich functional features derived from the UniProt protein database. The structural features include small molecule binding sites (including drug binding sites), protein-protein interaction interfaces, protein-nucleic acid interactions, solvent inaccessible protein "core" versus solvent exposed areas, and predicted small-molecule druggability. The UniProt-derived functional features represent high quality functional site annotations, including post-translational modification sites, functional domains and motifs, and regions of biological interest.

In addition, we explore the relationship between mutation occurrence in protein structural and functional sites, and recurrence in cancer. We found that recurrent missense mutations are significantly enriched in specific structural and functional environments, including small-molecule binding sites and predicted druggable pockets. These results provide promising hypotheses that recurrent, cancer driving mutations may be targeted by small-molecule drugs. We posit that further characterisation of the structure-function implications of each mutation in COSMIC-3D will increasingly aid mutation-guided target discovery and drug design.

#3286

A prototype Cancer Publication Portal (CPP) for summarizing and accessing cancer genomic publications.

Andrew Johnson,1 Roland DePratti,2 Garrett M. Dancik1. 1 _Eastern Connecticut State University, Willimantic, CT;_ 2 _Central Connecticut State University, New Britain, CT_.

A search of PubMed finds more than 71,000 publications with the keywords "cancer" and "gene" published since the beginning of 2015. This increasingly large volume of publications necessitates the development of text-mining tools to help cancer researchers navigate and summarize articles efficiently. Here, we present a prototype Cancer Publication Portal (CPP) that allows a user to search and summarize the biomedical literature for a gene of interest. CPP leverages the data generated by PubTator, a tool that uses robust text-mining methods and associates articles with gene names and Medical Subject Heading (MeSH) terms. Additionally, CPP associates articles with therapeutic agents and selected cancer-related terms from the NCI Thesaurus. To use CPP, the user selects a gene of interest, and is presented with a summary of MeSH terms, therapeutic agents, and cancer-related terms that are associated with the selected gene. Cancer-related terms are highlighted, so that, for example, a user can quickly ascertain the frequency of publications per cancer type, or how many articles are associated with "mutations" in the gene. The summaries are interactive, so that the user can click on a term of interest to filter the results, and update the summaries. The user can repeat this process to narrow in on a specific topic of interest. Following all searches and filters, relevant articles are listed and are linked back their PubMed entries so that the reference article can be accessed. CPP currently includes information for ~4 million publications associated with >10,000 human genes. The underlying data is stored in an Oracle database while R/Shiny is used to implement the web interface. CPP is available from the following link: https://gdancik.github.io/bioinformatics/

#3287

An integrated TCGA pan-cancer clinical data resource to drive high quality survival outcome analytics.

Jianfang Liu,1 Tara Lichtenberg,2 Katherine A. Hoadley,3 Laila M. Poisson,4 Alexander J. Lazar,5 Andrew D. Cherniack,6 Albert J. Kovatich,7 Christopher C. Benz,8 Douglas A. Levine,9 Adrian V. Lee,10 Larsson Omberg,11 Denise M. Wolf,12 Craig D. Shriver,7 Vesteinn Thorsson,13 The Cancer Genome Atlas Research Network, Hai Hu1. 1 _Chan Soon-Shiong Institute of Molecular Medicine at Windber, Windber, PA;_ 2 _Nationwide Children's Hospital, Columbus, OH;_ 3 _University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 4 _Henry Ford Health System, Detroit, MI;_ 5 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 6 _The Eli and Edythe L. Broad Institute of MIT and Harvard, Cambridge, MA;_ 7 _Uniformed Services University / Walter Reed National Military Medical Center, Bethesda, MD;_ 8 _Buck Institute for Research on Aging, Novato, CA;_ 9 _NYU Langone Medical Center, New York, NY;_ 10 _University of Pittsburgh Cancer Institute and Magee-Women's Research Institute, Pittsburgh, PA;_ 11 _Sage Bionetworks, Seattle, WA;_ 12 _University of California San Francisco, San Francisco, CA;_ 13 _Institute for Systems Biology, Seattle, WA_.

Background The Cancer Genome Atlas (TCGA) program collected detailed clinicopathologic annotation data along with multi-platform molecular profiles across 33 cancer types (>11,000 tumors). These data contain features unique to the democratized nature of this largest ever clinical-genomic dataset, but questions have been raised about the quality of its outcome measures. To address these questions, we produced and quality checked a standardized compendium named the TCGA Pan-Cancer Clinical Data Resource (TCGA-CDR).

Method Clinical data were downloaded from TCGA data portal. Commonly used data elements were quality assessed (QA), integrated and processed with the help from disease experts and TCGA Biospecimen Core Resource. Clinical outcome endpoints of overall survival (OS), progression-free interval (PFI), disease-free interval (DFI), and disease-specific survival (DSS) were derived. Each qualifying end point was subjected to multi-step assessment including evaluation for sufficient follow-up (Maller and Zhou, 1994; Shen, 2000), event number >20 and rate ≥ 30%, plateaued cumulative event plot, and median event time < median censored time. Validation and application examples used standard statistical methods including Cox proportional hazards regression.

Results Thirty-three enrollment and 97 follow-up data files were processed for 11,160 patients. Over 1000 QA issues were identified and resolved for 21 commonly used clinical data elements during derivation of the 4 outcome endpoints. Each endpoint was quality scored with tumor-specific use recommendations provided. Despite perceived short follow-up times, PFI was derived with high confidence and recommended as a clinical endpoint choice for 27 of the 33 pan-cancer types, but with caution for 5 others. OS is recommended as an appropriate endpoint for 23 cancer types, and with caution for 6 others. Guidance for using DSS and DFI is also provided. The value of TCGA-CDR was demonstrated through validation and application examples including, 1) patients with ER(-) breast tumors showing worse outcomes compared to those with ER(+) tumors; 2) patients with higher-stage (III, IV) tumors showing worse outcomes than those with lower-stage (I, II) tumors; and 3) patients achieving disease-free status after diagnosis with better outcomes than those never achieving disease-free status.

Conclusion We compiled and quality assessed a new TCGA-CDR containing 4 standardized outcomes for 11,160 cancer patients across 33 tumor types. This resource, generating endpoint results consistent with independent non-TCGA findings, provides new research opportunities to produce biologically insightful observations at unprecedented clinical scale.

Disclaimer The views expressed in this article are those of the authors and do not reflect the official policy of the department of Army/Navy/Air Force, Department of Defense, or U.S. government.

#3288

The landscape of circular RNA in cancer.

Josh N. Vo, Yajia Zhang, Sudhanshu Shukla, Lanbo Xiao, Dan Robinson, Yi-Mi Wu, Sisi Gao, Carl Engelke, Xuhong Cao, Alexey Nesvizhskii, Arul Chinnaiyan. _University of Michigan-Ann Arbor, Ann Arbor, MI_.

Circular RNAs (circRNAs) are an intriguing class of RNA due to their covalently closed structure, relatively high stability, and implicated roles in gene regulation. Here we used exome capture RNA-Seq, a non-poly(A) high-throughput RNA-sequencing protocol, to detect and characterize circRNAs across >800 cancer samples. When compared against the two gold-standard methods (Ribo-Zero and RNase-R), capture sequencing significantly enhanced the enrichment of circRNAs and preserved accurate circular-to-linear ratios. We compiled the detected circRNAs from capture sequencing into the public OncoCirc database, the most comprehensive catalogue of circRNA species to-date. With this database, we identified the best circRNAs to serve as biomarkers for prostate cancer and were able to detect a novel class of circRNAs involved two genes. OncoCirc will serve as a valuable resource for the development of circRNA for diagnostic or therapeutic purposes for other cancers, as well as the study of circularization as an intriguing RNA splicing process.

#3289

Implementing evolving clinical standards in a variant interpretation knowledgebase.

Kilannin Krysiak, Arpad M. Danos, Alex H. Wagner, Susanna Kiwala, Joshua F. McMichael, Adam C. Coffman, Erica K. Barnell, Yan-Yang Feng, Benjamin J. Ainscough, Cody A. Ramirez, Malachi Griffith, Obi L. Griffith. _Washington University in St Louis, St Louis, MO_.

The rapid expansion of clinical sequencing and targeted therapies has driven the accelerated evolution of clinical guidelines and regulatory standards surrounding sequence variants. Organizations such as the NCCN, FDA, ACMG, etc., continue to incorporate sequence variants into a variety of clinical contexts. The pace of these changes has led to several disparities between guidelines and resources used for the interpretation of clinically identified variants. 1) Despite the inherent lag in creating such standards through expert consensus, this evolution outpaces their incorporation into many resources used for clinical sequencing analysis. 2) Guidelines are often not readily accessible in programmatic, structured ways to promote rapid updates, including many guidelines only available in human-readable text formats. 3) These guidelines apply to various clinical contexts (drug response, prognosis, etc.), each requiring their own schema changes for their proper incorporation. 4) Levels of specificity provided by these guidelines are not conducive to matching specific genomic coordinates (e.g., FGFR2 fusion) or maintaining evidence provenance. We recently developed the Clinical Interpretations for Variants in Cancer (CIViC; civicdb.org) crowd-sourced, open-access knowledgebase, providing a structured framework for evaluating genomic variants of various types (e.g., fusions, SNVs) for their therapeutic, prognostic, predisposing or diagnostic utility. Since then, the CIViC resource has continued to evolve and has tackled the challenges above. This crowd-sourced model of both data entry and application development leverages a wide network of users with various expertise to adapt the resource at a pace beyond what a single organization could achieve. We have introduced a new entity to CIViC that aggregates individual evidence items into a single clinical assertion, allowing guideline incorporation at varying levels of specificity. For example, classification of germline variants by ACMG guidelines assigns individual evidence to specific codes (e.g., PVS1, PP1) that are aggregated to classify a variant as benign or pathogenic. The CIViC assertion provides the variant classification and supporting evidence that can be readily re-evaluated by any user, not just the initial submitter, and reviews and tracks any changes. An assertion can be created for highly specific variants (e.g., EGFR T790M), which can be submitted to ClinVar or more generic variants (ALK fusions) unsupported by many resources. Assertions support searchable, structured, and versioned annotations including FDA companion tests and drug approvals, germline ACMG categories, somatic AMP/ASCO variant levels, NCCN and WHO guidelines. The CIViC knowledgebase provides rapid integration of the latest guidelines and regulatory standards in a freely accessible resource that is flexible enough to evolve with this rapidly changing field.

#3290

The Side-Out Foundation Metastatic Breast Cancer Database, an open-access portal for multi-omic molecular data and more.

Elisa Baldelli,1 Shiva Ghaemi,1 Sanja Avramovic,1 Janusz Wojtusiak,1 Lance A. Liotta,1 Farrokh Alemi,1 Emanuel F. Petricoin,1 Bryant Dunetz,2 Mariaelena Pierobon1. 1 _GEORGE MASON UNIVERSITY, Manassas, VA;_ 2 _The Side-Out Foundation, Fairfax, VA_.

Introduction: With the high volume of molecular data being generated daily from human malignancies, there is an increasing need to create novel internet-based portals where this information is readily and easily accessible to physicians, scientists, and the general public. Although numerous databases have been created to capture the molecular characteristics of primary tumors, limited resources are available when it comes to the metastatic lesions. We have developed a novel open-access database to capture demographic, clinical, and pathological information, outcome data, and multi-omic based molecular profiles from metastatic breast cancer (MBC) patients.

Materials and Methods: The portal was created using the open-source relational database management system MySQL and the custom-codes were written using the PHP server-side scripting language. User interface, management, and authentication were created in WordPress. The database is primarily used to record information collected through the Side-Out clinical trials, a series of prospective Phase II trials targeting refractory MBC (NCT01074814, NCT01919749, NCT03195192). De-identified patients' information includes patients' demographics, treatment history, pathological characteristics of the primary tumor, outcome data, and multi-omic molecular profiles. Molecular information collected from each lesion include genomic (NGS-based whole/targeted exome sequencing), transcriptomic (RNA microarray/RNA Seq), proteomic (protein expression by IHC), and phosphoproteomic (protein pathway activation mapping by Reverse Phase Protein Microarray) data. Over 700 data fields are collected for each patient. A higher level of security for the recorded information is achieved by using a secondary database along with custom-codes during the data entry process. Investigators are exploring how Block-chain database design can be used to make portion of the data public while encrypting patient identifiers and key variables.

Results: We present an overview of the portal, its usability, how access can be requested by interested third party, the user-friendly interface for downloading clinical, pathological and molecular data, and a few examples of how these data can be used to explore different aspects of metastatic breast cancers.

Conclusions: To our knowledge, this is the first web-based publicly accessible database portal where broad multi-omic profiles are captured from the MBC patients along with demographic, clinical and pathological data. This open-access portal represents a unique and highly valuable tool as it integrates different aspects of the disease and can be used for correlative analyses and hypothesis-generating studies. Finally, this web-based portal allows free-of-charge dissemination of data from existing or upcoming clinical and translational studies targeting breast cancer patients.

#3291

The human cancer DNA methylation marker atlas.

Alexander Koch,1 Gerrit A. Meijer,2 James G. Herman,3 Wim Van Criekinge,4 Manon van Engeland1. 1 _Maastricht University, Maastricht, Netherlands;_ 2 _The Netherlands Cancer Institute, Amsterdam, Netherlands;_ 3 _University of Pittsburgh, Pittsburgh, PA;_ 4 _Ghent University, Ghent, Belgium_.

For decades, scientists have been searching for cancer biomarkers to improve the diagnosis and treatment of cancer. This resource-intensive and time-consuming endeavor has resulted in thousands of biomarker publications, but translation of these markers into clinical practice hardly takes place. Estimations by Poste (2011) and Kern (2012) put the number of published markers that are used in the clinic below one percent. This failure to translate biomedical findings into clinical applications has been termed the translational research valley of death (Butler, 2008).

If we are to cross this biomedical valley of death for DNA methylation markers, we have to increase the scientific quality and reproducibility of biomarker research, reduce the number of markers lost in translation, and accelerate the development of clinically useful markers. This will require a large-scale, coordinated effort from all stakeholders: scientists, funding organizations, scientific journals, private partners, and patients.

We have devised a strategy to tackle these issues and improve the reliability, efficiency and translation of cancer DNA methylation markers, while at the same time promoting data sharing. At the heart of this strategy is the construction of a database of all published markers. This marker atlas would provide researchers with a valuable resource where they can find and evaluate existing markers or, in collaboration with private partners, have a marker experimentally validated. Using the collected data, we also plan to develop a reporting standard for DNA methylation markers. The ultimate goal of our efforts is to offer cancer patients more and better biomarkers. We have summarized our approach in an article submitted to Nature Reviews Clinical Oncology for review.

While our submitted article announces our intent to create the marker atlas, we would like to present the first version of the database to the research community at AACR, together with the insights we will have gained by then and an update on our progress with the reporting standard. In our initial analyses for example, we counted over 14,000 publications describing an estimated 2,422 cancer DNA methylation markers (compared to fourteen commercially available markers (0.6%), a clinical translation rate in line with the numbers published by Poste and Kern). Based on the data collected between the submission of this abstract and the AACR conference, we will be able to improve this estimate, giving us a more accurate view of the current status of the cancer DNA methylation biomarker field.

The AACR conference would be the perfect platform to introduce the marker atlas to the wider cancer research community and to grow community support. Not only would it provide us with expert feedback and increase the number of collaborators, community-wide support will be indispensable for the longevity of what we believe to be a valuable resource. More information on the atlas can be found online at cancermatlas.com.

#3292

Revisiting TCGA data identifies key genes in lung adenocarcinoma.

Jianxiang Shi,1 Mengtao Xing,2 Chenglin Luo,2 Xiao Wang,3 Liping Dai,3 Jianying Zhang3. 1 _Zhengzhou University, Zhengzhou, China;_ 2 _The University of Texas at El Paso, El Paso, TX;_ 3 _Henan Academy of Medical and Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, China_.

Although the incidence rate of lung cancer has declined over the past few years, it remains the leading cause of cancer death worldwide. The Cancer Genome Atlas has provided invaluable resources for cancer research. The development of next generation sequencing technology and evolution of data analysis tools make it possible to interpret the underlying mechanism of oncogenesis. In current study, normalized FPKM data from 572 samples derived from patients with lung adenocarcinoma (LUAD) from TCGA was used to do weighted gene co-expression network analysis (WGCNA) to identify gene modules associated with lung adenocarcinoma. Raw counts data was used to identify differentially expressed genes in LUAD by using DESeq2 in R (version 3.4.2). WGCNA results shows that magenta (r = 0.47, p <0.001)), black (r = 0.40, p <0.001)) and yellow (r = -0.88, p <0.001) modules are top 3 modules that associated with LUAD and cancer stage. Gene Ontology over-representation analysis shows that genes in magenta modules are involved with transcript elongation; genes in black modules are enriched in cell meiosis related biological processes, including chromosome segregation, nuclear division, DNA replication and mitotic nuclear division et al; genes in yellow module are enriched in angiogenesis, leukocyte migration and hemostasis. It is noteworthy that genes in magenta and black modules are upregulated and in yellow modules are downregulated. GOLM1 and MUC4 in magenta module play certain roles in cancer development. Several genes in MEGA family, including MAGEA3, MAGEA6, MEGEA16 and MEGEA1, and IGF2BP1 in black module have been reported to be overexpressed in various kinds of tumors. However, whether these in silicon discovered genes can be used to distinguish LUAD from controls still needs to be further evaluated.

Keywords: lung adenocarcinoma; TCGA; gene expression analysis; bioinformatics; cancer biomarker

#3293

Solid tumors have frequent mutation, copy number variation and differential mRNA expression of GPCRs: Are such GPCRs functional oncogenes.

Krishna Sriram, Kevin Moyung, Ross Corriden, Paul Insel. _UCSD, La Jolla, CA_.

G protein-coupled receptors (GPCRs), the largest family of membrane receptors, regulate numerous cellular functions and are drug targets for many disorders but not generally in cancer. We analyzed The Cancer Genome Atlas (TCGA) for GPCR mutations, copy number variation (CNV) and mRNA expression in 20 categories of solid tumors. We found that numerous GPCRs are frequently mutated, in particular, certain adhesion GPCRs (e.g.,GPR98 and GPR112), which appear to have a higher density of mutation events than other genes of similar length. Tumors with high mutational burden show a high frequency of such GPCR mutations that may result from DNA damage. CNV (especially amplifications and single-copy deletions) of GPCRs is widespread. In certain tumor types, particular GPCRs (e. g., GPR160) show high-level amplification in >30% of tumors. However, CNV does not appear to be a major factor in determining differential expression (DE) of GPCRs in tumors.

Compared to RNA expression in normal tissues (GTEX database), most solid tumor types (including those accounting for the most annual deaths) differentially express >50 GPCRs, including 77 GPCRs with increased expression in at least one tumor type that are targets for approved drugs, providing opportunities for drug repurposing. DE of GPCRs is largely independent of tumor stage/grade, largely insensitive to specific driver mutations, and similar in primary and metastatic tumors, which both have high expression of numerous GPCRs in nearly all members within tumor populations. Cancer cells highly express most GPCRs overexpressed in tumors, making such GPCRs (especially those that are druggable) attractive candidates for further validation and functional studies. Multiple published studies provide functional data for certain such GPCRs.

These findings imply a previously unappreciated role for GPCRs in solid tumors. Overexpressed GPCRs in tumors may regulate tumor biology as "functional oncogenes" and are potentially novel biomarkers and therapeutic targets, including for approved drugs.

#3294

Functional annotation for alternative splicing to investigate disrupted splicing in cancer.

Daejin Hyung, Jihyun Kim, Soo Young Cho, Charny Park. _National Cancer Center, Goyaungsi Gyeonggi-do, Republic of Korea_.

Alternative splicing (AS) plays a key role to confer human genome complexity. The advent of mRNA high-throughput sequencing facilitates to identify precise differential alternative splicing analysis. Massive result estimated from RNA-Seq should be sorted out whether AS has functional impact or not. Previous studies investigated protein domain or nonsense-mediated decay of AS event region. However other functional sites on exon inclusion region also exist. For example, miRNA binding site, and post-translational modification of AS site already are reported to assign functional impact. Therefore we developed ASpedia (http://combio.snu.ac.kr/aspedia/) to annotation functional impact of AS event. AS events of our database were generated from human genome hg19 ENSEMBL and Refseq. ASpedia provides a database, explore system, and AS browser to annotate functional impact evidence encompassing DNA, RNA, and protein. The user could query multiple AS events from differential AS analysis result. Currently our system supports a jar execution file to convert input query file from MISO or rMATS result.

The variant of splicing factor (SF) disrupts normal splicing. It could be assumed that target genes of mutated SF could be transcribed to aberrant isoforms by changed splicing machinery. Therefore we investigated the comprehensive SF variant status from TCGA somatic mutation and copy number. SF3B1 (2% in BRCA) and U2AF1 (4% in AML, and 2% in LUAD) were shown to recurrent mutation including hotspot. ELAVL2 known dispensable splicing factor to brain tissue were highly recurrent in deletion (18% in GBM). In order to examine aberrant AS events caused SF variants, we estimated exon-inclusion proportion Percent-Spliced-In (PSI) values for identifiable AS events using TCGA RNA-Seq dataset. Using differentially AS event analysis, we identified AS events that were significantly differentially spliced in the presence of a U2AF S34F/Y mutation (119 in AML). And we purposed to finding In order to investigate functional site, the differentially spliced AS events were additionally annotated using ASpedia. Finally we identified the landscape of that AS events were altered by SF variants, and we proved that a part of the events was involved in cancer-associated functions.

#3295

CAGI: The Critical Assessment of Genome Interpretation, a community experiment to evaluate phenotype prediction: implications for predicting impact of variants in cancer.

Gaia Andreoletti,1 Roger A. Hoskins,1 Susanna Repo,1 Daniel Barsky,1 Steven E. Brenner,1 John Mult,2 Cagi participants3. 1 _University of California, Berkeley, Berkeley, CA;_ 2 _University of Maryland, Rockville, MD;_ 3 _Multiple locations, CA_.

Interpretation of genetic variants plays an essential role in cancer and other diseases. Needs for variant interpretation span from basic research to informing profound clinical decisions. The Critical Assessment of Genome Interpretation (CAGI, \'kā-jē\\) is a community experiment to objectively assess computational methods for predicting the phenotypic impacts of genomic variation. CAGI participants are provided genetic variants and make predictions of resulting phenotype. These predictions are evaluated against experimental or clinical characterizations by independent assessors. CAGI establishes objective reference standards on how well current computational methods do—and do not—meet clinical and research requirements. As such CAGI enables appropriate use of methods and promotes the development of improved approaches. There have been notable discoveries from each of the four CAGI experiments to date, and general themes have emerged. Some examples illustrating the themes: a challenge, in which participants had to blindly predict how missense variants in p16 affect proliferation in human cell lines, showed that individual variants prediction for even the top performing methods were not consistently accurate. The RAD50 challenge in which participants were asked to classify RAD50 variants in breast cancer cases and controls, along with other challenges, showed that missense methods tend to correlate better with each other than with experiment (for reasons that may reflect biases in the predictive methods but also in the experimental assays). Bespoke approaches often enhance performance, as seen for example in also the RAD50 challenge, where the use of the knowledge of which domains are involved in DNA repair resulted in more accurate performance. Results from a challenge in which participants had to predict whether p53 core domain mutations recue activity of inactive p53 showed that protein three-dimensional structure-based missense methods do well in a few cases, while sequence-based methods have more consistent performance on most challenges. A challenge in which predictors predicted the response of 54 breast cancer cell lines to a panel of cancer drugs showed that training the model with external data enabled to find the correct ballpark of drug sensitivity. Interpretation of non-coding variants shows promise but is not at the level of missense; one challenge showing this had predict the probability that variants in BRCA1 and BRCA2 collected by Free the Data are pathogenic. Two groups correctly predicted all the deletion and insertion mutations, but they were not able to correctly predict classification for all the intron mutations. CAGI findings suggest that running multiple uncalibrated methods and considering their consensus may result in undue confidence, making this procedure inadvisable.

### Systems and Computational Biology

#3296

SMMART: Serial measurements of molecular and architectural responses to therapy.

Brett Johnson, Jamie Keck, Max Morris, Kiara Siex, Annette Kolodzie, Swapnil Parmar, Jessica Riesterer, Koei Chin, Summer Gibbs, Laura Heiser, Paul Spellman, Kyle Ellrott, Ozgun Babur, Emek Demir, Adam Margolin, Jeremy Goecks, Lisa Coussens, Raymond Bergan, Joe Gray. _OHSU, Portland, OR_.

SMMART is a precision medicine research program focused on understanding the evolution of actionable biology and mechanisms of resistance in human tumors during therapy. This is accomplished through in depth functional, 'omic and multiscale image analysis of longitudinal samples acquired during treatment. Here we present a case report detailing the insights that can be gained from the comparative analysis of pre- and post-treatment biopsy specimens in a late-stage metastatic breast cancer patient. To understand the molecular evolution of cancer, we interrogated genomics with targeted and whole exome sequencing, transcriptomics with RNA and gene-fusion sequencing, and proteomics with reverse phase protein arrays. To understand cellular organization and architectural changes, we employed multi-scale imaging tools, including scanning electron microscopy (SEM), cyclic immunofluorescence, immune cell profiling with cyclic immunohistochemistry, and traditional pathological assessment. During the course of treatment, we monitored patient response to therapy with clinical imaging, circulating tumor DNA sequencing and cancer protein assessment.

Individual assays revealed key aspects of how this individual's cancer evolved under therapeutic pressure. For example, mutational profiling revealed the patterns of clonal evolution and the acquisition of new genetic driver events. 2D and 3D SEM showed changes in ECM organization, macropinocytosis, mitochondrion size, number and density and number and organization of filopodia-like protrusions. We used a 30-color cyclic immunofluorescence analysis to identify differences in cancer cell proliferation and differentiation state, as well as the composition and organization of infiltrating immune cells. In addition, integrative analyses of multiple data types provided insight into the evolution of actionable biology within this patient's disease. This included changes in the suitability of the patient for immune checkpoint inhibitors as well as specific tyrosine kinase inhibitors. The comprehensive molecular and architectural characterization of an individual patient's cancer at multiple time points provides biologically novel and clinically relevant insight into the ways in which cancers become resistant to treatment.

#3297

A tyrosine kinase interactome reveals network states that guide the use of targeted therapies in cancer.

Swati Kaushik, Gwendolyn Jang, Hsien-Ming Hu, Khyati Shah, Xin Zhao, John Jascur, John Von Dollen, Erik Verschueren, Jeffrey Johnson, Nevan Krogan, Sourav Bandyopadhyay. _University of California, San Francisco, San Francisco, CA_.

Characterization of the genomic landscapes of cancer patients has provided valuable insights into the key oncogenic drivers and revolutionized the concept of precision treatment of patients. However, a key limitation is that targetable alterations are only found in a small fraction of patients. This is due to the fact that the majority of cancer drugs are developed against specific oncogenes. However, oncogenes do not act in isolation but rather function as a part of complex protein interactions that can alter oncogene activity and dependence. We hypothesize that a systems approach to read the cellular activity of oncogenic proteins by mapping interaction network states of cancer cells can aid in patient stratification for targeted therapy. To identify interaction networks centered on the major class of cancer drug targets, we experimentally mapped protein-protein interaction (PPI) networks of all 90 human tyrosine kinases (TK) using proteomics approach of affinity purification and mass spectrometry. This analysis identified 1,458 high confidence interactors of TK in HEK293 cells. Detailed analyses of this interactome revealed the diverse cellular localizations and novel associations of TK with multiple protein complexes, suggesting a broader functional role in cellular signaling than previously appreciated. To map the cellular activity of TK in cancer patients, we developed a novel computational approach to integrate PPI networks with genomic data from cancer patients profiled in TCGA. Application to lung adenocarcinoma samples identified that activity of EGFR interactors could be used to define an EGFR network state that was highly predictive of the presence of EGFR mutation. Intriguingly, our analysis identified that 23% of EGFR wild-type samples were positive for the EGFR network state, suggesting a role for EGFR in lung cancer beyond EGFR mutant cases. Furthermore, this state was highly predictive of erlotinib sensitivity in EGFR wild-type lung PDX and cell lines. We identified that many KRAS and NF1 mutant NSCLC samples were EGFR network state positive and displayed evidence of EGFR activation identified by RPPA. These results indicate that a network state approach can precisely expand the pool of patients that may benefit from EGFR TK inhibitors (TKi). Finally, we tested whether components of TK networks were critical for their function by performing synthetic lethal RNAi screens in cell lines with mutation in key TK and identified many kinase interactors as drug sensitizers. Our results indicate that integration of high-throughput genomic datasets with the PPI networks provides an effective tool to understand complex oncogenic network states of cancer cells and provides a high-resolution readout of tumor cell dependence. This work provides the most complete interaction map for TK to date and is a valuable resource to probe mechanisms of oncogene addiction to improve the utility of TKi in cancer.

#3298

High-content phenotyping of somatic cancer mutations by functional variomics.

Nidhi Sahni, Patrick Kwok-Shing Ng, Kang Jin Jeong, Gordon B. Mills. _UT MD Anderson Cancer Ctr., Houston, TX_.

Cancer genomes are highly complex with numerous somatic mutations identified across patient populations. Previous studies on genomic mutations have shed light on new means for cancer therapeutic interventions. With rapid advances in next-generation sequencing, accumulating genotypic information in the absence of efficient and systematic functional analyses of genomic aberrations will create a bottleneck in understanding genotype-phenotype relationships in cancer. To address these challenges, here we report a systems-level functional variomics approach integrating high-throughput phenotyping with robust computational analyses to investigate mutation-specific effects. This systematic functional platform consists of massively parallel mutagenesis, sensitive survival assays using growth factor-dependent cell models, and functional network perturbation profiling of mutations on signaling effects. We profile several thousands of genomic aberrations, including point mutations, gene fusions and indels, and significantly expand the repertoire of characterized actionable mutations. This study represents a valuable resource and provides insights in prioritizing cancer-causing mutations, and uncovering patient-specific disease mechanisms at a high resolution, a critical step towards personalized precision medicine.

#3299

High-throughput combinatorial CRISPR-Cas9 gene knockout reveals most genetic interactions are context dependent.

John Paul Shen, Dongxin Zhao, Brenton Munson, Amanda Birmingham, Roman Sasik, Ana Bojorquez-Gomez, Katherine Licon, Kristin Klepper, Alex Beckett, Kyle Salinas Sanchez, Prashant Mali, Trey Ideker. _UCSD, San Diego, CA_.

Genetic interactions, in particular negative or "synthetic-lethal" interactions for which simultaneous disruption of two genes causes cell killing, have implications for therapeutic development as has been demonstrated by the clinical success of PARP inhibitors specifically for tumors with loss-of-function mutations in BRCA1/2. However, further applications of synthetic-lethal cancer therapy have been limited by poor understanding of the important genetic interactions in a cancer cell, and how these vary from one cancer type to another or from patient to patient. To enable systematic mapping of these genetic interaction networks, we recently developed a CRISPR-Cas9 screening methodology for knocking out single and pairs of genes in high throughput. Critical to this method is the precise determination of single-gene knockout effects, which is accomplished by serial measurement of the relative changes in gRNAs at days 3, 14, 21 and 28 post-transduction. To robustly quantify gene fitness and genetic interactions we developed a novel computational analysis framework that integrates all samples across the multiple days of the experiment; with said method we achieve Pearson correlation of 0.95 or greater between biologic replicates in the same cell line (p < 1x10-30). Additionally we demonstrate that our analysis method is robust to the compositional effects inherent in a pooled knockout experiment. To facilitate reproducibility of analyses and distribution to the scientific community, the code has been packaged into a modular series of python notebooks freely available on github. Evaluating all pairwise gene knockout combinations among a panel of 73 genes divided between tumor-suppressor genes (TSG) and cancer-relevant drug targets (DT) in a total of 5 cancer cell lines from diverse lineages (HeLa, A549, 293T, U2OS, LN229), we identified 226 synthetic lethal and 14 epistatic interactions at a Z-score cut-off of -3 (FDR ~0.3). Of the synthetic lethal interactions 203 (89.8%) were private to a single cell line, and no interaction was seen in more than 3 of 5 five cell lines. Thus far 10 (out of 16 tested) therapeutically relevant interactions have been replicated in low-throughput assays using either combinatorial drugs or CRISPR knockout of a TSG paired with a drug (71% precision or positive predictive value). The cell line specificity of interactions was also confirmed in low-throughput assays (75% negative predictive value). In summary, we have discovered many therapeutically relevant genetic interactions in cancer and identified the great importance of cellular context on the architecture of the genetic interaction network. Recognizing that there will be great diversity in genetic interaction between different tumors, it will be important to perform future studies across a large number of samples, which is enabled by the high-throughput method we have developed.

#3300

Network analysis of the human protein-protein interactome: Tumorigenic signaling mechanisms.

Diana Murray, Kamrun N. Begum, Andrea Califano, Barry Honig. _Columbia University Medical Center, New York, NY_.

The dysregulated signaling responsible for tumor cell state implementation and maintenance has been shown to be mediated by the concerted action of master regulator (MR) proteins. Highly connected MR protein modules serve as "tumor checkpoints" by translating upstream genetic alterations into aberrant protein activity that drives pathophysiological cellular phenotypes. Direct pathways for information flow among MR proteins, and between individual MR proteins and their upstream modulators and downstream effectors, are largely unknown. The Prediction of Protein-Protein Interactions (PrePPI) database contains 1.4M predictions for ~85% of the human proteome; of these, ~300K are predicted to be physical, i.e. direct one-to-one, interactions.We apply network analysis concepts, as implemented in the R package igraph, to the PrePPI interactome to predict structural mechanisms underlying tumorigenic signal transduction. Shortest path and random walk with restart algorithms elucidate 1) physical interactions between MR proteins, 2) physical interactions that connect MR proteins that do not directly bind to each other, 3) upstream signaling proteins with recurrent and patient-specific mutations that physically interact with MR proteins, and 4) downstream cofactors and transcription factors that may be involved in effecting a dysregulated phenotype.Our analysis predicts that physical interactions fully connect BACH2, BCL6, IRF8, and SPIB, established germinal center MR proteins. 1) BACH2 and BCL6 are predicted to interact directly through their BTB/POZ domains; BTB domains are known to mediate heterodimeric protein-protein interactions. 2) Additional proteins, including IBTK, ANFY1, PAX7, ZEB1, GABPB1/B2, connect SPIB to BACH2 and to BCL6, in some cases through ankyrin repeats, which are structural protein-protein interaction motifs. Several of the PrePPI-predicted proteins, e.g. IBTK, ZEB1, GABP, have previously been implicated in B-cell biology. The possible implications of our analyses for a wide range of MR proteins modules will be presented.The computational prediction of physical protein-protein interactions within the regulatory architecture of a tumor cell is a powerful approach to discovering novel tumorigenic signaling mechanisms, formulating specific experimentally testable hypotheses of function, including compound mechanisms of action, and prioritizing novel drug targets.

#3301

Using a novel single-cell lineage-tracing technique to uncover the mechanisms driving nongenetic cancer relapse.

Yaara Oren,1 Aviv Regev,2 Joan Brugge3. 1 _The Broad Institute and Harvard Medical School, Boston, MA;_ 2 _The Broad Institute, Cambridge, MA;_ 3 _Harvard Medical School, Boston, MA_.

Despite favorable initial response to platinum chemotherapy, the majority of patients with ovarian cancer will develop recurrent disease and succumb to it within 5 years of diagnosis. Initial recurrence is frequently platinum-sensitive and women can respond to multiple lines of platinum-based chemotherapy before eventually developing platinum-refractory disease. The observation that the tumor cells that survive the initial therapy give rise to a chemosensitive progeny is inconsistent with a simple Darwinian model of selection of genetically resistant clones. Tumor dormancy, stochastic cell state shifts and stem cell-like populations are among the mechanisms hypothesized to mediate re-emergence of platinum-sensitive ovarian cancer. However, given the lack of high-throughput methods to simultaneously track cell state and lineage, it is not currently feasible to distinguish the relative contribution of each of these factors. To address this need, we generated a new lentiviral barcode library, termed Watermelon. Unlike existing barcoding libraries that enable the tracking of multiple cancer cells solely at the DNA level, the Watermelon library encodes transcribed barcodes, which allows one to map the lineage to the transcriptional profile of each individual cell. This unique barcoding scheme, which is coupled with a genetic H2B retention system, enables simultaneous tracing of lineage as well as the transcriptional and proliferative states of each cell in the population during drug treatment. We have established and characterized an in vitro platinum-sensitive relapse model and are applying the Watermelon system together with time-lapse imaging to study the mechanisms underlying time-to-relapse variation and cell-fate decisions. By broadening our knowledge of the cellular and molecular pathways that affect noninherited drug resistance, our work will facilitate the future development of novel therapies that delay or even prevent the emergence of platinum-refractory ovarian cancer.

#3302

The molecular landscape of oncogenic signaling pathways in The Cancer Genome Atlas.

Francisco Sanchez-Vega,1 Marco Mina,2 Joshua Armenia,1 Walid K. Chatila,1 Augustin Luna,3 Konnor La,1 Sofia Dimitriadoy,4 David L. Liu,5 Havish S. Kantheti,6 Zachary Heins,1 Angelica Ochoa,1 Benjamin Gross,1 Jianjiong Gao,1 Hongxin Zhang,1 Ritika Kundra,1 Cyriac Kandoth,1 Istemi Bahceci,7 Leonard Dervishi,7 Ugur Dogrusoz,7 Wanding Zhou,8 Hui Shen,8 Peter W. Laird,8 Alice H. Berger,9 Trever G. Bivona,10 Alexander J. Lazar,11 Gary Hammer,12 Thomas Giordano,12 Lawrence Kwong,11 Grant McArthur,13 Chenfei Huang,14 Mitchell J. Frederick,14 Frank McCormick,10 Matthew Meyerson,3 The Cancer Genome Atlas Research Network, Eliezer Van Allen,5 Andrew D. Cherniack,5 Giovanni Ciriello,2 Chris Sander,3 Nikolaus Schultz1. 1 _Memorial Sloan Kettering, New York, NY;_ 2 _University of Lausanne, Lausanne, Switzerland;_ 3 _Dana-Farber Cancer Institute, Boston, MA;_ 4 _Princeton University, Princeton, NJ;_ 5 _Broad Institute of Harvard and MIT, Cambridge, MA;_ 6 _University of Texas at Dallas, Dallas, TX;_ 7 _Bilkent University, Ankara, Turkey;_ 8 _Van Andel Research Institute, Grand Rapids, MI;_ 9 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 10 _University of California San Francisco, CA;_ 11 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 12 _University of Michigan, Ann Arbor, MI;_ 13 _Peter MacCallum Cancer Centre, Melbourne, Australia;_ 14 _Baylor College of Medicine, Houston, TX_.

Over the past decade, The Cancer Genome Atlas (TCGA) has profiled more than 11,000 tumors spanning 33 distinct cancer types. The TCGA PanCanAtlas is a collaborative project by the TCGA Research Network that aims to address relevant overarching questions in oncology based on a cross-cancer analysis of the full, uniformly reprocessed TCGA data set. Here, we present results from our analysis of genetic alterations in mitogenic signaling pathways across cancer.

Genetic alterations in signaling pathways that control cell cycle progression, apoptosis, and cell growth are common hallmarks of cancer, but the extent, mechanisms, and co-occurrence of alterations in these pathways differ between individual tumors and tumor types. Using mutations and copy-number changes in 9,125 tumor samples profiled by TCGA, we analyzed the mechanisms and patterns of alterations in 10 canonical pathways: cell cycle, Hippo, Myc, Notch, beta-catenin / WNT, PI-3-Kinase / Akt, receptor-tyrosine kinase / RAS / MAP-kinase signaling, TP53, and TGF-beta signaling, as well as oxidative stress response. For each of these pathways, we propose an expert-curated description (or "template") that includes the relevant (altered) genes and the connections between them, as well as a detailed catalogue of the driver mutations and copy number changes with known oncogenic relevance. We provide a high-level map of pathway alteration frequencies across tissues and relevant cancer subtypes as well as detailed frequencies of alteration at the gene level for each individual pathway. We also investigate relationships of co-occurrence and mutual exclusivity across pathways and evaluate therapeutic implications, including drug combinations. Forty-nine percent of tumors had at least one potentially targetable alteration in the evaluated pathways, and 31% of tumors had multiple targetable alterations, making them candidates for combination therapy.

Our work delineates the full landscape of oncogenic alterations in mitogenic signaling pathways across cancer, and the pathway templates as well as the richly annotated data set that we provide will constitute an invaluable public resource for future use by the cancer genomics and precision oncology communities.

#3303

A comprehensive TCGA Pan-Cancer molecular study of gynecologic and breast cancers.

Ashton Berger,1 Anil Korkut,2 Rupa S. Kanchi,2 TCGA Pan-Gynecologic Group and Research Network, Gordon B. Mills,2 Douglas A. Levine,3 Rehan Akbani2. 1 _Broad Institute, Boston, MA;_ 2 _UT MD Anderson Cancer Ctr., Houston, TX;_ 3 _New York University, New York, NY_.

The Pan-Gynecologic (Pan-Gyn) molecular study analyzed data from 2,579 tumors representing five TCGA tumor-type projects: high-grade serous ovarian cystadenocarcinoma (OV), invasive breast carcinoma (BRCA), uterine corpus endometrial carcinoma (UCEC), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), and uterine carcinosarcoma (UCS). Principal aims were to identify shared and unique molecular features, clinically meaningful subtypes, and potential future therapeutic targets. We analyzed data of multiple types, including somatic copy number alterations (SCNAs), mutations, DNA methylation, mRNA expression (from mRNA-seq), microRNA (miRNA) expression (from miRNA-seq), long non-coding RNA (lncRNA) expression (from miRNA-seq), and protein expression (from reverse-phase protein arrays; RPPA).

We found multiple genomic and epigenomic features that were shared across the Pan-Gyn tumor types and distinguished them from other cancer types. Sixty-one somatic copy number alterations (SCNAs) and 46 significantly mutated genes (SMGs) were found in the Pan-Gyn cohort; 11 of the SCNAs and 11 of the SMGs had not been reported in previous TCGA single-disease papers on the same tumor types. Ten predominant mutation signatures were found that correlated with COSMIC signatures, whereas 10% of the samples had no SMGs. Most of the Pan-Gyn tumor types shared similar miRNA profiles. The exception, ovarian cancer, was extremely different from the rest, and, unexpectedly, the miRNA profiles of basal-like breast cancers closely resembled those of cervical cancers. Protein expression analysis (RPPA) revealed some OV and UCEC samples with the "reactive" signature previously identified in BRCA and shown to be prognostically relevant. It also revealed a subtype with low ER and AR expression levels (important markers for hormone therapy) that spanned all 5 tumor types. Novel lncRNA analysis showed several functionally significant ER-regulated lncRNAs and gene/lncRNA interaction networks. Pathway analysis identified subsets of tumor samples that had high levels of leukocyte infiltration, with potential implications for immunotherapy. Using 16 key molecular features across the Pan-Gyn cancers, we identified five novel cross-tumor and prognostic subtypes with possible clinical implications. Finally, we developed a decision tree that can classify patients into the five subtypes based on just 6 of the 16 molecular features. Those 6 features are potentially assessable in clinical laboratories.

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

### Epigenetic Changes as Molecular Markers of Cancer

#3304

The mutation landscape of cancers serves as a record of early malignant transformation.

Paz Polak,1 Rosa Karlic,2 Kirsten Kubler,3 William D. Foulkes,4 Gad Getz3. 1 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 2 _University of Zagreb, Zagreb, Croatia;_ 3 _Broad Institute, Cambridge, MA;_ 4 _McGill University, Montreal, Quebec, Canada_.

How the cell lineage influences a tissue's susceptibility to malignant transformation is a fundamental question in cancer biology, which has been barely addressed in cancer genomics thus far. Cell properties are encoded in the cell type-specific chromatin structure and we previously demonstrated that the cell-of-origin (COO) chromatin organization is a key determinant of the landscape of somatic mutations, which accumulated over lifetime serving as a memory of the historical cell lineage (Polak et al, Nature , 2015). We now show that this principle is generalizable to common tumor types and offers insights into the molecular events of cancer initiation. We extended our work to 2,641 genomes from 30 cancer types and epigenetic modifications from 98 normal tissues. In 25 cancer types, the tumor originated from a cell type that was its direct cellular counterpart or a close proxy; in only two, there was no match or a close proxy; and in the remaining three (esophageal, pancreatic ductal and biliary adenocarcinoma) the best matched cell type suggested metaplasia to stomack mucosa like tissue.The cellular context of breast tumor formation was investigated in more detail, showing that the COO, and not the gene inactivation event, determines the subtype. Basal-like tumors appeared to arise from luminal progenitor cells, while all other subtypes arose from mature luminal cells. Furthermore, irrespective of the inactivation mechanism (pathogenic germline, somatic truncating or epigenetic silencing event), all BRCA1/2- and RAD51C-altered basal-like tumors best matched to luminal progenitors while BRCA1/2- and CHEK2 -mutated luminal A/B subtypes best matched mature luminal cells. Finally, we observed that tumor type-specific driver genes reside in genomic regions that are defined by a highly active chromatin environment in their COOs. This highlights their essential role in cell type differentiation and implies the acquisition of somatic mutations early, when the chromatin architecture still reflected the COO. Taken together, our findings shed light on the crucial role of the COO in shaping the mutational landscape and tumor evolution.

#3305

Cell-of-origin differentiation stages define methylation-based subtypes of human colorectal adenomas and carcinomas.

Felix Bormann,1 Manuel Rodríguez-Paredes,1 Felix Lasitschka,2 Dominic Edelmann,1 Axel Benner,1 Yehudit Bergman,3 Claudia R. Ball,4 Hanno Glimm,4 Heinz G. Linhart,5 Frank Lyko1. 1 _German Cancer Research Center, Heidelberg, Germany;_ 2 _Institute of Pathology, Heidelberg University, Heidelberg, Germany;_ 3 _Cancer Research, Institute for Medical Research Israel-Canada, Jerusalem, Israel;_ 4 _National Center for Tumor Diseases, Heidelberg, Germany;_ 5 _University Hospital Freiburg, Freiburg, Germany_.

Introduction: Colorectal adenomas are precursor lesions of colorectal cancers and represent clonal amplifications of single cells from colonic crypts. DNA methylation patterns specify cell-type identity during cellular differentiation and therefore provide novel opportunities for tumor subclassification and patient stratification.

Methods: Infinium 450k and EPIC data from of non-malignant colorectal adenomas was applied to a consensus clustering algorithm. This defined epigenetic subtypes that were subsequently tested for clinico-pathological features, such as morphology and mutation status. Additionally, the subtypes were compared with Infinium 450k data from colonic crypt sections and also colorectal cancer samples provided by The Cancer Genome Atlas. Finally, clinical significance was addressed by testing for subtype specific overall survival rates.

Results: The analysis of the adenoma dataset defined 3 distinct epigenetic subtypes of human premalignant lesions. These 3 subtypes also reflect 3 of 4 epigenetic subtypes identified in colorectal cancer. The fourth, cancer-specific subtype represents the previously known subclass defined by microsatellite instability phenotype and the CpG island methylator phenotype. Comparisons to Crypt section methylomes and to other reference methylomes revealed that the other 3 subtypes represent epigenetic programs reflecting various intestinal crypt cell differentiation stages. Patient survival correlated with the differentiation stages with a particular poor prognosis for patients with stem cell-related epigenetic signatures.

Conclusion: Our results establish a novel and clinically relevant approach for colorectal adenoma and cancer classification and illustrate how differences in the cell-type of origin shape the tumor methylome.

#3306

The clinical importance of DNA methylation signatures in chronic lymphocytic leukemia patients treated with chemo-immunotherapy.

Tomasz K. Wojdacz,1 Harindra E. Amarasinghe,2 Matthew JJ Rose-Zerilli,2 Alice Beattie,2 Jade Forster,2 Latha Kadalayil,2 Stuart Blakemore,2 Helen Parker,2 Marta Larrayoz,2 Ruth Clifford,3 Zadie Davis,4 Monica Else,5 Dena Cohen,6 Andrew J. Steele,2 Richard Rosenquist,7 Andrew Collins,2 Andrew Pettitt,8 Peter Hillmen,8 Christoph Plass,9 Anna Schuh,3 Daniel Catovsky,5 David G. Oscier,4 Christopher C. Oakes,10 Jonathan C. Strefford2. 1 _Aarhus Univeristy, Aarhus, Denmark;_ 2 _University of Southampton, Southampton, United Kingdom;_ 3 _University of Oxford, Oxford, United Kingdom;_ 4 _Royal Bournemouth Hospital, Bournemouth, United Kingdom;_ 5 _The Institute of Cancer Research, London, United Kingdom;_ 6 _University of Leeds, Leeds, United Kingdom;_ 7 _Uppsala University, Uppsala, Sweden;_ 8 _University of Liverpool, Liverpool, United Kingdom;_ 9 _The German Cancer Research Center, Heidelberg, Germany;_ 10 _The Ohio State University, Columbus, OH_.

Variations in the CLL DNA methylome reflect modifications that occur during normal B cell maturation, along with IGHV mutated (M-CLL) and unmutated CLL (U-CLL), retaining an imprint of the DNA methylation signature of memory (m-CLL) and naive B cells (n-CLL), respectively, with a third intermediate epigenetic subgroup (i-CLL) (1-3). To further test the clinical utility of DNA methylation signatures, we performed the first analysis of patients entering clinical trials; we tested treatment-naive CLL patients [n=605] randomized to CLL4 (chemotherapy, CT) (4), ARCTIC and ADMIRE (both chemo-immunotherapy, CIT) (5, 6). We identified n-, i- and m-CLL in 49.3% (n=299), 32.0% (n=195) and 18.5% (n=112) of our patients, respectively. Fewer m-CLL patients were identified in our study compared to published data reflecting the progressive nature of our cohort, with 80% (n=245/305, P<0.001) of U-CLL cases exhibiting the n-CLL signature (i-CLL: 17% and m-CLL: 3%). For M-CLL cases, 9%, 50% and 41% exhibited the n-, i- and m-CLL epigenetic signature, respectively. 68% (80/117, p<0.001) of cases with del(11q), 77% (41/53, p<0.001) with trisomy 12, and 68% (38/56, p=0.03) with TP53 lesions were n-CLL. Cases with NOTCH1 (p=0.01) and SF3B1 (p=0.02) mutations were also enriched in n-CLL. Next, we investigated the impact of methylation signatures on progression-free (PFS) and overall survival (OS). In CT patients, n-, i- and m-CLL patients exhibited a median PFS of 23, 34 and 35 months, and OS of 63, 66 and 106 months, respectively. n-CLL showed significantly shorter PFS than i-CLL (HR 0.64, p<0.001) and m-CLL (HR 0.52, p<0.001), and had the shortest OS, again compared to i-CLL (HR 0.73, p=0.01) and m-CLL (HR 0.33, p<0.001). Ten-year OS differed according to epigenetic signature (P<0.001) and was reached by only 14% of n-CLL patients. Multivariate Cox proportional analysis, controlling for confounding variables (incl. clinical features, IGHV status, TP53, NOTCH1 and SF3B1) in 278 patients, showed that m-CLL was an independent prognostic factor for OS (HR 0.46, p<0.01). In 247 CIT patients, univariate analysis showed that the i- (HR:0.57, p=0.05) and m-CLL (HR:0.3, p=0.002) subgroups displayed longer PFS. In a multivariate model, including TP53 lesions and IGHV status (239 patients), the m-CLL subgroup retained independent prognostic significance (HR:0.25, p<0.001). In conclusion, we provide important evidence that DNA methylation analysis may aid in the identification of patients destined to demonstrate durable remissions when treated with these agents. References: 1. Kulis, M. et al. Nat Genet 44, 1236-1242 (2012). 2. Queiros, A.C. et al. Leukemia 29, 598-605 (2015). 3. Oakes CC, et al. Nat Genet. 2016 Mar;48(3):253-64. 4. Catovsky, D. et al. Lancet 370, 230-239 (2007). 5. Howard, D.R. et al. Leukemia Nov;31(11):2416-2425 (2017). 6. Munir, T. et al. Leukemia Oct;31(10):2085-2093 (2017).

#3307

BRCA1 promoter methylation analyzed in 1032 primary breast cancers predicts favorable response to chemotherapy independently of p53 mutations.

Olafur A. Stefansson,1 Holmfridur Hilmarsdottir,1 Kristrun Olafsdottir,2 Laufey Tryggvadottir,3 Asgerdur Sverrisdottir,2 Jon G. Jonasson,2 Jorunn E. Eyfjord,1 Stefan Sigurdsson1. 1 _University of Iceland, Reykjavik, Iceland;_ 2 _Landspitali University Hospital, Reykjavik, Iceland;_ 3 _Icelandi Cancer Registry, Reykjavik, Iceland_.

Individuals with germline mutations in the breast cancer susceptibility gene, BRCA1, are at increased risk of developing breast- and ovarian cancer. BRCA1 is involved in DNA double strand break repair (DSB) by homologous recombination (HR), an error-free DNA repair pathway that uses an intact sister chromatid for the repair. BRCA1 mutated tumors are therefore HR deficient and sensitive to drugs that stall DNA replication forks, resulting in DSBs. Tumor cells lacking the protein are characterized by genomic instability and a pattern of specific genome-wide mutation signature as a consequence of dependence on alternative error prone repair pathways. BRCA1 can also be inactivated epigenetically by CpG promoter hypermethylation. These BRCA1 epigenetically silenced tumors are more likely to be of the basal-like subtype and are strongly associated with this pattern of mutational signature characteristic for individuals with germline mutations in BRCA1. This suggests that the tumorigenesis in the sporadic cancer cases is similar to tumors where germline mutations are present, highlighting the importance of epigenetic silencing of BRCA1. The aim of the study was to examine the clinical significance of epigenetic silencing of BRCA1 in primary breast cancer, focusing on the response to chemotherapy. DNA methylation analysis of the BRCA1 promoter was performed by pyrosequencing tumor samples from 1032 patients with primary breast cancer of which 966 were sporadic, 61 were BRCA2 germline mutation carriers and 5 were BRCA1 mutation carriers. The p53 mutational status was also studied in the same primary tumors by DNA sequencing. We analysed the methylation status of BRCA1 in tumors derived from 1032 patients and found BRCA1 to be promoter hypermethylated in 29 sporadic tumors (29 out of 966, 3.0 %) whereas none of the tumors derived from BRCA mutation carriers were BRCA1 promoter hypermethylated. Patients with BRCA1 promoter hypermethylation receiving chemotherapeutic drug treatment show significantly better breast cancer specific survival (P=0.0192). The trend is opposite in patients not receiving chemotherapeutic treatment, were patients with BRCA1 hypermethylated tumors show worse survival (P=0.074). The impact of BRCA1 methylation with respect to survival in chemotherapy treated patients was found to be independent of p53 mutational status, ER status, tumorsize, lymph node status, year- and age at diagnosis (HR = 0.108; 95% CI = 0.015-0.794; P = 0.0288). BRCA1 promoter hypermethylation predicts improved disease outcome in anti-cancer drug treated patients. Therefore, BRCA1 promoter methylation status in primary breast cancer is an important factor to be considered when deciding treatment options.

#3308

Urine epigenetic biomarkers for NSCLC diagnosis.

Lane Lerner,1 Lily Zheng,1 Anastasia Kottorou,2 Chen Chen,3 Tomoaki Ito,4 Kristen Rodgers,4 Beverly Lee,4 Robert Winn,1 Enrico Benedetti,1 Tza- Huei Wang,5 Malcolm V. Brock,4 James G. Herman,6 Alicia Hulbert1. 1 _University of Illinois at Chicago, Chicago, IL;_ 2 _Medical School University, Patras, Greece;_ 3 _The Second Xiangya Hospital CSU, Xiangya, China;_ 4 _Johns Hopkins University School of Medicine, Baltimore, MD;_ 5 _Johns Hopkins Whiting School of Engineering, Baltimore, MD;_ 6 _University of Pittsburgh School of Medicine, Pittsburgh, PA_.

Purpose:

The National Lung Cancer Screening Trial showed mortality reduction on patients with NSCLC by the use of Low-dose CT screening. The study exhibits an unreasonably high false positive rate of 96.6%, which can to lead to significant morbidity and mortality from unnecessary tests. This study aim to determine if methylated promoter regions of a panel of genes that are correlated with Non-Small Cell Lung Cancer can be detected in urine from patients with lung cancer versus those without.

Materials and Methods:

We conducted a prospective case-control study recruiting subjects from the Lung Cancer Spore Trial. Urine and pre-operative CT scans were obtained from all patients. We processed the urine using the Methylation on Beads assay to isolate and bisulfite treat circulating DNA and then employed Quantitative Methylation Specific Real-Time PCR to detect promoter methylation status of the genes: CDO1, TAC1, HOXA7, HOXA9, SOX17 and ZFP42. Sensitivity, specificity, PPV and NPV values were calculated for each gene methylation status.

Results:

34 patients were studied, including 23 patients with NSCLC and 11 patients with benign non-cancerous lesions. The sensitivity, specificity, PPV and NPV values for lung cancer detection in urine are shown in Table 1. Sensitivities ranged 43-96%, Specificities 64-91%, PPV 71-100% and NPV 40-67%. Promoter methylation of the gene panel CDO1, TAC1, HOXA7, HOXA9, SOX17 and ZPF42 has a 78% sensitivity, 91% specificity, 95% PPV and 67% NPV for lung cancer detection.

Conclusion:

Our study suggests that urine can provide a highly sensitive and specific non-invasive route for lung cancer detection. These results are very promising and could potentially reduce unnecessary morbidity and mortality in people undergoing screening for NSCLC by use of a non-invasive method that could be more accessible on primary care centers. However, further studies and validation with larger sample sizes and different populations are necessary before its application to clinical practice.

Patients with positive gene methylation from urine samples and its accuracy for NSCLC detection.

---

|

Cancer (n=23) | Cancer (n=23) | Control (n=11) | Control (n=11) | |

|

n | Sensitivity | n | Specificity | PPV | NPV

CDO1 | 14 | 61 | 1 | 91 | 93 | 53

TAC1 | 15 | 65 | 0 | 100 | 100 | 58

SOX17 | 16 | 70 | 4 | 64 | 80 | 50

HOXA7 | 11 | 48 | 3 | 73 | 79 | 40

HOXA9 | 10 | 43 | 1 | 91 | 91 | 43

ZPF42 | 22 | 96 | 9 | 18 | 71 | 67

Panel of genes | 18 | 78 | 1 | 91 | 95 | 67

#3309

Circulating epigenetic biomarkers for the early detection of ovarian cancer.

Gerald C. Gooden,1 Christophe Legendre,2 Monique Spillman,3 Ajay Goel,4 Bodour Salhia1. 1 _Keck School of Medicine / USC, Los Angeles, CA;_ 2 _Translational Genomics Research Institute, Los Angeles, CA;_ 3 _Texas Oncology, Dallas, TX;_ 4 _Baylor Scott & White Research Institute and Sammons Cancer Center, Dallas, TX_.

Background Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Greater than 75% of cases are diagnosed with advanced stage, which is associated with 5-year survival rates of less than 20% compared with 92% if caught early. Previous attempts at discovering early detection biomarkers for EOC have largely failed because of reliance on sub-optimal discovery approaches and the lack of comprehensive patient cohorts. DNA methylation changes are among the earliest alterations in cancer and methylation represents a stable and robust modification on DNA. In addition, several groups including ours have reported the detection of tumor-associated methylation changes in cfDNA extracted from plasma. Accordingly, in this study, we performed comprehensive DNA methylation analysis by reduced representation bisulfite sequencing (RRBS) to identify DNA methylation biomarkers that can be detected in circulation and used for the early detection of ovarian cancer. Methods For the biomarker discovery phase, a cohort of 23 stage 1 serous EOC tissues, and 10 unmatched, adjacent normal tissues from fallopian tubes were analyzed by RRBS. We used Metilene, a binary segmentation algorithm combined with a two-dimensional statistical test, to detect differentially methylated regions (DMRs). For validation, we performed targeted bisulfite amplicon sequencing. Briefly, bisulfite primers were designed to regions of interest, optimized, multiplexed and sequenced to depths ranging from 500-25,000X on the Illumina MiSeq platform. The validation cohort consisted of cfDNA isolated from the plasma of 51 independent EOCs and 10 normal individuals as well as the genomic DNA from tissue used in the discovery cohort. Results We identified 10972 statistically significant (q-value <0.05) DMRs between stage 1 EOC and normal samples. Of these, 227 were hypermethylated and had an average differential methylation value of greater than 30% in tumors compared to normal. Unsupervised clustering analysis of these DMRs demonstrated clear separation of tumor from normal, with the possibility of three distinct molecular subtypes of serous EOC. Six DMRs were prioritized and further validated in both tissue and plasma. Targeted bisulfite amplicon sequencing performed revealed hypermethylation in greater than 50% of tumor samples across the DMRs. Additionally, the area under the curve (AUC) for the DMRs interrogated ranged from 0.91 and 0.94. Conclusion In conclusion, our RRBS based comprehensive biomarker discovery identified a six-DMR panel that demonstrated preliminary robust diagnostic accuracy in EOC samples. Further evaluation of these DMRs in multicenter retrospective and prospective cohorts might lead to the development of a non-invasive diagnostic, as well as a population-screening assay for OC patients.

#3310

Non-invasive diagnosis of colorectal cancer via targeted high-throughput DNA methylation sequencing of circulating tumor DNA (ctDNA).

Xianrui Wu,1 Jiacheng Chuan,2 Tuo Hu,1 Tingting Peng,2 Meng Yang,2 Hui Shen,3 Peter Laird,3 Yangbin Gao,2 Xuyu Cai,2 Weihong Xu,2 Jian-bing Fan,2 Ping Lan1. 1 _The Sixth Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China;_ 2 _AnchorDx Medical Co. Ltd., Guangzhou, China;_ 3 _Van Andel Institute, Grand Rapids, MI_.

Colorectal cancer (CRC) commonly develops from precancerous polyps. Most polyps are benign. The current screening method involves stool tests and colonoscopy, which is an unpleasant procedure. Even though stool DNA-based approach has been developed, it is still a clinical challenge to differentiate benign complications and malignant CRC.

We took the approach of high-throughput DNA bisulfide sequencing of tissue samples to identify cancer-specific methylation signatures. We learned methylation patterns by in-depth data mining and then applied pattern recognition approach to classify plasma samples. From a cohort of 70 pairs of tumor-normal matched tissue samples, we identified 1062 differential methylation markers. Next, we applied a greedy optimization algorithm to build a malignancy classification model using a training set of 118 plasma samples (malignant CRC: 56, benign complications: 23, healthy subjects: 39). In the training set, we achieved an AUC of 91.9% (95%CI: 87.3-96.5%). In an independent test set of 150 plasma samples (malignant CRC: 56, benign complications: 23, healthy subjects: 71), we achieved an AUC of 92.9% (95%CI: 88.3%~97.5%). Specifically, the model had a sensitivity of 91.1% for the identification of malignant CRC, and a specificity of 84.5% and 78.3% for healthy subjects and benign complications respectively. Our assay was demonstrated to be very sensitive towards early-stage CRC detection, with a sensitivity of 76.5% and 95% for stage I and II CRC respectively. The performance of this assay still awaits further validation in a large-scale clinical trial.

In summary, we have developed a highly sensitive blood-based non-invasive diagnostic assay for the identification of malignant CRC, which can aid clinical decisions for doctors. This approach can also be extended to non-invasive early screening for various other cancer types.

#3311

Loss of EZH2 at tumor invasive front is correlated with higher aggressiveness.

Julian Boehm,1 Julienne K. Muenzner,1 Aylin Caliskan,1 Benardina Ndreshkjana,1 Katharina Erlenbach-Wünsch,1 Susanne Merkel,1 Roland Croner,1 Tilman T. Rau,2 Carol I. Geppert,1 Arndt Hartmann,1 Adriana Vial-Roehe,3 Regine Schneider-Stock1. 1 _University Hospital, FAU Erlangen-Nürnberg, Erlangen, Germany;_ 2 _University Bern, Bern, Switzerland;_ 3 _Federal University of Health Sciences of Porto Alegre (UFCSPA), Erlangen, Brazil_.

Background: Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths, mainly due to its high metastatic rate, the acquisition of drug resistance and associated recurrences. Today, it is well known that not only genetic alterations but also epigenetic modifications play a major role in these complex processes. The enhancer of zeste homolog 2 (EZH2) represents the catalytic subunit of polycomb repressive complex 2 that preferentially methylates lysine 27 on histone 3 and is involved in epigenetic gene silencing. In addition to its function as a cell cycle regulator, EZH2 has also been associated with the epithelial-to-mesenchymal transition as well as angiogenic and metastatic processes. While an increase in EZH2 expression is well known for CRC and has been associated with both cancer stem-like cell properties and highly aggressive tumors, its prognostic value is still controversially discussed in literature.

Methods: Here we investigated the expression of EZH2 in a cohort of colon cancer patients, focusing on the role of EZH2 at the invasion front of CRC tumors. We established a semiautomated software workflow for a time-efficient and objective analysis of EZH2-stained patient samples. In addition, we analyzed HCT116 cells treated with the EZH2 inhibitor DZNep in vitro by crystal violet assay, Western blot and cell cycle analysis and in vivo by the chorioallantoic membrane (CAM) assay in comparison to untreated controls in order to experimentally simulate the clinical situation.

Results: While EZH2 expression in the tumor center did not have a prognostic value, EZH2 loss at the invasion front was significantly correlated with poor clinical outcome. Analysis by the trained semiautomated Definiens' Tissue Studio Software (TSS) and manual scoring showed a pronounced interobserver correlation when analyzing tumor centers of TMA punches or whole slides. We were able to mimic the observed EZH2 loss at the invasion front in human CAM xenografts. We found that DZNep-treated HCT116 cells strongly invade into the CAM and their tumors display a higher grade of vascularization, which demonstrates a more aggressive phenotype when EZH2 is lost.

Conclusion: From our clinical and experimental findings we propose a potential explanation for the highly contradictory literature data concerning EZH2 expression levels and prognosis in CRC. By establishing the TSS-based analysis for EZH2-stained specimens, we were able to generate a reliable tool to evaluate immunostainings of certain biomarkers to be applied in the daily routine.

#3312

Identification of tissue-of-origin in cancer of unknown primary site (CUPS) using methylation-specific targeted resequencing: A pilot study.

Jeong Mo Bae,1 Kwangsoo Kim,1 Hee Jun Chae,2 Xianyu Wen,3 Kang Yeol Kim,4 Hwang Kwan Gwon,4 Nam-Yun Cho,3 Gyeong Hoon Kang3. 1 _Seoul National University Hospital, Seoul, Republic of Korea;_ 2 _Sookmyung Women's University, Seoul, Republic of Korea;_ 3 _Seoul National University College of Medicine, Seoul, Republic of Korea;_ 4 _Korea University, Seoul, Republic of Korea_.

Purpose: Cancer of unknown primary site (CUPS) is a group of cancers for which the anatomic site of origin remains occult after detailed investigation. Until now, immunohistochemistry and tissue-specific RNA expression pattern have been used to predict tissue-of-origin in CUPS. However, these techniques do not fulfill the sensitivity and specificity for clinical practice. Recently, several studies found the tissue-specific methylation patterns in the genome. In this context, we tried to discover the tissue-specific methylation markers by analyzing genome-wide methylation data in The Cancer Genome Atlas (TCGA). Then, we aimed to develop the methylation-specific next-generation sequencing panel that predicts the tissue-of-origin in CUPS by validating panels in clinical samples.

Experimental Design: We selected every 17 mostly hypermethylated CpG sites in 31 cancer types by analyzing 8,350 cases of Infinium 450K methylation data in TCGA. With the selected 527 CpG sites as input variables, we constructed 465 decision tree models for all pairwise classification of 31 cancer types by using C50 package in R. Based on the two class classification models, we implemented an ensemble voting classifier for tissue-of-origin prediction test in fresh frozen tissue of 50 primary cancers of 8 tissue types using methylation-specific next-generation sequencing, which targeted 527 tissue-specific CpG sites by post-bisulfite method.

Results: In in silico analysis, we randomly selected 80% of the TCGA data for training decision tree models and used the remaining 20% of the data for testing those models. The sensitivity and specificity for the tissue-of-origin in 31 cancer types of testing data were 0.91 and 0.99 in decision tree models, respectively. In methylation-specific next-generation sequencing, our model predicted 40 cases (80%) of total 50 clinical samples, successfully.

Conclusions: Prediction of tissue-of-origin using methylation-specific next-generation sequencing might be promising. For clinical application, further study using more comprehensive targeted resequencing panel using formalin-fixed, paraffin-embedded tissues will be followed.

#3313

Epigenetic biomarkers of prognosis in stage IIA colon cancer.

Sara Pagotto,1 Elisa Porcellini,2 Maria G. Bacalini,2 Diletta Sarti,3 Ingrid Garajova,4 Elisabetta Broseghini,2 Noemi Laprovitera,2 Francesco Vasuri,4 Paolo Garagnani,2 Renato Mariani-Costantini,1 Michele Basso,3 Rosa Visone,1 Luigi M. Larocca,3 Manuela Ferracin,2 Angelo Veronese1. 1 _G. D'Annunzio University of Chieti-Pescara, Italy;_ 2 _University of Bologna, Bologna, Italy;_ 3 _Catholic University of Sacred Heart, Roma, Italy;_ 4 _S.Orsola-Malpighi University Hospital, Bologna, Italy_.

Adjuvant therapy is a systemic treatment administered after primary tumor resection to reduce the risk of relapse and death in colorectal cancer (CRC) patients. Generally, adjuvant treatment is recommended for stage III and 'high-risk' stage II CRC (NCCN guidelines). Nonetheless, adjuvant treatment is still debated for stage IIA (T3N0) patients, whose estimated recurrence rate is 15-20% in the absence of any further therapy after resection of the primary tumor. With the aim to find additional prognostic biomarkers that could improve current patient selection, we analyzed the global methylation profile of a discovery group of 10 relapsing/non-relapsing stage IIA colon cancer samples, without any other known risk factor (low-risk), by Illumina Epic 850K array. We identified a panel of 25 Illumina probes that were able to predict recurrence in chemotherapy-naïve patients. Then, we assessed the methylation of the most predictive Cytosine-Guanine (CpG) dinucleotides in 107 stage IIA colon cancer patients from two different cohorts, by EpiTYPER technology. We validated several CpGs as differentially methylated in relapsing vs. non-relapsing CRCs (p<0.05). For these CpG sites, we analyzed the methylation level also in the matching normal mucosa surrounding the tumor, to identify potential premalignant epigenetic lesions. In addition, we examined the association between the methylation level at specific CpG sites and prognostic clinical characteristics, overall survival and time-to-relapse. We concluded that the analysis of epigenetic alterations in archival colon cancer tissues could increase the panel of risk factors predictive for recurrence, and thus improve the selection of patients who could benefit from adjuvant therapy.

#3314

Heritable methylation marks associated with prostate and breast cancer risk.

Melissa C. Southey,1 Jihoon E. Joo,1 James G. Dowty,2 Roger L. Milne,3 EE Ming Wong,1 Pierre-Antoine Dugué,3 Dallas English,2 John L. Hopper,2 David E. Goldgar,4 Graham G. Giles3. 1 _Monash University, Clayton, Australia;_ 2 _The University of Melbourne, Melbourne, Australia;_ 3 _Cancer Council Victoria, Melbourne, Australia;_ 4 _Huntsman Cancer Institute, UT_.

While most epigenetic marks are reprogrammed during early embryogenesis, some studies have reported Mendelian-like inheritance of germline DNA methylation in particular in cancer susceptibility genes. For instance, individuals with MLH1 silenced throughout the soma fit the clinical criteria for hereditary nonpolyposis colorectal cancer that is indistinguishable from the syndrome resulting from germline mutations in MLH1. Research using multiple-case breast cancer families has shown that LINE-1 and Sat2 DNA methylation levels are lower in individuals with a strong family history. Family clustering of cancer could therefore be due to epigenetic as well as genetic and shared environmental factors. We have recently identified heritable methylation marks associated with breast and/or prostate cancer susceptibility by conducting a study involving 45 Australian multi-generational families with multiple cases of breast or prostate cancer who are not known to carry genetic mutations in cancer susceptibility genes. We developed and applied a new statistical method to identify heritable methylation marks based on complex segregation analysis and identified 24 and 41 methylation marks significantly associated with breast and prostate cancer risk respectively. Several marks across VTRNA2-1, a gene located in a differentially methylated region that is involved in imprinting and shows allele-specific methylation, were associated with heritable risk of both cancer types. A proportion of all identified marks were found to be associated with cancer risk in independent nested case-control studies (ie outside of the multiple-case family setting). We are expanding these successful studies to include additional families and to estimate HRs and age-specific cumulative risks of cancer associated with these marks to enable the incorporation of this information into clinical tools for risk prediction.

#3315

**Identification of ZFP42/REX1 as a regulator of cancer stemness in CD133** + **liver cancer stem cells by genome-wide DNA methylation analysis.**

Steve Tin Chi Luk,1 Man Tong,1 Kai Yu Ng,1 Kevin Yuk-Lap Yip,2 Xin Yuan Guan,1 Stephanie Ma1. 1 _University of Hong Kong, Hong Kong;_ 2 _Chinese University of Hong Kong, Hong Kong_.

The existence of a cancer stem cell (CSC) subset is well established in hepatocellular carcinoma (HCC). This subset can be characterized, among others, by its CD133 expression. Since the discovery of this marker, studies have revealed deregulated pathways and epigenetic alterations associated with this subset, but few have focused on changes in DNA methylation. This study aims to study the aberrant DNA methylation patterns governing CD133+ CSC-driven HCC. Results from Infinium HumanMethylation450 BeadChip (HuMet450 BeadChip) have led to the discovery of differential methylation patterns between CD133+ liver CSCs and matched CD133- differentiated samples. ZFP42/REX1 is one of the top-ranking differentially methylated candidates. Probes corresponding to its promoter are extensively hypermethylated in the CD133+ samples. We validated the down-regulation of REX1 mRNA levels in sorted CD133+ HCC cell lines (n=4) and HCC clinical samples (n=46, p<0.05), as compared to CD133- counterparts and non-tumor liver samples. REX1 and CD133 expressions are also significantly negatively correlated in HCC cell lines (n=8, R2=0.65). To explore the hypothesis that promoter DNA hypermethylation modulates REX1 expression, we treated HCC cells with 5-aza. Only low-REX1-expressing cells re-expressed REX1. Results of pyrosequencing of a CG island of interest located in the REX1 promoter show that REX1 expression appears to be negatively correlated with the degree of DNA methylation in HCC cell lines (n=4); and that HCC samples (n=15) exhibit a predominantly hypermethylated state compared to non-tumor samples (n=16). To add onto the clinical relevance of REX1, HuMet450 BeadChip datasets of HCC sample cohorts were obtained from Gene Expression Omnibus and the Cancer Genome Atlas. In these cohorts, the REX1 promoter region is significantly hypermethylated in HCC samples and this methylation is correlated to poor prognosis as well as increased expression of stemness markers. In addition to DNA methylation, we also discovered other regulatory mechanisms for REX1 expression involving histone methylation and acetylation. Functional studies with stable REX1 over-expressing and knock-down HCC cells suggest REX1 expression to negatively correlate with the cells' abilities to migrate, invade, and form foci in vitro; and initiate tumor formation, metastasize and self-renew in vivo. In summary, REX1 is a tumor-suppressor gene that is frequently down-regulated in human HCCs. The multiplicity of REX1 deactivating mechanisms together facilitates REX1 down-regulation to mediate various cancer and stem-like properties in the disease.

#3316

Hypermethylation at the SMPD3 promoter is frequently detected in hepatocellular carcinoma tumor tissue and corresponding plasma circulating DNA, and potentially differs by tumor subtype.

Sandi Alexander Kwee,1 Karolina Peplowska,1 Maarit Tiirikainen,1 Min-Ae Song,2 Linda L. Wong1. 1 _University of Hawaii Cancer Center, Honolulu, HI;_ 2 _The Ohio State University Comprehensive Cancer Center, Columbus, OH_.

Background: Circulating plasma DNAs may exhibit altered methylation reflective of aberrant epigenetics in tumor tissues, which may present a clinical opportunity for non-invasive assessment of tumor molecular phenotype. Hypermethylation at the promoter region of SMPD3 (Sphingomyelin phosphodiesterase 3), a tumor suppressor gene, has been identified in several genome-wide DNA methylation studies involving tissue specimens of hepatocellular carcinoma (HCC) (Shen et al., 2012; Song et al., 2013). The purpose of this study was to measure and compare SMPD3 promoter hypermethylation (specifically at cg10426893) in tumors, adjacent liver tissue, and circulating cell-free DNA (cfDNA) to examine its potential as a biomarker of HCC corresponding with tumor molecular subtype.

Methods: Blood and liver tissue specimens were collected following written informed consent from 54 patients undergoing partial liver resection for BCLC stage A HCC. DNA methylation was quantified through pyrosequencing analysis of bisulfite-treated DNAs from corresponding tumor tissue, adjacent liver tissue, plasma, and buffy-coat samples. Results were compared within patients using paired t-test. Transcriptomic molecular sub-classification based on the Hoshida classification system (2009) was performed on a subset of 39 tumors to explore potential associations between SMPD3 promoter hypermethylation and HCC molecular subtype.

Results: Levels of SMPD3 promoter methylation were significantly higher in tumor tissue compared to adjacent liver tissue (37.0% vs. 8.8%, p < 0.0001) as well as when compared to cfDNA samples (32.9% vs. 14.3%, p < 0.0001). SMPD3 promoter methylation levels in cfDNA and tumor tissues were also significantly higher than in corresponding buffy-coat samples (p = 0.0146 and p < 0.0001, respectively). Tumors belonging to the S3 molecular sub-class (n = 22) exhibited significantly higher SMPD3 promoter methylation levels than non-S3 tumors (n = 17) (44.1 % vs. 24.1%, p = 0.0166).

Conclusions: Significant increases in DNA methylation involving the promoter region of SMPD3 in HCC tumor tissues are reflected in cfDNA, supporting potential clinical liquid biopsy applications. Preliminarily, this epigenetic alteration appears to be associated with the S3 (differentiation) molecular subtype of HCC.

#3317

Genes encoding neuropeptide receptors are epigenetic markers in patients with head and neck cancer: A site-specific analysis.

Daiki Mochizuki,1 Kiyoshi Misawa,1 Atsushi Imai,1 Takeharu Kanazawa,2 Hiroyuki Mineta1. 1 _Hamamatsu University School Of Medicine, Hamamatsu-shi, Japan;_ 2 _Jichi Medical University, Tochigi, Japan_.

Staging and pathological grading systems are useful but imperfect predictors of recurrence in head and neck squamous cell carcinoma (HNSCC). The aim of this study was to determine the methylation status of eight GPCR-encoding genes in HNSCCs and its relationship to recurrence, survival, and clinical characteristics (e.g., tumor location and lymph node metastasis). All eight genes (NPFFR1, NPFFR2, HCRTR1, HCRTR2, NPY1R, NPY2R, NPY4R, and NPY5R) encode neuropeptide receptors and are in the Class A β subgroup. This study is the first to implicate neuropeptide receptors in the genesis of HNSCC. To identify potential prognostic markers, we examined the methylation status of eight neuropeptide receptor gene promoters in 231 head and neck squamous cell carcinomas. The NPFFR1, NPFFR2, HCRTR1, HCRTR2, NPY1R, NPY2R, NPY4R, and NPY5R promoters were methylated in 80.5%, 79.2%, 67.1%, 73.2%, 35.1%, 36.4%, 38.5%, and 35.9% of the samples, respectively. In a multivariate Cox proportional hazards analysis, the odds ratio for recurrence was 2.044 (95% confidence interval [CI], 1.323-3.156; P = 0.001) when the NPY2R promoter was methylated. In patients without lymph node metastasis (n = 100), methylation of NPY2R (compared with methylation of the other seven genes) best correlated with poor disease-free survival (DFS) (odds ratio, 2.492; 95% CI, 1.190-5.215; P = 0.015). In patients with oral cancer (n = 69), methylated NPY1R and NPY2R were independent prognostic factors for poor DFS, both individually and, even more so, in combination (odds ratio, 3.90; 95% CI, 1.523-9.991; P = 0.005). Similar findings were observed for NPY2R and NPY4R in patients with oropharyngeal cancer (n = 162) (odds ratio, 5.663; 95% CI, 1.507- 21.28; P = 0.010). The present study suggests that the methylation status of the NPY1R, NPY2R, and NPY4R genes is an independent indicator of DFS in patients with oral and/or oropharyngeal cancers. Our findings support the use of methylation markers in patient selection for adjuvant therapy after initial surgical treatment and may aid oropharyngeal cancer screening and surveillance programs. However, they are preliminary and hence need to be validated in larger and more homogeneous HNSCC patient cohorts.

#3318

Frequent promoter hypermethylation correlates with better prognosis in pharyngeal cancer.

Takuya Nakagawa,1 Keisuke Matsusaka,1 Kiyoshi Misawa,2 Masaki Fukuyo,1 Kiyoko Takane,1 Toyoyuki Hanazawa,1 Hisahiro Matsubara,1 Yoshitaka Okamoto,1 Atsushi Kaneda1. 1 _Chiba University Graduate School of Medicine, Chiba, Japan;_ 2 _Hamamatsu University School of Medicine, Hamamatsu, Japan_.

The incidence of Human Papilloma Virus (HPV) associated oropharyngeal cancer is dramatically increasing in developed countries, due to mainly HPV 16. Whereas viral infection status could affect molecular subtypes in other cancers, molecular features of HPV(+) and HPV(-) OPSCCs including epigenetic aberrations are not fully clarified. We here performed genome-wide DNA methylation analysis of OPSCC. We identified 144 candidate methylation-associated silencing genes using Infinium 450k analysis of 13 OPSCC samples, 4 normal mucosal samples and two HNSCC cell lines with/without 5Aza/TSA. Re-expression of the hypermethylated genes by 5-aza-2-deoxycytidine was confirmed by RNA-seq. Among the 144 candidate genes, 9 genes were selected and their methylation status was analyzed in 70 pharyngeal SCC cases including 55 OPSCC and 15 hypopharyngeal SCC cases using pyrosequencing. HPV(+) samples showed higher methylation level within all the samples (P=0.0002, t-test) and within OPSCC (P=0.001, t-test). While HPV infection significantly correlated with positive staining of p16 in immunohistochemistry, HPV(+) as well as p16(+) status significantly correlated with better prognosis (P = 0.005 and 0.002, log-rank test). The aberrant hypermethylation of an identified marker gene also correlated with better prognosis significantly when all the cases were analyzed (P = 0.04). Interestingly, the its hypermethylation status also correlated with better prognosis within p16(-) cases (P = 0.03). Our data suggest that aberrant DNA methylation might be involved in genesis of OPSCC, that DNA methylation might associate with HPV infection, and that methylation marker might work as prognostic marker. To elucidate the correlation between DNA methylation and OPSCC including HPV(+) and HPV(-) OPSCCs, we additionally performed genome-wide DNA methylation analysis of OPSCC using Infinium 450k analysis of 83 OPSCC samples, and integrated our data with 81 OPSCC data from the Cancer Genome Atlas (TCGA). HPV(+) and HPV(-) OPSCCs were mainly classified into two epigenotypes, respectively. Analyzing the correlation with clinicopathological data, we created a new classification strategy for OPSCC.

#3319

Epigenetic upregulation of E3 ubiquitin ligase CBLC enhances EGFR dysregulation and signaling.

Shiao-Ya Hong, Yu-Rung Kao, Te-Chang Lee, Cheng-Wen Wu. _Institute of Biomedical Science, Taipei, Taiwan_.

CBLC (CBL proto-oncogene c) belongs to the CBL protein family which has E3 ubiquitin ligase activity towards activated receptor tyrosine kinases. CBLC expression is physiologically restricted to normal epithelial cells but frequently upregulated in non-small cell lung cancer (NSCLC). Compared to its family members, very little is known about the functions of CBLC in tumorigenesis. Here, we demonstrated that CBLC is an epigenetically demethylated target and its expression can be upregulated after treatment with a DNA methylation inhibitor, 5'-azacytidine in NSCLC cells. CBLC depletion significantly inhibited cell viability and clonogenicity in vitro and reduced the tumor growth in a xenograft model. In addition, CBLC silencing further sensitized EGFR-mutated NSCLC cells to tyrosine kinase inhibitors treatment. On the other hand, after ligand stimulation, ectopic expression of CBLC enhanced the activation of EGFR and its downstream ERK1/2 signaling via competing with CBL for EGFR binding. By analyzing the ubiquitin linkages on activated EGFR (aEGFR), we found that CBLC ubiquitinated and positively regulated aEGFR stability through the conjugation of polyubiquitin by K6 and K11 linkages. The CBLC-mediated polyubiquitination promoted aEGFR preferentially recycling back to the plasma membrane or trafficking to the cell nucleus. In summary, we demonstrate a novel mechanism by which aEGFR escape lysosomal degradation in a CBLC/ubiquitin-dependent manner to sustain its activation. Our work identifies CBLC as a potential diagnostic biomarker and also points to its utilization as a novel therapeutic target for NSCLC therapy.

#3320

Discovery and development of DNA methylation biomarkers in human papillomavirus related oropharyngeal squamous cell carcinoma.

Shuling Ren,1 Daria A. Gaykalova,2 Theresa W. Guo,2 Alexander V. Favorov,2 Elana J. Fertig,2 Zubair Khan,2 Peter M. DeJong,3 Mizuo Ando,4 Chao Liu,1 Akihiro Sakai,5 Takahito Fukusumi,1 Sunny Haft,1 Sayed Sadat,1 J Silvio Gutkind,1 Wayne M. Koch,2 Joseph A. Califano1. 1 _University of California San Diego, La Jolla, CA;_ 2 _Johns Hopkins Medical Institutions, Baltimore, MD;_ 3 _Michigan State University, East Lansing, MI;_ 4 _Tokyo University, Tokyo, Japan;_ 5 _Tokai University, Isehara, Japan_.

Purpose: Human papillomavirus (HPV) related oropharyngeal squamous cell carcinoma (OPSCC) has an improved prognosis compared to cancers associated with tobacco and alcohol exposure, and also exhibits a different composition of epigenetic alterations. In this methylome-wide association study, we identified DNA methylation-based biomarkers with potential utility in screening for HPV positive OPSCC. Experimental Design: In the discovery stage, genome wide DNA methylation was measured using methyl-CpG binding domain protein-enriched genome sequencing (MBD-seq) in 50 HPV positive OPSCC tissues and 25 normal tissues. We defined 51 differentially methylated regions (DMRs) with maximal methylation specificity to cancer samples. We used TCGA methylation array data to evaluate the performance of the proposed candidates. Top 20 candidates were validated using quantitative methylation specific PCR (QMSP) in independent 24 HPV related OPSCCs and 22 controls. Results: Supervised hierarchical clustering of 51 DMRs based on TCGA methylation data found that HPV positive OPSCC had significantly higher DNA methylation levels compared to normal samples and head and neck squamous cell carcinoma (HNSCC) without HPV or with different tumor sites. The methylation levels of all top 20 candidates in HPV positive OPSCC were significantly higher than those in normal samples in TCGA data. 16 of these 20 candidates were found by QMSP to have significant higher methylation levels compared with controls. 2 candidates (CCDC181 and OR6S1) had a sensitivity of 100%, while 10 candidates (KCNA3, ITGA4, BEND4, ELMO1, SFMBT2, HOXB4, ZNF439, ZNF93, VSTM2B and ZNF773) had a specificity of 100%. Our 20 candidates were found by the receiver operating characteristic analysis to have a prediction accuracy ranging from 56.3% to 99.8%. Conclusions: This study discovered 20 epigenetic markers for detection of HPV related OPSCC, which can potentially be applied to develop a population based screening test and improve disease management.

#3321

Dysregulation of m6A RNA methylation regulators in colorectal cancer: Clinical implication as prognostic biomarkers and potential therapeutic targets.

Raju Kandimalla,1 Feng Gao,2 Chun Ruan,1 Michael Hsieh,1 Xin Wang,2 Ajay Goel1. 1 _Baylor Scott &White Research Institute and Charles A. Sammons Cancer Center, Dallas, TX; _2 _City University of Hong Kong, Hong Kong_.

Purpose Among more than 100 known post-transcriptional modifications, N6-Methyladenosine (m6A) represents the most prevalent internal modification in mammalian mRNAs. This epigenomic modification is orchestrated by a distinct group of enzymes and cofactors, categorized as writers, erasers and readers. Writers consist of a multicomponent complex consisting of at least three proteins, namely methyltransferase-like 3 (METTL3), METTL14, and Wilms' tumor 1-associating protein (WTAP1). These m6A marks are erased by two RNA demethylases, fat mass and obesity-associated protein (FTO) and alkylated DNA repair protein alkB homolog 5 (ALKBH5). Lastly, YTHDF1/2/3 family genes constitute the 'readers' that preferentially bind to RNA harboring m6A modification. Accumulating evidence suggests that m6a RNA methylation plays a crucial role in cancer progression. Herein, we for the first time elucidate the role of m6a regulators in colorectal cancer (CRC), in order to gain insights into their functional role and potential clinical significance in this malignancy. Experimental design Two large independent publicly available genome-wide mRNA expression datasets (N=509, TCGA and N=566, CIT) were used to study the expression of m6a RNA methylation regulators and their association with prognosis and gene expression based Consensus Molecular Subtypes (CMS) in CRC. Furthermore, we studied the tumorigenic properties of the RNA demethylase, FTO, by knockdown and overexpression strategies in CRC cells. Results The risk-score derived from a seven gene mRNA expression classifier consisting of METTL3, METTL14, WTAP, YTHDF1, YTHDF2, FTO and ALKBH5 was associated with poor disease-free survival in the training (TCGA) and validation (CIT) cohorts, with corresponding hazard ratios of 1.90 (95% CI: 1.24-2.92, p<0.002) and 1.86 (95% CI: 1.25-2.78, p<0.001) respectively. Furthermore, a high-m6a risk-score was significantly associated with a mesenchymal, CMS4 subtype, with FTO being the most overexpressed gene in CMS4 subtype of CRCs. Functionally, knockdown of the FTO gene in CMS4 cell lines (MDST8 and NCIH716) dramatically inhibited cellular proliferation, invasion and colony formation abilities. In contrast, overexpression of FTO in RKO cells resulted in enhanced proliferation, invasion and colony formation. Conclusions In summary, we for the first time report the dysregulation of m6a RNA methylation regulators in CRC, wherein we discovered their significant association with poor prognosis and a CMS4 CRC subtype, as well as elucidated the functional role of FTO in CRC cell lines. Besides the potential of utilizing this seven gene m6a regulator gene expression classifier to predict prognosis, our data suggest that targeting FTO might represent an attractive strategy for therapeutic intervention in patients with a mesenchymal CRC subtype with poor prognosis.

#3322

DNA hypermethylation plays an important role in the epithelial-mesenchymal transition in triple negative breast cancer.

Julia Santucci-Pereira, Yanrong Su, Jose Russo. _Fox Chase Cancer Center, Philadelphia, PA_.

We have developed an in vitro model of triple negative breast epithelial cell lines which represents the progression of the basal breast cancer subtype (Russo et al, FASEB 2006; Huang et al, Cancer Res 2007). This model consists of three cell lines, MCF10F, trMCF and bsMCF representing respectively the normal, transformed and tumorigenic phases of the cancer progression. There are genomic differences among these cells that are characteristics of epithelial-mesenchymal transition (EMT). In the present work, we evaluated the changes in methylation throughout the EMT by performing MBD-Cap sequencing of MCF10F, trMCF and bsMCF. Methylated DNA was separated using MethylMiner™ Methylated DNA Enrichment Kit (Life Technologies), and the samples were used for library preparation and sequencing using Illumina platform. To identify the changes in the methylation profile of these cell lines during the process of EMT we performed the comparisons MCF10F vs. trMCF; trMCF vs. bsMCF; and MCF10F vs. bsMCF. Genes were considered differentially methylated when they showed adjusted p-value (Benjamini and Hochberg) < 0.01 and at least twofold difference in methylation levels. We found that overall the methylation patterns increase as the EMT takes place. We observed 816 genes hypermethylated in trMCF compared to MCF10F, 229 genes in bsMCF compared to trMCF, and 1269 hypermethylated genes in bsMCF vs. MCF10F. Gene ontology and pathway enrichment analyses revealed that differentiation, morphogenesis, motility and adhesion were processes over-represented among the genes that were hypermethylated throughout the EMT process. One of the hypermethylated genes was NRG1 (neuregulin 1), which encodes a cell adhesion molecule and has been shown to be methylated in breast cancer tissue. Although in smaller numbers, we also observed some genes hypomethylated during the EMT. There were 29 hypomethylated genes in trMCF compared to MCF10F, 94 in bsMCF vs. trMCF, while 45 hypomethylated genes were observed in bsMCF compared to MCF10F. These data show that methylation is an important epigenetic molecular mechanism during the cancer progression in this in vitro model. Most of the DNA methylation changes occur during the first step of malignant transformation, from normal to transformed cells (845 genes with differentially methylated areas). Moreover, the additional epigenetic modifications during the EMT (323 genes in bsMCF vs. trMCF) contribute to the cellular development of tumorigenic properties. These results point us to the potential use of demethylating agents, such as DNMT inhibitors, as therapeutic drugs against triple negative breast cancer. (This work was supported by The Pennsylvania Cancer Cure Grant 6914101, the Barbara and Joseph Breitman donation, the Flyers Wives and by NIH core grant CA06927).

#3323

Role of RNA methyltransferase METTL3 in melanoma.

Ujwal Dahal, Kang Le, Mamta Gupta. _George Washington University, Washington, DC_.

Melanoma is the sixth most common cancer in the USA. Introduction of BRAF inhibitors and advances in cancer immunotherapy has greatly improved treatment of melanoma, however, the problem of tumour relapse and therapy resistance persists. Better understanding of the mechanism of development of the diseases is urgently needed to improve therapeutic intervention. Role of DNA and histone modifications have been well established in several diseases, however, RNA modifications, specifically, N6 methyl adenosine (m6A), has only recently begun to be shown to play critical role in cancer. m6A plays crucial roles in mRNA stability, splicing, export, translation and decay and is regulated by: methyltransferase complex proteins METTL3, METTL14, and WTAP; the demethylases FTO and ALKBH5, and the m6A reader proteins. In the present study, we uncover the role of RNA methyltransferase METTL3 in human melanoma cells. METTL3 expression was assessed in 8 melanoma cell lines (A375, A375MA2, A2058, WM164, 451Lu, WM793, SKMEL2, WM3918). Western blotting data revealed METTL3 to be upregulated in seven melanoma cell lines (except A2058) relative to normal melanocytes. To uncover the role of METTL3 in melanoma, we knockdown METTL3 with lentiviral shRNA and studied the effect on melanoma cells tumorigenesis. The colony formation assay revealed reduced colony number in the METTL3 knockdown A375, A375MA2 and WM793 cells relative to the scrambled control cells. The main cause of melanoma associated fatality is its metastatic dissemination we therefore, next sought to investigate whether METTL3 could affect melanoma cells migration and invasiveness. We performed migration and invasion assays to detect the migratory potential of control or METTL3 knockdown A375, A375MA2 and WM793 cells. Interestingly, both migration and invasion was found to be reduced in METTL3 knockdown cells in all the three cell lines examined. RNA dot blot assay demonstrated a decreased m6A level in total RNA of the METTL3 knockdown cells relative to control cells. p16 and p53 mRNA were found to be enriched in m6A immunoprecipitated samples as compared to IgG control, pointing towards a potential role of m6A in regulating expression of melanoma associated genes. Ongoing effort is aimed at identifying what known or novel regulators of the migratory/invasive cascade is regulated by METTL3 and whether it is through m6A mediated or independent mechanisms. In summary, we have shown that METTL3 is overexpressed in human melanoma and play an important role in metastasis.

#3324

Diagnosis and prognosis of hepatocellular carcinoma with ctDNA methylation markers.

Kang Zhang. _University of California San Diego, Irvine, CA_.

Abstract

An effective blood-based method for the diagnosis and prognosis of hepatocellular carcinoma (HCC) has not yet been developed. Circulating tumor DNA (ctDNA) carrying cancer-specific genetic and epigenetic aberrations may enable a noninvasive "liquid biopsy" for diagnosis and monitoring of cancer. Here, we identified an HCC-specific methylation markers by comparing HCC tissue and normal blood leukocytes and showed that methylation profiles of HCC tumor DNA and matched plasma ctDNA are highly correlated. Using cfDNA samples from a large cohort of 1098 HCC patients and 835 normal controls, we constructed a diagnostic prediction model that showed high diagnostic specificity and sensitivity (P<0.001) and was highly correlated with tumor burden, treatment response, and stage (P<0.001). Additionally, we constructed a prognostic prediction model that effectively predicted prognosis and survival (P<0.001). Together, these findings demonstrate in a large clinical cohort the utility of ctDNA methylation markers in the diagnosis, surveillance, and prognosis of HCC.

#3325

Epigenetics, regulation of cancer immunotherapy response genes, and race: A comparison of DNA methylation in non-small cell lung cancers from African Americans and European Americans.

Allison McHayle, Amy Boles, Lysa Diarra, Khadijah A. Mitchell. _Lafayette College, Easton, PA_.

African Americans (AA) have the highest non-small cell lung cancer incidence and lowest 5-year survival rates compared with any other population in the United States. Smoking is the strongest risk factor for lung cancer development. Paradoxically, AA smoke less than European Americans (EA). Even AA never-smokers have higher rates of lung cancer when compared with EA. This suggests other factors may contribute to lung cancer health disparities, both nonbiologic and biologic in scope. Biologic determinants are largely understudied. Recently, breakthroughs in cancer immunotherapy have revolutionized treatment paradigms. However, AA lung cancer patients may not benefit from these therapeutic advances as much as their EA counterparts if the underlying lung tumor biology differs in cancer immunotherapy response gene regulation. Previous work across cancers has shown clear immune-system differences in AA compared with EA patients. Additionally, smoking is known to mediate DNA methylation. We hypothesize that population-specific differences in DNA methylation regulate cancer immunotherapy response genes. Comparative integrative genomics was performed using clinical demographic and lung tumor tissue data from TCGA (Illumina 450K methylation array, mRNA-seq). The DNA methylation status of 554 recently identified essential genes for cancer immunotherapy response was assessed in both LUAD (n=53 AA, 367 EA) and LUSC (n=48 AA, 274 EA) patients. Although many genes varied significantly by race (P value + FDR ≤0.01) in both histologies (n=285), some gene signatures were LUAD-enriched (n=19), while others were LUSC-enriched (n=101). Significant DNA methylation decreases at a LUAD-enriched gene (SHISA4) in AA compared with EA corresponded with upregulation of this gene in 15% AA and 14% EA LUAD patients, respectively. Similarly, we observed significant decreases in DNA methylation at a LUSC-enriched gene (RAD1) in AA compared with EA, and this correlated with mRNA upregulation in 15% AA and 49% EA LUSC patients. Decreased immunotherapy response gene expression in AA suggests that targeted immunotherapy would be less effective in this population. To our knowledge, this study is the first to explore how differential DNA methylation between AA and EA with lung cancer may translate to differences in cancer immunotherapy drug response. Future studies include exploring other LUAD- and LUSC-enriched genes and their relationship with smoking, validating these findings in lung cancer cell lines from AA and EA for downstream mechanistic studies, and elucidating novel biologic mechanisms in diverse populations to advance therapeutic goals (e.g., PDX models in AA and EA).

#3326

Upregulation of epigenetic changes acquired by HPV16 E6/E7 oncoproteins in mouse keratinocytes: Targeting ATM/PI3K.

Eva McGhee,1 Mengtao Li,2 Yi-Ling Lin,3 Liliana Zarate,1 Naomi Long,1 Mai Do,1 Chinelo Ezechukwu,1 Nichelle Cox,1 Hyun Chung,1 Jenna Cormier,1 Meidrah Tyler,1 Victoria Vidal,1 Billy Ballard,1 Roland Pattillo,4 Jay Vadgama1. 1 _Charles R. Drew University of Medicine and Science, Los Angeles, CA;_ 2 _University of California Los angeles, Los Angeles, CA;_ 3 _University of California Los Angeles, Los Angeles, CA;_ 4 _Morehouse School of Medicine, Atlanta, GA_.

Human Papillomavirus (HPV), a non-enveloped epitheliotropic double stranded DNA virus, is an etiological agent of oral cancer. Oral cancer is related to the persistent infection by high-risk HPV type 16, E6/E7 oncoproteins. The E6/E7 oncoproteins significantly contribute to the carcinogenic genetic instability effect of high-risk HPV through the degradation of two gatekeeper proteins, p53 and pRB, respectively. The ATM gene, also known as a gatekeeper protein plays a central role in the complex processes that repair DNA strand breaks. The ATM gene is located at 11q22.3 and belongs to a protein family known as the PI3K. Many studies have tried to clarify the epigenetic instability role of ATM in cancer function and susceptibility. However, there are no studies addressing the relationship between HPV infection-oral cancer, ATM/PI3K expression, and genomic instability. The aim of this study is to analyze the influence of HPV16 E6/E7 oncoproteins in oral cancer and the epigenetic profile. In our experimental procedures, we proposed a novel approach to study HPV-induced epigenetic changes with reconstructed human oral epithelium in SCID mouse. We used HPV-16, which is the most common HPV type found in oral cancer. Two plasmids were used to produce HPV-16 E6/E7. The first plasmid p16sheLL expresses the two viral capsid proteins, L1 and L2. The second plasmid pBR322HPV16 contains the full length HPV-16 genome. This approach closely mimics the architecture of normal human oral epithelium. In our study, we first focused on analyzing selected cellular gene loci where DNA methylation profiles are known to be altered, either by HPV infection or in oral cancer. We examined the following gene promoters DcR1/DcR2 (apoptosis regulation), p16 (cell cycle control), DAPK (cancer metastasis), and MGMT (DNA repair). We also analyzed progressive morphological and cytopathic changes in the HPV-infected oral epithelia. These data indicate that HPV E6/E7 oncoproteins can promote increased upregulation of apoptosis, disrupt cell cycle regulation, and decrease DNA double strand break repair. Epigenetics and chromosomal instability events were noted, specifically, the deletion of the distal region of chromosome 11q22-23 in the E6/E7 transfected keratinocytes. These results indicate that HPV-16, E6/E7 oncoproteins induced epigenetic changes in mouse keratinocytes, which may further promote oral carcinogenesis.

#3327

Correlating blood-based DNA methylation markers and prostate cancer risk factors in African-American men.

Bernard Kwabi-Addo,1 Emmauel Moses-Fynn,2 Wei Tang,3 Desta Beyene,4 Victor Apprey,4 Yasmine Kanaan4. 1 _Howard Univ. College of Medicine, Washington, DC;_ 2 _University of Maine, Machias, ME;_ 3 _National Institute of Health, Bethesda, MD;_ 4 _Howard University, Washington, DC_.

Background: Detection of prostate cancer at an early stage is key for successful treatment and improvement of outcome. However, the limitations of PSA screening are well recognized and therefore novel approaches to prostate cancer screening are necessary. We have investigated the clinical significance of DNA methylation in the blood serum of African-American men with prostate cancer.

Method: We used pyrosequencing to examine the promoter methylation for a panel of 8 genes in blood samples extracted from 91 prostate cancer cases and 91 noncancerous control cases.

Results: Seven of the 8-gene panel--RARβ2, TIMP3, SPARC, CDH13, LINE1, CYB5R2 and DRD2--tested achieved significant difference between the cancer cases when compared to control cases. The RARβ2 and TIMP3 demonstrated significant association of DNA methylation with age for all cases analyzed. Associations of methylation status and various clinicopathologic features and patient survival were evaluated: we observed significant inverse association of serum vitamin D levels in the cancer cases and DNA methylation of CDH13 and also an inverse correlation between TIMP3 and DRD2 methylation with supplementary vitamin D in the cancer cases. On the other hand, we observed a direct correlation between HIN1 methylation and serum vitamin D level. In addition, we observed a direct correlation between RARβ2 and Sparc methylation with PSA level in the control but not the cancer cases.

Conclusion: Promoter methylation occurred more frequently in the serum of patients with prostate cancer than noncancerous cases, suggesting that epigenetic markers in serum have potential for diagnosis of prostate cancer in AA men.

#3328

Association between LOX expression, LINE-1 DNA methylation and prognosis in esophageal cancer.

Rebecca Tauzen Kalikawe, Yoshifumi Baba, Miyake Keisuke, Yagi Taisuke, Yuki Kitano, Sawayama Hiroshi, Hoyoshi Yukiharu, Iwatsuki Maasaki, Miyamoto Yuji, Yoshida Naoya, Ishimoto Takatsugu, Hideo Baba. _Kumamoto Univ. Graduate School of Medical Sci., Kumamoto, Japan_.

Background: Abnormal function of human body enzymes and epigenetic alterations such as DNA methylation lead to human carcinogenesis. Lysyl oxidase (LOX) is an extracellular matrix remodeling enzyme and its upregulation has been associated with poor prognosis in various cancers. LINE-1 methylation is considered as a surrogate marker of global DNA methylation and is attracting interest as a good predictor of cancer prognosis. Despite LOX involvement in tumor proliferation, migration and invasion in other types of tumors, the importance of LOX enzyme and its epigenetic regulation in pathogenesis of esophageal cancer has yet to be fully elucidated. Therefore, we constructed this study to examine the correlations between LOX expression, LINE-1 DNA methylation and the clinical pathological profile in patients with esophageal cancer.

Methods: mRNA expression array analysis was done using tissue biopsy and public array data. We then examined LOX expression in esophageal cancer using tissue biopsy by Real time PCR.LOX expression was evaluated in 284 surgically resected ESCC by immunohistochemistry, and the correlation was analyzed with LINE-1 methylation, obtained previously with pyrosequencing. In vitro we looked on LOXs role in tumor progression by growth and invasion assay using ESCC cell lines.

Results: Our microarray data reveled that LOX was the highly upregulated gene in hypo methylated tumors (fold change>2X). In tissue biopsies LOX was significantly higher in tumor part compared to normal epithelium. In immunohistological study, LOX was identified in tumor cytoplasm. High LOX expression correlated with decrease in Overall survival, cancer-specific survival and disease-free survival. (P <0.001**). Moreover, significant association with tumor size, invasion depth, tumor stage and lymph node metastases was also observed. In multivariate analysis LOX expression.

and tumor depth were an independent prognostic factor. Nevertheless, high LOX expression correlated to lower methylation levels (P=0.01). Invitro, we observed upstream of LOX expression by 5-AZA treatment but LOX knockdown had no influence in methylation levels. LOX knockdown suppressed growth in both TE6 and TE11 and reduced invasion and migration ability in TE 11 cell line.

Conclusions: Our findings show LOX is regulated epigenetically by genome wide hypomethylation and promote invasion and proliferation of ESCC. Hence, LOX can serve as a prognostic biomarker in ESCC patients and therapeutically targeting LOX could reverse progression of esophageal cancer.

2269/2600 characters

#3329

A newly developed Septin9 gene methylation assay dectecting double DNA strands for screening of colorectal cancer.

Qiuju Liang,1 Zhengbin Huang,2 Qingwen Xu,3 Feipeng Xu,3 Wenwei Huang,2 Wenfang Xie,1 Lei Zeng,1 Jiasu Liu,1 De-Hua Yu1. 1 _USK Bioscience, Shenzhen, China;_ 2 _Department of General Surgery, Hanchuan People's Hospital of Hubei Province, Hanchuan, China;_ 3 _Department of Gastrointestinal Surgery, Affiliated Hosptial of Guangdong Medical University, Zhanjiang, China_.

Colorectal cancer (CRC) is one of the most common malignant tumors and places an enormous burden on society. The US FDA approved the application of Epi proColon on 12 April 2016, and permitted the application of the Septin9 gene methylation assay as a CRC screening test for average-risk population over 50 years old. The effectiveness of methylated Septin9 for CRC detection has been reviewed in the newly published recommendation statement by US Preventive Services Task Force (USPSTF). The Septin9 gene methylation assay is more cost efficient with higher compliance than the stool DNA test, and has been proven to be an accurate, reliable, fast, and convenient molecular test. However, the currently FDA approved Epi proColon assay needs to perform triplicated PCR reactions for each sample which leads to high cost. In order to improve the sensitivity and specificity, simplify the operational procesure, and reduce cost, we have developed an new Septin9 gene methylation assay in which the specific sequences of both methylated DNA strands and control gene could be amplificated and detected in one reaction. We evaluated sensitivity, specificity, and clinical performance using plasma samples from colonoscopy negative individuals and CRC patients. The preliminary data showed that the sensitivity and specificity for CRC detection was 77.4%(41/53) ,97.8%(90/92) respectively. A large scale clinical study with more than 1000 subjects is currently in progress. In conclusion, we have developed a new Septin9 gene methylation assay detecting double DNA strands, which demonstrated improved performance for early detection of CRC in clinical setting.

#3330

Molecular surgical margin analysis of pancreatic cancers.

Masamichi Hayashi, Suguru Yamada, Hiroshi Tanabe, Yoshiyasu Kato, Katsuhito Tanaka, MItsuru Tashiro, Haruyoshi Tanaka, Tomonari Asano, Hideki Takami, Masaya Suenaga, Yukiko Niwa, Yasuhiro Kodera. _Nagoya university, Nagoya city, Aichi prefecture, Japan_.

Background: Pancreatic cancer is one of the incurable cancers among solid malignancies. Basically, R0 tumor resection could be one of the critical factors for their prognosis. However, pathological diagnosis of surgical margins is sometimes unreliable due to tissue shrinkage and skipped lesion like tumor budding. We applied our 'molecular surgical margin analysis' using tissue imprinting procedure to evaluate the precise surgical margin status of the surgical specimens after pancreatoduodenectomy.

Methods: Quantitative methylation specific PCR (QMSP) assay for 3 representative tumor-specific methylation markers (CD1D, KCNK12, PAX5) were established and validated by paired normal and tumor tissues from pancreatic ductal carcinoma cases (n=48). Then, these markers were applied to prospectively collected ①peritoneal lavage samples (n=16), ②drainage fluid samples (n=18) and ③surgical margin imprinting samples (n=11).

Results: QMSP values of tumor tissues were significantly higher than adjacent normal tissues and relatively high specificity to cancer cells in all three markers with optimal cut-off values (CD1D: P<0.001, 94%, KCNK12: P=0.001, 75%, PAX5: P<0.001, 87%). These marker positive cases also showed marginally or significantly poor overall survival in univariate analysis (CD1D: HR=1.78, P=0.083, KCNK12: HR=2.57, P=0.005, PAX5: HR=2.64, P=0.003). Since these markers were revealed to be very cancer-specific, we tried to use them as cancer detection markers in clinical samples. ①CD1D methylation was positive in 6 cases among 16 cases. Although 4 cases showed histopathologically positive, 2 cases were negative. These cases might have subclinically positive cancer cells in the fluid. Actually, one of them relapsed 5 months after the surgery. ②CD1D methylation was positive in 4 cases among 18 postoperative cases. These cases were all treated with surgery first, while neoadjuvant cases showed no methylation in postoperative drainage fluid. ③ Molecular surgical margin analysis revealed 4 positive cases and 7 negative cases. To find the meaning of molecular margin positive, we still following-up these cases.

Conclusion: Very cancer-specific methylation detection assay was established. Although we still need to follow-up the cases for a year or more, invisible cancer cells in the fluid or on the surgical margin might have an association with tumor recurrence after the surgery.

#3331

Development of a sensitive method to detect aberrant DNA methylation in cfDNA for pancreas cancer diagnosis.

Keiko Shinjo,1 Keisuke Katsushima,1 Miho Suzuki,1 Genta Nagae,2 Hiroyuki Aburatani,2 Kenji Yamao,3 Yutaka Kondo1. 1 _Nagoya Univ. Graduate School of Medicine, Nagoya, Japan;_ 2 _Tokyo University Research Center for Advanced Science and Technology, Tokyo, Japan;_ 3 _Aichi Cancer Center Hospital, Nagoya, Japan_.

Pancreatic cancer is a highly lethal malignancy despite recent clinical advances. Since pancreatic cancer is often difficult to diagnose, there is an urgent need to develop noninvasive biomarkers for detection. Epigenetic alterations have been recognized as a common hallmark of many types of human cancers as well as pancreatic cancer. The cell-free DNA (cfDNA) in blood possesses tumor-related genetic alterations as well as epigenetic alterations. Therefore, invention of blood-based DNA methylation biomarkers and their high-sensitive detection system may be a promising diagnostic tool for pancreatic cancer. In order to establish the diagnostic biomarkers, first we performed genome-wide DNA methylation analysis of 38 endoscopic ultrasound-guided fine-needle aspiration specimens of pancreatic cancer using Illumina Infinium HumanMethylation450 BeadChip. We further analyzed the DNA methylation profiles of other types of tumors in the public database TCGA (The Cancer Genome Atlas), and identified five marker genes, which were specifically methylated in pancreatic cancers. Analysis of DNA methylation status of pancreas cancers comparing 10 normal tissues in TCGA data set revealed that 99% of tumors were methylated in at least one of the five markers (sensitivity, 98.9%; specificity, 44%). We next examined the DNA methylation status of these markers in the validation cohort (n=46) by bisulfite pyrosequencing or quantitative methylation-specific PCR and found that 42/46 (91.3%) cases were methylated in at least one marker. Finally, we analyzed methylation status in cfDNA from pancreatic cancer patients. We performed bisulfite sequencing using next-generation sequencer and detected methylation in only 5/13 (38.5%) samples in one of the markers. Since the amount of DNA from cancer tissue is very small, we need to develop more a sensitive technique to detect aberrant DNA methylation to diagnose cancer patients. Now we are developing a new sensitive method to detect DNA methylation in cfDNA.

#3332

Genome-wide DNA methylation profiling of peripheral blood leukocytes identifies predictors of aggressive prostate cancer.

Yuyan Han,1 Jia Li,2 Mutian Zhang,2 Jeri Kim,1 Xifeng Wu,1 Deqiang Sun,2 Jian Gu1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Texas A &M University, Houston, TX_.

Background: Prostate cancer (PCa) is the most commonly diagnosed cancer for men in the United States. Most of the PSA screening-detected PCa are slow-growing and not life-threatening. However, the majority of PSA-detected PCa receive aggressive treatment, which is often associated with significant morbidity. Clinical variables, such as Gleason Score (GS), PSA level, and tumor stage, are not sufficient to distinguish aggressive from indolent diseases at diagnosis. Biomarkers are urgently needed for more accurate prediction of individual tumor behavior and patient prognosis. DNA methylation has shown profound impact on cancer development and progression. In this study, we performed a genome-wide DNA methylation profiling of peripheral blood leukocytes to identify predictors of aggressive PCa. Methods: We performed leukocyte DNA methylation profiling in 140 GS=6 PCa patients and 147 GS ≥ 8 patients using the Illumina's Methylation 450K BeadChip. The differential methylated CpG position (DMP) was identified using the ChAMP analysis package implemented in R. Top differentially methylated CpG sites between less aggressive (GS=6) and more aggressive (GS≥ 8) patients were used to develop a signature to best cluster these two groups using semi-supervised recursively partitioned mixture modeling (SS-RPMM) algorithm. The white blood cell subtype composition was analyzed with RefbaseEWAS method. Ingenuity pathway analysis was used to find the enriched pathways that may determine aggressive prostate cancer. Results: Analyzing all the assayed CpG sites together, the mean methylation level appeared to be lower in GS ≥ 8 patients than in GS=6 patients. This global DNA hypomethylation was only significant at CpG sites located within gene body, shore, shelf, opensea and 3' UTR regions, but not in promoter and regions close to transcription start sites. There were many individual hypermethylated CpG sites in GS ≥ 8 patients that are mostly located in CpG islands within promoter and in the vicinity of transcription start sites. We then split the patients into a training (70 GS=6 and 73 GS ≥ 8 patients) and testing set (70 GS=6 and 74 GS ≥ 8 patients) to identify and validate a CpG methylation signature that best differentiates GS=6 and GS≥ 8 patients. The signature can identify a subgroup of aggressive PCa patients with higher risks of biochemical recurrence (HR=2.57, 95% Cl, 0.99-6.68). We also analyzed the white blood cell subtypes in patients and found that natural killer (NK) cell proportion is significantly decreased in patients who experienced biochemical recurrence (P=0.045), indicating weakened immune response. Conclusion: These data suggest that DNA methylation in peripheral blood leukocytes may serve as valuable biomarkers to supplement clinical variables to predict aggressive PCa at diagnosis and worse prognosis of PCa patients.

#3333

Detection of IDH1 R132H mutation in situ in human astrocytoma and glioma FFPE samples.

Na Li, Mindy Wang, Hongzhe Sun, Zhifu Zhang, Xin Wang, Emily Park, Xiao-Jun Ma, Robert Monroe. _Advanced Cell Diagnostics, Newark, CA_.

IDH1 and IDH2 mutations have been shown to be early events in gliomagenesis. IDH1 mutations are present in ~33% of gliomas and constitute ~90% of all IDH mutations. Approximately 88% of IDH1 mutations involve the substitution c.395G>A leading to the R132H amino acid change. IDH1 mutations, including R132H, are important to recognize clinically as they are more frequent in younger patients and confer a survival advantage. Patients with IDH1 mutations show lower expression of the prognostic markers p53 and Ki-67 and achieve greater benefit from maximal tumor resection. In addition to their prognostic utility, IDH mutations including IDH1 R132H can be useful as diagnostic markers for low-grade gliomas. In difficult to diagnose glial lesions, the presence of IDH mutations strongly favors a diagnosis of glioma versus reactive gliosis. Although PCR and DNA sequencing-based approaches can detect the IDH1 R132H mutation, these techniques require destruction of tissue for nucleic acid isolation and destroy the morphologic context, highlighting the need for a methodology that can be performed on formalin-fixed, paraffin-embedded (FFPE) tissues. In this study, we developed an assay to specifically detect the IDH1 R132H mutation in FFPE samples in expressed IDH1 mRNA transcripts using the novel BasescopeTM technology. IDH1 wild-type (WT) and IDH1 mutation (MT) probes were tested in 6 astrocytoma and 3 glioma FFPE specimens from unique clinical cases. All samples exhibited positivity with the IDH1 WT probe. Two of six astrocytomas and one of three gliomas showed positivity with the IDH1 R132H mutation probe. Among the three positive samples, one astrocytoma exhibited positive signals for the IDH1 R132H mutation in almost all tumor cells, whereas one astrocytoma and one glioma showed only scattered positive signals with the IDH1 R132H mutation probe in a subset of tumor cells. Our findings indicate that the BasescopeTM assay is a novel RNA in situ hybridization assay to visualize point mutations in a highly specific and sensitive manner within the morphologic tissue context. In this study, we demonstrate that the BasescopeTM IDH1 R132H assay is capable of detecting the IDH1 R132H mutation in routine FFPE clinical specimens for the purpose of assisting in the diagnosis of gliomas and providing prognostic information. The BasescopeTM technology also allows for correlation of the frequency and distribution of IDH1 R132H mutations in glial tumors with patient outcomes and overall survival.

### Exploring Oncogenic Transcription Factors

#3334

**CREB is required for** KRAS **-driven lung tumorigenesis.**

Jong woo Lee,1 Frank J. Slack,2 Eric J. Nestler,3 Roy S. Herbst,1 Jaseok Peter Koo1. 1 _Yale School of Medicine, New Haven, CT;_ 2 _Harvard Medical School, Boston, MA;_ 3 _Icahn School of Medicine at Mount Sinai, New York, NY_.

The aberrant constitutive activation of the transcription factor, cyclic-AMP response element- binding (CREB) protein, has been associated with poor prognosis and chemotherapeutic resistance in KRAS-mutated human cancers, one of which includes non-small cell lung cancer (NSCLC). However, the role of CREB in KRAS-driven lung tumorigenesis has not been fully understood. Using adenoviral delivery of Cre specifically to the mouse lung, we generated a new transgenic lung cancer mouse model driven by Kras activating mutation G12D and concomitant with Creb loss (KrasLSL-G12D/+; CrebFlox/Flox). In this study, we used this model to show that a proto-oncogenic transcription factor CREB, is required for KRAS-driven lung tumorigenesis. Loss of Creb (Creb-/-) in the mice harboring Kras-mutated lung tumors significantly suppressed tumor development, and thus, dramatically prolonged mouse survival when compared to either wild (Creb+/+) or heterozygous (Creb+/-) deletion of Creb. Mechanistically, we identified a few novel CREB target gene. These data suggest that CREB is essential for KRAS-driven lung tumorigenesis and that CREB could be a potential target for therapeutic treatment for individuals suffering from KRAS-mutant lung cancers.

#3335

**Characterizing the MAX** H28R **secretome in endometrial cancer.**

Craig M. Rush, Miranda L. Gardner, Caroline E. Sapp, Michael A. Freitas, Paul J. Goodfellow. _The Ohio State University Comprehensive Cancer Center, Columbus, OH_.

We have previously shown that the recurrent endometrial cancer MAX p.His28Arg mutation (MAXH28R) is associated with a proangiogenesis phenotype in both in vitro and xenograft models. The purpose of this study is to characterize the MAXH28R secretome to identify the proangiogenic and other paracrine factors contributing to MAXH28R associated tumorigenesis. Bottom-up high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) of secreted proteins coupled with microarray analysis of global gene expression was performed in AN3CA endometrial cancer cell lines that stably express the long isoform of MAX-wild type (MAXWT) or MAXH28R. Orthogonal validation of differentially expressed proteins was performed by Western blot (WB) and enzyme-linked immunosorbent assay (ELISA) using MAXWT\- or MAXH28R-expressing AN3CA cell lines. Bioinformatic analysis of the proteins secreted into conditioned media (secretome) identified 114 proteins differentially expressed by MAXH28R compared to MAXWT-expressing AN3CA cells (P < 0.05). Differential expression of proteins was highly correlated with mRNA expression (Pearson r = 0.75, P < 0.001), indicating that a subset of the differentially expressed proteins are potential direct transcriptional targets of MAX. Secretome analysis revealed that Laminin-β1 expression in MAXH28R was reduced more than 100-fold compared to MAXWT (P < 0.001), while ALCAM (CD166) expression was increased 3.5-fold (P < 0.001). Decreased expression of TGFβ1 (5.9-fold decrease, P = 0.023) and TGFβ2 (3-fold decrease, P = 0.009) was seen for MAXH28R cells compared to MAXWT. Differential expression of Laminin-β1 and ALCAM was confirmed by WB analysis and TGFβ1 and TGFβ2 by ELISA. Our analyses revealed extensive changes in the MAXH28R secretome, as exemplified above. Reduced Laminin-β1 highlights alteration in the extracellular matrix with implications for cell adhesion and integrin signaling. Increased ALCAM expression indicates changes to cell-cell adhesion. Elevated levels of ALCAM in conditioned media are consistent with increased ALCAM shedding, which is a marker of more aggressive tumors in a variety of malignancies. Reduced TGFβ ligand levels suggest potential alterations in paracrine and autocrine TGFβ signaling. However, the impact on canonical and noncanonical TGFβ signaling in MAX mutant endometrial cancers remains unknown. ChIP-qPCR experiments are ongoing to assess MAXH28R binding at the promoters of differentially expressed genes. Biologic validation of MAXH28R effects on differential expression of secreted proteins is being performed in the Ishikawa and RL95-2 endometrial cancer cell lines that stably express MAXH28R and MAXWT. The effects of reduced Laminin-β1 on integrin signaling and anchorage independent growth will be reported, as will effects of altered TGFβ expression on canonical and noncanonical TGFβ signaling.

#3336

ARID5B activates the TAL1-induced core regulatory circuit and the oncogene MYC, thereby promoting T-cell leukemogenesis.

Wei Zhong Leong, Shi Hao Tan, Phuong Cao Thi Ngoc, Stella Amanda, Alice Wei Yee Yam, Wei-Siang Liau, Zhiyuan Gong, Lee N. Lawton, Daniel G. Tenen, Takaomi Sanda. _National Univ. of Singapore, Singapore, Singapore_.

The oncogenic transcription factor TAL1/SCL is abnormally expressed in 40-60% cases of T-cell acute lymphoblastic leukemia (T-ALL) cells. It induces an aberrant transcriptional program with its regulatory partners (E-proteins, LMO1/2, GATA3, RUNX1 and MYB) in malignant T cells. However, the critical factors that are directly activated by the TAL1 transcriptional complex and contribute to T-ALL pathogenesis are largely unknown. Here, we identified ARID5B, an AT-rich interactive domain (ARID) family DNA binding protein, as a collaborating oncogenic factor involved in the transcriptional program in T-ALL. Our result indicated that ARID5B expression is downregulated at the CD4, CD8 double negative 2-4 stages in normal thymocytes, while it is activated by the TAL1 complex in human T-ALL cells. The enhancer located approximately 135 kb upstream of the ARID5B gene locus is associated with a super-enhancer in multiple T-ALL samples but not in normal thymus. These data suggested that ARID5B is aberrantly activated in T-ALL cells. Interestingly, ARID5B-bound regions are predominantly associated with active transcription, as shown by the active histone marks (H3K27ac, H3K4me1 and H3K4me3) by ChIP-seq analysis. ARID5B and TAL1 frequently co-occupy target genes and coordinately control their expression. Notably, ARID5B positively regulates the expression of TAL1 and its regulatory partners (GATA3, RUNX1 and MYB). In addition, ARID5B activates the expression of the oncogene MYC. Importantly, ARID5B is required for the survival and growth of T-ALL cells in vitro, and forced expression of ARID5B in immature thymocytes results in thymus retention, radio-resistance and tumor formation in zebrafish. Our results indicate that ARID5B reinforces the oncogenic transcriptional program by positively regulating the core regulatory circuit and the oncogene MYC in T-ALL, thereby contributing to T-cell leukemogenesis.

#3337

Nuclear S1P binds PASB domain of HIFα to facilitate HIFα-HIF1β heterodimer formation in hypoxic TNBC cells.

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

Triple-negative breast cancers (TNBCs) have increased expression of hypoxia-inducible factor 1α (HIF1α) and HIF-direct genes that associated with decreased overall survival in patients. Previously we have shown that nuclear sphingosine-1-phosphate (S1P), a lipophilic pro-cancer signal mediator, a product of the catalytic reaction by sphingosine kinase 2 (SphK2), directly binds to and inhibits histone deacetylases-1 and -2 (HDAC1 and 2), thus, altered epigenetic landscape in breast cancer cells. SphK2/S1P signaling is essential for metastatic TNBC cell growth and survival. We hypothesize that SphK2/S1P epigenetically regulates HIFα functions in TNBC cells; TNBC subset with elevated SphK2 and HIF1α would be a marker for SphK2-mediated HIFα targeted therapy. METABRIC cohort gene expression analysis revealed that the mRNA expression of HIF1α mRNA and downstream angiogenesis gene, vascular endothelial growth factor B (VEGFB) are associated with overall poor survival for TNBC patients compared to non-TNBC patients. Here, we demonstrate that a subset of TNBC patient tissue samples nuclei has elevated HIF1α and SphK2 protein expressions. SphK2/S1P markedly enhances hypoxia-induced HIFα proximal promoter histone acetylation. SphK2/S1P is found in the nuclear protein complexes with HIFα. Moreover, S1P is docked well at the PASB domain of HIFα and facilitate HIFα-HIF1β heterodimer formation in hypoxia. SphK2/S1P markedly augmented HIF1α direct target gene HIF2α and VEGFA promoter occupancy in hypoxia. Down-regulation of SphK2 with siRNA markedly attenuated transcription of HIFα-direct target genes associated with cancer stem cells, angiogenesis, and metastasis. In agreement with an oncogenic role of SphK2, selective inhibition of SphK2 markedly reduced nuclear histone acetylation, HIF1α expression, and in vivo human TNBC tumor growth in mice. Our data strongly suggested that TNBC subset with elevated SphK2 and HIF1α would be a good candidate for SphK2 targeted therapy. Supported by Health Research, Inc. (HRI) Grant 71-4084-01 (NCH).

#3338

BP1 induces an epithelial to mesenchymal transition in breast cancer cells by modulating the Twist/IL6 pathway.

Farhad Vesuna,1 Bin-Jin Hwang,2 Jinguen Rheey,2 Mamta Giri,2 Mandeep Gill,2 Sidney W. Fu,2 Ashley Irving,1 Ala Lisok,1 Yehudit Bergman,1 Venu Raman,1 Patricia E. Berg2. 1 _Johns Hopkins University School of Medicine, Baltimore, MD;_ 2 _George Washington Univ. Medical Ctr., Washington, DC_.

Background. BP1 (Beta Protein 1) belongs to the Distal-less family of homeobox genes. We have demonstrated that BP1 is activated in over 80% of invasive ductal breast tumors, where it is associated with breast cancer progression. The mechanism(s) of BP1 involvement in breast cancer progression, invasion and metastasis are still not known. Homeobox genes contribute to the epithelial to mesenchymal transition (EMT). During EMT, epithelial cells acquire mesenchymal features which lead to motility, invasiveness and resistance to apoptosis. EMT is also characterized by changes in apico-basal polarity and a dramatic remodeling of the cytoskeleton. During progression toward metastasis, cancer cells acquire a mesenchymal gene expression phenotype and increased motility. This transition allows the tumor cells to metastasize and establish secondary tumors at distant sites. One of the drivers of the EMT is the transcription factor Twist. Twist is a member of highly conserved family of basic helix-loop-helix transcription factors and is involved in the specification and differentiation of mesenchymal tissue in embryos. Twist overexpression can induce EMT, generate cancer stem cells, and promote metastasis in vivo. We hypothesized that BP1 might promote cancer metastasis and invasiveness mediated though Twist and the EMT.

Methods. We generated BP1 overexpressing cell lines, MCF-7/BP1 and HS578T/BP1, along with the associated vector controls. Expression of BP1 was tested by both protein and transcript levels by Western blotting and qRT-PCR, respectively. BP1 binding to Twist was assayed by chromatin immunoprecipitation. Confocal microscopy was used to localize BP1 expression and Boyden chamber assays were employed to assess the migratory and invasive ability of these cells. ELISA assays were employed to study IL6 expression in BP1 expressing cells.

Results. BP1 expression led to an increase in Twist expression. Mechanistically, we demonstrated that BP1 bound proximally to the Twist promoter and regulated its expression. BP1 induced an EMT in both cell lines as seen by (a) lack of expression of E-cadherin, and an increase of vimentin and fibronectin expression, as well as (b) morphological changes, including a spindle-like, and more migratory phenotype. We also observed that BP1 induced expression of the cytokine IL6 in both cell lines. We conclude that BP1 regulates the EMT in breast cancer cells via the Twist and IL6 pathways.

#3339

Modulating TAK1 expression through the inhibition of GSK3 impairs YAP/TAZ oncogenic functions in pancreatic cancer.

Raffaela Santoro, Marco Zanotto, Geny Piro, Carmine Carbone, Giampaolo Tortora, Davide Melisi. _Univ. of Verona, Verona, Italy_.

Background: Resistance to chemotherapeutic drugs poses one of the greatest challenges in pancreatic cancer (PC) treatment. TGFβ-activated kinase 1 (TAK1) has been demonstrated to drive chemoresistance in PC through the phosphorylation-dependent activation of the NF-κB transcription factor. The transcriptional regulators Yes Associated Protein (YAP) and transcriptional coactivator with a PDZ binding domain (TAZ) are central determinants of malignancy and have been identified as a critical oncogenic effectors of KRAS in PC. We hypothesized that TAK1 could drive PC aggressiveness by sustaining YAP and TAZ oncogenic activities.

Methods: TAK1 expression was silenced by shRNA in AsPC1, Panc1, and MDA-Panc28 cell lines. GSK3 was targeted by using the small molecule inhibitor LY2090314. The expression of TAK1, YAP/TAZ and their target genes in PC cell lines was studied by both Western blot and qPCR. Cell migration was assessed by transwell assays, and stemness by both spheroid formation and percentage of cancer stem cells (CSC). SRB assays were used to assess the in vitro chemopotentiation of nab-paclitaxel, gemcitabine, oxaliplatin, and SN-38. In vivo activity of LY2090314 alone or in combination with nab-paclitaxel was evaluated in an orthotopic nude mouse model with luciferase-expressing AsPC1 tumors. Expression of YAP/TAZ target genes in tumor samples was measured by both qPCR and IHC.

Results: knockdown of TAK1 resulted in inhibition of K48-linked ubiquitination and proteasomal degradation of YAP and TAZ, which was independent on TAK1 kinase activity. Significant reduction of proliferation, migration and chemoresistance of shTAK1 PC cell lines was also observed. Treatment with LY2090314 induced silencing of TAK1, as well as a remarkable reduction of the expression of both YAP and TAZ and their targets. In combination, the GSK3 inhibitor strongly potentiated the cytotoxic activities of chemotherapeutic agents in all three PC cell lines. In nude mice, i.p. admnistration of LY2090314 plus nab-paclitaxel significantly reduced tumor burden.

Conclusions: Our study identified GSK3 and TAK1 as major modulators of YAP and TAZ stability and oncogenic activities, thus indicating that inhibition of TAK1 expression by modulating GSK3 activity could represent a valid approach to revert in vivo the intrinsic chemoresistance of PC.

#3340

Endogenous, CRISPR-mediated overexpression of MYC family members as a framework to discover MYC-specific vulnerabilities.

Marcel A. Dammert, Martin L. Sos. _University of Cologne, Köln, Germany_.

MYC activation reshapes cellular transcription, promotes cell growth, cell cycle progression and transformation and therefore MYC is considered a strong driver of tumor development. The MYC family includes c-MYC, MYCN, and MYCL that are typically associated with distinct tumor types. Based on the structural properties of these transcription factors MYC family members are considered undruggable targets. However, potential vulnerabilities associated with the cellular MYC status may provide a basis for therapeutic intervention. Currently, there is a lack of comparative studies assessing the specific contribution of each MYC family member to tumor development. Established cell lines in which MYC family members are overexpressed differ greatly in their genetic background, thereby impeding the analysis of MYC family member specific effects. Furthermore, the complexity of MYC regulation and MYC biology is not adequately recapitulated by traditional, heterologous MYC overexpression approaches.

Therefore, we set out to establish an endogenous overexpression system using a CRISPR activation (CRISPRa) approach to activate the individual MYC family members for a systematic comparison of their impact on target gene activation, cell growth and drug sensitivity. Using sgRNAs specific to the promoter regions of the particular MYC family members in combination with a nucleolytically inactive Cas9-VP64 fusion protein, we successfully achieved transcriptional upregulation of all three MYC family members in multiple cell lines including NIH3T3 and MEF cells. The increase in MYC mRNA resulted in markedly elevated MYC protein levels. In addition, MYC activation enhanced transcription of canonical MYC target genes like NPM1, FBL, and 45S pre-rRNA suggesting a functional impact of upregulated MYC. Moreover, activation of c-Myc led to increased anchor-independent growth of cells in a soft-agar colony formation assay indicating that high c-Myc levels induce transformation in contrast to N-Myc or L-Myc. In line with previous studies that implicated upregulated c-Myc as an initiator of genome instability, we observed elevated levels of DNA damage by monitoring phosphorylated histone variant H2AX (γH2AX) in cells with activated c-Myc whereas N-Myc or L-Myc failed to induce γH2AX. Activation of c-Myc further led to increased sensitivity to cisplatin and other chemotherapeutic agents.

In conclusion, we established an endogenous MYC overexpression model system that enables a systematic comparison of the different MYC family members and their specific contribution to tumor initiation, growth and drug sensitivity. The system successfully recapitulated previous observations of MYC activation and can serve as a platform to probe for MYC family member specific vulnerabilities and thereby pave the way for novel, targeted therapy approaches in MYC-driven cancer.

#3341

Inhibiting the destruction of the oncogene ZNF217 promotes breast cancer metastasis to lung.

Beth Facchine,1 Junmin Wu,1 Megan Fabry,1 Matt Messana,1 William Kaliney,2 Laurie Littlepage1. 1 _University of Notre Dame, South Bend, IN;_ 2 _South Bend Medical Foundation, South Bend, IN_.

The oncogene and transcription factor ZNF217 is overexpressed in 20-30% of breast cancers. Its overexpression correlates strongly with poor prognosis in patients and causes accelerated tumor progression, metastasis, and chemoresistance in vivo. While several studies have examined the regulation of ZNF217 at the gene and mRNA levels, little is known about how ZNF217 is regulated as a protein. In this study we identify the regions of ZNF217 that are required for protein turnover and other cancer phenotypes, including increased invasion and metastasis. Due to the high expression and aberrant localization of ZNF217 in some human breast tumors, both ZNF217 protein expression levels and localization of ZNF217 may be critical determinants of ZNF217's function in vivo. To investigate the role of ZNF217 protein on cancer progression, we first examined ZNF217 protein expression in human breast tumors by Western analysis. In addition to full-length ZNF217 protein, smaller ZNF217 proteins were detected and were even more prominent than full-length ZNF217 in both human breast tumors and cell lines, but the importance of these smaller isoforms remains unknown. To investigate the function of the smaller ZNF217 proteins and to determine the requirements of the regions of ZNF217 protein to promote breast cancer, we examined the consequences of removing regions of ZNF217 using truncation mutants overexpressed in human breast cancer cells. As a structure-function analysis, we tested both truncation mutants and full-length ZNF217 for their ability to promote phenotypes usually associated with ZNF217 overexpression, including protein turnover, increased branching of organoids, tumor burden, and metastasis. For protein turnover, these mutants were expressed in breast cancer cells and treated with cycloheximide to inhibit protein synthesis. This assay identified the regions of ZNF217 required for ZNF217 protein turnover. Further analysis of the sequence in this region identified a putative destruction box required for the protein turnover. We next determined which regions of ZNF217 are required for organoid branching and metastatic tumor burden. Both the N-terminus and C-terminus of ZNF217 were required for organoid branching. Interestingly, the same region identified to be required for ZNF217 protein turnover was not required for metastatic tumor burden. In fact, overexpression of breast cancer cells expressing stabilized ZNF217 truncation mutants significantly increased the metastatic tumor burden in vivo when assayed by tail vein injection. These results suggest that regulation of ZNF217 protein expression may be clinically valuable in generating a clinical assay used to generate personalized treatment strategies for patients with advanced metastatic breast cancer and high ZNF217 expression.

#3342

Truncated glioma-associated oncogene homolog 1 (tGLI1) is a novel mediator of mesenchymal subtype of glioblastoma and a novel transcriptional activator of CD44.

Tadas K. Rimkus. _Wake Forest University Health Sciences, Winston-Salem, NC_.

The subpopulation of stem-like cells, or glioma stem cells (GSCs), is a key driver of tumor initiation, recurrence, and chemoresistance. Recently, patient-derived GSC lines were identified to contain two mutually exclusive subtypes termed proneural and mesenchymal. Mesenchymal GBM and GSCs were found to be more aggressive and radioresistant; however, the molecular pathways supporting mesenchymal GBM and mesenchymal GSCs are not well understood. To address this knowledge gap, we examined truncated glioma-associated oncogene homolog 1 (tGLI1) for its role in mesenchymal subtypes of GBM and GSCs. Discovered in our laboratory, tGLI1 is a novel alternatively spliced, gain-of-function variant of GLI1 zinc-finger transcription factor with an in-frame deletion of 41 codons retains all of the known functional domains of GLI1, but gains the ability to activate genes not regulated by GLI1. In this study, we report for the first time that tGLI1 is a tumor-specific transcription factor that facilitates GBM growth, enriches in the mesenchymal subtype of GBM and GSCs, and promotes mesenchymal GSCs via transcriptionally activating CD44 expression. Using an orthotopic GBM xenograft mouse model, we observed that tGLI1-overexpressing tumors grew more aggressively, with increased proliferation and angiogenesis, compared to control and GLI1-overexpressing xenografts. IHC analysis using tGLI1- and GLI1-selective antibodies we developed showed that tGLI1 was significantly expressed in GBM specimens but undetectable in normal brains whereas GLI1 was expressed in both tissues. Datamining further showed that tGLI1 activation signature (tGAS) was correlated with glioma grade, tumor angiogenesis, and poor overall survival, and that GBMs with high tGAS were enriched with mesenchymal GBM and GSC gene signatures. Neurospheres contained increased levels of tGLI1, but not GLI1, compared to the monolayer culture. Mesenchymal GSCs expressed more tGLI1 than proneural GSCs. Ectopic tGLI1 expression significantly enhanced the ability of GSCs to yield neurospheres in vitro and form aggressive tumors in mouse brains. Finally, we examined the ability of tGLI1 to regulate stemness genes and found that tGLI1 binds to and transactivates the promoter of the CD44 gene, a marker and mediator for mesenchymal GSCs. Collectively, these findings advanced our understanding of GBM biology by establishing tGLI1 as a novel transcriptional activator of CD44 and a novel mediator of mesenchymal GBM and GSCs.

#3343

Identifying kinases and phosphatases that regulate STAT3 activity.

Elina Parri, Heikki Kuusanmäki, Arjan van Adrichem, Meri Kaustio, Laura Turunen, Krister Wennerberg. _Institute for Molecular Medicine Finland (FIMM), Helsinki, Finland_.

The STAT3 transcription factor is hyperactivated in many cancers where it induces cancer progression by driving drug resistance, metastasis and pro-cancer inflammation. We therefore sought new strategies to target the activation of STAT3. Many kinases and signalling pathways can cause STAT3 activation, and it is not well understood how this is regulated in cancer cells. In some rare solid and hematological cancers, such as NK-/T-cell large granular lymphocytic leukemia and inflammatory hepatocellular adenomas, STAT3 has also been found to be mutated, resulting in a constitutively active STAT3 protein.

In testing mock transfected vs. STAT3(Y640F)-transfected cells, as a means to introduce activated STAT3 in already transformed cells, in a screen against 525 anticancer agents, we identified that activated STAT3 protects the cells from cytotoxic effects of several classes of anticancer compounds. Therefore, we performed RNAi screens to identify kinases and phosphatases that regulate STAT3 activity and therefore may act as drug-sensitizing targets. Using STAT3 transcription-driven luciferase reporter cell lines expressing active wild type (WT) or mutant (Y640F) STAT3, we identified several candidate genes (CDC7, CDK8, CSK, CSNK2A1, PI4KII, PTPRH, DDR2) whose downregulation led to differential STAT3 transcriptional activity. Strikingly, none of the hits caused highly selective effect on either mutant or wild-type STAT3 expressing cells, suggesting that the phosphoregulation of hyperactive mutant and IL6 induced wild-type STAT3 are mechanistically similar. Using small molecule inhibitors targeting CDK8, CSNK2A1, DDR2 and CDC7, we confirmed that STAT3 transcriptional activity was inhibited in a dose and time dependent fashion without severely affecting cell viability. Small molecules targeting of CSNK2A1, DDR2 and CDC7 kinase activity caused a slow inhibition of STAT3 transcriptional activity where the strongest effects were only seen after 72 h. CDK8 kinase inhibition, on the other hand, reduced STAT3 activity effectively already after 4 h. This suggests that CDK8 regulates STAT3 activity more directly than CSNK2A1, DDR2 and CDC7. Inhibition of CDK8 and CSNK2A1 resulted in decrease of STAT3 phosphorylation in the STAT3(Y640F)-transfected cells, and knockdown of CSNK2A1 inhibited the nuclear localization of STAT3.

In conclusion, we have found that hyperactive STAT3(Y640F) can protect cancer cells from several drug classes, mimicking the case when hyperactivated wild type STAT3 is acting as malignancy promoting protein in cancers. In a functional genetics screen, we found six kinases and a phosphatase that regulate transcriptional activity of STAT3. Using small molecule inhibitors, we could confirm that targeting the hit kinases regulate STAT3 transcriptional activity. Together, our data suggest that there are multiple kinases that may be targeted to counteract STAT3 mediated drug resistance in cancers.

#3344

ASPSCR1-TFE3 directs epigenetic-induced autophagy in alveolar soft part sarcoma.

Jared J. Barrott,1 Ian Lock,2 Bodrie Jensen,2 Amir Pozner,2 Kevin B. Jones2. 1 _Idaho State University, Pocatello, ID;_ 2 _University of Utah, Salt Lake City, UT_.

Altered metabolism is considered to be one of the new hallmarks of cancer. Autophagy is one major avenue of altered cancer metabolism, enabling cell survival under metabolic stress and promoting chemoresistance. The nuclear localization of MiTF/TFE3 family transcription factors has associated with upregulated transcription of autophagy genes in pancreatic cancer. Alveolar soft part sarcoma (ASPS) is a rare but deadly soft-tissue sarcoma, with a predilection for adolescent and young adult victims. ASPS is noteworthy for its resistance to traditional cytotoxic chemotherapies. It consistently associates with a t(X;17) chromosomal translocation that produces the ASPSCR1-TFE3 target gene, bearing the DNA-binding domain from TFE3 and protein interaction domains from ASPSCR1. We have demonstrated that conditional expression of ASPSCR1-TFE3 is sufficient to drive alveolar soft part sarcomagenesis in the mouse with complete penetrance. Mouse tumors recapitulate human ASPS histology and transcriptomes. Our objective was to identify the direct targets of ASPSCR1-TFE3 and how these targets confer resistance to doxorubicin. The human cell lines ASPS-1 and FUUR-1, as well as mouse tumors driven by expression of ASPSCR1-TFE3 were subjected to nuclear fractionation and chromatin immunoprecipitation using antibodies against ASPSCR1 and RNAPol2. Cells and tumors were further characterized for their presence of auotphagic flux by detection of LC3-II and abundance of lysosomal proteins LAMP1 and CTSD. Furthermore, cells treated with doxorubicin were analyzed by gas-chromatography mass spectrometry (GC-MS) for metabolites involved in cellular respiration and glycolysis. Lastly, mice were treated with either control, monotherapy of chloroquine (15 mg/kg) or doxorubicin (10 mg/kg), or combination therapy for up to 5 months. Mice on combination therapy showed a statistical improvement in survival of 3 months over control and doxorubicin treatments. We report not only the first genome-wide localization of the ASPSCR1-TFE3 oncoprotein on chromatin from ASPS cell lines and mouse tumors, but also its association with actively transcribed genes. Among these are found many genes related to autophagy. We demonstrate high expression of autophagy-related proteins at baseline conditions in human tumors, cell lines and mouse tumors. We also demonstrate active autophagic flux even in the absence of stress conditions. Inhibition of autophagy strongly synergizes with chemotherapy to kill ASPS cells, suggesting a mechanism for resistance. Furthermore, mice treated with combination therapy of chloroquine and chemotherapy significantly extends life 3 months beyond control mice. We have therefore demonstrated the direct targets of ASPSCR1-TFE3 in ASPS, including a number of autophagy genes that are expressed in these tumors. Inhibition of autophagy in ASPS causes the tumor cells to be more susceptible to chemotherapeutic stress.

#3345

MAZ is a downstream target of CCN1 and promotes aggressive behavior of pancreatic cancer cells via CRAF-ERK signaling pathway.

Gargi Maity,1 Inamul Haque,1 Arnab Ghosh,1 Gopal Dhar,1 Vijayalaxmi Gupta,1 Sandipto Sarkar,1 Imaan Azeem,1 Douglas McGregor,1 Abhishek Choudhary,1 Donald R Campbell,2 Sushanta K. Banerjee,1 Snigdha Banerjee1. 1 _VA Medical Ctr., Kansas City, MO;_ 2 _Saint Lukes Hospital, Kansas City, MO_.

MAZ (Myc-associated zinc-finger protein) is a transcription factor with dual roles in transcription initiation and termination. Deregulation of MAZ expression has been shown to associate with the progression of pancreatic ductal adenocarcinoma cancer (PDAC). However, underlying mechanisms of action of MAZ in PDAC progression are largely unknown. Here we present evidence that MAZ mRNA expression and protein level were increased in human PDAC cell lines, tissue samples, subcutaneous tumor xenograft model in a nude mouse, and spontaneous cancer in the genetically engineered PDAC mouse model. We found that MAZ is predominantly expressed in pancreatic cancer stem cells. Functional analysis demonstrates that the depletion of MAZ in PDAC cells results in inhibition of invasive phenotypes such as phenotypic shift, migration, invasion, and the sphere-forming ability of PDAC cells. Mechanistically, we found no direct effect of MAZ on the expression of K-Ras mutants, but evidently MAZ increases the activity of CRAF-ERK-signaling, which is a downstream signaling target of K-Ras. Activation of CRAF-ERK-signaling by MAZ is mediated via p21-activated protein kinases (PAK) and protein kinase B (AKT/PKB) signaling cascades and promotes invasive phenotypes of PDAC cells. Moreover, a matricellular oncoprotein CCN1 regulates MAZ expression in PDAC cells. Thereby, we proposed that CCN1-induced expression of MAZ promotes invasive phenotypes of PDAC cells is not through direct K-Ras activation as presently thought but instead through the activation of CRAF-ERK signals. Collectively, these studies highlight key players that could help in clinical management, prognosis, and therapeutic strategies.

This project is funded by VA Merit Award grants (SB & SKB).

#3346

PRMT5-mediated methylation of YBX1 regulates NF-kB activity in colorectal cancer.

Antja-Voy Hartley,1 Benlian Wang,2 Masaru Miyagi,2 Rasika Mundade,1 James Hamilton,1 Tao Lu1. 1 _Indiana University School of Medicine, Indianapolis, IN;_ 2 _Case Western Reserve University, Cleveland, OH_.

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the US with a staggeringly less than 14% survival rate for patients with metastatic disease. This poor clinical outcome for CRC patients is largely attributed to the lack of understanding of the factors that drive CRC progression, leading to a dearth of available treatment strategies for this disease. Today, treatment modalities and clinical management of colorectal cancer (CRC) are fundamentally based on the success of controlling specific tumorigenic pathways. Interestingly, aberrant activation of the transcription factor nuclear factor ĸB (NF-ĸB) is increasingly recognized as a crucial player in CRC progression. Therefore, understanding the mechanisms underlying regulation of NF-ĸB holds great promise for devising new therapeutic strategies for CRC. Recently, we identified the oncogenic protein, Y-box binding protein 1 (YBX1), as a novel activator of NF-κB. Since YBX1 overexpression has also been linked to poor patient outcome in CRC, we sought to further understand whether this YBX1/NF-ĸB axis could be a potential target for CRC treatment. In this study, we discovered that YBX1 is methylated on arginine 205 (YBX1-R205me2), an event that is critical for YBX1-mediated NF-ĸB activation and target gene expression. Additionally, co-immunoprecipitation studies revealed that the R205 to alanine (A) mutant (YBX1-R205A) also significantly diminished the interaction between YBX1 and the p65 subunit of the transcriptionally active NF-ĸB, demonstrating a novel mechanism by which methylation of YBX1 mediates protein-protein interactions. Overexpression of YBX1-R205A significantly attenuated the migration, proliferation, and anchorage-independent growth of a panel of CRC cells, suggesting that YBX1-R205me2 is essential to the oncogenic functions exerted by the YBX1/NF-ĸB axis in CRC. Furthermore, we revealed that protein arginine methyltransferase 5 (PRMT5) is responsible for the methylation of YBX1-R205. Collectively, our novel findings present a complex picture of the sophisticated regulation of NF-ĸB through PRMT5-mediated YBX1-R205 methylation and suggest that pharmacological disruption of the YBX1/NF-κB axis using PRMT5 inhibitors could serve as the basis for new therapeutics that impede YBX1/NF-κB-driven CRC progression.

#3347

SOX9 is a critical regulator of triple-negative breast cancer growth and invasion.

Yanxia Ma, Jonathan Shepherd, Dekuang Zhao, Lakshmi Bollu, Jamal Hill, Yun Zhang, Abhijit Mazumdar, Powel H. Brown. _MD Anderson Cancer Center, Houston, TX_.

Background: Triple-negative breast cancers (TNBCs) have the worst prognosis of all breast cancers, and have few available therapies other than non-specific and toxic chemotherapy. To identify novel targets for TNBCs, we investigated expression levels of transcription factors (TFs) in TNBCs compared to those in Non-TNBCs. Our previous analyses identified the TFs highly expressed in TNBCs. Of these, six SRY (Sex Determining Region Y)-related HMG-box (SOX) TFs were highly expressed in TNBCs as compared to non-TNBCs. SOX genes belong to a superfamily of gene. There are approximately 20 SOX genes in humans and mice.

Hypothesis: SOX TFs are important regulators of TNBC cell growth and metastasis.

Material and Methods: We measured breast cancer cell growth using an automated cell counting assay. Cell migration and invasion were detected by transwell assays in non-TNBC (MCF7 and ZR75-1) and TNBC (MDA231 and MDA468) cells. DOX-inducible SOX9-knockout (KO) cell lines were established in MDA231, MDA468, and LM2 cell lines using an inducible Cas9-CRISPR system. A SOX9 expressing lentivirus was used to overexpress SOX9, and siRNAs were used to knockdown (KD) SOXs in the different breast cancer cells. Cell cycle phase and apoptosis were detected using flow cytometric analysis. Protein and mRNA levels of SOX9 in cell lines were examined by western blotting and qRT-PCR assays. SOX9 RNA expression data were obtained from the Oncomine Database. The Curtis dataset was used to analyze survival according to SOX9 expression using Kaplan-Meier survival curves and the statistical significance was determined using the log-rank test.

Results: We performed a screen using specific siRNA targeting the 6 SOX transcription factors (SOX4, 6, 8, 9, 10 and 11) that are high-expressed in TNBC, and measured the effect of loss on SOX expression on TNBC cell growth and invasion. SOX4, 6, 8, 9, and 10-KD using siRNA caused decreased cell proliferation (by 30% or more) of MDA231 and MDA468 TNBC cells, but not of MCF7 and T47D cell lines (non-TNBC). SOX11 or 17-KD had no effect on breast cancer growth. SOX9-KD and SOX9-KO decreased cell migration and invasion of MDA231 and MDA468 cells. Reduced expression of SOX9 also inhibited the in vivo growth and metastasis of MDA MB-231 and LM2 cells in mice. In contrast, overexpression of SOX9 in MCF7 and ZR75-1 cells increased cell migration and invasion. Our studies also demonstrated that loss of SOX9 induced a G1 to S cell cycle arrest and apoptosis. In addition, high expression of SOX9 was correlated with worse overall survival and 5-year disease-free survival in patients with TNBC breast cancer.

Conclusion: Our results demonstrate that the SOX9 acts as an essential molecule regulating TNBC growth and invasion. In the future, it may be possible to target SOX9 and its downstream genes to treat TNBC and prevent its metastasis.

Grant Support: These studies were supported by a Susan G. Komen for the Cure Promise Grant (PB).

#3348

The role of SOX2 in promoting resistance to AR-targeted therapies in prostate cancer.

Larischa de Wet,1 Anthony Williams,1 Marc Gillard,1 Steve Kregel,2 Tzintzuni Garcia,1 Erin McAuley,1 Ryan Brown,1 Donald Vander Griend1. 1 _University of Chicago, Chicago, IL;_ 2 _University of Michigan, Ann Arbor, MI_.

Prostate cancer is the most common type of cancer in men, with approximately 181,000 new cases diagnosed in 2016. Due to the central role of the androgen receptor (AR) in prostate development and more importantly prostate cancer cell survival and proliferation, strategies targeting AR have been the mainstay therapy for over 70 years. However, despite potent inhibition of AR pathway activation, many patients develop castration-resistant prostate cancer (CRPC). Second-line therapies, such as enzalutamide, has increased overall survival in CRPC, but resistance to these therapies inevitably emerges, suggesting that other pathways apart from AR signalling are contributing to the failure of treatments. We have previously demonstrated that SOX2 [SRY (sex determining region Y)-box 2] is an AR-repressed gene that is expressed in a large percentage of high Gleason grade prostate tumours, as well as in most metastases. Additionally, expression of SOX2 within a castration-sensitive cell line is sufficient to enable castration-resistant tumor formation in vivo, and enzalutamide resistance in vitro. In prostate cancer, SOX2 is not found with its normal stem cell co-factors, NANOG and OCT4, leading to the hypothesis that SOX2 is interacting with a novel co-factor in prostate cancer to regulate expression of genes promoting castration-resistance. A chromatin-immunoprecipitation experiment in a castration-resistant prostate cancer cell line was performed to determine if SOX2 binds the same genes and activates similar pathways in prostate cancer as in embryonic stem cells. Approximately half of the SOX2 bound genes in the CRPC cell line were shared with the embryonic stem cell line, and these genes are involved in signalling pathways and maintenance of stem cell pluripotency; the genes bound uniquely in the CRPC cell line are present in pathways involved with metabolic processes. To identify potential binding partners of SOX2, computational analysis of the sites bound by SOX2 in the promoter regions of target genes determined the FOXA1 motif is within close proximity to the SOX2 motif, and the physical interaction of these proteins was confirmed through co-immunoprecipitation. Further understanding of SOX2 target genes and the pathways that SOX2 activates in the presence of enzalutamide will be crucial to identify mechanisms of resistance and enable the development of novel therapies for castration-resistant prostate cancer.

#3349

TEAD4 expression in human hepatocellular carcinoma correlates with poor prognosis and promotes tumor growth and migration.

Nadia Tosti, Manuela Lanzafame, Viola Paradiso, Charlotte K. Ng, Salvatore Piscuoglio, Cristina Quintavalle, Luigi Maria Terracciano. _Institute of Pathology, Basel, Switzerland_.

Background: TEAD family is a group of four transcription factor members, including TEAD1, TEAD2, TEAD3 and TEAD4. These genes are known to be involved in the regulation of different genes associated with cell proliferation, differentiation and apoptosis. TEAD proteins represent the major downstream effectors of the conserved Hippo signalling pathway, known to be deregulated in different cancer entities. In the present study, we sought to explore the expression of TEAD4 in hepatocellular carcinoma (HCC) and its effect in in vitro models. Methods: We evaluated TEAD4 expression at mRNA level using data from The Cancer Genome Atlas (liver dataset) and at protein level using a tissue microarray containing 192 HCCs, 108 cirrhotic tissues and 79 normal liver samples. For in vitro experiments, two liver cancer cell lines were stably overexpressed and downregulated for TEAD4 to characterize its role in in vitro models, in terms of proliferation, migration and anchorage-independent growth. Results: TEAD4 was found overexpressed in a subset of HCCs in the TCGA dataset and its overexpression correlated with worst overall survival (p=0.03). The tissue microarray analysis showed a similar trend not only in the HCCs but also in cirrhotic tissues. The in vitro assays demonstrated that the overexpression of TEAD4 promoted tumour growth and migration as well as increased foci formation capacity. On the other hand, TEAD4 knockdown was able to revert the phenotype, suggesting a putative oncogenic role of TEAD4 in hepatocarcinogenesis. Conclusions: Finally, TEAD4 is frequently overexpressed in HCCs and in cirrhotic tissues and its expression is associated with poor prognosis. Furthermore, in vitro evidence suggests that TEAD4 may act as oncogene and may have a pivotal role in hepatocarcinogenesis.

#3350

Delineating the p63-driven oncogenic network and epigenomic signature in salivary gland tumors.

Akinsola Oyelakin, Christian Gluck, Satrajit Sinha, Rose-Anne Romano. _State University of New York at Buffalo, Buffalo, NY_.

Head and Neck Squamous Cell Carcinomas (HNSCCs) constitute a diverse group of cancers that share their origin from the epithelium-rich upper aero-digestive tract. These cancers include oro-pharyngeal, laryngeal and salivary gland carcinomas, each endowed with a wide-ranging spectrum of distinct pathological subtypes and defined molecular and genetic determinants. Salivary gland neoplasms in particular, epitomizes this diversity and remain relatively less studied and ill-understood. To address this knowledge void and to get a better understanding of the molecular etiology of salivary gland tumors, we have focused our studies on p63, an oncogenic master transcription factor that is overexpressed in HNSCC, including salivary glands tumors. By utilizing the A253 cells, a representative cell-line isolated from a human submandibular carcinoma, here we have performed a comprehensive transcriptomic and epigenomic characterization of the p63-driven and global transcriptional circuitry. Our ChIP-seq based studies have identified the complete repertoire of the genome-wide enhancer regions in the A253 cells and importantly have unearthed a cohort of H3K27Ac-enriched super enhancers specifically enriched in p63 binding, highlighting the important cell-lineage directing role for p63 in these cells. Furthermore, RNA-seq based examination of the underlying changes in gene expression following the loss of p63 have also unearthed key mediators of the p63-cistrome that are likely to impinge upon cellular and oncogenic functions. In parallel, we have leveraged these newly developed resources with complementary genomic information from TCGA, and specifically HNSCC datasets, to examine the relevancy of our findings in the tumor context. Taken together, our multipronged approach has revealed several novel p63 target-enriched gene regulatory networks that span the breadth of core and emerging hallmarks of cancer. In particular, we have unveiled a p63-dependent network of several E26 transformation-specific (ETS) family of transcription factors which act downstream of p63 and act in concert to generate pro-oncogenic and anti-tumor suppressive forces. Interestingly, our studies also reveal that p63 and its ETS co-partners serve as an upstream molecular switch favoring the repression of TGF-β and concurrent activation of BMP signaling pathways, which may contribute favorably to proliferative and survival circuitry in HNSCC. These observations and other broader genomic insights into cancer cell specific metabolism and transcriptional addiction that has emerged from our studies can be further exploited for the development of diagnostics and/or therapeutics against salivary gland cancers and more broadly HNSCCs.

#3351

Myc inhibition by Omomyc impairs melanoma growth and progression through genome-wide gene expression reprogramming.

Mariano F. Zacarias-Fluck, Génesis Martín, Daniel Massó-Vallés, Laia Foradada, Jonathan R. Whitfield, Marie-Eve Beaulieu, Laura Soucek. _VHIO, Barcelona, Spain_.

Myc encodes for a transcription factor deregulated in the vast majority of human cancers, mainly by amplification or constant upstream oncogenic signaling. Previous work from our laboratory showed that targeting Myc by transgenic expression of the Myc inhibitor Omomyc is an effective strategy to inhibit tumor progression and cause tumor regression in a wide array of mouse cancer models, without any evidence of toxicity in normal tissues. Our goal now is to assess whether Myc inhibition can be an effective approach to treat melanoma, the most dangerous form of skin cancer. For this purpose, we transfected human melanoma cell lines with a doxycycline-dependent vector expressing Omomyc-RFP and characterized the effect of Myc inhibition on proliferation and colony formation, as well as the expression of different cell cycle related proteins. We observed that Omomyc significantly reduced proliferation of a wide variety of cell lines, regardless of their driving mutations. In p53 wild type cells, Omomyc expression was accompanied by p53 stabilization and concomitant p21 upregulation, and in BRaf or NF1 mutated melanoma cells, by downregulation of cyclin D1. In order to elucidate the mechanism of action underlying this anti-tumorigenic effect, we performed a microarray analysis of A375 melanoma cells in the presence or absence of Omomyc-RFP expression. This analysis showed that Omomyc affected genome-wide gene expression. More in detail, Gene Set Enrichment Analysis showed that Omomyc significantly blunts the expression of both Myc and E2F targets, as well as RNA biogenesis, DNA replication and different cell cycle checkpoints, among many others relevant gene sets. Importantly, Omomyc significantly reduced the expression of genes related to melanoma metastases. Finally, in order to validate Omomyc therapeutic impact in vivo, A375 and SkMel147 cells were s.c. implanted into nude mice and the effect of Omomyc expression on tumor progression was evaluated. Our results show that Omomyc expression significantly impairs the growth of both cell lines, increasing the survival of tumor-bearing mice. These results combined clearly show that Omomyc-mediated Myc inhibition is an effective means to impair melanoma progression, by directly targeting Myc, inhibiting cell cycle and decreasing melanoma aggressiveness.

#3352

**HMGA1 chromatin remodeling protein induces** HOXB13 **to drive cancer stem cell properties and tumor progression in prostate cancer models.**

Lionel Chia, Lingling Xian, Guangjing Zhu, Mohammad Heydarian, William B. Issacs, Karen Reddy, Linda Smith Resar. _Johns Hopkins University, School of Medicine, Baltimore, MD_.

Increasing evidence suggests that cancer cells undergo chromatin remodeling and epigenetic reprogramming during tumor progression, although the underlying mechanisms remain poorly understood. The High Mobility Group A1 (HMGA1) chromatin binding protein is an architectural transcription factor that binds to AT-rich regions in DNA where it displaces histone HI and recruits transcriptional complexes to modulate gene expression. The HMGA1 gene is highly expressed during embryogenesis and in adult stem cells, but silenced postnatally in differentiated tissues. HMGA1 becomes re-expressed in most high-grade cancers and high levels portend adverse clinical outcomes. In prostate cancer (PCa), HMGA1 overexpression and protein immunoreactivity associates with high pathologic grade, although its function in this setting is unknown. To gain mechanistic into the role of HMGA1 in PCa, we silenced HMGA1 in 2 human PCa cell lines: 1) PC3-Epi, a PCa clone selected for epithelial properties, and 2) PC3-EMT, a more invasive PCa clone with mesenchymal properties. Silencing HMGA1 halts proliferation in both PC3-Epi and PC3-EMT cells. Cell morphology changed most dramatically in the PC3-EMT cells, transforming spindle-shaped, mesenchymal cells to more cuboidal, epithelial-like cells. Both migration and invasion were disrupted, but only in the more invasive PC-EMT cells. Colony formation and the stem cell property, three-dimensional (3D) sphere formation, were also blocked in cells with HMGA1 knock-down. To elucidate transcriptional networks downstream of HMGA1, RNA-seq was performed in both cell lines + HMGA1 silencing. We identified genes involved in cell signaling, protein synthesis, post-translational modifications, cell motility, mitotic spindle formation, and development. We focused on the HOXB13 developmental gene, which encodes a transcription factor involved in prostate development. Intriguingly, HOXB13 germline mutations are linked to familial PCa. We found that HOXB13 and HMGA1 are co-regulated in PCa cells by quantitative RT-PCR. HMGA1 occupies 2 sites within the HOXB13 promoter region by chromatin immunoprecipitation. Strikingly, silencing HOXB13 recapitulates HMGA1 phenotypes, impairing proliferation, colony formation, and 3D sphere formation. This work not only reveals a novel role for HMGA1 in regulating both cancer stem cell properties and tumor progression in PCa through HOXB13, but also suggests that targeting the HMGA1-HOXB13 pathway could be effective therapy in invasive PCa.

#3353

ZNF414 as a functionally relevant transcription factor in pancreatic and breast cancer cells.

Alejandra Rodríguez Martínez, Elisa M. Vuorinen, Anastasia Shcherban, Nina K. Rajala, Matti Nykter, Anne Kallioniemi. _Faculty of Medicine and Life Sciences, University of Tampere; BioMediTech Institute, Tampere, Finland_.

Background: The Zinc Finger Protein 414 (ZNF414) is a member of the krüppel C2H2-type zinc-finger protein family. ZNF414 is a cargo protein for Karyopherin α7 (KPNA7), a nuclear importer expressed during embryogenesis, absent in most adult tissues but re-expressed in cancer cells. KPNA7 is involved in promoting proliferation and maintaining nuclear morphology in several breast and pancreatic cancer cell lines. Similar effects on cell growth have been evidenced for ZNF414 using in vitro knock-down experiments. Other than this, the function of ZNF414 remains uncharacterized but, as a zinc finger protein with nuclear localization, it is likely to act as a transcription factor. This study aimed at identifying target genes and DNA binding motifs of ZNF414 in pancreatic and breast cancer cells. Methods: We used next generation sequencing methods on Hs700T and MCF-7 cell lines. RNA-seq was used to identify genes regulated by ZNF414 and ChIP-exo and ATAC-seq analyses to uncover its genomic binding regions. Samples for RNA-seq were collected 12h and 24h after transfection with siRNAs targeting ZNF414. For ChIP-exo and ATAC-seq experiments, cells were transfected to transiently overexpress V5-tagged ZNF414. Results: RNA-seq data analyses revealed 33 and 296 differentially expressed genes (DEGs) in Hs700T cells at 12h and 24h time points, respectively. The corresponding amounts of DEGs in MCF-7 cell line were 177 and 556. There were 23 and 108 DEGs in common to both cell lines at 12h and 24h, respectively. Interestingly, gene ontology analyses revealed enrichment of many functional categories related to cellular proliferation in both cell lines, providing a molecular explanation for the observed ZNF414-elicited phenotype. ChIP-exo data was processed using MACE tool and then combined with the ATAC-seq open chromatin signal to identify the most reliable peaks. We looked at the genomic location of these peaks and observed clear enrichment in the promoter and 5'UTR regions, indicating that ZNF414 is a classical transcription factor. Using MEME for de novo motif discovery analyses, we identified several putative binding motifs common to both cell lines, some of which were also found in promoters of DEGs. Conclusion: This study uncovered the transcriptional regulation that underlies the role of ZNF414 as inducer of cellular proliferation in pancreatic and breast cancer cells.

#3354

Chemically induced degradation of the transcription factor BCL6.

Nina Kerres,1 Steffen Steurer,1 Stefanie Schlager,1 Gerd Bader,1 Maureen Caligiuri,2 Christian Dank,1 John R. Engen,3 Peter Ettmayer,1 Daniel Gerlach,1 Thomas Gerstberger,1 Bingsong Han,2 Roxana E. Iacob,3 Dirk Kessler,1 David R. Lancia,2 Mayer Moriz,1 Nikolai Mischerikow,1 Klaus Rumpel,1 Renate Schnitzer,1 Tilman Voss,1 Xiaozhang Zheng,2 Andreas Zoephel,1 Norbert Kraut,1 Darryl McConnell,1 Mark Pearson,1 Manfred Koegl1. 1 _Boehringer-Ingelheim, Vienna, Austria;_ 2 _FORMA Therapeutics, Watertown, MA;_ 3 _Northeastern University, Boston, MA_.

The transcription factor BCL6 is a known driver of oncogenesis in lymphoid malignancies, including diffuse large B-cell lymphoma (DLBCL). Disruption of its interaction with transcriptional repressors interferes with the oncogenic effects of BCL6. We have used structure-based drug design to develop highly potent compounds that block this interaction. A subset of these inhibitors also cause rapid ubiquitylation and degradation of BCL6 in cells. These compounds display significantly stronger induction of expression of BCL6-repressed genes and antiproliferative effects than compounds that merely inhibited co-repressor interaction. The fact that the magnitude of effects elicited by this class of BCL6 degrading compounds greatly exceeds that of our equipotent non-degrading inhibitors offers exciting opportunities for the development of BCL6-based lymphoma therapeutics. To support further research, the most potent BCL6 degrading inhibitor is made freely available to the research community as an in vitro tool compound. Please see http://www.opnMe.com for further infos.

#3355

Clock genes DEC1 and BMAL1 regulate the expression of stem cell marker genes Sox2 and c-Myc in cervical cancer.

Fuyuki Sato, Yasueru Muragaki. _Wakayama Medical Univ., Wakayama city, Japan_.

It has been reported that abnormalities of clock genes are associated with tumor progression. Clock genes CLOCK, BMAL1/2, Differentiated embryonic chondrocyte gene (DEC)1/2, PER1/2/3 and CRY1/2 play important roles in the regulation of cell proliferation, cell cycle, apoptosis, epithelial to mesenchymal transition (EMT) and metastasis. We have shown that DEC1 is highly expressed in pancreatic cancer and oral cancer cells compared with non-tumor cells. However, the expression of clock genes is not well understood in cervical cancer. In this study, we focused on the expression of DEC1, DEC2 and BMAL1 in human cervical cancer tissues, examining their expression in biopsy and surgical resection samples by immunohistochemistry. As a result, DEC1 was strongly positive in tumor cells compared with adjacent non-tumor cells. Especially, the positive cells were observed in the front lesions of the tumors, suggesting that DEC1 is associated with invasion. The positive staining of vimentin was also observed in the same lesions where DEC1 was positive. On the other hand, no obvious significant difference in DEC2 expression was observed between tumor cells and non-tumor cells. For BMAL1, we found a week staining in the tumor cells compared with non-tumor cells. In addition, we examined the expression of stem cell marker Sox2 and c-Myc in cancer tissues. They were highly expressed in tumor cells compared with the adjacent non-tumor cells. Furthermore, Sox2 was expressed in metastatic lesions higher than the primary sites. These results suggest that clock genes DEC1 and BMAL1 may promote progression of cervical cancer, regulating Sox2 and c-Myc expression.

#3356

The EWS/FLI1 transcriptome is characterized by marked heterogeneity across Ewing sarcoma cell lines.

Susan M. Kitchen-Goosen, Megan J. Bowman, Marie Adams, Penny Berger, Patrick J. Grohar. _Van Andel Research Inst., Grand Rapids, MI_.

BACKGROUND: Ewing sarcoma (ES) is the second most common pediatric bone cancer. ES cells are dependent on the activity of the EWS/FLI1 transcription factor for cell survival. EWS/FLI1 drives proliferation, dysregulates the cell cycle and establishes a block in differentiation. Despite the known dependence of Ewing sarcoma on EWS/FLI1, the exact identity of all the downstream targets of EWS/FLI1 is not known. While gene signatures have been reported for EWS/FLI1, these fail to capture the marked heterogeneity in the EWS/FLI1 transcriptome or correlate these expression changes with cellular phenotype. METHODS: Next generation RNA sequencing with deep coverage was used to generate a comprehensive analysis of the EWS/FLI1 transcriptome by silencing EWS/FLI1 in 5 different ES cell lines at two different time points. These results were organized into gene expression networks and signaling nodes were identified by weighted correlation network analysis (WGCNA). The networks were validated by a 500-gene signature of EWS/FLI1 targets using a next generation sequencing capture assay and the Illumina platform. The cellular endpoint of EWS/FLI1 silencing was evaluated by caspase 3,7 cleavage to measure apoptosis, BGAL staining to measure senescence and scratch assays to capture migration. RESULTS: Next generation RNA sequencing with siRNA silencing of EWS/FLI1 revealed marked heterogeneity in the EWS/FLI1 transcriptome among cell lines. Targets of EWS/FLI1 that were common across cell lines exhibited small magnitude gene expression changes (log fold <2), while large magnitude gene expression changes were unique to individual cell lines (log fold>2). When the log fold change in expression was restricted to greater than 2, only 13 common repressed targets and 4 common induced targets were identified in all 5 cell lines. In addition, silencing of EWS/FLI1 induced dramatically different phenotypes across cell lines, where p53 wild-type cell lines underwent senescence and the p53 mutant cell lines did not. Most cell lines did not continue to proliferate with EWS/FLI1 silencing with the exception being the widely used A673 cell line. Cellular migration did not change with EWS/FLI1 silencing in many of the cell lines in contrast to A673 cells. CONCLUSIONS: EWS/FLI1 drives different gene expression signatures and different phenotypes in unique cell lines. This diversity needs to be considered as EWS/FLI1 directed therapies are developed.

#3357

Genetic regulation of RUNX2 induce apoptotic cell death through regulating the expression of SOX2 in CRPC-NE cells.

Yuki Noguchi,1 Natsuki Wariishi,1 Shiina Iwai,1 Gengo Kashiwazaki,1 Junichi Taniguchi,1 Toshikazu Bando,1 Masaya Baba,2 Souichi Adachi,1 Hiroshi Sugiyama,1 Yasuhiko Kamikubo1. 1 _kyoto university, kyoto, Japan;_ 2 _kumamoto university, kumamoto, Japan_.

Although runt-related transcription factor 2 (RUNX2) is well known for its bone-specific transcriptional regulator in highly metastatic prostate cancer, there is little knowledge about its role of RUNX2 in castration-resistant neuroendocrine prostate cancer (it is named CRPC-NE). It is well known that expression of Androgen Receptor (AR) in CRPC-NE cases is lower than other types and AR suppresses RUNX2 expression by binding it and abrogating its recruitment to DNA. This fact suggests that in CRPC-NE, low expression of AR, the addiction of RUNX2 is relatively higher than that of other prostate cancer types. Extensive analysis of the clinical datasets revealed that RUNX2 is one of the most consistently up-regulated genes in CRPC-NE and the overexpression of SOX2 confers an accelerated disease progression and shortened overall survival periods to CRPC-NE patients. We also found that the silencing RUNX2 induced apoptotic cell death in CRPC-NE cells independent on p53, but through transcriptionally down-regulating SOX2 expressions. Mechanistically, ChIP-qPCR assay and luciferase reporter experiments confirmed that RUNX2 positively regulates SOX2 expression by binding to the consensus RUNX2 binding sequence (5'-TGTGGT-3') located in the proximal promoter region of SOX2. Short hairpin RNA (shRNA)-mediated knockdown of RUNX2 in the CRPC-NE cell line PC-3 induced the decreased expression of SOX2 and the increased expression of cleaved form of caspase-3 and PARP in a p53 independent manner. Silencing of SOX2 in PC-3 cells suppressed the proliferation of these cancer cells and induced apoptosis, which phenotypes were rescued by restoring SOX2 in the cells. These data indicates that inhibition of SOX2-apoptosis axis via RUNX2 could be a better therapeutic choice in CRPC-NE. Lastly, we examined the efficacy of our novel molecule Chlorambucil-conjugated Pyrrole Imidazole Polyamide (we named it as Chb-M'), which specifically binds to the consensus RUNX2 binding sequence and inhibits RUNX2 target gene. Surprisingly, Chb-M' had tremendous inhibitory effect on PC-3 cells (IC₅₀ value at 620 nM) through effective SOX2 inhibition. Additionally, it was exceptionally well-tolerated in mice and exerted excellent efficacy against xenograft mice models of CRPC-NE. Taken together, our work identified a novel interaction of RUNX2 and SOX2-apoptosis axis, offering a new strategy for the management of poor-prognostic advanced stage CRPC-NE cancer patients.

#3358

FOXF1 defines the core-regulatory circuitry in gastrointestinal stromal tumor (GIST).

Leili Ran,1 Yuedan Chen,1 Wai Pung E. Wong,1 Dan Li,1 Zhen Cao,1 Shipra Shukla,1 Yuanyuan Xie,1 Deyou Zheng,2 Richard P. Koche,1 Cristina R. Antonescu,1 Yu Chen,1 Ping Chi1. 1 _Mem. Sloan Kettering Cancer Ctr., New York, NY;_ 2 _Albert Einstein College of Medicine, New York, NY_.

The cellular context that integrates upstream signaling and downstream nuclear response dictates the oncogenic behaviour and shapes treatment responses in distinct cancer types. Here, we uncover that in GIST, the forkhead family member, FOXF1, directly controls the transcription of two master regulators, KIT and ETV1, both required for GIST precursor-interstitial cells of Cajal (ICC) lineage-specification and GIST tumorigenesis. Further, FOXF1 co-localizes with ETV1 at enhancers and functions as a pioneer factor that regulates the ETV1-dependent GIST-lineage specific transcriptome through modulation of the local chromatin context, including chromatin accessibility, enhancer maintenance and ETV1 binding. Functionally, FOXF1 is required for human GIST cell growth in vitro and murine GIST tumor growth and maintenance in vivo. The simultaneous control of the upstream signaling and nuclear response sets up a unique regulatory paradigm and highlights the critical role of FOXF1 in enforcing the GIST cellular context for highly lineage-restricted clinical behaviour and treatment response.

#3359

Elucidating the oncogenic mechanism of the TAZ-CAMTA1 and YAP-TFE3 fusion proteins.

Nicole Merritt,1 Dushyandi Rajendran,2 Zhen-Yuan Lin,2 Xiaomeng Zhang,3 Katrina Mitchell,3 Colleen Fullenkamp,1 Anne-Claude Gingras,2 Kieran Harvey,3 Munir Tanas1. 1 _University of Iowa, Iowa City, IA;_ 2 _Mount Sinai Hospital, Toronto, Ontario, Canada;_ 3 _Peter MacCallum Cancer Centre, Melbourne, Australia_.

Introduction: Sarcomas are cancers that originate from mesenchymal-derived tissues and represent 1% of adult cancers and 15% of pediatric cancers. No effective medical therapy exists for sarcomas, and the 5-year survival of metastatic sarcomas is 16%. Epithelioid hemangioendothelioma (EHE) is a vascular sarcoma where 90% of tumors contain a WWTR1-CAMTA1 gene fusion, whereas the other 10% contain a YAP1-TFE3 gene fusion. TAZ (WWTR1) and YAP are oncogenic transcriptional coactivators and paralogues of one another that are negatively regulated by the Hippo signaling pathway. Constitutive activation of TAZ/YAP is observed in many types of cancer, including sarcomas, however genetic alterations within the pathway are rare. The precise mechanism by which TAZ-CAMTA1 and YAP-TFE3 transform cells is poorly understood.

Methods: NIH-3T3 and human sarcoma cell lines were transduced with TAZ-CAMTA1 or YAP-TFE3. Western blot and immunofluorescence were used to evaluate expression and localization of the fusion proteins. In vitro assays were used to assess hallmarks of cancer. Protein-protein interactions were identified through BioID-tandem mass spectrometry. Xenograft experiments in NSG mice with cell lines expressing the fusion proteins were performed to study in vivo hallmarks of cancer.

Results: The TAZ-CAMTA1 and YAP-TFE3 fusion proteins contain the N-terminus of TAZ and YAP fused in frame to the C-terminus of CAMTA1 and TFE3, rendering the N-terminus of TAZ and YAP unresponsive to negative regulation by the Hippo pathway. Immunofluorescence revealed that the fusion proteins are localized within the nucleus in both sparse and confluent states, compared to TAZ and YAP which are only localized to the nucleus under sparse conditions. TAZ-CAMTA1 and YAP-TFE3 also promote anchorage-independent growth and increased proliferation. Mass spectrometry revealed differential protein-protein interactions between TAZ-CAMTA1 and/or YAP-TFE3 and the Crumbs complex (MPDZ, PATJ, RASSF8, AMOT, AMOTL1), SWI/SNF complex (ARID1A, ARID1B, SMARCC2, SMARCE1), the Mediator complex (MED12), and chromatin remodeling complexes, as compared to full length TAZ and YAP. Xenografts of transduced NIH-3T3 and SW872 cells showed that the fusion proteins drive tumorigenesis and metastasis in NSG mice.

Conclusions: The data indicate that the oncogenic properties of TAZ-CAMTA1 and YAP-TFE3 can be explained by their nuclear enrichment, dysregulation of the upstream Hippo pathway, and interaction with key transcriptional regulatory proteins. Future in vitro and in vivo studies will elucidate additional mechanisms by which these fusion proteins transform cells and promote sarcomagenesis, potentially resulting in new therapeutic targets. 

### Genomic Instability

#3360

Origins of mutations in colorectal cancer.

Barry Gold. _Univ. of Pittsburgh, Pittsburgh, PA_.

The origins of most human cancers remain unclear. In selected cases, exposure to potent environmental mutagens or familial germ line mutations, which affect tumor suppressor genes or oncogenes, have been identified with specific cancers. Wnt signaling due to mutations in APC is compromised as an early event in 75% of human colorectal cancer (CRC), with truncating nonsense mutations (65%) dominating the mutation spectrum. Because 43% of the nonsense mutations occur at Arg CGA codons, the C→T mutation is attributed to hydrolytic deamination of 5-methylC. The enhanced incidence of colorectal cancer (CRC) associated with the Western diet has been epidemiologically linked to heterocyclic aromatic amines (HAA) produced by high temperature cooking of meat. Analysis of the mutation spectra in CRC indicated that the G→T transversions, which are produced by this class of mutagens were not enriched but were actually lower than what would be statistically anticipated based on random mutations. Moreover, the APC mutation patterns in the U.S.A. vs. China are indistinguishable despite differences in diet. However, dissection of the APC mutation pattern in tumors that arise in the different anatomical regions of the large intestine shows that the nonsense mutation pattern in APC differ in the different regions: there is a statistically significant increase in G→T transversions in the rectum vs. the other regions, albeit, the percent of G→T mutations still remains lower than predicted based on random mutagenesis. Therefore, it appears that HAA's may contribute to the incidence of rectal cancer.

#3361

Are mismatch repair (MMR) defects and microsatellite instability (MSI) relevant in renal cell and prostate carcinomas? Their significance for future therapies.

Xu Naizhen, Marston Linehan, Peter A. Pinto, Maria J. Merino. _NIH/NCI, Bethesda, MD_.

Background Few studies have investigated mismatch repair (MMR) defects in renal and prostatic cancers. Normally, MMR accurately detects and corrects mistakes made during replication of a DNA strand. But, a defective MMR system is very likely to miss copy-number errors, particularly in microsatellites (short tandem base repeats). Alterations in function of MMR genes can result in microsatellite instability (MSI), known to facilitate tumor progression and poor prognosis. MMR deficiency is observed frequently in colonic and endometrial carcinoma as part of Lynch's syndrome, as well as occasionally in other neoplasms. In our study, we assayed MMR expression in a series of kidney tumors representing the spectrum of renal malignancies and in a wide-ranging group of prostate cancers (for which clinical information and Gleason grade was obtained in all cases).

Materials and methods Immunohistochemical (IHC) staining of FFPE sections from 45 kidney cancers and 30 prostatic tumors was performed. The main MMR proteins of interest are MSH2 and MLH1, plus their heterodimer partners MSH6 and PMS2; staining was directed at these 4 targets. Antibody sources were Abcam, Cambridge, MA 02139 (MLH1, MSH6, PMS2) and Agilent Technologies, Wilmington, DE 19808 (MSH2). Also, PD-L1 expression was evaluated using antibody from Cell Signaling, Danvers, MA 01923.

Results Forty-five cases of renal cell carcinoma (RCC) comprised 12 cases of clear cell VHL (von Hippel Lindau) related cancer, 11 papillary type 1, 11 chromophobe, and 11 HLRCC (hereditary leiomyomatosis and renal cell carcinoma). Only one case of chromophobe RCC with sarcomatoid differentiation showed complete loss of MLH1 and PMS2 (2% of 45 renal tumors). Of prostate cancers, only one of 30 tumors (3%) was completely negative for both MLH1 and PMS2; that tumor was Gleason grade 8. The remaining 29 cases were positive for all MMR proteins.

Discussion Our study suggests that neither kidney nor prostate cancers are likely to possess significant MMR defects. In our series, clear cell, papillary and HLRCC tumors did not lack MMR expression; one chromophobe case negative for MMR (and presumed liable to MSI) may have developed genomic changes as disease progressed to a more aggressive, sarcomatoid pattern. Prostate cancer with MMR defects also appears to be rare, and we found no correlation with grade and stage. MMR defects lead to highly unstable microsatellite sequences and altered gene expression, so while only a small proportion of these tumors exhibited loss of MMR proteins, it is possible that in these few cases the loss may be significant and that such patients may benefit from new modalities of treatment. IHC detection of MMR defects (as predictors of probable MSI) may prove a useful prognostic tool in identification of rare renal and prostatic cancers likely to exhibit greater genetic instability and neoplastic advance.

#3362

Mice with a "deletor" phenotype allow high resolution detection of lymphoid leukemia tumor suppressor genes.

Mianmian Yin,1 Timour Baslan,2 Amy Freeland,3 Steven C. Pruitt,3 Peter D. Aplan1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 3 _Roswell Park Cancer Institute, Buffalo, NY_.

Mice that are homozygous for a deficiency allele of the DNA replication factor minichromosome maintenance protein 2 (designated Mcm2def) are born viable and are healthy for the first 2 months of life. Beginning at three months, these mice develop precursor T-cell lymphoblastic leukemia/lymphoma (pre-T LBL). Copy number aberration (CNA) analysis showed that these pre-T LBL samples had 8-14 small (100-1000 kb) interstitial deletions per sample. Remarkably, all mice had two or more deletions that encompassed genes known to be relevant for human pre-T LBL, including Pten, Cdkn1a, Tcf3, and Tcf12.

Mice that express a NUP98-HOXD13 (NHD13) transgene develop a wide array of leukemias, most commonly myeloid, less commonly T-cell, and, rarely, B-lineage. To identify myeloid tumor suppressor genes, we crossed the NHD13 transgene onto the Mcm2def background. All Mcm2def:NHD13+ mice developed pre-T LBL by 3 months of age, reflecting the highly penetrant nature of the Mcm2def phenotype. None of the Mcm2def:NHD13+ mice developed myeloid leukemia. Surprisingly, approximately 30% of the Mcm2def:NHD13+ mice developed concurrent B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and pre-T LBL. The thymus was typically infiltrated with pre-T LBL cells, whereas the bone marrow and spleen were infiltrated with BCP-ALL cells, characterized by clonal VDJ rearrangement and CD19 staining. Parenchymal organs (lung, kidney, liver) were variably infiltrated with pre-T LBL, BCP-ALL, or both. CNA analysis showed that the pre-T LBL were characterized by Pten, CDkn1a, Tcf3, and Tcf12 deletions, similar to the Mcm2def pre-T LBL, whereas the BCP-ALL were characterized by homozygous or heterozygous deletions of Pax5 and a 400 kb region encompassing Cebpb and Ptpn1. There were no shared deletions present in both BCP-ALL and pre-T LBL from the same mouse, indicating that the BCP-ALL and pre-T LBL arose independently, and not from a common precursor. The high frequency (5/7 samples) of acquired Pax5 deletions in the BCP-ALL samples serves to validate the model; whereas the finding of Cebpb/Ptpn1 deletions (5/7 samples) suggests an unanticipated role of either Cebpb or Ptpn1 in BCP-ALL.

#3363

PUM3 **is a triple-negative breast cancer dependency gene that functions in replication fork restart and repair.**

Daniel Weekes,1 Elodie Noel,1 Callum Walker,2 Nick Balan,2 Vandna Shah,1 Bhavna Sidhu,1 Anna Pardix,1 Stephen Pettitt,2 Christopher J. Lord,2 Anita Grigoriadis,1 Tutt N. Andrew1. 1 _Kings College London, London, United Kingdom;_ 2 _Intititute of Cancer Research, London, United Kingdom_.

Triple-Negative Breast Cancers (TNBC) are characterized by high levels of structural chromosome alterations and harbor mutational scars of defects in homologous recombination. Importantly, by acting either as drivers, or as tumor specific dependencies mutated genes in these regions represent potential therapeutic targets

In search of such genes we identified that knockdown of the PUM3 gene, which is contained in a 9p24 recurrent ampliicon, impaired the growth of a subset of breast cancer but not mammary epithelial cell lines. Furthermore, the growth defects observed upon PUM3 knockdown in SUM149, a BRCA1 deficient cell line, were reversed by reactivation of BRCA1's function suggesting a BRCA1-deficiency associated dependency. PUM3 knockdown led to an increase in gH2Ax and 53BP1 nuclear foci, impaired restart of stalled replication forks, and decreased RAD51 mediated repair of collapsed replication forks. As a result PUM3 depleted cells have reduced DNA replication rate and slowed cell cycle progression. Knockdown of PUM3 also impaired replication stress induced PARylation and PAR foci formation and PUM3 and PARP1 knockdown showed phenotypic epistasis with respect to cell growth suggesting a potential mechanism for PUM3s replication fork related functions.

PUM3 forms a complex with gH2Ax and the replication stalling agent hyroxyurea and PARP-inhibitors increases the nuclear concentration of PUM3. Furthermore PUM3 knockdown increased the sensitivity of breast cancer cells to these agents indicating a role for PUM3 in the repair of lesions generating by these DNA damaging agents.

By studying PUM3 we have identified a novel player in replication fork restart and repair whose activity may be particularly relevant in homologous recombination deficient TNBCs. A greater understanding of PUM3's function and the pathways that regulate it may identify novel therapeutic targets for TNBCs. Importantly, direct or indirect modulation of PUM's functions may potentiate the effects of replication stress inducing therapies such as PARP-inhibitors.

#3364

RAI2 maintains genome stability of breast cancer cells.

Lena Boettcher,1 Bernd Zobiak,2 Kerstin Borgmann,3 Alexandra Zielinski,3 Antonio Virgilio Failla,2 Klaus Pantel,1 Harriet Wikman,1 Stefan Werner1. 1 _University Medical Center Hamburg-Eppendorf, Institute of Tumorbiology, Hamburg, Germany;_ 2 _University Medical Center Hamburg-Eppendorf, UKE Microscopy Imaging Facility, Hamburg, Germany;_ 3 _University Medical Center Hamburg-Eppendorf, Department of Radiotherapy and Experimental Radiooncology, Hamburg, Germany_.

Genome instability is considered as an enabling hallmark of cancer formation and progression as well as a source of intratumoral heterogeneity. Retinoid acid-induced protein 2 (RAI2) was initially identified as a new metastasis-associated protein especially related with the presence of disseminated tumor cells in the bone marrow of breast cancer patients. In primary breast tumors low RAI2 gene expression correlates with mutant TP53 status and moreover, RAI2 depleted luminal breast cancer cell lines show decreased expression of key regulators of G2/M transition. We hypothesize that RAI2 has an influence on mitotic fidelity and is involved in maintaining genomic stability in breast cancer cell lines. To analyze consequences of RAI2 depletion on mitotic fidelity in luminal breast cancer cells, we followed mitotic cells by live cell imaging and analyzed chromosomal segregation by immune fluorescence staining and karyotyped RAI2 depleted cells. For analysis of cell cycle dependent RAI2 protein expression we executed Western blot analysis after cell synchronization and treatment with genotoxic agents. Localization of ectopically expressed HA-tagged RAI2 protein was analyzed in breast cancer cells treated with genotoxic agents using immune fluorescence staining. Finally, we performed in silico validation in different breast cancer data sets. Our findings show that RAI2 depletion in luminal breast cancer cell lines leads to increased chromosomal instability, associated with loss of acentric chromosomal fragments during ana- and metaphase as well as a prolonged mitosis. Furthermore, karyotyping revealed a significant reduction of chromosome number from 78 to 73 in RAI2 depleted cells. In synchronized parental breast cancer cells we could show an increased RAI2 expression during S/G2 phase compared to unsynchronized cells. Interestingly, genotoxic treatment resulted in a significant upregulation of RAI2 protein, which can also be seen as an increased formation of nuclear speckles (p<0.05). Finally, in silico analysis of breast cancer data sets revealed a significant correlation between low RAI2 expression and an expression signature of chromosomal instability (p>0,001). Our findings show that RAI2 depletion in luminal breast cancer cell lines leads to increased chromosomal instability. Our results show that RAI2 depletion causes pre-mitotic DNA lesions. Moreover, in breast cancer patients a low RAI2 expression was significantly associated with chromosomal instability. We conclude that the RAI2 protein preserves mitotic fidelity and chromosomal integrity in luminal breast cancer cell lines by affecting either the DNA damage response or DNA replication during S- and/or G2-phase.

#3365

MAP3K10 **mutation as a biomarker for predicting response to chemotherapy in gastric cancer patients.**

Chae Hwa Kwon,1 Sun Jin Lee,2 Yuri Choi,2 Do Youn Park3. 1 _Biomedical research Institute, Pusan Naitonal University Hospital, Busan, Republic of Korea;_ 2 _Cancer research Institute, Pusan Naitonal University Hospital, Busan, Republic of Korea;_ 3 _Department of Pathology and biomedical research Institute, Pusan Naitonal University Hospital, Busan, Republic of Korea_.

Gastric cancer is one of most common malignant tumors. Although chemotherapy can improve the survival of gastric cancer, the benefit is limited because most cancer cells eventually become irresponsive to chemotherapeutic drugs. To investigate mechanism of resistance to chemotherapy, we performed whole-exome sequencing (WES) on resistant tumor and paired normal samples from 14 patients received adjuvant chemotherapy. We found that 3 patients had novel somatic mutations in mitogen-activated protein kinase kinase kinase 10 (MAP3K10), an upstream kinase of mitogen activated protein kinase (MAPK). MAP3K10R304C, MAP3K10H211R, or MAP3K10A30E were detected each patient, respectively. The survival rate was higher and apoptosis rate was smaller in the cells expressing each MAP3K10 mutations compared with parental controls and cells expressing wild-type MAP3K10, when treated with cisplatin or 5-FU. Moreover, cells expressing each MAP3K10 mutations exhibited higher levels of phosphorylated MEK1/2 and p38MAPK at baseline and when treated with cisplatin or 5-FU than wild-type AGS or MKN45 cells or those expressing wild-type MAP3K10. In summary, MAP3K10R304C, MAP3K10H211R, and MAP3K10A30E MAP3K10 confer resistance to cisplatin or 5-FU via activation of MAPK pathway in gastric cancer cells. Thus, somatic MAP3K10 mutations could be a potential biomarker for predicting resistance to chemotherapy for gastric cancer patients

#3366

APOBEC3 family of cytidine deaminases in sensitizing triple-negative breast cancer cells to cisplatin and carboplatin.

Kayla L. Conner, Asra N. Shaik, Jordan White, Wen Lei, Michele L. Cote, Steve M. Patrick. _Wayne State school of Medicine and Barbara Ann Karmanos Cancer Institute, Detroit, MI_.

Cisplatin is a DNA damaging chemotherapeutic which forms interstrand crosslinks (ICL) and intrastrand adducts that induce replication fork collapse and lead to apoptosis. These adduct sites can be repaired through several repair pathways, the most important being nucleotide excision repair (NER). Cisplatin and carboplatin ICLs form a unique structure that forces cytosines flanking the adduct site to become extrahelical. Our lab has previously shown that base excision repair (BER) and mismatch repair (MMR) mediate sensitivity to cisplatin and carboplatin as a consequence of nonproductive processing of DNA flanking the ICL structure. The APOBEC3 (A3) protein family of cytidine deaminases have recently been implicated in cancer development and have potential to deaminate the extrahelical cytosines formed by cisplatin ICLs. Polβ knockdown MDA-MB-231 cells are resistant to cisplatin in vivo compared to wild-type cells (p<0.001). Knockdown of APOBEC3 family members in MDA-MB-231 breast cancer cells results in a resistant phenotype to cisplatin and carboplatin treatment compared to the control. Utilizing CRISPR-Cas9 genome editing, we generated knockouts of A3 members. A3D knockdown in the APOBEC3C (A3C) knockout results in resistance to cisplatin compared to the control, suggesting that multiple A3 enzymes can deaminate the ICL induced extrahelical cytosine. APOBEC3A (A3A) knockdown in a cell line with high expression of A3A (SK-BR-3) shows no difference in cisplatin and carboplatin treatment, suggesting that A3A does not deaminate extrahelical cytosines. Overexpression of A3C or A3D in cell lines with low expression of A3 proteins (SK-BR-3) results in sensitivity to cisplatin. These results together suggest that A3C, A3D, and A3G can deaminate the ICL extrahelical cytosines, resulting in the activation of BER. The subsequent nucleotide misincorporation by Polβ followed by MMR protein binding would physically block NER proteins from repairing the ICL, therefore conferring sensitivity to cisplatin and carboplatin.

#3367

USP7 regulates CSB protein recovery after UV-induced DNA damage.

Shengcai Wei. _The Ohio State University, Columbus, OH_.

Cockayne syndrome group B (CSB) protein functions in transcription-coupled repair (TCR), a sub-pathway of nucleotide excision repair (NER). We previously demonstrated that ultraviolet radiation (UVR) induces a valosin-containing protein (VCP)/p97-dependent ubiquitin-mediated CSB degradation. In this study, we further revealed that CSB protein is ubiquitinated immediately after ultraviolet-light irradiation and undergoes proteasomal degradation. The recovery of CSB protein level is dependent on USP7. Disruption of USP7 function sensitizes CSB degradation to lower UVR doses. We further demonstrated USP7's interaction with CSB in vivo by immunoprecipitation and in vitro by GST pulldown assays. Interestingly, in vitro CSB strongly binds to TRAF domain, which also interacts with UV stimulated scaffold protein A (UVSSA). Additionally, USP7 also interacts with VCP/p97 using its TRAF domain and C-terminal in in vitro. Furthermore, VCP/p97 was demonstrated to associates with USP7 in vivo. These results suggest that USP7 rescues CSB from ubiquitin-mediated proteolysis after (VCP)/p97-mediated CSB extraction from chromatin, preventing the CSB-ubiquitin conjugates being presented to proteasome for degradation.

#3368

Insertion and deletion frameshift rates and mutational spectra of tetranucleotide microsatellites in DNA mismatch repair-deficient human cells.

Maide O. Raeker, Jovan Pierre-Charles, John M. Carethers. _Univ. of Michigan, Ann Arbor, MI_.

Background. Oxidative DNA damage and inflammatory cytokines cause loss of function of the MMR protein MSH3. MSH3-MSH2 (MutSβ) function is required for correction of postreplicative polymerase errors in the form of frameshifts at tetranucleotide microsatellite sequences to prevent elevated microsatellite alterations at selected tetranucleotides (EMAST). EMAST is increasingly being used as a biomarker for MSH3 deficiency in subtyping colorectal cancers due to its association with metastasis and poor patient outcome. Here, we sought to understand frameshift mutational behavior of tetranucleotide sequences in the absence of MMR.

Methods. We generated EGFP-based mutation reporter model systems containing the D9S242 tetranucleotide microsatellite at its native length of (AAAG)18 and at modified lengths of (AAAG)15 and (AAAG)12 to represent other endogenous microsatellite lengths. Plasmid vectors were constructed for each length that placed EGFP +1 bp or -1 bp out-of-frame for protein translation, enabling us to measure deletion and insertion frameshifts. These constructs were stably-integrated into MMR-deficient HCT116 cells, and monoclonal cell lines containing the constructs were isolated to measure mutation rates and spectra. Deletion and insertion frameshift mutations restored the EGFP reading frame to become detectable by flow cytometry. Genomic DNA from mutated cells were isolated and sequenced for insertion/deletion frameshift mutational biases.

Results. Combined insertion/deletion frameshift mutation rates at D9S242 locus ranged from 31.6 X 10-4 to 71.1 X 10-4 mutations/cell/generation and was strongly correlated with longer length of tetranucleotide microsatellites (r2 = 0.986, P = 0.0375). We observed slightly higher deletion mutation rates over insertion rates at longer microsatellites, but equivalent insertion and deletion rates at shorter microsatellites. However, accumulation of more deletion frameshifts over time contributed to a distinct mutational bias for deletions for each length, likely due to continual frameshift mutation at insertions. About 79% of all observed frameshifts (insertion or deletion) were one- repeat (e.g., four nucleotides), 16% two-repeat, and 5% three or more repeat mutations that were consistent with a slipped strand mispairing mutation model.

Conclusions. Our results suggest that intact MMR recognizes and repairs shorter length tetranucleotide frameshifts more efficiently than larger ones, and preferentially corrects loops on the template strand (preventing deletions) over loops on the newly-synthesized strand (preventing insertions) in human cells. MMR deficiency thus manifests in human tissue with a deletion mutation bias at tetranucleotide sequences (EMAST). Longer tetranucleotide microsatellites have increased propensities for larger DNA slippage errors.

#3369

Mosaic chromosome X copy-number aberrations in leukocytes of never-smoking lung cancer patients: The Female Lung Cancer Consortium in Asia (FLCCA).

Jason Y. Wong,1 Mitchell Machiela,1 Weiyin Zhou,1 Wei Jie Seow,1 Bryan Bassig,1 Jinming Zhang,1 Meredith Yeager,1 Wei Zheng,2 Xiao-Ou Shu,2 Hongbing Shen,3 Keitaro Matsuo,4 Chao Agnes Hsiung,5 Maria P. Wong,6 Yun-Chul Hong,7 Jiu-cun Wang,8 Yi-Long Wu,9 Baosen Zhou,10 Robert Klein,11 Zhihua Yin,10 Tangchun Wu,12 Pan-Chyr Yang,13 Yong-Bing Xiang,14 Adeline Seow,15 Yu-Tang Gao,14 Chen Wu,16 Jianjun Liu,17 Zhibin Hu,18 Laurie Burdett,1 Qiuyin Cai,2 Juncheng Dai,18 Dongxin Lin,16 Kexin Chen,19 Stephen Chanock,1 Nathaniel Rothman,1 Qing Lan1. 1 _National Cancer Institute, Rockville, MD;_ 2 _Division of Epidemiology, Department of Medicine, Vanderbilt University Medical Center and Vanderbilt-Ingram Cancer Center, TN;_ 3 _Department of Epidemiology, School of Public Health, Nanjing Medical University, China;_ 4 _Division of Molecular Medicine, Aichi Cancer Center Research Institute, Japan;_ 5 _Institute of Population Health Sciences, National Health Research Institutes, Taiwan;_ 6 _Department of Pathology, Queen Mary Hospital, Hong Kong;_ 7 _Department of Preventive Medicine, Seoul National University College of Medicine, Republic of Korea;_ 8 _Ministry of Education Key Laboratory of Contemporary Anthropology, School of Life Sciences, Fudan University, China;_ 9 _Guangdong Lung Cancer Institute, Medical Research Center and Cancer Center of Guangdong General Hospital, Guangdong Academy of Medical Sciences, China;_ 10 _Department of Epidemiology, School of Public Health, China Medical University, China;_ 11 _Icahn School of Medicine Mount Sinai, NY;_ 12 _Institute of Occupational Medicine and Ministry of Education Key Lab for Environment and Health, School of Public Health, Huazhong University of Science and Technology, China;_ 13 _Department of Internal Medicine, National Taiwan University Hospital, Taiwan;_ 14 _Department of Epidemiology, Shanghai Cancer Institute, China;_ 15 _Saw Swee Hock School of Public Health, National University of Singapore, Singapore;_ 16 _Department of Etiology & Carcinogenesis and State Key Laboratory of Molecular Oncology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, China; _17 _Lung Cancer Center, Kyungpook National University Medical Center, Daegu, Republic of Korea;_ 18 _Department of Epidemiology and Biostatistics, Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Collaborative Innovation Center for Cancer Personalized Medicine, School of Public Health, Nanjing Medical University, China;_ 19 _Department of Epidemiology and Biostatistics, Tianjin Medical University Cancer Institute and Hospital, China_.

Lung cancer (LC) is a global health burden that accounted for 1.69 million deaths worldwide in 2015. Asian women have high incidence rates of LC, with about half of all diagnoses occurring among never-smokers. Chromosomal copy-number (CN) aberrations can arise de novo in somatic cells with progressing age. These genomic alterations can change the dosage of critical genes and impact biological processes. Gains and losses of Chromosome X in a proportion of cells (ChrX mosaicism) are markers of genomic instability; however, their relationship with LC is unclear. We characterized leukocyte ChrX mosaicism among never-smoking Asian female LC patients and cancer-free controls.

We investigated 5,137 LC cases (4,477 adenocarcinomas (AC) and 660 squamous cell carcinomas (SCC)) and 4,535 controls from 13 case-control studies and one cohort study in the Female Lung Cancer Consortium in Asia. Subjects were aged 19-91 years from China, Singapore, Taiwan, South Korea, and Japan. In case-control studies, blood was drawn after diagnosis and mostly before treatment. Leukocyte DNA was genotyped on Illumina 660W arrays, with a small subset on 610Q and 370K arrays. ChrX mosaicism was detected using normalized log R ratio (LRR) and B allele frequency (BAF) values from probes covering ChrX. ChrX was segmented for mosaic events (gains, losses, and copy-neutral) using circular binary segmentation on BAF values. Segments <2 Mb were excluded. Event CN state was assigned based on LRR values. LRR deviations of 0.01 and −0.01 were used to classify events as gain and losses, respectively, for ChrX-spanning mosaic events; while thresholds of 0.05 and −0.05 were used for those spanning a portion of ChrX. Mosaic proportions were estimated using deviation from the expected BAF given the LRR-defined CN state. Fisher's Exact tests on 2x3 and 2x2 tables were used to compare ChrX mosaic events between LC cases and controls. Exact logistic regression was used to assess associations between combined gain and loss events and AC.

We found 18 detectable mosaic ChrX events (0.19%) in 12 women. LC cases had 5 gains, 3 losses, and 4 copy-neutral mosaic events; while controls had no gains, 1 loss, and 5 copy-neutral events (p=9.8E-2). AC patients had significantly more combined gains and losses (n=7) and fewer copy-neutral events (n=0) compared to controls who had 1 combined gain and loss and 5 copy-neutral events (p=4.7E-3). Notably, all 5 gains were among AC and none among controls (p=3.5E-3). Women with combined gain and loss events had 7 times the odds of AC compared to those without them (p=3.8E-2).

Our preliminary findings suggest a possible role of leukocyte ChrX mosaicism, particularly CN gains, in the biological mechanism of LC development. However, given the rarity of these events and possible disease-effect, larger studies are needed to further evaluate ChrX mosaicism as a risk factor for future occurrence of LC.

#3370

Mad2l2 inhibits colorectal cancer growth by promoting ncoa3 ubiquitination and degradation.

yixin li, ge qin, qian long, changlin zhang, dingbo shi, miao chen, wuguo deng. Sun Yat-sen University Cancer Center;State Key Laboratory of Oncology in South China, guangzhou, China.

Nuclear receptor coactivator 3 (NCOA3) is a transcriptional coactivator that has elevated expression in multiple tumor types, including colorectal cancer (CRC). However, the molecular mechanisms that regulate the tumorigenic functions of NCOA3 in CRC remain largely unknown. In this study, we aimed to discover and identify the novel regulatory proteins of NCOA3 and explore their mechanisms of action. Immunoprecipitation coupled with mass spectrometry analysis was used to detect, identify and verify the proteins that interacted with NCOA3 in CRC cells. The biological functions of the candidate proteins and the underlying molecular mechanism were investigated in CRC cell lines and a mouse model in vitro and in vivo. The clinical significance of NCOA3 and its interaction partner protein in CRC patients was also studied. We discovered and identified mitotic arrest deficient 2-like protein 2 (MAD2L2, also known as MAD2B or REV7) as an interaction partner of NCOA3 by immunoprecipitation coupled with mass spectrometry analysis. Overexpression of MAD2L2 suppressed the cell proliferation, migration and clonogenecity in CRC cell lines and a mouse model by inducing the degradation of NCOA3. We also found that increased MAD2L2 expression in CRC cells activated p38, which was required for the phosphorylation of NCOA3 that led to its ubiquitination and degradation by the proteasome. Moreover, we demonstrated that MAD2L2 predicted favorable prognosis in CRC patients. These results indicate that MAD2L2 inhibits colorectal cancer growth by promoting NCOA3 ubiquitination and degradation. Our study have discovered a novel role of MAD2L2 in the regulation of NCOA3 degradation, and proposed that MAD2L2 serves as a potential tumor suppressor in CRC.

#3371

**Analysis of telomere length and dysfunction in normal breast tissue from** BRCA2 **mutation carriers.**

Birna Thorvaldsdottir,1 Beth A. Cimini,2 Morgan E. Diolaiti,2 Katrin Olafsdottir,3 Jon G. Jonasson,3 Jorunn E. Eyfjörd1. 1 _University of Iceland, Reykjavik, Iceland;_ 2 _University of California-San Francisco, San Francisco, CA;_ 3 _Landspitali University Hospital, Reykjavik, Iceland_.

The tumor suppressor protein BRCA2 is important for maintaining genomic stability. BRCA2 is essential for homologous recombination repair of DNA double strand breaks and also has a role in protection of stalled replication forks as well as in telomere protection and maintenance. Disruptions in telomere homeostasis can result in excessive telomere shortening and drive chromosome instability, a hallmark of BRCA2-related cancers. Germline mutations in the BRCA2 gene are associated with highly increased risk of breast cancer. A single deleterious founder mutation, BRCA2999del5, has been detected in the gene in the Icelandic population, making it feasible to study the influence of a single mutation at a population level. The BRCA2999del5 mutation is found in 6-7% of female breast cancer patients in Iceland and is associated with complex chromosomal changes in tumor tissue. Furthermore, the BRCA2999del5 mutation is associated with poor prognosis, although mutation carriers seem to differ with respect to age of onset and severity of disease. It is therefore of great interest to identify factors that influence breast cancer risk within the mutation carrier group. The aim of the study was to measure telomere length (TL) and levels of telomere dysfunction in tumor-adjacent normal breast tissue from BRCA2 mutation carriers and non-carriers and investigate their potential relationship with breast cancer risk. The study group consisted of 186 Icelandic breast cancer patients; BRCA2999del5 mutation carriers and sporadic cases, matched with respect to age and year of diagnosis. For TL measurements, paraffin embedded normal breast tissue samples were analyzed by Quantitative Fluorescence in Situ Hybridization. Telomere dysfunction induced foci (TIFs) can be detected where co-localization of telomere and DNA-damage signals occur. For this purpose, the samples were also immunofluorocently stained for the DNA damage repair protein 53BP1. In concordance with previously reported data in the literature, luminal epithelial cells showed the shortest TL and highest number TIFs of the cell types in normal breast tissue. This observation is highly relevant as these are the cells from which most breast cancers originate. No difference was detected in age-adjusted TL between BRCA2 mutation carriers and non-carriers. However, consistent with our recently published data from TL measurements in blood from the same study group, shorter TL is correlated with younger age at breast cancer diagnosis in BRCA2 mutation carriers but not in non-carriers. In conclusion, these results indicate that in BRCA2 mutation carriers, shorter TL in normal breast tissue is associated with earlier breast cancer occurrence.

#3372

Mutations of mismatch repair genes in Her2 overexpressed breast cancer.

Mohammed A. Baghdadi, Turki M. Sobahy, Hosam A. Alardati, Jamal E. Zekri. _King Faisal Hospital & Research Center, Jeddah, Saudi Arabia_.

The importance of Mismatch Repair (MMR) in therapeutic selection is an area of recent research. Minority of unselected breast tumors exhibit mutations indicative of deficient MMR. Here, we present our preliminary data investigating 7 MMR genes exclusively in Her2-overexpressed (Her2OE) breast cancer (BC). Twenty-five archival formalin-fixed paraffin-embedded samples from 25 Her2OE BC patients underwent targeted next generation sequencing for 7 MMR genes (MLH1, MLH3, MSH2, MSH6, PMS2, POLD1 and EPCAM). The analysis was performed through the Saudi Human Genome Project bioinformatics pipeline. Data filtration was performed to rank genetic variants based on their predicted pathogenicity. Methods of filtration included mutation effect, ClinVar findings, ExAC loss of function index (pLI) and collective proteomics index. Twenty-four out of 25 (96%) tumors contained significant mutations in one or more MMR genes. Three cases (12%) contained mutations in 3-4 MMR genes while 21 (84%) had 1-2 alteration(s) in these gene(s). Only one case showed no significant mutations in any of the studied MMR genes. MLH1, MLH3, MSH2, MSH6, PMS2, POLD1 and EPCAM were mutated in 6(24%), 1(4%), 5(20%), 18(72%), 2(8%), 6(24%) and 3(12%) respectively. In this preliminary report, mutations in MMR genes affect up to 96% of Her2OE BC tumors. Such result may impact on therapeutic choices. This result is to be confirmed in larger cohort.

#3373

Analysis of the "centrosome-ome" reveals potential causes of centrosome amplification in human cancer.

Ryan A. Denu, Justin C. Jagodinsky, Mark E. Burkard. _University of Wisconsin-Madison, Madison, WI_.

The centrosome is the microtubule organizing center of human cells and facilitates a myriad of cellular functions. This includes the organization of the mitotic spindle for faithful chromosome segregation during mitosis, cell polarization and migration, and primary cilia formation. A numerical increase in centrosomes, or centrosome amplification (CA), is common in cancer and correlates with aggressive clinical features and poor patient outcomes. CA is thought to arise by two major mechanisms: (1) centriole overduplication and (2) cell doubling events. To better assess the relative contributions of these two mechanisms, we analyzed 79 melanomas compared to 17 benign nevi and 60 prostate cancers compared to 20 benign prostate samples. We probed these samples for pericentrin (to mark all centrosomes) and CEP170 (to mark centrosomes with mature centrioles). CEP170 is expected on most or all amplified centrosomes with cell doubling, but not with overduplication (1 per cell expected). We find fewer CEP170-positive centrosomes in cancer samples compared to benign samples, indicating that centriole overduplication is the predominant mechanism leading to CA in human cancer. Given this finding, we next sought to identify the predominant molecular mechanisms leading to centriole overduplication. To address this question, we analyzed mutations, copy number alterations, and RNA expression data in the 366 proteins reported to localize to the centrosome using TCGA data. We identified a list of candidate centrosome proteins that are most frequently altered in cancer. Furthermore, given that cells with CA undergo cell cycle arrest unless other compensatory alterations are made, such as loss of p53, we ranked alterations in centrosome genes by the fold enrichment in p53 mutant versus p53 wild-type tumors. We identified the following candidates: CEP19, CEP72, NDRG1, PTK2, SPATC1, and TBCCD1 (gain-of-function mutations) and AKAP9, CEP76, CTNNB1, MCPH1, NEURL4, and NPM1 (loss-of-function mutations or deletion). In cellulo analysis of these candidates reveals that loss of MCPH1 (microcephalin) causes the most robust increase in centriole number. MCPH1 mutations are known to give rise to microcephaly and CA. MCPH1 gene deletions and/or mRNA downregulation (Z score < 2) are seen in 5-15% of human cancers, depending on the anatomic site of the tumor. We conclude that a myriad of different alterations in centrosome genes can give rise to CA in human cancer, and that loss of MCPH1 is a common and penetrant cause of CA in human cancer.

#3374

CRISPR/Cas9 mediated genome editing induces exon skipping by alternative splicing or exon deletion.

Haiwei Mou,1 Jordan Smith,1 Lingtao Peng,1 Hao Yin,2 Jill Moore,1 Xiao-Ou Zhang,1 Chunqing Song,1 Ankur Sheel,1 Qiongqiong Wu,2 Deniz Ozata,1 Yingxiang Li,3 Daniel Anderson,2 Charles Emerson,1 Melissa Moore,1 Zhiping Weng,1 Wen Xue1. 1 _Univ. of Massachusetts Medical School, Worcester, MA;_ 2 _Massachusetts Institute of Technology, Boston, MA;_ 3 _Tongji University, Shanghai, China_.

CRISPR is widely used to disrupt gene function by inducing small insertions and deletions. Here, we show that some single-guide RNAs (sgRNAs) can induce exon skipping or large genomic deletions that delete exons. For example, CRISPR-mediated editing of β-catenin exon 3, which encodes an autoinhibitory domain, induces partial skipping of the in-frame exon and nuclear accumulation of β-catenin. A single sgRNA can induce small insertions or deletions that partially alter splicing or unexpected larger deletions that remove exons. Exon skipping adds to the unexpected outcomes that must be accounted for, and perhaps taken advantage of, in CRISPR experiments.

#3375

The chromosomal instability in tumorigenesis of esophageal squamous cell carcinoma.

Ting Yan, Xiaolong Cheng, Yongping Cui. _Shanxi Medical University, Taiyuan, China_.

Chromosomal instability (CIN) is the leading cause of the cell genome instability and is a common trait of more than 70% of human cancers, therefore is implicated as an initiator of tumorigenesis. More importantly, CIN may facilitate the adaptation of tumors to environmental or stromal stress and is implicated in determining tumor progression and associated with poor outcome, tumor relapse, and multi-drug resistance across a range of cancer types. The latest studies suggest that cancer is the result of micro-evolution altermate with macro-evolution. That means the cells get mutations through micro-evolution and expansion by the mutational accumulation; the cells begin to undergo macro-evolution instead of micro-evolution when encounter tumorigenic factors, and CIN will be the main driver for tumor evolution. However, studies focus on genomic signatures, especially for macro-evolutionary events, in esophageal squamous cell carcinoma (ESCC) are limited. We integrated 31 whole genomic sequencing data of ESCC samples from China to investigate underlying evolutionary characteristics in ESCC.

We combined copy number profiles of 1660 cancer specimens from 5 types of gestrointestinal tumors. Different from colorectal carcinoma, liver hepatocellular carcinoma, pancreatic adenocarcinoma and stomach adenocarcinoma, ESCC possesses largely copy number alterations, and frequent copy-number changes were not clustered in specific chromosome. In our cohort, we found whole genome doubling (WGD) that acts as one of mechanisms for polyploidy was predicted as a late event in the majority of ESCC genome. Moreover, loss of heterozygosity events was more likely to occur in chromosomes harboring tumor suppressor genes in ESCC. The 40% of neutral loss of heterozygosity events was not a result of genome doubling suggesting an alternative mechanism for neutral loss of heterozygosity formation. Importantly, deconstruction of copy number alterations extending to telomere revealed that telomere-bounded copy number alterations play a critical role for amplification/deletion of oncogenes/suppressor genes. For well-known genes SOX2, PIK3CA and TERT, almost all of their amplifications were telomere bounded, which was further confirmed in a Japanese ESCC cohort. Furthermore, we provide evidence that karyotype evolution was mostly punctuated in ESCC. Collectively, our data reveal the potential biological role of whole genome doubling, neutral loss of heterozygosity and telomere-bounded copy number alterations, and highlight macro-evolution in ESCC tumorigenesis.

#3376

Early onset of chromosomal instability in breast preneoplasia detected by single-cell genomics.

Katerina Fagan-Solis,1 Dennis A. Simpson,1 Luciano Martelotto,2 Jorge S. Reis-Filho,2 John H. Petrini,2 Gaorav P. Gupta1. 1 _The University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Chromosomal rearrangements and copy number aberrations are a major mechanism for driver gene alterations in breast cancer. However, the origins of chromosomal instability during breast tumorigenesis are poorly understood. We have recently shown that oncogene expression in normal mammary epithelial cells induces replication stress and a DNA damage response (DDR). The double-strand break sensor Mre11 is required for the oncogene-induced DDR, and suppression of Mre11 is sufficient to promote the development of Her2/Neu-driven breast cancer in a transgenic mouse model. Here we have applied single-cell genomics to primary mammary epithelial cells from this mouse model and demonstrate a profound effect of oncogene expression on chromosomal instability in preneoplastic cells, which is further augmented in Mre11-mutant cells. Her2/Neu-induced chromosomal breakpoints are highly enriched in the vicinity of large genes, suggesting a possible role for gene transcription and/or R-loops in the origin of oncogene-induced genomic fragility. Mre11 hypomorphism does not affect the number of chromosomal breakpoints but alters the genomic aberration signature to one that is over-represented by larger-size deletions. These findings indicate that chromosomal instability is an early event during Her2/Neu-initiated breast neoplasia. Furthermore, DDR perturbation—in the form of Mre11 hypomorphism—is not required for oncogene-induced genomic instability but rather alters the pattern of observed copy number aberrations and promotes the proliferative expansion of genomically unstable clones. Thus, single-cell genomics applied to murine models of breast preneoplasia reveals insights into the origins of chromosomal instability in breast cancer that may inform novel strategies to prognosticate or prevent the progression of high-risk premalignant breast lesions.

#3377

Extensive subclonal mutations in human colorectal cancers detected by duplex sequencing.

Lawrence A. Loeb,1 Kaitlyn J. Loubet-Senear,1 Brendan F. Kohrn,1 Michael W. Schmitt,1 Mary P. Bronner,2 Robert A. Beckman3. 1 _Univ. of Washington, Seattle, WA;_ 2 _University of Utah, Salt Lake City, UT;_ 3 _Georgetown University Medical Center, Washington, DC_.

The accumulation of somatic mutations is a defining hallmark of cancer. Human colorectal cancers (CRC) contain thousands of mutations that are detected at frequencies greater than 1%. Some of these are "driver mutations" that have been positively selected during tumor progression. In addition, subclonal mutations—those occurring only in a fraction of malignant cells—are randomly distributed and contribute to phenotypic and morphologic heterogeneity of cancer cells within a tumor. Cells containing specific subclonal mutations may be present prior to therapy and can be positively selected and account for the rapid emergence of therapeutic resistance. The extent of subclonal mutations in cancer, however, has been difficult to quantify, as the high error rate of next-generation sequencing precludes reliable detection of mutations present in fewer than 1-5% of cells. Here, we apply the highly accurate duplex sequencing methodology to quantify the extent of subclonal mutations in CRC. We analyzed somatic mutations in genes encoding replicative DNA polymerases (POLδ and POLε) as well as in genes reported to be frequently mutated in CRC. Duplex sequencing was carried out at depths varying from 500X-30,000X and an accuracy of 10-8. We find that CRCs with DNA repair deficits harbor an unexpectedly large complement of unique subclonal mutations; indicating that the mutational diversity of CRCs has been greatly underestimated. The frequency of subclonal mutations in normal colonic mucosa and CRC is greater than reported for clonal mutations. The burden of tumor-associated subclonal mutations does not correlate with age and the spectrum of single-nucleotide substitutions is different from that in nonmalignant cells, indicating that different mechanisms of mutation accumulation are operative in normal and CRC. Our data are consistent with a mutation rate of 1.1 x 10 per base pair, indicating that each malignant cell and its daughter encoded genomic DNA differs by more 3,000 nucleotides. The linearity of subclonal mutation accumulation as a function of sequencing depth, using DNA obtained from five different tumors, is in accord with a neutral model of tumor evolution. We calculate that the probability of mutations at any type is so high that it is likely that every possible somatic point mutation is present by the time a tumor is clinically detectable, and this could account for the high frequency by which tumors become resistant to therapeutic agents.

#3378

Variance in the distance between chromosome territories 9 and 22 in lymphocytes from peripheral blood healthy subjects: A proof of concept for a possible risk factor in chronic myeloid leukemia.

David G. Vallejo, Eunice Fabian, Luis A. Herrera. _Instituto Nacional de Cancerología, Ciudad de México, Mexico_.

The objective of our study was to assess the variations in the 3D organization of Chromosome Territories (CT) of chromosomes 9 and 22 in lymphocytes from healthy subjects. The differences in the distances of CTs 9 and 22 between subjects may provide a new perspective in the predisposition for specific chromosome translocations. We quantified the intra- and inter-subject variation in the distance of CT 9 and 22 as a proof of concept for new questions on the physiological and pathological effects of these differences. Methods: Blood samples from healthy subjects were processed for the isolation of lymphocytes with Ficoll as the gradient media. After the isolation, we fixed the cells directly onto coverslips for a 3D Fish protocol with 9 and 22 whole chromosome probes. Our protocol has been standardized to maintain the integrity of the nucleus with only minor changes in its volume. After the hybridization, we used the Elyra System of Zeiss to obtain super resolution images in Z stack. The images were processed with the Imaris software for its 3D reconstruction, measurement (distances, volumes and colocalization) and analysis. Results: The CTs 9 and 22 behave differently in peripheral blood lymphocytes. The CT 22 has a more internal position (closer to the center of the nucleus) in comparison with the CT 9, which can be seen most of the times in contact with the border of the nucleus. The CT 22 occupies a smaller volume than CT 9, which has almost a twice as larger volume. The CTs 9 have a polarized distribution inside the nucleus, therefore the distance between the CT 9-9 was larger (~7.3 μm ± 0.9) than the distance between CT 22-22. Although the position of the CTs was highly variable in cells from the same individual, we found a statistically significant difference in the inter-individual distance between CT 9-9 and the minimal distance between CT 9-22. Conclusion: Our study suggests that there are differences in the distribution of CT in lymphocytes isolated from peripheral blood between our subjects. These differences could prove to be a new individual characteristic with important implications in the predisposition for specific diseases, mainly to those related with chromosomal rearrangements.

#3379

Predictors of mosaic chromosome Y loss and associations with mortality in 223,338 men of the UK Biobank.

Erikka Loftfield, Weiyin Zhou, Meredith Yeager, Stephen J. Chanock, Neal D. Freedman, Mitchell J. Machiela. _National Cancer Institute, Bethesda, MD_.

Mosaic loss of the Y chromosome (mLOY) in peripheral leukocytes is an age-related event commonly observed in men older than 50 years of age in which a fraction of leukocytes loses the Y chromosome. In addition to age, previous studies have indicated an association between smoking and increased risk of mLOY. The relationship between mLOY and other health-related exposures (e.g., obesity and alcohol consumption) and with disease outcomes (e.g., cancer risk and overall mortality) remains unclear. We therefore investigated the causes and consequences of mLOY in a large collection of 223,338 men from the UK Biobank. We scanned Y chromosomes for deviations in genotyping array log R ratio intensities for evidence of mLOY. A total of 3,789 (1.7%) men showed evidence for mLOY with median chromosome Y LRR values less than -0.15 and 596 (0.3%) men had high proportions of cells affected with mLOY with median LRR values less than -0.40. Our analysis robustly confirms prior associations of increasing mLOY risk with increasing age (Padj<4.9×10-324). In addition, we observe a strong association between mLOY and smoking (Padj=7.1×10-193), with a higher percentage of current smokers (3.4%) affected by mLOY than former smokers (2.1%) and non-smokers (0.9%). As reported previously for autosomal mosaicism, we observed less mLOY in men of African ancestry (0.4%) compared to men of European ancestry (1.8%, Padj=0.002). Surprisingly, obese men were less likely to have mLOY, with a lower percentage of men with a BMI≥35 having mLOY than men in the normal range (1.3% vs 1.7%, Padj=0.0007). Self-reported health was also associated with mLOY, with a higher percentage of mLOY among men reporting poor health than men reporting excellent health (2.1% vs 1.4%, Padj=0.008). Although no associations were observed between mLOY and prevalent non-melanoma skin cancer (Padj=0.54), suggestive associations were observed with prevalent diabetes (Padj=0.003) and prevalent cardiovascular disease (Padj=0.01). With respect to mortality, we observed an association with mLOY in men with high proportions of cells affected only (LRR<-0.40, Padj=0.002). By cause of death, we noted an association for cancer-specific mortality (HR=1.53, 95% CI=1.13-2.06, Padj=0.0056) in men with high proportions of cells affected, but not for cardiovascular disease mortality, although the number of deaths among men with mLOY was modest (LRR<-0.15= 334 deaths, LRR<-0.40= 75 deaths). Overall, we see evidence for an association of mLOY with several health-related exposures as well as with mortality among men with high proportions of cells affected. Future functional studies related to the physiologic effects of chromosome Y loss in circulating leukocytes are needed to better understand how mLOY may impact disease risk.

#3380

BRIP1/FANCJ is vital for efficient response to and repair of replication-associated DNA damage.

David W. Clark, Chinnadurai Mani, Komariah Palle. _University of South Alabama Mitchell Cancer Institute, Mobile, AL_.

BRCA1-Interacting Protein 1 (BRIP1), a DNA helicase and tumor suppressor also known as FANCJ, is implicated in breast and ovarian cancers as well as the genetic instability syndrome Fanconi anemia (FA) which predisposes patients to multiple neoplasms. This study investigates the role of FANCJ in repair of replication-associated DNA damage which can lead to DNA strand breaks, chromosomal instability, and tumorigenesis. FANCJ has long been known to function in repair of DNA crosslinks; however, its role in protecting cells from replication-associated DNA damage is less well known. We have shown that loss of FANCJ sensitizes cells to damage caused by compounds that block DNA replication, such as hydroxyurea (HU) and the topoisomerase I poison camptothecin (CPT). Loss of FANCJ disrupts the cell's response to replication-associated DNA damage by impairing Chk1 and Chk2 phosphorylation and stability and activation of multiple DNA repair proteins. FANCJ-deficient cells exposed to low levels of CPT failed to progress through S-phase indicating that FANCJ is important for intra-S phase DNA damage checkpoint and recovery from replication fork blocks. FANCJ depletion leads to a 90% decrease in homologous recombination repair in assays designed to measure repair of DNA double strand breaks as well as a loss of expression of repair-associated proteins and DNA repair foci in response to damage from HU and CPT. Loss of FANCJ impairs DNA replication and leads to accumulation of gross chromosomal aberrations. Importantly, some of the DNA damage response functions of FANCJ are independent of its helicase activity, as a helicase-deficient mutant is able to compensate for loss of FANCJ in activation and stability of FANCD2 and other HR proteins. Collectively our findings indicate a novel role for FANCJ as a key regulator of the DNA damage response to errors in DNA replication potentially leading to mutagenesis and tumorigenesis.

#3381

Determining the relationship between a measure of stress-induced mutagenesis and patient survival in cancer.

Luis Cisneros,1 Charles Vaske,2 Kimberly J. Bussey1. 1 _NantOmics, LLC, Tempe, AZ;_ 2 _NantOmics, LLC, Santa Cruz, CA_.

Background: Clustering of mutations observed in cancer cells is reminiscent of the clustering caused by the stress-induced mutagenesis (SIM) response in bacteria. SIM occurs when double-strand breaks (DSB) in DNA are accompanied by cellular stress, leading to mutations concentrated around the DSB with a characteristic decay in abundance with distance from the DSB. We hypothesized that clustered mutations in cancer are caused by SIM and measures of SIM correlate with patient survival.

Methods: Data from whole genome sequencing of 1939 tumors from the International Cancer Genomics Consortium portion of the Pan-Cancer Analysis Working Groups (PCAWG) were analyzed. Clusters were defined as 3 or more single nucleotide variants (SNVs) with inter-variant distances of less than 25kb and a < 1% probability of occurrence based on the negative binomial regression given by the total rate of observed mutations in the genome. Cluster shape was measured by computing a normalized cumulative probability distribution of SNVs within 250kb of cluster centroids. A stress-introduced heterogeneity (SItH) score was calculated to quantify the difference between the observed distribution and a uniform, random distribution (the null hypothesis). This score can be computed on a per cluster basis as well as a single overall score on a per patient basis.

Results: Overall SItH scores ranged from 0.145 to 0.999 and varied significantly by organ site (ANOVA, F=141.9, p<2.2x10-16). After controlling for organ site, we found that in primary tumors, more positive SItH scores predicted increased patient survival (Cox Proportional Hazard Regression [CPHR]; Hazard Ratio (HR) = 0.431, 95% CI: 0.2033-0.9138, p=0.0282). However, when recurrent and metastatic tumors were considered as a group, higher SItH scores predicted decreased patient survival (CPHR; HR = 7.87, 95% CI: 1.229-50.49, p= 0.0295). When we looked at the diversity of SItH scores computed on a cluster basis, the type of tumor sample was no longer relevant. The inner quartile range (IQR) of the per interval SItH score associated with worse survival, with a HR of 4.59 (Cox Proportional Hazard Regression, 95% CI: 1.261-16.7, p= 0.0208).

Conclusions: The heterogeneity represented by the IQR is likely derived from both the strength of SIM as mutational process within a tumor and the clonal diversity of the tumor, both of which would be expected to impact disease outcome. The relationship between overall SItH and the IQR with respect to survival suggests that cluster heterogeneity predominantly represents the amount of time SIM has been active during carcinogenesis while overall SItH represents the ratio of SIM relative to other mutational processes.

#3382

Methylation status and microRNA expression changes in women with breast cancer stratifying by DNA repair capacity.

Carmen M. Ortiz-Sanchez,1 Jarline Encarnacion,1 Rafael Guerrero,2 Jaime Matta1. 1 _Ponce Health Sciences University-Ponce Research Institute, Ponce, Puerto Rico;_ 2 _Johns Hopkins School of Medicine, Baltimore, MD_.

The American Cancer Society estimates 252,710 new breast cancer (BC) cases and 40,610 cancer related-deaths in the US, only in 2017. In addition to the well-established BC risk factors, our studies during the last decade have shown that women having a low DNA repair capacity (DRC) as measured through the nucleotide excision repair pathway is associated with higher BC risk. This finding allows us to study BC etiology based on DRC rather than using the ordinary stratification; for example, we found an association between DRC levels and estrogen receptor status. Since DRC has been associated with different clinical aspects of BC and it is known that it can be regulated through epigenetic mechanisms, more studies regarding the effect of epigenetic changes on DRC in BC are needed. Therefore, the main objective of this study is to identify epigenetic mechanisms that could be influencing DRC levels in women with BC through gene silencing. The main hypothesis consists in testing whether the epigenetic changes such as methylation and microRNA (miRNA) levels can be used to stratify BC patients by DRC levels. Plasma and tumor samples were randomly selected from our BC cohort. DRC values were previously measured in lymphocytes using the host cell reactivation assay. BC cases and controls were divided in two categories: low (≤3.8%) and high (>3.8%) DRC. MicroRNAs were extracted from 56 plasma samples (27 BC cases and 29 controls) to perform an expression profile analysis using the Human MicroRNA A Cards v 2.0 assay. Relative miRNA expression was calculated using the 2-ΔCt method. Methylation status of tumors was obtained through an epigenome-wide DNA methylation analysis stratifying by DRC using the Illumina Human Methylation EPIC 850K array. After unbiased selection and pathologist evaluation, 56 formalin-fixed paraffin-embedded tumors were selected and sectioned for DNA extraction. Based on the DRC, the samples were divided in low (n=24) and high (n=32) using the established cut-off. A significant association between miR-323-3p, miR-29b, and miR-159a expression and the BC outcome was found (p<0.01). Using the DRC categories, all three miRs were found to be differentially expressed when comparing cases and controls with high and low DRC (p<0.05, KW). The methylation data was analyzed using the bump hunting method. The epigenome-wide analysis of the BC methylome revealed 23 regions that were significantly differentially methylated between tumors with low and high DRC (p<0.05). Although DRC levels are low when the BC malignancy is developed, whether these changes occur during carcinogenesis or due to a preexisting epigenetic disposition is still unknown. However, our results lead us to conclude that epigenetic changes such as methylation and miRNA expression can influence the DRC levels in BC. This study was supported by: S06GM008239-20, 9SC1CA182846-04, U54CA163068, 2U54CA163071-06, and R003050/MD007579.

#3384

ER-α: The paramount facet predisposing BRCA1 defective tumors.

Arathi Rajan, Neetha Rajan Latha, Priya Srinivas. _Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, India_.

BRCA1 and BRCA2 are key tumor-suppressor proteins that aid in DNA damage repair and hence, play a pivotal role in ensuring the cellular genomic stability. Though BRCA1 protein is indispensable for all the cells, there is a restriction of cancers due to BRCA1 mutation, largely to the breast and the ovary. To date, a valid explanation for this predisposition does not prevail; nevertheless, a link between estrogen receptor-α (ER-α) and BRCA1 has been reported. Thus, it could be speculated that BRCA1 functions are being manifested through numerous interacting partners, which once identified, might unravel the enigma behind the BRCA1 defective selective tumorigenesis. These factors thus led us to the current study, which aims to analyze the role of ER-α in BRCA1-mediated DNA damage repair and its association with breast tumorigenesis. The direct interaction between BRCA1 and ER-α has been extensively elucidated and BRCA1 mutation has been well known to be associated with cancers in tissues where ER-α is overexpressed; consequently, its role in DNA damage repair could be a definite possibility. To deduce the exact function of ER-α in BRCA1-mediated DNA damage repair, ER-α proficient and BRCA1 wild-type breast cancer cell line, MCF-7, was used for the study. Stable clones of MCF-7 cell lines deficient in ER-α as well as BRCA1 were generated by independent shRNA transfections. Repairable DSBs were created in the cell lines using specific concentration of the platinum-based drug, cisplatin (cis-diamminedichloridoplatinum). Nuclear damage (DNA-DSB) and the ability of cells to sense and repair the DSBs due to cisplatin treatment in ER-α/ BRCA1 deficeint and proficient cells was detected by the scoring of phosphorylation in the histone variant H2AX (γ-H2AX) by immunocytochemistry and Western blot analysis. Damage repair events like phosphorylation of BRCA1 and its recruitment to the nuclear damage site as foci and direct interaction between these proteins during damage repair were analyzed by varying experimental procedures. Finally, the DNA DSB repair efficiency of these cells were directly measured using specific DNA DSB repair assays. The results obtained from microscopy, immunocytochemistry, Western blot analysis and other assays noticeably indicated that the DNA damage repair efficiency was significantly reduced in the ER-deficient condition. Together, these results supply clues to the indispensable role of ER-α in DNA DSB repair. The current study acts as basic evidence in understanding the molecular biology behind the predisposition of BRCA1 mutations to breast or ovarian cancers, which otherwise remains elusive. Unveiling these hidden mechanisms might pave the way for the identification of new target,s which would enable better therapeutics for these cancers.

#3385

ETNK1 **mutations promote ROS production and DNA damage through increased mitochondrial activity.**

Diletta Fontana,1 Mario Mauri,1 Antonio Niro,1 Luca Massimino,1 Mayla Bertagna,1 Giovanni Zambrotta,1 Mario Bossi,1 Stefania Citterio,2 Barbara Crescenzi,3 Giovanni Signore,4 Vincenzo Piazza,4 Cristina Mecucci,3 Guido Cavaletti,1 Delphine Rea,5 Carlo Gambacorti-Passerini,1 Rocco Piazza1. 1 _University of Milano-Bicocca, Monza, Italy;_ 2 _University of Milano-Bicocca, Milan, Italy;_ 3 _University of Perugia, Perugia, Italy;_ 4 _Istituto Italiano di Tecnologia, Pisa, Italy;_ 5 _Hopital Saint-Louis, Paris, France_.

Atypical chronic myeloid leukemia (aCML) is a clonal disorder belonging to the myelodysplastic/myeloproliferative syndromes. About 13% of aCML cases carry somatic mutations in ETNK1 gene, encoding for H243Y, N244S, and G245V substitutions. We previously showed that ETNK1 mutations cause a decreased catalytic activity of the enzyme. Despite this evidence however, their oncogenic role remained largely unexplained. Since ETNK1 activity is essential for the synthesis of phosphatidylethanolamine (PE) and given that PE is one of the most abundant phospholipids in the inner membrane of mitochondria, we focused our attention on mitochondrial activity. In order to characterize the oncogenic effect of ETNK1 variants we generated CRISPR/Cas9 clones carrying heterozygous N244S mutation and homozygous ETNK1 deletion (KO cells) on the 293 Flp-In™ cell-line. Both N244S and KO cells showed a significant increase in mitochondrial activity (1.78 and 2.13 fold increase, respectively; p= 0.0096 and p=0.0050) compared to WT, as assessed by MitoTracker™ Red. In line with this finding, electron microscopy revealed a significant modification in mitochondria morphology for N244S and KO cells, changing from an elongated, tubular shape to a round, swollen one. ATP (1.67 and 1.68 fold; p<0.0001; ATPlite Luminescence Assay System) and ROS production (1.66 and 1.74 fold increase; p<0.0001; CellROX™ Green Reagent) were similarly increased. Histone H2AX phosphorylation (γ-H2AX) analysis revealed a higher number of foci in N244S and KO cells (2.60±0.22 and 2.89±0.27; p<0.0001) compared to WT (0.56±0.08). A similar increase in γ-H2AX (3.6 fold; p=0.0037) was present in primary samples from aCML patients carrying ETNK1 mutation compared to ETNK1-WT ones.

In line with these data, a higher mutation rate was detected in N244S and KO cells (8.09*10-7±9.6*10-8 and 8.20*10-7±1.28*10-7; p=0.0060 and p=0.0264) compared to WT (2.98*10-7±8.2*10-8) by 6-thioguanine assay.

The reconstruction of the hierarchy of somatic mutations in ETNK1-mutated aCML patients revealed that ETNK1 variants invariably occur very early in the evolution history of the aCML patients.

Taken together, our results show that impairment of ETNK1 function causes an increase in mitochondrial activity, which in turn leads to increased ROS production driving the accumulation of DNA mutations. Since the characterization of aCML subclonal architecture indicates ETNK1 mutations as a very early event in the history of the disease, we hypothesize that ETNK1 could contribute to the onset of aCML through the activation of a mutant phenotype, which in turn would accelerate the accumulation of further oncogenic mutations.

#3386

The oncogenic role of CD133 in hepatocellular carcinoma.

Yick Pang Ching, Wing Lim Chan. _The University of Hong Kong, Hong Kong, Hong Kong_.

Liver cancer (hepatocellular carcinoma, HCC) is one of the most prevalent malignancies worldwide. Because of the late diagnosis of the disease, the prognosis of HCC patient is often undesirable. Cancer Stem Cell (CSC), which represents a subpopulation of cancer cells with stem-like properties, plays a critical role in tumorigenesis and contributes significantly to the relapse and chemo-drug resistance of cancers. The cell surface marker, CD133/Prominin-1, has been identified as a biomarker for CSC in various cancers, including HCC. To understand the role of CD133 in hepatocarcinogenesis, we expressed CD133 in HCC cells and showed that CD133 significantly promoted cell growth. Interestingly, our data also indicated that expression of CD133 led to chromosomal instability indicated by elevation of aneuploidy population and centrosome numbers in CD133 expressing cells. To understand the molecular mechanism, we observed that a putative tumour suppressor in HCC, called TAX1-binding protein 2 (TAX1BP2), which functions as a centrosome over-duplication suppresser, was downregulated by CD133. Taken together, these data suggest that CD133 plays an oncogenic role in HCC and may downregulate TAX1BP2 to promote chromosome instability, centrosome duplication and hepatocarcinogenesis.

#3387

The pliancy of plasma cell differentiation status conceals a gradient of chromosomal instability in newly diagnosed multiple myeloma patients.

Marina Martello, Rosalinda Termini, Barbara Santacroce, Enrica Borsi, Vincenza Solli, Chiara Benni, Andrea Poletti, Lucia Pantani, Beatrice Zannetti, Serena Rocchi, Elena Zamagni, Paola Tacchetti, Francesca Ulbar, Mario Arpinati, Gabriella Chirumbolo, Nicoletta Testoni, Giulia Marzocchi, Giovanni Martinelli, Michele Cavo, Carolina Terragna. _Univ. of Bologna, Bologna, Italy_.

The pliancy of Multiple Myeloma (MM) plasma cells differentiation status acts as adaptive strategy to exogenous stress (e.g. in response to therapy). However, the genomic background that supports any diverse plasma cell differentiation phenotype has not yet been inferred.

Aim: to correlate the genomic background with the phenotypic plasticity of MM clone(s) at diagnosis, in order to stratify patients (pts) according to both the level of chromosomal instability (CIN) and their PC differentiation stages, and ultimately to evaluate the impact of this stratification on the disease outcome.

64 newly diagnosed pts were included in the present study. Most pts (54/64) received a PI-based treatment as front-line therapy. Each patient was characterized by 6-color multiparametric flow citometry analysis, combining CD138-PE, CD38-PE-Cy7, CD20-APC, CD19-APC-Cy7, CD27-FITC, CD45-FITC, CD28-APC, CD44-FITC, CD54-APC, CD81-PerCP-Cy5.5, CD56-APC and SHH-PE, as functional marker of Hedgehog pathway activation (Miltenyi Biotech). Whole CNAs characterization of CD138+ purified BM PCs was carried out by SNPs array ( Cytoscan HD ,Affymetrix).

According to the detected CIN, as described both by total CNAs and portion of genome changed (GC), 3 major subgroups were identified: the first one with high CIN (21 pts; medium tot. CNAs = 550, % GC ≥ 25%); the second one with a intermediate CIN (25 pts; medium tot. CNAs = 315, % GC = 10-25%) and the third one with low CIN (18 pts; medium tot. CNAs = 105, % GC ≤ 10%). As expected, in pts with high CIN, hyperdiploidy explains more than a quarter of unstable genome; however, they were also characterized by a higher prevalence of high-risk features, such as 1p deletion (FAF1), 16q deletion (WWOX, FANCA) and 17p deletion (TP53), which were almost exclusively associated to high CIN pts (p<.05).

A detailed immunophenotypic analysis of the three subgroups of pts showed that high CIN background mainly characterized mature PCs (17/21 = 81%), as described by: a) a significant deregulation of both CD19 and CD81; b) a higher expression of CD28 and CD44; c) a reduced expression of CD20, CD27 and CD45.

Finally, the presence of more mature PCs with high CIN characterizes pts carrying baseline clinical features associated to bad prognosis (e.g. PET lesions, k/l ratio, ISS III, β2-microglobulin; p<.05). In addition, these pts tend to obtain high quality response rates (≥CR) to PI induction therapy.

In conclusion, a high level of genomic complexity correlates with an advanced PCs differentiation stages in newly diagnosed MM patients; this is associated with a prevalence of poor prognosis features. Chromosomal instability, together with cellular phenotypic pliancy, represents an important, yet poorly defined, mechanism by which MM clone(s) accelerate their own evolution and survival.

Acknowledgements: AIRC, AIL, Fond. Berlucchi.

#3388

Molecular characterization of HPV type 18 cervical cancer: Upregulation of telomerase expression and induced chromosomal instability by E6 and E7 oncoproteins.

Mai Do, Nichelle Cox, Naomi Long, Liliana Zarate, Chinelo Ezechukwu, Jenna Cormier, Hyun Chung, Meidrah Tyler, Judith Okoro, Diondra Harris, Victoria Vidal, Jerica Watson, Ellie Canty, Shyam Arya, Benjamin Liu, Roland Pattillo, Billy Ballard, Jay Vadgama, Eva McGhee. _Charles R. Drew University of Medicine and Science, Los Angeles, CA_.

Human papillomavirus (HPV), high-risk type 18, is directly associated with approximately 98% incidence of invasive cervical cancer. Epithelial cells infected with HPV-18 become transformed and exhibit overexpression of telomerase activity and chromosomal instability. As a result of this transformation, E6 and E7 oncoproteins are perpetually expressed: E6 degrades the tumor suppressor p53; E7 inhibits the tumor suppressor pRB, leading to uncontrollable growth and the extension of telomere length. While a few studies explored the chromosomal instability induced by HPV E6 and E7 oncoproteins, the full scope of the problem has not been clearly characterized. In this research we investigated HPV-18 acquired genomic instability by E6 and E7 oncoproteins. HPV-positive cervical cancer cells, HeLa, were studied through spectral karyotyping (SKY), Giemsa banding (G-banding), telomere length, and telomerase activity using PCR ELISA assay and the TRAPeze XL Kit, which uses fluorescence energy transfer. With the use of SKY, G-banding, and chromosomal instability analysis, HeLa cells exhibited many aneuploidies. In our results, there were consistent translocations on chromosomes 4 and 11, with deletions on chromosomes 11 and 20. There were also other translocations on chromosomes 9 and 20, with deletions on chromosome 10. Additionally, copies of chromosome 5 and unidentifiable marker chromosomes were noted. Moreover, telomerase data suggest that upregulated expression of telomerase activity correlates with the increase in chromosomal instability. The aforementioned aneuploidy is demonstrative of the induced chromosomal instability from HPV-18 infection. In addition to the tumor-suppressor disruption acquired by E6 and E7 oncoproteins, HeLa cells showed gene deletion on chromosome 11. The ATM gene, which is specifically located between distal regions 11q22.3 and 11q23 of chromosome 11, is known to help identify breaks in DNA and plays a crucial role in DNA repair. Damage to the ATM gene may further play an important role in increasing cancer progression.

### Genomic Profiling of Tumors 1

#3389

Clonal evolution of atypical proliferative lesions into breast cancers.

Tomomi Nishimura,1 Kenichi Yoshida,1 Yukiko Kawata,1 Yasuhide Takeuchi,1 Nobuyuki Kakiuchi,1 Yusuke Shiozawa,1 Kosuke Aoki,1 Masahiro Hirata,2 Tatsuki R. Kataoka,2 Takaki Sakurai,2 Yuichi Shiraishi,3 Kenichi Chiba,3 Kengo Takeuchi,4 Hironori Haga,2 Satoru Miyano,3 Masakazu Toi,1 Seishi Ogawa1. 1 _Kyoto University, Kyoto, Japan;_ 2 _Kyoto University Hospital, Kyoto, Japan;_ 3 _The Institute of Medical Science, University of Tokyo, Japan;_ 4 _Japanese Foundation for Cancer Research, Kyoto, Japan_.

[Introduction] Non-malignant proliferative lesions in the breast have been implicated in the development of invasive breast cancer. Previous studies showed that adjacent atypical proliferative lesions and breast cancers shared common genetic alterations, suggesting that these evolved from the same ancestral cell. However, the clonal structure of atypical proliferative lesions and their clonal dynamics during progression to cancer are poorly understood. In this study, we compared genetic profiles of normal ducts, non-malignant proliferative lesions and cancers in the same patients to illustrate the clonal evolution of cancer from a non-malignant epithelial cell.

[Methods] Multiple samples were collected from different atypical proliferative lesions within the cancer-borne breast, including invasive cancers, using micro-dissection from formalin-fixed, paraffin-embedded surgical specimens. Somatic mutations and copy number alterations (CNAs) were then evaluated by whole exome sequencing.

[Results] A total of 34 samples from 5 premenopausal females carrying estrogen receptor-positive cancers were analyzed, where the samples were obtained from normal ducts (N = 6), non-atypical (N = 1) and atypical (N = 8) proliferative lesions, and invasive (N = 3) and non-invasive (N = 16) cancers. The number of somatic mutations per sample was ranging from 1 to 276 and increased with an elevation of atypical-level. Two cases with bilateral cancers had a pathogenic germline mutation of either BRCA2 and TP53, where no somatic mutations or CNAs were shared by individual proliferative lesions, suggesting multifocal independent cancerous evolutions. By contrast, in the remaining three unilateral cases, no pathogenic germline mutations were detected, but all proliferative lesions, which were separated by a distance of 7-25 mm, shared one or more driver alterations, such as an AKT1 mutation (UID: KU01), concurrent 1q gain and 16q loss (der(1;16)) (UID: KU02), and a GATA3 mutation and der(1;16) (UID: KU03), while harboring private mutations/CNAs of their own. The analysis of phylogenic trees based on the number of shared mutations predicted an early origin of these founder mutations, which frequently predated decades before the onset of cancer.

[Conclusions] Early breast cancer development is thought to be shaped by the simultaneous evolution of multiple precancerous clones. It may be multi-focally initiated by a germline mutation, frequently terminated in bilateral cancers. Otherwise, in unilateral cases, cancer clones might be derived from a common ancestral clone, which has acquired a driver founder mutation long before the onset of cancer, and undergo independent evolution, giving rise to multiple proliferative lesions, from which invasive cancer finally evolves. Our findings provide unique insight into the early development of breast cancer.

#3390

Comprehensive analysis of tumor immunity by integrating transcriptome and methylome.

Xuefeng Wang,1 Cong Liang2. 1 _Moffitt Cancer Center, Tampa, FL;_ 2 _Yale University, New Haven, CT_.

Immunotherapy has produced promising results in treating cancers. Further understanding of the crosstalk between tumor and its immune microenvironment is likely to have an immediate impact on therapeutic intervention as well as the development of markers of drug resistance. One main difficulty in quantifying the components of tumor microenvironment is that currently there are considerable technological and analytical barriers to assessing and measuring tumor immunity in situ. In this work, we propose to improve the prediction of cell mixtures by both gene expression and epigenetic information. The vast amount of DNA methylation data has been collected in public domain but remains under-analyzed and largely disconnected. No studies have yet investigated the value of using existing methylation panels for immune cell deconvolution in tumor tissues. In our preliminary data, we have observed a strong association between DNA methylation probes and immune signatures such as total TCR expression and tumor infiltrates estimation in melanoma and breast cancer. We have built a more rigorous prediction model based on methylation markers and to extend the analyses to all major cancer types. The results will be externally validated with pooled data from published studies with eligible methylation assay and immune signature measurements. We work closely with immunology scientists to perform a critical evaluation of the robustness and reproducibility of the predictive models. Results of the study, if validated, will have immediate clinical application in screening patient immunity, and will further support the hypothesis that the DNA methylome adds a new dimension to the pan-omic snapshot of cancer immunity and tumor microenvironment. Based on the proposed analytical framework, future prospective studies can be developed using newer methylation platforms—to compare patient groups with different immune conditions and patients in different stages of treatment.

#3391

Comparison of gene copy number variations (CNV) in primary and metastatic lung cancers.

Yunfei Shi,1 Naiquan Mao,2 Xiao Ding,3 Jianji Guo,4 Gang Guo,5 Zheng Chen,6 Weiguang Gu,7 Lei Dai,4 Shiwang Wen,8 Hui Jia,9 Ziqiang Tian,10 Junping Shi,11 Taiyan Guo,11 Jicheng Yao,11 Fei Pang,11 Gungwei Chirn,11 Ming Yao11. 1 _First Affiliated Hospital of Kunming Medical University, China;_ 2 _Affiliated Tumor Hospital of Guangxi Medical University, China;_ 3 _Shandong Provincial Hospital Affiliated to Shandong University, China;_ 4 _First Affiliated Hospital of Guangxi Medical University, China;_ 5 _The Third Affiliated Hospital of Kunming Medical University,Yunnan Tumour Hospital, China;_ 6 _Shanghai Shandong Provincial Hospital Affiliated to, China;_ 7 _The People's Hospital of Nai Hai, China;_ 8 _Fourth Hospital of Hebei Medical University, China;_ 9 _Shandong Cancer Hospital Affiliated to Shandong University, Shandong Academy of Medical Sciences, China;_ 10 _OrigiMed Inc. Fourth Hospital of Hebei Medical University, China;_ 11 _OrigiMed Inc., Shanghai, China_.

Background: Characterization of genomic alterations that drive patient disease development is critical in cancer management. The genomic alteration and population characteristics between the primary tumors and metastases in patients with non-small cell lung cancer (NSCLC) is still unclear. Here we evaluated the frequency and distribution of all classes of genomic alterations in both primary and metastatic NSCLC by comprehensive genomic profiling approach.

Methods: FFPE tumor and matched blood samples of 1135 Chinese NSCLC patients (female:male is 670: 465), including 954 primary tumors and 181 metastases, were collected for next-generation sequencing (NGS) based panel assay. Gene copy number variations (CNV) including gene amplifications and deletions, short variants, and gene fusions were assessed.

Results: On average, 0.65 gene copy number variations were detected in the 181 metastatic lesions, which is higher than 0.43 CNVs per sample in the primary lesions (p value is 0.003). No significant difference was found in short variants (2.48 vs 2.31), fusions (0.27 vs 0.20) and germline mutations (0.26 vs 0.31) between the primary and metastatic NSCLC. In the top-ranked genes with copy number alterations, EGFR amplifications were detected in 11% of the primary and 15% in metastases. MET amplifications, CDKN2A/B deletions, and PMS2 deletions were more frequently found in metastatic lesions: MET (7.7% vs 1.7%, p=0.0001), CDKN2A (5.5% vs 1.5%, p=0.002), CDKN2B (5.0% vs 1.9%, p=0.0007), and PMS2 (3.9% vs 1.2%, p=0.016).

Conclusions: Copy number variations are common driver mutations in NSCLC. We reported here that the prevalence of copy number variations between primary and metastatic lung cancers was different, including driver genes MET, CDKN2A/B, and PMS2. No difference was observed in terms of frequency on short variants or gene fusions in NSCLC.

#3392

Comprehensive molecular profiling of melanoma brain metastases (MBMs) and patient (pt)-matched extracranial metastases (ECMs).

Grant M. Fischer,1 Ali Jalali,2 Aron Joon,1 Michael T. Tetzlaff,1 Alexander J. Lazar,1 Fernando Carapeto,1 Mariana P. Macedo,3 Courtney W. Hudgens,1 Jennifer L. McQuade,1 Khalida Wani,1 Brandy Conner,1 Bhavana Singh,1 Michael A. Davies1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Baylor College of Medicine, Houston, TX;_ 3 _A.C. Camargo Cancer Center, São Paulo, Brazil_.

BACKGROUND: MBMs are a common and lethal complication of advanced melanoma. An improved understanding of the molecular features of MBMs could facilitate the development of more effective treatments for patients (pts). Further, identification of differences between MBMs and ECMs would indicate the need for organ-specific treatment strategies.

METHODS: Transcriptomic capture and Illumina RNA-sequencing (RNA-seq) were performed on (i) surgically resected formalin-fixed, paraffin-embedded (FFPE) MBMs (98 tumors from 84 pts) and (ii) surgically-resected ECMs from the same pts (54 from 38 pts). The EdgeR/limma/voom pipeline was used to perform differential gene expression (DGE) analyses. Pathway analyses were performed via Ensemble Gene Set Enrichment Analysis (EGSEA). The ESTIMATE and MCPCounter R packages were used to assess immune infiltrates from voom-transformed counts. Illumina whole exome sequencing (WES) was performed on 27 matched pairs of MBMs and ECMs from 17 pts. MuTect algorithm was utilized to assess non-synonymous, stop-gain, and stop-loss mutations. DNA methylation profiling was performed on 16 pt-matched pairs of MBMs and ECMs by the Illumina MethylationEPIC BeadChip platform; data was analyzed via the ChAMP pipeline. Quantitative analysis of CD3 and CD8 immune markers was performed by immunohistochemistry (IHC) on 31 MBMs and 49 pt-matched ECMs.

RESULTS: Overall patterns of mRNA expression, mutational burden, and gene methylation were largely similar between pt-matched pairs of MBMs and ECMs, as unsupervised hierarchical clustering was driven primarily by patient identification. However, EGSEA of RNA-seq data identified significant (FDR q-value < 0.10) differences in immune networks (decreased in MBMs) and neuronal differentiation factors (increased in MBMs) in the pt-matched tumors. MCPCounter analysis revealed significant (p < 0.05) depletion of all classes of immune cells except neutrophils in MBMs compared to ECMs. IHC confirmed decreased TCD3 (p = 0.005) and TCD8 (p = 0.013) infiltrating cells in MBMs. ESTIMATE ImmuneScores among MBMs inversely correlated with expression of VSIG4, a relative of the B7 family believed to inhibit T-cell proliferation, and were significantly (p = 0.0088) increased in MBMs previously treated with radiation (XRT). Increased ImmuneScores also associated with significantly (p = 0.00002) improved overall survival (OS) from surgery for MBM. Significant correlations in mean promoter region methylation changes and RNA-seq log fold-changes were identified in 5/10 (50%) of the neuronal factors overexpressed in MBMs, suggesting an epigenetic mechanism for their differential expression.

CONCLUSIONS: Significant differences in immune and neuronal gene networks were detected in MBMs compared to patient-matched ECMs, and expression of immune genes in MBMs positively correlated with previous XRT and OS.

#3393

Characterizing recurrent high-grade serous ovarian cancer through whole-genome sequencing.

Elizabeth L. Christie,1 Swetansu Pattnaik,2 Sian Fereday,1 Australian Ovarian Cancer Study, Heather Thorne,1 Andrea Bild,3 David D. Bowtell1. 1 _Peter MacCallum Cancer Centre, Melbourne, Australia;_ 2 _Garvan Institute of Medical Research, Darlinghurst, Australia;_ 3 _City of Hope, Duarte, CA_.

Acquired drug resistance is the major obstacle in controlling high-grade serous ovarian cancer (HGSC) and leads to poor overall survival. Comparatively little massively parallel sequencing data exist from HGSC patients with recurrent disease who have been extensively treated with chemotherapy or newer targeted agents, such as anti-angiogenics or PARP inhibitors (PARPi). We previously identified four mechanisms of acquired resistance from whole-genome sequencing (WGS) of 23 HGSC patients with recurrent disease; a resistance mechanism of any kind was only identified in half of the patients. Since 2012, we have collected tumor samples from 16 HGSC patients through our rapid-autopsy program. On average, we collect 17 tumor sites per patient during the autopsy, and to date we have performed WGS on 28 autopsy samples from 8 patients. These data are allowing us to understand HGSC at end-stage, by examining the complete catalog of resistance mechanisms within an individual patient and reconstructing the natural history of HGSC. One acquired resistance mechanism we identified in HGSC involves transcriptional fusion of the drug efflux pump ABCB1 to an upstream gene that causes its overexpression. ABCB1 encodes the multidrug resistance transporter MDR1, also known as P-glycoprotein. Through WGS and targeted RNA sequencing on end-stage tumor samples as well as recurrent ascites samples, we identified multiple fusion partners to ABCB1, with more than 15% of recurrent HGSC patients harboring ABCB1 transcriptional fusions. A number of HGSC patients carry multiple ABCB1 fusions, demonstrating convergent evolution within patients and the strong selective advantage of ABCB1 overexpression by gene fusion. Extending our analysis to recurrent and autopsy samples from breast and prostate cancer patients also identified ABCB1 transcriptional fusions as a resistance mechanism in these cancers. Our findings suggest that ABCB1 transcriptional fusions are a common mechanism of acquired chemotherapy resistance in ovarian, breast and prostate cancer patients, and that characterizing end-stage disease is particularly informative for understanding resistance mechanisms.

#3394

Actionable fusion kinases in microsatellite instability-high colorectal cancers.

Kazuhito Sato,1 Masahito Kawazu,1 Yoko Yamamoto,1 Toshihide Ueno,1 Shinya Kojima,1 Genta Nagae,1 Manabu Soda,1 Takafumi Oga,1 Shinji Kohsaka,1 Eirin Sai,1 Yoshihiro Yamashita,1 Shoichi Hazama,2 Hisae Iinuma,3 Hiroyuki Aburatani,1 Hiroyuki Mano1. 1 _University of Tokyo, Tokyo, Japan;_ 2 _Yamaguchi University, Yamaguchi, Japan;_ 3 _Teikyo University, Tokyo, Japan_.

Colorectal cancer (CRC) patients who are microsatellite instability-high (MSI-H) due to mismatch repair (MMR) deficiency have poor prognosis after relapse by consensus molecular subtyping. In response to reports of some MSI-H CRC resistance to immune checkpoint blockade and immune-related adverse events, we sought to identify a new therapeutic target. MSI testing was done on ~2,800 resected colorectal tumors; next-generation sequencing was done on 149 MSI-H tumors showing MSI in both BAT25 and BAT26 loci. MSI-H CRCs are conventionally divided into hereditary and sporadic categories; in this study whole-exome sequencing, transcriptome sequencing, and methylation array revealed that sporadic MSI-H CRCs are further classifiable into somatic MMR gene mutation (Lynch-like) or MLH1 promoter methylated (MM) types. Methylation of other MMR genes (MSH2/MSH6/PMS2) was not detected. The mutational properties of sporadic MSI-H CRCs harboring somatic MMR gene mutations more closely resembled those of Lynch syndrome-associated MSI-H CRCs than those of MM CRCs, which were more likely to develop in the right-sided colon and harbor more insertions/deletions, more recurrent mutations of BRAF/RNF43, and fewer recurrent mutations of KRAS/APC than Lynch syndrome-associated/Lynch-like CRCs (P < 0.001, Fisher's exact test). Loss of heterozygosity of the locus encompassing TGFBR2, MLH1, and CTNNB1 was specific to Lynch syndrome-associated/Lynch-like MSI-H CRCs; this loss appears to be instrumental in the inactivation of TGFBR2/MLH1. Pathway-level analyses of MSI-H CRCs identified DNA damage-sensing and histone H3 methylation-associated pathways as well as Wnt signaling and RAS/RAF/mitogen-activated protein kinase pathways. One mutational signature, Mutational Signature A, accounted for a median percentage 2.3 times larger of mutations in tumors with mutated PMS2 than in tumors with intact PMS2 (P = 1.5 × 10-5, Mann-Whitney U test). Although it is believed that fusion kinases are found in less than 1% of all CRCs, clinically actionable fusion kinases were detected in 11/19 (58%) MM MSI-H CRCs lacking KRAS/BRAF oncogenes. A 3T3 transformation assay confirmed the tumorigenicity of identified fusion kinases; small-molecule inhibitors that suppress the activity of the kinases identified in fusion products significantly attenuated malignant transformation of 3T3 cells in a concentration-dependent manner. Patients with MSI-H CRCs harboring fusion kinases had worse post-relapse prognoses than did patients with MSI-H CRCs harboring KRAS oncogenes. These findings promise to advance MSI-H CRC precision medicine.

#3395

A scalable microfluidic platform for determining cellular heterogeneity by copy number detection.

Andrew Price, Jon Sorenson, Kamila Belhocine, Claudia Catalanotti, Zeljko Dzakula, Susana Jett, Viajy Kumar, Bill Lin, Tony Makarewicz, Alaina Puleo, Mohammad Rahimi, Sanjam Sawhney, Joe Shuga, Maengseok Song, Katie Sullivan-Bibee, Tobias Wheeler, Yifeng Yin, Michael Schnall-Levin, Rajiv Bharadwaj. _10x Genomics, Inc., Pleasanton, CA_.

The ability to sequence entire genomes at the level of single cells (SCs) is an essential tool for mapping heterogeneity in cancer. Knowledge of this variability provides insight into cancer dynamics and the efficacy of therapeutics. Copy number variant (CNV) analysis requires only sparse coverage creating an affordable means for measuring cellular differences genome-wide. However, current approaches to SC DNA sequencing suffer from low throughput, cumbersome workflows and remain accessible to only a few select laboratories. We have developed a microfluidic, partitioning-based solution that allows sensitive CNV detection of thousands of SCs concomitantly. Isolated cells or nuclei are treated to gain complete accessibility to genomic DNA then co-partitioned along with gel beads supporting oligonucleotide barcodes. Barcoded fragments, representing genomic abundance, are generated and converted into Illumina-compatible libraries. The workflow supports various sample types including primary cells, cultured cells, tissue-dissociated cells and nuclei extracted from flash-frozen tissues. Accompanying this platform we have also developed a scalable bioinformatics pipeline for genome alignment, normalization, copy-number detection, and clustering of SC profiles. Using clusters for CNV analysis we can reliably detect 100 kb scale events and, in large clones, identify CNV events down to tens of kilobases with high confidence. Analysis of our SC whole-genome sequencing data shows excellent coverage uniformity, comparable to leading plate-based SC amplification techniques. To validate the accuracy of CNV detection, calls are measured against orthogonal data from well-characterized cell lines including RPMI8226, HCC1954, and HCC1143 and high concordance is observed. Furthermore, we are able to observe multiple clones including CNVs in normal cell lines GM12878 and BJ suggesting absolute cell line validation requires whole-genome SC sequencing. Evaluating nuclei extracted from stage 1 breast carcinoma and stage 1 renal cell carcinoma we find clones exhibiting common somatic mutations. Furthermore, many cells exhibit extensive genomic heterogeneity, effects we attribute to DNA replication, with the frequency of such events elevated in fast growing samples such as tumors or fresh cell cultures.

In conclusion, we demonstrate a novel droplet-based system that permits scalable CNV calling at the SC level enabling high resolution characterization of intra-tumor heterogeneity. It is our intention that a reliable and cost-effective system will encourage widespread adoption of SC analysis as a means of characterizing the progression of cancer, lead to more effective treatments and unveil genomic diversity in other genetically heterogenous systems such as neuronal cells, high-passage cell lines and samples from aging populations.

#3396

Characterization of oral squamous cell carcinoma transcriptome through long read sequencing technology.

Neetu Singh,1 Dinesh Kumar Sahu,1 Ratnesh Kumar Tripathi,2 Archana Mishra,1 Satyendra Kumar Singh,1 Rebecca Chowdhry,1 Sameer Gupta,1 Divya Mehrotra,1 Preeti Agarwal,1 Madhu Mati Goel,1 Sudhir Singh,1 Arun Chaturvedi,1 Akshay Mishra,1 Satya Prakash Agarwal,1 Manish Bajpai,1 Ravi Kant,1 Madan Lal Bhatt1. 1 _King George's Medical University, Lucknow, India;_ 2 _Imperial Life Sciences, Gurgaon, India_.

Large and increasing public-databases of oral squamous cell carcinoma (OSCC) transcriptome sequencing data has been generated through next-generation which has limitation for novel full-length transcript isoforms. Hence, our objective was to identify differential specific OSCC-related novel full length transcript isoforms, long noncoding RNA and fusion genes compared to non-matched oral-cavity control samples through PacBio-RSII platform. The OSCC, patients from different anatomical sites (gingivobuccal region, retro-molar trigone and lower gum) were processed through human transcriptome array 2.0 (HTA2.0). Heatmap generated expression patterns showed two distinct subgroups against control. Both tumor and control samples were individually run on Oncoscan array. On aggregate analysis significant copy number gain of Ch 7p11.2 EGFR gene; and Ch11q13.3-13.4 (FGF19, FGF4, FGF3, LOC101928443, ANO1-AS2, ANO1, FADD, MIR548K, PPFIA1, CTTN, SHANK2) were identified. Based on HTA2.0 and Oncoscan array we pooled six oral cancer and three oral control samples for Isoseq analysis. Differentially expressed full length transcripts between OC and OT were generated through GFOLD and were processed through Reactome Pathway. On considering more than 20 transcript-entities, Metabolism of RNA (22), Developmental Biology (25), Cytokine Signaling (25), Innate Immune System (25), Metabolism of proteins (31), Immune System (35), Metabolism (29), and Signal Transduction (21) pathways were identified. Differential regulation was also validated through HTA2.0. The identified high quality full length transcripts were annotated and classified through Blast2Go in different sub-group under Biological-Process, Cellular-Process and Molecular-function in Level 2. Out of these most highly up-regulated were Type I-Keratins (KRT)-KRT17, -KRT16, -KRT14 and Type-II keratins-KRT6A and -KRT6B showing involvement of formation of cornified envelope, Keratinization, Cell-cell communication, Type I hemi-desmosome assembly and developmental biology pathways. Long non-coding RNA-NMD candidates ARL2-SNX15, RAB4B-EGLN2, SENP3-EIF4A1 and fusion genes-ACTA2--ACTB, ACTB--ACTC1, ACTB--ACTG2, CALML3--CALM3, CKM--CKB, ENO1--ENO3, IGKV1-27--IGKV3-15, IGKV4-1--IGKJ1, IGKV4-1--IGKJ2, IGKV4-1--IGKJ3, IGKV4-1--IGKJ4, KRT6B--KRT6A were also differentially expressed. Additionally, 457 novel full length transcript isoforms including 289 from OC and 168 from OT datasets were identified. Hence, differentially regulated-KRT17, KRT16, KRT14, KRT6A, KRT 6B; long non-coding RNA and identified fusion genes and full length novel transcript isofoms may be the characteristic of these tumors after validation in histo-pathologically characterized FFPE-Keratinized OSCC and may also prove as early detection marker for Keratinized OSCC if identified in pre-neoplastic conditions.

#3397

Clinical significance of 3q amplification in squamous cell carcinoma of lung.

Eunjung Lee, Chungyeul Kim, Han-Kyeom Kim, Baek-hui Kim. _Korea University, Seoul, Republic of Korea_.

Squamous cell carcinoma (SqCC) is major subtype of non-small cell lung cancer (NSCLC). One of the molecular characteristic of SqCC is amplification of distal portion of 3q. The aim of this study is to find out the clinical significance of the 3q amplification in SqCC of lung. Method : We extracted DNA from 90 cases of FFPE blocks of lung SqCC tissue. Ten genes (PIK3CA, SOX2, KLHL6, MCF2L2, LAMP3, MCCC1, ABCC5, ATP11B, TP63, YEATS2) included in 3q were selected and the amplification of each gene was analyzed by using NanoString nCounter analysis system (PhileKorea). The hierarchical clustering was performed with the resulting values. Results : We identified two groups in all 90 cases of SqCC by hierarchical clustering. One group with 3q amplification was 50 cases, and the other group without 3q amplification was 40 cases. There was no significant difference in sex, age, stage, smoking history, histologic grade and the results of immunohistochemical staining (p63, p40 and p53) between the two groups. It showed the tendency that the group with 3q amplification presented poor disease free survival (P=0.193) and overall survival (P=0.34) than the group without 3q amplification. Among the ten genes, PIK3CA and YEATS2 were significantly associated with survival. Each genes with amplification showed poor disease free survival (PIK3CA; P=0.036, YEATS2; P=0.025). Conclusion : The amplification of 3q can help sorting out two subsets of SqCC of lung whose clinical outcomes are likely to diverge. Among the genes included in 3q, PIK3CA and YEATS2 were associated with prognosis. These finding demonstrate the role of these genes in biologic behavior of tumor and suggest opportunities for targeted therapies.

#3398

Analysis of whole transcriptome RNA-seq of large numbers of clinical FFPE samples.

Yulia Newton,1 Justin Golovato,2 Mark Johnson,2 Shahrooz Rabizadeh,2 Zack Sanborn,1 Steve Benz,1 Charles J. Vaske1. 1 _NantOmics, Santa Cruz, CA;_ 2 _NantOmics, Culver City, CA_.

Background: We evaluate the feasibility, quality, and analytical potential of whole-transcriptome RNA-seq on over one thousand clinical FFPE tumor samples. Transcriptional profiling of RNA is used for clinical decision making in many tumor types. Clinical analysis of RNA is complicated by the common use of formalin-fixed paraffin-embedded (FFPE) tissue storage, which can cause low yield and RNA degradation.

Methods: RNA was extracted from FFPE material using commercially available kits using an RNAseH based ribodeplete. Multiple libraries per sample are sequenced using standard Illumina sequencing. Bowtie2, RSEM, and custom software are used for alignment, transcript quantification, fusion detection, and variant expression analysis.

Results: We observe a >85% success rate on whole transcriptome RNA-seq on our cohort of more than 1000 samples. We find reliable transcript quantification upon successful sequencing, and on a subset of samples quantify differences between FFPE and fresh-frozen material when using ribodeplete. We also compare transcriptional profiles of clinical FFPE samples to an independent set of fresh frozen, poly-A capture samples from The Cancer Genome Atlas (TCGA) and show differences between poly-A capture and ribodeplete RNA isolation methods as well as FFPE vs. FF effects. We present a robust mapping methodology for comparison of public FF poly-A dataset to our FFPE ribodeplete samples. We demonstrate that we are able to utilize this joint gene expression space to perform site of origin prediction on FFPE samples. This is especially important for clinical application in analyzing Cancer of Unknown Primary (CUP) samples as well as detecting outlier samples whose molecular features may suggest additional therapeutic avenues. Finally, we show reliable detection of fusion transcripts from FFPE RNA-seq material from whole-transcriptome analysis and expression of somatic tumor variants detected from DNA sequencing.

Conclusions: Large scale sequencing of RNA from clinical FFPE materials provides reliable transcriptomic results comparable to existing public databases of RNA, enabling research on cohorts of tumors that are FFPE-banked and unavailable as frozen tissue.

#3399

Comparative genomic analyses of osteosarcoma etiology reveal a chromosomal structural rationale for the increased incidence of osteosarcoma in dogs.

Aaron L. Sarver,1 Lauren Mills,1 Nuri Temiz,1 MIlcah Scott,1 Anne Sarver,1 Logan Spector,1 Jinhua Wang,1 Mathew Breen,2 Subbaya Subramanian,1 Branden Moriarity,1 Jaime Modiano,1 David Largaespada1. 1 _University of Minnesota, Minneapolis, MN;_ 2 _North Carolina State University College of Veterinary Medicine, Raleigh, NC_.

Risk of osteosarcoma is significantly higher in large and giant breed dogs than in any other animal, including humans. To identify the reason for this observation, we used a comparative genomic approach to identify aberrations responsible for osteosarcoma etiology in a Sleeping Beauty transposon-accelerated mouse model, in human patients, and in naturally occurring canine tumors, using RNA-Seq and exome paired tumor normal analyses. Fusions identified in Sleeping Beauty-mutagenized tumors revealed a role for Cdkn2a disruption in Trp53 signaling, and for numerous genes which cooperate with disrupted TrpP53 signaling, indicating the existence of many diverse routes to osteosarcoma tumor formation. Similarly, human tumors showed TP53 pathway disruption associated with a high level of diversity of additional driver mechanisms, supporting multiple independent routes to tumor formation. However, in the majority of canine tumors, observed TP53 pathway aberrations co-occurred with loss of both copies of the region containing the PTEN tumor suppressor. In human osteosarcoma, only heterozygous PTEN loss was observed, and in both humans and mice, PTEN aberration was observed in a much smaller percentage of the tumor population. We hypothesize that increased osteosarcoma incidence in dogs is partly due to a syntenic rearrangement of the peri-PTEN locus in the canine genome. The PTEN gene is part of a small synteny block that localized in the distal end of canine chromosome 26 (CFA 26) during evolution. We hypothesize that this location change creates a high risk of loss in the context of cytogenetic instability caused by disruption of TP53, thereby providing a structural genetic rational for the higher incidence of osteosarcoma in dogs. Consistent with these results and with the powerful nature of PTEN as a tumor suppressor, canine osteosarcomas with homozygous loss of PTEN are associated with worse outcomes than canine osteosarcomas with intact PTEN. These results suggest that engineering genomes to minimize cancer risk may be a realistic approach to the prevention of cancer.

#3400

Characterizing genomic variation and tumor heterogeneity in cancer.

Claudia Catalanotti, Sarah Garcia, Kamila Belhocine, Vijay Kumar, Zeljko Dzakula, Andrew Price, Shamoni Maheshwar, Yifeng Yin, Michael Schnall-Levin, Rajiv Bharadwaj, Sara Agee Le, Deanna M. Church. _10x Genomics, Pleasanton, CA_.

Cancer genomes are highly unstable with new genetic variations emerging even within a single metastatic site, making it difficult to track the causal changes that drive metastasis and treatment resistance. Here we present a two-pronged approach for analyzing the full spectrum of genetic variations present in cancer samples. The first approach allows for comprehensive and high-resolution characterization of a broad range of variant types on bulk tumor sample. While the second approach characterizes structural variation at the level of the single cell, allowing for the exploration of tumor clonality and heterogeneity. At the core of our approaches is a microfluidics platform that enables the production of hundreds of thousands to millions of partitioned barcoded reactions. This platform can partition high-molecular weight DNA or single cells. Together, these complementary approaches provide a more complete picture of the genomic variation and clonal structure present in a tumor.

For bulk tumor analysis, we obtained high molecular weight DNA from known cancer cell lines and used the 10x Chromium Genome solution to produce Illumina-ready sequencing libraries. In this workflow, partitioning of a limited amount of genomic DNA allows for haplotype-level dilution of genome equivalents, which are then barcoded to create a novel data type referred to as "Linked-Reads". These molecular barcodes are used to identify reads originating from the same input molecule providing long range information on highly accurate short reads. In addition to highly accurate SNP calling, this further enables identification of complex structural rearrangements in tumor genomes. To gain insight into tumor heterogeneity and clonal structure, we performed single cell DNA sequencing and analysis using 10x Chromium scDNA solution. This platform integrates single cell encapsulation, cell lysis and DNA barcoding into a streamlined workflow. Molecular barcodes are used to associate reads with individual cells allowing for copy number variant (CNV) detection. We applied our scDNA sequencing method to a variety of cancer cell lines revealing their clonal structure, as identified by CNVs, with the capability to identify as few as 10 cells in a sample size of one thousand cells. Using cluster analyses we were able to detect 100kb scale events and by aggregating reads in large clones we were able to confidently identify smaller CNV events down to tens of kilobases. Using whole genome bulk sequencing we identified more than 500 large structural variants in HCC1954, including balanced and unbalanced events. In this presentation, we will integrate this Linked-Read data with single cell genome analysis on the same samples, and compare the genetic variation revealed by these two approaches. We will further explore the power of combining these data types for a more complete picture of tumor genome dynamic

#3401

Comprehensive analysis of genetic alterations and intratumor heterogeneity in myxofibrosarcoma.

Yasuhide Takeuchi,1 Annegret Kunitz,2 Hiromichi Suzuki,1 Kenichi Yoshida,1 Nobuyuki Kakiuchi,1 Yusuke Shiozawa,3 Akira Yokoyama,1 Yoichi Fujii,1 Tetsuichi Yoshizato,1 Kosuke Aoki,1 Keisuke Kataoka,1 Yasuhito Nannya,1 Yuichi Shiraishi,3 Teppei Shimamura,4 Kenichi Chiba,3 Hiroko Tanaka,3 Hideki Makishima,1 Satoru Miyano,3 Hironori Haga,1 Frederik Damm,2 Seishi Ogawa1. 1 _Kyoto University, Kyoto, Japan;_ 2 _Charite University Hospital, Berlin, Germany;_ 3 _The University of Tokyo, Tokyo, Japan;_ 4 _Nagoya University, Nagoya, Japan_.

Myxofibrosarcoma (MFS) is a rare subtype of soft tissue sarcomas (STSs) preferentially affecting elderly. Histologically, MFS is distinct from other STSs, in that it is characterized by the proliferation of pleomorphic spindle cells with varying degrees of myxoid components. However, the molecular pathogenesis of MSF is poorly understood.

In this study, we conducted an integrated molecular analysis of 44 samples from primary MFS patients, in which samples were analyzed by whole-genome sequencing (WGS) (n=2), whole-exome sequencing (WES) (n=44), RNA sequencing (n=3), DNA methylation array (n=16), and immunohistochemistry (IHC, n=27). Copy number alterations (CNAs) were identified by sequence based copy number analysis. The obtained genomic data were combined with those from STS cases from the Cancer Genome Atlas (TCGA) cohort, including 17 MSF samples and compared to the data from other STS samples (n=189). To further investigate the genetic basis of mixed histological components and chronological changes in MFS, we performed analysis from multi-regional and/or multi-time-point samples from 8 MSF cases.

A total of 4,613 mutations were identified by WES in 61 primary MFS samples with a median of 44.0 mutations/sample. Mutations were dominated by age-related C to T transitions at CpG sites. Most frequently mutated genes included TP53 (n=21, 34.4%), ATRX (n=9, 14.8%), and RB1 (n=3, 4.9%). A fusion gene associated with TRIO was detected by RNA sequencing in a single case. Among other STSs, undifferentiated pleomorphic sarcoma (UPS) harbored the most similar genetic abnormalities (most frequently mutated genes included TP53 (40.9%), ATRX (29.5%), and RB1 (11.3%)), suggesting that these two subtypes are genetically closely related. Also combined cases with CNAs (n=35) and strong staining in IHC (n=13), TP53 abnormality was found in most cases (n=56, 91.8%). Two MSF cases evaluated by WGS showed complex structural abnormalities, where 491 and 198 somatic structural variations were detected suggestive of increased genetic instability. Multi-regional sampling (n=5) disclosed a high level of intratumor heterogeneity with less than 29.0% of mutations being shared by different samples. Multi-time points sampling (n=6) revealed that the number of mutations was significantly higher in relapse samples (odds ratio 1.6, p = 0.03). While in all cases, TP53 lesions were observed at the initial time-point, others were subclonal and acquired during the clinical course. Finally, in methylation analysis, 3,817 differentially methylated regions were detected (Stouffer's p < 0.05), based on which MFS were clustered into two distinct subtypes.

In summary, the genetic profile of MFS is characterized by frequent abnormalities in TP53, ATRX, and RB1 and closely related to other STSs, especially to UPS. Clonal TP53 abnormalities resulted in complex chromosomal structure and a high degree of intratumor heterogeneity.

#3402

Castration resistance transcriptome in prostate cancer revealed by single-cell RNA-seq.

Aaron M. Horning,1 Che-Kuang Lin,1 Yao Wang,1 Brandon Lieberman,2 Devalingam Mahalingam,1 Ming Gao,1 Pei Wang,1 Chiou-Miin Wang,1 Zhijie Liu,1 Jianhua Ruan,3 Michael A. Liss,1 Victor X. Jin,1 Tim H-M Huang,1 Chun-Liang Chen1. 1 _UT Health Science Ctr. at San Antonio, San Antonio, TX;_ 2 _Trinity University, San Antonio, TX;_ 3 _UT at San Antonio, San Antonio, TX_.

Fatal metastatic castration-resistant prostate cancer (mCRPC) remains without sensitive early detection biomarkers and effective therapeutic targets. Among 2.5 millions of prostate cancer patients, the majority will face a dilemma, to treat or not to treat, at a point as cancer progresses. Biomarkers for mRCPC at an early stage represent an unmet need. With early identification, clinicians could design new treatment strategies to reduce metastasis-related morbidity and to extend survival of patients. In this study, we deployed single-cell RNA-seq on prostate cancer cells (LNCaP, ABL and PC3) to determine the transcriptome in androgen independency and castration resistance of prostate cancer. We identified potential 336 androgen-independence specific genes and 2396 castration resistance specific genes in ABL and PC3 cells respectively, while only 136 genes were shared in both cells. These genes, mostly upregulated were enriched in 43 and 166 signaling pathways that implicated the complexity of the castration resistance transcriptomic systems and networks. The signaling pathways are involved in advanced and metastatic malignancies including WNT, TGFB, ITGA/B, STAT, EPH, focal adhesion, adherens junction, regulation of actin cytoskeleton, gap junction, tight junction and EMT. Malignant potencies of ~ 40 pathways were validated by in silico analysis of the RNA-seq data from the prostate cancer cohort of The Cancer Genomic Atlas (TCGA) using Kaplan-Meier disease free and survival curve analyses. The transcriptomic regulation of these genes was further validated and correlated with ATAC-seq data. In order to further verify the functions of those signaling pathways in castration resistance, 9 major signaling pathways were evaluated using small molecule inhibitors. Castration resistant prostate cancer cells showed significant defective cell proliferation, migration, invasion and sphere formation in the presence of inhibitors, whereas LNCaP and ABL cells displayed limited or non-significant changes. Interestingly, 4 small molecule inhibitors showed significant suppression on the growth of stem-like circulating tumor cells that were derived from clinical blood samples of prostate cancer patients. Our data suggest that those castration resistance specific genes and signaling pathways revealed by single-cell RNA-seq may serve as potential markers and therapeutic targets.

#3403

**Conservation of colorectal cancer cell-intrisinc transcriptional traits in paired** in vitro **/** in vivo **cellular models.**

Claudio Isella,1 Consalvo Petti,2 Carlotta Cancelliere,2 Daniela Cantarella,2 Roberta Porporato,2 Livio Trusolino,1 Andrea Bertotti,1 Alberto Bardelli,1 Enzo Medico1. 1 _University of Turin, Candiolo Cancer Institute - IRCCS, Italy;_ 2 _Candiolo Cancer Institute - IRCCS, Italy_.

The best clinical and molecular criteria currently adopted in the diagnosis of colorectal cancer (CRC) often fail to predict the natural history of the disease, or to provide information regarding the molecular mechanisms inducing cancer onset and progression. To address these issues, molecular taxonomies based on transcriptional CRC subtypes have been developed; however, since the CRC transcriptome is heavily affected by mRNAs of stromal origin, these transcriptional traits are not specifically associated with cancer cell-intrisic features. To overcome this limitation, we recently employed gene expression profiles of CRC tissues specifically deprived of stromal transcripts to define "colorectal cancer cell-intrisinc subtypes" (CRIS). CRIS classification is not only bestowed with high prognositc and predictive value: it also conveys biological and molecular information on the subtypes, that may be tailored for specific therapies. To assess reliability of CRIS subtyping in preclinical CRC models, we designed a "XenoLine" platform, composed of 60 CRC cell lines representative of each CRIS class, grown in vitro and implanted in NOD-SCID mice to obtain xenograft tissues. After subcutaneous injection of 5 milion cells, xenografts were allowed to grow until they reached 2500 mm3 and were explanted. Engraftments were performed in duplicate and had a success rate around 85%. Further engraftments are ongoing. RNA-seq analysis was employed to compare in vivo- and in vitro-grown cells, and evaluate the stability of CRIS subtypes. The XenoLine RNA-seq profiles were in-silico microdissected, discriminating transcripts of epithelial cancer cell origin (human reads) from transcripts of stromal origin (mouse reads). Comparing paired in vitro / in vivo gene expression profiles, we observed that: 1) matched model pair correlation was systematically higher that random pair correlations; 2) for most genes, variation of expression across cells was consistent between in vitro and in vivo profiles; 3) CRIS classification was substantially maintained upon xenograft propagation. In conclusion, the molecular features distinguishing CRIS subtypes are resilient to major changes in cell growth conditions, such as changes in the microenvironment and acquisition of supporting stroma. The XenoLine platform was successfully established and will be further extended to better ascertain differences between in vitro- and in vivo- grown CRC cells of variable molecular makeup.

#3404

Clinical significance of APOB inactivation in hepatocellular carcinoma.

Gena Lee,1 Min Jun Kwak,1 Do Won Kim,1 Jiwon Koh,2 Eun Wook Joo,3 Susie Kah,3 Yeong-Eun Sim,3 Sun Young Yim,4 Ju-Seog Lee1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Seoul National University, Seoul, Republic of Korea;_ 3 _Kyunghee University, Seoul, Republic of Korea;_ 4 _Korea University, Seoul, Republic of Korea_.

Hepatocellular carcinoma (HCC) is the seventh most common cancer and second the most lethal cancer globally. 5-year OS rates in US are only 11%. Sequencing of HCC genome revealed unexpected frequent mutations in serum proteins such as albumin (ALB) and apolipoprotein B (APOB), which are not frequently mutated in other cancers. However, clinical significance and underlying biology of loss of these serum proteins in HCC are currently unknown. Here we show that loss of APOB, major carrier of lipid in blood, in HCC is significantly associated with poor survival of HCC patients by applying comparative genomics approach that integrate genomic data from mouse models and human HCC tumors. We further show that loss of APOB leads to shifting balance of lipid metabolisms favoring for tumor growth. For development of genomic signature reflecting hepatic APOB activity and test and validation of its association with prognosis, we used unsupervised approach combined with supervised prediction models. First, gene expression signature were generated from Apob-silenced mouse livers and hepatic Apob-specific signature was identified by applying statistical analysis (P < 0.005 and 1.5-fold difference between Apob-silenced vs. Apob-Wt). Second, by applying unsupervised hierarchical clustering of mouse hepatic Apob signature in human HCC tumors first and constructing Bayesian prediction models later, HCC tumors with low APOB activity were identified. When HCC patients were stratified into APOB-high and APOB-low subgroups, overall survival (OS) rate and recurrence free survival (RFS) rate of patients in APOB-low group is significantly lower than those in APOB-high (P < 0.001), indicating that APOB activity in HCC is significantly associated with prognosis of patients. This association was validated in 4 independent cohorts of HCC patients (n=88, 240, 242, and 371 in total of 941 patients). Since APOB is one of major carrier proteins for lipid, loss of APOB in HCC would lead to accumulation of intracellular fatty acid. Many studies showed that fatty acid are necessary for proliferation of cancer cells as essential for cell membranes and de novo biogenesis rate of fatty acid is well associated with prognosis of cancer patients. APOB expression is negatively correlated with methylation of APOB promoter. Our study suggested potential novel strategy of cancer cells to increase supply of fatty acids for fast proliferation. HCC cells increase supply of intracellular fatty acids by inhibiting export of fatty acids through inactivation of APOB. Further analysis of multiple genomic data revealed oncogenic YAP1 are highly active in APOB-low subgroup as evidenced by expression of downstream targets such as FOXM1 and TEAD4, suggesting that alteration in lipid metabolism could lead to activation of YAP1. For the first time, we showed that APOB inactivation has a clinical impact in HCC patients with significant alteration in regulators associated with tumorigenesis.

#3405

A mutational signature associated with alcohol consumption and prognostically mutated driver genes in esophageal squamous cell carcinoma.

Xiangchun Li,1 Mengyao Wang,1 Meng Yang,1 Hongji Dai,1 Baifeng Zhang,1 Wei Wang,1 Xinlei Chu,1 Xin Wang,1 Hong Zheng,1 Ruifang Niu,1 Wei Zhang,2 Kexin Chen1. 1 _Tianjin Medical University Cancer Institute & Hospital, Tianjin, China; _2 _Wake Forest Baptist Comprehensive Cancer Center, Wake Forest Baptist Medical Center, Wake Forest, NC_.

Aim: Esophageal squamous cell carcinoma (ESCC) is often diagnosed at advanced and incurable stage. Information on driver genes and prognosticators in ESCC remains incomplete. Herein, we elucidated significantly mutated genes (SMGs), mutational signatures, and prognosticators in ESCC.

Methods: Three MutSig algorithms (i.e. MutSigCV, MutSigCL and MutSigFN) and "20/20" ratio-metric were employed to identify SMGs. Nonnegative matrix factorization was used to decipher mutational signatures. Kaplan-Meier survival analysis, multivariate Cox and logistic regression models were applied to analyze association between mutational features and clinical parameters.

Results: We identified 26 SMGs, including 8 novel (NAV3, TENM3, PTCH1, TGFBR2, RIPK4, PBRM1, USP8 and BAP1) and 18 previously reported. Three mutational signatures were identified to be prevalent in ESCC including clocklike C>T at CpG, APOBEC overactive C>T at TpCp[A/T], and a signature featured by T>C substitution. The T>C mutational signature was significantly correlated with alcohol consumption (OR: 3.59; 95% CI: 2.30-5.67; P < 0.001). This alcohol consumption signature was also observed in liver cancer and head and neck squamous cell carcinoma, and its mutational activity was substantially higher in samples with mutations in TP53. Survival analysis revealed that TENM3 mutations (HR: 5.54; CI: 2.68-11.45; P < 0.001) and TP53 hotspot mutation p.R213* (HR: 3.37; CI: 1.73-8.06; P < 0.001) were significantly associated with shortened survival outcome. The association remained statistically significant after controlling for age, gender, TNM stage and tumor grade.

Conclusions: We have uncovered several new SMGs in ESCC and defined an alcohol consumption related mutational signature. TENM3 mutation and TP53 hotspot mutation p.R213* are independent prognosticators for poor survival in ESCC.

#3406

Comprehensive genomic profiling of hematologic malignancies identifies recurrent somatic splicing factor mutations in non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM).

Lee A. Albacker,1 Silvia Buonamici,2 Garrett M. Frampton,1 Peter Smith,2 Philip J. Stephens,1 Markus Warmuth,,2 Ping Zhu,2 Lihua Yu2. 1 _Foundation Medicine Inc, Cambridge, MA;_ 2 _H3 Biomedicine Inc., Cambridge, MA_.

Somatic mutations of splicing factors have been reported in multiple tumor types and have been recognized as a new hallmark of cancer. Although these mutations have been observed in myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML) and chronic lymphocytic leukemia (CLL), the frequency of these mutations in other hematological malignancies is unknown.

We surveyed somatic mutations of several splicing factors (SF3B1, SRSF2, U2AF1, ZRSR2, DDX3X, ZMYM3, PCBP1 and U2AF2) in 6,235 patients across 15 hematological malignancies. 405 genes were analyzed by DNAseq at >500X coverage using FoundationOneHeme. Consistent with prior reports, we found that the hematopoietic malignancies with the most frequent splicing factor mutations were CMML (48.3%), MDS (36.9%), AML (25.3%) and CLL (22.5%). However, we also identified splicing factor mutations in NHL (13.8%) and MM (9%). In addition to mutations found across the different hematopoietic malignancies in SRSF2 (6%), SF3B1 (4.5%), U2AF1 (3.3%) and ZRSR2 (2.2%), we found DDX3X to be the fifth most frequently mutated gene at 1.6%, followed by ZMYM3 (0.8%), PCBP1 (0.5%) and U2AF2 (0.4%), indicating the importance of splicing dysregulation in hematological malignancies.

Within NHL, diffuse large B cell lymphoma (DLBCL) has the highest frequency of splicing factor mutations (18.3%), and these patients exhibited increased tumor mutation burden (TMB, 13.7 vs. 10.0 mutations per Mb, P < 0.05). Among the splicing factors, the RNA helicase DDX3X is the most frequently mutated in NHL (5.2%). DDX3X is a X chromosome gene and its mutations in NHL are associated with male gender (P = 0.006). Consistent with the reported mutations in CLL and natural killer/T-cell lymphoma, the majority of mutations are loss of function or missense mutations clustered in the two helicase domains. This suggests a pathological relevance of DDX3X in lymphoid malignancies.

In MM, SF3B1 and SRSF2 are two most frequently mutated genes at 3.8% and 1.9%, and patients with these mutations are associated with increased TMB (4.2 vs. 2.5, P < 0.001). Moreover, SF3B1 mutations occurred in 5.3% of samples with IGH-CCND1/2/3 or IGH-MAF/MAFB translocations, but <1% of samples with IGH-WHSC1/FGFR3 translocations. Although the most common SF3B1 mutation in hematopoietic malignancies is p.K700E, in MM the most frequent SF3B1 mutation is p.K666 (36.9% vs p.K700E 12.3%).

Here, we identify splicing factor mutations in NHL and MM, including hotspot somatic mutations of SF3B1, U2AF1 and SRSF2 and loss of function or missense mutations in DDX3X. Overall, our results broaden the disease association of splicing factor mutations in hematological malignancies and strengthen their role in disease pathogenesis.

#3407

A high performance target-enrichment strategy for liquid biopsy.

Jilong Liu, Yan Song, Lei Liu, Meihua Tan, Han Liu, Jingjing Wang, Mingzhi Ye. _BGI, Guangzhou, China_.

For abroad application of liquid biopsy in routine clinical settings, it is essential to use a high performance target-enrichment technique that able to detect all major classes of mutations including single nucleotide variants (SNVs), insertion/deletions (InDels), copy number alternations (CNVs) and rearrangements with affordable costs. The regular enrichment strategies employ unique molecular index (UMI) to suppress sequencing error are limited by several factors for their widespread adoption in clinical application. As an instance for hybridization based methods, Cancer Personalized Profiling by deep Sequencing (CAPP-SEQ) is expensive due to low on target rate. Oncomine, as an example for multiple PCR based technique, is unable to detect DNA rearrangements. Herein, we propose a novel target-enrichment strategy based on the innovated sequencing platform BGISEQ-500. The probes used here are blocked by ddNTPs at 3' end which suit for pyrophosphorolysis-activated polymerization. Modified probes in 3' end consist of complementary sequences targeting SNVs, InDel's hotspots, frequently rearranged intron regions, and a sequencing platform-specific tail in 5' end. In a multi-cycle hybridization and copy procedure with prepared library for enrichment, probes work in two roles: hybridization baits and primers for successively extension; while in Post-PCR, they act as universal PCR sequence to produce sequencing library. Since each molecule containing target locus as well as forward UMI adapters will be captured and copied, we could distinguish the origin of each reads by pull-down probes among huge sequencing data, and select probes with high performance to construct clinical NGS panels. Meanwhile, intermediate and final products from the capture process will be gathered for quality control to evaluate the annealing efficiency and on-target ratio. QPCR primers for EGFR and KRAS are included in the panel to represent areas with normal/low GC content. The collected data were useful to monitor the loss of molecules during hybridization and provide key information for further improvement of the panel. For a customized sequencing panel of non-small-cell lung carcinoma (NSCLC) with sequencing regions about 30k, the sequencing results showed overall very high percentage of reads on target (>95%) and very high data utilization rate (>75%). With 40ng DNA used as input for library generation and 4 Gbyte data size, we can achieve 8000X de-duplicate sequence depth, of which 5000X is error corrected depth, thereby ensure the stable detection of mutation with frequency as low as 0.1%. We believe this novel capture and analysis technique is also applicable to other capture-based sequencing product and will achieve similar performance. Cancer patients, especially NSCLC will benefit from this new sequencing technique.

#3408

Characteristics of circulating tumor DNA in lung cancer patients.

Tomonori Abe, Chiho Nakashima, Akemi Sato, Eisaburo Sueoka, Shinya Kimura, Naoko Aragane. _Saga University, Saga, Japan_.

Liquid biopsy using circulating tumor DNA (ctDNA) has been spread world-wide. We established fully automatic sensitive mutation detection system, mutation-biased PCR and quenched probe system (MBP-QP) method, and accomplished multicenter prospective study to investigate the utility of ctDNA in lung cancer patients who acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI). The results of the clinical study showed that ctDNA was frequently detected in lung cancer patients with distant metastasis, and detection of ctDNA was associated with poor prognosis. In metastatic animal model using immunodeficient mice, NOJ mice, the amount or ctDNA was associated with tumor progression such as tumor burden and metastasis. Based on these evidences, we hypothesized that ctDNA has some biological effects on tumor progression. In order to examine that, we analyzed biological and clinical characteristics of ctDNA. ctDNA was extracted from 1000μl plasma by automated DNA extraction system using cellulose magnetic beads. Compared to circulating free DNA (cfDNA) isolated from healthy volunteers, size distribution of ctDNA or cfDNA using a capillary electrophoresis system was different, that is one peak around the size of 170 bp in healthy individuals, and two peaks, 170 bp and 5 kb in advanced lung cancer patients. We next examined 130 plasma samples from 92 lung cancer patients in addition to 18 benign pulmonary disease patients and 20 healthy individuals at Saga University Hospital. The DNA concentration quantified by Quantus®, the fluorescent measurement of dsDNA intercalated dye, was higher in lung cancer patients compared to those in benign pulmonary disease patients and healthy individuals. Among lung cancer patients, DNA concentration was increased in those with advanced stages, especially in presence of metastasis. In addition, 5 kb fragments were significantly increased in these cases compared to 170 bp fragments. To investigate which fragment contained tumor-derived DNA, 170 bp and 5 kb fragments were separately isolated, and EGFR mutation, L858R was examined. L858R was detected in both ctDNA fragments, 170 bp and 5 kb, indicating that both sized DNA fragments contain tumor-derived DNA. Although the 170 bp short fragments of ctDNA are well known as an apoptotic product, the origin of 5 kb long fragments has not been clarified. We have examined whole ctDNA sequence using next generation sequencing, and compared two fragments. In addition, origins of each fragment have been investigated, and biological effects on tumor progression will be analyzed.

#3410

Copy number estimation from whole-exome sequencing in tumors.

Shawn Anderson,1 Zhiwei Che,2 Raja Keshavan,2 Andrea O'Hara,2 Dong Lin,1 Yuzhuo Wang,1 Colin Collins,1 Soheil Shams2. 1 _The Vancouver Prostate Centre, Vancouver, British Columbia, Canada;_ 2 _BioDiscovery, Inc., El Segundo, CA_.

At the Vancouver Prostate Centre (VPC), a personalized medicine approach is the focus to the understanding and treatment of prostate and other cancers. As a part of this approach, the xenograft program is used to identify and validate mechanisms and drivers of progress and therapeutic targets. VPC frequently uses Next-Generation Sequencing (NGS) technologies for copy number (CN) analysis. Many CN from NGS algorithms require a matched normal reference for CN estimation, however, a matched normal may not be possible for many reasons including unavailability of patient blood sample and poor quality of the matched sample in question. The correct CN algorithm for NGS data is critical to overcome these obstacles. Here we will share our results from analysis of Whole Exome Sequencing (WES) of prostate cancer patient-derived xenograft (PC PDX) samples using Nexus Copy Number software with BioDiscovery's BAM MSR algorithm. A pooled reference can be created using unmatched normal samples; however, when normal samples are not available, tumor samples from within the batch and/or run can be used to create a reference. BAM MSR derives CN and B-Allele Frequency (BAF) from WES, whole-exome sequencing (WGS), and targeted panels using a pooled reference. Due to an absence of normal samples, randomly selected tumor samples were used to build a reference file, which was used for baseline CNV results. By using a recursive refinement method, which incorporated evaluation of B-allele frequency (BAF) patterns among the pooled samples, a subset of tumor samples was selected to create a refined pooled reference using BAM MSR. All tumor samples were then subjected to CN estimation, which included adjustment based on tumor ploidy. While creating a normal reference including PC PDX tumor samples may seem counter intuitive due to enrichment of cancer cells and replacement of stromal component with mouse origin (e.g. fibroblasts, endothelium and immune cells), and high early recurrent copy number signatures, the BAM MSR algorithm was able to get results comparable to previous aCGH results. This is of special importance because the lack of normal tissues is commonplace in cancer research.

#3411

Biological subtypes of nasopharyngeal carcinoma by genomic profiling.

Kai Xun Joshua Tay, Chunfang Zhu, Sujay Vennam, Sushama Varma, Quynh-Thu Le, John Sunwoo, Robert West. _Stanford University School of Medicine, Stanford, CA_.

Objective:

Nasopharyngeal carcinoma (NPC) is a common Epstein-barr virus-associated epithelial malignancy in several parts of the world including Southeast Asia. While the majority of patients are treated uniformly with a combination of chemo and radiation therapy, 20% of patients experience recurrence, most commonly in the form of distant metastasis. NPC subtypes based on underlying differences in biology are unexplored and are likely responsible for the heterogeneous clinical response. As NPC biopsies are small and have significant stromal infiltrate, obtaining pure epithelial cells for genomic profiling is a challenge. We aim to overcome these limitations to identify biological subtypes and dysregulated molecular pathways in NPC.

Methods:

We first evaluated 217 whole exomes sequences, including 102 microdissected tumors, from three previous studies for single nucleotide variants and copy number changes, following GATK standards and using next generation sequencing copy number callers. We then applied a novel method for gene expression profiling developed in our lab to a new cohort of EBV-positive primary NPC cases from our institution. We performed laser capture microdissection, separately dissecting tumor, normal and microenvironment for each case. We applied Smart-3SEQ, a novel 3' end RNA-Seq technique which allows for the accurate quantification of transcript abundance in dissected FFPE samples comprising only a few hundred cells.

Results:

We achieved a per-base concordance of 80.4% between copy number profiles by SNP array and whole exome sequencing. Unsupervised clustering identified three distinct copy-number groups of NPC tumors, with a low copy-number group demonstrating an 18.7% better 5-year disease-specific survival, not attributable to stage. Apart from broad cytogenetic changes, narrow regions of amplifications (e.g. 1q21, 11q13) and deletions (e.g. 9p21, 11q22) were important for defining copy-number subtypes.

Preliminary differential gene expression analysis showed that genes involved in cell cycle and cellular differentiation were significantly dysregulated in tumor cells (p < 0.001 and p = 0.03 respectively), while genes involved in cilia assembly and flagella transport were upregulated in normal cells (p < 0.001 for both). Our further analysis includes defining tumor subtypes based on gene expression, identifying key driver pathways, and correlating with EBV-latent gene expression, the immune environment, as well as clinical outcome.

Conclusion:

NPC tumors are biologically heterogeneous and can be classified based on their mutational and gene expression profiles. This provides an important basis for the consideration of escalation and de-escalation of therapy in selected patient groups.

#3412

Androgen receptor binding sites are highly mutated in prostate cancer.

Tunc Morova,1 Mehmet Gonen,1 Attila Gursoy,1 Ozlem Keskin,1 Nathan A. Lack2. 1 _Koc University, Istanbul, Turkey;_ 2 _Koc University/Vancouver Prostate Centre, Istanbul, Turkey_.

Androgen receptor (AR) signaling is essential to nearly all prostate cancer cells. Any alterations to AR-mediated transcription can have a profound effect on prostate carcinogenesis and tumor growth. While the AR protein has been extensively studied, little is know about those mutations to non-coding regions where AR binds to DNA. Using whole genome sequencing from 197 primary prostate cancer patients, we demonstrate that AR binding sites have a dramatically increased rate of mutations that is greater than any other transcription factor. This is not due to the specific chromosomal region as increased mutation were only observed in prostate cancer. The rate of mutations correlated with AR occupancy and did not cluster with any histone mark or transcription factor. Suggesting that this may occur with other lineage-specific transcription factors, we observed that estrogen receptor binding sites also had an elevated rate of mutations in breast cancer. Interestingly, the mutations at AR binding sites had a high frequency of TpG->ApG. These uncommon purine transversions have been shown to occur when abasic sites are incorrectly repaired. Based on the mutations observed at the binding site of AR and other related transcription factors, we propose that AR occupancy impairs access of base excision repair enzymes to endogenous DNA damage. Overall, this work demonstrates that non-coding AR binding sites are frequently mutated in prostate cancer and may potentially act as driver mutations.

#3413

A pan-cancer atlas of genomic, epigenomic and transcriptomic alterations in the TGF-β pathway.

Anil Korkut,1 Sobia Zaidi,2 Rupa Kanchi,1 Ashton C. Berger,3 Gordon Robertson,4 Lawrence N. Kwong,1 Mike Datto,5 Jason Roszik,1 Shiyun Ling,1 Andre Schultz,1 Visweswaran Ravikumar,1 Ganiraju Manyam,1 Arvind Rao,1 Simon Shelley,6 Yuexin Liu,1 Zhenlin Ju,1 Donna Hansel,7 Guillermo de Velasco,8 Arjun Pennathur,9 Jesper B. Andersen,1 Colm J. O'Rourke,10 Kazufumi Ohshiro,2 Wilma Jogunoori,2 Nancy Gough,2 Shulin Li,1 Hatice Osmanbeyoglu,11 Andres Houseman,12 Shuyun Rao,2 Maciej Wiznerowicz,13 Jian Chen,1 Shoujun Gu,2 Wencai Ma,1 Jiexin Zhang,1 Pan Tong,1 Andrew D. Cherniack,3 Chuxia Deng,2 Linda Resar-Smith,14 Jaffer Ajani,1 The Cancer Genome Atlas Research Network, John N. Weinstein,1 Lopa Mishra,2 Rehan Akbani1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _George Washington University, Washington, DC;_ 3 _Broad Institute, Boston, MA;_ 4 _‎BC Cancer Agency Genome Sciences Centre, Vancouver, British Columbia, Canada;_ 5 _Duke Cancer Institute, Durham, NC;_ 6 _University of Wisconsin, Madison, WI;_ 7 _University of California, San Diego, San Diego, CA;_ 8 _Dana-Farber Cancer Institute, Boston, MA;_ 9 _University of Pittsburgh Medical Center, Pittsburgh, PA;_ 10 _University of Copenhagen, Copenhagen, Denmark;_ 11 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 12 _Oregon State University, Corvallis, OR;_ 13 _Poznan University of Medical Sciences, Poznan, Poland;_ 14 _The Johns Hopkins University School of Medicine, Baltimore, MD_.

The TGF-β pathway is a multifunctional signaling cascade with context-dependent roles in diverse biologic processes, including tumor promotion or suppression, metastasis, stem cell homeostasis, and immune suppression. Due to its highly context-dependent nature, decoding functional outcomes of the TGF-β pathway in specific tissues is highly challenging. Here, we present comprehensive genomic, transcriptomic and epigenomic analyses of the TGF-β pathway identified by 44 core pathway genes across 33 TCGA tumor types and 9125 samples. The core pathway genes involve TGF-β like ligands, receptors, intracellular SMAD molecules and adaptors. Although individual core pathway genes were rarely mutated or copy number altered in different cancer types, 41% of all samples have at least one genomic alteration in the TGF-β pathway, predominantly in the form of mutations. We identified a highly conserved TGF-β downstream gene expression signature associated with alterations in core pathway genes, suggesting that the alterations in the pathway have shared functional consequences. We observed a significant enrichment of the genomic alterations in gastrointestinal cancers (GI) with a distinct gene expression signature. The newly identified gene expression signature (over- or downregulation of key TGF-β downstream genes) in pan-cancer cohort was associated with significantly poor prognosis, particularly when it co-occurred with genomic alterations in the core pathway. Analysis of mutational hotspot sites revealed 6 genes with hotspots recurring in at least 9 (up to 78) mutational incidences. The hotspot mutations were also highly enriched in GI cancers. We identified previously characterized cancer mutation sites on SMAD4 and SMAD2 as hotspots mainly in GI cancers. We hypothesized novel functions to two of the newly identified hotpot sites through structural and trancriptomic analyses, and two other novel hotspot sites in the pathway await functional characterization. miRNA and epigenomic analyses revealed that TGF-β pathway activity is limited by epigenetic silencing or miRNA expression, especially in cancers with very low pathway gene expression levels. This multidimensional study provides the multifacefed landscape of TGF-β signaling in both individual disease and pan-cancer settings to guide future functional and therapeutic studies of this key cancer pathway.

#3414

Determining microsatellite instability (MSI) status of colorectal cancers through next-generation sequencing (NGS).

Alex So, Shile Zhang, Shannon Kaplan, Joyee Yao, Phillip Le, Christine Glidewell-Kenney, Kristina Kruglyak, Jenny Jie Chen, Ali Kuraishy, Ina Deras, Karen Gutekunst. _Illumina, San Diego, CA_.

Introduction: The MSI status of a tumor is often a marker of deoxyribonucleic acid (DNA) mismatch repair deficiency. Recent clinical trials have shown MSI-High (MSI-H) tumors are more likely to respond to checkpoint inhibitor immunotherapy. The United States Food and Drug Administration recently granted approval for a checkpoint inhibitor in metastatic solid tumor that demonstrates high microsatellite instability. Here we show the accuracy of determining the MSI status of colorectal cancers (CRC) using either TruSight™ Tumor 170 (TST170), a NGS-based 170 gene panel for solid tumor profiling, or whole exome sequencing (WES). Further, we report the accuracy of identifying the MSI status through sequencing of tumor-only samples without the subject-matched normal DNA.

Experimental Method: The MSI status of 52 CRC tumors (51 formalin-fixed, paraffin embedded and 1 fresh-frozen sample) were evaluated using a microsatellite instability (MSI) assay (Promega), which is a detection method that uses capillary electrophoresis to analyze PCR-products. For comparison to WES, the tumors and subject-matched normal DNA libraries were prepared and enriched using TruSight™ Oncology reagents (Illumina). Targeted NGS libraries were prepared and enriched for the TST170-gene panel using the TST170 library kit (Illumina). NGS libraries were sequenced with the HiSeq™ 2000, HiSeq™ 2500, or NextSeq™ 550 instruments (Illumina). MSI scores of the 52 tumors were calculated through a novel internally developed bioinformatics algorithm.

Data Summary: From the 52 subjects assessed through Promega's MSI detection kit, 28 and 24 were found to be MSI-H and microsatellite stable (MSS), respectively. These data were used as the benchmark for the MSI status of these subjects. Both the NGS-based tumor-normal data from WES and TST170 achieved a 100% concordance with PCR-based MSI detection. In addition, when only the sequencing data of the tumor samples were used without the subject-matched normal DNA, the Illumina MSI algorithm could identify the MSI status of the 52 subjects with 98% concordance with Promega's MSI detection kit.

Conclusion: Collectively, this data indicates that NGS can be utilized for determining tumor MSI status using WES or the TST170 panel. In addition, a bioinformatics algorithm was developed that successfully categorized tumors according to their MSI status without requiring subject-matched normal DNA sequence data.

For Research Use Only. Not for use in diagnostic procedures.

#3415

Cytogenetics of renal oncocytomas identify three distinct and mutually exclusive diagnostic classes of chromosome aberrations.

Christopher B. Anderson,1 Michael Lipsky,1 Subhadra V. Nandula,2 Freeman E. Christopher,1 Matthews Thomas,1 Caitlin E. Walsh,1 Gen Li,1 Matthias Szabolcs,1 Mahesh M. Mansukhani,1 James M. McKiernan,1 Murty V. Vundavalli1. 1 _Columbia Univ. Medical Center, New York, NY;_ 2 _Cancer Genetics, Inc, Rutherford, NJ_.

Christopher B. Anderson1, Michael Lipsky1, Subhadra V. Nandula2,3, Christopher E. Freeman2, Thomas Matthews2, Catlin Walsh2, Gen Li1, Matthias Szabolcs2, Mahesh M. Mansukhani 2, James M. McKiernan1, Vundavalli V. Murty 2 1Departments of Urology, 2Pathology and Cell Biology, Columbia University Medical Center, New York, New York; 3Cancer Genetics Inc, Rutherford, New Jersey

The cytogenetic alterations in renal oncocytoma (RO) are poorly understood. We analyzed 130 consecutive RO for karyotypic alterations. Clonal chromosome abnormalities were identified in 63 (48.5%) cases, which could be categorized into 3 classes of mutually exclusive cytogenetic categories. Class 1 RO had diploid karyotypes with 11q13 rearrangement in balanced translocations with ten or more different chromosome partners in all 20 (31.7%) cases. We identified recurrent translocation partners at 5q35, 6p21, 9p24, 11p13-14 and 11q23, and confirmed that CCND1 gene rearrangement at 11q13 utilizing fluorescence in situ hybridization (FISH). Class 2 RO exhibited hypodiploid karyotypes with loss of chromosome 1 and concurrent losses of Y in males and X in females in 25 (39.7%) cases. The class 3 tumors comprising of 18 (30.2%) cases showed diverse types of abnormalities with the involvement of 2 or more chromosomes exclusive of abnormalities seen in Class 1 and 2 tumors. Furthermore, karyotypically uninformative cases were subjected to FISH analysis to identify class 1 and 2 abnormalities. In this group we found similar frequencies of CCND1 rearrangement, loss of 1 or Y as with karyotypically abnormal cases. We validated our results against 91 tumors from the Mittleman database. Correlation of clinical data with all the 3 classes of RO showed no clear evidence of overall patient survival. Our findings support the hypothesis that RO exhibit 3 principal cytogenetic categories, which may have different roles in initiation and/or progression. These cytogenetic markers provide a key tool in the diagnostic evaluation of RO.

#3416

Customizable gene panels overcome challenges associated with targeted resequencing.

Andrew John Barry,1 Kruti M. Patel,2 Amy B. Emerman,2 Scott Adams,2 Sarah Bowman,2 Evan Mauceli,2 Fiona Stewart,1 Eileen Dimalanta,1 Salvatore Russello,1 Charles Elfe,2 Theodore Davis,1 Cynthia Hendrickson2. 1 _New England Biolabs, Inc., Ipswich, MA;_ 2 _Directed Genomics, Ipswich, MA_.

Efficient utilization of targeted gene panels for clinical research is challenged by the wide variation in gene constituents specific to a given study. While focused gene panels efficiently provide the necessary depth of coverage for low-frequency variant detection, the high costs and technical requirements associated with panel design present obstacles to delivering assays that are focused on the specific targets of interest. The NEBNext DirectTM technology utilizes a novel approach to selectively enrich nucleic acid targets ranging from a single gene to several hundred genes, without sacrificing specificity. The approach rapidly hybridizes both strands of genomic DNA with biotinylated probes prior to streptavidin bead capture, enzymatic removal of off-target sequences, and conversion of captured molecules into sequence-ready libraries. This results in an exceptionally uniform coverage profile for a given target. Unlike alternative hybridization methods, this 1-day workflow does not necessitate upfront library preparation, and instead converts the captured molecules into libraries compatible with Illumina sequencing. We have designed and optimized baits specific to the full exonic content of >800 genes associated with cancer as well as a variety of other disease areas. These are designed, balanced, and pooled on a per gene basis, and can be readily combined into customized panels, allowing rapid turnaround of specific custom gene subsets. Here, we present the ability to rapidly deploy custom gene panels across a variety of panel sizes and content, while maintaining high specificity, uniformity of coverage across target content, and sensitivity to detect nucleic acid variants from clinically relevant samples.

#3417

Detection and analysis of oncovirus integration sites in FFPE-derived human tumor samples using hybrid capture and massively parallel sequencing.

Michael K. Slevin,1 Winslow T. Powers,1 Robert T. Burns,1 Bruce M. Wollison,1 Haley A. Coleman,1 Amanda L. Paskavitz,1 Anwesha Nag,1 Danielle K. Manning,2 Elizabeth Garcia,2 Matthew D. Ducar,1 Aaron R. Thorner,1 Laura E. MacConaill1. 1 _Dana-Farber Cancer Inst., Boston, MA;_ 2 _Brigham and Women's Hospital, Boston, MA_.

Viral genome integration into the host's genetic material can provide cells with selective advantages that make critical contributions to unregulated growth, cell division, and genomic instability. Current clinical tests include in situ hybridization, immunohistochemical, and real-time PCR assays that are unable to identify viral integration points in relation to oncongenic somatic mutations. This precludes a complete picture of how such mutations work together to promote, maintain, and disperse tumor progression. Precision cancer medicine employs genomic technologies (e.g., massively parallel sequencing) for high-throughput genomic profiling to molecularly define patient tumors, allowing identification of clinically actionable mutations. We created a custom probe set for targeted hybrid capture enrichment of several oncoviruses and have tested it in combination with our clinical tumor DNA profiling probe set, OncoPanel v3.

Selected viruses were chosen due to their serving as causative cancer agents that disrupt tumor suppressors, facilitate genomic instability, and increase oncogene expression. The full viral genomes of hepatitis B virus and high-risk human papilloma virus (HPV) strains, 16, 18, 33, and 45 were targeted. Additionally, regions coding for E6 and E7 oncoproteins were targeted for several of the low-risk HPV strains, as was the LANA region of Kaposi's sarcoma-associated virus.

FFPE-derived human tumor samples of known infection status were fragmented to 250 bp and converted into Illumina libraries. Pooled libraries underwent hybrid capture with custom, Agilent-designed OncoPanel and viral probes, and resulting captures were sequenced on an Illumina HiSeq2500 sequencer.

SvABA (Structural variation and indel analysis by assembly) was used to perform de novo sequence assembly on soft-clipped and discordant aligned reads, and viral integration sites were identified by realigning contigs to the host genome to obtain breakpoint coordinates. Reduction of false positives was facilitated using dual-matched sample barcodes (IDT), which virtually eliminate barcode cross-talk, allowing for confident detection of low allele frequency events. Furthermore, inclusion of unique molecular identifiers (UMI) permitted discrimination of PCR duplicates and errors.

Our results demonstrate high concordance with clinical samples of known infection status and provide viral integration break point locations in association with genomic tumor mutations. Viral integration sites are often associated with deletions or amplifications of the host's flanking genomic regions. Furthermore, viral integration may disrupt or enhance expression of tumor suppressors and oncogenes, respectively. Coupled with tumor molecular profiling, this information will better inform patient treatment.

#3418

Cross-comparison of targeted gene expression technologies for patient stratification.

Raghavee Venkatramanan,1 Tuuli Saloranta,1 Inah Golez,1 Elliot Swanson,1 Kimberly Kruse,1 Vickie Satele,1 Saman Tahir,1 Sally Dow,1 Evan Anderson,1 Briana Hudson,1 Spencer Chee,1 Kerry Deutsch,1 Steve Anderson,2 Fang Yin Lo,1 Anup Madan1. 1 _Covance, Redmond, WA;_ 2 _Labcorp, Minneapolis, MN_.

Colorectal cancer (CRC) is one of the major causes of global cancer mortality. Until recently, KRAS has been the only predictive biomarker for anti-EGFR therapy for metastatic CRC, and yet predicting prognosis in clinical practice is still poor. Therefore, a more accurate method for prognosis of CRC patients is needed. Gene expression profiling has shown great promise in predicting prognosis of individual patients in diverse cancers. The development of RNA-sequencing has greatly facilitated identification of biomarkers that can be used to stratify patients for targeted therapies. Despite the decrease in cost of sequencing in last few years, the time and the resources needed for analysis limit its use in clinical trials for patient selection. Targeted gene expression technologies like qPCR and NanoString enable highly customizable assays that can be conveniently performed for patient recruitment. The aim of this study was to investigate potential alternatives for gene profiling using a novel NanoString Plex Set technology. The Plex Set system comes with prepackaged and custom code sets in identifying genetic markers. Up to 8 samples can be pooled to each nCounter cartridge lane, enabling a total of 96 samples per run, thus making the total cost relatively affordable. For this study, gene expression signature was developed using RNA-Seq data where we have profiled 74 CRC samples, 20 of which have matching normal samples. A RAS signature score based on expression profile was calculated for each sample. In order to look for potential gene signatures, differential gene expression analysis was performed between the following groups: (a) samples with high versus those with low RAS signature scores in the 54 CRC, (b) KRAS mutant versus wild-type samples, and (c) tumor versus normal samples in the clinical study. We hypothesized that our genes of interest are most likely significantly differentially expressed in one of these groups. The counts of significantly expressed gene for the groups (a-c) are 1560 and 34, respectively, and we are working on the third case. Therefore, significantly deferentially expressed genes between groups were selected and ranked based on frequency of occurrence. These genes of interest are being analyzed using NanoString Plex Set and qPCR to evaluate the potential of using NanoString Plex Set system for targeted gene expression profiling. Results of these analyses will be presented. 

### Genomic Profiling of Tumors 2

#3419

Genomic characterization of mutation hotspots related to chemotherapy responses in esophageal squamous-cell carcinoma with archived formalin-fixed paraffin-embedded tissues.

Chen Guo,1 Josephine Ko,1 Wei Dai,1 Lihua Tao,1 Simon Law,2 Anthony Wing Ip Lo,1 Maria Li Lung1. 1 _University of Hong Kong, Hong Kong, Hong Kong;_ 2 _University of Hong Kong, Queen Mary Hospital, Hong Kong, Hong Kong_.

Introduction: Currently neoadjuvant chemoradiation therapy (CRT) prior to surgery is the most common treatment for Esophageal Squamous-cell Carcinoma (ESCC) patients. Given the fact that traditional TNM staging of the patients before treatment could not reflect their overall survival, a recent study conducted in Hong Kong demonstrated that the percentage of viable tumor cells remaining in the primary tumor after the CRT therapy was a prognostic marker for overall survival. Clinicians defined patients with no viable tumor cells (0%) as good responders, while the others having greater than 50% tumor cells remaining (50-100%) were considered poor responders. They observed that good responders show significantly better five-year survival rate. However, little is known about the mutation profiles between good and poor responders stratified in this way.

Methods: Taking advantage of archived formalin-fixed paraffin-embedded (FFPE) tissues with comprehensive clinical outcomes, I apply a novel approach to systematically screen for possible somatic mutations that are associated with patients' responses to CRT in ESCC. I obtained 100 archived FFPE blocks from Queen Mary Hospital, Hong Kong, 50 each representing either good responders or poor responders. DNA libraries were constructed to target 131 cancer-related genes with TOMA Os-seq, which adopted single-stranded ligation to minimize the loss of input FFPE DNA. Then, libraries were sequenced on Illumina HiSeq X Ten. The final list of somatic mutations was filtered with in-house pipeline, excluding both germline variants from public databases and in-house Hong Kong database. Copy number variations (CNVs) were also identified with a pre-designed control sample in the same sequencing batch.

Results: One test run on samples with good quality DNA showed high sensitivity and specificity up to 100% for pre-designed mutations. For CNV, the sensitivity was 87.5% and the specificity was 97.5%. Trials of sequencing with several ESCC FFPE samples revealed deleterious mutations from genes including TP53, BRCA2, and those mutations were successfully validated by Sanger sequencing. Groups of critical protein-altered mutations and CNVs that distinguish good and poor responders of ESCC will be presented in detail.

Conclusion: In all, FFPE blocks are valuable sources to uncover the biomarkers for CRT response in ESCC. I expect those biomarkers can aid future evaluation of newly diagnostic patients to avoid unnecessary efforts in treating patients that are unsuitable for the traditional therapy and thus facilitate effective treatments.

Acknowledgements: Theme-based Research Scheme grant provided by the Hong Kong Research Grants Council (T12-701/17-R to MLL).

#3420

Discovery of genetic alterations governing sinonasal squamous cell carcinoma tumorigenesis.

Adrian D. Schubert, Nyall R. London, Justin A. Bishop, Esther C. Broner, David Sidransky, Evgeny Izumchenko. _Johns Hopkins University, School of Medicine, Baltimore, MD_.

Tumors of the sinonasal tract are uncommon, and comprise 5% of all cancers of the head and neck (H&N). Approximately 50-80% of sinonasal malignancies are sinonasal squamous cell carcinoma (SNSCC). Unlike H&N cancers, tobacco smoking does not seem to play a key role in the development of sinonasal tumors and the exact etiology and molecular mutations governing SNSCC tumorigenesis are not well understood. Inverted sinonasal papilloma (ISP) is a locally aggressive, benign epithelial neoplasm arising in the paranasal sinuses which has a high recurrence rate and transforms to SNSCC in 10% to 25% of cases, and therefore represent an intermediate step in SNSCC progression. Due to its uncommon nature, there is still a significant knowledge gap in our understanding of SNSCC pathogenesis and genetic mutations associated with SNSCC progression have not yet been characterized. To this end, we performed whole exome sequencing (WES) and RNA-Seq on 4 ISPs and matched SCCs samples collected from the same patients. For each patient, DNA extracted from matched lymphocytes was used as a DNA sequencing control, whereas matched uninvolved mucosa was used as a tissue specific control for RNA-Seq. 285 and 602 non-synonymous aberrations were identified across the preneoplastic lesions and tumors respectively. Interestingly, genes associated with adverse survival in HNSCC patients (such as EGFR, FAT1 and IGF1R) were mutated in more than 1 dysplastic lesion. PIK3CA was among the most mutated genes in invasive neoplasms. Notably, in three patients we identified mutations that were present in both preneoplastic and invasive neoplasms, which included cancer driver genes actionable in HNSCC and other solid malignancies (such as PIK3CA, TP53, FAT1 and EGFR). RNA-Seq analysis of the same samples revealed a subset of genes whose expression level sequentially increased or decreased from normal tissue, to early lesions to SNSCC. Interestingly, expression of genes associated with survival, migration, invasiveness and inhibition of apoptosis (such as SET, RAD21, NPM1, PTPN11, CLIP1, KIF5B, EGFR, NRAS, KRAS, MET, MYCL and CCND1) consecutively increased along the tumor progression in all three analyzed, whereas expression of tumor suppressors (such as PTCH1, IKZF1, GAS7, FOXP1, NTRK3 and LRP1B) was downregulated. Although our data suggest that preneoplastic ISP lesions may be clonally related to SCC in some cases, validation of these observations in a larger specimens' cohort is warranted to further delineate the mechanisms underlying the SNSCC tumorigenesis.

#3421

Genomic correlates of patient age in diffuse-type gastric cancer.

Hark K. Kim, Soo Y. Cho. _National Cancer Ctr. Korea, Goyang, Republic of Korea_.

Background: Gastric cancer is increasing in incidence among young white populations in the US. Gastric cancer in young patients is notable for its enrichment of diffuse histology as well as female predominance and aggressive clinical course, whereas gastric cancer is generally more frequent in males. Thus, clinicopathological characteristics of diffuse-type gastric cancer differ according to patient age, but the molecular mechanisms for early-onset gastric cancer's unique clinicopathological characteristics have not been elucidated. While the incidence of intestinal-type gastric cancer is decreasing worldwide, that of diffuse-type gastric cancer remained constant. Nonetheless, relatively small numbers of diffuse-type gastric cancers have been represented in whole exome sequencing studies such as TCGA project. We therefore investigated germline and somatic mutation profiles of diffuse-type gastric cancers with regards to patient age.

Methods: We conducted whole exome and targeted sequencing and SNP6.0 array analyses of resected tumor tissue and blood samples collected from young (45 years old or younger) Korean patients with diffuse-type gastric cancer, and compared the genomic data with those from older Korean patients with diffuse-type gastric cancer. Genomic data of Korean diffuse-type gastric cancers were also compared with TCGA diffuse-type gastric cancers, 70% of which were from Caucasians.

Results: Among 84 young Korean patients with DGC, 7 patients (8.3%) harbored germline mutations in TP53, CDH1, ATM, RAD51D, or PALB2. Three tumors with strong mutation signatures for inherited DNA repair defects harbored germline mutations in either RAD51D or PALB2, with loss-of-heterozygosity in the tumors. The most significant somatic mutations in young patients with diffuse gastric cancer were CDH1, TP53, ARID1A, KRAS, PIK3CA, ERBB3, TGFBR1, FBXW7, RHOA, and MAP2K1. Within the Korean population and within populations of other ethnicities, mutations in CDH1 (42.2%) or TGFBR1 (7.3%) were more frequent in younger patients than in older patients (17.4% and 0.9%, respectively). In contrast, the RHOA mutation (9.2%) was less frequent in younger patients than in older patients (19.1%). CDH1 alterations, but not RHOA mutations, were associated with poor prognosis (hazard ratio, 3.4 (95% CI, 1.5-7.7)), which may be associated with the aggressive clinical course of diffuse-type gastric cancers in young patients.

Conclusions: Our largest-ever genomic analysis of diffuse-type gastric cancer reveals young age-specific mutation profiles (supported by National Cancer Center Grant 1710809 and Multi-omic Research Program).

#3422

Genomic landscape of Japanese cancer patients across multiple tumor types: Prospective molecular profiling study of 3,022 patients at Shizuoka Cancer Center.

Masakuni Serizawa,1 Takeshi Nagashima,2 Keiichi Ohshima,3 Keiichi Hatakeyama,3 Yuji Shimoda,2 Shumpei Ohnami,4 Kouji Maruyama,5 Takashi Sugino,6 Tohru Mochizuki,3 Yasuto Akiyama,7 Kenichi Urakami,4 Masatoshi Kusuhara,8 Medical staff and experimental staff of the Shizuoka Cancer Center, Ken Yamaguchi9. 1 _Drug Discovery and Development Division, Shizuoka Cancer Center Research Institute, Nagaizumi-cho, Sunto-gun, Shizuoka, Japan;_ 2 _SRL Inc., Shinjuku-ku, Tokyo, Japan;_ 3 _Medical Genetics Division, Shizuoka Cancer Center Research Institute, Nagaizumi-cho, Sunto-gun, Shizuoka, Japan;_ 4 _Cancer Diagnostics Division, Shizuoka Cancer Center Research Institute, Nagaizumi-cho, Sunto-gun, Shizuoka, Japan;_ 5 _Experimental Animal Facility, Shizuoka Cancer Center Research Institute, Nagaizumi-cho, Sunto-gun, Shizuoka, Japan;_ 6 _Division of Pathology, Shizuoka Cancer Center, Nagaizumi-cho, Sunto-gun, Shizuoka, Japan;_ 7 _Immunotherapy Division, Shizuoka Cancer Center Research Institute, Nagaizumi-cho, Sunto-gun, Shizuoka, Japan;_ 8 _Region Resources Division, Shizuoka Cancer Center Research Institute, Nagaizumi-cho, Sunto-gun, Shizuoka, Japan;_ 9 _Shizuoka Cancer Center, Nagaizumi-cho, Sunto-gun, Shizuoka, Japan_.

Background: Cancer genome sequencing enables efficient identification of genetic alterations as potential therapeutic targets, representing a fundamental component of precision oncology to make therapeutic decisions based on individualized genetic signatures. In January 2014, the Shizuoka Cancer Center of Japan launched Project HOPE, which is the first prospective molecular profiling study centered on genome sequencing across multiple tumor types. Herein, we describe the distributions of the tumor mutation burden (TMB; number of mutations/Mb), genetic alterations frequency, mutational signature across tumor types, and their associations with tissue of origin, histological type, and carcinogenic factors.

Methods: Between January 2014 and April 2017, 3,174 tumor samples from 3,022 patients who underwent surgery at the Shizuoka Cancer Center were collected with informed consent and subjected to whole-exome sequencing (WES) with an Ion Proton system. Corresponding peripheral blood samples were also subjected to WES for identification of tumor-specific genetic alterations. Oncogenic fusions were detected by targeted RNA sequencing. Samples with estimated tumor purity < 20% were excluded because of the risk of false negatives, leaving 2,899 samples (91%) in the final analysis.

Results: Overall, 2,642 samples were derived from the primary tumor, with a median TMB of approximately 2.7. The principal tumor types of ≥40 primary tumors were as follows (N, TMB): colorectal adenocarcinoma (CRAD; 905, 3.3), lung adenocarcinoma (LUAD; 324, 1.6), gastric adenocarcinoma (GAD; 272, 3.0), head and neck squamous cell carcinoma (HNSC; 164, 2.6), breast invasive ductal carcinoma (BIDC; 138, 1.1), hepatocellular carcinoma (HCC; 124, 3.6), lung squamous cell carcinoma (LUSC; 79, 5.6), and gastrointestinal stromal tumor (GIST; 41, 0.7). Approximately 7.8% of the samples (160/2,047) showed a high mutation burden above the threshold defined by the TMB distribution in each principal tumor type, and the mutation signature was related to a defect of DNA repair and exposure to environmental mutagens. Dominant oncogenic pathways based on the profile of genetic alterations in each tumor type were as follows: Wnt (CRAD), receptor tyrosine kinase (LUAD, GIST), TP53 (GAD, HNSC, LUSC), PI3K (BIDC), and chromatin modification (HCC). GIST was the tumor type with the highest proportion of clinically actionable genetic alterations, followed by BIDC and LUAD.

Conclusions: This is the first report of a prospective genome sequencing analysis across multiple tumor types in Japan. The resulting mutational profile can provide a landscape of commonalities and differences in genetic profiles among tumor types in Japanese patients with cancer, and can contribute to planning a basket study for expanding the patients suitable for treatment with molecular-targeted drugs.

#3423

Determining neoadjuvant cisplatin-based chemosensitivity in muscle invasive bladder cancer through differential gene and miRNA expression analysis.

Neal Murphy*,1 Paras Shah*,2 Annette Lee,3 Thomas Bradley,4 Manish Vira,5 Ilya Korsunsky,3 Andrew Shih,3 Oksana Yaskiv,1 Zachary Kozel,5 Anthony Liew,3 Houman Khalili,3 Xinhua Zhu4. 1 _North Shore Long Island Jewish Medical Center, Manhasset, NY;_ 2 _(*contributed equally) Smith Institute for Urology, Northwell Health System, New Hyde Park, NY;_ 3 _Feinstein Institute for Medical Research, Manhasset, NY;_ 4 _Monter Cancer Center, Lake Success, NY;_ 5 _Smith Institute for Urology, Northwell Health System, New Hyde Park, NY_.

Background: Muscle invasive bladder cancer (MIBC) neoadjuvant responders as opposed to nonresponders demonstrated significantly improved 5-year cancer specific survival and reduced nodal positivity rates. Identification of chemo responsive cohorts would not only facilitate targeted delivery of chemotherapy to those most likely to benefit, but also avoid morbidity and delayed surgical intervention in patients unlikely to derive benefit. We evaluated the role of gene and miRNA expression profiles in initial TURBT (transurethral resection of bladder tumor) specimens of patients before undergoing neoadjuvant chemotherapy and subsequent radical cystectomy. Pathological response at time of cystectomy was used to classify initial TURBT specimens as responders (pT0) versus non-responders (≥pT2). Differential expression of gene and miRNA between the two groups may help stratify patients being considered for neoadjuvant chemotherapy.

Methods: TURBT specimens from patients with MIBC were preserved in FFPE tissue blocks before patients subsequently received cisplatin-based neoadjuvant chemotherapy. A total of 13 pT0 and 17 ≥pT2 patients were selected and matched for age, gender and tumor stage. RNA was extracted from FFPE blocks using an Ambion total nucleic acid isolation kit. mRNA was sequenced using Illumina TruSeq RNA Access Library and NextSeq technology. Gene counts were assessed by ht-seq counts and differential expression using DESeq2. Pathway analysis was performed using GAGE. Differential expression of 754 miRNAs was analyzed using the TaqMan openarray miRNA panel.

Results: In responders, a gene-set enrichment analysis revealed significant upregulation of genes in the hsa00190 pathway (implicated in oxidative phosphorylation), hsa03040(spliceosome function). In non-responders, there was significant upregulation of genes in the hsa04510 pathway (focal adhesion), hsa04512 (ECM-receptor interaction), hsa04310(Wnt signaling), hsa04350(TGF-beta signaling) and hsa04010 (MAPK signaling). For the miRNA analysis, significant upregulation of miR-23a, miR-27a, miR-135b, miR-145, miR-422a was seen in responders, and miR-93, miR-107, miR-339-3p, miR-532 in non-responders. Using a random forest classification, a significant proportion of non-responders were correctly identified compared to responders.

Conclusion: Gene and miRNA expression in initial MIBC TURBT specimens may be used as an aid in predicting neoadjuvant non-responders. Increasing the sample size is needed to further validate our findings and may lead to a successful chemotherapy prediction model.

#3424

Genomic alterations in clonal hematopoiesis.

Kuan-Lin Huang, Mingchao Xie, Yige Wu, Reyka Jayasinghe, Rajees Varghese, R. Jay Mashl, Song Cao, Matthew Wyczalkowski, Wen-wei Liang, Michael C. Wendel, Ryan Fields, Michael D. McLellan, Daniel C. Link, Feng Chen, Li Ding. _Washington Univ. St. Louis, Saint Louis, MO_.

Cancer development is an incremental process. Mutations that initiate clonal hematopoiesis often arise in individuals many years before disease symptoms are apparent. We discovered blood somatic mutations in 8,612 germline blood samples in The Cancer Genome Atlas, drawing fundamental distinctions between these events and germline variants. In a preliminary scan of 5,949 samples, 13,345 blood-specific mutations were identified, preferentially distributed across 26 genes, including ones frequently mutated in hematologic malignancies (e.g., DNMT3A, ASXL1, TET2, JAK2, IDH2, SF3B1, MLL3, and KDM6A) and genes rarely mutated in primary hematologic tumors, including EGFR, PIK3CA, NOTCH2, and PPM1D. Further, we utilized both exome sequencing and SNP array data to conduct the first large-scale survey of blood somatic copy number variation, discovering potentially oncogenic events such as JAK2 amplification. Finally, elderly individuals and smokers have increased likelihoods of undergoing clonal expansion. Our analysis of genomic alterations reveals the origins of hematologic malignancies and can facilitate advancement in early detection and prevention.

#3425

Exome-sequencing derived mutations of endocrine treated ER-positive early breast cancer.

Werner Schroth,1 Siarhei Kandabarau,1 Peter Fritz,1 Thomas Mürdter,1 Liza Bacchus,2 German Tamoxifen Study Group,1 Matthias Schwab,3 Hiltrud Brauch4. 1 _Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology Stuttgart, and University of Tuebingen, Germany;_ 2 _Robert Bosch Hospital, Stuttgart, Germany;_ 3 _Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology Stuttgart, Department of Clinical Pharmacology, University Hospital Tuebingen, Cancer Consortium DKTK, and German Cancer Research Center DKFZ, Heidelberg, Germany;_ 4 _Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology Stuttgart, University of Tuebingen, German Cancer Consortium DKTK, and German Cancer Research Center DKFZ, Heidelberg, Germany_.

Anti-hormonal treatment of ER-positive early breast cancer with tamoxifen and/or aromatase inhibitors (AI) significantly improves patient outcome, however there is a lack of markers that predict the one third of treated patients who relapse or die. Although ER positive (luminal) tumors are known to be genetically highly diverse, the relevance of somatic mutations with regards to endocrine treatment outcome and course of the patients' disease is not fully understood. We conducted a prospective observational clinical trial for the investigation of treatment outcome determinants including 1219 tamoxifen and/or AI-treated postmenopausal ER-positive early breast cancer patients (DRKS00000605, recruitment between 2005-2010, median follow up 5.1 years). Of these, 40 tumors divided into 20 patients each with and without disease recurrence matched by prognostic variables (age, tumor size, grade, nodal status, treatment) were analyzed for their mutation pattern. We performed whole-exome sequencing of tumor and normal DNA (Agilent SureSelect, Illumina) for the identification of single nucleotide variants and small indels. Somatic mutations were identified following subtraction of germline single nucleotide polymorphisms (SNP), filtering based on raw-read quality parameters, in silico predicted biological function, and disease relatedness using the Ingenuity variant analysis software package (Qiagen). Clinical outcome associations were tested either on gene or on variant level. Somatic mutations with predicted deleterious effects were identified in 18 out of 40 known mutation-driver genes including PIK3CA, GATA3, MAP3K1, and TP53 (45%). Thirteen patients carried a mutation in the most frequently affected PIK3CA (32.5%). Of note, three out of four patients with a E542K mutation belonged to the breast cancer recurrence group, while 5 of 6 tumors with the H1047R mutation originated from patients without recurrence suggesting differential effects of these PIK3CA hotspot mutations. Common germline SNPs at IL17RB, ANK2, POM121, PGR, PTPRB, RAD51D, RFPLA4, and SUMO3 provided first hints for a possible association with relapse status (p<0.05). We will report on the extended genotype-outcome-correlations to further evaluate the relevance of somatic mutations for endocrine treatment outcome of early ER-positive breast cancer.

#3426

Genetic architecture of esophageal squamous cell carcinoma in African American veterans.

Robert Wadleigh,1 Krithika Bhuvaneshwar,2 Gustavo Marino,3 Vincente Notario,4 Jack Lichy,5 Yuriy Gusev,2 Hayriye Verda Erkizan6. 1 _Hematology and Medical Oncology Section, Veterans Affair Medical Center DC, Washington, DC;_ 2 _Innovation Center for Biomedical Informatics, Georgetown University, Washington, DC;_ 3 _Gastroenterology and Hepatology, Veterans Affair Medical Center DC, Washington, DC;_ 4 _Radiation Medicine, and Biochemistry and Molecular and Cellular Biology, Georgetown University, Washington, DC;_ 5 _Pathology and Laboratory Medicine, Veterans Affair Medical Center DC, Washington, DC;_ 6 _Institute for Clinical Research, Veterans Affair Medical Center DC, Washington, DC_.

Background: Esophageal carcinoma (EC) is one of the most common malignancies of the gastrointestinal tract worldwide and is unresponsive to therapy. Esophageal squamous cell carcinoma (ESCC) comprises the majority of EC in African Americans, Asians and other ethnic groups, while esophageal adenocarcinoma (EAC) predominates among Caucasians. Poor prognosis of EC in Caucasians and African Americans is reflected by five-year survival rates of 21% and 14 %, respectively. The aggressiveness and lower survival rate of African American ESCC patients than that from patients of other ethnic groups are evident even after adjusting for treatment modalities and socioeconomic factors. These characteristics suggest the existence of an ethnic- or race-dependent component of EC etiology. However, the genetic architecture of ESCC in AA is not well studied, and thus, mostly undefined. Our study aims to identify exonic mutations that may represent critical genetic changes in African American ESC carcinogenesis. Methods and Materials: Whole exome sequencing (WES) of matched tumor and normal tissue DNA from endoscopic biopsies from late-stage ESCC of nine African American Veteran male patients was conducted. This study was approved by the DC VAMC Institutional Review Board, and a written informed consent was obtained from each patient prior to biopsy. Biopsies were de-identified, freshly frozen and stored at -80 °C. To proceed to exon capture, we enriched our samples by using Agilent SureSelect XT Human All Exon V6+UTR. Paired-end sequencing at a read depth of 100X was performed on the exon libraries using the Illumina HiSeq 4000 sequencer. We applied Genome Analysis Toolkit's best practices WES pipeline to identify both germline and somatic variations in the dataset. The Seven Bridges Cancer Genomics Cloud platform was used for processing the data and annotating the variants. Variants were mapped to genomic regions and further aggregated at the gene level, pathways, and biological processes relevant to disease by using Reactome, Pathway Studio, and Ingenuity Variant Analysis. Results: In all samples, 27,586 variants consisted of 86% single nucleotide variation, 8.5% insertions and 5.5% deletions. Approximately half of the variants caused missense changes while 0.12% created nonsense mutations. High impact mutations occurred in genes that have a role in DNA damage repair, stress response, detoxification pathways, keratinization and immune surveillance. Conclusion: We found unique genomic variation in African American ESCC, that reveals a potential molecular signature for the aggressiveness and lethality of ESCC in this population. Future functional studies will be conducted to elucidate the role of these mutations in ESCC pathogenesis.

#3427

Gatekeeper inactivation drives glioma progression into secondary glioblastoma.

Kirsi Johanna Granberg,1 Matti Annala,1 Serafiina Jaatinen,1 Joonas Haapasalo,2 Olli Yli-Harja,3 Hannu Haapasalo,2 Wei Zhang,4 Matti Nykter1. 1 _University of Tampere, Tampere, Finland;_ 2 _Tampere University Hospital, Tampere, Finland;_ 3 _Tampere University of Technology, Tampere, Finland;_ 4 _Comprehensive Cancer Center of Wake Forest Baptist Medical Center, Winston-Salem, NC_.

<Glioblastoma (GBM) is the most common and lethal form of brain cancer in humans. Median survival is 15 months with best available treatment. Most GBMs arise de novo (primary GBM), but 5 - 10% progress from lower grade gliomas (secondary GBM). As progression of low grade glioma into secondary GBM significantly impacts prognosis, a better understanding of this process is paramount for treatment and monitoring of affected patients. In this study, we applied whole genome and transcriptome sequencing to primary glioma and relapsed secondary GBM tissue from seven patients with progression. All primary gliomas carried IDH1 mutations, and in all cases the mutation was inherited by the secondary GBM. ATRX alterations in all five astrocytomas and TERT promoter mutations in both 1p19q-codeleted oligoastrocytomas were also inherited in progressed tumors. In five patients, progression was associated with increased genomic instability, whereas mutation load was significantly increased in two other patients. One of them exhibited a hypermutation signature caused by a mutation in the proofreading domain of DNA polymerase epsilon, while the second had lost both copies of the DNA mismatch protein MSH2. In addition, both 1p19q-codeleted tumors had acquired focal inactivating deletions of the protein tyrosine phosphatase PTPRD at progression, suggesting a novel driver mechanism for GBM progression. The most common progression-related genomic alterations were CDKN2A deletions, TP53 mutations, RB1 deletions, PTEN deletions, and deletions of genes crucial to the double strand break repair pathway. Taken together, progression into secondary GBM was significantly related to deletions in tumor suppressor genes as well as TP53 mutations. Disruption of these gatekeepers appears to be a significant mechanism for glioma progression.>

#3428

Development and validation of the plasma monitor test system.

Kevin J. Kelly, Jasmina Uvalic, Daniel Bergeron, Shelbi Burns, Melissa Soucy, Guruprasad Ananda, Andrew Hesse, Pavalan Panneer Selvam, Honey V. Reddi. _The Jackson Laboratory, Farmington, CT_.

Introduction: Non-invasive monitoring of variants in a cancer patient for minimal residual disease, recurrence and/or resistance has tremendous clinical utility and warrants the development of a cell-free circulating tumor DNA panel, which involves the use of a simple blood draw. Towards this end, JAX has validated a new liquid biopsy assay called the Plasma MonitorTM that focuses on 84 clinically significant hotspots across 14 genes to complement our current clinical test menu, with a focus on comprehensive profiling of cancer.

Methods: Post development and optimization of a custom amplicon panel, which included running multiple batches of samples through the assay to determine appropriate extraction methods, input DNA QC metrics, sequencing batch size, and sequencing loading concentrations, clinical validation of The Plasma MonitorTM was initiated. Validation included the evaluation of 20 uncharacterized plasma samples and 14 known controls and was executed in 5 phases: (1) Sample Processing for Validation Parameter determination; (2) LOD, sensitivity, specificity and accuracy (3) inter-assay concordance; (4) intra-assay concordance; (5) clinical validity in terms of interpretation and reporting of variants identified.

Results: The final clinical protocol was developed using an input of 10ng of ctcfDNA quantified and qualified by a custom qPCR assay. Wet lab results of the first validation batch can be seen in Table 1. Using samples containing variants with known allele frequencies and a droplet digital PCR (ddPCR) based confirmation of novel variants identified by our assay, we established the assay's limit of detection (LOD) to be 0.9%. At this LOD, sensitivity, specificity, and accuracy were found to be 96.6%, 100%, and 98% respectively.

Conclusion: Based on the results of this validation, the Plasma Monitor assay will be incorporated into the JAX clinical test menu. This new assay allows for comprehensive profiling and monitoring of cancer progression, response to therapy and minimal residual disease, and a significant benefit to both patients and clinicians.

#3429

Genome and epigenome profiling of high stage neuroblastoma.

Miki Ohira-Ichikawa,1 Ryuichi P. Sugino,1 Hisanori Takenobu,1 Zhenghao Li,1 Masayuki Haruta,1 Genta Nagae,2 Claire Renard-Guillet,2 Akira Nakagawara,3 Hiroyuki Aburatani,2 Takehiko Kamijo1. 1 _Research Institute for Clinical Oncology, Saitama Cancer Center, Saitama, Japan;_ 2 _RCAST, University of Tokyo, Tokyo, Japan;_ 3 _Saga Medical Center Koseikan, Japan_.

Neuroblastoma (NB) is the most common pediatric extracranial solid tumor with a wide range of clinical phenotypes from spontaneous regression to highly resistant to chemotherapy. Loss of 1p and 11q and genomic alterations of MYCN, ALK, ATRX and TERT have been known to be involved in oncogenesis and unfavorable phenotype of NB, however, only small numbers of recurrent driver mutations have been observed by NGS-based studies. This implies epigenetic alterations also influence the pathogenesis of NB. In this study, to elucidate the molecular basis of malignant subtype of NB, we conducted methylome analysis for 145 primary NBs (stage 1:3; stage 2:8; stage 3:16; stage 4:105; stage 4s:13) by Infinium Human Methylation 450 BeadChip. By hierarchical clustering of methylome signature, the tumors were classified into four clusters. Cluster-1 shows good similarity with nerve cell controls and, together with cluster-3 (very young, early tumor stage), exhibited favorable prognosis. Unfavorable cluster-2 and -4 exhibited hyper-methylation phenotypes in CpG islands (CGI) and the former was enriched by MYCN-amplified tumors. These two clusters (n=73) showed strong correlation with poor patient prognosis (p=0.0055). 1400 hyper-methylated CGI locus which corresponded 550 genes were picked up and annotated by GO database. The term "cell adhesion", "cell-cell signaling", "transmission of nerve impulse" and "cellular component morphogenesis" were enriched. No 1p or 11q genes were enriched in hyper-methylated genes but several genes such as ANKK1 on chr11q whose lower expression levels are strongly correlated with unfavorable prognosis of the patient (p=4.0e-07) were included. Further analyses by comparing the influences in gene expression profiles by methylation and by histone modification of PRC2 are ongoing. Our recent data indicated that NTRK1, whose higher expression is a strong favorable marker, is transcriptionally downregulated in MYCN-amplified tumors by both methylation and H3K27me3 on distinct promoter regions (Li Z et al). Since application of epigenetic drugs are one of the possible therapeutic choices for NB, transcriptional regulation by the epigenetic alterations are needed to be intensively studied.

#3431

Evaluation of genetic mutations and copy number alterations in biliary tract cancer using targeted exome sequencing.

Heejung Chae, Changhoon Yoo, Deokhoon Kim, Seonmin Lee, Kim Dan Bi, Jae Ho Jeong, Heung-Moon Chang, Baek-Yeol Ryoo, Kyu-pyo Kim. _Asan medical center, Seoul, Republic of Korea_.

Background: Biliary tract cancer (BTC) is a heterogeneous group of cancers anatomically divided into the gallbladder cancer (GBC), intrahepatic cholangiocarcinoma (ICC) and extrahepatic cholangiocarcinoma (ECC). None of molecular target agents has been proven to improve the prognosis of BTC yet. To gain a deeper understanding of BTC's pathophysiology and find its new potential therapeutic targets, this study is aimed to investigate genetic profiles of BTC and their clinical implications.

Methods: A total of 69 patients who had been pathologically diagnosed as adenocarcinoma of biliary tract from January 2014 to November 2016 and had available surgical or biopsy specimen were enrolled for this study. With the DNA specimen extracted from previously-collected tumor tissue, somatic mutations and copy number variation (CNV) analyses were performed using targeted exome sequencing. Data regarding the baseline patient characteristics and treatment outcomes were retrospectively obtained by reviewing the patients' medical records.

Results: Nineteen patients with GBC (28%), 24 patients with ICC (35%) and 26 patients with ECC (37%) were included in this analysis. Genetic mutation and copy number alteration (CAN) were observed in 59 (85%) and 35 (51%) patients, respectively while 5 patients (7%) did not have any genetic alterations. The most commonly mutated gene was TP53 (n=33, 47.8%), followed by KRAS (n=18, 26.1%), ARID1A (n=10, 14.5%) and IDH1 (n=9, 13.0%). IDH1 mutation appeared more frequently in patients with ICC (n=7, 29.2%, P=0.015) compared to those with GBC (n=1, 5.3%) or ECC (n=1, 3.8%) while ERBB2 and ERBB3 mutation were found only in GBC and ECC. HER2 amplification was observed in 7 patients (4 with GBC and 3 with ICC). Among those, one patient showed 3+ for HER2 test by immunohistochemistry (IHC) while four patients were 2+. All somatic mutations and CNVs documented in the present study were classified into five pathway modules which were identified by Nakamura et al. (Nature Genetics 2015). Majority of genetic alterations (n=45, 65.2%) belonged to Kinase-RAS module, followed by TP53 (n=41, 59.4%), TGF-β-SWI/SNF-MYC (n=22, 31.9%), Epigenetic (n=16, 23.2%) and RB-cell cycle (n=10, 14.5%) modules. Alterations in Epigenetic module were most common in ICC (vs. GB vs. ECC, 50.0% vs. 15.8% vs. 3.8%, P=0.002) while alterations in TGF-β-SWI/SNF-MYC module were most frequent in ECC (vs. GB vs. ICC, 46.2% vs. 3.6% vs. 31.6%, P=0.031).

Conclusion: Genetic profile of BTC is heterogeneous according to its anatomic location. Our results indicate that subgroup of BTC patients may have benefit from targeted therapy such anti-IDH and anti-HER2 inhibitors.

#3432

Evolving genomes of breast cancer aromatase inhibitor resistance models.

Reiner Hoppe,1 Siarhei Kandabarau,1 Ping Fan,2 V Craig Jordan,2 Hiltrud B. Brauch3. 1 _Dr. Margarete Fischer-Bosch - Institute of Clinical Pharmacology, Stuttgart, and University of Tuebingen, Germany;_ 2 _MD Anderson Cancer Center, University of Texas, Houston, TX;_ 3 _Dr. Margarete Fischer-Bosch - Institute of Clinical Pharmacology, Stuttgart, and University of Tuebingen, German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany_.

Acquired endocrine resistance is a severe barrier in the anti-hormonal treatment of breast cancer and is responsible for its failure in about half of the treated patients. In vitro models of long-term estrogen deprived breast cancer cells serve as laboratory tools to explore new therapeutic strategies. The models MCF-7:5C and MCF-7:2A mimic clinical AI resistance but regress with E2 due to the reconfiguration of survival signaling known as E2-inducible apoptosis (5C rapid, 2A delayed reaction). Alterations of stress related pathways including the accumulation of endoplasmic reticulum stress, oxidative stress, and inflammatory stress that occur prior to E2-induced apoptosis (Ariazi et al. 2011; Fan et al. 2013, 2015; Sweeney et al. 2014) and specific miRNA expression profiles (Hoppe et al Oncotarget 2016) have been linked with these resistance and E2-inducible apoptosis phenotypes. Little is known on their underlying genetic variability. Here we compare the mutational profiles of 5C and 2A clones with those of the estradiol stimulated growth sensitive MCF-7:WS8 and standard MCF-7 (ATCC) cells. Whole genome libraries were sequenced on the Illumina HiSeq platform (2x150bp reads). Alignments were manually inspected (bcftools mpileup) to avoid false negative variant calling in poorly (<20x) covered regions. Loci with at least 10x coverage and absence of any variant reads were classified as wildtype. The coding genome positions defined as mutant or wildtype in all four cell types served as a basis to select candidate mutations for the verification of MCF-7 variants via comparison to the COSMIC database (http://cancer.sanger.ac.uk/cosmic) and identification of novel mutations by variant calling (GATK HaplotypeCaller). Putative protein effects of variants were predicted by the SnpEff tool. We verified 421 point mutations in MCF-7 (COSMIC) of which 203 mutations were lost in our models, with additional specific losses of 4, 4, and 18 mutations in WS8, 2A and 5C, respectively. In 5C cells new mutations affected 261 genes with 31 high impact mutations based on splice site alterations in 7 genes (i.e. CERS2, SEMA3A, TDRD7, ATP5H) and gain of stop codons in 24 genes (i.e. CACNA1S, NOTCH4, SUN1, JPH3, SERPINB2). Similarly, in 2A cells there were 12 high impact mutations among 122 affected genes (e.g. ARHGEF5, ARHGAP35, ARAP2). We will report on the integrative genomic analysis including larger structural variants (short indels and CNVs) towards the evolution of these models for a comprehensive understanding of the biology of E2-inducible apoptosis.

#3433

Genomic analysis of patient-derived xenograft (PDX) model from metastatic breast cacner.

Dongjin Shin,1 Seock-Ah Im,2 Seongyeong Kim,3 Minjung Kim,1 Yu Jin Kim,3 Jinjoo Kang,4 Ahrum Min,3 Jieun Lee,4 Giyong Jang,4 Deukchae Na,5 Kyung-Hun Lee,2 Jongil Kim1. 1 _Department of Biochemistry and Molecular Biology, Seoul National University, Seoul, Republic of Korea;_ 2 _Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea;_ 3 _Cancer Research Institute, Seoul National University, Seoul, Republic of Korea;_ 4 _Department of Life Science, Ewha Womans University, Seoul, Republic of Korea;_ 5 _Ewha Institute of Convergence Medicine, Ewha Womans University Mokdong Hospital, Seoul, Republic of Korea_.

Background: Patient-derived xenograft (PDX) models have been shown to be predictive of clinical outcomes and are being used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Molecular subtyping of metastatic breast cancer is specifically important for clinical decision for targeted therapy, endocrine therapy, and chemotherapy. The PDX model, reflecting the same molecular characteristics of a patient's tumor, can provide valuable information prior to treatment. We explored the possibility of generating PDX from metasttic breast cancer and examined whether the primary or metastatic cancer of the patient and the tumor implanted in the mouse have the same genetic characteristics.

Method: We established PDX models using small biopsy samples of tumors from primary breast or metastatic cancer using NSG mouse. Clinicopathologic characteristics and outcome were collected from the electronic medical records. Whole-exome sequencing was performed using Illumina platform. Single-nucleotide polymorphisms (SNPs) and small insertions and deletions (Indels) were called using muTect(1.1.7) and IndelGenotyper provided by GATK (3.6.0). We also investigated whether copy number variations (CNVs) are maintained in the PDX models. Conifer was used for counting the read framents. The read counts of normal and tumors were normalized by a log2 scale.

Results: We established 24 PDX models from 34 biopsy samples (11 primary breast cancer and 23 metastatic cancer). 11 samples were hormone receptor-positive luminal type, 9 samples were HER2-positive type and 13 samples were triple-negative subtype. Our results indicate that the most frequent mutated genes are TP53, ARAF, GNAQ, ATRX, and PIK3CA in total samples. TP53 was the most frequently altered gene in all subtypes. Other mutated genes were slightly different for each subtype, followed by GNAQ and GATA3 in hormone receptor-positive subtype. ARAF and ATRX were in TNBC subtype. In HER2+ subtype, GNAQ, BRCA2, PIK3CA and ARID1B were same number of mutations. EIF3E and IKZF3 were the most frequently amplified genes and USP6 and SSX4 were the most frequently deleted genes. In addition, we compared the results in 5 cases that had all of the patient's tumor sample and the xenografted tumor sample; the same mutation was found and reflected the molecular features of the patient.

Conclusion: In this study, it was possible to establish PDX from biopsy tissues and PDX models maintain the genomic characteristics of the patient tumor. We believe that by using PDX models and bioinformatics analysis, it is possible to find new druggable targets and eventually the proper drug for personalized medicine.

#3434

Digital spatial profiling platform allows for spatially resolved, high-plex quantification of mRNA distribution and abundance on FFPE and fresh frozen tissue sections.

Daniel Zollinger, Kristina Sorg, Jill McKay-Fleisch, Kristi Barker, Karen Nguyen, Chris Merritt, Joseph Beechem. _Nanostring Technologies, Inc., Seattle, WA_.

Characterization of spatial mRNA expression across multiple targets in a single tissue section has proven difficult to accomplish. To address this unmet need, we have developed a novel imaging platform, Digital Spatial Profiling (DSP), designed to simultaneously analyze 10s to 100s of RNAs (or proteins) from discrete regions by detecting oligo-conjugated probes. We show that this system can resolve mRNA expression panels in a highly multiplexed and automated manner on human FFPE and fresh frozen tissues. DSP RNA probes consist of antisense target-recognition sequences and UV-cleavable tags that can be released in a precise manner by the DSP instrument and digitally counted using nCounter® reagents. For this, probes are first hybridized overnight in situ to human FFPE or fresh frozen tissue sections. After hybridization, tags are released by nondestructive exposure of UV light, automatically collected by the DSP Instrument, and quantified using nCounter® technology. The nCounter PlexSet™ reagents are used to enable the analysis of 96 regions of interest with a single nCounter run. To demonstrate the regional specificity of RNA detection on DSP, we profiled distinct regions in human tonsillitis tissues using a 48-plex of RNA target probes. Our results show that genes are detected in the expected region-specific manner. Using an orthogonal in situ hybridization (ISH) approach, we validate the RNA DSP results in tonsil across multiple targets. To determine the regionally specific RNA expression in tumor area and nontumor area in non-small cell lung cancer (NCSLC), we used a high-plex probe mix of relevant Immuno-oncology RNA targets. These results show that the DSP platform can be used to obtain high-plex, spatial RNA expression data on tissue sections. The protein profiling assay on DSP, presented elsewhere, depends upon FFPE-compatible antibodies, which can be difficult to generate for some targets. Utilizing spatial mRNA profiling provides an alternative for cases where the necessary antibodies do not exist. In all, simultaneous, high-plex RNA and protein detection with DSP on serial sections could provide a novel method that can bridge the gap between translational research discovery and clinical applications.

#3435

Differential genomics and transcriptomics between tyrosine kinase inhibitor sensitive versus resistant BCR-ABL dependent chronic myeloid leukemia.

Neetu Singh,1 Anil Kumar Tripathi,1 Dinesh Kumar Sahu,1 Archana Mishra,1 Margaret Linan,2 Bianca Argente,3 Julia Varkey,3 Rebecca Chowdhry,1 Avinash Agarwal,1 Chris Yoo,3 Ravi Kant,4 Madan Lal Bhatt1. 1 _King George's Medical University, Lucknow, India;_ 2 _Systems Imagination, Scottsdale, Arizona, USA, Scottsdale, AZ;_ 3 _Systems Imagination,, Scottsdale, AZ;_ 4 _All India Institute of Medical Sciences- Rishikesh, Rishikesh, India_.

Previously it has been stated that BCR-ABL1 fusion protein is sufficient to induce CML, but additional genomic changes are required for disease progression. Based on this notion, we profiled control and Tyrosine Kinase Inhibitors (TKI)-treated-CML-samples in different-phases supported by copies of BCR-ABL-transcripts. Molecular-profiling was done through Molecular Inversion Probe (MIP) based array, Human Transcriptomics Array2.0, and Axiom Biobank genotyping-arrays. Transcriptionally, clusters of control, TKI-resistant and -sensitive cases were identified which showed correlation with BCR-ABL transcript-levels with the involvement of 228 pathways. On a comparison between TKI-sensitive versus resistant cases, at the exonic level Cassette Exon splicing event was observed in LAPTM4B (0.46), PIEZO2 (0.32), ANGPT1 (0.29), CFH (0.28), HLTF (0.28), SPTLC3 (0.26); Alternative 3' Acceptor Site splicing (0.22); and Junctional splicing in CD109 and ZNF711. These splicing events led to up-regulation of these transcripts. On further processing through Reactome Pathway, we identified over-expression of Hemostasis pathway in CML-resistant cases with specific involvement of Tie2 and Basigin signalling pathway. Based on CNV profiling, we identified low CNVs in CP-new and CP-UT-TKIs-sensitive cases with undetectable or <10% BCR-ABL copies. While, high CNVs (previously reported gain of 9q34) was observed in BCR-ABL-independent and -dependent TKIs-non-sensitive-CP-UT/AP-UT/B-UT and B-new samples. Further, genotyping CML-CP-UT cases with BCR-ABL 10 to 77.02% copies, we identified and validated rsIDs, rsID239798 and rsID9475077 in linkage with FAM83B which may act as potential therapeutic resistance candidate. BCR-ABL1, additional genetic events, over-expression of Tie-2 and Basigin signalling pathway and rsIDs in linkage with FAM83B in TKI resistant cases can be used for developing a signature profile which may help in monitoring therapy.

#3436

Early mutated genes and subsequent mutational trajectory in breast cancer subtypes.

Yee Him Cheung,1 Jie Wu,1 Vinay Varadan,2 Nevenka Dimitrova3. 1 _Philips Research North America, Cambridge, MA;_ 2 _Case Western Reserve University, Cleveland, OH;_ 3 _Philips Healthcare, Valhalla, NY_.

Current analyses on somatic mutations mainly involve counting samples as either carriers or non-carriers. Such a binary approach can identify the frequently mutated genes, but may miss the early mutated genes that are critically involved in tumorigenesis, but only in a small specific group of patients. This work is an attempt to identify such early mutated genes that drive the etiology of each breast cancer subtype by analyzing the variant allele frequency (VAF) of somatic mutations, which is a good proxy for mutational timeline. We further studied their pathway associations and subsequent trajectory of mutational progression. In this study, we analyzed the 72,084 non-synonymous somatic mutations in 16,164 genes of 817 TCGA breast cancer samples (HER2+: 65, Luminal A: 415, Luminal B: 176, Basal: 136 and Normal-like: 25) using VAF adjusted by sample purity derived from multiple methods. Gene allele frequency (GAF) is obtained by choosing the maximum VAF per gene per sample. For each subtype, we shortlisted 100 genes with highest number of mutation carriers and ranked them by decreasing average GAF across the carriers. We assume genes with higher average GAF are more likely to harbor clonal mutations that occur during early tumor progression. We then performed Inflo Network Analysis on RNA-Seq expression data to identify the dysregulated pathway interactions associated with the early mutated genes. The top two genes with the highest presence of mutations within each subtype are as follows: (a) HER2+: ERBB2 (VAF = 0.39 ± 0.21, n = 4), KAT6A (0.31 ± 0.09, 4), (b) Luminal A: CTCF (0.41 ± 0.19, 13), MAP2K4 (0.37 ± 0.14, 24), (c) Luminal B: MAP2K4 (0.46 ± 0.11, 5), TP53 (0.46 ± 0.17, 64), and (d) Basal: SCN10A (0.46 ± 0.29, 5), MYH9 (0.43 ± 0.16, 5). Among them, the genes with the strongest support are MAP2K4 (n = 24) for Luminal A and TP53 (n = 64) for Luminal B. Network analysis with Inflo on the same samples reveals that MAP2K4 mutation carriers are most strongly associated with the down regulations of "Regulation of Androgen receptor activity (target: JUN)" and "regulation of p38-alpha and p38-beta (target: DUSP8)" in Luminal A, with a difference in average Inflo score of -2.24 and -2.22 respectively; whereas TP53 mutation carriers are most strongly associated with the down regulations of "direct p53 effectors (target: RPS27L)" and "RB tumor suppressor/ checkpoint signaling in response to dna damage (target: CDK2)" in Luminal B, with a difference of -2.98 and -2.67 respectively. We found further that both PIK3CA and TTN were frequently co-mutated with TP53 in all subtypes. However, TTN mutations in general have lower VAF, indicating their late emergence in tumor progression. Our work shows the potential of VAF analysis for identifying driver genes, understanding tumor progression and evaluating the impact of a mutation on a patient. As future work, we may improve the VAF estimates by adjusting for CNVs and weight each variant by pathogenicity.

#3437

Genomic alterations, mutation burden, and microsatellite instability status of Chinese intrahepatic cholangiocarcinoma.

Jingyu Cao,1 Bo Jiang,2 Zimin Liu,1 Weibin Shu,3 Lei Li,4 Han Yang,5 Hua Li,6 Zhiyu Xiao,7 Maolin Yan,8 Jie Lin,9 Siqin Liu,10 Peng Zhang,10 Milind Javle,11 Ming Yao,10 Kai Wang,10 Haitao Zhao12. 1 _The Affiliated Hospital of Qingdao University, Qingdao, China;_ 2 _People's Hospital of Hunan Province, Changsha, China;_ 3 _Shandong Provincial Hospital Affiliated to Shandong University, Ji'nan, China;_ 4 _Shandong Cancer Hospital Affiliated to Shandong University, Shandong Academy of Medical Sciences, Ji'nan, China;_ 5 _First Affiliated Hospital of Guangxi Medical University, Nanning, China;_ 6 _The Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China;_ 7 _Sun Yat-sen Memorial Hospital,Sun Yat-sen University, Guangzhou, China;_ 8 _Fujian provical hospital, Fuzhou, China;_ 9 _The Second Affiliated Hospital of Kunming Medical University, Kunming, China;_ 10 _Origimed Inc, Shanghai, China;_ 11 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 12 _Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing, China_.

Background: Intrahepatic cholangiocarcinoma (ICC) is often diagnosed at an advanced stage with limited therapeutic options and poor prognosis. Targeted and immune-therapy have yielded promising therapeutic effects in various solid tumor patients (pts). Thus the understanding of ICC genomic profiling, especially which of Chinese pts, seems to be necessary for the treatment strategy for ICC.

Methods: Comprehensive genomic profiling of molecular alterations and predictive biomarkers to personalized treatment genes were performed on Formalin Fixed Paraffin Embedded (FFPE) samples and matched blood samples of 69 Chinese ICC pts. Genomic alterations (GAs) including substitutions, short and long insertion/deletions (indels), copy number variations and gene rearrangements were assessed. Tumor mutation burden (TMB) of total somatic substitutions and indels per megabase after filtering known driver mutations was calculated. Microsatellite instability (MSI) status was determined by identifying and scoring multiple mono-nucleotide repeats loci.

Results: Median age of the 69 ICC pts was 58 years (range: 38-88), and male vs female was approximately 3:1. Average of 3.7 GAs /sample and 0.9 actionable GAs/sample were detected. The most widespread genomic alterations among the pts were revealed as TP53 (44.9%), BRCA2 (26.1%), ARIDIA (24.6%), TSC2 (18.8%), KRAS (18.8%), FGFR2 (15.9%), LRP1B (14.5%), CFTR (15.9%), BAP1 (15.9%), ATM (15.9%), FAT3 (14.5%), FANCA (14.5%). The actionable mutations of the PI3K/mTOR pathway were found in 18.8% of the pts. In total, pts with one or more actionable GAs were 62.3%. Pts presented MSI-high were 7.2% of the total. Proportion was 10.1% for the pts with TMB value higher than 20 mut/Mb, and 32.8% for those higher than 10 mut/Mb. The TMB range of the 69 pts was 1.1 to 58.4 mut/Mb, and median was 7.4 mut/Mb.

Conclusions: In our study, 62.3% of Chinese ICC pts were detected to have actionable GAs which could potentially guide and influence their personalized treatments. The identification of more frequent high TMB level than the published western populations indicated that Chinese ICC pts may more likely derive therapeutic benefits from immune checkpoint inhibitors.

#3438

Genetic analysis of melanoma from an albino patient.

Candelaria Bracalente,1 Piyushkumar Mundra,1 Adriana Rinflerch,2 Pablo Garcia Martinez,1 Victoria Volonteri,2 Lucas Trucco,1 Gaston Galimberti,2 Nathalie Dhomen,1 Valeria Pavet Rodriguez,1 Richard Marais1. 1 _Cancer Research UK, Manchester Insitute, Manchester, United Kingdom;_ 2 _Hospital Italiano de Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina_.

Melanoma, the deadliest type of skin cancer, derives from the transformation of melanocytes, which protect the skin from ultraviolet radiation (UVR) through the synthesis of melanin. UVR is the major risk factor for melanoma. Intriguingly, in people with oculocutaneous albinism (OCA), who lack or have a reduction in melanin synthesis melanoma is surprisingly rare, whereas other UVR-driven skin cancers are comparatively high. Since UVR exposure and light skin complexion increase melanoma risk, it is unclear why melanoma is rare in OCA. Therefore, we evaluated how the absence of melanin impact the mutation burden in OCA and identify driver mutations by whole exome sequencing (WES) analysis of melanomas from a patient with OCA.

The patient (in their 50s) presented cutaneous basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) in addition to melanoma. The histopathology of one tumor (sample A), located on the left forearm, indicated a desmoplastic nodular melanoma, Clark level III, Breslow of 1 mm and a high mitotic index. The second tumor (sample B) was located on the right knee and presented a superficial spreading melanoma, Clark level II, a Breslow of 0.31 mm and a low mitotic index. For WES, genomic DNA was purified from formalin-fixed paraffin-embedded tissue sections and compared against germline blood. Analysis confirmed a G47D mutation in the TYR gene, which has previously been reported in OCA1B patients. Consistent with sample A coming from an area habitually exposed to UVR, it presented 4874 SNVs and 1556 missense SNVs and a predominant signature 7 mutational process. Similarly, consistent with sample B coming from an area that is habitually protected, it presented only 45 SNVs and 16 missense SNVs and did not present a predominant signature 7 mutational process. Notably, despite the histopathology of the skin adjacent to sample B appeared normal, it presented 39 SNVs and 10 missense SNVs. Intriguingly, all samples harbor NRASQ61K mutations and sample B additionally presented a mutation in NOTCH1, which is more common in SCC. Thus, we show that in OCA, melanomas from a UVR exposed areas present high mutation burden and UV mutational signature, whereas melanomas from UVR protected skin present a low mutation burden. Our data suggest that NRASQ61K is a driver oncogene in melanomas from albinos and that these patients can develop both UVR-driven and UVR-independent melanomas.

#3439

GATA3 mutation of luminal-type breast cancer patients in Taiwan.

Shih Feng Tsai. _National Health Research Institutes, Zhunan, Miaoli, Taiwan_.

To investigate the mutation landscape of luminal-type breast cancer in Taiwan, we have conducted whole-genome sequencing on 20 cases of breast cancer, including 15 cases of those positive for estrogen receptor and 5 cases negative for estrogen receptor. Specifically, we searched for mutations in TP53, GATA3, and ESR1 genes. Additionally, we designed an amplificon sequencing scheme to interrogate the GATA3 coding sequences. GATA3 mutation has been previously reported to be correlated with suppression of proliferation upon aromatase inhibitor treatment. Also, the prevalence and clinical significance of GATA3 mutation-positive breast cancer differed between the TCGA cohort and the Fudan cohort. In our current series that focuses on breast cancer patients of Han Chinese living in Taiwan, we have identified 21 GATA3 mutations among 72 breast cancer tumor specimens screened by the amplicon sequence, and another 3 samples with GATA3 mutation by whole-genome sequencing. Notably, Pro409Alafs mutation occurred in six cases and this truncation mutation showed an average VAF of 0.155. A specific antibody was raised against the additional c-terminal amino acid sequence for the detection of the abnormally expressed proteins in these cases. As GATA3 is required for epithelial cell differentiation and as both normal breast luminal epithelium and estrogen-receptor-positive tumors showed high GATA3 expression, we are conducting further studies to demonstrate the clinical relevance of GATA3 mutation and the potential value of the Pro409Alafs as an outcome predictor for luminal-type breast cancer.

#3440

Genomic evolution of synchronous and metachronous breast cancer metastasis revealed from a prospective clinical trial.

Keltouma Driouch, Zakia Tariq, Sylvain Baulande, Virginie Raynal, Virginie Bernard, Francois-Clement Bidard, Vanessa Benhamo, Ivan Bieche, Brigitte Sigal, Rosette Lidereau, Paul Cottu. _Institut Curie, Paris, France_.

Background: Tumor progression is an evolutionary process associated with the accumulation of somatic genomic alterations. Even though metastasis is a fundamental process and a challenging issue for the progress in targeted treatment, the molecular drivers of cancer metastasis remains poorly understood. Despite the advance in high throughput sequencing and the characterization of hundreds of breast tumor genomes, it is still unclear whether cancer genomes evolve by neutral processes or whether genetic alterations that favorable tumor progression are selected under pressures of cancer therapy or both.

To gain insights into the molecular processes driving breast cancer metastasis and to identify targetable genetic events associated with early and late stages of the disease, we studied the mutational profiles of a series of paired primary breast tumors and subsequent metastases collected as part of the ESOPE clinical Trial.

Methods: Matched primary breast tumors, first metastases and germline DNAs were obtained from 31 patients, divided in 10 therapy-naïve synchronous primary cancers and metastases and 21 metachronous primary tumors and metastases that undergone first line adjuvant treatments. On Illumina platform, WES was performed and three variant callers were used in parallel to predicted variations. After annotation, variants with low 1000Genome frequency and absent in germline samples, were validated by IGV visualization and then considered as somatic variants. To identify significantly mutated genes, we used the Mutation Significance (MutSigCV) tool and the Cancer Gene Census list of relevant genes. Analysis of mutational process signatures was also performed though DeconstructSigs R packages.

Results: Synchronous and metachronous metastases globally exhibited similar mutation rates, however a significant difference was observed in the number of accumulated private mutations in both types of metastases. With few exceptions, our data indicate that the genomic profiles of metastases mainly diverge late in breast cancer progression, with relatively few private mutations in the primary tumors. In contrast, an increase of somatic variants was found in the metachronous metastases as compared to their primary tumors of origin. Most of driver mutations were shared by primary tumors and metastases but additional drivers were highlighted in the metastatic lesions, such as variants of PIK3CA, IGF1R, CDH1, NF1 as well as ESR1. Moreover, recurrent mutated genes were also identified in metastases that have not been previously described (FDR<0.1). Analyses of mutational signatures revealed a shift towards APOBEC, POLE, MMR and HR activity. In conclusion, synchronous and metachronous breast cancers differ in their genomic evolution and the characterization of driver mutations in metastases might open new avenues for novel therapeutic strategies in metastatic breast cancer.

#3441

Genome-wide gene expression analysis in asbestos-related non-small cell lung cancer.

Eeva Kettunen,1 Veer Singh Marwah,2 Henrik Wolff,1 Dario Greco,2 Kirsti Husgafvel-Pursiainen1. 1 _Finnish Inst. of Occupational Health, Helsinki, Finland;_ 2 _Universities of Tampere and Helsinki, Tampere and Helsinki, Finland_.

Recently we examined genome-wide DNA methylation in lung cancer from patients occupationally exposed to asbestos (Kettunen et al 2017 Int J Cancer). Distinct methylation changes in lung tumors were shown in relation to asbestos exposure and tobacco smoking. Now we have performed genome-wide gene expression microarray experiments with Agilent SurePrint G3 Human GE 8x60K v3 Microarrays using same samples. Patients had been interviewed for work and smoking histories and their pulmonary asbestos fiber counts were measured by electron microscopy. Fresh-frozen lung cancer tissues (LC, n=42) and paired microscopically normal peripheral lung tissues (NT, n=21) were studied. After data processing and analyses for differential gene expression, data were filtered for adjusted p < 0.05 and fold change > 1.5. Principal component analysis showed distinct clusters of normal lung and cancer tissues. In lung cancers compared with their paired microscopically normal lung, 5931 genes such as those involved in neutrophil degranulation and immune system were differentially expressed, with the lowest adjusted p=6.71E-37. In our preliminary analyses, 34 genes in NT and 44 in LC, and 152 genes in NT and 12 in LC showed differential expression when the asbestos-exposed were compared to non-exposed and current smokers to ex-smokers, respectively. The genes differentially expressed in relation to smoking or to asbestos exposure had the lowest adjusted p=8.50E-06 and 9.83E-04, respectively. Within differential gene expression related to asbestos exposure, chemokine activity and regulation of cellular protein metabolic processes were implicated whereas microtubules and positive regulation of transcription were related with tobacco smoking burden. In conclusion, comparison of gene expression patterns between lung cancer tissue and paired normal lung showed distinct principal component analysis clusters. Our preliminary data suggest different major biological processes in gene expression for these exposures. Financial support from the Cancer Society of Finland (2014 and 2015) is gratefully acknowledged.

#3442

Genome-wide association study reveals potential biomarkers after pulmonary metastasectomy for head and neck cancer.

Hsueh-Ju Lu,1 Chih-Cheng Hsieh,2 Chi-Chun Yeh,3 Yi-Chen Yeh,2 Muh-Hwa Yang,4 Chi-Ying F. Huang,5 Peter Mu-Hsin Chang2. 1 _Chung Shan Medical University Hospital, Taichung, Taiwan;_ 2 _Taipei Veterans General Hospital, Taipei, Taiwan;_ 3 _Jin An Clinic, New Taipei City, Taiwan;_ 4 _Institute of Clinical Medicine, National Yang Ming University, Taipei, Taiwan;_ 5 _Institute of Biopharmaceutical Sciences, National Yang Ming University, Taipei, Taiwan_.

Introduction: At least 10% of head neck cancer squamous cell carcinoma (HNSCC) progress to pulmonary metastases with overall survival (OS) around 12 months. Whether aggressively pulmonary metastasectomy (PM) improving survival or not is controversial.

Method and materials: Different head neck cancer patients receiving PM from thoracic surgeon were enrolled from 2003 to 2012. Matched HNSCC and adenoid cystic carcinoma (ACC) samples as control were selected to perform whole-exon sequencing (WES) and identify potential biomarkers between short- and long- term surviving groups after metastasectomy. Identified mutant genes were mapped into possible signaling pathways by using DAVID ( https://david.ncifcrf.gov/).

Result: A total of 31 head and neck cancer patients were enrolled and the survivals were different significantly among cancer types. Eight matched PM samples (3 short-term post-metastaectomy survival (PMS) HNSCC, 3 long-term HNSCC, and 2 ACC), with differently clinical outcomes (PMS, P = 0.016; OS, P = 0.015), were enrolled for WES. Eighteen percentages (18%, 306/1706) of identified genetic alternations were mapped into DAVID. Cancer-associated signal pathways were top-ranked related to short-term HNSCC; hematopoietic cell lineage associated pathways for long-term HNSCC; complement and coagulation cascades associated pathways for ACC. The overlapped genes between short-term HNSCC and ACC were relative to metabolic-associated pathway, such as branched-chain amino acid consumption (P < 0.001, fold enrichment (FE) = 7.97) and the metabolism of arginine and proline metabolism (P < 0.001, FE = 4.96). The overlapped genes between long-term HNSCC and ACC were relative to focal adhesion (P =0.008, FE = 2.84) and ECM-receptor interaction (P = 0.015, FE = 4.08).

Conclusion: Genome-wide association study effectively separated clinical outcome of head neck cancer patients and successfully identified specific mutations for prognosis after PM.

Basic characteristics of patients receiving whole-exon sequencing for pulmonary metastatic tumor.

---

|

Group | Age | Gender | Primary tumor site | Histological type | Initial pathologic staging | Brief history | Overall survival | Post-metastasectomy survival

Case 1 | Short-term survival | 86.0 | Male | Hypopharynx | Squamous cell carcinoma | pT4aN2b | Surgical resection followed by adjuvant radiotherapy; one metastatic nodule at right lower lung was resected. | 23.9 | 14.4

Case 2 | Short-term survival | 60.9 | Male | Oropharynx | Squamous cell carcinoma | pT4aN1 | Surgical resection followed by adjuvant chemoradiotherapy; two metastatic nodules at right and left upper lung were resected. | 26.0 | 11.9

Case 3 | Short-term survival | 47.1 | Male | Oropharynx | Squamous cell carcinoma | pT4aN2b | Induction chemotherapy followed by definitive surgical resection and adjuvant chemoradiotherapy; one left upper lung metastasis was resected. | 22.8 | 7.1

Case 4 | Long-term survival | 38.9 | Male | Hypopharynx | Squamous cell carcinoma | pT2N2b | Surgical resection followed by adjuvant radiotherapy; multiple bilateral lung metastases with sequential wedge resection for left and right side lesions | 52.1 | 40.2

Case 5 | Long-term survival | 51.3 | Male | Hypopharynx | Squamous cell carcinoma | pT3N2b | Surgical resection followed by adjuvant chemoradiotherapy; 3 metastatic lesion at right upper, middle and left lower lung were resected. | 89.8 | 42.7

Case 6 | Long-term survival | 54.4 | Male | Hypopharynx | Squamous cell carcinoma | pT4aN2b | Surgical resection followed by adjuvant chemoradiotherapy; 2 metastatic lesions at bilateral lung apices with sequential wedge resection | 134.8 | 91.1

Case 7 | Control | 39.6 | Female | Oral cavity | Adenoid cystic carcinoma | pT4aNo | Surgical resection followed by adjuvant chemoradiotherapy; 2 metastatic nodules at right upper and middle lung were resected | 130.1 | 87.0

Case 8 | Control | 46.2 | Female | Right maxillary sinus and nasal cavity | Adenoid cystic carcinoma | Unknown | Surgical resection followed by adjuvant radiotherapy; 2 metastatic nodules at right upper and middle lung were resected | 195.0 | 79.9

#3443

Full-length characterization of transcript isoforms to investigate cancer-associated mutations.

Alison Tang. _UC Santa Cruz, Santa Cruz, CA_.

Mutations in the splicing factor SF3B1 in various cancers have been associated with characteristic alterations in splicing. SF3B1 is one of the most frequently mutated genes in chronic lymphocytic leukemia and is associated with poor patient prognosis. While alternative splicing patterns caused by mutations in SF3B1 on a junction level have been observed, these patterns have not been systematically examined on an isoform level. Isoform-level analyses are imperative for building a better understanding of altered splicing in a cell; exon connectivity is best resolved using long reads. As such, we have resequenced cDNA from CLL samples with and without K700E mutation in SF3B1, as well as a normal B cell, with nanopore sequencing technology. By converting changes in current caused by blockage of DNA threading through a nanopore into sequence, nanopore sequencing can sequence complete molecules of DNA that exceed 200kb in length. We have developed a novel workflow to perform isoform-level differential splicing analyses leveraging the full-length transcript data that nanopore affords. We report results from nanopore sequencing data that are concordant with known SF3B1 biology from short read sequencing as well as altered splicing patterns that are only observed definitively using long reads. Splicing analysis of nanopore reads between the SF3B1WT and SF3B1K700E identifies a bias toward increased alternative 3' splice sites than alternative 5'. In addition, we observe a relative decrease in the expression of genes with intron retention events in CLL samples compared to B cell. We also discover a previously unreported finding of enrichment in intron retention events in SF3B1WT relative to SF3B1K700E and no enrichment between CLL SF3B1MT and B cell, suggesting that mutated SF3B1 in CLL modifies the intron retention landscape to be more similar to a normal phenotype. Gene ontology analyses reveal enrichment in nuclear export in genes associated with intron retention events, and we postulate that many of these are detained introns of transcripts that are kept in the nucleus. With full-length cDNA sequence, we are also able to better detect premature termination codons and estimate the proportion of transcripts subject to nonsense-mediated mRNA decay. As nanopore sequencing has yet to become a routine tool for characterization of the transcriptome, our work demonstrates the utility of nanopore sequencing for cancer and splicing research.

### Kinases 2

#3444

EGFR-dependent matriptase activation in ErbB2-amplified breast cancer.

Darius Gaymon. _Georgetown University, Washington, CT_.

Of the nearly 250,000 new breast cancer cases expected annually, nearly 25% will have ErbB2 (HER2) gene amplification, which is associated with increased growth signaling, more aggressive disease, and poor patient prognosis. Matriptase is a type II transmembrane serine protease that plays a role in normal epithelial cell biology. The expression and activation of matriptase are tightly regulated in normal cells partly through co-expression with endogenous inhibitor, HAI-1, which rapidly inhibits the protease, once activated, by the formation of a high affinity complex. Matriptase has oncogenic potential when its regulation is deranged and it activates downstream substrates known to play a role in tumor progression such as pro-HGF. ErbB2 activity can regulate matriptase activity in prostate cancer and so we set out to elucidate the relationship between ErbB2 and matriptase in ErbB2-amplified breast cancer. SKBR3, AU565, and ErbB2 transfected MCF7 cells (MB7) were used to explore the relationship between ErbB2 levels and matriptase activation as they express higher levels of ErbB2 compared with MCF7 cells. Under standard growth conditions, high level of ErbB2 activation was associated with extensive matriptase activation. Activation of ErbB2 by stimulating EGFR with EGF resulted in increased matriptase activation. Inhibition of ErbB2 activity by serum deprivation or Lapatinib (a dual ErbB2 and EGFR kinase inhibitor) treatment resulted in decreased matriptase activation. Treatment with Lapatinib or the EGFR-specific inhibitor, Gefitinib, blocked matriptase activation whereas the ErbB2 inhibitor, AG825, has no effect. Using inhibitors of downstream signaling pathways, EGFR-induced matriptase activation was shown to be mediated by PI3K signaling. Further elucidation of the pathway is ongoing. In summary, our data suggest that EGFR signaling in this context modulates matriptase activation, which may also contribute to breast cancer progression.

#3445

Functional interaction between SEPT9_v1 and EGFR in triple-negative and inflammatory breast cancer.

Amanda Agosto, Jose G. Cirino, Kimberleve Rolón, Gabriel E. Figueroa, Gabriela Martinez, Katixa Montañez-Negrón, Keishla Rodríguez-Mártir, Xavier Bittman, Esther A. Peterson. _University of Puerto Rico, San Juan, PR_.

The poor prognosis for patients with inflammatory breast cancer (IBC) emphasizes the need to better understand the molecular signature of this disease with the goal of developing effective targeted therapeutics. Importantly, 20-40% of IBC cases are triple-negative breast cancers (TNBC) in which targeted hormone therapy is not effective. Interestingly, approximately 1/2 of cases of IBC overexpress the epidermal growth factor receptor (EGFR), a member of the ErbB family of receptors involve in cell proliferation, migration, and cytoskeleton remodeling. Recently, SEPT9, a member of a large family of cytoskeletal proteins important in diverse biological processes, was found to interact with EGFR. Dysregulation of SEPT9 isoforms have been associated in different cancer types, including breast cancer. To date, no studies have looked at the potential pro-oncogenic role of SEPT9 isoforms in IBC or TNBC. We will examine the functional relationship between SEPT9_v1, cytoskeleton remodeling and EGFR signaling in the IBC cell lines SUM149, SUM190 and TNBCs. Our hypothesis is that SEPT9_v1 contributes to oncogenesis by regulating the activity of ErbB receptors in IBC and/or TNBC. To test our hypothesis, generation of stable cell lines with knockdown or overexpression of SEPT9_v1 using lentiviral and delivering vectors will be developed. Establishment of 2D and 3D cultures of the cell lines will be performed to analyze if the acquisition of pro-oncogenic phenotypes and the effects on cytoskeleton dynamics are SEPT9_v1 and/or EGFR dependent. To assess these phenotypes, we will use cell proliferation assays using alamar blue to determine the viability of cells over time, in vitro transwell migration, invasion assays and fluorescent microscopy to determine if there is aberrant morphology or cytoskeleton remodeling. We will determine the specific effect of SEPT9_v1 overexpression and knockdown to the activation of the EGFR signaling. Stability of ErbB receptors upon SEPT9_v1 overexpression or knockdown will be also measured by protein stability assays and by looking directly in the plasma membrane of cells with fluorescent microscopy. Downstream activation of the EGFR pathway will be assessed by Western blot analysis of activated effector proteins. Co-immunoprecipitation analysis will be performed to test if there is a specific interaction between EGFR and SEPT9_v1. Our preliminary data shows that approximately 50% of breast cancer cell lines overexpress SEPT9_v1 mRNA when compared to a panel of human mammary epithelial cell lines. Also, by WB analysis we determined that SUM149 expresses two isoforms, SEPT9_v1 and SEPT9_v3. This work will provide new insights into the functional relationship between SEPT9_v1 and EGFR and their contribution to the aggressive metastatic phenotype in IBC. Furthermore, it will help us to identify new potential therapeutic targets for the better management of this disease.

#3446

Deciphering key cancer and inflammation signaling pathways with homogeneous bioluminescent cell-based kinase activity assays.

Hicham Zegzouti, Brian Hwang, Nidhi Nath, Said Goueli. _Promega Corporation, Madison, WI_.

Signaling pathways activation leads to a multitude of cellular responses including modulation of enzyme activity, altered gene expression, and protein translocation or degradation. Specific phosphorylation events by specific kinases in the cell constitute important nodes in signaling pathways. Altered normal kinase activity leads to several undesirable consequences, causing diseases such as cancer and inflammation. Thus, monitoring these signaling events is essential to better understand normal cell behavior and disease states. Current assays to analyze cellular kinase activities can be tedious, nonhomogeneous, and not easily adaptable to HTS or suffer from generation of false hits due to fluorescence interference, signal quenching, or require special instruments for detection. To overcome these shortcomings, we developed a simple homogeneous assay platform to detect phosphorylation of specific endogenous proteins in cells. We took advantage of the NanoLuc Binary Technology (NanoBiT), a two-subunit system based on NanoLuc luciferase that was successfully demonstrated for protein-protein interaction (PPI) detection. We generated a detection system containing secondary antibody pairs linked to the two Nanoluc components, NanoBiT small and large fragments (11 aa peptide and 18 kDa). Cell lysate containing the phosphorylated protein is incubated with two primary antibodies that recognize separate epitopes on a single protein. These will bring NanoBiT secondary antibodies into proximity to form an active NanoLuc luciferase that makes light in proportion to the amount of target protein. When the primary antibody pair includes a phosphospecific antibody, the luminescence reflects the level of target protein phosphorylation. We tested this system by monitoring the activation of signaling pathways involved in cancer, immune and inflammatory responses (NF-κB, BTK and JAK/STAT). In very few steps we detected the expected biologic response of these pathways either by monitoring BTK autophosphorylation, assessing phosphorylation and degradation of IκB or phosphorylation of STAT3 upon activation of the cells with TNFα, or IL6, respectively. We also tested different small- or large-molecule inhibitors of these pathways or their corresponding node kinase (BTK, IKK, or JAKs) and obtained the expected pharmacology. The bioluminescent system presented here has many advantages including "Add and Read" format, no cell engineering required as the phosphorylation of endogenous substrate is detected in any cell type, and only a simple luminometer is required for detection. Our results demonstrate that this technology can be adapted to any signaling pathway analysis, allowing scientists to analyze signaling pathways of interest, study the kinase cellular activity and regulation or identify specific kinase or pathway inhibitors.

#3447

IL-6 promotes tumor metastasis by promoting anoikis resistance and stemness phenotype in cancer cells via the activation of FAK and nanog stabilization.

Arti Yadav, Bhavna Kumar, Pawan Kumar. _The Ohio State University, Columbus, OH_.

The process of tumor metastasis is highly complex and involves dissemination of tumor cells from the primary tumor through the vascular system. However, most of the cancer cells, particularly epithelial cancer cells, have very low survival rates in circulation and undergo rapid anoikis. Tumor cells that acquire malignant potential have developed mechanisms to resist anoikis and thereby survive while travelling though the lymphatic or circulatory systems. Recently, we have shown that IL-6 promotes epithelial-mesenchymal transition (EMT) and tumor metastasis. A number of studies have shown that FAK signaling pathways play an important role in inducing anoikis resistance in tumor cells. In this study, we examined if IL-6 promoted tumor metastasis by enhancing stemness and anoikis resistance in cancer cells via the activation of FAK signaling. Our results demonstrate that IL-6 significantly enhanced stemness phenotype in cancer cells as measured by tumorsphere formation and ALDH positive cells by upregulating nanog protein levels. Nanog knockdown significantly reversed IL-6-mediated cancer stem cell (CSC) phenotype. IL-6 treatment of cancer cells induced FAK phosphorylation in a time dependent manner. Interestingly, FAK knockdown did not significantly decrease nanog mRNA levels but markedly decreased nanog protein levels. FAK depletion completely blocked IL-6-mediated nanog phosphorylation and enhanced nanog protein ubiquitination and degradation. These results suggest that FAK may be modulating nanog protein levels by stabilization nanog protein through phosphorylation. Furthermore, FAK knockdown significantly decreased IL-6-mediated tumorsphere formation, anoikis resistance and tumor metastasis. Taken together, our results show that IL-6 promotes tumor metastasis by enhancing stem cell phenotype and anoikis resistance in head and neck cancer cells via FAK activation and nanog protein stabilization.

#3448

**Targeting the dopamine receptor 2 in** BRAF **mutant colorectal cancer.**

Arman Javadi,1 Nicholas Forsythe,1 Alaa Refaat,1 Jessica-Ann Weir,1 Hajrah Khawaja,1 David Waugh,1 Rohinton Tarapore,2 Joshua E. Allen,2 Patrick Johnston,1 Sandra Van Schaeybroeck1. 1 _Queens University Belfast, Belfast, United Kingdom;_ 2 _Oncoceutics Inc, Philadelphia, PA_.

Background: BRAFV600E mutations occur in ~10% of colorectal cancer (CRC), are associated with poor survival and have limited responses to BRAF/MEK inhibition with or without EGFR inhibition. There is an unmet need to understand the biology of poor prognostic BRAFMT CRC. Using gene expression data from BRAFMT CRC patient samples, we recently identified that dopamine receptor degradation and unfolded protein response are dominant pathways deregulated in the BRAFMT subgroup with the poorest outcome. The aim of this study was to investigate the role of the dopamine receptor 2 (DRD2) pathway as novel target in BRAFMT CRC cells. Methods: Small molecule DRD2 antagonists ONC-201 and ONC-206 (Oncoceutics Inc) and a panel of isogenic paired and non-isogenic BRAFMT and BRAFWT cells were used. MTT, Flow Cytometry, Western blotting and caspase- 8, 3/7 activity assays were used to measure cell survival/death. DR5 cell localization was performed using flow cytometry. A compound library including small molecules approved by the FDA was used. Results: BRAFMT CRC cells were highly sensitive to the DRD2 antagonists ONC-201 and ONC-206 with IC50 values between 1.9-4.5μM and 0.16 and 0.24μM respectively. Treatment with ONC-201 and ONC-206 resulted in marked increases in expression levels of the endoplasmic reticulum stress proteins ATF4, CHOP and the active (spliced) form of XBP1 (sXBP1) (indicators of activation of the PERK and IRE1α UPR branches), and this was associated with apoptosis induction as indicated by PARP cleavage, caspase-9 cleavage and increased caspase-3/7 activity in the BRAFMT VACO432 cell line but not in the WT VT1 clone. Importantly, no significant effect on proliferation or apoptosis was obtained in the normal colon CCD-18 fibroblast cell line following treatment with ONC-201 or ONC-206. Using a small molecule compound library, we found that the taxanes paclitaxel and docetaxel resulted in strong synergy and apoptosis when combined with ONC-201 or ONC-206, in particular in BRAFMT CRC cells. Mechanistically, we found that the apoptosis induced by combined ONC-201/paclitaxel treatment was dependent on caspase-8 activation and on up-regulation of the death receptor 5 (DR5). Conclusions: Taken together, we have identified a role for DRD2 signalling in the survival of BRAFMT CRC cells. Our data support the development of DRD2 antagonists, in particular in combination with taxanes, for the treatment of BRAFMT CRC tumours.

#3449

AGL loss promotes anchorage independent growth of non-small cell lung cancer by activating FAK.

Sunny Guin, Craig Richmond. _Gundersen Medical Foundation, La Crosse, WI_.

Loss of glycogen debranching enzyme (AGL) has been shown to promote growth of bladder and non-small cell lung cancer. AGL loss predominantly assists anchorage independent growth of non-small cell lung cancer (NSCLC) cells. However the mechanism driving this anchorage independent growth with AGL loss is not well understood. To identify the driver pathways which contributions to increased anchorage independent growth with AGL loss, we carried out a phospho-kinase screen in A549 NSCLC cells with and without AGL expression. The kinase screen identified an increase in phospho-FAK (Tyr 576/577) levels with loss of AGL. The result of the screen was validated using separate independent Western blot experiments in A549 and other NSCLC cell lines which showed an increase in FAK activation at Tyr 576/577 with loss of AGL. We next treated NSCLC cells with and without AGL with FAK specific inhibitor (FAK inhibitor 14). Treatment with FAK inhibitor 14 had a greater cytotoxic effect (p<0.05) on NSCLC cells that have AGL loss. These data suggest that activation of FAK is a major driver of anchorage independent growth in NSCLC cells that have lost AGL expression and inhibition of FAK activity can serve as a therapeutic avenue for NSCLC patients with low tumor AGL expression.

#3450

Elucidating the role of mixed lineage kinase 3 (MLK3)-β-catenin axis in hepatocellular carcinoma.

Karan S. Saini, Taher Gheewala, Subhasis Das, Gautam Sondarva, Ajay Rana, Basabi Rana. _University of Illinois at Chicago, Chicago, IL_.

Hepatocellular carcinoma (HCC) is one of the leading cause of cancer-related deaths worldwide. Of the estimated 40,710 new cases of liver cancers diagnosed in 2017, about three-fourths were HCCs. Surgery, liver transplantation, tumor ablation and chemotherapy are available treatment options if detected at an early stage. However, the only therapeutic option currently available for advanced HCCs is Sorafenib which is only effective for a few months. There is thus an urgent need for the development of safe and more efficacious treatment options for HCC. MLK3 is a member of the mitogen-activated protein kinase (MAPK) family that can activate JNK pathway to induce cell death in neuronal and non-neuronal cells. Several studies have shown that MLK3 plays a significant role in cancer invasion, migration and metastasis. Earlier studies from our laboratory have demonstrated that MLK3 can induce the expression of β-catenin and regulate β-catenin downstream signaling. The current studies were designed to obtain further mechanistic insight on MLK3-β-catenin crosstalk and elucidate the detailed mechanism. To address this, our first goal was to map the domain(s) of β-catenin that mediate MLK3-induced stabilization and interaction by creating β-catenin deletion constructs with N terminal or C-terminal deletions. Overexpression studies designed with these revealed that AA 51-130 of β-catenin was important to mediate MLK3-induced stabilization. In addition, initial Immunoprecipitation and Immunoblotting combination studies also suggest this domain of β-catenin might be mediating the interaction with MLK3. Since our earlier studies suggested that MLK3-induced stabilization of β-catenin was dependent on its kinase activity, studies are currently underway to identify potential phosphorylation sites that might be mediating this stabilization of β-catenin. To obtain a broader picture on the potential effects of MLK3 in HCC disease progression, studies were performed with HCC cell lines following treatment with a pan MLK inhibitor CEP-1347 (CEP). These studies revealed a decrease in PCNA and c-myc expression along with increased PARP cleavage upon CEP treatment, in a time-dependent manner, indicative of a role in antagonizing cell proliferation and induction of cell death respectively. Cell cycle analysis revealed that CEP arrests HCC cells at G2/M phase of cell cycle. Flow cytometric studies also revealed a time-dependent increase in cell apoptosis and increased mitochondrial depolarization following treatment with CEP, associated with increased cleavage of Caspases 3, 8 and PARP. Further validation

following siRNA-mediated knockdown of MLK3 showed a potentiation of the apoptotic response following CEP treatment. Taken together these findings indicate that MLK3 plays a major role in HCC progression and CEP-induced targeting of this axis might be efficacious in the management of HCC.

#3451

Testing signaling algorithm in platinum-resistant ovarian carcinomas: A simultaneous inhibition of RAS-RAF and cell-cycle pathways signals by trametinib with paclitaxel or ribociclib/abemaciclib.

Nandini Dey, Casey Williams, Kirstin Williams, Jessica Klein, Jennifer H. Carlson, David Starks, Luis Rojas Espaillat, Pradip De, Brian Leyland Jones. _Avera Research Inst., Sioux Falls, SD_.

INTRODUCTION: Ovarian cancer (OC) frequently harbors genetic or epigenetic alterations that aberrantly activate CDK4/CDK6 pathway (Nam & Kim. Int J Gynecol Cancer 2008). Ribociclib (R) is being used in Phase I clinical trial in metastatic epithelial ovarian cancer (NCT03294694). CDK4/6 inhibitor, abemaciclib (A) is effective in KRAS mutant NSCLC. Platinum-taxol chemotherapy is the part of the standard of care for epithelial OC patients. However, acquired resistance to the platinum therapy can poorly affect prognosis (Jayson et al., Lancet, 2014; Sonego et al., Sci. REP. 2017). Additionally, the recurrent resistant EOCs almost invariably grow as metastatic disease (Jayson et al., Lancet, 2014).

AIM: We interrogated the genomic alterations of the RAS-RAF (RR) and the cell-cycle (CC) pathways to generate a signaling algorithm in precision medicine for testing rational combinations of RR pathway inhibitor, Trametinib (T) with Paclitaxel (P) or CDK4/6 inhibitor (R/A) in platinum-resistant OC.

METHODS: We analyzed genomic alterations (FoundationOne) of 90 tumor samples from 75 consecutive OC patients (Avera Cancer Institute; '14-'17). The genomic changes in FoundationOne results were resolved into RR and CC pathways. Parental and cis-platin resistant RAF mutated A2780 iso-genic cell lines were used to study time-dependent effect of T (MEK1/2 inhibitor) and A/R (CDK4/6 inhibitor), single or in combination with P on (1) proliferative signals (WB), (2) cell cycle (Flowcytometry), (3) live-dead staining, (4) apoptosis (Flowcytometry; TMRE-based Mitochondrial potential; Triple IF by MitoView Blue+NucView488 Casp3 substrate + CF 594 AnnexinV) and (5) 3D clonogenic growth.

RESULTS: 100 genes were altered in a total of 87% metastatic and 11% adjuvant tumors with a predominant histological type of high grade serous (58%). 7 genes including NF1> KRAS>MAP3K1>NF2>BRAF>MAP2K4>NRAS of the RR pathway were altered in 33% of tumors. 10 CC pathway genes including CCNE1, CDKN2A/B, in addition to TP53 and MYC were altered. We observed that 20-24% tumors had co-alterations of the two pathways and importantly, all tumors with co-alterations were metastatic. Although treatment of R and P as single agent significantly increased pRSK in resistant cell line at 6 hours, a combination of T + P or T + A/R was effective in inhibiting proliferative signals and inducing apoptosis abrogating downstream pERK and pRSK. A combination of T+P was most effective in inducing cl-casp3 at 24 hours. 3D ON-TOP assay for 10 days demonstrated a significant blockade of clonogenic growth with T+P and T+R in resistant cells.

CONCLUSION: Plotting an in-depth information about co-alterations of genes of the RR and the CC pathway on the landscape of cell signaling is a useful tool to crack the code of cis-platin-resistance for improved treatment options.

#3452

TAK228, a kinase inhibitor of mTORC1 and mTORC2, is highly effective in ER+ breast cancer model.

Pradip De, Jennifer H. Carlson, Tyler Jepperson, Casey Williams, Nandini Dey, Brian Leyland Jones. _Avera Research Inst., Sioux Falls, SD_.

Background: mTOR is an atypical Ser/Thr kinase involved in regulating major cellular functions, such as nutrients sensing, growth, proliferation and angiogenesis. Current FDA approved clinical mTOR inhibitors only inhibit mTORC1 and have several limitations. First, inhibiting mTORC1 releases the negative feedback of PI3K-AKT signaling and therefore activates upstream AKT signaling. Secondly, allosteric inhibitors of rapalogs block the phosphorylation of some but not all mTORC1 downstream substrates. In particular phosphorylation of 4EBP1 is largely insensitive to rapalogs. In order to address these issues, "second generation" ATP competitive catalytic inhibitors including TAK228 against mTORC1/C2 have also been developed and are now in active in various stages of clinical trials.

Purpose: Resistance of breast cancers (BC) to targeted hormone receptor inhibitors often occurs through deregulation of the PI3K-AKT-mTOR pathway. Mutations or loss of function of upstream regulators such as TSC1/2, LKB1, or components of the PI3K pathway such as PIK3CA, AKT, or PTEN have been reported in most types of human tumors including BC. Here, we investigated the effects of TAK228 alone or in conjunction with letrozole in ER+ BC cell lines.

Methodology: Anti-proliferative, apoptotic, cell cycle, and intracellular signaling effects of TAK228 alone and in combination with letrozole were evaluated in ER +/PIK3CA mutated and ER+/PTEN mutated BC cell lines.

Results: 1) TAK228 caused a strong differential growth inhibition in ER+/PIK3CA or ER+/PTEN mutated or ER+ (WT of PI3K pathway molecules) BC cell lines by 3D-ON-TOP clonogenic assay and real-time monitoring in an IncuCyte Zoom. Inhibition is greater when TAK228 was combined with letrozole. 2) Administration of TAK228 induced cell cycle G0/G1 arrest and resulted in increased apoptosis in a dose-dependent manner. 3) qRT-PCR data showed that pro-apoptotic transcript, PUMA and cell survival transcripts SURVIVIN & STATHMIN mRNA expressions were significantly increased and decreased respectively following the treatment of TAK228. 4) CYCLIN D1 mRNA expression was significantly decreased in both 24 & 48 hrs following the treatment of TAK228. 5) TAK228 is mechanistically distinct from everolimus by a more potent and comprehensive inhibition of the PI3K-AKT-mTOR signaling network: a) avoided S6K inhibition-mediated feedback activation of PI3K-AKT and b) abrogated 4EBP1 phosphorylation.

Conclusion: Data showed that TAK228 effectively inhibited proliferation and enhanced apoptosis via inhibition of the PI3K-AKT-mTORC1/C2 signaling pathway and dual TORC1/2 inhibition also mitigated the feedback activation of AKT caused by selective inhibition of mTORC1, and because of this, TAK228 may yield greater therapeutic benefit in BC patients.

#3453

PAK6 localizes to centrosome and modulates centrosome homeostasis.

Houjun Jia,1 Danda Chapagai,2 John Mallow,2 Michael D. Perttunen,2 Vijaya Iragavarapu,2 Jianning Wei,2 Michael L. Lu2. 1 _The First Affiliated Hospital of Chongqing Medical University, Chongqing, China;_ 2 _Florida Atlantic University, Boca Raton, FL_.

Centrosome abnormalities are linked to genomic instability and are considered one possible cause of cancer progression. In prostate and breast cancer, centrosome over-duplication is correlated with lymph node and distant metastasis. In prostate cancers, centrosome amplification is a useful predictor of tumor recurrence, highlighting its potential role in promoting advanced disease. PAK6 was identified as an androgen receptor (AR) interacting protein. It has been determined that activated PAK6 plays an active role in promoting hormone-regulated prostate cancer metastatic phenotypes such as upregulating cell motility and invasiveness. Several lines of evidence indicate that PAK6 is overexpressed in advanced cancers including prostate, colon and liver cancers. The up-regulated expression of PAK6 in advanced diseases underscores its potential role in disease progression.

Using a novel anti-PAK6 antibody readily recognizes the native PAK6 protein, we determined that PAK6 localizes at centrosome and in some cell types also at plasma membrane. This observation revised our previous view of PAK6 as a soluble cytoplasmic protein. Ectopically expression of PAK6 leads to hyperploidy in LNCap cells. We hypothesized that aberrant PAK6 expression plays a critical role in the development of hyperploidy. In current study, we characterized the role of PAK6 in the dysregulation of centrosome-triggered hyperploidy in cancer cells. PAK6 centrosome localization was determined by co-staining of PAK6 with centrosome marker γ-tubulin using immunofluorescence microscopy of a pair of mouse embryo fibroblasts (MEF) derived either from WT mice (MEF-WT) or from PAK6-null mice (MEF-KO). PAK6-centrosome association was also confirmed biochemically by co-fractionation of PAK6 with γ-tubulin in centrosomal subcellular-fractions using a sucrose gradient. Down-regulation of PAK6 expression by lentivirus mediated shRNA promotes centrosome amplification with increased centrosome size in LNCap and MCF7 cells. Accelerated microtubule aster regrowth following nocodazole-induced depolarization was observed in MEF KO cells in as compared to MEF WT cells. Reciprocally, the inhibitory effects of PAK6 overexpression on aster growth was demonstrated in tetracycline inducible U2OS cells.

Our results indicate that PAK6 on one hand promotes aneuploidy, but on the other hand its expression suppresses centrosome amplification and microtubule dynamics. These apparent incompatible results suggest that PAK6 functions at multiple levels that in one hand it regulates centrosome amplification and microtubule dynamics, and on the other hand it may be involved in regulating the chromosome segregation and cytokinesis. The net effects of PAK6 expression may be context dependent.

#3454

HIV-1 envelope protein gp120 promotes glioma tumor growth through the Akt and MAP kinase pathways.

Gabriel Valentin-Guillama, Yuriy Kucheryavykh, Lilia Kucheryavykh. _Universidad Central del Caribe, Bayamon, Puerto Rico_.

The median survival of glioblastoma (GBM) patients with HIV is significantly shorter compared to HIV-negative GBM patients, despite the fact that they receive the same treatments. This indicates that HIV infection is associated with more aggressive tumor behavior. Earlier we shown that gp120, a glycoprotein found in the HIV shell, stimulates up-regulation of glycolysis in glioma cells. The purpose of this study was to evaluate the underlying signaling mechanisms of gp120 dependent up-regulation of glycolysis in glioma cells.

Using antibody array and western blot we have identified that U87 glioma cells treated with gp120 (200 ng/ml) for 5 consequent days showed increased activation of MAP kinase and Akt/mTOR pathways. Increase of pAkt(pS473), pGSK3b(pS9), pMKK3(S189), pmTOR(S2448), p38(T180/Y182), pJNK(T183) has been identified. This data coincides with previously obtained results showing that glioma cells treated with gp120 exhibit higher proliferation rates and increased cell survival compared to un-treated glioma cells.

In summary, we conclude that gp120 triggers activation of regulatory kinases Akt and MAPK involved in cell growth, cell proliferation, cell survival, protein synthesis, transcription, glycolysis, and cell migration.

#3455

Biologic functions of BMX/BTK in non-small cell lung cancer.

Wen-Chien Huang. _Taipei Mackay Memorial Hospital, Taipei, Taiwan_.

Non-small cell lung cancer (NSCLC), a leading cause of tumor-related deaths in Taiwan and worldwide, remains not only a health care but also a social burden in Taiwan. EGFR and KRAS mutations and deregulation of the EGFR/KRAS pathways are critical for lung tumorigenesis and progression; however, our knowledge of the genes responsible for the heterogeneity of lung carcinoma is inconclusive. Despite increasing documentation of the role of the Bruton's tyrosine kinase (BMX/BTK) in tumorigenesis, as well as increasing use of BMX/BTK inhibitors as new cancer drugs, to the best of our knowledge, this is the first study on the identification and characterization of BMX/BTK in lung cancer. Our preliminary data show increased expression of BMX/BTK protein in lung cancer cells compared to their nontumor counterparts, using immunohistochemical (IHC) staining and Western blot. Further, using clinicopathologic analyses of large prognoscan database cohorts, we established a negative correlation between BMX/BTK expression and patients' outcome. However, this bioinformatics-based clinical significance of BMX/BTK and its functional mechanisms in the tumorigenesis of NSCLC remains to be verified. In this project, we will comprehensively investigate the role of the BMX/BTK gene in lung tumorigenesis and progression, with its underlying molecular mechanisms. We will also develop and evaluate the therapeutic effect of in-house small-molecule inhibitors of BMX/BTK in lung cancer using animal model. In aim 1, we will explore the expression of BMX/BTK and its related upstream regulators and downstream effectors in lung cancer using IHC staining of primary NSCLC tissue samples. These data will be collated and statistically correlated with patients' clinical data. In aim 2, we will investigate the role of BMX/BTK in lung cancer cell proliferation, epithelial-to-mesenchymal (EMT), and cancer stem cell phenotype, as well as determine the underlying molecular mechanisms, using loss and gain of BMX/BTK function, and functional assays such as the colony formation, invasion, migration and spheroid formation assays in lung cancer tissues and cell lines. In the aim, we will validate the in vitro findings by examining BMX/BTK activities in vivo, evaluate the therapeutic effect of novel BMX/BTK inhibitors and investigate its clinical significance, using tumor xenograft NOD-SCID mice models. We anticipate that this current study will demystify the role and molecular mechanisms of BMX/BTK in the tumorigenesis of lung cancer and provide relevant information for effective anti-lung cancer therapeutic strategy, including combined targeted therapy.

#3456

Molecular mechanisms of RSK2-ELK3 signaling axis in neoplastic cell transformation.

Seon-Yeon Cho, Cheol-Jung Lee, Sun-Mi Yoo, Seung-Min Kim, Juhee Park, Yong-Yeon Cho. _College of Pharmacy, The Catholic University of Korea, Bucheon, Republic of Korea_.

Constitutive active Ras mutation (RasG12V) plays a key role in cell proliferation and transformation in many solid human cancers. RSK2, a member of p90RSK superfamily, controlled by ERKs is an intermediate signaling molecule transducing Ras activation signal to downstream substrates including transcription factors, epigenetic factors and some kinases. Since RSK2 shows wide range of substrate spectrum, identification of RSK2's novel substrates is important to understand the role of RSK2 in cancer. We found that ELKs including ELK1, ELK3 and ELK4 are novel interaction partners of RSK2 by mammalian two-hybrid assay. In vitro kinase assay and immunoprecipitation assay indicated that ELK3 is a major binding partner of RSK2. Luciferase reporter assay demonstrated that ELK3 transactivation activity was increased by RSK2, resulted in the increase of c-fos promoter activity. Importantly, constitutive active (CA)-RasG12V-mediated foci formation in NIH3T3 cells were more increased by ectopic co-expression of CA-RasG12V and RSK2. Additionally, signaling blockage of the CA-RasG12V/ELK3 by RSK2 knockdown suppressed foci formation. These results demonstrated that RSK2-ELK3 signaling axis plays an important role in RasG12V-mediated neoplastic cell transformation.

Key words : MAPK, RSK2, ELKs, cell proliferation, cell transformation

#3457

Chemotherapy enables Brk/PTK6-dependent survival of triple-negative breast cancer cells via induction of an AhR/GR/HIF signaling axis.

Tarah M. Regan Anderson,1 Shihong Ma,2 Carlos J. Perez Kerkvliet,1 Taylor M. Helle,1 Raisa Krutilina,3 Ganesh V. Raj,2 John A. Cidlowski,4 Kathryn L. Schwertfeger,1 Tiffany N. Seagroves,3 Carol A. Lange1. 1 _University of Minnesota, Minneapolis, MN;_ 2 _University of Texas Southwestern Medical Center, Dallas, TX;_ 3 _University of Tennessee Health Science Center, Memphis, TN;_ 4 _National Institute of Environmental Health Sciences, Research Triangle Park, NC_.

The metastatic cascade is a complex process that requires cancer cells to survive despite exposure to conditions of high physiologic stress. We previously showed that breast tumor kinase (Brk; also known as PTK6), a mediator of aggressive breast cancer phenotypes, is induced in breast cancer cells in response to a convergence of cellular and hormonal stress signals mediated by cross talk between hypoxia-inducible factors (HIFs) and glucocorticoid receptors (GR). Specifically, p38-MAPK dependent phosphorylation of GR-Ser134 (p-GR) and p-GR/HIF transcriptional complexes mediated heightened Brk gene expression in response to multiple inputs to stress pathway activation. Following studies in mice to demonstrate that Brk is a GR target gene in the mammary gland, immunohistochemistry (IHC) was performed on human primary breast tumor tissues using total and p-GR antibodies. To model stress-induced p-GR action relevant to tumor progression, molecular markers of stress signaling were measured in triple negative breast cancer (TNBC) cell lines treated with chemotherapy (Taxol and 5-flourouricil) and following growth in suspension (ultra-low attachment (ULA)). Co-immunoprecipitation and ChIP assays were used to demonstrate association of p-GR with novel co-regulatory factors in transcription complexes at the Brk promoter. Cell viability and migration assays were performed following Brk knock-out using CRISPR/Cas9 gene editing. Systemic Dex administration in mice confirmed that Brk is a GR target gene in vivo. In human breast tumor samples, phospho-GR was significantly associated with TNBC relative to luminal cancers. Chemotherapy and ULA induced activation of p38 MAPK, phosphorylation of GR, and upregulation of HIFs as well as the Aryl hydrocarbon receptor (AhR), a known mediator of cancer cell survival under cellular stress. Moreover, AhR and GR co-purified constitutively, and following chemotherapy or ULA culture, these factors assembled at the Brk promoter in a HIF-dependent manner. Brk induction was critical for TNBC cell survival during Taxol treatment or during ULA culture and for cancer cell migration, acquired biological phenotypes that enable cancer cells to successfully complete the metastatic cascade.These studies define AhR as a novel p-GR binding partner and show that increased p-GR/AhR and Brk expression drive a migratory phenotype relevant to TNBC progression. Strategies to target p-GR signaling may ameliorate stress-induced acquisition of aggressive cancer phenotypes required for metastatic cancer spread that are linked to high breast cancer patient mortality.

#3458

Neurotensin increases breast cancer proliferation through activation of AMPK.

Jeremy Johnson, Jing Li, B. Mark Evers. _University of Kentucky, Lexington, KY_.

Introduction. Neurotensin (NT), a thirteen amino acid peptide predominantly localized to enteroendocrine cells of the small intestine, is released after fat ingestion. While its major role involves regulating lipid metabolism and storage, NT, acting through its high-affinity receptor (NTR1), stimulates the growth and progression of a variety of NTR1-positive cancers. Proneurotensin—a stable precursor produced in equimolar amounts as NT—has been associated with breast cancer development in women, and prior work has implicated the NT/NTR1 axis in breast cancer progression; however, very little is known about the underlying NT signaling pathways that stimulate breast cancer growth. The purpose of this study is to elucidate mechanisms by which NT leads to increased proliferation and aggression of breast cancer—with particular emphasis placed on the role of AMP-activated protein kinase (AMPK). Methods. MCF-7 (estrogen-dependent) and MDA-MB-231 (triple negative) are breast cancer cell lines that express NTR1. (i) Proliferation assays were performed by treating both cell lines with NT (0 or 100 nM) in serum-free media for 24, 48, and 72 h and then counting the viable cells. (ii) Next, to assess signaling pathways mediating the effects of NT, both cell lines were treated with NT (0 or 100 nM) in serum-free media for 15 min, 30 min, 1 h, 6 h, 12 h, and 24 h; immunoblotting was performed for phosphorylated and total forms of AMPK and its downstream effector acetyl CoA carboxylase (ACC). Results. (i) Increased proliferation of MCF-7 cells treated with NT (100 nM) was observed after serum-starvation for 48 h. While MDA-MB-231 cells treated with NT demonstrated improved survival after serum-starvation for 48 h, the effects were not significantly different. (ii) NT induced activation of AMPK and ACC in MCF-7 cells after treatment for 6 and 12 h, but these effects were not observed in MDA-MB-231 cells. Conclusions. Our findings suggest a possible linkage between NT and increased proliferation of estrogen-dependent breast cancer cells through activation of AMPK and its downstream effector proteins. These effects were minimal in NTR1-expressing triple negative breast cancer cells, suggesting that the molecular classification of the tumor plays an important role in NT signaling and downstream effects. Further delineation of the differential effects of NT in specific breast cancer phenotypes has the potential to identify novel therapeutic targets in the treatment of this disease.

#3459

Dual targeting of BTK and PLK1 causes lethal synergistic effects in mantle cell lymphoma.

Changying Jiang, Shengjiang Huang, Yang Liu, Krystle Nomie, Leo Zhang, Michael Wang. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Mantle cell lymphoma (MCL) is an incurable, aggressive B cell malignancy. Ibrutinib, an oral irreversible Bruton's tyrosine (BTK) inhibitor, was FDA-approved in 2013 for patients with relapsed/refractory MCL. In further support of BTK inhibition for the treatment of MCL, acalabrutinib, an irreversible second-generation BTK inhibitor, was recently approved by the FDA for previously treated MCL. Although BTK inhibition has resulted in remarkable MCL patient outcomes, primary resistance and acquired resistance to ibrutinib remains a challenging hurdle to the treatment of MCL patients. In this study, we aimed to discover alternative therapies to overcome resistance to ibrutinib for MCL patients. To identify therapies that will overcome resistance to BTK inhibition, we performed a combinational screen for ibrutinib with FDA-approved drugs and novel agents and found that combination treatment of ibrutinib with the polo-like kinase 1 (PLK1) inhibitors volasertib or GSK461364 achieved synergistic growth inhibition in both ibrutinib-sensitive and -resistant MCL cells. PLK1, a master regulator of entry into mitosis, is overexpressed in many types of cancer, is correlated with poor clinical outcome, and plays an important role in the development of drug resistance. Volasertib, a second-generation PLK1 inhibitor, has been intensively studied, and there is an ongoing phase III trial to further determine the efficacy and safety of this agent in a large AML cohort. Phase I study of GSK461364 has been completed, and promising antitumor activity, especially in non-Hodgkin lymphoma and AML, has been determined. We found that PLK1 is highly expressed in MCL cells, and treatment with the PLK1 inhibitors volasertib and GSK461364 as single agents significantly arrested the cell cycle in the G2/M phase, potently reduced cell viability with IC50 values ranging from 6-28 nM and 8-33 nM, respectively, via induction of apoptosis in both ibrutinib-sensitive (n=3) and -resistant (n=6) MCL cells. Interestingly, treatment with the BTK inhibitor alone induced PLK1 expression, suggesting a compensatory mechanism in MCL cells to combat BTK inhibition. Dual BTK and PLK1 inhibition caused cell cycle arrest in the G2/M phase at a similar level to that of the PLK inhibitors alone; however, synergistic effects on cell viability and cleavage of PARP and caspase 3 were observed. The detailed mechanisms underlying these synergistic effects are currently under investigation. We are also validating the in vitro findings using patient-derived xenograft models in vivo. These data suggest that targeting PLK1 in MCL is a promising therapy and that dual targeting of BTK and PLK1 is a promising therapeutic strategy to improve MCL patient outcomes and to overcome ibrutinib resistance.

#3460

p38-p14ARF-CtBP2 axis as a novel regulator of CSC phenotype and tumor cell dormancy.

Rio S. Boothello,1 Nirmita J. Patel,1 Priyadarshan K. Damle,1 Chetna Sharon,2 Kranthi Kumar Chougoni,1 Umesh R. Desai,1 Steven R. Grossman,1 Bhaumik B. Patel1. 1 _Virginia Commonwealth University, Richmond, VA;_ 2 _McGuire VA Medical Center, Richmond, VA_.

Introduction: Cancer cell dormancy and the cancer stem cell (CSC) phenotype are interlinked and play a crucial role in disease relapse. The pp38/pERK signaling ratio positively regulates cellular dormancy. We observed that heparin hexasaccharide (HS06) inhibited colon CSC self-renewal through isoform specific induction of pp38α/β, increasing the pp38/pERK ratio, and downregulation of C-terminal binding protein-2 (CtBP2) levels which is known to play a critical role in CSC growth. Hence, we hypothesized that HS06 inhibits CSCs self-renewal and prevents dormancy escape through regulation of CtBP2 in a manner dependent on p38 activation. Method: Spheroid culture of HT29 and HCT116 colon cancer cells was used for CSC self-renewal monitoring. Transient transfection with p38α, p14ARF, CtBP2 shRNA and/or expression vector with appropriate scrambled or vector controls was used to modulate cell signaling through this pathway. Standard western blotting, Q-PCR, flow cytometry, and immunoprecipitation were used to examine protein levels, their phosphorylation status and protein-protein interactions. Short term exposure of colon cancer cells (HT29) to chemotherapy (5-Fluorouracil (125μM) and oxaliplatin (5μM) (5-FUOX)) was used to study a transient dormancy-like state followed by growth escape. HT29 CSC (1x105 CD133+/CXCR4+ cells) induced xenografts were established s.c. in NCr nude mice were treated with HS06 (150mg/kg 3xwk., 3 weeks) and monitored for tumor growth, and xenografts were analyzed for CSC makers and p38-p14ARF-CtBP2 axis status. Results: HS06 inhibited CtBP2 levels but increased the expression of its negative regulator p14 ARF in vitro and in vivo in a manner dependent on p38 activation, as SB203580, a selective p38α/β inhibitor, as well as p38α shRNA, both near-completely reversed the effects of HS06 on p14ARF, CtBP2, and CSC self-renewal. This regulation of p14ARF and CtBP2 was post-transcriptional in nature. Moreover, reciprocal coimmunoprecipitation studies revealed a novel physical interaction between endogenous pp38 and p14ARF, which resulted in serine phosphorylation of p14ARF in a p38 activation-dependent manner that was critical for regulation of CtBP2 levels and CSC self-renewal. Using a chemotherapy induced cellular dormancy model, we observed that activation/upregulation of p38 and p14ARF prevented escape from dormancy in vitro, whereas increased CtBP2 had the opposite effect. Conclusion: We have discovered a novel p38-p14ARF-CtBP2 axis that plays a dual role in colon cancer CSC self-renewal and cellular dormancy. Agents targeting this pathway e.g., HS06 and CtBP2 inhibitors, represent novel therapeutic strategies to inhibit CSCs and prevent dormancy escape leading to reduced tumor relapse and/or cure.

#3461

PID1, a novel brain tumor growth suppressor, is a target for phosphorylation by a newly identified binding partner.

Anup S. Pathania,1 Xiuhai Ren,2 Min Mahdi,2 Eslam Nouri Nigjeh,3 Yang Fu,4 Ebrahim Zandi,3 Xiaojiang Chen,4 Gregory M. Shackleford,2 Anat Erdreich-Epstein5. 1 _Children's hospital Los Angeles/University of Southern California, Los Angeles, CA;_ 2 _Children's hospital Los Angeles, Los Angeles, CA;_ 3 _Keck School of Medicine/University of Southern California, Los Angeles, CA;_ 4 _Norris Comprehensive Cancer Center/University of Southern California, Los Angeles, CA;_ 5 _Children's hospital Los Angeles/Norris Comprehensive Cancer Center/University of Southern California, Los Angeles, CA_.

Background: Our group were first to establish a link between PID1 (Phosphotyrosine Interaction Domain containing 1) and cancer, showing PID1's tumor inhibitory effect in medulloblastoma, glioblastoma and atypical teratoid rhabdoid tumor (ATRT) brain tumor cell lines and correlation of higher PID1 mRNA with longer patient survival. PID1 was first identified in 2006 and is associated with obesity-related insulin resistance and mitochondrial dysfunction. Here we present a novel interacting partner of PID1 which phosphorylates and modulates its growth suppressive functions.

Methods: In silico predictions, in vitro kinase assay, phosphoproteomic analysis, co-immunoprecipitation, SDS-PAGE, western blotting, flow cytometry. Cells: CHLA-07-BSGBM pediatric glioblastoma, U87 glioblastoma and D283 medulloblastoma cell lines, HEK293FT, primary pooled mouse embryo fibroblasts (MEFs).

Results: Using prediction algorithms we found that PID1 was a likely phosphorylation target of a member of the G protein-coupled receptor kinase (GRK) family. In vitro kinase assays coupled with phosphoproteomic analysis confirmed this phosphorylation. Overexpressed HA-PID1 and this GRK could be reciprocally co-immunoprecipitated in 293FT cells and endogenous GRK was co-IPd by endogenous PID1 in CHLA-07-BSGBM. A kinase-dead GRK mutant co-IP'd with PID1 similar to the wildtype GRK indicating that the GRK-PID1 interaction did not require the kinase function of this GRK. Deletion mutants of PID1 differentially bound the GRK in co-IPs in a manner that corresponded to the differential growth inhibition these PID1 deletion mutants exerted in glioblastoma and medulloblastoma cell lines. Functionally, siRNA silencing of this GRK caused apoptosis (annexin-PI positive cells and PARP-1 cleavage) in Pid1wt MEFs suggesting that this GRK mediates survival in MEFs. Apoptosis by the GRK siRNA was mitigated in Pid1ko compared to Pid1wt MEFs, suggesting that PID1 functions downstream of the GRK and negatively regulates this GRK's pro-survival signaling. Importantly, we found similar results in glioblastoma cells using gain/loss of function of the two proteins.

Conclusions: We found that PID1 is phosphorylated by a member of the GRK family and interacts with it, affecting this GRK's pro-survival activity. Experiments are now uncovering the molecular ordering of PID1 and this GRK in mediating the growth-suppressive effect of PID1 in brain tumors.

#3462

EGFRvIII-Stat5 signaling enhances glioblastoma cell invasion.

Alison Roos,1 Harshil D. Dhruv,2 Sen Peng,2 Landon J. Inge,3 Serdar Tuncali,1 Michael Pineda,2 Nghia Millard,4 Jeffrey A. Winkles,5 Joseph C. Loftus,1 Nhan L. Tran1. 1 _Mayo Clinic, Scottsdale, AZ;_ 2 _Translational Genomics Institute, Phoenix, AZ;_ 3 _Norton Thoracic Institute, Scottsdale, AZ;_ 4 _Translational Genomics Insitute, Phoenix, AZ;_ 5 _University of Maryland School of Medicine, MD_.

Glioblastoma Multiforme (GBM) is the most common malignant brain tumor in adults. Most GBM patients succumb to the disease less than one-year post diagnosis due to the highly invasive nature of the tumor, which prevents complete surgical resection and gives rise to tumor recurrence. The invasive phenotype also confers radio and chemoresistant properties to the tumor cells; therefore, there is a need to develop new therapeutics that target drivers of GBM invasion. Amplification of EGFR is observed in over 50% of GBM tumors, of which half concurrently overexpress the variant EGFRvIII, and expression of both receptors confers a worse prognosis. EGFR and EGFRvIII cooperate to promote tumor progression and invasion, in part, through activation of the Stat-signaling pathway. Here we report that EGFRvIII activates Stat5 and GBM invasion, in part, by inducing the expression of a previously established mediator of glioma cell invasion and survival, fibroblast growth factor-inducible 14 (Fn14). EGFRvIII-mediated induction of Fn14 expression is dependent upon Stat5 and requires activation of Src, whereas EGFR regulation of Fn14 is dependent upon MEK/ERK-Stat3 activation. Notably, treatment of EGFRvIII expressing GBM cells with the FDA approved Stat5 inhibitor pimozide blocked Stat5 phosphorylation, Fn14 expression, and cell migration. Since EGFR inhibitors display limited therapeutic efficacy in GBM patients, we hypothesize that the EGFRvIII-Stat5-Fn14 signaling pathway represents a node of vulnerability in the invasive GB cell population and that targeting critical effectors in this pathway will limit GBM tumor dispersion, mitigate therapeutic resistance, and increase survival.

#3463

Crosstalk between PKCalpha and TGF-beta signaling suppresses inhibitor of DNA binding 1 (Id1) expression in intestinal epithelial cells.

Xinyue Li, Michelle Lum, Adrian Black, Jennifer Black. _University of Nebraska Medical Center, Omaha, NE_.

Inhibitor of DNA binding (Id) proteins (Id1-Id4) are dominant negative antagonists of basic helix-loop-helix (bHLH) transcription factors with recognized functions in development, stem cell self-renewal, and cell fate determination. Recent studies have identified Id proteins as master regulators of cancer-initiating cells and tumor aggressiveness, with critical roles in regulation of central hallmarks of cancer, including cell proliferation, survival, senescence, angiogenesis, migration, metastasis, and chemoresistance. In colorectal cancer (CRC), Id1 overexpression is associated with aggressive clinical behavior and poor prognosis, while decreasing sensitivity to chemotherapeutic agents. Protein kinase C α (PKCα) is a serine/threonine kinase that functions as a tumor suppressor in CRC. PKCα signaling induces cell cycle arrest in nontransformed IEC-18 intestinal epithelial cells via an ERK-dependent mechanism. A major target of PKCα negative control in these cells is Id1. To further characterize mechanisms underlying the tumor-suppressive effects of PKCα, we modulated PKCα expression and activation in CRC cells and nontransformed IEC-18 cells and performed RNA-seq analysis to identify changes in gene expression. This analysis identified transforming growth factor beta receptor 1 (TGFβR1) as an mRNA upregulated by PKCα signaling. Subsequent RT-PCR and Western blot analysis confirmed that PKCα upregulates TGFβR1 at both the mRNA and protein levels. The MEK inhibitors U0126 and PD98059 blocked PKCα upregulation of TGFβR1, pointing to an ERK-dependent mechanism. Notably, inhibition of TGFβR1 with GW788388 prevented PKCα-mediated Id1 downregulation, linking TGF-β signaling to the tumor-suppressive effects of PKCα. Further studies indicated that TGFβR1 activates a p38-mediated noncanonical pathway to downregulate Id1 in response to PKCα activity. Collectively, these findings identify a PKCα→Erk→TGFβR1→p38⟞Id1 signaling axis in intestinal epithelial cells. Both PKCα and TGF-β function as tumor suppressors in the intestinal epithelium and both pathways are inactivated during intestinal tumor progression. By providing a direct link between two tumor-suppressive signaling pathways with established effects on intestinal tumorigenesis, our findings broaden our understanding of CRC and open avenues for potential novel therapeutic approaches to this disease. (Supported by NIH grants DK60632, CA54807, CA036727 and CA191894.)

#3464

MUC1 regulates TGF-β in pancreatic cancer.

Priyanka Grover,1 Mahboubeh Yazdanifar,1 Mohammad Ahmad,1 Ru Zhou,1 Angat Puri,1 Kajal Grover,2 Xinghua Shi,1 Pinku Mukherjee1. 1 _Univ. of North Carolina - Charlotte, Charlotte, NC;_ 2 _Univ. of North Carolina - Chapel Hill, NC_.

Pancreatic Cancer (PC) is the 3rd leading cause of cancer-related deaths in the US. 90% of PC is Pancreatic Ductal Adenocarcinoma (PDA). 85% of PDA overexpress tumor associated Mucin-1 (tMUC1), a membrane bound glycoprotein that is aberrantly glycosylated. tMUC1 overexpression in PDA plays a critical role in tumor progression and metastasis, however, the mechanism remains elusive. Transforming growth factor-β (TGF-β) is a cytokine with a dual functionality. Within normal cells and early carcinogenesis, TGF-β functions as a tumor suppressor and induces apoptosis. This effect is mediated by activation of the canonical SMAD pathway via the TGF-β Receptor 1 (TGF-βRI). However, during later stages of cancer, TGF-β becomes a tumor promoter and stimulates EMT, migration, and invasion. This effect is thought to be mediated by the non-canonical Erk1/2 pathway. We have recently shown that high tMUC1-expressing PDA cells respond to TGF-β via activation of the non-canonical pathway and undergoes EMT whereas, low tMUC1 expressing PDA cells respond to TGF-β via activation of the canonical SMAD pathway and undergoes apoptosis. Hypothesis: tMUC1 and TGF-β molecular pathways are interconnected and that tMUC1 plays a key role in regulating TGF-β's dual function. Method: The effects of TGF-β1 to induce apoptosis versus invasiveness in a variety of tMUC1 high and low PDA cell lines were determined. Tissue specific gene expression dataset from healthy subjects were collected from the GTEx Project (n=171). Tumor-specific gene expression data from PDA patients were collected from the TCGA-PAAD project (n=178). The Pearson's correlation coefficient was calculated for TGF-β1 canonical and non-canonical pathway genes and MUC1 and the p-values adjusted using Bonferroni multiple-testing correction. Finally, we determined the anti-tumor efficacy of neutralizing TGF-β1 in vivo in high and low tMUC1-expressing PDA tumors. Results: In response to TGF-β treatment, tMUC1 high PDA cells resisted apoptosis, enhanced their invasiveness, and activated the Erk pathway, while tMUC1 low cells became apoptotic and activated the SMAD pathway. The signaling was dependent upon tyrosine phosphorylation of MUC1 cytoplasmic tail domain by Src. RNA-Seq analysis in human populations reveal eight genes that show significant correlation (low p-value OR p-value < 0.05) in the TCGA PDA tissue which were insignificant in the GTEx healthy tissue. A moderate positive correlation was identified for MAPK3/ERK1(r= 0.549, corrected p-value = 4.47x10^-14) and SRC (r=0.592, corrected p-value = 6.3x10^-17). In contrast, a moderate negative correlation coefficients were identified for PLCG1 (r=-0.406, corrected p-value =3.86x10^-07), SMAD2(r=-0.38, corrected p-value=3.38x10^-06), and TGFβ3 (r=-0.32, corrected p-value =2.3x10^-06). Finally, in mice, tMUC1-high PDA tumors responded to the neutralization of TGF-β1 by significantly reducing tumor growth, while having no effect on tMUC1-low PDA tumors.

#3465

Biased signaling downstream of epidermal growth factor receptor regulates proliferative versus apoptotic response to ligand.

Remah Ali,1 Wells Brown,2 Jo Davisson,1 Michael Wendt1. 1 _Purdue Univ., West Lafayette, IN;_ 2 _University of British Colombia, British Columbia, Canada_.

Inhibition of EGFR signaling by small molecule kinase inhibitors and monocloncal antibodies has proven effective in the treatment of multiple metastatic cancers. In contrast, metastatic breast cancer (BC) derived from EGFR-driven mammary tumors is inherently resistant to EGFR-targeted therapies. Mechanisms that contribute to this inherent resistance remain poorly defined. Here we show that in contrast to primary tumors, ligand-mediated activation of EGFR is dominated by signaling transducer and activator of transcriptional 1 (STAT1) signaling in metastatic BC. This change in downstream signaling leads to apoptosis and growth inhibition in response to EGF in metastatic BC cells. Mechanistically, these changes in downstream signaling result from an increase in the internalized pool of EGFR in metastatic cells, increasing physical access to STAT1. Along these lines, an EGFR mutant that is defective in endocytosis is unable to elicit STAT1 phosphorylation and apoptosis. Additionally, inhibition of endosomal EGFR signaling using an EGFR inhibitor linked to a nuclear localization signal specifically prevents EGF-induced STAT1 phosphorylation and cell death without affecting EGFR:MAPK signaling. Pharmacologic blockade of MAPK signaling through the use of the MEK1/2 inhibitor trametinib led to a dramatic increase in EGF-mediated STAT1 phosphorylation and enhanced EGF-induced apoptosis in metastatic BC cells. Importantly, use of a trametinib:EGF combination also facilitated the apoptotic switch in EGFR function in primary tumor cells, but not normal mammary epithelial cells. These studies reveal a fundamental switch in EGFR in metastatic BC. Furthermore, they demonstrate that pharmacological biasing EGFR signaling toward STAT1 activation is capable of revealing the apoptotic function of this critical pathway.

#3466

Growth inhibitory MEK-ERK signaling in the intestinal epithelium.

Navneet Kaur, Robert Lewis, Adrian Black, Jennifer Black. _University of Nebraska Medical Center, Omaha, NE_.

Increasing evidence points to members of the PKC family of serine/threonine kinases as important tumor suppressors; however, the signaling pathways involved remain poorly defined. Our previous studies have determined that PKCα, which acts as a tumor suppressor in the epithelium of the intestine/colon, is activated coincident with growth arrest in intestinal crypts in vivo. PKCα activity induces cell cycle withdrawal in intestinal epithelial cells, a process that involves downregulation of cyclin D1 and Id1, upregulation of the cyclin dependent kinase inhibitors p21cip1 and p27kip1, and characteristic changes in pocket proteins and DNA licensing factors. Pharmacological inhibitors of the ERK pathway revealed that these changes are dependent on activation of MEK-ERK signaling, thus identifying a growth inhibitory ERK-mediated signaling axis in the intestinal epithelium. Given the interest in inhibitors of the ERK pathway for treatment of colon and other cancers, characterization of this tumor suppressive pathway will be essential for rational development of this treatment modality and to avoid and/or manage potential adverse effects of ERK inhibition in patients. To this end, the mechanistic basis for the growth suppressive effects of the PKCα→ERK module on cyclin D1, Id1 and p21cip1 are being investigated in non-transformed intestinal epithelial cells and colon cancer cells. Use of selective inhibitors and siRNA-mediated knockdown has determined that PKCα activates multiple components of the ERK pathway, including RAS, RAF, MEK and ERK. Notably, tumor suppressive effects are seen in colon cancer cells that harbor activating mutations in KRAS, indicating that growth inhibitory ERK signaling can override oncogenic signals arising from RAS mutation. PKCα signaling is associated with activation of ARAF, BRAF and CRAF and with induction of RAF heterodimers. A requirement for RAF activity was confirmed by the ability of LY3009120 or sorafenib to block growth inhibitory effects of PKCα. However, knockdown of individual RAF proteins fails to block these effects of PKCα, indicating some redundancy between the RAF proteins in mediating growth inhibitory signals. While the scaffolding protein KSR1 has been shown to be required for oncogenic RAS-ERK signaling, PKCα was still able to elicit growth inhibitory effects in KSR1 knockdown HCT-116 colon cancer cells. These data indicate that differential use of scaffolding proteins may explain both the growth inhibitory effects of PKCα-ERK signaling and their ability to override oncogenic RAS signals. A better understanding of these mechanisms may serve to guide the use of ERK inhibitors for colon cancer therapy. Supported by NIH grants DK60632, CA54807, CA157774 and CA191894.

#3467

Growth factor receptor trafficking in neuroblastoma differentiation.

Jacqueline Lesperance,1 Divya Subramonian,1 Sarah Woodfield,2 Rongjun Guo,3 Andrew Bean,4 Dolores Lopez-Terrada,2 Michael Ittmann,2 Sara Hakim,1 Peter E. Zage1. 1 _University of California San Diego, La Jolla, CA;_ 2 _Baylor College of Medicine, Houston, TX;_ 3 _University of Alabama-Birmingham, Birmingham, AL;_ 4 _University of Texas Medical School, Houston, TX_.

Background/Objectives: UBE4B is an E3/E4 ubiquitin ligase involved in growth factor receptor (GFR) trafficking. The UBE4B gene is located in the chromosome 1p36 region commonly deleted in high-risk neuroblastoma tumors. We have previously identified associations of UBE4B expression with neuroblastoma patient outcomes. However, the functional roles of UBE4B in neuroblastoma tumor differentiation are not known.

Design/Methods: We analyzed the association of UBE4B gene expression with expression of known markers of neuroblastoma tumor differentiation using publicly available databases and screened cell lines and neuroblastoma tumor samples for UBE4B protein expression using Western blot and quantitative immunohistochemistry. We measured UBE4B expression by Western blot in cell lines before and after induction of differentiation with 13-cis-retinoic acid treatment and determined the effects of UBE4B overexpression and depletion on retinoic acid-induced differentiation using continuous live-cell imaging of neurite morphology, immunohistochemistry, and Western blot for markers of differentiation. Effects on signaling through the Ras/MAPK pathway were measured using Western blots.

Results: UBE4B expression was associated with neuroblastoma differentiation in patient tumors, and UBE4B gene and protein expression were associated with expression levels of known markers of neuroblastoma differentiation. Retinoic acid treatment resulted in increased UBE4B expression in retinoic acid-sensitive, but not -resistant, neuroblastoma cells, and UBE4B depletion was associated with increased ERK phosphorylation, increased proliferation, and inhibition of retinoic acid-induced neuroblastoma differentiation.

Conclusion: We have demonstrated associations between UBE4B expression and tumor differentiation in gene expression databases and in neuroblastoma tumor samples, and our data suggests that UBE4B expression is required for retinoic acid-induced differentiation, potentially through effects on activation of the Ras/MAPK pathway. These associations between UBE4B and neuroblastoma differentiation combined with the established role of UBE4B in GFR trafficking suggest that UBE4B-mediated receptor trafficking may contribute to the responses of neuroblastoma tumors to therapy and to the outcomes of patients with neuroblastoma.

#3468

Antitumor effect by inhibition of PI3K/AKT/mTOR and RAS/MAPK pathways in human oral squamous cell carcinoma cells.

Norihiko Tokuzen, Koh-ichi Nakashiro. _Ehime Univ. Graduate School of Medicine, Toon, Ehime, Japan_.

Molecular target drugs are used in the treatment of a variety of malignant tumors, but this approach to developing novel therapies for oral squamous cell carcinoma (OSCC) has lagged behind the progress seen for other cancers. The PI3K/AKT/mTOR and RAS/MAPK pathways are important pathways for the growth and survival of cancer cells. These pathways also have been activated such as AKT and ERK overexpression in OSCC. In this study, we investigated the antitumor effect of PI3K/mTOR inhibitor, NVP-BEZ235 and MEK1/2 inhibitor, Trametinib, in human OSCC cells. First, we confirmed the inhibitory effect of NPV-BEZ235 and Trametinib in PI3K/AKT/mTOR and RAS/MAPK pathways by Western blotting. NPV-BEZ235 and Trametinib were reduced phosphorylation of AKT, p70S6K, and MEK1/2 in OSCC cells. Next, we examined the anti-proliferative effect of NVP-BEZ235 and Trametinib using 4 human OSCC cell lines by WST-8 assay. NVP-BEZ235 and Trametinib significantly inhibited the growth of all OSCC cells in a dose-dependent manner in vitro. Further, we investigated potential synergistic effects of inhibiting both the PI3K/AKT/mTOR and RAS/MAPK pathways, by treating with NVP-BEZ235 and Trametinib. The anti-proliferative effect of this combination was measured in OSCC cells by calculating the Combination Index. Combination treatment with NVP-BEZ235 and Trametinib showed a synergistic effect on the cell growth of all OSCC cells in vitro. Subsequently, we analyzed the effect of NBP-BEZ235 and Trametinib on the cell cycle of green fluorescent protein (GFP)-SAS cells by flow cytometry. Treated with NVP-BEZ235 showed no enhanced effect compared to control. Treatment of Trametinib resulted in the decrease in percentage of cells in G2 phase and increase in G1 phase. This indicated that the anti-proliferative effect of Trametinib was due to G1 arrest. Furthermore, treatment of Trametinib increased apoptosis cells in GFP-SAS cells. Next, we assessed the effect of NVP-BEZ235 and Trametinib on the in vivo growth of GFP-SAS cells. GFP-SAS tumor-bearing mice were treated with single, combination, or vehicle by oral daily. Treatment with NVP-BEZ235 and Trametinib significantly reduced the size of tumors compared with vehicle control. However, combination treatment showed no synergistic effect compared to single treatment. Finally, we investigated the gene expression changes with combination treatment using microarray analysis. The suppression of PI3K/AKT/mTOR and RAS/MAPK pathways caused overexpression of genes related to cell cycle and DNA-repair. These results suggest that activation of PI3K/AKT/mTOR and RAS/MAPK pathways support the growth of OSCC cells and NVP-BEZ235 and Trametinib may be a useful therapeutic strategy for OSCC.

#3469

Dual inhibition of the EGFR and Aurora A pathways in Kras mutated non-small cell lung cancers.

Tetyana Bagnyukova,1 Brian Egleston,1 Mackenzie Kramer,2 Igor Astsaturov,1 Erica Golemis,1 Hossein Borghaei1. 1 _Fox Chase Cancer Ctr., Philadelphia, PA;_ 2 _Philadelphia College of Osteopathic Medicine, Philadelphia, PA_.

Lung cancer is the leading cause of cancer related death in the US. Patients with advanced disease generally have a poor prognosis with a median survival of around 10 to 12 months with standard chemotherapy. In patients with non-squamous non-small cell lung cancers (NSCLC) incidence of KRAS mutations reach ~22%. Specific treatments for Kras mutated NSCLC have not been developed although recent reports suggest an advantage for treatment with immunotherapy over chemotherapy in the second line setting. Published reports using a synthetic lethal screening method show that the combined inhibition of the EGFR and Aurora A kinase pathways could have synergistic effect on viability in head and neck and colon cancer cell lines suggesting a potential benefit of such combination therapy in these patients (Astsaturov and colleagues, 2010). Our study aims to examine the effects of combination of erlotinib (E), an EGFR small-molecule inhibitor, and Alisertib (MLN8237, A), an inhibitor of Aurora A kinase, in lung cancer models with different KRAS status. Human NSCLC cell lines with both wild type and mutated KRAS showed weak or moderate sensitivity to both drugs in vitro. Treatment with both E and A had synergistic activity in all examined cell lines, wild type KRAS (H2228, H1299) or mutated KRAS (A549, H1299, H358, H460, and H62). Western blot analysis confirmed that combination treatment enhanced apoptosis compared to single drug use as evidenced by cleaved PARP. In xenograft models, CB17 SCID mice were treated with single drug or E+A for three weeks. H322M xenograft tumors bearing wild type KRAS were moderately sensitive to growth inhibition by E (10 mg/kg/day) and A (20 mg/kg/twice daily) alone and the combination had an additive effect. A549 and H358 xenografts were insensitive to E because of the mutated KRAS and moderately sensitive to A. However, the combination of E+A had a synergistic effect and significantly inhibited tumor growth. Western blot analysis of signaling pathways downstream of the EGFR and Aurora A in tumor xenografts shows synergistic inhibition after two-drug treatment. Our results suggest that combined inhibition of both pathways might be an effective treatment strategy for lung cancers harboring KRAS mutations.

#3470

Ibrutinib and sorafenib synergistically inhibit HCC growth in preclinical models.

Kalpana Ghoshal, Cho-hao Lin, Khadija Elkholy, Nissar A. Wani, Ding Li, Juan M. Barajas, Peng Hu, Xiaoli Zhang, Lianbo Yu, Tasneem Motiwala. _Ohio State Univ., Columbus, OH_.

Hepatocellular carcinoma (HCC) is the second major cause of cancer-related death worldwide with limit therapeutic options. Thus, there is an urgent need to develop novel alternative therapies for HCC. In this study, we report that ibrutinib, recently approved for the treatment of B cell malignancies, and a covalent inhibitor of TEC (BTK, ITK etc.) and ERBB (EGFR, Her2 etc.) family of tyrosine kinases, inhibits tumorigenic functions of human HCC cells in vitro and in xenografts. More importantly, co-treatment with ibrutinib and sorafenib, an approved targeted therapy for advanced HCCs that marginally improve patients' survival, synergistically inhibited proliferation and clonogenic survival of HCC cells including those with acquired sorafenib resistance. Mechanistically, ibrutinib inhibits Akt and ERK signaling pathways through inactivating EGFR, its irreversible substrate in HCC cells. Besides, tumor sphere formation and expression of cancer stem cell markers were suppressed by ibrutinib and sorafenib co-treatment in HCC cells. Ectopic expression of the constitutively active Akt mutant abrogated the synergism of these two kinase inhibitors on HCC cell survival. Ibrutinib and sorafenib combination therapy significantly suppressed tumor growth of highly aggressive HCCLM3 subcutaneous xenografts in NSG mice. Collectively, these results demonstrate that ibrutinib could be a re-purposed anti-HCC drug, and our data provides the evidence for the therapeutic potential of ibrutinib and sorafenib combination as an effective and attractive strategy for treating HCCs including those with sorafenib resistance.

#3471

Expression of the DEK oncogene in breast cancer cells promotes M2 polarization of tumor associated macrophages.

Miranda S. Shephard,1 Nicholas A. Pease,2 Jon Cheek,1 Lisa M. Privette Vinnedge1. 1 _Cincinnati Children's Hospital Medical Center, Cincinnati, OH;_ 2 _University of Washington, Seattle, WA_.

Breast cancer is the most common cancer and the second leading cause of cancer deaths among women. DEK is a known oncoprotein found to be highly expressed in more than 60% breast cancers and is found to be an independent marker of poor prognosis. However, the molecular mechanisms by which DEK promotes tumor progression are poorly understood. To elucidate the oncogenic functions of DEK, we performed RNA-seq analysis on isogenic Dek-knockout and complemented murine breast cancer cells, which indicated dysregulation of immune signaling. Among the target genes identified and confirmed was an upregulation of thymic stromal lymphopoietin (TSLP) in Dek expressing breast cancer cells. TSLP was previously shown by Han et al to amplify the alternative (M2) activation of macrophages. M2-like macrophages are tumor promoting, they recycle iron for cell growth and encourage tissue remodeling and repair activities like angiogenesis. Classically (M1) activated macrophages are tumor suppressing and are typically associated with response to infection. To test the immune modulating functions of Dek-expressing breast cancer cells, we treated bone marrow derived macrophages (BMDM) with tumor cell conditioned media. We found that in vitro, Dek expressing cancer cells induced an M2-like polarization of macrophages, as determined by the expression of M2-associated genes, enhanced migration, and iron recycling phenotypes, which was accompanied by inhibited ERK1/2 sighnaling. We found this phenotypic trend to be true in vivo, as well. In sectioned mammary tumors from MMTV-RontgDek+/+ and MMTV-RontgDek-/- mice, we see lower levels of iron staining in Dek+/+ tumors than in Dek-/- tumors using Prussian blue staining that co-localized with F4/80 macrophage marker staining. This suggested that Dek expression in the tumors induced an iron recycling, M2-like phenotype in tumor associated macrophages. These findings suggest that tumor Dek expression may promote breast cancer progression by inducing M2-like macrophage polarization in a murine breast cancer model.

#3472

Dual PI3K/mTOR inhibition in PIK3CA mutant pancreatic cancers.

Susan Payne,1 Mitchell Depke,1 Alex Yueh,1 Joseph T. Sharick,2 Peter F. Favreau,2 Cheri A. Pasch,1 Linda Clipson,1 Kristina A. Matkowskyj,1 Melissa Skala,2 Dustin A. Deming1. 1 _Univ. of Wisconsin, Madison, WI;_ 2 _Morgridge Institute for Research, Madison, WI_.

Background: Pancreatic cancer is one of the most deadly cancer diagnoses with limited long-term survival for patients, even with early-stage disease. 3-5% of these cancers have mutations in the phosphoinositide 3-kinase (PI3K) signaling pathway. There is growing interest in targeting cancers with this mutation using specific pathway inhibitors. We have previously proven that dual PI3K/mTOR inhibition is sufficient to decrease tumor growth in mice with a PI3K p110* transgene. Here we aim to determine if this treatment can induce a response in pancreatic cancers with the human PIK3CA H1047R hotspot mutation.

Methods: Transgenic mice were developed using a cre lox-p system (Pdx1-cre) to develop PIK3CA H1047R (PCPK mice) pancreatic adenocarcinomas. Tumors were harvested and grown in spheroid culture. These were treated with increasing concentrations of BEZ235, a dual PI3K/mTOR inhibitor. Treatment groups were compared using spheroid diameter changes and metabolic activity via NADH and FAD+ quantitation. Additionally, immunofluorescence staining was performed and quantified on treated spheroid cultures. PET/CT diagnostics were performed and analyzed pre- and post- treatment with BEZ-235 on a cohort of these experimental mice. Treated tissues were resected for immunohistochemistry and Western blot analyses.

Results: Organotypic PCPK spheroid cultures were treated with BEZ235 in the culture media for 48 hours. Pre- and post-brightfield imaging demonstrated a modest reduction in the percent change in spheroid diameter compared to control (control: 41.8%, BEZ235 100nM: 9%, BEZ235 200nM: -7.7%, BEZ235 400nM: -14.3%; p<0.001). This stability of the spheroid size correlated with minimal differences between the control and treated spheroids using the optical metabolic imaging, including nonsignificant changes in NADH lifetime, FAD+ lifetime and the optical redox ratio (p=NS). PCPK mice (n=10) aged until they developed cancers and then were treated with BEZ235 for 14 days. Pre- and post-positron emission tomography/computed tomography (PET/CT) were performed. Nonsignificant changes in tumor volume and PET avidity were seen between the treatment groups. Both in vitro and in vivo treatments with BEZ235 proved to have no significant change in tumor avidity in this model. Immunoblotting demonstrated persistent activation of the PI3K pathway despite BEZ235 treatment in these spheres and mice as measured by persistent phosphorylation of RPS6 and 4EBP1.

Conclusions: Despite significant responses being previously detected in mice with pancreatic tumors initiated by the PI3K p110* transgene, BEZ235 did not result in a similar treatment effect in PCPK mice possessing the human PIK3CA H1047R hotspot mutation. Further studies will examine the potential resistance mechanisms of these tumors to this therapy and potential ways in which to overcome this resistance.

#3473

Investigating RAS and RTK signaling in MPNST progression and drug resistance.

Candace N. King,1 Carrie Graveel,2 Matthew Steensma2. 1 _Van Andel Institute Graduate School, Grand Rapids, MI;_ 2 _Van Andel Research Institute, Grand Rapids, MI_.

Neurofibromatosis, type 1 (NF1) is a tumor predisposition syndrome affecting about 1 in 2,500 live births in the United States. The protein product of the NF1 gene, neurofibromin, loses its function as a negative regulator of the RAS protein, leading to the development of painful and disfiguring benign tumors known as a neurofibromas. Eight to thirteen percent of these neurofibromas will transform into malignant peripheral nerve sheath tumors (MPNSTs). MPNSTs are aggressive soft tissue sarcomas that do not respond to the standard sarcoma treatment of chemotherapy and surgical resection. There is a critical need for efficacious targeted therapy to increase the average life span of these patients. Current targeted therapy efforts have shown modest efficacy clinically due to the establishment of compensatory feedback mechanisms that propagate tumor progression and recurrence. These feedback mechanisms are not completely understood in MPNSTs. In lung cancer, inhibition of MEK in the RAS pathway has been shown to activate upstream receptor tyrosine kinases (RTK). Genomic studies of MPNST samples have shown that 30% of tumors have amplification of the RTKs MET and EGFR. Furthermore, in a study of the genomic progression of a NF1 patient pre- and post-treatment, we found amplification of MET and EGFR that increased following treatment. In a study on triple-negative breast cancer cells, it has been shown that MET and EGFR crosstalk may be responsible for the reactivation of the inhibited cancer pathways and drug resistance. Therefore, we hypothesize that coordinated deregulated RAS signaling and MEK inhibition leads to MET and EGFR amplification, expression, activation, and crosstalk, which drives MPNST progression, drug resistance, and tumor recurrence. To test our hypothesis, we will use both in vitro and in vivo experimental methods to pursue the following specific aims: (1) determine the effect of MEK inhibition on establishing compensatory feedback loops that increase RTK activation and downstream signaling, (2) determine the role of MET-EGFR crosstalk signaling in MPNST tumor progression and resistance to kinase inhibition, and (3) determine the efficacy of combination RTK and MEK inhibition in vivo. 

### Metabolomics

#3474

Integrative metabolomic and genomic analysis identifies fatty acid oxidation as a metabolic node in glioblastoma.

Shiva Kant, Antony Prabhu, Pravin Kesarwani, Praveen Kumar, Stewart F. Graham, Prakash Chinnaiyan. _Beaumont Health, Royal Oak, MI_.

Glioblastoma (GBM) represents an aggressive brain tumor with limited treatment options. Although considerable progress has been made in understanding molecular alterations unique to these tumors, the diverse metabolic programs driving their aggressive phenotype are only beginning to be recognized. As an initial investigation, using a library consisting of over 3000 biochemicals, we performed global metabolomic profiling in patient-derived GBM (n=80) and low-grade glioma (LGG; n=28). Alterations in fatty acid β-oxidation (FAO) emerged as a key metabolic node differentiating GBM from LGG, as demonstrated by an accumulation of acyl carnitines. Metabolic heterogeneity was observed within GBM that could further define tumors as FAO 'high' and 'low'. To begin to understand the molecular underpinnings of this metabolic heterogeneity, gene expression profiling on matched tumor samples was performed. We determined that established molecular subtypes of GBM correspond with FAO phenotypes, with FAO 'high' and 'low' tumors enriched with mesenchymal (MES) and proneural (PN) GBM subtypes, respectively. These findings were metabolomically and functionally recapitulated in molecular subtype-specific preclinical models, with an accumulation of acyl carnitines and enhanced FAO, contributing to nearly 60% of baseline cellular respiration, in MES glioma tumor initiating cells when compared to PN cells. Analysis of gene expression profiles from these lines uncovered an orchestrated transcriptional program designed to promote fatty acid uptake and activation. Consistent with gene expression findings, BODIPY labeling and fluorescent microscopy uncovered the unique capacity of MES cells to uptake fatty acids from the media, which was perturbed following FATP inhibition. Chemical and molecular inhibition of mediators of FAO, including fatty acid uptake, acylation, and CPT-1 demonstrated selective inhibition of proliferation in MES cells and inhibition of FAO using etomoxir demonstrated anti-tumor activity in vivo in an orthotopic model. Studies designed to determine the biologic consequence of enhanced FAO in GBM were not able to attribute this metabolic node to ATP synthesis. Systemic evaluation of the intermediary metabolism of FAO identified accumulation of the endogenous HDAC inhibitor 3HB and histone acetylation as consequence to enhanced FAO in GBM, which was also elevated in patient samples. The addition of 3HB rescued MES cells from the anti-proliferative effects of FAO inhibition and gene expression profiling identified FAO-mediated transcriptional programs contributing to the aggressive phenotype of MES in GBM. Collectively, our findings suggest that FAO represents a metabolic phenotype in GBM driven by enhanced fatty acid uptake and acylation, providing insight into both mechanisms driving the aggressive phenotype of this tumor and novel therapeutic targets.

#3475

Novel correlation-based network analysis of breast tumor metabolism identifies the glycerol channel protein Aquaporin-7 as a regulator of breast cancer metastasis.

Chen Dai,1 Jennifer Arceo,1 James Arnold,2 Junmin Wu,1 Norman J. Dovichi,1 Arun Sreekumar,2 Jun Li,1 Laurie E. Littlepage1. 1 _University of Notre Dame, South Bend, IN;_ 2 _Baylor College of Medicine, Houston, TX_.

The complex yet interrelated connections between cancer metabolism, gene expression, and oncogenic driver genes have the potential to identify novel biomarkers and drug targets with prognostic and therapeutic value. Using GC-MS, LC-MS/MS, and capillary zone electrophoresis (CZE)-MS platforms, we quantified and compared the levels of 374 metabolites in breast tumor tissue from normal tissue and transgenic mouse breast cancer models overexpressing a panel of oncogenes (PyMT, PyMT-DB, Wnt1, Neu, and C3-TAg). Comparison of the metabolite profiles from the tumors identified oncogene-induced metabolic reprogramming of the tumor tissues. To develop a higher order understanding of the driver genes and metabolites in breast cancer, we next developed a discovery-based correlation network analysis that captured interactions between both metabolite and gene expression data. This analysis uniquely identified a metabolic network of metabolites and genes with prognostic value in breast cancer patients, identifying 35 metabolite and 33 gene hubs that are likely integral to breast tumor metabolism. Further MALDI-MS based imaging revealed heterogeneous distribution of hub metabolites between stromal and epithelial tissue in breast tumors from transgenic mouse models, as well as heterogeneity within the tumor epithelium, suggesting complex metabolic landscape even within the tumor epithelium. We initially focused on the gene hub aquaporin-7 (Aqp7), a water and glycerol channel protein, as a novel regulator of breast cancer. AQP7 deficiency in animal models is associated with adipocyte hypertrophy, increased glycerol and triglyceride accumulation, insulin resistance, and obesity. We discovered that AQP7 is a prognostic marker of overall survival and metastasis in breast cancer patients. Aqp7 is expressed in the epithelium and adipocytes in normal and tumor breast tissue. Reduced Aqp7 expression in mouse breast cancer cells decreased both proliferation and lung metastatic burden. These data suggest AQP7 promotes invasive phenotypes of breast cancer progression. Using an unbiased, discovery-based approach, this study shed light on important players in breast cancer metabolism from a new perspective that complements current guided network analyses.

#3476

Chemotherapy modulates proteomic pathways and neuronal metabolites in the brain.

Justin B. Davis,1 Grace N. Lawrence,2 Michael G. Harpole,1 Robin D. Couch,1 Lance A. Liotta,1 Theodore C. Dumas,2 Virginia A. Espina1. 1 _George Mason University, Manassas, VA;_ 2 _George Mason University, Fairfax, VA_.

Chemotherapy modulates proteomic pathways and neuronal metabolites in the brainChemotherapy saves lives but diminishes the quality of life and functional ability of cancer patients by impairing cognitive function in a phenomenon called "chemobrain". Elucidating the neural substrates modulated by chemotherapy is critical for deciphering the molecular underpinnings of cognitive dysfunction. Animal models have been used to investigate the impact of numerous chemotherapy drugs on cognitive ability and revealed negative effects of nearly all drugs tested, with oxidative stress, inflammation, apoptosis, and a reduction in hippocampal neurogenesis observed after treatment. Still, the underlying proteomic and metabolic mechanisms of chemotherapy-related cognitive dysfunction is not fully understood. We developed an animal model to assess the effects of cyclophosphamide (CTX) and docetaxel (DTX), two chemotherapy agents commonly delivered to breast cancer patients. Our study goals were to determine the proteomic effects of chemotherapy in specific brain regions and the associated global metabolomic shifts in the brain. Healthy, female Long Evans rats were treated with CTX and/or DTX, in four weekly doses. Controls received injections of vehicle alone. Following chemotherapy, fresh brain tissue was dissected medially. The right brain region was frozen for global metabolomic analysis via tandem mass spectrometry. The left brain was fixed in TheraLin for proteomic analysis of post-translationally modified proteins using reverse phase protein microarrays and immunohistochemistry. Fixed brain tissue was sectioned, and the hippocampus, cerebellum, olfactory bulb, and cerebral cortex were procured using laser capture microdissection. We identified biochemical shifts associated with chemotherapy that impact neurobiological processes underlying memory dysfunction. Hierarchical clustering of signal transduction proteins revealed that chemotherapy resulted in elevated levels of Calpain 1 and 2 in the cerebellum, Calpain 2 in the cerebral cortex, and autophagy proteins in the hippocampus and cerebellum. Doublecortin, a neurogenesis protein, was elevated in the cerebellum. Docetaxel raised the abundance of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), while all chemotherapies diminished levels of glutathione, a neuromodulator of excitatory NMDA receptors. This animal study provides molecular information that offers empirical support for future intervention research that may help prevent cognitive decline and preserve cognitive health in cancer survivors.

#3477

Metabolic pathway analyses identify proline biosynthesis as a promoter of liver carcinogenesis.

Zhaobing Ding,1 Russell E. Ericksen,1 Michael Steckel,2 Andrea Haegebarth,2 Sylvia Gruenewald,2 Weiping Han1. 1 _Singapore Bioimaging Consortium, A*STAR, Singapore, Singapore;_ 2 _Bayer AG, Berlin, Germany_.

Cancer cells undergo dramatic metabolic reprogramming, largely to meet the requirements of sustained proliferation. These metabolic pathways are tightly regulated by various oncogenes, such as Myc and Ras. However, it has been difficult to directly and effectively inhibit these oncogenic signaling cascades with pharmacological compounds. Therefore, focusing on the downstream metabolic pathways that enable indefinite growth may provide a therapeutic opportunity. To identify metabolic enzymes required for hepatocellular carcinoma (HCC) tumorigenesis, we compared gene expression profiles of normal liver tissue to the Morris Hepatoma and DEN (Diethylnitrosamine)-induced HCC models as well as a liver regeneration model. PYCR1 (Pyrroline-5-Carboxylate Reductase 1), an enzyme in the proline biosynthesis pathway, was identified as one of the top up-regulated genes in the HCC models. An increase in PYCR1 protein levels in tumor samples versus normal liver tissue was confirmed by Western blot. shRNA-mediated knockdown of PYCR1 in HCC cell lines (Huh-7, Hep3B, HepG2 SNU-398, and MH3924a) potently reduced cell proliferation in vitro and tumor growth in vivo. Conversely, overexpression of PYCR1 enhanced the proliferation of Hep3B and AML12 cells. Importantly, PYCR1 expression was not elevated in regenerating tissues, and knockdown in a non-tumorigenic cell line did not influence proliferation. Additional enzymes in the proline biosynthesis pathway were also altered in tumors. For instance, ALDH18A1 was up-regulated and knockdown by shRNA decreased proliferation of human HCC cell lines. Metabolomic analyses suggested that the metabolism of glucose was disrupted in PYCR1 knockdown cells, with reduced fluxes through glycolytic and pentose phosphate pathways. Clinical data demonstrated that PYCR1 expression was increased in HCC, and expression levels correlated with increasing tumor grades and were independent predictors of clinical outcome. Overall, our data suggest that enhanced expression of proline biosynthetic enzymes promotes cell proliferation, and that inhibiting PYCR1 or ALDH18A1 may be a novel therapeutic strategy to target HCC.

#3478

Integrative metabolomic and genomic analysis of glioblastoma.

Antony Dayalan, Pravin Kesarwani, Shiva Kant, Prakash Chinnaiyan. _Beaumont Health, Royal Oak, MI_.

Normal 0 false false false EN-US JA X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:Cambria; mso-ascii-font-family:Cambria; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Cambria; mso-hansi-theme-font:minor-latin;} Despite considerable progress in understanding the molecular alterations in Glioblastoma (GBM), the diverse metabolic programs driving their aggressive phenotype remains unclear. We performed global metabolomic profiling in patient-derived GBM (n=80) and low-grade glioma (LGG; n=28). Hierarchical clustering of profiles identified clear metabolic programs differentiating LGG from GBM. GBM had an accumulation of metabolites that appeared mutually exclusive compared to LGG, a majority of which was involved in lipid and peptide metabolism. We next examined metabolic heterogeneity within GBM. Hierarchical clustering identified unique metabolic subtypes in GBM. The first subtype, which represented <10% of the tumors analyzed, was defined by an accumulation of lysolipids, a second subtype, by alterations in amino acid, nucleotide, and lipid metabolism. The third subtype, which comprised the majority of tumors, had a unique accumulation of dipeptides in addition to a heterogeneous accumulation of the metabolites of the first two subtypes. To understand the molecular underpinnings of the metabolic heterogeneity in GBM, integrative analysis using gene expression profiles of matched tumors was performed. Preliminary analyses determined that metabolic heterogeneity in GBM is associated with known molecular subtypes, demonstrating mutual exclusivity of the proneural and mesenchymal metabolic profiles, consistent with their molecular signatures. MGMT methylated and IDH mutated tumors were evenly distributed within the metabolic subtypes, indicating that conserved metabolic programs associated with phenotypic changes are required for gliomagenesis. As aberrant fatty acid metabolism, amino acid metabolism, and accumulation of dipeptides represented core metabolic pathways differentiating LGG from GBM and appeared to contribute towards metabolic heterogeneity, we studied these pathways in further detail. Integrated cross-platform analyses uncovered a tightly orchestrated and highly redundant transcriptional program designed to drive the observed metabolic phenotype, also observed in preclinical models. Collectively, integrated metabolomic and genomic analyses helps in both, understanding biologic processes associated with gliomagenesis and identification of novel therapeutic targets.

#3479

Predicting metabolic vulnerabilities in cancer patients through integrated analysis of patient genomic data and RNAi screens.

Abhinav Achreja,1 Anjali Mittal,1 Ziwen Zhu,1 Frank Weinberg,1 Reva Kulkarni,2 Deepak Nagrath1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _Huron High, Ann Arbor, MI_.

Genomic instabilities, especially tumor suppressor gene (TSG) deletions, are commonplace in tumors. Homozygous and heterozygous deletions of genomic locus of TSGs are essential for cancer progression. However, their deletion is accompanied by loss of neighboring housekeeping genes required for essential cellular functions. Oftentimes, genes that encode metabolic enzymes are lost leading to blockades in crucial metabolic functions. This forces cancer cells to become dependent on paralogous metabolic pathways to compensate for the loss of metabolic function. Identifying such metabolic dependencies present novel opportunities for therapeutic intervention; since targeting genes in these compensatory pathways exposes metabolic vulnerabilities in cancer cells by inducing a collateral lethal effect. Our novel algorithm utilizes genomic information available in publicly available cancer patient databases to obtain candidate metabolic genes. The algorithm then predicts collateral lethal genes involved in compensatory metabolic pathways using (i) elementary flux modes, (ii) flux variability analysis and (iii) multiple metabolic objectives. The predicted genes are validated using RNA interference (RNAi) screening databases of cancer cell-lines. Our integrated algorithm was able to predict previously unexplored metabolic vulnerability in non-small cell lung cancer. These predictions were validated empirically to demonstrate the potential of metabolic collateral lethality in cancers. The algorithm is widely applicable to all types of cancers and takes advantage of publicly available clinical data and empirical screens to predict therapeutic metabolic targets.

#3480

COX-2 alters the metabolic secretome in triple negative human breast cancer xenografts.

Santosh Kumar Bharti, Paul T. Winnard, Yelena Mironchik, Louis Dore-Savard, Balaji Krishnamachary, Zaver M. Bhujwalla. _The Johns Hopkins University School of Medicine, Baltimore, MD_.

Cyclooxygenase-2 (COX-2) is an active mediator of the inflammatory response of cells and plays an important role in the development, progression, invasion, and metastasis of cancers including breast cancer [1]. Tumor interstitial fluid (TIF), the milieu that contains the tumor secretome, is one of the least examined aspects of the TME because of the difficulty in sampling this fluid from tumors. Here, for the first time, we have sampled TIF from COX-2 overexpressing triple negative SUM-149 human breast cancer xenografts and empty vector SUM-149 xenografts.

The cloning, construction of a lentivirus vector expressing COX-2 gene, and the establishment of SUM-149 cells stably overexpressing COX-2 (SUM-COX-2) were reported by us previously[2]. A home-built TIF collection chamber was inserted subcutaneously in female SCID mice with 4-6 1-2 mm tumor pieces packed around the chamber. Once tumors were ~ 400 mm3, TIF was collected from chambers. Each chamber yielded ~50μL of TIF that was analyzed with high-resolution 1H magnetic resonance spectroscopy (MRS) at 750 MHz.

SUM-COX-2 tumors showed consistently higher COX-2 expression compared to SUM-EV tumors. COX-2 overexpression resulted in a significant increase of lactate, glutamate, acetate, and succinate, and a significant decrease of glucose, glutamine, citrate, formate, and lipids; pyruvate tended to decrease. The changes in lactate and lipids are consistent with our earlier observations where COX-2 downregulation in triple negative MDMB-231 human breast cancer cells resulted in a significant decrease of lactate and an increase of lipids and lipid droplets in intact perfused cells[3]. Here, COX-2 overexpression increased glycolysis. Depletion of pyruvate observed here in COX-2 overexpressing cells would limit production of acetyl-CoA and consequently citrate to diminish fatty acid synthesis/lipids. Increased succinate parallels the increase in glutamate/α-ketoglutarate, the upstream intermediate to succinate in the tricarboxylic acid cycle [1], and indicates increased utilization/depletion of glutamine to supplement the TCA cycle. Importantly, accumulation of succinate inhibits HIF-1α prolyl hydroxylase that stabilizes HIF-1α driven cancer promoting metabolic pathways such as enhanced glycolysis and increased ROS[4]. Moreover, increased succinate also through the succinate/succinate dehydrogenase reaction, provides necessary electrons to the electron transport chain upstream of the COX-2 reaction[5]. These data provide new insights into the role of COX-2 in the metabolic secretome and tumor metabolism, and identify metabolic pathways as potential targets for reducing the effects of COX-2 expression in cancer.

Ref.

1. Wang et. al. Nat. Rev. Can. 2010

2. Krishnamachary et al. Oncot. 2017

3. Shah et al. NMR Biomed. 2012

4. Selak et al. Can Cell. 2005

5. Mills et al. Trd in Cell Bio. 2014

#3481

Elucidating the metabolic crosstalk between lymphatic endothelial cells and breast cancer using 1H NMR metabolomics.

Suehelay Acevedo-Acevedo, Douglas C. Millar, Sean P. Palecek. _University of Wisconsin-Madison, Madison, WI_.

Breast cancer is the second most commonly diagnosed cancer among women worldwide. Patient death is typically caused by metastasis development rather than the primary tumor. Metastasis in breast cancer has been shown to occur via blood and lymphatic vessels. Research shows that breast cancer cells 'educate' lymphatic and blood endothelial cells to support tumor growth by stimulating growth factor secretion and directing tumor dissemination. In addition, cell metabolism is altered during malignant transformation. Cancer cells have increased energy and macromolecule biosynthesis requirements to sustain rapid proliferation. However, it remains unclear how tumor endothelial cell metabolism is altered and how metabolism contributes to tumor metastasis. Therefore, this study aims to use 1H NMR metabolomics to identify breast cancer-endothelial cell metabolic interactions in a high-throughput manner. Firstly, we studied if in vitro co-culture with breast cancer altered endothelial cell metabolism. We identified distinct metabolic profiles for lymphatic endothelial cells (LECs) grown in monoculture or co-culture with three different breast cancer cell lines. Principal component analysis revealed LECs co-cultured with breast cancer cells clustered separately from control LECs. Interestingly, LECs co-cultured with triple negative breast cancer cells clustered together with LECs co-cultured with HER2+ breast cancer cell lines indicating that the metabolic changes occurring in these LECs were similar. One-way ANOVA with Tukey's HSD post-hoc analysis revealed 17 significantly different metabolites between all five conditions analyzed including: lactate, glucose, phosphocholine, aspartate, acetate, glycerophosphocholine, lysine and methionine. Quantitative metabolite set enrichment analysis revealed 13 metabolic pathways that were significantly enriched between control LECs and LECs co-cultured with breast cancer cell lines. Enriched pathways included: glycolysis, gluconeogenesis, pyruvate metabolism and protein biosynthesis. Together, these results indicate that culturing LECs with certain breast cancer lines causes significant changes in endothelial metabolism. Alterations in endothelial cell metabolism in response to breast cancer co-culture can further our understanding of tumor-vascular interactions and may lead to identification of metabolic biomarkers and a better understanding of breast cancer metastasis.

#3482

Cachexia has profound metabolic consequences in the heart and skeletal muscle.

Santosh Kumar Bharti, Paul Winnard, Yelena Mironchik, Marie-France Penet, Zaver M. Bhujwalla. _The Johns Hopkins University School of Medicine, Baltimore, MD_.

Cachexia is an underexplored and yet devastating consequence of cancer that is the cause of 20% of all cancer related deaths [1]. Cachexia is associated with poor treatment outcome [3], fatigue, and extremely poor quality of life [2, 3]. Tissue wasting is one of the characteristics associated with cachexia syndrome. Here we have used 1H MRS to characterize the metabolic profile of skeletal muscle and heart muscle obtained from normal mice and noncachexia (Panc1) and cachexia inducing (Pa04C) human pancreatic cancer xenograft bearing mice to further understand this syndrome.

Male severe combined immunodeficient (SCID) mice were inoculated in the right flank with cancer cells (2 × 106) and in the right hind leg muscle with cachexia reporter myoblasts (2×106) [4]. Mice were sacrificed once tumors were ~400 mm3, and the heart and skeletal muscle were harvested for dual phase solvent extraction. 1H magnetic resonance (MR) spectra of the water phase were acquired with a 750 MHz MR spectrometer.

Quantitative changes in metabolite levels were obtained from 1H MR spectra. Leucine, creatine, lactate and glucose were significantly lower, and acetate and formate were significantly higher, in the muscle of cachectic mice compared to non-cachetic mice. Cachectic mice had significantly lower alanine, succinate, glycine, lipid and PUFA (poly unsaturated fatty acids), and significantly higher acetate, pyruvate, and formate compared to normal mice. Non-cachectic mice had significantly lower alanine, succinate, phosphocreatine, and glycine, and significantly higher leucine, isoleucine, valine, pyruvate, creatine, taurine and glucose compared to normal mice.

In the heart, leucine, isoleucine, valine, aspartate, glucose, lipid and PUFA were significantly lower, and glutamine was significantly higher, in cachectic mice compared to non-cachetic mice. Cachectic mice had significantly lower leucine, isoleucine, valine, lactate, alanine, glutamate, and PUFA, and significantly higher glutamine and glucose compared to normal mice. Non-cachectic mice had significantly lower alanine, and significantly higher glucose and lipid compared to normal mice.

These data highlight, for the first time, the profound metabolic changes that occur in skeletal muscle and the heart with cachexia, identifying potential in vivo 1H MRS indices to detect the onset of cachexia from changes in branched chain amino acids, glucose, and PUFAs. Our data also provide new insights into the effects of cachexia as well as noncachexia inducing tumors on skeletal muscle and heart metabolism that may lead to metabolic interventions in 'metabolotheranostic' strategies to reduce the morbidity associated with cancer and cachexia.

Supported by NIH R01CA193365, R35CA209960, and P30CA06973.

Ref.

1. Argiles et al: Nat. Rev. Can 2014, 14(11):754

2. Fearon et al: HPB (Oxford) 2010, 12(5):323

3. Ozola et al: Pancreatology 2015, 15(1):19

4. Winnard et al: Can Res. 2016, 76(6):1441

#3483

1 **H MRS characterization of the cachetic brain metabolome induced by human pancreatic pancer xenografts.**

Santosh Kumar Bharti,1 Paul T. Winnard,1 Yelena Mironchik,1 Marie-France Penet,1 Anirban Maitra,2 Zaver M. Bhujwalla1. 1 _Johns Hopkins University School of Medicine, Baltimore, MD;_ 2 _The University of Texas, Houston, TX_.

The uncontrollable extreme weight loss due to cachexia results in a particularly poor quality of life causing profound weakness, listlessness, and an inability to function. A major characteristic of cachexia is accelerated skeletal muscle and fat storage wasting causing nutrient mobilization both directly as lipid and amino acids, and indirectly as ketone bodies and glucose derived from liver keto- and gluconeogenesis with systemic distribution including to the tumor through the bloodstream. Previously, we have reported the initial characterization of a myoblast optical imaging reporter that allowed real-time longitudinal monitoring of the early onset of cancer induced wasting and measured plasma metabolic changes associated with pancreatic ductal adenocarcinoma (PDAC)-induced cachexia. Here, for the first time, we have performed high-resolution quantitative 1H MR spectroscopy (MRS) of brain tissue to characterize the brain metabolome of normal mice and mice with human PDAC xenografts that induce cachexia (Pa04C cells) or are noncachectic (Panc1 cells).

Male severe combined immunodeficient mice were inoculated in the right flank with cancer cells (2×106) and in the right hind leg muscle with reporter myoblasts (2×106). Once the mice were sacrificed, brains were harvested, freeze clamped and stored in -80°C until 1H MRS analysis. Dual phase solvent extraction was performed on brain tissues and MR spectra were acquired on 750 MHz (17.6T) spectrometer.

As anticipated, mice with cachexia-inducing Pa04C tumors showed significant weight loss with time. For the first time, we found that brains from Pa04C tumor bearing mice exhibited a profound reduction in water soluble metabolites as compared to Panc1 and nontumor bearing normal mice. Significant decreases in neurotransmitters: γ-aminobutyric acid (GABA), N-acetyl aspartate (NAA), and taurine as well as lactate, myo-inositol, phosphocholine, glycerophosphocholine, creatine, formate, and essential amino acids: Leu, ILe, Val, and Phe were observed in brains from cachectic mice compared to noncachectic and healthy mice. Non-cachexia inducing Panc1 tumors also induced a significant decrease of brain GABA, glutamate, aspartate, total choline and tyrosine compared to normal brains. These results provide new insights into profound changes in brain metabolism during cachexia that are likely indicative of compromised CNS function and may be a major contributing factor to the systemic control of cachectic wasting. These data provide strong evidence to support investigating new metabolic interventions to reverse CNS injury and cachexia. and provide new CNS targets for early detection using in vivo MRS techniques.

Acknowledgement: This work was supported by NIH R01CA193365, R35CA209960, and P30CA06973.

#3484

In vivo **metabolomics reveals a potentially potent combination therapy for MYC-driven medulloblastoma.**

Allison R. Hanaford, Brad Poore, Jesse Alt, Barbara Slusher, Charles G. Eberhart, Eric H. Raabe. _Johns Hopkins Univ. School of Medicine, Baltimore, MD_.

The MYC oncogene is associated with aggressive forms of the pediatric brain tumor medulloblastoma. MYC promotes oncogenesis in part by altering cellular glucose and glutamine metabolism. We hypothesized that MYC-driven medulloblastoma would be sensitive to the glutamine metabolic inhibitor 6-diazo-5-oxo-l-norleucine (DON). In MYC-driven medulloblastoma cell lines, 10uM DON treatment increases apoptosis by up to 280% (p<0.04) as compared to vehicle control. In human neural stem cells transformed with MYC, but not in untransformed cells, DON treatment caused up to a 127% increase in apoptosis compared to vehicle (p<0.001). Once-weekly DON therapy increased median survival by up to 246% (p<0.004) in three different MYC-driven medulloblastoma orthotopic xenograft models. To elucidate the mechanism of DON, we performed stable isotope resolved metabolomics (SIRM) on two MYC-driven medulloblastoma tumor models. SIRM revealed that tumors from DON treated animals had decreased production of asparagine (p<0.016). Production of aspartate was not decreased (p>0.4), suggesting that DON was inhibiting asparaginase synthetase, the enzyme that transfers the ammonia group from glutamine to aspartate to generate asparagine. We hypothesized that DON efficacy could be enhanced by asparaginase (ASNase), an enzyme that breaks down asparagine into aspartate and ammonia. ASNase is a commonly used therapy in pediatric patients with leukemia and lymphoma. In MYC-driven medulloblastoma cell lines, treatment with low-dose DON or ASNase as single agents did not significantly increase apoptosis by cleaved caspase-3 immunofluoresence or cleaved-PARP western blot. The combination of low-dose DON and ASNase increased apoptosis by up to 577% (p<0.0001). Similarly, in human neural stem cells transformed with MYC, the combination of low-dose DON and ASNase increased apoptosis by up to 523% (p<0.0001). We hypothesized that depletion of asparagine from both intracellular and extracellular pools would induce the uncharged tRNA/endoplasmic reticulum stress response. Western blotting revealed that the combination of DON and ASNase increased expression of the transcription factor ATF4 and the pro-apoptotic protein CHOP, which are critical components of the uncharged tRNA response. ATF4 is a known regulator of endoplasmic reticulum stress induced apoptosis through transcription of pro-apoptotic proteins. These data suggest that DON and ASNase could be a powerful therapeutic combination for treating MYC-driven medulloblastoma and possibly other MYC-driven malignancies.

#3485

Transcriptomic and metabolomic profiles in renal cell carcinoma (RCC) tumors reflect ontogeny of RCC subtypes.

Pascale Fisel,1 Florian Büttner,1 Anna Reustle,1 Verena Klumpp,1 Stefan Winter,1 Steffen Rausch,2 Jörg Hennenlotter,2 Stephan Kruck,2 Arnulf Stenzl,2 Judith Wahrheit,3 Denise Sonntag,3 Marcus Scharpf,4 Falko Fend,4 Abbas Agaimy,5 Arndt Hartmann,5 Jens Bedke,2 Matthias Schwab,1 Elke Schaeffeler1. 1 _Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology Stuttgart and University of Tuebingen, Stuttgart, Germany;_ 2 _Department of Urology, University Hospital Tuebingen, Tuebingen, Germany;_ 3 _Biocrates Life Science AG, Innsbruck, Austria;_ 4 _Institute of Pathology and Neuropathology, University Hospital Tuebingen, Tuebingen, Germany;_ 5 _Institute of Pathology, University Erlangen-Nuernberg, Erlangen, Germany_.

Introduction

The renal cell carcinoma (RCC) subtypes clear cell RCC (ccRCC), papillary RCC (papRCC) and chromophobe RCC (chRCC) are proposed to arise from distinct cell types of the nephron. The subtypes differ not only in their incidence, but also in terms of metastasis risk and prognosis. The most common form ccRCC, accounting for ~75% of the cases, is characterized by reprogramming of tumor metabolism and is therefore assumed to be a metabolic disease. In contrast, little is known about the metabolic alterations in non-ccRCC subtypes. In this study, we applied transcriptomics and metabolomics profiling to identify key pathways altered in specific RCC subtypes and in ccRCC-derived metastases. In addition, the potential of metabolites to improve subtype classification and to identify promising drug targets for new therapeutic interventions was elucidated.

Experimental Procedures

Genome-wide expression analyses were performed in ccRCC (n=58), chRCC (n=19), papRCC (n=14), matching non-tumor tissues, and ccRCC-derived metastases (n=9) using HTA 2.0 gene expression arrays. The transcriptomic profiles were compared to expression data of RCC cohorts of TCGA (KIRC, n=452; KIRP, n=260; KICH, n=59). In addition, a targeted metabolomics approach using LC/MS technology was applied in these tissues.

Results

Pathway enrichment analyses based on metabolic gene expression signatures revealed metabolic alterations in proximal vs. distal cells of the nephron. Accordingly, the respective RCC subtypes arising from proximal (ccRCC, papRCC) or distal (chRCC) tubular cells showed major differences in transcriptional activity of metabolic pathways, reflecting their cell type of origin. Based on metabolite levels, hierarchical cluster analyses separated the samples into three groups, differentiating non-tumor tissues, chRCC, as well as ccRCC, ccRCC-derived metastases and papRCC, indicating differences depending on RCC ontogeny also for metabolic profiles. ccRCC-derived metastases clustered with primary ccRCC tumors, irrespective of affected organs. When comparing ccRCC and chRCC subtypes, major differences were found, e.g. for certain biogenic amines of the polyamine pathway. Differential regulation of this pathway was also reflected on transcriptome level. In vitro experiments revealed that interfering with the polyamine pathway by inhibiting the ornithine-decarboxylase with difluoromethylornithine, reduced cell viability and mitochondrial activity in RCC cells.

Conclusion

In this study, we elucidated the transcriptomic and metabolomic profiles of RCC tumors, which confirm the distinct ontogeny of RCC subtypes from different parts of the nephron. Furthermore, we found that identification and quantification of differentially regulated metabolites can improve RCC classification and represent an attractive opportunity to reveal subtype-specific therapeutic targets.

#3486

Metabolomics in estrogen receptor or triple-negative breast cancer.

Jennifer J. Hu,1 Cristiane Takita,1 Eunkyung Lee,2 Jean Wright3. 1 _Univ. of Miami Sylvester Comp. Cancer Ctr., Miami, FL;_ 2 _University of Central Florida, Orlando, FL;_ 3 _Johns Hopkins University, Baltimore, MD_.

Breast cancer is the most frequently diagnosed cancer in women. Metabolic syndrome and obesity may contribute to triple-negative breast cancer (TNBC) and there are obesity-related alterations in metabolites. Therefore, we pilot-tested metabolomics in ER-negative or TNBC using urine samples from 60 breast cancer cases. In total, 475 metabolites were quantified in different super-pathways: 147 in amino acid, 38 in carbohydrate, 27 in cofactors and vitamins, 12 in energy, 50 in lipid, 31 in nucleotide, 17 in peptide, and 153 in xenobiotics. Our data in 13 ER- and 47 ER+ showed that urinary beta-alanine is higher (3.35+3.73 vs. 1.82+2.26) in ER- breast cancer patients. Our data also show, the first time, that the urinary beta-alanine was higher in TNBC (3.60+4.33; N=8) compared to other subtypes (1.93+2.32; N=52). Beta-alanine is a nonessential amino acid that has previously been shown to be metabolized into carnosine, which functions as an intracellular buffer. In breast cancer cells, beta-alanine reduces both tumor cell migration and proliferation without acting in a cytotoxic fashion. It also significantly increased breast cancer cell sensitivity to doxorubicin. Our data on xanthine are also consistent with the previous study on breast tumor that slightly higher level was observed in ER- patient (1.41+1.35 vs. 1.16+0.88). The level of xanthine was significantly higher in TNBC compared to other subtypes (1.67+1.69 vs. 1.14+0.84). More importantly, our pilot data also showed that urinary mannose levels were significantly higher in ER- tumor (2.21+1.90 vs. 0.90+1.21 p=0.032) and TNBC (2.19+1.77 vs. 1.03+1.38, p=0.037), which is consistent with a previous study that reported that serum mannose levels can differentiate patients with metastatic and localized breast cancer. Metabolic drugs for cancer, such as glutaminase inhibitors, are currently under development and being tested in clinical trials. Therefore, if our data are validated, metabolomics of ER- or TNBC will contribute to innovative discoveries that will impact breast cancer prevention and precision medicine targeting metabolomic pathways.

#3487

Bioenergetic profiling of cancer cell lines: Quantifying the impact of glycolysis on cell proliferation.

Natalia Romero, Pamela M. Swain, Yoonseok Kam, George Rogers, Brian P. Dranka. _Agilent Technologies, Lexington, MA_.

Fast proliferating cells require tight regulation to achieve a balance between the use of nutrients for ATP production (through glycolysis and oxidative phosphorylation) and the use of intermediate metabolites to sustain the increased biosynthetic activity. Cancer cells, but also high proliferative non-transformed cells exhibit high glycolytic activity during rapid proliferation even in the presence of normal oxygen concentrations in culture. However, despite the high glycolytic activity, the role of glycolysis is not necessary as a major contributor of ATP but to allow nutrient assimilation into biosynthetic precursors. Using Agilent Seahorse extracellular flux analysis, we have developed a cell-based assay which allows simultaneous measurement of the two-main cellular metabolic pathways to calculate the total rate of cellular ATP production as well as the fractional contribution from each pathway. The assay allows for real time changes in total ATP production rate to be quantified, and also the relative source of that ATP after exposure to drugs or changes in extracellular fuel supply. When we applied this new assay to a panel of 20 cancer and highly proliferative cell lines, we found that even in cell lines considered highly glycolytic, ATP production from glycolysis never represents more than 65% of total energy production and between 30-50% for most of the cell lines analyzed. The correlation between glycolytic ATP contribution to total ATP production and other cell phenotypes such as proliferation rate and motility was also analyzed. The use of this assay will allow for improved characterization of the bioenergetic profile of cancer cell variants, discrimination between normal and cancer cell types, and allow researchers to better understand the role of aerobic glycolysis in cell proliferation.

#3488

Elucidation of Warburg effect and cell cycle regulations through analysis of cancer tissue omics data and kinetic modeling.

Tao Sheng. _Univ. of Georgia, Athens, GA_.

Cancer cells tend to depend more on anerobic glycolysis to generate adenosine 5'- triphosphate (ATP), which is an inefficient energy producing process termed as "The Warburg effect". The causes or functions of Warburg effect have been extensively studied, however, the growth advantage it confers to cancer cells still remains unclear. In this study, we propose that the metabolic shift in cancer cells would facilitate the accumulation of nutrients and biomass (e.g., nucleotides, amino acids and lipids), which would promote the cell cycle initiation and progression as unicellular organisms do. Specifically, by comprehensively analyze the gene expression and mutational profile in 16 cancer types, we identified (1) the gene expression profile of cancer cells is evolutionarily more unicellular like when compared to normal; and (2) certain cell cycle associated mutations are associated with the metabolic alternations in cancer cells, such as nucleotide synthesis pathways and lipids metabolism. Moreover, we developed a system of kinetic equations to capture the metabolic relations between nutrient metabolism and cell cycle initiation and progression. The kinetic model shows that the rapid nucleotide synthesis as well as high influx of glutamate would affect the activities of cell cycle signaling and CDKs genes. Our study provides a novel mechanistic view in linking the Warburg effect and cancer cell cycle initiation and progression.

#3489

Comprehensive characterization of the SLC transporters in human cancer.

Ruiping Wang, Shaojun Zhang, Andy Futreal, Linghua Wang. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Metabolic reprogramming has now been well recognized as one of 10 hallmarks of cancer. The plasma membrane transporters, that enable efficient cellular metabolism and aid in nutrient sensing, are receiving increasing attention in recent years. Membrane transporters include 3 main classes: ABC transporters, p-type ATPases, and the solute carrier family (SLC). The SLC group of transporters includes nearly 400 members organized into 52 families that mediate the transport of a wide variety of substrates across biological membranes. Until recently, little was known about the role of these SLC transporters in the mechanisms of cancer development and progression. Yet, the genomic landscape and clinical relevance of SLC transporters in human cancer have not been investigated systematically. We here performed a comprehensive characterization of 396 SLC transporters by integrating multi-platform data across over 30 tumor types from The Cancer Genome Atlas. We observed frequent dysregulation of SLC transporters across cancer types and identified cancer type- and subtype-specific expression signatures. We identified significant correlations of these SLC transporters with the activity of cancer metabolic pathways and explored their complex regulatory networks. We also investigated the role of SLC transporters in tumor metastasis. We further evaluated the potential clinical relevance of these SLC transporters and identified multiple SLC transporters that were significantly associated with patients' clinical outcomes. Our study provided an in-depth understanding of the role of SLC transporters in cancer metabolic reprogramming, elucidated their mechanisms in cancer development and metastasis, and provided novel biomarkers and potential therapeutic targets that can be further developed for future clinical applications.

#3490

Mechanism of action for antioxidant Twendee X on hepatic cancer cell line HepG2.

Haruhiko Inufusa. _Gifu University, Gifu, Japan_.

[Purposes of the study] Reactive oxygen species (ROS) are associated with clinical condition of cancer patient, and closely involved in cancer growth and metastasis. Twendee X (TWX) is a composition consisting of Vitamin C, Glutamine, Cystine, Riboflavin, Succinic acid, Fumaric acid, Coenzyme Q10, and Niacin. TWX strongly reduces oxygen radicals or ROS in vitro and in vivo (World Intellectual Property Organization WO2013/072441 A1 COMPOSITION FOR PROTECTION AGAINST CELL-DAMAGING EFFECTS). TWX has significant effects of reducing tumor growth and inhibition metastasis. Hydrogen peroxide level (d-ROMs test) of tumor bearing nude mouse in serum was significantly reduced and increased NK-activity (2016 AACR meeting). Mechanism of action was examined for TWX using hepatic cancer cell line HepG2.

[Experimental procedures] Human hepatic cancer cell line HepG2 was used in this study. Cell proliferation experiments of antioxidant by ICDD (France). Metabolome analysis was by Human Metabolome Technologies (Tokyo). In both experiments, HepG2 cell line treated one hour with TWX (60ug/ml). [Data] TWX inhibits the RAF-MEK-ERK pathway starting from ERK. Metabolome analysis shows total shift of metabolism, and especially increase mitochondrial metabolism and ATP production. [Conclusion] Effects of TWX on cancer cell line are not only increase immune activity, but also inhibit proliferation pathway, and mitochondrial metabolism change. Several publications using Vitamin-E or Cysteine as anti-oxidant showed cancer progression or increase metastasis. However, Vitamin-E or Cysteine has limited anti-oxidant effects and hydrogen peroxide level (d-ROMS test) dose not change. On the other hand, TWX has significant effects reduce hydrogen peroxide level, and Japanese Association for Dementia Prevention already started clinical trial for MCI and after stroke patients. For the cancer patients, clinical trial using TWX combination with standard therapy will be start soon.

### Oncogene Growth Factors and Their Receptors

#3491

Heterogeneous extrachromosomal amplification of mutant PDGFRA is associated with an aggressive phenotype in glioblastoma.

Artem D. Berezovsky,1 Andrea D. Transou,1 Susan M. Irtenkauf,1 Laura A. Hasselbach,1 Julie Koeman,2 Hoon Kim,3 Roel G. Verhaak,3 Tom Mikkelsen,1 Laila M. Poisson,1 Ana C. deCarvalho1. 1 _Henry Ford Health System, Detroit, MI;_ 2 _Van Andel Institute, Grand Rapids, MI;_ 3 _The Jackson Laboratory for Genomic Medicine, Farmington, CT_.

Background & Objective: Receptor tyrosine kinase (RTK) signaling is altered in over 80% of glioblastoma (GBM) cases, frequently through gene amplification. About 14% of GBMs carry amplification in the gene coding for platelet-derived growth factor receptor A (PDGFRA), according to TCGA. PDGFRA plays a key role in brain development and is associated with GBM proneural (PN) subtype. Despite the frequency of oncogenic RTK signaling in GBMs, RTK inhibitors have not yet achieved sufficient efficacy in clinical trials to earn FDA approval. Imatinib, a multi-kinase inhibitor that targets PDGFRA, has not shown efficacy in earlier clinical trials for GBM, in which amplification status was not an inclusion criteria. It has been reported that treatment of GBMs carrying extrachromosomal (ecDNA) amplification with EGFR inhibitors leads to a decrease in ecDNA copy number as a mechanism of resistance. Here, our objective was to assess the heterogeneity of ecDNA PDGFRA amplification in a newly diagnosed PN GBM tumor (HF3253), and to evaluate whether similar modulation of ecDNA copy number could be attained by Imatinib treatment of patient-derived xenografts (PDX). Experimental Approaches & Results: PDGFRA amplification was detected by low pass whole genome sequencing and confirmed to be extrachromosomal by fluorescent in situ hybridization (FISH) in metaphase spreads using PDGFRA and centromere 4 labeled DNA probes. The amplified PDGFRA also harbored a novel missense mutation corresponding to the extracellular domain. PDGFRA FISH signals/number ranged from 3 to 100 in the HF3253 samples, with high signal (> 20) evident in 67%, 39%, and 43% and low amplification signal (6 < x < 9) evident in only 0%, 6%, and 19% of nuclei from biopsy, neurosphere, and xenograft respectively. HF3253 neurospheres were orthotopically implanted into the brains of immunocompromised nude mice. Imatinib mesylate was administered by oral gavage at a 75 mg/kg/day dosage in 5-day cycles with 2-day drug holiday intervals, starting 2 weeks post implant. Control mice received vehicle gavage under the same schedule. Mice were monitored daily and sacrificed on the basis of weight loss or neurological symptoms. HF3253 PDX treated with Imatinib mesylate did not have a significant survival advantage (median survival: 47.2 days) relative to control mice (median survival: 45.8 days; log rank test p value = 0.7825). The untreated and treated PDXs exhibited high levels of phospho-PDGFRA by IHC. Two independent in vitro dose response assays showed no difference in the IC50 for Imatinib between PDGFRA positive and negative GBM neurospheres: (9.447 µM, 8.384 µM) and (8.972 µM, 6.896 µM). Conclusions: We have developed a patient derived model of glioblastoma that retains ecDNA PDGFRA amplification and high levels of expression and RTK activation. Although PDGFRA is a driver of malignancy, our results show that better inhibitors are needed.

#3492

Wnt5a induces ROR1 to associate with DOCK1 and promote growth of breast cancer cells.

Md Kamrul Hasan, Victoria Tripple, George F. Widhopf, Suping Zhang, Barbara A. Parker, Thomas J. Kipps. _Moores Cancer Center, University of California San Diego, San Diego, CA_.

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic protein expressed on breast cancer, but not on virtually all normal adult tissues. Studies have found that high-level expression of ROR1 in tumors of breast cancer patients associated with enhanced cell growth, aggressive disease, and shorter overall survival compared to that of patients with tumors that had low expression of ROR1 (Zhang S., et al., PLOS ONE, 7(3): e31127, 2012). Analysis of ROR1-immune precipitates of primary breast cancer patient-derived-xenograft (PDX) lysates via immunoblot analyses revealed that ROR1 was associated with DOCK1 (Dedicator of Cytokinesis 1, also known as DOCK180) in breast cancer PDX tumors. DOCK1 is a member of the DOCK-A subfamily of guanine exchange factors (GEFs) specific for Rac1 that is expressed in breast cancer PDX tumors or cell lines. DOCK1 also contains a N-terminal SH3 domain. We found that ROR1 dissociated from DOCK1 in PDX cells cultured in serum-free medium, unless the cells were stimulated with exogenous Wnt5a. Wnt5a could induce ROR1 to complex with DOCK1 and cause activation of Rac1; this effect could be inhibited by silencing DOCK1 in PDX cells. Furthermore, these effects of Wnt5a on PDX cells also could be blocked by treatment of the PDX cells with cirmtuzumab, a humanized anti-ROR1 mAb, which is undergoing clinical evaluation in patients with cancer. We corroborated these findings using the breast cancer cell-line MCF7, which also expresses DOCK1, but does not express ROR1. MCF7 cells can be transduced to express ROR1 or various mutant forms of ROR1, allowing us to examine the structure-function relationships required for ROR1-DOCK1 interactions. We confirmed that DOCK1 complexes with ROR1 in response to Wnt5a in MCF7-ROR1 cells. We find that silencing DOCK1 specifically impaired the capacity of Wnt5a to enhance growth of MCF7-ROR1 cells in vitro. We generated truncated forms of ROR1 and found the cytoplastmic proline-rich domain (PRD) of ROR1 was required for ROR1 to complex with DOCK1 and activate Rac1 upon stimulation with Wnt5a. We next introduced single amino-acid substitutions of proline (P) to alanine (A) in the ROR1-PRD at positions 784, 808, 826, or 841 in potential SH3-binding sites. In contrast to wild-type ROR1, or other ROR1P->A mutants, ROR1P(808)A had impaired capacity to recruit DOCK1 to ROR1 in response to Wnt5a. Moreover, unlike MCF7 cells transfected with wild-type ROR1 or ROR1 with P->A substitutions at positions 784, 826, or 841, MCF7 cells transfected to express ROR1P(808)A did not have a growth advantage over that of MCF7 cells that do not express ROR1. This study reveals that the recruitment of DOCK1 may be critical for the capacity of Wnt5a to enhance breast cancer cell growth, which may contribute to the observed increased tendency for disease progression in breast cancer patients who have tumors that express high-levels of ROR1.

#3493

Promoting role of AKR1B10 in HER2+ breast cancer at early stage.

Ramina Khoshaba, Deliang Cao. _SIU School of Medicine, Springfield, IL_.

Background:

Breast cancer is the second most common cause of female cancer-related death in the United States. Aldo-keto reductase 1B10 (AKR1B10) is a 35-kDa protein that is highly expressed in breast cancer in infiltrative and Ductal Carcinoma in Situ (DCIS) stages. Human epidermal growth factor receptor2 (HER2) is a tyrosine kinase receptor which is highly expressed in 15-20% of infiltrative breast cancer but over 60-70 % in DCIS.

Methods:

We targeted AKR1B10 and HER2 expression using AKR1B10 and HER2 expression lentiviruses in a panel of breast cancer cell lines that have different endogenous expression status of AKR1B10 or HER2, including MCF-7(AKR1B10-/HER2 low), ZR-75-1(AKR1B10-/HER2+), MDA-MB-468 (AKR1B10+/HER2-) and the normal human mammary cells MCF10A (AKR1B10-/HER2-). We investigated the effects of AKR1B10 or HER2 expression on cell proliferation, clonogenicity, adhesion, migration, and invasion. Western blots were performed to check integrin α5, δ-catenin expression and the phosphorylation of signaling proteins in Raf/MEK/ERK and AKT/P-mTOR pathways.

Results:

Our data showed that AKR1B10 promoted the proliferation of HER2+ human breast cancer cell. The results of colony forming and soft agar assays demonstrated that AKR1B10 stimulated clonogenic efficiency of HER2+ cells. Also, AKR1B10 promoted HER2+ cells adhesion to extracellular matrix, facilitated their migration and significantly increased their invasion capability. Moreover, it upregulated integrin α5, δ-catenin expression, and promoted HER2+ cells growth and invasiveness through the activation of AKT/mTOR and Raf/MEK/ERK pathways

Conclusion:

AKR1B10 promotes HER2+ human breast cancer cell proliferation and invasiveness.

#3494

Downregulation of SREBPs inhibited colon cancer cell growth and tumor initiation by alteration of metabolism.

Yang-an Wen, Xiaopeng Xiong, Yekaterina Zaytseva1, Tianyan Gao. _Markey Cancer Center, Lexington, KY_.

Sterol regulatory element binding proteins (SREBPs) belong to a family of transcription factors that regulate the expression of genes required for the synthesis of fatty acids and cholesterol. There are three SREBP isoforms, SREBP1a, SREBP1c and SREBP2, have been identified in mammalian cells. SREBP1a and SREBP1c are derived from a single gene through the use of alternative transcription start sites. Increasing evidence suggests that increased lipogenesis is needed for cancer cells to accommodate high rates of proliferation. Activation of SREBP1 and enhanced expression of its target genes have been observed in different types of human cancers. However, the functional importance of SREBPs in colon cancer remains to be fully elucidated. In this study, we investigated the role of SREBP-mediated lipid biosynthesis in promoting tumor growth and initiation by regulating cellular metabolism in colon cancer. Knockdown of SREBP-1 or SREBP-2 decreased the expression of target genes required for lipid biosynthesis and levels of fatty acids and triacylglycerides in colon cancer cells. Bioenergetic analysis using Seahorse XF96 Extracelluar Flux Analyzer revealed that silencing SREBP-1 or SREBP-2 expression reduced the mitochondrial respiration, glycolysis, as well as fatty acid oxidation suggesting an inhibition of cellular metabolism. As a result, the rate of cell proliferation and the ability of cancer cells to form tumor spheroids in suspension were decreased. Similar results were obtained in colon cancer cells in which SCAP, an upstream protease responsible for activating SREBP-1 and SREBP-2, was silenced. Mechanistically, downregulation of SREBPs inhibited the activation of AMPK and Akt signaling. Furthermore, knockdown of SREBP-1 or SREBP-2 inhibited xenograft tumor growth in vivo and significantly decreased the expression of genes associated cancer stem cells. Taken together, our findings establish the molecular basis of SREBPs-dependent metabolic regulation and provide a rationale for targeting fatty acid synthesis as a promising approach in colon cancer treatment.

#3495

Trip13, a target of miR192, facilitates colorectal cancer progression through WNT/β-catenin signaling.

Sumit Agarwal, Balabhadrapatruni V. Chakravarthi, Darshan S. Chandrashekar, Rong Jun Guo, Pran Datta, Sooryanarayana Varambally, Upender Manne. _University of Alabama at Birmingham, Birmingham, AL_.

Most colorectal cancers (CRCs) exhibit genomic instability or chromosomal instability (CIN) in the form of aneuploidy. These aberrations are associated with chromosomal gains or losses. In human malignances, levels of a member of the AAA-ATPases family, TRIP13, correlate with CIN. In the present study, we investigated the expression and biological function of TRIP13 in CRC. In silico gene expression profiling using the UALCAN (http://ualcan.path.uab.edu/) web portal showed elevated TRIP13 expression in CRCs as compared to normal/benign colonic epithelial tissues. Studies employing immunoblot analyses confirmed elevated expression of the TRIP13 protein in CRC tissues compared to their matched normal/benign colorectal tissues. Concurrent immunohistochemical analysis of tissue microarrays, containing normal and tumor tissues, further validated cytoplasmic TRIP13 expression in CRCs as compared to their contiguous normal/benign epithelial components. To understand the functional significance of TRIP13 in CRCs, particularly in regard to progression of cancer cells, experiments were performed by utilizing lentivirus-mediated TRIP13 silencing in CRC cell lines that express high levels of TRIP13. Depletion of TRIP13 impeded cell proliferation, colony formation, invasion, cell motility, and spheroid-forming capacity. TRIP13 depletion also showed suppression of EGFR activation and WNT/β-catenin signaling. Furthermore, bioinformatic analysis revealed that TRIP13 is a predicted target of miR192. Dual-luciferase reporter assays showed that miR192 binds to the 3' UTR of TRIP13. Also, overexpression of miR192 in HCT116 cells lowered TRIP13 expression and reduced cell proliferation, colony formation, and invasion, suggesting that it is a tumor suppressor. Thus, these findings demonstrate a function of TRIP13 in the progression of CRCs and indicate that TRIP13 inhibition by small molecules is a possible strategy for treatment of CRC.

#3496

**GC1118, a novel anti-EGFR andtibody, shows more potent antitumor activity regardless of KRAS mutation or high-affinity lignad stimulation compared with cetuximab in gastric cancer.**

Ji Eun Park,1 Mei Hua Jin,1 Ah-Rong Nam,1 Ju-Hee Bang,1 Do-Youn Oh,2 Yung-Jue Bang2. 1 _Cancer Research Institute,Seoul National Univ. College of Medicine, Seoul, Republic of Korea;_ 2 _Department of Internal Medicine,Seoul National Univ. College of Medicine, Seoul, Republic of Korea_.

Background: EGFR overexpression in gastric cancer (GC) has been reported in about 30% of patients. However, cetuximab and panitumulab, anti-EGFR antibodies, have failed to improve overall survival of GC patients compared with standard chemotherapy alone. GC1118 is a novel anti-EGFR antibody with distinct binding epitope compared to cetuximab or panitumumab, and has superior inhibitory activity against high-affinity EGFR ligands (Mol Cancer Ther 2016). In this study, GC1118 was tested to evaluate the antitumor effects in gastric cancer cells.

Methods: Using a total of 15 kinds of GC cell lines (SNU-1, SNU-5, SNU-16, SNU-216, SNU-484, SNU-601, SNU-620, SNU-638, SNU-668, SNU-719, AGS, MKN-28, MKN-45, NCI-N87, and KATO-), GC1118, cetuximab, cisplatin, and 5-FU were tested. Cells were treated with or without high-affinity EGFR ligands, EGF 20ng/mL or HB-EGF 100ng/mL.

Result: GC1118 exhibited more potent antigrowth effects in the majority of GC cells than cetuximab in colony forming assay and MTT assay, regardless of KRAS mutation status of cells. The synergistic efficacy was observed when cells were treated with GC1118 in combination with cytotoxic chemotherapeutic agents (cisplatin or 5FU) in colony formation assay and migration assay compared to each treatment alone, even under the EGFR-ligand stimulation condition. GC1118 significantly blocked the p-AKT or p-ERK signaling under the presence of ligand induced-EGFR activation. However, cetuximab failed to block p-AKT or p-ERK that was upregulated by EGF or HB-EGF. Moreover, this signal-blocking activity was more potent in case of GC1118 compared with cetuximab in the chemotherapy combination experiment.

Conclusion: GC1118 showed more potent antitumor effects in GC compared with cetuximab. In combination with cytotoxic chemotherapy, GC1118 also has more potent activity compared with cetuximab regardless of KRAS mutation status or high-affinity ligand stimulation condition. This study supports further clinical development of GC1118 in gastric cancer.

#3497

Fibroblast growth factor receptor 3 (FGFR3) aberrations in muscle-invasive urothelial carcinoma.

Young Saing Kim,1 Soon Il Lee,2 Se Hoon Park3. 1 _Gachon University Gil Medical Center, Incheon, Republic of Korea;_ 2 _Dankook University Hospital, Cheonan, Republic of Korea;_ 3 _Sungkyunkwan University Samsung Medical Center, Seoul, Republic of Korea_.

Purpose: Muscle-invasive urothelial carcinoma (UC) has a poor prognosis and novel approaches to systemic therapy are required. Recent studies show that FGFR3 activation is an important contributor to tumor development and angiogenesis in UC. Therefore, FGFR3 is a potential therapeutic target in UC. The purpose of this study was to evaluate the rates and types of FGFR3 aberrations in invasive UC.

Methods: We analyzed primary tumor samples from 74 UC patients who had received radical cystectomy (n=40) or ureteronephrectomy (n=34). Ion AmpliSeq Cancer Hotspot Panel v2 and nCounter Copy Number Variation Assay were used to detect FGFR3 aberrations.

Results: Fifty-four patients (73%) had high-grade tumors, and 62% had lymph node involvement. Sixteen patients (22%) harbored FGFR3 alterations, the most common of which was FGFR3 mutations (n=13). These mutations included Y373C (n=3), N532D (n=3), R248C (n=2), S249C (n=2), G370C (n=1), A797P (n=1), and 746_747insG (n=1). In all three patients with FGFR3 rearrangement, the fusion partner was TACC3. The frequency of FGFR3 aberrations was higher in bladder UC (28%) than in UC of the renal pelvis and ureter (15%), but the difference was not statistically significant (P=0.183). Genes that were co-aberrant with FGFR3 included APC (88%), PDGFRA (81%), RET (69%), and TP53 (69%).

Conclusions: FGFR3 abnormalities are less frequently seen in upper tract UC than bladder UC. Patient selection could be critical in designing new approaches for anti-FGFR3 therapy in the perioperative setting.

#3498

Regulation of AR through an FGF5 driven mechanism.

Dalton McLean. _University of Wisconsin, Madison, WI_.

Purpose

To investigate the role of increasing FGF5 expression in the progression of prostate cancer and emergence of castration resistant prostate cancer.

Methods

Utilizing a cell line model representing different stages of prostate cancer progression, we assessed FGF5 expression and function in vitro and in vivo. We analyzed both an outcomes and progression tissue microarray (TMA) containing ~400 prostate cancer patient cores to assess FGF5 expression in a clinical setting.

Results

There was a significant increase in the both RNA and protein expression of FGF5 in tumorigenic cell lines compared to nontumorigenic. In TMAs that were analyzed, there was a significant increase of FGF5-positive cells across progression of prostate cancer from benign to metastatic, with the highest percentage occurring in cases of cancer.

When performing gain of function experiments with recombinant FGF5 (rFGF5) or overexpression plasmids, we found that FGF5 significantly increased the proliferation of AR-positive cell lines. Conversely, in AR-negative cell lines, FGF5 inhibited proliferation. Because FGF5 is associated with stem cells and differentiation events, we next asked whether the population of FGF5 cells that increases through cancer progression could lead to molecular changes in the total population. In addition to inhibiting proliferation of AR-negative nontumorigenic BPH1 cells, rFGF5 resulted in a robust AR expression. In LNCaP, C4-2, and C4-2B rFGF5 resulted in an increase in AR expression. In C4-2 and C4-2B cells an increase in AR protein coincided with an increase in nuclear YAP1, a potent transcriptional regulator.

We hypothesized that androgens could be regulating FGF5 expression in the prostate. When androgen dependent LNCaP cells are subjected to an androgen-depleted environment, FGF5 protein and RNA significantly increased within 24hrs. Additionally, when AR is stimulated using dihyrotestosterone, FGF5 protein and RNA levels significantly decreased. These effects are not observed in cell lines that do not express AR.

Conclusions

These results suggest that FGF5 is increased in prostate cancer progression cell lines as well as patient samples, with highest expression in aggressive cancer cores. Data also suggests that AR plays a role in the regulation of FGF5 and vice versa. These data suggest that FGF5 driven AR expression could be driven by YAP1 nuclear localization. This mechanism will be further interrogated.

#3499

The mutational status of residue Y842 in FLT3 predicts the drug response in acute myeloid leukemia.

Julhash U. Kazi, Lars Rönnstrand. _Lund University, Lund, Sweden_.

The type III receptor tyrosine kinase FLT3 plays important roles in the development of early hematopoietic progenitor cells and is found to be mutated in around 35% of all cases of acute myeloid leukemia (AML). AML is a heterogeneous disease that affects the myeloid lineage of blood cells. Wild-type FLT3 needs its ligand, FL, for signal transduction. However, the oncogenic mutants are constitutively active even in the absence of ligand. Similar to the other type III receptor tyrosine kinases, such as KIT, CSF1R, PDGFRA and PDGFRB, ligand binding to the FLT3 induces dimerization, activation of the receptor and auto-phosphorylation. Tyrosine phosphorylation sites in FLT3 create docking sites for interacting proteins which transduce signals linked to survival and proliferation, among other things. Although the most common mutations in FLT3 includes the internal tandem duplication (ITD) mutations in the juxtamembrane domain of FLT3, point mutations in the kinase domain also occur. The so-called activation loop tyrosine residue is well conserved in all receptor tyrosine kinases and has long been known to be essential for the activity of some but not all receptor tyrosine kinases. Recently we have shown that activation loop tyrosine residue in KIT is not essential for its activation but plays an important role in regulating receptor stability and downstream signaling. The activation loop tyrosine residue in FLT3 (Y842) has been found to be mutated in some AML patients and acquired mutations of this residue results in drug resistance when combined with FLT3-ITD. We observed that different Y842 mutants (Y-to-F, Y-to-C and Y-to-H) display differential binding affinity for kinase inhibitors. Expression of Y-to-F mutant in myeloid cells resulted in impaired activation of ERK1/2 through reduced recruitment of SHP2 to the receptor. Furthermore, mice xenografted with myeloid cells expressing this Y-to-F mutant of FLT3, in FLT3-ITD background, resulted in delayed tumor formation. Gene set enrichment analysis (GESA) demonstrated that the Y-to-F mutation causes suppression of anti-apoptotic genes. Taken together, our data suggest that the activation loop tyrosine residue in FLT3 plays an important role in FLT3 downstream signaling and drug sensitivity.

#3500

A pipeline to identify driver mutations.

Deepankar Chakroborty,1 Kari Kurppa,2 Ilkka Paatero,3 Laura Elo,3 Klaus Elenius1. 1 _University of Turku, Turku, Finland;_ 2 _Dana Farber Cancer Institute, Boston, MA;_ 3 _Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland_.

Introduction: Cancer tissues harbor thousands of mutations while the current list of clinically validated actionable variants contains only about a dozen genetic markers. Most of the somatic mutations in cancer are expected to be inconsequential passenger mutations that reflect the general instability of the tumors. The discovery of most of the currently known driver mutations has been facilitated by their accumulation in mutation hot-spots within their respective genes. However, a great majority of mutations in cancer tissues are rare and no information is currently available about their functional significance. Several lines of in vitro and in vivo clinical evidence also indicate that there is a significant number of, as yet unidentified, activating driver mutations that could serve as predictive markers for oncology.

Method: To identify functional driver mutations, we have established a functional genetics screen based on expression of random gene variants and the ability of driver mutations to promote a growth-advantage in vitro, as compared to passenger mutations. The screen was set up using a randomly mutated expression library encoding thousands of variants of epidermal growth factor receptor (EGFR), a well-known oncogene, as a model. The library was retrovirally introduced into murine lymphoid Ba/F3 cells, that normally require interleukin-3 (IL-3) for survival but can exploit ectopic expression of activated variants of oncogenic kinases to compensate for the deficiency of exogenous IL-3. While expression of wild-type EGFR did not promote IL3-independent survival of the Ba/F3 cells, as expected, transduction of the mutant EGFR library did, indicating that the surviving Ba/F3 pool included EGFR mutations with ligand-independent activity. To identify and quantify the frequency of these mutations, targeted next-generation sequencing of the EGFR inserts was carried out.

Results: Using the method we were able to identify approximately 20 candidate activating mutations out of the 7000 random EGFR mutations in the original library. The 20 candidates included the well-known activating EGFR mutation, L858R, validating the pipeline. A previously unidentified activating mutation was also found, and the growth promoted by the mutant was susceptible to inhibition by clinically available EGFR inhibitors.

#3501

Adipocytes sustain pancreatic cancer progression through a non-canonical WNT paracrine network inducing ROR2 nuclear shuttling.

Carmine Carbone, Geny Piro, Raffaela Santoro, Francesca Simionato, Valeria Merz, Camilla Zecchetto, Aldo Scarpa, Giampaolo Tortora, Davide Melisi. _University of Verona, Verona, Italy_.

Background: Solid epidemiological evidences connect obesity with incidence, stage, and survival in pancreatic cancer. However, the underlying mechanistic basis linking adipocytes to pancreatic cancer progression remain largely elusive. We hypothesized that factors secreted by adipocytes could be responsible for epithelial-to-mesenchymal transition (EMT) induction and, in turn, a more aggressive phenotype in models of pancreatic preneoplastic lesions.

Methods: We studied the role of factors secreted by two adipogenic model systems from primary human Bone Marrow Stromal Cells (hBMSC) in an in vitro experimental cell transformation model system of human pancreatic ductal epithelial (HPDE) cell stably expressing activated KRAS (HPDE/KRAS).

Results: We measured a significant induction of EMT and aggressiveness in HPDE and HPDE/KRAS cell lines when cultured with medium conditioned by fully differentiated adipocytes (ADIPOCM) if compared with the same cells cultured with medium conditioned by hBMSC (hBMSCCM) from two different healthy donors. Several genes coding for soluble modulators of the non-canonical WNT signaling pathway, including FRZB, SFRP2, RSPO1, WNT5A and 5B were significantly overexpressed in fully differentiated adipocytes than in their respective in hBMSC. ADIPOCM induced the overexpression and the nuclear translocation of the Frizzled family member Receptor tyrosine kinase-like orphan receptor (Ror) 2 in HPDE and HPDE/KRAS cells. Vantictumab, an anti-Frizzled monoclonal antibody, reduced ROR2 nuclear translocation and in turn the EMT and aggressiveness in HPDE and HPDE/KRAS cells.

Conclusions: We demonstrated that adipocytes could induce EMT and aggressiveness in models of pancreatic preneoplastic lesions by orchestrating a complex paracrine signaling of soluble modulators of the non-canonical WNT signaling pathway that determine, in turn, the activation and nuclear translocation of ROR2. This signaling pathway could represent a novel target for pancreatic cancer chemoprevention. Most importantly, these factors could serve as novel biomarkers to

select a risk population among obese subjects for screening and, thus, early diagnosis of pancreatic cancer.

#3502

Chemoproteomics provide insights into cell surface reprogramming during KRAS-mediated transformation.

Neethi Nandagopal, Léo Aubert, Sami Nourreddine, Geneviève Lavoie, Philippe Roux. _University of Montreal, Montreal, Quebec, Canada_.

KRAS is among the most frequently mutated genes driving human cancers, including ~45% of colorectal cancers (CRC). Despite decades of research, mutant KRAS is considered undruggable, and is frequently associated with resistance to chemotherapy and other targeted therapies. While the effect of mutant KRAS on regulating intracellular signaling events is well studied, much less is known about its impact on the cell surface proteome (surfaceome), which constitutes the predominant therapeutic target for FDA-approved drugs. To study the cell surfaceome, we optimized a chemoproteomics approach that selectively isolates cell surface proteins and allows their relative quantification using mass spectrometry. When comparing normal intestinal cells to their counterparts expressing oncogenic KRAS (G12V), we identified 104 (~13%) and 170 (~22%) cell surface proteins that were significantly upregulated or downregulated, respectively, in KRAS-transformed cells. Overall, a myriad of proteins, including receptors, solute carriers, adhesion molecules, etc., were modulated by mutant KRAS, and their potential roles in KRAS-mediated transformation are currently being studied. We also found that most of the cell surface proteins were altered in response to transcriptional changes induced by KRAS signaling, and due to increased global endocytosis in KRAS-mutated cells. Our study suggests potential cell surface proteins that could be targeted in KRAS-mutant cells, which will be verified in subsequent studies using human CRC models. Our work helps assemble a global view of how oncogenic KRAS alters the spatial distribution of several cell surface proteins, and thereby potentially impacts tumor functions and therapeutic response. 

### Therapeutic Approaches

#3504

A chemical biology screen identifies a unique vulnerability of neuroendocrine cancer cells to SQLE inhibition.

Christopher Mahoney, David Pirman, Victor Chubukov, Taryn Sleger, Anil Padyana, Stefan Gross, Sebastian Hayes, Zi Peng Fan, Gabrielle McDonald, Yu Chen, Joshua Murtie, Giovanni Cianchetta, Raj Nagaraja, Rohini Narayanaswamy, Sung Choe, Stuart Murray, Shengfang Jin, Scott Biller, Thomas Roddy, Gromoslaw A. Smolen. _Agios Pharmaceuticals, Cambridge, MA_.

Numerous reports have described the differential metabolism of cancer cells as compared to their normal counterparts. However, only relatively few metabolic genes with cancer-specific mutations have been reported and the identification of cancer subsets with particular metabolic vulnerabilities remains a challenge.

To explore potential cancer-specific dependencies, we conducted a chemical biology screen utilizing a collection of small molecule inhibitors targeting diverse metabolic pathways in a large panel of cancer cell lines. A subset of neuroendocrine tumors, particularly small cell lung cancers (SCLC), displayed a striking dependence on squalene epoxygenase, SQLE, an enzyme in the cholesterol biosynthetic pathway. To develop further confidence in these findings, we have determined the first three-dimensional SQLE structure and further advanced a pharmacological toolbox for SQLE. Using these tools, we showed that the observed effects are on target and that the patterns of cellular sensitivity observed in vitro display excellent translation to in vivo xenografts studies. Interestingly, using a variety of orthogonal approaches, we demonstrated that SQLE sensitivity appears not to be related to overall inhibition of the cholesterol pathway but rather to specific and toxic accumulation of the SQLE substrate, squalene.

Collectively, these findings highlight the utility of chemical biology screens and identify SQLE as a potential therapeutic target in a subset of neuroendocrine tumors, particularly SCLC.

#3505

Tumor radiosensitization by suppression of hexosamine biosynthetic pathway.

Elena V. Efimova, Oliver K. Appelbe, Steve Seung-Young Lee, Natalia Ricco, Aishwarya Ramamarthy, Nicolas Rymut, Tamica Collins, Donald J. Wolfgeher, Sara Warrington, Stephen J. Kron. _University of Chicago, Chicago, IL_.

The elevation of glucose and glutamine metabolism characteristic of cancer has been ascribed to increased demand for metabolic intermediates that provide building blocks for rapid cell growth. However, recent discoveries have highlighted complementary effects mediated by metabolites serving as cofactors for chromatin modifying enzymes and providing metabolic intermediates for proteins posttranslational modification. Along with epigenetic regulation of gene expression, multiple chromatin modifiers modulate DNA damage repair, raising the question of whether metabolic reprogramming may thereby promote genomic instability and cell immortality. In our prior work, we uncovered a role for the glycolysis hexosamine biosynthetic pathway (HBP) in DNA repair, ionizing radiation induced foci (IRIF) resolution, and prevention of radiation induced cancer cell senescence. Here we report that the targeting of key enzymes in the HBP pathway, O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) or O-GlcNAcase (OGA/ MGEA5), have contrary effects on the DNA damage response in tumors. shRNA knockdown of OGA resulted in increased GlcNAc protein modification, protection of cancer cells from irradiation through improved double strand break (DSB) repair and IRIF resolution in tumor cells, increased proliferation, and prevented radiation-induced senescence. In turn, silencing of OGT diminished O-GlcNAc modification, prevented DSB repair, increased IRIF persistence, reduced proliferation, and promoted radiation-induced senescence in tumor xenografts. These findings reveal an important relationship between cancer cell metabolism, DSB repair, and senescence and provide a rationale for

targeting metabolic reprogramming, specifically O-GlcNAc modification, to block cancer cell immortality and overcome resistance to genotoxic stress.

#3507

Targeting the insulin-like growth factor-1 receptor in MTAP-deleted renal cell carcinomas.

Jihao Xu, Wen-Hsin Chang, Carissa Huang, David Yang, Ching-Hsien Chen. _UC Davis, Davis, CA_.

Renal cell carcinoma (RCC) has emerged as a metabolic disease characterized by dysregulated expression of metabolic enzymes. Patients with metastatic RCC have an unusually poor prognosis and near-universal resistance to all current therapies. To ultimately improve cancer treatment and survival rate, there is an urgent need to reveal the mechanisms by which metabolic enzymes and aberrant pathways regulate oncogenic signaling. Through an integrated two-step analysis of RCC metabolic pathways, we previously identified dysregulated expression of methylthioadenosine phosphorylase (MTAP) in aggressive RCC. An accumulation of cellular methylthioadenosine has been observed in MTAP-deleted cancer cells. Interestingly, we found a decreased of protein-methylation level with concomitant with an increase in tyrosine phosphorylation after MTAP knockout. Next, we performed a phospho-kinase antibody array screen and identified the type 1 insulin-like growth factor-1 receptor (IGF1R) as the top-one candidate with upregulated tyrosine phosphorylation in response to MTAP loss. IGF1R phosphorylation acted upstream of Src and STAT3 signaling in MTAP-knockout RCC cells. Reduction of phospho-IGF1R by a selective inhibitor of IGF-1R, linsitinib, inhibited the cell migration and invasion capability of MTAP-deleted cells. Data from cell viability assays showed that MTAP loss enhanced linsitinib-mediated cytotoxicity in RCC cells. Our data suggest that IGF1R signaling is a driver pathway to confer aggrieves nature of MTAP-deleted renal cell carcinomas.

#3508

Separate and combined effects of caloric restriction mimetics and autophagy inhibition on KRAS-driven pancreatic adenocarcinoma.

Xuewen Chen, Ciara H. O'Flanagan, Michael Coleman, Channing J. Der, Stephen D. Hursting. _Univ. of North Carolina at Chapel Hill, Chapel Hill, NC_.

Calorie restriction (CR) is one of the most effective dietary interventions for reducing cancer risk and progression. However, maintaining a dramatically reduced calorie intake over an extended time period is very challenging for most individuals. Thus a major effort is under way to identify mechanism-based pharmacologic approaches that mimic the anticancer effects of CR without the requirement of reduced calorie intake. We have established that CR exerts antitumor effects at least in part through altered metabolism, reduced circulating growth factors (such as insulin and IGF-1) and reduced signaling downstream of the insulin receptor and IGF-1R along the phosphoinositide 3-kinase/mTOR axis. We have previously shown CR reduced growth of KRAS-driven tumors in vivo, and the antitumorigenic effect was enhanced when combined with autophagy inhibition. Here we evaluated the effects of in vitro CR via glucose and serum modulation, as well as separate and combined effects of CR mimetics and autophagy inhibition on the KRAS-driven murine pancreatic adenocarcinoma (PDAC) cell line, FC1242. Glucose modulation alone via glucose restriction, metformin or AICAR treatment showed no effect on clonogenic growth of FC1242. In contrast, both cell viability and clonogenicity were significantly reduced by serum starvation, rapamycin treatment, IGF-1R/insulin receptor inhibition by BMS-754807, and sirtuin inhibition with nicotinamide, suggesting these cells are dependent on mTOR/IGF-1 signaling. The growth-suppressive effects of these CR mimetics were enhanced when combined with autophagy inhibition via chloroquine or verteporfin. Together, the study indicated the targeting of mTOR or IGF-1 signaling may most closely mimic the growth-suppressive effects of CR, and that co-treatment of these CR mimetics with autophagy inhibition may be an effective strategy for PDAC anticancer therapy.

#3509

The glutaminase inhibitor CB-839 synergizes with CDK4/6 and PARP inhibitors in pre-clinical tumor models.

Ethan D. Emberley, Mark Bennett, Jason Chen, Matthew Gross, Tony Huang, Amani Makkouk, Gisele Marguier, Alison Pan, Sandra M. Spurlock, Susanne Steggerda, Francesco Parlati. _Calithera Biosciences, Inc., South San Francisco, CA_.

Many tumor cells utilize the amino acid glutamine to meet the elevated bioenergetic and biosynthetic demands of rapid cell growth. The enzyme glutaminase converts glutamine to glutamate, which is used to fuel the TCA cycle, synthesize amino acids and nucleotides, and balance cellular oxidative stress. We developed CB-839, a potent and orally bioavailable small molecule inhibitor of glutaminase, that blocks production of glutamate and generation of downstream metabolites glutathione, malate and aspartate. Mice treated with CB-839 had decreased levels of nucleotides in their tumors, likely due to glutamine-derived aspartate being required for nucleotide biosynthesis. Consistent with our finding that CB-839 decreases nucleotide pools, CB-839 treatment delayed cancer cells from either entry into S-phase or progression through S-phase. Based on this observation, the ability of CB-839 to synergize with therapies that block cell cycle progression was tested. CB-839 synergized with the CDK4/6 inhibitor palbociclib in colorectal carcinoma (CRC), triple negative breast cancer (TNBC) and ER+ breast cancer cell lines resulting in anti-proliferative activity. The combination of CB-839 with palbociclib also led to decreased cell cycle progression through S-phase and caused an accumulation of cells in G1 as measured by an EdU DNA incorporation assay. In vivo, the combination of CB-839 with palbociclib resulted in enhanced anti-tumor activity in both an ER+ breast cancer and CRC xenograft tumor model. We next investigated whether CB-839 treatment would enhance the anti-tumor effects of DNA repair inhibitors, given the ability of CB-839 treatment to decrease nucleotide pools. CB-839 treatment in combination with the PARP inhibitors niraparib and talazoparib led to synergistic anti-proliferative activity in TNBC, CRC, non-small cell lung carcinoma, ovarian and prostate cancer cells. In vivo, the combination of CB-839 with PARP inhibitors showed enhanced anti-tumor activity compared to single agent treatment in a CRC tumor xenograft model. CB-839 is currently undergoing evaluation for efficacy in the treatment of cancer in several phase I/II clinical trials. These encouraging pre-clinical results support the testing of CB-839 with CDK4/6 or PARP inhibitors in cancer patients.

#3510

Small molecule ACSS2 inhibitors target acetate metabolizing tumor cells in hypoxic conditions.

Monali Banerjee, Sourav Basu, Debjani Chakraborty, Ritesh Shrivastava, Sandip Middya, Rajib Ghosh, Dharmendra Yadav, Arghyotri Sinha, Avratanu Das, Navin Pandit, Mohinder Naiya, Sabyasachi Debnath, Sangeeta Dey, Debottam Dasgupta, Nirmal Das, Anuj Singh, Dipayan Sarkar, Sukanya Sarkar, Ganesh Narasipuram, Nagaswami Mane, Nicky Singhal, Nidhi Rawat, Anindita Middya, Arjun Surya. _Curadev Pharma Pvt. Ltd., Noida, India_.

BACKGROUND

Tumors such as glioblastoma proliferating under hypoxic, hypoglycemic and hypolipidemic conditions derive almost half of their nutritional requirement from the short chain fatty acid acetate. Acetyl CoA synthetases (ACSS 1 to 3) are a family of enzymes that metabolize acetate to the multifunctional metabolite acetyl-CoA. Acetyl-coA is a key precursor of cellular biomass and is required for epigenetic modification. There is an increasing body of clinical evidence that places acetate and ACSS2 at a critical metabolic node in tumors. We have developed potent small molecule enzyme inhibitors of ACSS2 that block the functional activity of the enzyme in vitro and in vivo. Furthermore, these inhibitors synergize with known chemotherapeutics to eliminate cancer cells in hypoxic conditions.

METHODS

A pharmacophore based medicinal chemistry campaign yielded initial hits which were refined in subsequent rounds of structure activity relationships. ACSS2 activity was measured in cell free and cellular assays by monitoring the conversion of radiolabelled acetate to biomass in depleted oxygen environments and in combination with chemotherapeutics such as doxorubicin.

RESULTS

Our medicinal chemistry campaign yielded several highly potent ACSS2 specific enzyme inhibitors with good pharmacokinetic properties in the rodent. Our lead compound CRD1400 is an ACSS2 specific inhibitor with low nanomolar potency and kills acetate dependent tumor cells and sensitizes several tumor cell lines to the chemotherapeutic doxorubicin. CRD1400 is has been selected for further assessment in patient derived xenograft models based on its potency for increased cell killing under hypoxic conditions.

CONCLUSIONS

Inhibition of the enzyme ACSS2 depletes tumor cells of the nutrient acetate and provides a new modality of attacking metabolic vulnerabilities in tumors. When used in combination, CRD1400 reduces chemotherapeutic resistance that arises in low oxygen environments common in tumors and enhances the potency of commonly used cancer medication.

#3511

The mechanism of antitumor effect of metformin forgemcitabine-resistant pancreatic adenocarcinoma.

Keiichi Suzuki,1 Osamu Takeuchi,1 Yukio Suzuki2. 1 _Kitasato Institute Hospital, Tokyo, Japan;_ 2 _Kitasato University School of Pharmacy, Tokyo, Japan_.

Metformin (MET) is the first-line treatment for type 2 diabetes mellitus. Several epidemiological studies have reported anti-cancer effects of MET, including against pancreatic ductal adenocarcinoma (PDAC), which mainly acts through induction of AMP activated protein kinase (AMPK). Gemcitabine (GEM) has become the standard chemotherapy for PDAC but tolerance to GEM has become a burdensome issue. We evaluated the anti-tumor effects of MET for GEM-resistant PDAC in a xenograft mouse model. For this in vivo study, BxG30 (the cell line for GEM-resistant PDCA) was implanted into both flanks of female BALB/c nude mice. Mice were divided into four groups: (i) control (no treatment); (ii) the GEM-treated group (100 mg/kg); (iii) the MET-treated group (600 mg/kg); and (iv) the combined treatment group (G+M). Mice were fed for 4 weeks. Estimated tumor volumes and body weights were measured each week. Treatments were initiated 2 weeks after implantation. MET was administrated orally once per day. GEM was given by intraperitoneal injection once per week. Compared with the control, the final tumor volumes were decreased significantly only in the G+M group. [SCW1] The treated control ratio (T/C%) was calculated: GEM, 80.2%; MET, 54.0%; G+M, 47.2%. The anti-tumor effect of GEM for BxG30 was clearly limited[SCW2] . MET group showed satisfactory anti-tumor effects, but T/C% was <50% only in the G+M group. This result revealed that combination therapy had excellent anti-tumor effects even for GEM-resistant PDAC. The phosphorylation of ribosomal proteins S6 and 4E-BP, important targets of the mammalian target of rapamycin (mTOR) signaling pathway, was surveyed by western blot analysis. Western blot analysis showed inhibition of S6 and 4E-BP phosphorylation by co-incubation with MET, but not with GEM. Hypoxia-inducible factor 1 (HIF-1) is the one of the most important target of mTOR signaling pathway influencing tumor cell to progress, thus the expression level of HIF-1 was evaluated by western blot analysis as well. The results showed significant inhibition of HIF-1 expression by MET treatment, but not by GEM incubated under hypoxia (95%N2, 5%O2). Then the production of VEGF was evaluated by ELISA under hypoxia. The result showed suppression of VEGF production by MET treatment, but not by GEM. Our data showed that MET develops a different anti-tumor effect from GEM by suppression of mTOR-HIF-1 signaling. These results are of great clinical interest and reveal the potential of another anti-tumor agent for treatment of PDAC.

#3512

Impact of IDO inhibitor on tryptophan and kynurenine pathway reflected in the tumor microenvironment and highlighted using quantitative mass spectrometry imaging.

Lauranne Poncelet,1 Rima Ait-Belkacem,1 Bruno Gomes,2 Stefan Linehan,3 Gregory Hamm,1 Jonathan Stauber1. 1 _Imabiotech, Loos, France;_ 2 _Roche, Basel, Switzerland;_ 3 _Imabiotech, Billerica, MA_.

Introduction: Indoleamine-2,3-dioxygenase (IDO1) is an enzyme which converts tryptophan (Trp) into kynurenine (Kyn) in many cell types. IDO1 has a critical role in immunosuppressive mechanisms that permit tumor cells to escape the immune system when Trp and Kyn level decrease and increase in the microenvironment, respectively. Therefore, IDO1 has been one of the first targets that have really been purposed for immuno-oncology (I-O) applications and immunotherapies development. A potent and selective IDO1 inhibitor such as Epacadostat (EPA) can significantly enhance the antitumor activity by restoring the body's natural ability to recognize and to fight cancer. In this study, using quantitative mass spectrometry imaging (QMSI, we went further than only quantifying the metabolites and the drug. Both target exposure and engagement studies were handled in CT26 mouse tumor model, plasma and whole blood.

Methods: Undifferentiated colon carcinoma CT26 cell line was implanted into mice. Tumors, plasma and blood were sampled from two groups: one treated group (3 mice, 100mg/Kg of EPA 2 hours before sampling), and one control group of 2 mice. All samples were stored at -80°C until use. 1,5-diaminonaphthalene (1,5-DAN) matrix mixed to internal standards was sprayed onto tumor tissue sections of 10 µm thickness using the automatic TM Sprayer (HTX Technologies, LLC) prior analysis. Data acquisition was performed using 7T MALDI-FTICR (SolariX XR, Bruker Daltonics) at 120 µm spatial resolution in full scan negative mode. Acquired data were treated with MultimagingTM software (ImaBiotech). Histological staining was applied afterwards. Quantitative results of Trp, Kyn and EPA in whole tumors, plasma and blood were then confirmed by LC-MS/MS analysis.

Results: Using both QMSI and LC-MS/MS technologies, quantification was realized for the two endogenous metabolites and the drug. Thus, using a potent and selective IDO1 inhibitor, EPA, CT26 model validation was possible. As expected, a decrease of Kyn/Trp ratio was noticed in treated CT26 tumors (-24%), plasma (-57%) and blood (-45%) compared to control samples. By inhibiting IDO1 and decreasing Kyn in tumor cells, EPA increases and restores the proliferation, activation and regulation of various immune cells. This promoted infiltration of active immune cells into the tumor microenvironment is an important feature of many immunotherapies. It is marked by glycolysis signatures and adenosine-inosine metabolism.

#3513

**Curcumin induces a fatal energetic impairment by inhibiting ATP-synthase activity and decreasing ATP generation and oxygen consumption in** in vitro **and** in vivo **tumor models.**

Ulrich Pfeffer,1 Giovanna Bianchi,2 Silvia Ravera,3 Chiara Traverso,2 Adriana Amaro,1 Francesca Piaggio,1 Laura Emionite,1 Tiziana Bacchetti,2 Lizzia Raffaghello2. 1 _Ospedale Policlinico San Martino, Genova, Italy;_ 2 _Istituto G. Gaslini, Genova, Italy;_ 3 _University of Genoa, Genova, Italy_.

Curcumin has been reported to inhibit inflammation, tumor growth, angiogenesis and metastasis by decreasing cell growth and by inducing apoptosis mainly through the inhibition of nuclear factor kappa-B (NFkB), a master regulator of inflammation. Recent reports also indicate potential metabolic effects of the polyphenol and we therefore analyzed whether and how it affects the energy metabolism of tumor cells. We show that curcumin inhibits the activity of ATP-synthase in isolated mitochondrial membranes leading to a dramatic drop of ATP and a reduction of oxygen consumption in in vitro in several murine tumor cell lines (CT26 colon cancer, B16 melanoma, L1210 lymphocytic leukemia, 4T1 breast cancer) and in vivo in syngeneic tumor models. The effects of curcumin on ATP-synthase are independent of the inhibition of nuclear factor kB (NFkB) since the IkB Kinase inhibitor, SC-514, inhibits the expression of the NFkB target gene, BCL2, but does not affect the activity of the ATP-synthase. The activities of the glucose metabolism enzymes hexokinase, phosphofructokinase, pyruvate kinase and lactate dehydrogenase are only slightly affected in a cell type specific manner. The energy impairment translates into decreased tumor cell viability. Apoptosis is induced by promoting the generation of reactive oxygen species and malondialdehyde (MDA), a marker of lipid oxidation. Tumor autophagy is induced by curcumin at least in part due to the activation of the AMP-activated protein kinase (AMPK). These activities translate into a significant delay of in vivo tumor growth likely due to a reduction of tumor angiogenesis since we observe reduced number and size of tumor vessels in vivo. These results establish the ATP-synthase, a central enzyme of the cellular energy metabolism, as a target of the anti-tumoral polyphenol leading to inhibition of cancer cell growth and a general reprogramming of tumor metabolism.

#3514

Differential metabolic targeting of PDAC cells with Zaprinast.

Yi Rao, Seth Gammon, David Piwnica-Worms. _MD Anderson Cancer Center, Houston, TX_.

Many pancreatic cancers consume excessive glutamine as a preferable source for anaplerosis, maintenance of proper redox state, and synthesis of intermediates for cancer cell growth and proliferation. Abrogating glutamine metabolism is under investigation to inhibit the proliferation/ growth of glutamine-addicted cancers. In addition, inhibition of the mitochondrial pyruvate carrier (MPC1) can potentially induce cancer cells to become more dependent on glutaminolysis. In this regard, Zaprinast, originally developed as a phosphodiesterase 5 (PDE5) inhibitor, has recently been identified as an inhibitor of both glutaminase as well as MPC1. We compared the growth inhibition effect of Zaprinast against BPTES, a targeted glutaminase inhibitor, and UK5099, a targeted MPC1 inhibitor, as well as the combination of BPTES and UK5099. Using pancreatic ductal adenocarcinoma (PDAC) cells, KRAS mutant (Mia Paca2) and KRAS wildtype (Panc03.27), KRAS mutant neuroendocrine (QGP1), and normal pancreatic epithelial cells (HPNE), Zaprinast alone showed more potent growth inhibition in all tested cells lines than single agent treatment with BPTES or UK5099. Further, Zaprinast effectively depleted mitochondrial reserve respiratory capacity similar to UK5099, rendering mitochondria more vulnerable to oxidative stress or pharmacological inhibitors targeting the electron transport chain. In addition, Zaprinast treatment of cancer cells differentially elevated ROS levels greater than 10-fold compared to normal HPNE cells (ROS levels: Mia Paca2 ~ Panc03.27 > QGP1 > HPNE). However, the increase in ROS levels did not correlate with Zaprinast-induced growth inhibition (IC50: Mia Paca2=300 µM, Panc03.27=110 µM, HPNE=650 µM, QGP1 >1 mM). Although Zaprinast-induced growth inhibition was selective toward adenocarcinoma cells but not the neuroendocrine subtype, the selective Zaprinast-induced elevation of ROS levels toward all cancer subtypes renders possibilities for potential synthetic lethality when combined with other metabolic inhibitors.

#3515

Down the rabbit hole with structural effects of FK866 in primary CLL B cells.

Cheryl Peltier,1 Eileen McMillan-Ward,1 Elizabeth Henson,1 Nabanita Chatterje,1 Donna Hewitt,1 Danielle Desautels,1 Matthieu Bourrier,1 Cornelia Mutz,2 Iris Gehrke,3 Eric Bouchard,4 Heinrich Kovar,5 Shantanu Banerji,1 Spencer Gibson,1 Sara Israels,1 Versha Banerji1. 1 _University of Manitoba, Winnipeg, Manitoba, Canada;_ 2 _St. Anna Kinderkrebsforschung Children's Cancer Research Institute, Vienna, Austria;_ 3 _Bayer AG, Cologne, Germany;_ 4 _Université de Saint-Boniface, Winnipeg, Manitoba, Canada;_ 5 _St. Anna Kinderkrebsforschung Children's Cancer Research Institute, Austria_.

FK866 (Daporinad or APO866) is a specific inhibitor of NAMPT (nicotinamide phosphoribosyltransferase), a rate limiting enzyme involved in NAD generation. It is a prospective cancer treatment utilized in several clinical trials and has generated much interest. It has been previously demonstrated that FK866 (10nM) induces cell death in CLL patient derived B-cells (CLL-B cells) by early depletion of NAD (1 day after treatment) followed by a subsequent decrease in cellular ATP levels (2 days after treatment).

Upon further study we observed that CLL-B cells have structural changes after treatment with FK866 for either 6 or 18-20 hours via electron microscopy. There is a presence of bound structures as well as nuclear invaginations. These structures are only observed in cells that were treated with FK866 and not with any other drugs used (fludarabine, bendamustine, chlorambucil) nor with the DMSO or untreated controls.

We stained f-actin (a cytoskeletal protein) with FITC labelled phalloidin in CLL-B cells with and without FK866 treatment and then viewed with confocal microscopy to exclude the chance that these structures were artifacts. Changes in the f-actin were only observed in the FK866 treated cells. Phalloidin staining was performed at 6 and 18 hours. We observed holes in the f-actin framework that passed through the entire cell and in some instances, went through the nuclei too.

A comparison of the phalloidin staining was performed in non-CLL based cell lines that were known to be sensitive to treatment with FK866 though structural differences had not been observed at the same time points (6 and 18-20 hours). Two Ewing's sarcoma cell lines and two small cell lung cancer cell lines were used. All four cell lines showed no changes in the f-actin.

Labeled dextran beads were observed via confocal microscopy to be taken up more readily by the FK866 treated CLL -B cells as compared to the vehicle control. This would imply that the treated cells are actively removing nutrients from the environment.

A lysosome stain, LysoTracker, was employed to determine if there was any involvement of lysosomes in this phenomenon. No change was observed between the untreated, vehicle control and the FK866 treated CLL patient derived B-cells.

We were curious about the mitochondrial dynamics in the FK866 treated cells so they were stained with MitoTracker. We were able to detect mitochondria in the cytoplasm of the cells in all treatments but the FK866 treated cells displayed a more distinct punctate pattern. Interestingly, we also observed the mitochondria were appearing at or near the holes in the cytoskeleton.

As the FK866 treated cells are deprived of their NAD, the CLL cells may be undergoing macropinocytosis, an mTOR mediated phenomenon used by cells to extract nutrients from the environment. We have shown that FK866 causes an increase in 4EBP1 phosphorylation compared to controls.

In conclusion, FK866 may mediate macropinocytosis in CLL cells in an mTOR dependent manner.

#3516

Discovery of novel SHMT small molecule inhibitors for cancer treatment.

Anna Bartosik, Pawel Guzik, Marta Sowinska, Karolina Gluza, Marcin Krol, Anna Wrobel, Agnieszka Dreas, Faustyna Iwanska, Magdalena Zastawna, Urszula Kulesza, Nicolas Boutard, David Schultz, Justyna Wujkowska, Karolina Pyziak, Agnieszka Sroka-Porada, Agnieszka Przybylowicz, Agnieszka Adamus, Magdalena Sieprawska-Lupa, Przemyslaw Golik, Piotr Kowalczyk, Krzysztof Brzozka, Tomasz Rzymski, Mateusz Nowak. _Selvita S.A., Krakow, Poland_.

Over-activation of the serine synthesis pathway, upregulation of SHMT2 has been described in over 20% of solid tumors (e.g. breast, lung, colorectal cancers). Such cancer cells are highly dependent on serine. Serine hydroxymethyltransferase (SHMT) plays a key role in a so-called one-carbon pathway, a group of biochemical reactions involved in amino acid metabolism. SHMT catalyzes the conversion of serine to glycine and also plays a role in the folate (vitamin B9) cycle. Antagonists of folate metabolism or antifolates are an established chemotherapy in certain cancers. Folate antagonism disrupts cell division, DNA/RNA synthesis and protein synthesis. Pemetrexed (for non-small cell lung carcinoma, mesothelioma) and methotreaxate (for autoimmune conditions like rheumatoid arthritis and certain cancers) are two well established and effective antifolates. The main drawback with antifolates in cancer treatment, however, is the development of resistance. In this study we report development of a series of small molecule SHMT1/2 inhibitors. Synthetized compounds exert potency in SHMT1/2 biochemical assay as well as in cellular assay (measured by the C13 serine to glycine conversion) with the low nanomolar range. Therapeutic effect of the compounds was investigated in the panel of cancer cell lines with different genetic background as well as with different SHMT2 levels. We identified several cell lines in which tested compounds inhibited cancer cell grow with nM GI50 values. Taken together, presented data supports our rationale for using SHMT1/2 inhibitors as a novel and interesting approach for the cancer therapy.

#3517

Targeting pH regulators to modulate human hepatocellular carcinoma microenvironment.

Alessandra Tuccitto,1 Olga Kuchuk,1 Davide Citterio,1 Veronica Huber,1 Chiara Camisaschi,1 Massimo Milione,1 Barbara Vergani,2 Antonello Villa,2 Licia Rivoltini,1 Chiara Castelli,1 Vincenzo Mazzaferro1. 1 _Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy;_ 2 _Consorzio MIA, Microscopy and Image Analysis, University of Milan Bicocca, Monza, Italy_.

Hepatocellular carcinoma (HCC) arises in a hypoxic/acidic microenvironment in which the pO2 is about 0.8%. This condition favors tumor progression and fosters immunosuppression. Tumor cells survive this obstile environment by overexpressing pH regulators such as carbonic anhydrase (CA) IX, XII and V-ATPase complex. We investigated the expression and functional properties of these molecules in HCC cell lines and patients, to evaluate their potential role as novel therapeutic agents against HCC. The expression of CAs and V-ATPase subunits was assayed by qRT-PCR, western blot and confocal microscopy in HCC cell lines exposed to normoxia (21% O2) or hypoxia (1% O2). The effect of pH regulatory inhibitors on HCC cell viability was evaluated by MTT assay. Paired tumoral and non-tumoral liver tissues (n=57) from HCC patients were analyzed for the expression of pH regulatory molecules by qRT-PCR and by immunohistochemistry. Multi-paramentric flow cytometry analysis was performed on patient-derived HCC cell suspensions exposed to V-ATPase inhibitor or vehicle for 24h (n=6). Human HCC tissue explants (n=8) were cultured in presence of V-ATPase inhibitor for 24h and gene expression was analysed by qRT-PCR. CAs and V-ATPase are expressed by HCC cell lines grown under normoxia (21% O2) and a selective increment of CAIX and CAXII was observed after hypoxia (1% O2) exposure. These molecules were involved in cancer cell survival, as demonstrated by the significant reduction of tumor viability by CAIX and V-ATPase inhibitors. Ex vivo analyses showed a focal and intense plasma membrane immunoreactivity for CAIX in discrete nests of hepatocytes inside HCC lesions and in cholangiocytes within normal and non-tumoral tissues. Tumor cells selectively expressed CAXII in the cytoplasm. In vivo and ex vivo we observed that in the hypoxic microenvironment of HCC CAXII was retained in the endoplasmic reticulum of hepatocytes. Significant, albeit heterogeneous, positivity for V-ATPase was detected in most of HCC lesions analyzed. Importantly, infiltrating immune cells compatible with M2 macrophage phenotype expressed the V-ATPase. The inhibiton of V-ATPase by omeprazole in cell suspensions obtained from clinical HCC tissues showed a decreased production of CCL22 paralleled by an increment of IFNγ by M2 macrophages. Short-term treatment of HCC tissue explants with omeprazole exerted important effects on tumor microenvironment reducing the immunosuppressive gene CCL22 together incrementing IFNG, but also affected the aggressiveness of tumoral cells inducing a down-modulation of epithelial to mesenchymal transition associated genes VIM and MYC and increasing the transcription of CDH1. In conclusion our data indicate that pH regulators, overexpressed in tumor and immune cells composing the HCC microenvironment, play a role in maintaining an aggressive and immunosuppressive milieu in HCC and likely they represent a promising target.

#3518

Piperlongumine, a novel treatment option to overcome BRAF inhibitor resistance in melanoma.

Sharavan Ramachandran, Neel Fofaria, Sanjay Srivastava. _Texas Tech Univ. Health Sciences Ctr., Amarillo, TX_.

Tumor cell metabolism is a hallmark of chemoresistance and its role has been well-defined in BRAF inhibitor resistance in melanoma. Melanoma cells are highly proliferative and are shown to develop addiction towards the metabolic pathways like glycolysis and mitochondrial respiration to meet energy demands. Melanoma cells predominantly utilize cytosolic aerobic glycolysis or Warburg effect to develop resistance towards BRAF inhibitors (vemurafenib and dabrafenib). Our observations indicated that vemurafenib resistant cells possessed 1.5 fold higher extra-cellular acidification rate (ECAR), an indicator of glycolysis when compared to sensitive cells. We also observed that STAT3, a known regulator of glycolysis was up-regulated in A375 and SKMEL28 BRAF inhibitor resistant cells when compared to the parental cell line. In addition, LDH-A, a key glycolytic enzyme was significantly overexpressed in A375 vemurafenib resistant cells when compared to its parental counterpart. Consequently, we observed that vemurafenib and dabrafenib treatment induces the expression of STAT3 in A375 and SKMEL-28 melanoma cells. Our results showed that piperlongumine inhibits the survival of A375 vemurafenib resistant (A375VR) melanoma cells. Our observations indicated that the combination of piperlongumine with vemurafenib and dabrafenib shows synergistic effects by suppressing 75% - 85% of cell survival when compared to vemurafenib/dabrafenib and piperlongumine (25% - 45%) treatment alone in A375 and SKMEL-28 BRAF inhibitor resistant melanoma cells. Moreover, the combination of piperlongumine with vemurafenib or dabrafenib induced 2-8 fold apoptosis when compared to vemurafenib/dabrafenib and piperlongumine treatment alone in A375 and SKMEL-28 BRAF inhibitor resistant melanoma cells. Piperlongumine treatment reduced the ECAR (glycolysis) in a concentration dependent manner after 12h of treatment in A375VR cells and suppressed nearly 50% of ECAR parameters glycolysis and glycolytic capacity. Treatment of A375VR cells with piperlongumine for 48h inhibited the expression of LDH-A and its upstream regulators pSTAT3 (Y705), β-catenin, c-Myc and HIF-1α as evaluated by western blot. In addition, oral administration of piperlongumine and vemurafenib combination suppressed 86% of tumor growth when compared to vemurafenib treatment. Taken together, our study thus indicate that piperlongumine potentiates the effects of BRAF inhibitors by inhibiting Warburg effect and its upstream regulators. [Supported in part by R01 grant CA129038, awarded to (S.K.S) by the National Cancer Institute.

#3519

Disrupting glutamine metabolism depletes glutathione and sensitizes atypical teratoid/rhabdoid tumor to carboplatin.

Sabrina Z. Wang,1 Brad Poore,1 Jesse Alt,1 Rana Rais,1 Sariah Allen,2 Brent Orr,2 Barbara Slusher,1 Charles Eberhart,1 Eric Raabe,1 Jeffrey Rubens1. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _St Jude Children's Research Hospital, Memphis, TN_.

Atypical teratoid rhabdoid tumors (AT/RT) are the most common malignant brain tumors of infancy. Recent high-throughput molecular analyses identified 3 distinct subgroups of AT/RT. Preliminary studies show that subgroups with elevated MYC expressions are particularly aggressive, with 18.5% 5-year survival. MYC is challenging to directly target, but MYC increases tumor cells' reliance on glutamine for their metabolic demands. We hypothesize that MYC-driven ATRT can be targeted by inhibiting glutamine metabolism. 6-Diazo-5-oxo L-norleucine (DON) is a glutamine analogue that was well tolerated in a phase I pediatric clinical trial, but DON has never been tested in MYC-driven pediatric tumors. Approximately 1/3 of human AT/RT express high MYC protein (immunohistochemistry for c-MYC expression on 22 human AT/RT, H-score > 1 standard deviation over the median for high c-MYC expression). DON slows cell growth and induces apoptosis in high MYC-expressing AT/RT while low MYC-expressing cells are resistant (MTS assay and Western blot for c-PARP, respectively). Transducing low MYC-expressing cell lines with lentivirus with MYC plasmid to force MYC expression confers sensitivity to DON therapy (decreased cell growth per MTS assay, increased apoptosis per Western for c-PARP). DON administered intraperitoneally 30mg/kg weekly nearly doubles median survival in orthotopic xenograft models of AT/RT (p<0.001 by log rank test; median survival extended from 21 to 36 days). Metabolic flux experiments tracing 15N and 13C labeled glutamine reveal that DON reduces intracellular glutathione. We combine DON with carboplatin because glutathione is critical for detoxification of carboplatin and find that the combination works synergistically to further slow cell growth and induce higher rates of apoptosis (MTS assay, MUSE Cell Viability Assay). This study takes a novel approach to targeting cancer metabolism and serves as the first targeted therapeutic strategy addressing the MYC-expressing subgroup of AT/RT. These findings support the rationale for potential future clinical trials testing glutamine analogues against the MYC subgroup of AT/RT as well as other deadly MYC-driven tumors.

#3520

Targeting lipid metabolism eradicates peritoneal metastases of ovarian cancer.

Run Chen, David Chan. _University of Hong Kong, Hong Kong, Hong Kong_.

Ovarian cancer is one of the deadliest women malignancies in the world. The chemoresistance accompanied with metastasis lead to high mortality of this disease. Different from other solid tumors, the advanced ovarian cancers prefer transcoelomic route in metastasis. Clinical observation indicates ovarian cancer patients with intraperitoneal metastases are closely association with poor prognosis. Emerging evidence has indicated that ascites fluids which acts as a reservoir providing plenty of growth factors, chemokines and importantly, free fatty acids etc. However, the significance of ascites microenvironment in governing ovarian cancer metastatic progression remain unclear. Herein, we report that the ascites microenvironment alters metabolism that in turn, enhances ovarian cancer cell aggressiveness. By mimicking the ascites microenvironment, we established omental conditioned medium (OCM). Ovarian cancer cells co-cultured in OCM showed enhanced ovarian cancer oncogenic capacities as observed in ascites. By RNA-Seq and proteomics analyses, we found the activities of lipid metabolism were elevated in ovarian cancer cells cultured in OCM. Depletion of Glut-1/-3/-4 by shRNAi approach or treatment of STF31, a glycolysis inhibitor, showed no or slight effect on ATP production and cell growth of ovarian cancer cells. In contrast, knockdown of Acetyl-CoA Carboxylase-α or -β (ΑCC1/2) or treatment of FASN inhibitor, Orlistat, significantly inhibited the ATP production and cell growth, suggesting ovarian cancer cells undergone a metabolic shift from aerobic glycolysis to β-oxidation when co-cultured in fatty acid enriched OCM. On the other hand, AMP-activated protein kinase (AMPK) is a key energy sensor and affects oncogenic capacities of many cancer cells. We found that its activity was induced initially for triggering fatty acid oxidation in ATP production. But the AMPK activity was gradually reduced due to the negative feedback loop of high ATP content. The lowered AMPK activity led to mTOR and TAK1/NF-κB signaling activation that result in the increased oncogenic capacities of ovarian cancer cells. Notably, inhibition of the above signaling pathways by a combined cocktails of AMPK activator, TAK1 and FASN inhibitors could remarkably impair OCM-mediated oncogenic properties of

ovarian cancer cells in vitro and in vivo. Taken together, these findings suggest that the ascites microenvironment induces metabolic reprogramming for ovarian cancer cells in production of energy supporting their aggressiveness, while targeting the lipid metabolism and associated signalings could impede peritoneal metastases of ovarian cancer.

#3521

Glutamine metabolic inhibition synergizes with L-asparaginase in MYCN-amplified neuroblastoma.

Micah J. Maxwell, Brad Poore, Allison Hanaford, Jesse Alt, Rana Rais, Barbara S. Slusher, Charles G. Eberhart, Eric H. Raabe. _The Johns Hopkins University School of Medicine, Baltimore, MD_.

Neuroblastoma is the most common extracranial solid tumor in children. Though it accounts for about 10% of pediatric cancers, it is disproportionately responsible for 15% of pediatric cancer deaths. MYCN is amplified in 20% of neuroblastomas and correlates with adverse outcome. MYCN is involved in the maintenance of cancer stem cells, as well as in driving tumor cell growth, proliferation and tumorigenesis. Attempts to inhibit MYCN directly have been largely unsuccessful. MYCN is known to drive tumor cell reliance on glutamine for cellular metabolism. 6-Diazo-5-oxo-L-norleucine (DON) is a well-characterized glutamine analogue that inhibits glutamine metabolism by irreversibly inactivating multiple glutamine-utilizing enzymes. DON was well tolerated in a previous phase I clinical trial in pediatric patients, but it has never been systematically tested in neuroblastoma patients. We show that MYCN-amplification confers sensitivity to DON therapy in in vitro models of neuroblastoma, and that DON administered by intraperitoneal injection twice weekly significantly reduces flank tumor volume in orthotopic mouse models of MYCN-amplified neuroblastoma (mean tumor volume 1715 mm3 vs. 207 mm3 in control animals, p-value = 0.00017 by t-test). We have also developed an orally bioavailable DON prodrug, JHU083, and we found that this drug administered orally three times weekly was similarly able to suppress neuroblastoma tumor growth in mice (mean tumor volume 1,115 mm3 vs. 217 mm3 in control animals, p-value = 0.0000088 by t-test). In metabolic flux experiments, tracing glutamine and glucose donation of 13C and 15N via liquid chromatography/mass spectrometry, DON prevents asparagine synthesis, depleting intracellular asparagine levels by 40% compared to control treated cells (p-value = 0.006). DON combined with L-asparaginase synergistically inhibits growth of MYCN-amplified neuroblastoma cell lines (CI = 0.25 by the Chou-Talalay method, indicating strong synergy; p-value = 0.00011). We conclude that DON depletes cellular pools of asparagine, and combination therapy with DON and L-asparaginase synergistically inhibits the growth of MYCN-amplified neuroblastoma. These studies provide the preclinical justification for potential clinical trials for the use of DON or DON prodrugs in combination with L-asparaginase as new therapeutic options for patients with MYCN-amplified neuroblastoma.

#3522

Both mitochondrial function and composition of BCL2 family proteins determines sensitivity to Cisplatin in ovarian cancer cells and are promising targets to overcome Cisplatin resistance in ovarian cancer.

Markus Kleih,1 Simon Heine,1 Kathrin Böpple,1 Meng Dong,1 Heiko van der Kuip,1 Walter E. Aulitzky2. 1 _Dr Margarete-Fischer-Bosch Institute, Stuttgart, Germany;_ 2 _Robert Bosch Hospital, Stuttgart, Germany_.

Mitochondria are critical target structures of platinum drugs. To study whether mitochondrial activities of Cisplatin are critical for its cytotoxic function we studied mitochondrial mass, mitochondrial function and composition of pro- and anti-apoptotic BCL2 family proteins in three resistant and three sensitive high-grade serous carcinoma (HGSC) cell lines.

Sensitive cell lines differed from resistant cells by higher mitochondrial mass (MM), higher levels of mitochondrial reactive oxygen species (mtROS) and higher basal oxygen consumption rate (OCR) levels as well as a higher ratio of pro- vs. anti-apoptotic BCL2 family proteins. Cell death upon Cisplatin in sensitive cells was dependent on induction of reactive oxygen species (ROS), loss of mitochondrial membrane potential (MMP), induction of BAX-BAK pores and caspases. Genetically alteration of the BCL2 family protein ratio by siRNA mediated knockdown of pro-apoptotic NOXA reduced acute response in sensitive cell lines, whereas knockdown of anti-apoptotic BCLw rendered resistant cells sensitive. In analogy, pharmacological inhibition of anti-apoptotic proteins by ABT737 also completely re-sensitized Cisplatin resistant cell lines.

Cisplatin exposure increased MM, mtROS and OCR in both sensitive and insensitive HGSC cell lines. However, these parameters reached higher peak levels in cell lines sensitive to induction of cell death by Cisplatin. Knockdown of PGC-1α (the master regulator of mitochondrial biogenesis) partially rescues sensitive cells from apoptosis. In addition, pharmacological inhibition of ATP synthase by Oligomycin A blocks MMP and mtROS induction by Cisplatin and inhibits Cisplatin induced cell death. This supports the hypothesis that induction of mitochondria leading to increased release of mtROS contributes to cell death by Cisplatin.

We finally tested whether other modulators of mitochondrial function restore sensitivity to Cisplatin in resistant cells. Whereas Complex-I and Complex-III inhibitors did not affect cytotoxicity of Cisplatin, the iron chelator VLX600 had significant synergistic activity with Cisplatin in resistant cancer cells. VLX600 inhibits oxygen consumption almost completely in combination with Cisplatin whereas both compounds had no significant effect on survival when used as monotherapy.

In conclusion, cytotoxicity of Cisplatin in HGSC cell lines depends on efficient induction of MM and mtROS. Resistance can efficiently be targeted by modulating the mitochondrial function by VLX600. These observations support the view that mitochondria are attractive targets for increasing Cisplatin activity on cancer cells.

#3523

Uncoupling oxidative phosphorylation reduces tumor growth in a murine colon cancer model.

Rachel J. Perry,1 Yongliang Wang,1 Xian-Man Zhang,1 Michael N. Pollak,2 Gerald I. Shulman1. 1 _Yale University School of Medicine, New Haven, CT;_ 2 _McGill University, Toronto, Quebec, Canada_.

Obesity predisposes to colon cancer, but the underlying mechanism is not well understood. Here we confirm that diet-induced obesity promotes MC38 colon adenocarcinoma tumor growth in mice, with a high-fat diet (HFD) doubling MC38 tumor growth rates; however, we show that this effect is reversed by oral administration of either a controlled-release mitochondrial protonophore (CRMP), which acts as a liver-specific uncoupler of oxidative phosphorylation, or metformin, the most commonly prescribed antidiabetic agent worldwide. Both agents lowered circulating insulin by ~70% in HFD-fed mice. To examine the role of this reversal of hyperinsulinemia in modulating tumor growth, we performed chronic insulin infusions in mice treated with CRMP or metformin and found that the reduction of tumor growth was abrogated by restoring plasma insulin concentrations in CRMP- or metformin-treated mice to levels observed in control HFD animals. These data implicate hyperinsulinemia-and not hyperglycemia, because plasma glucose concentrations were if anything lower in mice that received replacement insulin treatment-in driving obesity-associated tumor growth. Importantly, neither CRMP nor metformin had any impact on MC38 cell number in vitro when tumor cells were incubated in the concentrations of each agent measured in tumors in vivo, arguing against a direct effect of either agent to slow tumor growth. We also demonstrate for the first time that diet-induced obesity increases both glucose uptake and oxidation, measured using a novel combined radioisotope and stable isotope tracer method, in MC38 tumors in vivo, and that both CRMP and metformin reverse these effects. However, chronic infusion of insulin to restore plasma insulin concentrations to those measured in control HFD mice increased both tumor glucose uptake and oxidation-the latter effect observed for the first time to our knowledge in the current study. These data provide new evidence that perturbations of whole organism energy balance or hepatic energy metabolism can influence neoplastic growth through modulation of hyperinsulinemia. Furthermore, the data show that glucose uptake and utilization by cancers in vivo is not necessarily constitutively high, but rather may vary according to the hormonal milieu.

#3524

Novel prodrugs of the glutamine antagonist 6-diazo-5-oxo-norleucine (DON) as treatment for malignant peripheral nerve sheath tumor.

Kathryn Lemberg,1 Ying Wu,1 Jesse Alt,1 Liang Zhao,1 Alexandra J. Gadiano,1 Chabely Rodriguez,1 Rana Rais,1 Pavel Majer,2 Jaishri Blakeley,1 Barbara Slusher1. 1 _Johns Hopkins School of Medicine, Baltimore, MD;_ 2 _Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic_.

Neurofibromatosis Type I (NF1) is a heritable tumor predisposition syndrome in which up to 10% of patients develop malignant peripheral nerve sheath tumor (MPNST), an aggressive sarcoma. For MPNST that is incompletely resected at diagnosis, traditional cytotoxic chemotherapeutic strategies offer a 5 year event-free survival of less than 40%; thus new therapeutic strategies are desperately needed. Reprogramming of energy metabolism, whereby tumor cells take up more glutamine than healthy cells and direct this substrate to replenish metabolites for proliferation, is a hallmark of several cancers that has not been effectively leveraged for treatment of MPNST. Our group has recently described JHU 395, a nervous system targeted prodrug of the glutamine antagonist 6-diazo-5-oxo-norleucine (DON) which delivers DON preferentially to the brain resulting in less gastrointestinal toxicity, which was the main toxicity of DON in past clinical trials. The primary goals of this study were to evaluate glutamine antagonism and JHU 395 activity in MPNST. Using multiple Schwann and MPNST cell lines we investigated cell proliferation in culture under glutamine deprivation and antagonism. Mass spectrometry (MS)-based metabolomic profiling was used to characterize differences between MPNST cells treated with vehicle versus DON. MS-based bioanalytical methods were also used to investigate DON delivery to tumor cells by JHU 395. We found that growth of MPNST cells in culture is preferentially inhibited by glutamine deprivation and DON treatment when compared to immortalized Schwann cells derived from non-tumored nerve (IC50 of 8-9 micromolar versus >30 micromolar). Targeted metabolomics analyses of DON treated human MPNST cells demonstrated multiple differences in downstream glutamine-dependent metabolites including intermediates in purine synthesis and amino acid synthesis, suggesting that DON acts broadly within the tumor cell to inhibit growth. While DON showed limited partitioning into MPNST cells versus plasma, JHU 395 preferentially delivered DON into MPNST with over 5-fold higher cell-to-plasma ratio. Prodrug pharmacokinetic studies in mouse demonstrated that oral administration of the prodrug safely delivered DON at micromolar concentrations to peripheral nerve. In conclusion, compared to healthy Schwann cells, MPNST cells have unique vulnerability to antagonizing glutamine utilization. The nervous system directed prodrug JHU 395 enhances DON delivery to MPNST and represents a novel potential therapeutic approach for these aggressive tumors. Based on these results we have initiated efficacy studies of JHU 395 in the murine NPcis (NF1+/-;p53+/-) model of MPNST.

#3525

N1-hexyl-N5-benzyl-biguanide promotes proliferation of CD4+ and CD8+ T lymphocytes.

Zhijun Guo, David Owen, Pamela Rosato, David Masopust, Michael A. Farrar, David A. Potter. _Univ. of Minnesota, Minneapolis, MN_.

Introduction: Solid tumors are often resistant to immunotherapy due to a hostile microenvironment for immune cells, characterized by hypoxia and low nutrients. Metformin has been shown to potentiate PD-1 blockade and improve intratumoral T-cell function and tumor response through reduction of hypoxia (1). A novel biguanide (neo-biguanide) that potentiates immunotherapy similar to metformin, but is more potent, would be more ideal for combination with immunotherapy. Recently, we have discovered that metformin binds to the mitochondria associated enzyme CYP3A4 and thereby suppresses the electron transport chain (ETC) and oxygen consumption rates (OCR) (2). Structural biology approaches led to discovery of N1-hexyl-N5-benzyl-biguanide (HBB), which binds to the CYP3A4 heme with higher affinity than metformin and much more potently inhibits the ETC and OCR (2). HBB potently and specifically inhibits CYP3A4 arachidonic acid (AA) epoxygenase activity, causing OCR and growth inhibition of breast cancer cell lines and established mouse mammary tumors (IC50=3-30 uM). Because lymphocytes express relatively lower levels of the CYP3A4 target of HBB (3), we hypothesized that HBB may permit T lymphocyte growth at concentrations that inhibit tumor epithelia. Therefore, effects of HBB on T cell proliferation and activation were tested with mouse splenocytes. Methods: HBB effects on T cell growth were tested across a range of concentrations (0 to 50 uM). C57BL/6J mouse splenocytes were incubated with or without biguanides in the presence of anti-mouse CD3 and anti-mouse CD28 antibodies. After 24 hours, cells were labeled with fluorescently tagged antibodies to mouse Thy-1, CD4, CD8, CD25, CD44, CD62L and CD73 surface markers and T cell subsets were analyzed by flow cytometry. Results: At low concentrations (0.09 to 1.5 uM) HBB did not inhibit T cell growth, but at higher concentrations (3 to 25 uM) HBB caused proliferation of CD4+ and CD8+ T lymphocytes with optimum concentrations of 6 to 12 uM (1.9-fold for CD4+ and 1.6-fold for CD8+; P<0.05 for both), conditions that inhibited breast cancer cells. Notably, CD4+FoxP3+ regulatory T cells were not increased by HBB. Metformin treatment caused an increase of CD4+ and CD8+ T lymphocytes only at concentrations >5 mM. Conclusions: HBB potently increases the proliferation of CD4+ and CD8+ mouse T lymphocytes stimulated with anti-CD3 and anti-CD28 antibodies at concentrations that suppress human and mouse mammary carcinoma cells. In contrast, metformin failed to show proliferation-inducing activity for T cells at concentrations below 5 mM. HBB therefore is a potent T lymphocyte activator, while inhibiting breast cancer epithelial cells, supporting further study of HBB to facilitate immune therapy in animal models. 1. Cancer Immunol Res. 2017 Jan 1; 5 (1) 9-16. 2. Cell Chemical Biology. 2017 Oct 19; 24(10) 1259 - 1275. 3. Drug Metab Dispos. 2008 Jan;36(1):182-9.

#3526

Beta-oxidation inhibition as a novel therapy for cisplatin-resistant ovarian cancer.

Miran Rada,1 Jennifer Cha,2 Jessica Sage,1 Bo Zhou,3 Wei Yang,3 Zahra Ashkavand,1 Kenneth Norman,1 Sandra Orsulic,3 Dong-Joo Cheon1. 1 _Albany Medical College, Albany, NY;_ 2 _Rensselaer Polytechnic Institute, Troy, NY;_ 3 _Cedars-Sinai Medical Center, Los Angeles, CA_.

Ovarian cancer is the most lethal gynecological cancer and affects one in 70 females in the United States during their lifetime. Cisplatin resistance is the major challenge in the treatment of ovarian cancer but the underlying mechanisms are not fully understood. The purpose of this study is to develop novel therapeutic strategies to treat cisplatin-resistant ovarian cancer. We identified collagen type XI alpha 1 (COL11A1) as a novel biomarker associated with cisplatin resistance in ovarian cancer. COL11A1 is highly expressed by cancer-associated fibroblasts adjacent to cancer cells as well as A2780CIS cisplatin-resistant ovarian cancer cell line. Our data show that COL11A1 binds to DDR2 in ovarian cancer cells and inhibits cisplatin-induced apoptosis in ovarian cancer cells. To obtain mechanistic insights underlying COL11A1-driven cisplatin resistance, we performed Tandem Mass Tag proteomic analysis of A2780CIS ovarian cancer cells before and after COL11A1 knockdown. Our proteomics data revealed that mitochondrial fatty acid β-oxidation was the most upregulated pathway by COL11A1 in ovarian cancer cells. Mitochondrial fatty acid β-oxidation is the major pathway that breaks down fatty acids to produce excess ATP and NADPH to support cell survival. Several studies suggested the association of β-oxidation with tumor cell proliferation in various cancers. However, the role of β-oxidation in ovarian cancer chemoresistance is largely unknown. Our results show that COL11A1 enhances the expression of not only fatty acid receptor CD36, but also key enzymes of fatty acid β-oxidation, such as CPT1A, HADHB, ACSL1 and ACAA2. Significantly, COL11A1-mediated overexpression of these proteins was diminished in the presence of shRNA against DDR2.We also confirmed that ovarian cancer cells increase both fatty acid uptake and oxygen consumption rate in response to palmitate in the presence of COL11A1. Importantly, inhibition of fatty acid β-oxidation using shRNA against CPT1A attenuated the function of COL11A1 in cisplatin resistance. Taken together, our results suggest that COL11A1 confers cisplatin resistance by increasing fatty acid β-oxidation in ovarian cancer cells. Our study uncovers fatty acid β-oxidation as a promising therapeutic target to treat cisplatin-resistant ovarian cancer, particularly cisplatin-resistant recurrent ovarian cancers which typically express high levels of COL11A1.

#3527

Inhibition of mitochondrial pyruvate transport selectively sensitizes cancer cells to metabolic oxidative stress.

Shane R. Solst, Samuel N. Rodman, Melissa A. Fath, Eric B. Taylor, Douglas R. Spitz. _University of Iowa, Iowa City, IA_.

To determine if mitochondrial pyruvate transport could represent a therapeutic target for sensitizing cancer cells to oxidative stress, lung and breast cancer cells were treated with 5 µM UK5099 to inhibit the mitochondrial pyruvate carrier (MPC). Treatment with UK5099 selectively sensitized lung and breast cancer cells to clonogenic cell killing when combined with depletion of glutathione using 1 mM buthionine sulfoximine (BSO; a glutathione synthesis inhibitor) for 48 h and 72 h, relative to normal lung and breast epithelial cells. Furthermore, cancer cell killing mediated by UK5099 combined with BSO was inhibited by the thiol antioxidant, N-acetylcysteine (NAC; 20 mM), independent of GSH levels, indicating a mechanism of toxicity involving reduced thiols and metabolic oxidative stress. In addition, treatment with UK5099 alone for 48 h also decreased levels of total glutathione in cancer cells that could be reversed by NAC. Using oxidation-sensitive fluorescent dyes (CDCFH2 and MitoSOX), treatment of lung and breast cancer cells for 24 h and 48 h with UK5099 induced increases in steady state levels of pro-oxidants (presumably hydroperoxides and mitochondrial superoxide), which were further increased with BSO. Finally, treatment of breast cancer cells with UK5099 for 24 and 48 hours significantly sensitized breast cancer cells to clonogenic cell killing mediated by paclitaxel. These data support the hypothesis that inhibition of the MPC selectively causes an impairment of antioxidant capability in cancer cells that is enhanced by depletion of glutathione. Furthermore, these results also support the hypothesis that inhibition of the MPC represents a significant target for sensitizing human breast cancer cells to chemotherapy agents thought to induce oxidative stress.

Supported by R01CA182804.

#3528

Anti-CD47 immunotherapy regulates T cell metabolism and hypoxia in the tumor microenvironment.

Yismelin R. Feliz-Mosquea, Elizabeth Stirling, Katherine L. Cook, Adam Wilson, Manish Bharadwaj, Anthony J. Molina, Liliya Yamaleyeva, Pierre L. Triozzi, David R. Soto-Pantoja. _Wake Forest School of Medicine Comp. Cancer Center, Winston Salem, NC_.

Dysfunction of infiltrating CD8+ effector T cells can be induced by hypoxia and aberrant tumor metabolite uptake in the microenvironment causing an "exhausted" T cell phenotype that limits anti-tumor immunosurveilance. Hence- therapeutic strategies aimed at improving T cell bioenergetics have the potential to reinvigorate T cell responses to reduce tumor burden. CD47 is a widely expressed receptor that controls phagocytic activity by engaging its counter receptor, SIRPα, in macrophages. Also autonomously or by binding to its ligand Thrombospondin-1 CD47 activation can control cell fate under stress. Our prior work shows that targeting CD47 on CD8+ T cells enhanced cytotoxicity against cancer cells. Moreover, depletion of CD8+ T cells in murine models reversed the anti-tumor effect of anti-CD47 therapy. Our new data shows that targeting CD47 reduced the growth of B16 melanoma tumors by approximately 50% (1607 ± 213.7, saline vs. 815.1 ± 67.8, CD47 (-) *p<0.02 n=5-6). Further examination of tumors by live photoacoustic imaging showed that tumors of animals treated with anti-CD47 had reduction in oxygen tension and reduced vascularity when compared to control. This suggests that the reduction in tumor hypoxia by CD47 may shift metabolic competition in the microenvironment to increase metabolite availability allowing optimal activation of cytotoxic T cells. Cell respirometry measurements using an Agilent Seahorse bioanalyzer showed increased levels of mitochondrial function and glycolytic flux in CD47 null T cells when compared to WT. Moreover, these cells showed increased mitochondrial density and increased levels of the mitochondrial biogenesis regulator, PGC1-a. Treatment with CD47 antibody enhanced Pmel-1 CD8+ T cell effector function by reducing B16 melanoma target cell viability by over 60%. Anti-CD47 treatment of these T cells also resulted in upregulation of cell bioenergetics, suggesting that targeting CD47 may impact T cell metabolism to enhance cytotoxic activity against cancer cells. Therefore, our studies show a new role of CD47 immunotherapy regulating immunometabolism of T cells to enhance effector function which may lead to improvement of clinical outcomes in melanoma patients.

#3529

Modulation of MDM2 alters the metabolomic programming of dedifferentiated liposarcoma and its sensitivity to cholesterol inhibition.

Bryce Demoret,1 Andrew Patt,1 John Hays,2 Ewy Mathé,1 James L. Chen2. 1 _The Ohio State Univ., Columbus, OH;_ 2 _The Ohio State Univ. Wexner Medical Ctr., Columbus, OH_.

BACKGROUND: Dedifferentiated liposarcoma (DDLPS) is a highly morbid, mesenchymal tumor characterized by amplification of the 12q chromosomal loci. Although DDLPS are lipid tumors, they produce little lipid. Life expectancy is typically less than 15 months in the advanced setting and standard treatments remain highly toxic. Superior treatment options are clearly needed. Amplification of the MDM2 oncogene that resides in these loci is observed in 100% of all DDLPS; however, to variable levels. Through a negative feedback loop, MDM2 inhibits the tumor suppressive functions of p53 that halts cell growth after cellular stress. Previously, we have reported that MDM2 levels correspond with cellular growth and drug metabolism, but little is known about the effects of MDM2 alterations on global metabolomic profiles. These profiles will help us pinpoint dysregulated pathways that explain, at least partially, the functional effects associated with MDM2 amplification. METHODS: Six DDLPS cell lines were brought directly into culture from patients. MDM2 levels were determined via Western blot and RNA-sequencing. Metabolomics data was generated using the Metabolon platform. Cell viability assays were performed in ZOOM IncuCyte or measured by XTT. Atorvastatin was used to inhibit cholesterol synthesis. MDM2 levels were altered using SAR405838 to raise MDM2 levels and MI-192 to lower MDM2 levels. Synergy was calculated via Chou-Talalay method to determine the combination index (CI) using Compusyn software. RESULTS: MDM2 levels are inversely correlated with metabolites in the lipid and cholesterol pathway (Fisher's, p < 0.001). The lipid metabolism pathway was also the top deregulated pathway (pathway enrichment p-value = 0.03) in transcriptionally profiled DDLPS cell lines treated with MDM2 elevating agent SAR405838. MDM2 low DDLPS cell lines were exquisitely sensitive to HMGA-CoA reductase inhibitors in the low micromolar range. Lipid metabolite profiling of MDM2 low versus high cell lines treated with atorvastatin demonstrated that twice as many lipid metabolites were altered in MDM2 low versus high cells (Chi-square, p < 0.001). MDM2 levels by RNA-seq demonstrated significant correlation between MDM2 gene expression and atorvastatin IC50 doses (r=0.963). MDM2 modulation by use of SAR405838 and MI-192 respectively in combination with atorvastatin displayed antagonism (average CI = 1.5) and synergy (average CI = 0.63), respectively. CONCLUSION: Modulation of MDM2 alters cholesterol metabolism in DDLPS and may serve as druggable target.

#3530

Coenzyme Q10 (BPM31510-IV in clinical trials) increases mitochondrial Q-pool and modulates electron transport chain function to elicit cell death in pancreatic cancer cells.

Pallavi Awate, Tulin Dadali, Ryan Ng, Saie Mogre, Anne R. Diers, Hannah Rockwell, Justice McDaniel, Emily Chen, Fei Gao, Michael Kiebish, Stephane Gesta, Vivek Vishnudas, Niven R. Narain, Rangaprasad Sarangarajan. _BERG, LLC, Framingham, MA_.

Mitochondria play a multifaceted role in tumorigenesis through regulation of energy production, biomass, redox state, and engagement of cell death pathways. The mitochondrial Q (coenzyme Q10)-pool facilitates electron transport from Complexes I and II to III and is essential for regulating these activities. Therefore, altering mitochondrial Q-pool homeostasis represents a potential therapeutic strategy in cancer. BPM 31510 is a Coenzyme Q10 containing lipid nanodispersion currently in clinical trials for pancreatic cancer. Here, we used BPM 31510 to assess how modulation of mitochondrial Q-pool homeostasis impacts mitochondrial electron transport chain function to activate regulated cell death. Pancreatic cancer cell lines (MIA PaCa-2 and Panc-1) represent models with sensitivity to BPM 31510 both in vitro and in vivo. Treatment with BPM 31510 (EC50 dose, 24 h) resulted in significant mitochondrial enrichment of CoQ10 compared to other subcellular compartments, and quantitatively, CoQ10 levels were 100 times higher in mitochondria isolated from BPM 31510 treated cells over untreated controls (MIA PaCa-2 untreated, 0.31 nmol/mg; treated, 39.4 nmol/mg; Panc1 untreated, 0.21 nmol/mg; treated, 29.3 nmol/mg). Alterations in mitochondrial respiration characterized by dose-dependent decreases in succinate- or glycerol-3-phosphate-fueled respiration were observed in cells treated with BPM 31510, while pyruvate or TMPD/ascorbate-fueled respiration was only modestly affected, suggesting that BPM31510 specifically impairs respiratory responses dependent on Complexes II/III. Moreover, in the presence of multiple mitochondrial substrates, total respiratory capacity was decreased and reliance on pyruvate (Complex I)-fueled respiration was increased with BPM 31510 treatment, indicative of bioenergetic remodeling. Concomitantly to BPM 31510-dependent changes in mitochondrial respiratory responses, BPM 31510 exposure increased oxidation of the reactive oxygen species (ROS) probes, CellROX Green and DCF-DA, increased oxidized glutathione, and decreased levels of the cellular reducing equivalent NADPH. Importantly, BPM 31510-induced death could be partially rescued by agents which alleviate electron transport chain impairment linking respiratory function to the anti-cancer mechanism of action of BPM 31510. Together, these data indicate that BPM31510 directly impairs the mitochondrial Q-pool and respiratory function resulting in oxidative stress and consequential cell death and thus provide mechanistic understanding of the anti-cancer activity of BPM31510.

#3531

Glycolysis inhibition and its effect in doxorubicin resistance in neuroblastoma.

Fei Chu, Yiyong Qiu, Sandra Clark, Kathryn Solka, Mary Beth Madonna. _Ann & Robert H. Lurie Children's Hosp. of Chicago and Northwestern University Feinberg School of Medicine, Chicago, IL_.

Most cancer cells exhibit increased glycolysis and use this metabolic pathway to generate ATP (Adenosine triphosphate) as the main source of their energy supply. This phenomenon is known as the Warburg effect and is considered a fundamental metabolic alteration during malignant transformation. Increased glycolysis results in elevated production of lactate, which leads to acidification of tumor tissue and provides a microenvironment that promotes and selects cellular resistance to drugs. Thus, inhibition of glycolysis may lead to more effective treatment strategies for dealing with drug resistance. 3-Bromopyruvate (3-BrPA), an inhibitor of hexokinase II and an inhibitor of glycolysis, has been shown to be a potent chemotherapeutic agent. Despite many studies showing the efficacy of 3-BrPA against various cancer cells, there have been relatively few studies examining the effects of 3-BrPA on the anti-cancer effects of doxorubicin (Dox) in doxorubicin-resistant (DoxR) human neuroblastoma. The purpose of this study is to examine the effects of 3-BrPA upon DoxR human neuroblastoma cells to treatment with doxorubicin. In present study, we have found that 3-BrPA enhances the response of DoxR neuroblastoma to treatment with Dox and this enhancement exists in both normoxic conditions as well as hypoxic conditions. The results also suggested that this increased response of DoxR cells to treatment with Dox is mediated through decreased intracellular levels of ATP. Additionally the lactate assay revealed that intracellular lactate levels were decreased in neuroblastoma lines when glycolysis was inhibited with 3-BrPA. In order to determine if 3-BrPA, either alone or in combination with cytotoxic drugs, will lead to maximal efficacy of treating of drug-resistant tumor xenografts in vivo. Animal experiments were carried out to study the effects of 3-BrPA in drug-resistant neuroblastoma in nude mice. We have found that 3-BrPA combined with doxorubicin provided significant reduction in tumor size and the rate of tumor growth in mice. The efficacy of doxorubicin itself (88% reduction in tumor volume) overshadowed any effect of cotreatment with 3-BrPA on the Wild type tumors. However, in the DoxR tumors, doxorubicin alone had no efficacy as expected, but co-treatment with 3-BrPA suppressed tumor growth with an 80% reduction in tumor volume. These studies pave the way to determine whether inhibition of glycolysis may be an effective strategy to kill neuroblastoma cells and overcome drug resistance associated with hypoxic conditions. This novel approach may have broad applications in cancer treatment, considering the prevalent Warburg effect observed in a wide array of human cancers.

#3532

Bitter melon extract suppresses triple-negative breast cancer growth by inhibiting ACAT1.

So Hee Shim, Robert Steele, Ratna Ray. _Saint Louis University, Saint Louis, MO_.

Breast cancer is the most common malignant cancer diagnosed in women worldwide each year. Triple-negative breast cancers (TNBC), defined by the lack of expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), account for approximately 15% of breast cancers. Moreover, TNBC is usually more aggressive and metastatic with a worse prognosis. Bitter melon (Momordica charantia) is well known as a dietary supplement. Recently, we have shown an anti-cancer activity of bitter melon extract (BME) in breast cancer mouse models, although the mechanism is poorly understood. Furthermore, we became interested to understand the role of BME on cholesterol and lipid metabolism in TNBC. Our mass spectrometry data suggested that stored as a cholesteryl esters (CE) in Lipid droplets (LD) were much lower in BME treated TNBC cells. We found that not only CE but also LD was reduced in BME treated TNBC cells. The acyl-CoA:cholesterol acyl transferase-1 (ACAT-1) catalyzes the formation of CE from free cholesterols together with long-chain fatty acyl coenzyme A. Therefore, inhibition of ACAT-1 resulted in downregulation of CE, and upregulation free cholesterols. The attenuated CE is known to reduce cancer proliferation. Subsequent study demonstrated that BME treatment highly inhibited ACAT-1 in TNBC cells. The upregulated free cholesterol negatively regulates sterol regulatory element-binding proteins (SREBPs), which regulates lipogenesis protein such as fatty acid synthase (FASN) associated with cancer cell proliferation. We also observed that SREBP-1 and -2 were barely detectable, and FASN and low-density lipoprotein receptor (LDLR) were reduced in BME treated TNBC cells as compared to control cells. To our knowledge, this is the first report demonstrating that BME suppresses TNBC cell growth through ACAT-1 inhibition. BME supplement may potentially be an additional regimen in human breast cancer treatment.

#3533

Elevated expression of heme related proteins in viable tumor cells treated with vascular disrupting agent, combretastatin A-4 phosphate in xenograft models of NSCLC.

Sanchareeka Dey,1 Sharda Kumari,2 Sarada Preeta Kalainayakan,1 Poorva Ghosh,1 Li Zhang,1 Li Liu2. 1 _UT Dallas, Richardson, TX;_ 2 _UT Southwestern Medical Center, Dallas, TX_.

Tumors cannot grow without a supply of oxygen and nutrients. Hence, targeting tumor vasculature with Vascular Disrupting Agents (VDAs) represents a promising therapeutic strategy, especially for the treatment of solid tumors. However, VDAs cause central necrosis, leaving a rim of viable tumor cells that often eventually lead to tumor recurrence and development of resistance. Here, we aim to better understand the underlying molecular mechanisms leading to the acquisition of VDA-resistance and its correlation with the extent of hypoxia and respiratory functions.

A well-known VDA, Combretastatin A-4 phosphate (CA4P), was used for the study. First, a NSCLC cell line expressing luciferase was implanted into the right flanks of female SCID mice. Next, CA4P was administered intraperitoneally. The effect was monitored 3 and 24 hours post injection of CA4P through BLI (Perkin Elmer's IVIS Lumina III) after subcutaneous injection of luciferin. After sacrificing the mice, the tumors were harvested, formalin-fixed, and paraffin-embedded for Immunohistochemistry (IHC).

Our BLI data indicated significant decrease in radiance 3 hour post treatment (total flux in photons per second) which remained so at 24 hours, when luciferin was re-administered. Our IHC data indicated enhanced levels of enzymes involved in heme biosynthesis, uptake and degradation, and hemoproteins involved in oxidative phosphorylation. Furthermore, we examined the effect of a mitochondrial targeting agent (MTA) that undermined the expression of these proteins, on the progression of subcutaneous tumor xenografts.

### Ubiquitylation, Vesicles, and Membranes

#3534

Protein acylation mediates encapsulation of Src kinase into exosomes.

Ru Wen, Qianjin Li, Sungjin Kim, Yongjie Ma, Jin Xie, Houjian Cai. _Univ of Georgia, Athens, GA_.

Exosomes are nanosized vesicles (30-150 nm) secreted from almost all cell types (1). Exosomes play important roles in cell-cell communication through the transfer of exosomal proteins, mRNA, and microRNAs, mediating immune escape and promoting tumor progression (2,3). However, it remains unclear how oncogenic proteins are selectively disseminated through exosomes. Myristoylation is lipid modification of protein involved in the attachment of N-terminal glycine of proteins with a myristoyl group. It is critical for various biologic functions including cell signaling, protein localization, and cell-cell communication (4). In this study, we evaluated the role of protein acylation for the encapsulation of Src family kinases in exosomes. Myristoylation promotes the enrichment of Src kinase in exosomes whereas palmitoylation inhibits the encapsulation process. Additionally, phosphorylated Src protein was more favored to be capsulated in exosomes. Mechanistically, knockdown of TSG101 decreased expression levels of Src kinase in exosomes, indicating that Src encapsulation into exosomes is associated with endosomal sorting complexes required for transport (ESCRT). Finally, Src kinase can be translocated to normal cells via exosomes cargo to induce pathologic signaling. These results provide new insights of Src kinase encapsulation mechanism into exosomes, and therapeutic approaches for inhibiting the transfer of oncogenic proteins through targeting protein myristoylation process.

References:

1. Simons M, Raposo G. Exosomes--vesicular carriers for intercellular communication. Curr Opin Cell Biol 2009;21(4):575-81.

2. Skog J et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nature Cell Biol 2008;10(12):1470-6.

3. Abusamra AJ et al. Tumor exosomes expressing Fas ligand mediate CD8+ T-cell apoptosis. Blood Cells Mol Dis 2005;35(2):169-73.

4. Casey PJ. Protein lipidation in cell signaling. Science 1995;268(5208):221.

#3535

Differential expression and localization of the E3 ubiquitin ligase Nrdp1 in African American versus Caucasian American prostate cancer cells.

Neelu Batra,1 Sheryl Krig,1 Anhao Sam,1 Katelyn Macias,1 Rosalinda Savoy,2 Salma Siddiqui,3 Frank Melgoza,3 Leandro D'Abronzo,2 Sidhartha Hazari,1 Blythe Durbin-Johnson,4 Paramita Ghosh,3 Ruth L. Vinall1. 1 _California Northstate University, Elk Grove, CA;_ 2 _University of California, Davis, Sacramento, CA;_ 3 _VA Northern California Health System, Mather, CA;_ 4 _University of California, Davis, Davis, CA_.

Despite a recent decrease in overall incidence of prostate cancer (CaP), CaP incidence continues to be much higher among men of African origin living in the United States (223.9 men per 100,000) compared to those of European origin (139.9 men per 100,000). Also, the CaP age-specific mortality rates are 2.4 times greater among African-American (AA) compared with European American (EA) men. The causes for these differences are multifactorial, but include genetic effects that contribute to CaP-associated health disparity. We previously showed that the androgen receptor (AR) can suppress levels of the receptor tyrosine kinase ErbB3, a molecule that is known to drive CaP progression, by stimulating the E3 ubiquitin ligase Nrdp1 (also called RNF41 or FLRF). The goals of the current study were to determine whether differential expression and/or localization of Nrdp1 contribute to CaP-associated health disparities, and to further elucidate the mechanisms by which regulation of Nrdp1 levels and localization occurs. Immunohistochemistry (IHC) in prostate tissue determined that nuclear Nrdp1 levels are significantly lower in AA CaP patients (n=19) versus CA CaP patients (n=121) with localized disease (p=0.008). A similar association was observed in CaP cell lines; immunofluorescence (IF) analyses demonstrated MDAPCa2b and E006 (cell lines derived from AA CaP patients) express significantly lower levels of nuclear Nrdp1 compared to LNCaP, CWR22Rv1, C4-2, and C4-2B (cell lines derived from CA CaP patients). Western blot and qPCR determined that Nrdp1 levels are lower in AA versus CA CaP cells (MDAPCa2b express ~2-fold less Nrdp1 protein and mRNA levels compared to LNCaP), and that androgen withdrawal has a bigger impact on Nrdp1 expression levels in AA CaP cells (2-fold decrease in MDAPCa2b compared to ~1.2-fold decrease in LNCaP). Proteasomal regulation of Nrdp1 also occurs; treatment with MG132 resulted in accumulation of Nrdp1 in both AA and CA CaP cell lines. Interestingly, altering androgen and/or AR levels also affected Nrdp1 localization (IF analyses) as well as the ability of Nrdp1 to mediate CaP cell apoptosis (flow cytometry). In summary, we demonstrate that Nrdp1 is differentially expressed in AA versus CA CaP patients as well as in AA versus CA CaP-patient derived cell lines, and that androgen withdrawal has a bigger impact on Nrdp1 expression in AA CaP cell lines. We also demonstrate that androgen/AR is involved in determining Nrdp1 localization, and that Nrdp1 can mediate CaP cell apoptosis in the presence of AR. The combined data, and the knowledge that Nrdp1 regulates ErbB3 levels, suggest that differential expression of Nrdp1 in AA versus CA CaP may contribute to CaP-associated health disparities and may occur as a result of dysregulation of the regulation of Nrdp1 by androgen/AR.

#3536

SENP1 is a p53 deSUMOylating enzyme and its depletion induces p53 activity.

Mushui Dai, Krishna Chauhan, Yingxiao Chen, Xiao-Xin Sun. _Oregon Health & Sciences Univ., Portland, OR_.

SUMOylation plays important roles in diverse cellular processes by regulating protein-protein interaction, protein localization, trafficking, stability and activity. p53 can by SUMOyalted at lysine (K) 384. Yet, how SUMOylation regulates p53 activity remain unclear. SUMOylation can be revered by a group of proteins called deSUMOylating enyzme including the SUMO specific protease (SENP) family proteins. How p53 is regulated by deSUMOylation is also not well understood. Here, we show that SENP1 directly interacts with and desumoylates p53 in cells and in vitro. Depletion of SENP1 by siRNA-mediated knockdown drastically increased the p53 transactivation activity without inducing p53 protein levels, resulting p53-depndent cell cycle arrast. Further, SENP1 knockdown significantly promoted p53 transactivation activity in cells following DNA damage. Additionally, we found that SENP1 binds to and deSUMOylates PML and aborageted the enhancing effect of PML on p53 SUMOylation. Thus, SNEP1 is novel p53 desumoylating enzyme that regulates p53 activity but not its stability.

#3537

FBXW7-mediated extracellular signal-regulated kinase 3 stability in cancer development.

Seung-Min Kim, Cheol-Jung Lee, Sun-Mi Yoo, Seon-Yeon Cho, Juhee Park, Yong-Yeon Cho. _College of Pharmacy, The Catholic University of Korea, Bucheon, Republic of Korea_.

Extracellular signal-regulated kinase (ERK) is an atypical member of the mitogen-activated protein kinase (MAPK) family. Initial studies have demonstrated that ERK3 was highly expressed in the central nervous system, skeletal muscle, lung, thymus, and testis. Interestingly, unlikely with classical MAPKs including ERK1/2, JNKs and p38s, ERK3 is known to have narrow substrate specificity. Although the function of ERK3 has studied for last three decades, some information for the role of ERK3 is currently available. Recent studies indicate that ERK3 is upregulated in diverse cancer tissues including lung cancer and colon cancer and ERK2 promotes cancer cell migration and invasion in triple-negative breast cancer. Interestingly, ERK3 showed short protein half-life about 30-60 min and ubiquitin-specific protease 20, a deubiquitinase, induced the ERK3 protein level. Thus, identification of E3 ubiquitin ligase is important to understand ERK3 function in cell transformation and cancer development. In this study, we identified that FBXW7, a member of F-box protein complex, bind with ERK3. Moreover, we found that ectopic expression of FBXW7 decreased ERK3 protein level in a dose dependent manner. By proteome analysis, we found that ERK3 contained FBXW7 specific degron motif in C-terminal variable region. Importantly, FBXW7 depletion in HCT-116 restored ERK3 protein level. Taken together, we concluded that FBXW7 is a specific binding partner of ERK3 and may regulate ERK3 protein stability.

Key words: MAPK, ERK3, FBXW7, ubiquitination, protein stability

#3538

Targeting the E3 ubiquitin ligase RNF20 in ovarian cancer.

Alexander J. Cole,1 Kristie-Ann Dickson,2 Roderick Clifton-Bligh,2 Deborah J. Marsh2. 1 _University of Michigan, Ann Arbor, MI;_ 2 _Kolling Institute, University of Sydney at Royal North Shore Hospital, Sydney, Australia_.

E3 ubiquitin ligases are responsible for the final stage of the enzymatic ubiquitination cascade, where they transfer ubiquitin from an E2 conjugating enzyme to a lysine on the recognized substrate. The most common family of E3 ligases are RING (Really Interesting New Gene) domain proteins. Using immunohistochemistry, we have shown that the ring finger protein RNF20 is strongly expressed in 87% (379 of 424) of high-grade serous ovarian cancer (HGSOC) (1). In contrast, other tumors, including breast, metastatic prostate and colorectal cancer, have been reported to show a decrease in RNF20 transcript levels. The Cancer Genome Atlas has reported < 1% (3 of 316) HGSOCs with RNF20 missense variants of unknown pathogenicity. Given the high level of RNF20 expression in HGSOC, it is unlikely that RNF20 transcript levels are reduced in these tumors.

RNF20 functioning in a heterodimer with RNF40 is the main E3 ligase complex responsible for monoubiquitination of histone H2B at lysine 120 (H2Bub1) (2). This active histone mark leads to an open chromatin configuration favouring DNA access by transcriptional complexes and DNA repair proteins. The RNF20/RNF40 complex also monoubiquitinates and stabilizes the Eg5 motor protein that functions in spindle assembly during mitosis, preventing spindle assembly defects, cell cycle arrest and apoptosis. RNF20 has also been implicated in polyubiquitination of the tumor suppressor Ebp1, promoting its degradation via the 26S proteasome in malignant cells.

We have down-regulated RNF20 in ovarian cancer cell lines to investigate the potential of targeting this E3 ligase to overcome resistance to platinum therapies that is a major problem for the clinical management of women with ovarian cancer. Cell lines studied included the HGSOC lines; COV318, OVCAR-3, OVCAR-4, CaOV3, HEY and OVSAHO, as well as the endometrioid line A2780. Cells were studied as monolayers and spheroid cultures. We have shown that down-regulation of RNF20 leads to lower levels of H2Bub1, consistent with a more closed chromatin configuration. Clonogenic cell survival assays following the treatment of ovarian cancer cell lines with cisplatin showed an impaired ability of cells in which RNF20 had been down-regulated to form colonies. This work provides preliminary evidence that developing strategies to therapeutically down-regulate RNF20 may increase the efficacy of standard-of-care chemotherapy for women with ovarian cancer.

(1) Dickson KA et al., Hum Mol Genet (2016)

(2) Cole AJ et al., Endocr Relat Cancer (2015)

#3539

Establishing ubiquitylation patterns in cells: Efficient monitoring of oncogenic ubiquitylation activity.

Peter K. Foote, Xiaolong Lu, Rajesh Singh. _LifeSensors, Malvern, PA_.

Cells regulate their internal functions by the ubiquitylation of many key proteins, especially onco-proteins and tumor suppressors. As a result, ubiquitin proteasome system (UPS) substrates and the relevant UPS enzymes have been identified as attractive anti-cancer targets. However, traditional methods for monitoring ubiquitylation patterns, including by immunoprecipitation coupled with immunoblot analysis (IP/IB) or by mass spectrometry methods, are either insensitive, low throughput, or highly intensive. Here, we describe three Tandem Ubiquitin Binding Entity (TUBE)-based assays, UbiTest, HT-UbiTest, and UbiQuant S, which provide a platform to efficiently detect ubiquitylation of anti-cancer targets. UbiTest is an immunoblot-based assay, which can be used to easily identify ubiquitlyated proteins. Compared to traditional ubiquitin IP/IB, this assay avoids epitope masking of antibodies caused by ubiquitin modification. HT-UbiTest extends this concept using a TUBE pulldown followed by two-antibody detection in a 96-well plate, and allows high-throughput absolute quantification of a target of interest. UbiQuant S is a high-throughput assay to quantify ubiquitylation status in cells or tissue. Ubiquitylated proteins are identified by energy transfer between AlphaLISA bead-labeled TUBE and antibody against the endogenous protein of interest in a homogenous plate-based assay. Furthermore, we have extended these technologies to identify K63- and K48-linked poly-ubiquitylated proteins using poly-ubiquitin linkage-selective TUBEs. Several prominent therapeutic targets in oncology and immuno-oncology have been used to verify these assays, including Cbl-b and its substrates, p53, and USP7. The efficiency and sensitivity of these assays have wide applications in drug discovery targeting ubiquitylation-dependent signaling pathways. Examples using DUB and ubiquitin ligase inhibitors illustrate the power of this system. These novel assays are versatile tools for understanding the role of ubiquitylation in cancer treatment, and provide platforms to monitor disease biomarkers in response to drug action.

#3540

Ubiquitin code: Accelerating the discovery of poly-ubiquitylated proteins by novel chain-selective affinity matrices.

Apurva Chaturvedi, Peter Foote, Sarah Julius, Essam Abed, Rajesh K. Singh. _LifeSensors Inc, Malvern, PA_.

Ubiquitin (Ub) is attached via isopeptide bonds to lysine residues in the target protein or to another Ub to form poly-Ub chains. The reversibility, heterogeneity, and diversity of these modifications combined with the lack of suitable tools have made it difficult to properly isolate and characterize poly-ubiquitylated cellular proteins. However, in 2009, a novel technology called TUBEs (Tandem-repeated Ub-Binding Entities) was developed. TUBEs have revolutionized the Ub field by allowing poly-ubiquitylated proteins to be enriched/purified from cellular extracts. Development of TUBEs that selectively bind K48-, K63-, and M1-linked poly-ubiquitylated proteins has helped understand the role of modified proteins in cell physiology. However, the roles of other Ub-linkages remain obscured mainly due to the lack of tools that specifically recognize them. Structure-based approach to engineer unique high affinity Ub binding domains(UBDs) for rare Ub-linkages has been implemented to design highly selective affinity matrices. Biochemical and biophysical experiments demonstrate highly selective interactions of the matrices to distinct lysine-linked poly-Ub chains. Power of these highly selective affinity matrices has made it possible to perform Ub proteomics bypassing SILAC mass spectrometry. Application of these affinity matrices in Far Western detection, pull-down and imaging will be described in the poster. To conclude, we have identified unique binding partners for K6-, K11-, K27-, K29-, K33- and K48-linked poly-Ub chains. Development of chain selective affinity matrices (TUBEs) that selectively bind to poly-ubiquitylated proteins will dramatically accelerate the pace of discovery in this important area of biology.

#3541

Epigenetic regulation of KPC1 ubiquitin ligase has a regulatory role on the NF-κB pathway in metastatic melanoma.

Yuuki Iida,1 Aaron Ciechanover,2 Diego M. Marzese,1 Keisuke Hata,1 Matias Bustos,1 Shigeshi Ono,1 Jinhua Wang,1 Matthew P. Salomon,1 Kevin Tran,1 Yelena Kravtsova-Ivantsiv,2 Gordon B. Mills,3 Michael A. Davies,3 Dave S.B. Hoon1. 1 _John Wayne Cancer Institute at Providence Saint John's Health Center, Santa Monica, CA;_ 2 _Technion-Israel Institute of Technology, Haifa, Israel;_ 3 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Purpose: Abnormal activation of the NF-κB pathway promotes a more aggressive phenotype of cutaneous malignant melanoma. Understanding the mechanisms regulating the NF-κB pathway in melanoma is of critical importance. KPC1 (RNF123) is an E3 ubiquitin ligase that leads to proteasomal processing of precursor NF-κB1 p105 into mature p50, one of the most important steps in the NF-κB pathway regulation. We demonstrated novel epigenetic mechanisms affecting KPC1 expression that lead to an abnormal activation of the NF-κB pathway, which was significant during tumor progression in melanoma patients.

Experimental Design: Initially using melanoma cell lines, we investigated the functional interactions between KPC1 and NF-κB, and the epigenetic regulations of KPC1, including microRNA targeted interaction and DNA methylation. The clinical impact of KPC1 expression and these epigenetic regulations were further assessed in large cohorts of clinically well-annotated melanoma tissues (tissue micro-arrays; n=137, JWCI cohort; n=40) and melanoma TCGA database cohort (n=370). High-throughput RNA sequencing, reverse-phase protein array and human methylation 450k platform were utilized for comprehensive analyses.

Results: Initially using metastatic melanoma cell lines, we verified that KPC1 promotes processing of NF-κB1 p105 into p50, thereby modulates NF-κB-target gene expression and suppresses melanoma cell proliferation. Concordantly in melanoma tissue, KPC1 expression was down-regulated in AJCC stage IV melanoma compared to early stages (JWCI cohort stage I/II p=0.013, stage III p=0.004), whereby low KPC1 expression was significantly associated with poor overall survival in stage IV melanoma (tissue micro-arrays, n=137, Hazard Ratio 1.810, p=0.006). Furthermore, epigenetic mechanisms regulating KPC1, particularly miR-155-5p and

DNA methylation level at its promoter region, was shown to be significant in melanoma lines. This regulatory mechanism was validated in clinical melanoma tissues. High miR-155-5p expression, which is negatively regulated by its promoter DNA methylation level (melanoma TCGA database cohort; Pearson's r -0.455, p<0.001), is significantly associated with KPC1 down-regulation (JWCI cohort; p=0.028, melanoma TCGA database cohort; p=0.003).

Conclusions: We identified miR-155-5p, which is epigenetically controlled by its promoter methylation, has a regulatory role on KPC1 expression. These interactions promote to down-regulation of KPC1 and abnormal NF-κB pathway activation, leading to highly proliferative melanoma cells and poor clinical outcomes. These findings suggest utility of KPC1 expression level for stratification of stage IV melanoma patients, and the importance of the miR-155-5p-KPC1-NF-κB-axis in controlling melanoma proliferation.

#3542

Missense mutations in USE1 promote lung tumorigenesis.

Peter C. W. Lee. _University of Ulsan College of Medicine, Seoul, Republic of Korea_.

The UBA6-USE1 enzyme cascade is a poorly characterized arm of the ubiquitin-proteasome system. We found that UBA6 and USE1 proteins are frequently overexpressed in lung cancer patients (83.01% and 92.45%, respectively; n = 106). Stable overexpression of UBA6 or USE1 significantly increased cell proliferation, migration, and invasiveness in lung cancer cells and xenograft models, whereas their knockdown significantly reduced cell proliferation, migration, and invasion. USE1 has a conserved D-box domain and the level of the protein was regulated by the anaphase-promoting complex through its interaction with CDC20 and CDH1. Furthermore, several missense mutations in USE1 identified in patients prolong the half-life and stability of the protein. Our findings reveal novel roles for USE1 in lung cancer and the possible use of USE1 as a novel biomarker and therapeutic target for lung cancer treatment.

#3543

USP7 inhibitor P5091 inhibits esophageal squamous cell carcinoma growth by inducing NOXA-mediated apoptosis.

Tao Hu, Jing-Yang Zhang, Bei-Bei Sha, Miao-Miao Li, Xing-Ge Liu, Yi Zhang, Zi-Ming Dong, Pei Li, Ping Chen. _Zhengzhou University, Zhengzhou, China_.

Recently, deubiquitinase USP7/HAUSP was reported to be an attractive anti-cancer target. However, its expression and role in esophageal cancer remain elusive. Here, immunohistochemistry and western blot assay were used to detect the expression of USP7/HAUSP in esophageal squamous cell carcinoma (ESCC) tissues. Specific inhibitor P5091 was used to inhibit the activity of USP7 and explore the role and mechanism of USP7 in KYSE450, KYSE510 and KYSE30 ESCC cell lines.

Result: Staining-intensity analysis by immunohistochemistry demonstrated that USP7 was expressed higher in ESCC tissues compared with their corresponding adjacent tissues (P<0.01), which was confirmed by western blot analysis. CCK-8 assay, colony formation assay and tumor growth assay in subcutaneous transplantation tumor model indicated that P5091 could significantly inhibit ESCC cell growth in vitro and in vivo. Moreover, P5091 induced apoptosis by triggering unfolded protein reaction, accumulating the protein of ATF4 and thus transcriptionally upregulating the expression of NOXA.

Conclusion: These results showed that P5091 effectively inhibited ESCC cell growth in vitro and in vivo and supported the clinical investigation of P5091 for the treatment of ESCC.

Keywords: P5091, esophageal squamous cell carcinoma, ATF4, apoptosis, NOXA

* T H and J-Y Z contributed to this work equally.

#3544

Ras GTPases are modified by SUMOylation.

Byeong Hyeok Choi,1 Changyan Chen,2 Mark Philips,3 Wei Dai1. 1 _NYU School of Medicine, Tuxedo Park, NY;_ 2 _Northeastern University, Boston, MA;_ 3 _NYU School of Medicine, New York, NY_.

RAS proteins are GTPases that participate in multiple signal cascades, regulating crucial cellular processes including cell survival, proliferation, differentiation, and autophagy. Mutations or deregulated activities of RAS are frequently the driving force for oncogenic transformation and tumorigenesis. Given the important roles of the small ubiquitin-related modifier (SUMO) pathway in controlling the stability, activity, or subcellular localization of key cellular regulators, we investigated here whether RAS proteins are post-translationally modified (i.e., SUMOylated) by the SUMO pathway. We observed that all three RAS protein isoforms (HRAS, KRAS, and NRAS) were modified by the SUMO3 protein. SUMOylation of KRAS protein, either endogenous or ectopically expressed, was observed in multiple cell lines. The SUMO3 modification of KRAS proteins could be removed by SUMO1/sentrin-specific peptidase 1 (SENP1) and SENP2, but not by SENP6, indicating that RAS SUMOylation is a reversible process. A conserved residue in RAS, Lys-42, was a site that mediates SUMOylation. Results from biochemical and molecular studies indicated that the SUMO-E3 ligase PIASγ specifically interacts with RAS and promotes its SUMOylation. Moreover, SUMOylation of RAS appeared to be associated with its activation. In summary, our study reveals a new post-translational modification for RAS proteins. Since we found that HRAS, KRAS, and NRAS can all be SUMOylated, we propose that SUMOylation might represent a mechanism by which RAS activities are controlled.

#3545

Identifying the effects of lung cancer exosomes on non-tumorigenic human bronchial epithelial cells.

Hana Kubo, Sean E. Humphrey, Sarunya Kitdumrongthum, Feng Tian, Sarah Allen, Andrea L. Kasinski. _Purdue University, West Lafayette, IN_.

Lung cancer is the leading cause of death from cancer in most industrialized countries and many lung cancer patients remain undiagnosed until the development of locally advanced or metastatic lung cancer. Despite extensive research, universal biomarkers for the early diagnosis of lung cancer remain elusive, and therapies for treating lung cancer patients remain ineffective and unfocused. This makes the identification of new diagnostic biomarkers and therapeutic targets for the treatment of lung cancer a critical need for patients. Exosomes, nanovesicles secreted by a variety of cell types, are known to transfer bioactive molecules such as proteins and nucleic acids that induce physiological changes in recipient cells. Exosomes secreted by cancer cells have been shown to promote tumor initiation, progression, and metastasis in various cancer types, but have been understudied in lung cancer. Our study aims to identify the effects lung cancer secreted exosomes may have on non-cancerous cells of the tumor microenvironment by assessing their tumorigenic effects on normal lung epithelial cells. To that end, we treated normal lung epithelial cells with exosomes derived from a panel of human non-small lung cancer cell lines. Treated cells acquired the ability to invade and migrate. As a proxy for metastasis, cancer exosomes are also being assayed for their ability to permeabilize a monolayer of normal lung epithelial cells. Further assessing the molecules within cancer exosomes critical for promoting these oncogenic behaviors in non-tumorigenic cells could lead to the discovery of potential diagnostic biomarkers and therapeutic targets for inhibiting tumor growth in affected patients.

#3546

Extracellular vesicles and exosome-associated microRNAs in squamous cell carcinoma of the head and neck disrupt dendritic cell differentiation and maturation.

Luciana C. Marti,1 Elisangela P. Silva,1 Mariana B. Rizzo,2 Rossana V. Lopez,3 Otavio A. Curioni,2 Raquel A. Moyses,4 Fabio D. Nunes,4 Patricia Severino1. 1 _Hospital Israelita Albert Einstein, São Paulo - SP, Brazil;_ 2 _Hospital Heliopolis, São Paulo - SP, Brazil;_ 3 _Instituto do Câncer do Estado de São Paulo, São Paulo - SP, Brazil;_ 4 _Universidade de São Paulo, São Paulo - SP, Brazil_.

Squamous cell carcinomas (SCCs) remain among the most common malignancies of the head and neck region. Plasma microRNAs (miRNAs) are appealing biomarker candidates for diagnosis or therapy response monitoring since they may be associated with clinical and pathological features of the disease. Increasing evidence also points to a role for these molecules, mostly when transported by extracellular vesicles (EVs), in signaling between cancer and immune cells. The aims of this study are to search for potential miRNA biomarkers for SCCs in plasma, to evaluate the presence of these markers within EVs using SCC-derived cell lines as study models and to study the effect of EVs on monocyte-derived dendritic cells functions. Total RNA was isolated from plasma samples from head and neck SCC patients and healthy donors following optimized protocols. EVs from the culture medium of SCC cell lines were collected by differential centrifugation and visualized by transmission electron microscopy. The expression profile of miRNAs in plasma and EVs was evaluated by qRT-PCR. CD14+ cells from healthy donors were selected from peripheral blood using magnetic selection columns. Monocyte-derived immature dendritic cells were obtained following CD14+ cells treatment with human recombinant IL-4 and GM-CSF for six days and the maturation signal consisted on treatment of immature dendritic cells with LPS for 24 hours. Both treatments were carried out either in the presence or absence of EVs. Immunophenotyping of monocytes and monocyte-derived dendritic cells was based on the detection and expression levels of cell surface markers CD14, CD33, CD45, CD80, CD86, CD209 and HLA-DR by flow cytometry. A subset of miRNAs was consistently detected in SCC patients' plasma and EVs. Among these, miR-24 is of special interest since it has been associated with antigen processing and presentation by dendritic cells. The percentage of CD14+ cells expressing CD33 when cultured in the presence of EVs was significantly lower, and the same effect was seen for the CD209 and CD80 markers, suggesting alterations of antigen presentation functions. The percentage of cells expressing HLA-DR was similar both when CD14+ cells were cultured in the presence or without the addition of EVs, but the level of HLA-DR expression, as measured by fluorescence intensity, was lower when EVs were present, following both differentiation and maturation signals. The detection of miRNAs both in plasma and in association with EVs corroborates their potential as cancer biomarkers but also their possible implication in cell signaling. Differences in surface markers of monocyte-derived dendritic cells in the presence of EVs suggest an effect of EV content, including miRNAs, on differentiation and maturation processes, essential aspects for the proper function of dendritic cells in the tumor microenvironment.

#3547

Properties of lipid rafts in two epigenetically distinct subtypes of the oncogenic cell-line SW13.

Luis Espejo, Kathryn J. Leyva, Elizabeth E. Hull. _Midwestern Univ. - Glendale Campus, Glendale, AZ_.

The epithelial SW13 cancer cell line has two naturally occurring well-defined cell subtypes. The SW13(Vim-) has a phenotype that resembles typical epithelial tumors, displaying increased growth and proliferation with a low level of invasiveness. The SW13(Vim+) subtype has a mesenchymal-like phenotype, commonly associated with more invasive and metastatic states of epithelial cancers. SW13(Vim-) can be converted to a pseudo SW13(Vim+) phenotype when treated with histone deacetylase inhibitors (HDACi). This model is used to study the epigenetic phenotypic differences between tumor-like cancer cells and cells that display a more metastatic phenotype. Gene chip data obtained from the SW13 treated and untreated cells displays differential expression in many genes, specifically genes for cellular membrane proteins and genes involved in cholesterol, sphingolipid, and GPI-anchored protein metabolism; all of which are essential components of lipid rafts. Additionally, recent improved outcomes noted in patients using statin drugs further implicates that cholesterol plays a role in cancer progression, although the mechanism for this improvement is not well understood. This suggests lipid rafts are a candidate for a physical structure that manifests the differential effect of cholesterol perturbations in cancer phenotypes. We hypothesize that lipid raft protein composition is distinctly different between the two SW13 cell subtypes, and that the differences in the composition of lipid rafts may play a role in the unique oncogenic phenotypes of the two subtypes. Utilizing low-temperature detergent-resistant lipid raft isolation techniques, western blotting, and mass spectrometry we investigate protein composition differences between the two SW13 cell subtypes. Cholesterol assays are applied to assess the distribution and total cholesterol content of each SW13 subtype, this also provides translational confirmation of the gene chip data obtained from the subtypes. Cholesterol depletion techniques utilizing cyclodextrin for acute disruption and simvastatin for chronic disruption are being implemented to perturb lipid rafts and observe their role in the oncogenic phenotypes of the SW13 cell subtypes. Specifically, metastatic qualities are measured after lipid raft and cholesterol perturbations. Initial findings support differential cholesterol representation in the two SW13 cell subtypes. Potential findings may explain a mechanism in which cholesterol perturbations are capable of decreasing metastasis and improving the outcome of patients with epithelial cancers.

#3548

EPAC-RAP1, the alternative cyclic AMP signaling pathway, regulates cell cycle progression in primary melanoma cells.

Aishwarya Krishnan, Kirthana Prabhakar, Mary Ndiaye, Nihal Ahmad, Carlos Ivan Rodriguez, Vijayasaradhi Setaluri. _Univ. of Wisconsin-Madison, Madison, WI_.

Cyclic AMP (cAMP) is an important second messenger that acts as a key mediator in a wide range of cellular pathways. In melanocytes, the G-Protein Coupled Receptor (GPCR) Melanocortin-1-Receptor (MC1R), an important regulator of melanocyte proliferation and melanogenesis, activates cAMP signaling. Polymorphisms in MC1R lead to cAMP signaling impairments which are accompanied by higher risk for melanoma development. Despite the importance of cAMP signaling in melanocytes, very little is known about its role in melanoma. Exchange Protein directly Activated by cAMP (EPAC) constitutes an alternative pathway to cAMP signaling and is known to mediate the crosstalk between the cAMP and MAPK pathways. In this study, we show that pharmacological inhibition of EPAC, delays cell cycle progression and inhibits the growth of human primary melanoma cells. Flow cytometry and western blot analysis of cell cycle proteins showed that inhibition of EPAC arrests cells in G1-S and delays their mitotic progression. Paradoxically, inhibition of EPAC also caused an increase in AKT phosphorylation, suggesting a cell survival response elicited upon the cell cycle arrest caused by inhibition of EPAC allows continued survival of primary melanoma cells. Inhibition of EPAC in metastatic melanoma cells, on the other hand, stimulated their growth. These data show differential role of EPAC in primary and metastatic melanoma cells and provide a basis for targeting EPAC for prevention of cutaneous melanoma progression.

#3549

Exosome-mediated transfer of sh-CD99 is sufficient to modulate cell differentiation in Ewing sarcoma.

Alessandra De Feo,1 Marika Sciandra,1 Federica Felicetti,2 Manuela Ferracin,3 Alessandra Carè,2 Katia Scotlandi1. 1 _Rizzoli Orthopaedic Institute, Bologna, Italy;_ 2 _Istituto Superiore di Sanità, Rome, Italy;_ 3 _University of Bologna, Bologna, Italy_.

Background

Ewing sarcoma (EWS) is an aggressive childhood bone tumor with still highly unmet clinical needs. The tumor is characterized by the fusion oncoprotein EWS-FLI1 and the high expression of the membrane glycoprotein CD99. CD99 has been found to be released by EWS cells linked to exosomes (EXOs), small vesicles able to influence cellular behavior and surrounding microenvironment by transferring their contents into nearby or distant recipient cells. The delivery of EXOs devoid of CD99 was sufficient to induce neural differentiation in recipient EWS cells through miR- 34a-dependent inhibition of Notch-NF-kB signaling, thus indicating a possible therapeutic potential.

Aims

The study has the ambition to:

1. Evaluate the capabilities of EXOs released by Ewing's sarcoma, to influence cancer growth and/or metastasis through the horizontal transfer of their cargos.

2. Characterize EXOs secreted by CD99-silenced EWS cells in terms of miRNAs and protein and evaluate their general antimetastatic function, if any, taking advantage of their capabilities to be transferred into a broad panel of different Ewing's sarcoma cell lines, as recipients.

Results

In the present study we identified the role of Ewing sarcoma-derived EXOs as mediators of signals involved in cancer growth, metastases and differentiation. In particular, we analyzed the ability of EXOs, expressing or not CD99, to modulate the phenotype of EWS cells. Delivery of EXOs from CD99-silenced cells was sufficient to induce neural differentiation in recipient EWS cells by neurite outgrowth and increased expression of β-III tubulin marker of neural differentiation accompanied in vitro by a significant reduction of proliferation, invasion and chemotaxis convey the same phenotype obtained by stable CD99 silencing. Moreover, microarray analysis revealed a signature of 56 miRNAs differentially expressed in EXOs derived from CD99-silenced cells compared to EWS-derived EXOs. We focused our attention on miR-214 and miR-199. Those miRNAs are involved in the regulation of CD99 itself like, EWS-FLI1 or CD99-mediated pathways related to cytoskeleton and membrane organization. As a next step we searched for any tumorigenic effect possibly associated with miR-199 and mir-214. The restored expression of miR-199 and miR-214 in two Ewing Sarcoma cell lines was accompanied by a significant reduction of proliferation, invasion and chemotaxis in vitro. The outcomes deriving from miR-199 and miR-214 enforced expression were also evaluated on capability of forming foci in agar semisolid medium. Results showed a significant decrease of the number of foci in both cell lines.

Conclusions

Exosomes deprived of CD99 can alter the balance of important cellular components necessary for the metastatic process of EWS cells, thus representing a novel exciting therapeutic possibility for the design of novel therapy against metastatic EWS. AIRC IG18451 to KS; IG18815 to AC.

#3550

Lipid rafts contribute to ligand-induced signaling and survival in pediatric AML.

Padmini Narayanan,1 Julien Dubrulle,1 Robert B. Gerbing,2 Todd A. Alonzo,2 Fabio Stossi,1 Michele S. Redell1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Children's Oncology Group, Monrovia, CA_.

Pediatric acute myeloid leukemia (pAML) is an aggressive malignancy with high mortality due to therapy resistance and relapse. We previously reported that the failure of pAML cells to phosphorylate STAT3 upon G-CSF or IL-6 stimulation was associated with inferior survival. The defect in STAT3 signaling was not due to low expression of receptors or of STAT3. We hypothesized that the signaling failure seen in some pAML samples is not specific for STAT3, but rather a generalized dysfunction due to abnormal lipid raft function. Lipid rafts (LR) are highly ordered plasma membrane microdomains rich in glycosphingolipids and cholesterol. Two types of LR - flat planar and invaginated caveolar - contain different membrane proteins and serve distinct but overlapping cellular functions. Both types of LR organize receptors, downstream mediators, and repressors for efficient signaling. The role of LRs in signaling and clinical outcome is not yet established in pAML. Primary samples (n=95) from the Children's Oncology Group (COG) were stimulated with HS5 stromal cell conditioned medium (HS5CM), which contains IL-6 and dozens of physiological ligands. Basal and induced phospho-STAT3 (pSTAT3), pSTAT5, pAKT and pERK1/2 were measured by intracellular flow cytometry, and the fold change in the mean fluorescence intensity (ΔMFI) was calculated. The HS5CM-induced ΔMFI for pSTAT3 was positively correlated with that for pSTAT5 (p<0.0001, r=0.5), and pAKT (p=0.0002, r=0.4). Samples that failed to increase pSTAT3 failed to activate other pathways, suggesting a generalized signaling dysfunction. In AML cells with intact signaling, disruption of LRs with methylβcyclodextrin markedly reduced HS5CM-induced pSTATs, recapitulating the dysfunctional phenotype. LRs were assessed by immunocytochemistry in 29 primary samples with and without HS5CM stimulation. Choleratoxin B, which binds ganglioside GM-1, stained LRs. Antibodies to caveolin-1 (cav-1) marked caveolar LRs. We also stained for gp130, the signaling subunit of the IL-6 receptor. Images were captured by deconvolution fluorescence microscopy. Expression was quantified by pixel intensity for average of 30 cells per condition. Colocalization of gp130 with LR markers was determined by the Manders Overlap Coefficient (MOC). Intensities and MOCs varied between samples. The MOC of gp130 with cav-1 correlated significantly with pSTAT3 ΔMFI (p=0.02, r=0.43). MOC of gp130 with GM-1 did not correlate with pSTAT3 ΔMFI (p=0.85, r=0.03), suggesting that cav-1 facilitates IL-6/STAT3 signaling. Interestingly, patients whose samples demonstrated basal cav-1 and GM-1 intensities below the median had inferior 5-year event-free survival compared to those with intensities above the median (log-rank p=0.079 for cav-1; p=0.013 for GM-1). Our results suggest that LRs contribute to ligand-induced signaling and possibly to chemotherapy response. Further studies of LR biology in AML are warranted.

#3551

Lipid raft integrity is essential for metastasis and chemoresistance properties of CD133+ cancer stem cells in pancreatic cancer.

Vineet K. Gupta,1 Nikita S. Sharma,1 Kousik Kesh,1 Patrcia Dauer,2 Alice Nomura,1 Bhuwan Giri,1 Vikas Dudeja,1 Ashok Saluja,1 Sulagna Banerjee1. 1 _University of Miami, Miami, FL;_ 2 _University of Minnesota, Minneapolis, MN_.

Pancreatic cancer remains a significant health burden associated with limited patient survival and poses a major therapeutic challenge. Among cancer cells, Cancer stem cells (CSCs) are instrumental in inducing chemoresistance and metastasis in pancreatic cancer. CD133 has been identified as a CSC surface marker in several malignancies including pancreatic cancer. However, the functional role of CD133 in CSCs still not clear. The present study investigates the role of caveolar lipid raft integrity in functional properties of CD133+ CSCs. Current study showed first time that CD133 localizes to caveolar lipid rafts in pancreatic cancer cells, and associates with Caveolin1 (Cav-1) and cholesterol to form an integral signaling complex which drives the downstream processes of chemoresistance and metastasis. Further analysis showed that the integrity of the lipid-raft is crucial for maintenance of the functional properties of pancreatic CSCs, and its disruption leads to increased chemo-sensitivity to chemotherapeutic compound paclitaxel both in vitro as well as in vivo. Additionally, disruption of lipid raft results in decreased invasiveness and metastatic ability of the cells by deregulation of FAK signaling. The study also reveals that pancreatic cancer cells that lack the CSC population marker, CD133, did not respond to lipid raft disruption. Our results indicated that targeting the lipid-raft integrity in pancreatic cancer can specifically eradicate the CD133+ CSCs in pancreatic tumors, leading to a better prognosis for the disease.

#3552

Induction of store operated calcium entry (SOCE) suppresses glioblastoma growth by inhibiting the Hippo pathway transcriptional coactivators YAP/TAZ.

Zhijun Liu. _Penn State Hershey College of Medicine, Hershey, PA_.

Glioblastomas (GBM) are the most aggressive brain cancers without effective therapeutics. The Hippo pathway transcriptional coactivators YAP/TAZ were implicated as drivers in GBM progression and could be therapeutic targets. Here, we found in an unbiased screen of 1650 compounds that amlodipine is able to inhibit survival of GBM cells by suppressing YAP/TAZ activities. Instead of its known function as an L-type calcium channel blocker, we found that amlodipine is able to activate Ca2+ entry by enhancing store-operated Ca2+ entry (SOCE). Amlodipine as well as approaches that cause store depletion and activate SOCE trigger phosphorylation and activation of Lats1/2, which in turn phosphorylate YAP/TAZ and prevent their accumulation in the cell nucleus. Furthermore, we identified that protein kinase C (PKC) beta II is a major mediator of Ca2+-induced Lats1/2 activation. Ca2+ induces accumulation of PKC beta II in an actin cytoskeletal compartment. Such translocation depends on inverted formin-2 (INF2). Depletion of INF2 disrupts both PKC beta II translocation and Lats1/2 activation. Functionally, we found that elevation of cytosolic Ca2+ or PKC beta II expression inhibits YAP/TAZ-mediated gene transcription. In vivo PKC beta II expression inhibits GBM tumor growth and prolongs mouse survival through inhibition of YAP/TAZ in an orthotopic mouse xenograft model. Our studies indicate that Ca2+ is a crucial intracellular cue that regulates the Hippo pathway, and that triggering SOCE could be a strategy to target YAP/TAZ in GBM.

#3553

Manuka honey induced apoptosis in hepatocellular carcinoma through inhibition of Wnt/b-catenin and ERK1/2.

Heba R. Al Refaey, Ahmed S. Sultan. _Faculty of Science, Alexandria University, Egypt_.

Hepatocellular carcinoma (HCC) is one of the most malignant tumors that affect individuals worldwide and most therapies are still badly guaranteed due to its potential toxicity. Nowadays, there is increasing awareness focused on identification of naturally occurring anticancer agents derived from food or natural products, as honey which possess a high content of polyphenols and flavonoids.

Manuka honey (MH) has been recognized by its biological properties as antimicrobial agent, anti-inflammatory activity, antioxidant efficacy and has a potent role in wound healing. Also Manuka honey possesses anticancer property in inhibiting proliferation of malignant tumor. However, its antiproliferative and apoptotic effect on human hepatocellular carcinoma cells (HepG2) remain unclear. Therefore, the current study focused mainly on investigating the anticancer properties of MH and its mechanism of action. Cell viability was evaluated by MTT assay, microscopic examination was undertaken to monitor the morphological alternation of HepG2 cells after treatment. Furthermore, the antiproliferative and apoptosis mechanism was estimated by the level of caspase-3 activity and a possible cross talk between protein expression of total ERK1/2, oncogenic β-catenin, cyclin D1, proapoptotic protein, Bax and poly (ADP-ribose) polymerase (PARP) is explained by observing their expression levels by western blot analysis. The combination effect of MH with doxorubicin (DOX) was also investigated.

Our results revealed that MH treatment for 48 h showed a substantially proliferation inhibition of HepG2 cells in a dose dependent manner with IC50 concentration of (6.92± 0.005%) and induces dramatic morphological changes of cells, which considered as characteristics of apoptosis. Consistently, MH or combined treatment showed a significant increase in caspase-3 activity in treated HepG2 cells when compared to untreated cells. In addition, the expression levels of total ERK1/2, oncogenic β-catenin and cyclin D1 were significantly downregulated with MH treatment, whereas the expression level of Bax and PARP cleavage were remarkably upregulated after 48h. In addition, a combination of MH and doxorubicin led to a significant synergistic effect.

Collectively, for our knowledge, the present study provides the first report on the anticancer activity of MH on HCC cells. Our study may provide a targeting therapy for HCC by inhibition of Wnt/ β-catenin and ERK1/2 signalling pathways and induction of apoptosis was detected by Bax up-regulation and PARP cleavage after 48h. Moreover, MH can be considered as a promising naturally occurring anticancer compound that can be used as an effective adjuvant therapeutic for treatment of HCC.

#3554

The role of P2RY14 in acute leukemia.

Kinjal Shah, Julhash Kazi. _Lund University, Lund, Sweden_.

Leukemia is a group of blood cancers that originate in the bone marrow and involves uncontrolled proliferation of different types of blood cells. The PI3K/mTOR pathway is the second most frequently deregulated pathway in a majority of cancers such as breast cancer, lung cancer and melanomas as well as leukemia. There are several promising drugs targeting this pathway for use in the clinic. However, acquired resistance to the drugs remains a major challenge to overcome. Mutations in the genes coding for receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) are quite common in all forms of acute leukemia. This can be a major cause of deregulation of the PI3K-mTOR pathway. To understand how cells display resistance to the dual PI3K/mTOR inhibitor, we used a panel of acute leukemic cell lines. We observed that while a number of cell lines displayed sensitivity to the dual PI3K/mTOR pathway inhibitor PKI-587, many cells displayed substantial resistance. Cells sensitive to PKI-587 also showed aberrant activation of the PI3K/mTOR pathway and also displayed sensitivity to a panel of various other PI3K/mTOR inhibitors. Using RNA sequencing data from the COSMIC database, we compared gene expression between the cell lines that are either resistant or sensitive to PKI-587. We found that the expression of P2RY14 was around 9-fold upregulated in cells showing resistance to PKI-587. Thus, we identified Purinergic receptor 14, P2RY14, as the target gene. P2RY14 is a G protein-coupled receptor that has not been much studied in hematologic malignancies. However, this receptor seems to have a role in the localization of Hematopoietic Stem Cells (HSCs) and in promoting regenerative capabilities following injury. Acute lymphoblastic leukemia patients with higher expression of P2RY14 mRNA displayed relatively poor survival compared to patients carrying the lower expression of P2RY14. To understand the role of this receptor in leukemia, we stably transfected the murine pro B cell line Ba/F3 with P2RY14. Activation of P2RY14 by its ligand, UDP glucose, resulted in selective induction of ERK1/2 phosphorylation. Taken together, our data suggest that the GPCR P2RY14 couples to ERK signaling which probably has a significant role in acute leukemia. Nevertheless, further in vitro testing of the receptor functionality with various biochemical and molecular biology techniques is required before this receptor, P2RY14, can be utilized as a therapeutic target in the future treatment of acute leukemia.

#3555

TREM2 is upregulated and plays oncogenic role in gastric cancer.

Yulong Zheng, Lihua Jiang, Cheng Xiao, Nong Xu, Qiqi Gao, Yinan Yao, Yongxian Hu, Yun Sun, Xin Xu. _ZHEJIANG UNIVERSITY, HANGZHOU, China_.

Background

Triggering receptor expressed on myeloid cells-2 (TREM2) is thought to be invovled in innate immunity and inflammation regulation. More recently, the importance of TREM2 has been highlighted by identification in several kinds of solid tumors. Nonetheless, the oncogenic roles and potential molecular mechanisms of TREM2 in gastric cancer (GC) remain unknown.

Methods

The expression levels of TREM2 were determined by quantitative PCR (qPCR) and immunohistochemistry (IHC). MTT, colony formation, 5-Ethynyl-2′-deoxyuridine (EdU) incorporation, and in vivo tumorigenicity assays were performed to determine the pro-proliferative role of TREM2 in GC cells. mRNA sequencing, qPCR, and Western blotting were used to explore the underlying molecular mechanism of pro-proliferative role of TREM2 in GC cells.

Results

The mRNA expression of TREM2 was markedly up-regulated in tumor tissues (n=16) in comparison with that in corresponding non-tumorous tissues. Immunohistochemical study were performed to determine the protein expression in 108 patients with surgically resected GC. Its expression was positively associated with TNM stage (P=0.023) and T classification (P=0.021), and was inversly correlated with the poor clinical outcome of the patients.

The human GC cell lines MGC-803 and BGC-823 were used for proliferation analysis. The transfected GC cell lines with stable overexpression of TREM2 were established. Ectopic overexpression of TREM2 in GC cells promoted cell proliferation,

whereas loss of TREM2 function inhibited proliferation. In vivo animal experiments further validated the pro-proliferative role of TREM2. Deep sequencing to profile global gene expression of MGC-803 cells with TREM2 overexpression. In the biological process analysis, these differentially expressed genes were mainly involved in the MAPK signaling. Molecular mechanistic investigations explored that TREM2 upregulated GREM1 expression, thereby activating ERK1/2 signaling via VEGFR2-PLC-γ-PKC axis and promoting GC cell proliferation.

Conclusion

High TREM2 expression predicts poor prognosis in GC and TREM2 promotes GC cells proliferation. Our findings suggest that GREM1-VEGFR2-PLC-γ-PKC-ERK1/2 signaling axis contributes to the oncogenic roles of TREM2 on GC cells. TREM2 could be an effective prognostic biomarker and a potential therapeutic target.

#3556

Sam68 is required for the growth and survival of non-melanoma skin cancer.

Kai Fu, Fengyi Wan. _Johns Hopkins Univ., Baltimore, MD_.

Skin cancer is the most commonly diagnosed malignancy in the United States, and the incidence of skin cancer has increased dramatically over the last few decades. Non-melanoma skin cancer (NMSC), which includes squamous cell carcinomas (SCC) and basal cell carcinomas (BCC), is the most common type of skin cancer with substantial associated morbidity and mortality. Whereas targeting DNA repair signaling pathways has emerged as a promising therapeutic for skin cancer, the relevance of DNA damage responses (DDR) in the development and survival of non-melanoma skin cancer (NMSC) remains obscure. Here we report that Src-associated substrate during mitosis of 68 kDa (Sam68), an early signaling molecule in DDR, is elevated in skin tumor tissues derived from NMSC patients and skin lesions from Gli2-transgenic mice. Downregulation of Sam68 impacts the growth and survival of human tumor keratinocytes and genetic ablation of Sam68 delays the onset of basal cell carcinomas (BCC) in Gli2-transgenic mice. Moreover, Sam68 plays a critical role in DNA damage-induced DNA repair and nuclear factor kappa B (NF-κB) signaling pathways in keratinocytes, hence conferring keratinocyte sensitivity to DNA damaging agents. Together our data reveal a novel function of Sam68 in regulating DDR in keratinocytes that is crucial for the growth and survival of NMSC.

#3557

Beta-hydroxybutyrate inhibits oncogenic signaling and cellular motility in pancreatic cancer cells.

Surendra K. Shukla, Nina V. Chaika, Pankaj K. Singh. _UNMC Eppley Inst., F &P Buffett Cancer Ctr., Omaha, NE_.

Pancreatic ductal adenocarcinoma is a devastating malignancy with a five-year survival rate less than 7%. It is characterized by a plethora of metabolic and signaling pathways, which plays a critical role in the pathogenesis of the disease. Caloric restriction and ketogenic diets have been shown to be beneficial in the management of several kinds of malignancies including pancreatic cancer. During caloric restriction or ketogenic diet supplementation, blood level of ketone bodies (beta-hydroxy butyrate or BHB, acetoacetate, acetone) gets elevated in the body. Among, these ketone bodies, BHB, the most abundant ketone body in mammals has been shown to act as the modulator of several signaling and inflammatory pathways. We have evaluated the effect of BHB on oncogenic signaling pathways and motility of pancreatic cancer cells. We observed reduced activation of key oncogenic signaling pathways, including mTOR. We also observed inhibition of cellular motility in a dose-dependent manner. Furthermore, we observed reduced expression of c-Myc, a key oncogene and a downstream effector molecule of mTOR. Correspondingly, we also observed reduced expression of several c-Myc target genes. We have also evaluated the effect of BHB on epithelial-mesenchymal transition (EMT) markers and observed altered expression of EMT markers like N-cadherin and E-cadherin. We also evaluated the effect of BHB on the expression of inflammatory cytokines in the pancreatic cancer cells and observed that BHB treatment leads to the reduction of expression of several inflammatory cytokines including IL6 and IL1-beta. Overall, our findings suggest that BHB inhibits inflammatory signaling in pancreatic cancer cells and reduces the cellular motility by modulating the expression of EMT regulators.

#3559

**Galectin-1 upregulates CXCR4 in glioblastoma through upregulation of CXCR4 via NF-** κ **B activation** **.**

David Cachia,1 Rachel Malley,1 William A. Vandergrift,1 Sunil Patel,1 Gabriel A. Rabinovich,2 Arabinda Das1. 1 _Medical Univ. of South Carolina, Charleston, SC;_ 2 _Institute of Biology and Experimental Medicine, Buenos Aires, Argentina_.

Background: Despite aggressive treatment, including surgical resection, radiation and chemotherapy, over 90% of glioblastoma (GB) patients experience tumor recurrence. This may be due to high migratory potential, abnormal angiogenesis, molecular heterogeneity and a strong immunosuppressive environment. An earlier report from our laboratory demonstrated that GB cells express high levels of carbohydrate-binding galectin protein: galectin-1 (Gal-1). However, the role of galectin-1 in GB remains elusive. The aim of this study was to evaluate whether galectin-1 promotes tumor progression through upregulation of CXCR4 via NF-κB in in vitro GB models. Methods: To investigate the molecular mechanisms of Gal-1 on tumor progression, we used RT-PCR and western blots techniques. Gal-1 was stably over-expressed in the U118 and U87MG cell line. Gal-1 (+/-) sub-clones were assayed for attachment, proliferation, migration, and tube formation. Results: Gal-1 protein expression was assessed in human GB cells. We found increased Gal-1 protein expression in human GB cells and tissue. To investigate the effects of Gal-1 silencing on angiogenesis in vitro, we collected conditioned media from Gal-1-knockdown U118 and U87MG cells then applied this to human umbilical vein endothelial cells (HUVEC) culture to analyze endothelial cell proliferation, migration, and tube formation. Proliferation rate, migrating capability, and tube formation decreased significantly when HUVECs were cultured with concentrated conditioned media from Gal-1-knockdown cells. This data suggests that Gal-1 in GB cells may promote tumor angiogenesis both in vitro and co-culture. Results showed that silencing of Gal-1 in U118 and U87MG cells significantly repressed CXCR4 expression at the mRNA level and protein levels. To further explore the role of CXCR4 in GB cells, CXCR4 knockdown resulted in significant inhibition of cell migration and clonogenic ability in vitro. Thus, Gal-1 might upregulate CXCR4 expression to promote tumor progression. A previous laboratory study indicated that NF-κB promotes cancer cell motility by upregulating CXCR4 expression. Luciferase reporter assay was used to determine the involvement of NF-κB in Gal-1-induced CXCR4 expression in U118 and U87MG cells. NF-κB luciferase activity was significantly decreased in Gal-1-knockdown cells. Moreover, decreased CXCR4 promoter activity and protein expression in Gal-1-knockdown cells were observed. This data indicates that Gal-1 might modulate CXCR4 expression via NF-κB activation. Conclusion: Our in vitro cell culture results suggest that upregulation of galectin-1 and CXCR4 might at least in part explain the aggressive infiltrative biology of GB and the Gal-1-CXCR4 axis may serve as a therapeutic target. Further studies in different animal models are warranted.

#3560

Targeting the MIF receptor CD74 in bladder cancer tumorigenesis.

Justin Penticuff,1 Sambantham Shanmugam,1 Joshua Warrick,2 Brian Chase,1 Erika Abbott,1 Benjamin L. Woolbright,1 John A. Taylor1. 1 _University of Kansas Medical Center, Kansas City, KS;_ 2 _Penn State Medical Center, Hershey, PA_.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that can stimulate cellular proliferation in multiple tumors. Our previous data indicates genetic deletion of MIF results in T1 stage arrest in the N-Butyl-N-(4-hydroxybutyl) nitrosamine (BBN) induced bladder cancer model. Similarly, pharmacological inhibition of MIF results in lower stage tumors and reduced bladder weights when given therapeutically. CD74 is a human leukocyte antigen class II trafficking and processing protein that also functions as an extracellular receptor for MIF. Additionally, CD74 complexes with CD44 to act as the cognate MIF receptor complex, and upregulation of CD74 has been noted in high grade bladder cancer. We hypothesized CD74 would be upregulated in malignant bladder cancer cell lines, and knockout of CD74 would reduce bladder cancer tumorigenesis in the murine BBN model. The objectives of this study were to determine the effect of CD74 knockout on development and progression of urothelial cell carcinoma; as well as evaluate expression of the putative MIF signaling axis in BCa cell lines. BCa cell lines HTB-9, HTB-5, RT-4, T24 and HT-1376 and benign urothelial UROtsa cells were analyzed for MIF, CD74, and CD44 expression via quantitative PCR or western blot to assess expression of the MIF signaling axis. CD74 and CD44 expression by q-PCR were dramatically elevated in BCa cell lines vs. benign cell lines. Protein levels of CD74 and CD44 were elevated in multiple cell lines as well. MIF expression levels did not change between cell lines. To ascertain differences in BCa tumorigeneis, WT C57Bl/6J mice or CD74-/- mice were given 0.05% BBN in the drinking water for 22 weeks. Bladders were weighed and evaluated for pathologic staging by histology. After exposure to BBN, bladders from CD74-/- mice had a 33% reduction in weight vs. WT bladders (p=.062). Pathological analysis revealed lower stage tumors in bladders of CD74-/- mice (33% incidence; 6.7% pTa, 0% pTis, 6.7% pT1, 20% pT2, 6.7% pT3) as compared to bladders of WT mice (81.3% incidence; 0% pTa, 6.3% pTis, 25% pT1, 12.5% pT2, 37.5% pT3), which was a statistically significant reduction in tumor stage. Lower tumor stage correlated significantly with the CD74-/- genotype (p=0.02, Fisher's Exact Test). These data suggest CD74 likely mediates a portion of the effects of MIF in BCa, raising the possibility of both CD74 dependent, and independent MIF signaling pathways.

#3561

Functional expression of BK channel subunits in LNCaP prostate cancer cells under androgen-deprivation conditions.

Bailey Evans, Conor Breen, Niamh McKerr, Richard Kennedy, Karen D. McCloskey. _Queens University Belfast, Belfast, United Kingdom_.

Introduction: Prostate cancer (PCa) is the most commonly diagnosed cancer in men. Treatment for PCa includes androgen deprivation therapy (ADT) which while initially effective in reducing tumour growth and volume, resistance develops and relapse typically occurs within 24 months. Ion channel proteins are differentially expressed in normal prostate epithelium and PCa cells. We have previously reported differences in expression of the BK channel subunits BKα and BKγ, in a panel of PCa cell lines representing early androgen-sensitive disease compared with late castration-resistance. Here, we hypothesised that BK subunit expression was sensitive to ADT conditions and aimed to elucidate the functional expression of BK subunits in androgen-sensitive LNCaP cells under ADT. Methods: qPCR, Western blot, immunofluorescence and patch-clamp assays were performed. The BK channel blocker paxilline (300nM), enzalutamide (10 µM) and dihydrotestosterone (DHT, 100nM) were used (vehicle DMSO/ethanol <0.1%). ADT conditions were mimicked by substituting normal media with charcoal stripped, phenol-red free media for up to 10 days. Data are expressed as mean±SEM; ANOVA/paired Student's t-tests were performed; P≤0.05 considered as significant. Results: LNCaP cells were observed to have an epithelial morphology that became distinctly neuronal during ADT (N=3). Western blots showed decreased androgen receptor (AR) and increased neuron-specific enolase (NSE) protein expression, consistent with a neuroendocrine phenotype. BKα (KCNMA1) gene expression was decreased in LNCaP during ADT (n=3, P≤0.05) although protein expression was similar. BKγ (LRRC26) gene expression was reduced during ADT (N=4, P≤0.05). LNCaP had paxilline-sensitive BK currents, where current density was significantly reduced during ADT (N=9, N=7 respectively; P≤0.05). DHT treatment over 48hrs (following a 24hr serum starvation) increased KCNMA1 expression (N=2); this effect was attenuated with co-treatment of enzalutamide (10 µM) indicating that KCNMA1 in LNCaP may be partly modulated by DHT and the AR pathway. Interestingly, reintroduction of 10% FBS-containing media for 48hrs after the 24hr serum starvation period increased KCNMA1 expression. Conclusion

The findings suggest that BKα and BKγ gene expression in LNCaP cells is sensitive to ADT. The canonical role of BK channels to facilitate potassium ion transport was diminished during ADT, evidenced by reduced BK currents in ADT-treated cells. Modulation of BK subunit gene expression by DHT indicates regulation by the AR.

#3562

Merkel cell polyomavirus t-antigen regulates aggresome formation and autophagy in merkel cell carcinoma.

Hao Shi. _Karolinska Institute, Stockholm, Sweden_.

Merkel cell carcinoma (MCC) is an aggressive and deadly skin cancer. About 80% of MCCs harbor integrated Merkel cell polyomavirus (MCV) genome with a mutation in the large T (LT) gene, leading to expression of truncated LT and intact small T (sT). The molecular mechanism how the virus contributes to MCC development is still not fully understood. Paranuclear dot-like staining of cytokeratin 20 is a typical characteristic of MCC. However, it is unexplored whether these dot-like structures are functionally important in MCC. In this study, we aimed to characterize the identity of these dot-like structures and to determine whether MCV T-antigens play a role in the formation of these structures. Using immunostaining, we showed that these structures were co-localized with aggresome markers (including intermediate filaments, BAG3 and HSP70) and were localized at the microtubule organization center, as demonstrated by the presence of γ-tubulin. These structures became dispersed to the periphery upon microtubule depolymerization using colcemid. Similarly, silencing of BAG3 in MCV-positive MCC cells resulted in aggresome clearance. Interestingly, we observed that these structures were associated with MCV-positive MCCs and silencing of MCV T-antigens led to dispersion of the aggresome. Additionally, ectopically expressed truncated, but not wild-type, LT could facilitate aggresome formation in MCV-negative cells. We also showed that silencing of MCV T-antigens induced autophagy in MCV-positive MCC cells, while ectopically expressed MCV T-antigens suppressed autophagy in MCV-negative MCC cells. In conclusion, we uncover a novel role of MCV T-antigen in hijacking cellular degradation pathways, suggesting that the autophagy-aggresome pathway plays important roles in MCV infection and MCC tumorigenesis.

## CLINICAL RESEARCH:

### Adoptive Cell Therapy 3

#3563

Impact of the affinity of chimeric antigen receptor on immune activation profiles of T cells.

Chungyong Han,1 Seon-Hee Kim,1 Beom K. Choi,1 Byoung S. Kwon2. 1 _National Cancer Center, Goyang-si, Republic of Korea;_ 2 _Eutilex Co., Ltd., Seoul, Republic of Korea_.

Background: Chimeric antigen receptor (CAR) generally uses a single chain antibody fragment (scFv) of high affinity as an antigen-binding domain. Recent studies implied that T cells engineered with CAR (CAR T cells) of an extremely low affinity have the potential to reduce on-target off-tumor toxicity that is a serious side effect in CAR T cell therapy. Defining the relation of CAR affinity to immune activation profiles is important for understanding the nature of CAR T cells.

Methods: We examined the functional variation of CAR T cells when stimulated with target antigens of various affinities. HLA-DR-specific MVR CAR T cells that were previously developed in our group were used in this study. Since MVR CAR recognizes HLA-DR, Epstein-Barr virus-transformed lymphoblastoid cell lines (EBV LCLs) which stably express HLA-DR, stimulated MVR CAR T cells. Importantly, owing to the broad spectrum of MVR CAR affinity against polymorphic epitope of HLA-DR, six different EBV LCLs each of which express unique HLA-DRs, stimulated MVR CAR T cells with different magnitude based on the binding affinities. Using the combination of MVR CAR T cells and six different EBV LCLs, we assessed the extent of effector functions and gene expression induced by different CAR affinity.

Results: The cytolytic activity of MVR CAR T cells was correlated with the affinity between MVR CAR and HLA-DR. The induced polyfunctionality was highest in intermediate MVR CAR-target affinity and was decreased in weak and strong affinities. Gene expression analysis of the stimulated MVR CAR T cells identified a panel of genes of which the expression levels were correlated with CAR-target affinity. Gene ontology analysis revealed that the genes are mainly involved in a T cell activation pathway. Interestingly, we found that the genes involved in type I interferon signaling were upregulated following the stimulation with strong affinity antigens, while those expression levels were unchanged following the stimulation with weak and intermediate affinity antigens.

Conclusions: Here we describe the affinity-associate functional variation of CAR T cells defined by the use of the combination of MVR CAR T cells and EBV LCLs of various affinities. Using those effector/target cell combinations, we investigated the relation between CAR-antigen affinity and the effector functions which include cytolytic activity and polyfunctionality. Furthermore, we identified that type I interferon signaling is a distinct characteristics of strong affinity-induced activation of CAR T cells. These effector/target cell combinations and the observations will help to understand the nature of CAR T cells.

#3564

A combination of chimeric switch-receptor T cells targeting both PD1 and CTLA4 suppresses tumor growth.

Peng Li, Le Qin, Xinru Wei, Ruocong Zhao, Yunxin Lai. _Guangzhou Insts. of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China_.

Chimeric antigen receptor (CAR) T cells have been successfully used to treat B cell malignancies, but faces many obstacles in solid tumors. A major challenge is the immune-suppressive effects induced by PD-L1 expression in tumors and CD80/86 that ligate with CTLA-4 and are expressed in myeloid-derived immune suppressive cells. Dual blockade of PD-1 and CTLA-4 with monoclonal antibodies has achieved promising anti-tumor effects in clinics. Here, we designed two switch receptor constructs. One contains the truncated extracellular domain of human PD1, costimulatory domains of 4-1BB and TLR2, and the CD3z signaling domain (cPD-1). cPD-1 T cells specifically lysed PD-L1+ tumor cell lines and augmented cytokine secretion in vitro. In addition, cPD-1 T cells were able to suppress the growth of lung cancer, gastric cancer, and osteosarcoma in xenografts. The other construct, similarly, is composed of the truncated extracellular domain of murine Ctla4, human CD28, TLR2, and CD3z signaling domains (cmCtla-4). Because CD80/CD86 are expressed in murine myeloid cells rather than human tumor cells in PDX models, cmCtla4 T cells targeted mouse myeloid cells that were abundant in tumors but not human tumor cells. We found that cmCtla4 T cells hindered tumor growth in gastric cancer PDX. In combination, cPD-1 and cmCtla4 T cells synergistically induced superior anti-tumor effects in lung cancer PDX. Taken together, our findings demonstrated the anti-tumor function of switched immune checkpoint receptors and revealed a strategy of combining T cells targeting both tumor cells and tumor microenvironment, promoting clinical investigation of these chimeric switch-receptor T cells for cancer treatment.

#3565

T cell receptor signaling-responsive single chain IL-12 and IL-15 superagonist nanogel "backpacks" to enhance adoptive cell therapy in solid tumors.

Michael Fichter. _Koch Institute for Integrative Cancer Research at MIT, Cambridge, MA_.

Adoptive cell therapy (ACT) using patient-derived tumor-specific T cells is a promising approach for cancer treatment especially for the treatment of hematological malignancies. However, therapeutic approaches using ACT in patients with solid tumors, such as hepatocellular carcinoma (HCC), has not shown substantially improved clinical outcomes in particular due to immunosuppressive tumor microenvironments. Therefore, strategies to enhance ACT T cell functionality in vivo and to overcome the immunosuppressive microenvironments in solid tumors are strongly needed. We developed a strategy combining nanomedicine with ACT, based on the chemical conjugation of cytokine-loaded nanoparticles (NPs) as synthetic "backpacks" to the surfaces of live lymphocytes for adoptive therapy. Novel protein nanoparticles were synthesized by crosslinking of single chain IL-12 (scIL-12)- or IL-15 superagonist through disulfide-containing crosslinkers, forming nanogels that when bound to the surface of T cells, release the supporting cytokines in response to antigen receptor signaling-mediated changes in cell surface redox state. This TCR-mediated drug release allowed drug delivery to the T cells to be focused in tumors and tumor-draining lymph nodes. Coupling of cytokine nanogels to T cell membranes induced significant cell activation in terms of interferon-γ production or proliferation in a concentration-dependent manner. Antigen-specific CD8+ T cells "backpacked" with scIL-12 or IL-15SA nanogels were capable of substantial tumor cell killing in a B16F10 melanoma cancer model in vivo, while avoiding lethal systemic toxicities triggered by systemically-administered immunomodulatory cytokines. Ongoing studies are seeking to apply this approach in HCC, and this approach bears the potential for the development of successful immunotherapeutic strategies against this and other solid tumors where effective treatment options are still lacking.

#3566

Enhanced expansion and tumor targeting of adoptively transferred T cells with NKTR-214.

Giulia Parisi,1 Justin Saco,1 Felix Bergara,1 Paige Krystofinski,1 Ruixue Zhang,1 Cristina Puig Saus,1 Siwen Hu-Lieskovan,1 Begonya Comin-Anduix,1 Anna Wu,1 Deborah H. Charych,2 Antoni Ribas1. 1 _UCLA, Los Angeles, CA;_ 2 _Nektar Therapeutics, San Francisco, CA_.

NKTR-214 is a CD122-biased cytokine agonist designed to provide sustained signaling through the heterodimeric IL-2 receptor pathway (IL-2Rβγ) to preferentially activate and expand CD8 T and natural killer cells (NK) over Tregs in the tumor. We evaluated the tumor immunology, biodistribution and anti-tumor activity of NKTR-214 combined with ACT in the pmel-1 ACT/B16F10 melanoma tumor model. NKTR-214+ACT provides a robust and durable anti-tumor response compared to IL-2+ACT with less frequent dosing in the aggressive B16F10 model. NKTR-214+ACT led to significant tumor growth inhibition at day 14 compared with IL-2+ACT, 174mm3 vs 484mm3 tumor volume, respectively (p<0.05, n=12). Tumors of animals receiving IL-2+ACT grew to the endpoint of 1,500 mm3 18 days after treatment, while NKTR-214+ACT significantly improved the survival to 35 days (p<0.0001). Bioluminescence imaging (BLI) showed that NKTR-214+ACT treatment significantly increased T cells expansion in the spleen from day 5 to day 9 compared to IL-2+ACT (p<0.0001, n=5). At day 5, quantification of BLI of serial images with region of interest analysis at the site of spleen revealed an average radiance 14 folds higher in NKTR+ACT than in IL-2+ACT treated mice (1.3*10^7 vs 9.5*10^5 p/s/cm²/sr). BLI showed a stronger peak of activity of tumor-infiltrating effector T cells in the ACT+NKTR-214 group versus the ACT+IL-2 from day 5 to day 7 (p<0.0001, n =5). The second dose of NKTR-214 at day 9 triggered a second expansion of effector T cells in the spleen and tumor from day 12 to day 17, while no effect in the group treated with 3 doses IL-2 was observed (p<0.0001, n =5). The peak of signal was reached at day 14 with an average radiance 10 folds higher in the NKTR-214+ACT group compared to the IL2+act group (4.7*10^6 vs 4.5*10^5 p/s/cm²/sr). These data are supported by immuno-PET imaging using minibody (Mb) targeting CD8 in vivo. Ex vivo biodistribution analysis showed a signal in the spleen 5-folds higher in the ACT+NKTR-214 group compared with the ACT+IL-2 group on day 5 after treatment, 87% and 17% injected dose per gram, respectively (p<0.05, n=3). Flow cytometry analysis performed at the same time point showed that NKTR-214 treatment significantly increased pmel-1 CD8 T cells and amplified the CD8/Treg ratio compared to IL-2 both in spleen and tumor (3 and 6 folds, respectively, p<0.05, n=3). In conclusion, NKTR-214 + ACT is well tolerated and robustly mobilizes T cells into tumors where they durably persist, supporting NKTR-214 potential use in combination with cell-based therapeutics.

#3567

Novel approaches to high-affinity TCR isolation for clinical translation enabled by single cell RNA sequencing.

Megan S. McAfee,1 Kelly Paulson,1 Thomas Schmitt,1 Natalie Miller,2 Daniel Hunter,1 Jason Bielas,1 David Koelle,2 Valentin Voillet,1 Raphael Gottardo,1 Phillip Greenberg,1 Paul Nghiem,2 Aude Chapuis1. 1 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 2 _University of Washington, Seattle, WA_.

The success of T cell therapy for treatment of leukemia and melanoma provides rationale to pursue similar approaches for other cancers, particularly solid tumors. Merkel cell carcinoma, a highly aggressive skin cancer, is a model tumor for developing immunotherapies, as it is immune sensitive and most cases require obligate expression of conserved viral antigens. Like many solid tumor antigens, Merkel cell polyomavirus (MCPyV) oncoproteins are intracellular and thus not accessible to CAR-T cells, but can be targeted with T cell receptor (TCR) based therapy. TCRs for therapeutic translation for cellular immunotherapy must have sufficient affinity to bind the limited number of peptide-MHC molecules commonly expressed on tumor cells. Isolating high affinity natural TCRs offers a safety advantage to mutating lower affinity TCRs since the former TCRs have undergone selection in the thymus. However, identifying such TCRs by limited dilution cloning has been constrained not only by the time and effort required but also by the rarity of the highest affinity clones. Single cell RNA sequencing techniques allow for TCR identification from tiny, clonally diverse samples. We hypothesized this technique would be useful for isolating high-affinity TCRs from the peripheral repertoire of healthy donors or tumor bearing patients and allow for rapid clinical translation. We derived TCRs specific for the HLA-A*0201 restricted MCPyV viral epitope KLLEIAPNC from two sources: 1) the peripheral repertoire of 6 healthy HLA-A*02:01 donors stimulated 3 times in vitro with KLL peptide, and 2) polyclonal MCPyV-specific T cells that were adoptively transferred in a previous clinical trial and shown to localize to tumor and mediate a durable complete remission in a 60-year-old man with metastatic MCC. A total of 20,000 T cells were loaded onto the 10x Genomics 5' V(D)J scRNAseq platform; 14,022 cells (70.1%) were recovered, of which 11,417 (81% of recovered cells) yielded productive TCR alpha-beta pairing. Recovery was similar between healthy peripheral repertoire and patient TIL sources. TCR gene segments were codon optimized and assembled into a PRRLSIN lentiviral vector that is currently in clinical use. Initial screening in TCR-transduced cells revealed 5 TCRs that strongly bind KLL-pMHC tetramer independent of the CD8 co-receptor, including TCRs identified from both healthy donors and the immunotherapy responder. The candidate TCRs selected for further pre-clinical analysis produced inflammatory cytokines and specifically lysed HLA matched fibroblasts expressing physiologic levels of endogenously presented MCPyV oncoproteins. Safety analyses are ongoing. In summary, single cell RNA sequencing allows for the rapid identification from small, polyclonal human samples of functional highly avid paired TCRs, despite their presence at low frequency, thus facilitating rapid therapeutic translation.

#3568

Phosphatidylserine targeting antibody enhances antitumor activity of CAR T cells in mouse melanoma.

Sara Schad,1 Daniel Hirschhorn-Cymerman,1 Sadna Budhu,1 Hong Zhong,1 Xia Yang,1 Joseph Shan,2 Steven King,2 Taha Merghoub,1 Jedd Wolchok1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Peregrine Pharmaceuticals, Inc., Tustin, CA_.

A viable strategy to treat advanced cancers includes transferring of tumor-specific T cells. T cells that recognize tumor antigens can be expanded and reinvigorated ex-vivo. Furthermore, autologous T cells can be genetically modified to express antitumor T cell receptors or chimeric antigen receptors (CARs). Although the potency and specificity of tumor-specific T cells can be manipulated ex vivo, once reinfused into patients, the T cells are subjected to immunosuppressive mechanisms established by the tumor. An important immune checkpoint regulator within tumors is phosphatidylserine (PS), a phospholipid that is exposed on apoptotic cells, tumor cells and tumor endothelium. Innate immune cells exposed to PS secrete suppressive cytokines and chemokines that can significantly impair the function and activation of antitumor T cells. Antibodies that target PS have been shown to reactivate antitumor immunity by polarizing tumor-associated macrophages into a proinflammatory M1 phenotype, reducing the number of MDSCs in tumors and promoting the maturation of dendritic cells into functional APCs. Our lab has previously shown that a PS targeting monoclonal antibody (mch1N11), in combination with transgenic CD4+ T cells that recognize the melanoma antigen Trp1, can regress very advanced melanomas in all treated mice. Here, we further those studies with data showing that a 2nd-generation CAR T cell that binds Trp1 on the surface of B16 melanoma, in combination with mch1N11 can improve antitumor activity and overall survival in B16 tumor-bearing mice. Additionally, in vitro killing assays with antigen-specific T cells sorted from the tumor reveal that mch1N11 enhances the cytolytic function of these T cells against B16 melanoma. Flow cytometry analysis of local immune responses in the tumors of animals treated with tumor-specific T cells and mch1N11 showed a decrease in anti-inflammatory (M2) macrophages and FoxP3+ regulatory T cells. These findings highlight that diminishing suppressive mechanisms locally with mch1N11 can enhance the efficacy of transgenic TCR and CAR T cells to improve the outcome in patients with advanced-stage melanoma. Our studies may inform the design of clinical trials combining PS targeting antibodies with CAR T cell therapy in solid tumors.

#3569

Humanized mouse model of MAGE-A1-targeted anti-melanoma T cell therapy.

Megan S. McAfee, Trisha Sippel, Daniel Hunter, Jean Campbell, Thomas Schmitt, Robert Pierce, Anthony Rongvaux, Aude Chapuis. _Fred Hutchinson Cancer Research Center, Seattle, WA_.

Adoptive cellular therapy (ACT) has resulted in durable anti-tumor responses for both solid and hematological malignancies, mediated by the persistence and activation of transferred T cells. Unfortunately most patients, especially those with solid tumors, continue to progress despite persistent T cells. Thus revealing challenges specific to the hostile microenvironment of solid tumors and a critical need to identify factors that limit ACT efficacy. Furthermore, serial patient biopsies are challenging to obtain and accessible peripheral blood does not reflect relevant cell interactions at the tumor site. Xenograft models that lack infiltrating human macrophages, NK cells and lymphocytes cannot faithfully recapitulate critical interactions within the tumor microenvironment. To overcome this limitation, we initiated ACT in the MISTRG mouse that supports hematopoiesis after transplantation of human CD34+ stem cells resulting in functional human innate and adaptive immune cells. Co-transplantation of the melanoma cell line (Me275) results in tumor infiltration by human macrophages (TAMs) supporting tumor progression. The cancer/testis antigen Melanoma associated Antigen Gene (MAGE)-A1 is an attractive ACT target due to broad expression in tumors and limited expression in normal tissues rendering off-tumor tissue toxicities unlikely. We have identified a high-affinity HLA-A*02:01-restricted TCR that recognizes MAGE-A1278-286 peptide (TCRMA1) and confers effector functions in transduced CD8 and CD4 T cells when co-transduced with CD8αβ. Ex vivo analysis of MISTRG T cell cytolysis and proliferation suggest that CD4+ T cell help is required in this model. After two weeks of in vitro expansion, CD4+ and CD8+ T cells from humanized MISTRG spleens were transduced and transferred into MISTRG littermates harboring MAGE-A1+ Me275. Although TCRMA1 treated mice subtly controlled the growth of Me275, the changes were not significant. However, compared to an irrelevant TCR, only TCRMA1 CD8+ T cells localized to tumor and persisted in blood and spleen 8 and 28 days post transfer. TCRMA1 CD8 T cells displayed an effector phenotype, with high levels of PD-1 expression characteristic of recently activated and/or exhausted T cells. These results suggest that TCRMA1 cells recognized the tumor and were consequently activated, but could not overcome suppression in the microenvironment, mirroring human studies. Additional studies are being initiated to overcome the specific factors associated with the TAMs that limit in vivo T cell function. This model provides an ideal environment to test next generation T cell therapies, such as addition of intrinsic co-stimulation, to enhance responses against solid tumors. Humanized MISTRG mice are a powerful tool to study suppression of ACT in vivo, offering streamlined approaches to overcome hostile tumor microenvironments before clinical translation.

#3570

Adoptive transfer of tumor antigen-specific CTLs requires anti-CTLA-4 and anti-PD-1 to drive tumor eradication.

Lewis Zhichang Shi,1 Jianjun Gao,2 Luis Vence,2 Jorge Brando,2 James Allison,2 Padmanee Sharma2. 1 _Case Western Reserve University, Cleveland, OH;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Adoptive transfer of tumor-reactive T cells (ACT) has led to significant clinical responses in hematologic malignancies. However, its efficacy in solid tumors has been dismal, likely due to the immunosuppression imposed by the tumor microenvironment (TME) of solid tumors. A proven effective strategy to revert the immunosuppressive TME is immune checkpoint blockades (ICBs), including anti-CTLA-4 and anti-PD-1/PD-L1, which have been recently approved by FDA for cancer therapy. Here, using the orthotopic B16-F10 melanoma model, we combine ACT of PMEL-1 Tg+ CTLs that recognize the endogenous melanoma antigen gp100 with dual blockade of CTLA-4 and PD-1, the highly pursued effective combinatorial ICB, to treat established tumors. Although ACT alone transiently suppresses tumor growth, complete tumor regression requires additional dual anti-CTLA-4 and anti-PD-1 blockade. Mechanistically, we demonstrate that the ICOS-Eomes-IFN-γαμμα axis, but not the ICOS-T-bet-IFN-γαμμα axis, in CTLs mediates the therapeutic effects of ACT and ICBs. Our data provide direct evidence that combining ACT of CTLs with ICBs confers long-term protection to tumor challenges, which largely depends on CTL-intrinsic expression of ICOS.

#3571

Use of a universal CAR T cell plus bispecific adapters to eradicate antigenically heterogeneous tumors.

Yong Gu Lee,1 Isaac Marks,1 Madduri Srinivasarao,1 Ananda Kumar Kanduluru,2 Sakkarapalayam M. Mahalingam,1 Haiyan Chu,3 Philip S. Low1. 1 _Purdue University, West Lafayette, IN;_ 2 _On Target Laboratories, West Lafayette, IN;_ 3 _Endocyte Inc., West Lafayette, IN_.

Chimeric antigen receptor (CAR) T cell therapies have attracted considerable attention because of their abilities to focus the killing power of T cells specifically onto tumor antigen expressing cancer cells. Despite impressive successes in the clinic, CAR T cell technologies still suffer from an inability to kill cancer cells that mutate to avoid expression of the CAR T cell recognized antigen. While current CAR T cell technologies would require the engineering of a new CAR to recognize a second tumor-specific antigen, we have instead developed a universal CAR that can recognize any tumor antigen. In this modification of standard CAR T cell technology, we fuse an scFv that binds fluorescein to the exoplasmic domain of a T cell receptor that contains the usual 4-1BB and CD3 zeta activation domains within its cytoplasmic domain. We then promote engagement of the resulting CAR T cell with the desired cancer cell by administering a bispecific adapter molecule that contains fluorescein linked to a tumor-specific ligand via a short spacer. Upon addition of this adapter, simultaneous binding of the fluorescein to the CAR and the tumor-specific ligand to the cancer cell mediates multivalent binding of the CAR T cell to the cancer cell. With the simultaneous administration of a cocktail of different tumor-specific adaptors, each comprised of a fluorescein linked to a distinct tumor-specific ligand, multiple orthogonal cancer cells within a heterogeneous solid or liquid tumor can be simultaneously eliminated by a single CAR T cell. To test the ability of our proposed universal CAR T cell to eradicate solid tumors with multiple orthogonal tumor antigens, we first demonstrate the capacity of our anti-fluorescein CAR T cell to kill a variety of cultured human cancer cells in vitro upon addition of the optimal adapter. In this effort, we show that the same anti-fluorescein CAR T cell can kill: i) multiple folate receptor-expressing cancer cells upon addition of folate-fluorescein, ii) PSMA-expressing cancer cells upon addition of a PSMA ligand-fluorescein conjugate, iii) carbonic anhydrase IX-expressing cancer cells upon addition of a CA9 ligand-fluorescein conjugate, and iv) neurokinin-1 receptor expressing cancer cells upon addition of a NK1R ligand-fluorescein conjugate. We then demonstrate that this same universal killing potency is also effective in vivo by transfecting separate MDA-MB-231 breast cancer cell cultures with one of the above tumor antigens and then implanting the MDA-MB-231 cells expressing the orthogonal tumor antigens in NSG mice and showing that our universal CAR T cell can eradicate the resulting tumors upon administration of the appropriate bispecific adapter. Taken together these results establish that a single anti-fluorescein CAR T cell can be exploited to eliminate antigenically heterogeneous tumors upon addition of the correct cocktail of ligand-fluorescein conjugates.

#3572

CAR T-cells targeting the kappa myeloma antigen for the treatment of multiple myeloma.

Kavitha Gowrishankar,1 Rosanne Dunn,2 David Gottlieb,1 Kenneth Micklethwaite1. 1 _University of Sydney, Sydney NSW, Australia;_ 2 _HaemaLogiX Pty Ltd, Sydney, Australia_.

This study describes the development of highly specific and effective CAR T-cells expressing novel Chimeric Antigen Receptors targeting the Kappa Myeloma Antigen. Multiple myeloma (MM) is an incurable malignancy of differentiated plasma cells accompanied by severe bone lesions, cytopenia and hypercalcemia. The majority of MM patients produce excess monoclonal immunoglobulin and/or isotype-restricted free light chains (FLC). The monoclonal antibody KappaMab (kMAb-previously-MDX-1097) binds to a unique conformational epitope on the Kappa Myeloma Antigen (KMA). KMA consists of kFLC that is not associated with Ig heavy chain but is non-covalently associated with sphingomyelin on the cell membrane. KMA is present on k-restricted MM, some k-type lymphomas and B cells associated with Waldenstroms macroglobulinemia and not present on normal B cells, immune cells or normal human tissue. The restricted expression of KMA makes it an ideal target and the unique specificity of KMAb suggests that this antibody is an excellent candidate for the development of a novel treatment for myeloma patients.The scFv gene from the kMAb was fused with the co-stimulatory domains from either CD28 or CD137 (41BB), along with the activation domain of the CD3ƶ subunit to construct several second generation CARs in the piggyBac transposon vector. In addition, a novel chimeric construct of KMA fused to mCherry was designed, to enable the creation of stable antigen presenting cell lines. Transfected CD3+Tcells were enriched and expanded using irradiated autologous PBMCs and the irradiated KMA-mCherry cell line with the addition of 200IU of IL-15 every 48 hours. After a three week expansion, the CAR T-cells were phenotyped and analysed for functional specificity. Release of IFN-γ upon co-culture with KMA+ or KMA- cells indicated target specific activation. The potency of the CAR T-cells was confirmed in cytotoxicity assays, using 51Cr labelled cells. Pre-clinical data indicated that the KM.CAR T-cell containing the CD8 spacer was the most efficacious among all the KM.CAR T-cells generated, even when there were moderate levels of antigen presentation. The CD28 co-stimulatory domain containing KM.CAR T-cells expressed and expanded better than the ones containing the 41BB co-stimulatory domain. All the KM.CAR T-cells were effective in clearing target cells that expressed KMA. There was a mix of central and effector memory cells at the end of culture which indicated a potential for long term survival and function in vivo. Most of the KM.CAR T-cells showed negligible expression of the exhaustion marker PD-1. Myeloma is becoming the next haematological malignancy after leukaemia to demonstrate phenomenal response rates to CAR therapy. Our in vitro data indicates that the KM.CAR T-cell is likely to be effective in kappa restricted myeloma patients. In vivo testing of these CAR T-cells in a Rag2-/- myeloma mouse model system is currently underway.

#3573

Novel DNA vectors encoding a chimeric antigen receptor mediate long term expression without genomic integration.

Patrick Schmidt,1 Matthias Bozza,2 Aileen Berger,1 Claudia Luckner-Minden,1 Alexandra Tuch,2 Inka Zörnig,1 Dirk Jäger,1 Richard Harbottle2. 1 _National Ctr. for Tumor Diseases, Heidelberg, Germany;_ 2 _German Cancer Research Center, Heidelberg, Germany_.

Adoptive immunotherapy is one of the most encouraging therapeutic strategies for the treatment of a range of cancers. One particularly promising avenue of research is the functional introduction of Chimeric Antigen Receptors (CARs) into naive Human T-Cells for autologous-immunotherapy. Currently, the genetic engineering of these cells is achieved through the use of proprietary integrating vector systems such as lentiviruses or the sleeping beauty transposon system which present a risk of genotoxicity associated with their random genomic integration.

We have invented a novel DNA Vector platform for the safe and efficient generation of genetically engineered T-Cells for Human Immunotherapy. This DNA vector system contains no viral components and comprises only clinically approved sequences, it does not integrate into the target cell's genome but it can replicate autonomously and extrachromosomally in the nucleus of dividing human primary cells. These DNA Vectors offer several advantages over currently used vector systems; they are not subject to commercial licences, they are cheaper and easier to produce, and they can more quickly genetically modify human cells without the inherent risk of integrative mutagenesis.

In preclinical experiments we have successfully generated genetically engineered human T-Cells expressing the CAR receptor against several epitopes and have demonstrated their viability and capability in targeting and killing human cancer cells which express these epitopes. The long term anti-tumor activity of our DNA-CAR-T cells has been confirmed in vivo using xenotransplanted cell lines in immunodeficient mice.

We believe that this novel DNA Vector system provides a unique and innovative approach to this exciting therapeutic strategy for cancer therapy. We estimate that this novel methodology will provide a simpler method of CAR T-cell manufacturing, resulting in a 10-fold reduction in the cost of the CART-product.

#3574

Cytokine-autonomous, CAR-directed, off-the-shelf natural killer cells derived from a clonal engineered master pluripotent cell line.

Tom T. Lee,1 Ye E. Li,2 Ryan Bjordahl,1 Robert Blum,2 Sajid Mahmood,1 Huang Zhu,2 Gregory B. Bonello,1 Bahram Valamehr,1 Dan S. Kaufman2. 1 _Fate Therapeutics, Inc., San Diego, CA;_ 2 _University of California, San Diego, San Diego, CA_.

Natural killer (NK) cells are potent antitumor effectors that play an important role in innate and adaptive immunity. Despite recent clinical advances in the therapeutic use of NK cells, significant opportunities remain to harness their full potential in adoptive immunotherapy. For example, achieving consistent manufacturing of cancer immunotherapies using patient- and donor-sourced cells remains a significant challenge to delivering therapies to all patients who may benefit. There is also the need to improve the efficacy and persistence of adoptively transferred lymphocytes to promote favorable patient outcomes. We have previously demonstrated that induced pluripotent stem cells (iPSCs) can be genetically edited at the single-cell level and expanded to create clonal master engineered pluripotent cell lines, representing a renewable and reliable starting material for the creation of off-the-shelf (OTS) adoptive NK cell therapy. Using this unique platform, we evaluated the combination of NK cell-specific chimeric antigen receptors (CARs) with an autonomous protein to create a highly effective, persistent, and targeted NK cell therapy. The NK cell optimized CAR (NK-CAR) backbone contains the NKG2D transmembrane domain and the 2B4 co-stimulatory and the CD3ζ signaling domains to mediate a strong increase in NK cell signaling. To provide directed antitumor activity, anti-mesothelin (meso) and anti-CD19 scFvs were added to the NK-CAR backbone, and then engineered into the iPSC and subsequently differentiated to NK cells expressing NK-CARmeso or NK-CAR19. Using an ovarian cancer xenograft model in the initial study, a single dose of NK-CARmeso NK cells markedly inhibited tumor growth and mediated enhanced survival (84 days) compared to various controls, including NK cells harboring a 3rd-generation T-cell CAR construct with CD28 and 4-1BB costimulatory domains (p < 0.002). We next engineered an IL-15RF fusion protein to provide self-stimulating signals to support NK cell function and persistence. The IL-15RF construct was created by fusing mature IL-15Rα to IL-15 at the C-terminus through a flexible linker. The design mimics the trans-presentation of IL-15 bound to IL-15Rα that is presented to IL-15Rβ/γC dimer to initiate signaling. While both iPSC-derived NK cells (iNKs) and iNKs bearing IL-15RF expanded in vitro in a similar manner in the presence of soluble IL-15 and IL-2 (2040- and 3615-fold expansion in 14 days, respectively), only the iNKs bearing IL-15RF significantly proliferated in the absence of cytokines (10- vs. 701-fold expansion in 14 days, respectively). We are currently investigating the combination of NK-CAR and IL-15RF to determine the potential synergy between the two modalities to create cytokine-autonomous, OTS NK cell cancer immunotherapies, including those that are CAR-targeted against solid and liquid tumors.

#3575

Cell therapy with surface-tethered IL-12 improves anti-tumor efficacy and activates the endogenous immune system.

Jon Nardozzi, Elena Geretti, De-Kuan Chang, Douglas W. McMillin, Jesse Lyons, Ulrik Nielsen, Thomas Andresen, Douglas Jones. _Torque Therapeutics, Cambridge, MA_.

Adoptive cellular therapy (ACT) has shown impressive clinical responses in some hematologic malignancies. Progress for ACT in solid tumors, however, has been comparatively limited. A key challenge has been overcoming the immunosuppressive microenvironment of solid tumors, which limits T cell activation and persistence. To overcome this barrier, we have further developed our Deep PrimingTM platform to support delivery of therapeutic payloads that activate both the adaptive and innate immune system in the tumor microenvironment. Our Deep PrimingTM technology was designed to function predominantly in an autocrine fashion. To enable transpresentation and paracrine activity we 'tethered' cytokines to the T cell surface by constructing a fusion between an immunostimulatory cytokine and an antibody targeting abundant receptors on the T cell surface. This approach is versatile and enables tunable loading and persistence of multiple cytokines on the T cell surface. It also complements transgenic TCR and CAR-T cell therapies without the additional complexities or potential risks associated with further genetic engineering. Interleukin-12 (IL-12) is a potent cytokine with the potential to reshape the anti-inflammatory environment in solid tumors. However, its clinical utility has been limited by severe toxicities both from soluble administration or from adoptively transferred T cells engineered to secrete IL-12. By linking IL-12 to antibodies targeting T cell surface receptors such as CD45 we have improved control of dose and distribution. In in vitro model systems, surface-tethered IL-12 activated signaling on both the loaded T cells and on co-incubated, non-tethered cells (p<0.002), confirming their ability to signal in both cis and trans. Utilizing the Pmel model, which contains CD8 T cells specific to the gp100 antigen expressed in B16-F10 melanoma cells, we evaluated the ability of the IL-12 tethered fusion to augment ACT in vivo. Pmel T cells that were loaded ex vivo with surface-tethered IL-12 significantly improved anti-tumor activity against established B16 tumors compared with Pmel cells alone. The tethered IL-12 additionally activated the endogenous immune system, inducing a rapid transient lymphopenia of both transferred and endogenous CD8 T cells as well as endogenous NK cells (p<0.0001). This was followed by robust proliferation and activation of NK cells (p<0.0001) and proliferation and differentiation of CD8 T cells (p< 0.001). These effects were transient and returned to baseline within two weeks and resulted in minimal overt toxicity (3.3% avg body wt loss). Further analysis of preclinical safety will be discussed. This approach leverages the ability of T cells to traffic and accumulate within the tumor and holds potential to overcome the immunosuppressive environment associated with solid tumors.

#3576

FT500, an off-the-shelf NK cell cancer immunotherapy derived from a master pluripotent cell line, enhances T-cell activation and recruitment to overcome checkpoint blockade resistance.

Ryan Bjordahl, Sajid Mahmood, Svetlana Gaidarova, Ramzey Abujarour, Raedun Clarke, Laurel Stokely, Paul Rogers, Moyar Ge, Megan Robinson, Betsy Rezner, Tom Tong Lee, Bahram Valamehr. _Fate Therapeutics, San Diego, CA_.

The development of PD1/PDL1 targeting checkpoint inhibitors (CI) has transformed the oncology landscape, providing long term remissions in multiple indications. However, many tumor subtypes are resistant to checkpoint blockade therapy, and relapse remains a significant concern. Novel therapeutic approaches with the ability to overcome CI resistance are needed, and there is significant opportunity for therapies capable of additively or synergistically enhancing T-cell activation and recruitment when combined with CI.

Adoptive transfer of NK cells from healthy donors has the potential to recruit T cells to the tumor microenvironment and augment T-cell activation at the tumor site. NK cells have both direct anti-tumor activity and the capacity to secrete inflammatory cytokines and chemokines upon activation, enabling the cells to play a unique and critical role in regulating anti-tumor T cell activity. We sought to determine whether FT500, an off-the-shelf NK cell product derived from a clonal master pluripotent cell line, could synergize with CI to relieve local immunosuppression and enhance T-cell activation and recruitment to the tumor site.

FT500 is universally negative for cell surface PD1, and expression of PDL1 on tumor lines had no discernable effect on FT500 cytotoxicity. Similarly, addition of PDL1 blocking antibody had no effect on FT500 cytotoxicity or degranulation, suggesting that FT500 is inherently resistant to PDL1-PD1 mediated inhibition. Additionally, activation of FT500 induced the secretion of soluble factors capable of enhancing T-cell activation, as evidenced by increased upregulation of CD69.

We hypothesized that FT500 might also enhance CI by promoting recruitment of T cells to the tumor site. Using conventional in vitro transwell migration assays, we found that FT500 produced soluble factors that promoted the migration of activated T cells. Additional profiling confirmed FT500 production of a range of chemokines, including CCL3, CCL4, CXCL10 and CCL22. Furthermore, using an in vivo recruitment model, FT500 was able to recruit T cells out of the circulation and into the peritoneal cavity. Similarly, utilizing a three-dimensional tumor spheroid model in vitro, infiltration of T cells into tumor spheroids was significantly enhanced when combined with FT500, suggesting that FT500 can enhance tumor infiltration of T cells.

Our data suggest that FT500 is a potent producer of chemokines and can facilitate the recruitment of T cells to the tumor site. In addition to its direct cytotoxic potential, FT500 is also able to enhance T-cell activation, suggesting an ability to synergize with CI to reduce tumor burden. Together, our data provide evidence supporting the combination of FT500, an off-the-shelf NK cell cancer immunotherapy, with CI to overcome checkpoint blockade resistance.

#3577

Application of deep IL-15 backpacks to human T cells demonstrates tunable loading with enhanced cell proliferation and antitumor activity.

Pengpeng Cao, De-Kuan Chang, Andy Rakestraw, Amy Shaw, Ferdinando Pucci, Fabio Fachin, Christine McInnis, Shawn Carey, Austin Boesch, David Chirgwin, Carlos Tassa, Aaron Handler, Khristianna Jones, Philip D. Bardwell, Elena Geretti, Jon Nardozzi, Doug Jones, Jesse Lyons, Jonathan B. Fitzgerald, Becker Hewes, Ulrik B. Nielsen, Thomas Andresen. _Torque, Cambridge, MA_.

Cell therapies have demonstrated clinical benefit for some patients; however, in more resistant tumors, efficacy, persistence, and cell expansion may be improved with co-administration of an inflammatory cytokine such as interleukin 15 (IL-15). Clinical use of IL-15 has been limited due to systemic toxicity resulting in a narrow therapeutic index. Torque has advanced a "backpack" technique to attach IL-15 to the surface of T cells for adoptive transfer. IL-15 backpacks are ~120 nanometer particles consisting of multiple chemically crosslinked human IL-15/IL-15Rα/Fc heterodimers. Following crosslinker cleavage the IL-15 fusion proteins are released and drive T cell division in an autocrine fashion, providing a targeted, controllable and time-dependent immune stimulus which we term Deep Priming. Torque's lead program, TRQ15-01, consists of antigen-directed, autologous T cells carrying IL-15 backpacks. To explore the effects of IL-15 backpacks on T cells, we characterized backpack-carrying capacity of T cells, IL-15 heterodimer release kinetics, and T cell expansion by incubating IL-15 backpacks with human T cells activated by CD3/CD28 beads. Across 4 donors, T-cells were loaded in a reproducible manner proportional to the backpack labeling solution concentration. Backpacks promote the exponential expansion of activated T-cells for over one week through sustained release of IL-15 into the extracellular space. To explore the effect of IL-15 backpacks on CAR-T cell activity, IL-15 backpacks were loaded onto anti-EGFR CAR-expressing human CD3 cells. NSG mice bearing human H1299 tumor xenografts received CAR-T therapy with and without backpacks 7 days after tumor implantation. IL-15 backpacks significantly improved in vivo expansion of CAR-T cells (p < 0.008, at Day 10) and improved tumor growth inhibition (p < 0.003, 2-way ANOVA). To enable exploration of the clinical utility of backpacking using antigen-directed T-cells, we developed a novel, fully-closed, semi-automated cell manufacturing process with a yield of up to several billion antigen-directed cells. This process utilizes monocyte-derived dendritic cells pulsed with peptides from multiple tumor-associated antigens to expand cytotoxic T lymphocytes (CTLs). In a final step the antigen-directed CTLs are loaded with IL-15 backpacks to generate the TRQ15-01 cell product. Characterization of TRQ15-01 reveals controllable, dose-dependent loading of IL-15 backpacks on the antigen-directed T cells. Compared to non-backpacked antigen-directed cells, TRQ15-01 products displayed significantly higher expansion levels both in vitro (10-fold increase) and in NSG mice (20-fold increase). IL-15 backpack loading on human T cells is tunable and produces slow IL-15 release with sustained cell expansion and enhanced anti-tumor activity. Clinical trials with TRQ15-01 will be initiated in 2018.

#3578

Unmasking T-cell heterogeneity via single cell transcriptomic profiling.

Mukta Dutta, Tuuli Saloranta, Inah Golez, Kerry Deutsch, Cara Lord, Vickie Satele, Steve Anderson, Anup Madan. _Covance Genomics Laboratory, Redmond, WA_.

Single-cell gene expression analysis has been shown to delineate cellular heterogeneity. Such analyses result in the discovery of genes that identify subtypes of cells, or that mark intermediate states during a biological process. Cellular heterogeneity, plasticity, and diversity of T cells facilitate a wide range of functional flexibility that give rise to a remarkable breadth of potential responses against many different pathogens and in various disease settings. Understanding how T cells leverage these cellular characteristics particularly during dynamic processes such as development, differentiation, and antigenic response is important and well suited for single-cell analysis. Unbiased approaches to evaluate cellular states and subpopulations of single T cells can identify important aspects that may be concealed by targeted approaches such as FACS sorting on cell-surface antigens, or bulk expression analysis. Resolving these complexities in T cells is of particular interest as classical phenotypic criteria has been deemed insufficient for distinguishing different T cell subtypes and transitional states, particularly in the context of dysfunctional T cell states in autoimmunity and tumor-related exhaustion. We activated purified 2.5 million CD4+ T cells from Cynomolgus monkey (M. fascicularis) using equal amounts of biotinylated antibodies against CD2 (human), CD3 (primate) and CD283 (human). To determine the single cell transcriptomic profile of these activated T cells, we performed drop seq sequencing at 0 hours and 24 hours post activation. Cells at both time points were split into four technical replicates to determine variability in the sequencing process. In addition, RNA was also isolated from these cells and whole transcriptome analysis was performed using Illumina's Next Generation Sequencing technology. Our analysis show how cellular subpopulations can be identified from transcriptional data, and derive characteristic gene expression signatures that distinguish these states. We ordered single cells in pseudotime, placing them along a trajectory corresponding to individual cell's asynchronous progression during activation. Monocle was used for this purpose as it orders cells by learning an explicit principal graph from the single cell genomics data with Reversed Graph Embedding, an advanced machine learning technique, which robustly and accurately resolves complicated biological processes. This information could not have been obtained from bulk RNA-Seq analysis. Our results show that single-cell RNA-Seq is a powerful technique to study the cellular heterogeneity in T cells, a paradigm that will be of great value in the development of immunotherapeutic strategies.

#3579

**RegCAR-T** TM **: Enabling next-generation CAR-T therapy.**

Xu Zhang, Ke Tang, Qingxuan Song, Si Chen, christopher Kasbek, Tianjiao Wang, Jun Huang. _Admera Health, South Plainfiled, NJ_.

Chimeric Antigen Receptor (CAR) T-Cell Therapy has been emerging as a means of powerful cancer immunotherapy with its exceptional therapeutic efficacy, however, the full potential of CAR-T therapy is hampered by its severe side effects. Thus, CAR-T therapy that is controllable for optimal efficacy and safety is in need developing. To enable controllable CAR-T therapy, we have established a technology platform, RegCAR-TTM. In RegCAR-TTM, aptazymes are generated by in vitro selection/evolution of combinatory nucleic acid libraries and are used as molecular switches for controlling CAR expression. Aptazymes are RNA molecules consisting of an aptamer module and a ribozyme module, in which ribozyme self-cleavage can be allosterically regulated by the aptamer-ligand (e.g. a small molecule drug) interaction. With an aptazyme switch incorporated into the untranslated region (UTR) of a CAR gene, CAR expression can be temporally and dose-dependently controlled at mRNA level by a ligand, which may result in controllability of CAR-T efficacy and safety. Using RegCAR-TTM technology, we have generated aptazyme switches responsive to FDA-approved small molecules drugs with IC50 at micromolar level. Thus, next generation CAR-T therapies with aptazyme switches would be controllable by safe oral small molecules drugs. In conclusion, we have developed a technology platform (RegCAR-TTM) to generate aptazyme switches for enabling controllable CAR-T therapy.

#3580

Enhancing adoptive cell therapies through exogenous regulation.

Celeste Richardson, Kutlu Elpek, Michelle Ols, Tariq Kassum, Steve Shamah. _Obsidian Therapeutics, Cambridge, MA_.

Adoptive cell therapy with chimeric antigen receptor (CAR) modified T cells has demonstrated remarkable clinical efficacy in the treatment of certain B cell malignancies and more recently in multiple myeloma. However, in solid tumors, CAR-T therapy has been far less successful, likely due to the lack of robust CAR-T cell expansion, the immunosuppressive microenvironment, and clonal heterogeneity within these tumors. The trafficking of CAR-T cells to tumor sites provides an opportunity for selective delivery of cargo that can enhance CAR-T cell activity at the site of the tumor. Interleukin-12 (IL12) and membrane tethered interleukin-15 (mbIL15) have previously been shown to enhance CAR-T activity, yet the unregulated expression of either compromises safety and/or efficacy. The Obsidian Therapeutics platform equips engineered immune cells with new functionalities whose activity can be regulated via the administration of FDA-approved small-molecule drugs, putting dosing control back in the hands of the treating physician. We utilize small, fully human protein sequences called destabilizing domains (DDs) that confer reversible destabilization to a fused target protein. In the absence of ligand the fusion protein is degraded, whereas the presence of small molecule ligand restores expression and functionality. Furthermore, stabilization is titratable with small molecule ligand dose, providing finely tuned control over target protein expression and function. We have applied our technology to create DD-IL12 and DD-mbIL15 "cassettes" that provide exogenous regulation of cytokine activity when transduced into CAR-Ts for enhanced cellular function. We describe here the successful implementation of the DD technology in engineered primary human T cells and show small-molecule ligand controlled regulation of DD-IL12 and DD-mbIL15 protein expression in vivo in mouse models. These data demonstrate the feasibility of exogenous control over protein expression in primary human T cells for the development of next-generation CAR-T cell products with enhanced efficacy and more favorable safety profiles.

#3581

Convergence of the specificity and cross-reactivity of multiple T cell receptors isolated against a single cancer peptide/HLA complex.

Preeti Sharma, Daniel T. Harris, Marlies V. Hager, David M. Kranz. _University of Illinois, Urbana, IL_.

Adoptive T cell therapy using T cell receptors (TCRs) or chimeric antigen receptors (CARs) requires careful selection of the receptor so as to target only the specific cancer antigen, without cross-reactivity toward structurally similar antigens. In this study, we used two TCRs (T1 and RD1) that bind to the HLA-A2 restricted melanoma antigen MART-1 (ELAGIGILTV). TCR-T1 was initially isolated from a melanoma-reactive T cell line specific for MART-1 whereas TCR-RD1 was derived completely by in vitro engineering, switching the specificity of the well-studied A6 TCR from its cognate viral peptide (Tax) to MART-1 by directed evolution. Despite their distinct origins, engineered high-affinity forms of both TCRs had remarkably similar fine-specificities as determined using a panel of MART-1 variant peptides. This binding signature allowed us to conduct a bioinformatic scan of the human and mouse proteomes for peptides that were homologous to MART-1. Our analysis resulted in identification of a self-peptide (TM74B: IIAGLGILTV) that also bound to both T1 and RD1 TCRs. T1-transduced T cells (CD4+) from HLA-A2/Db transgenic (AAD) mice were potently activated by MART-1, and cross-reacted (at 1000-fold higher doses) with the TM74B peptide. CD8+ T cells transduced with the T1 TCR were activated by AAD+ antigen presenting cells, indicating recognition of an endogenous peptide, possibly TM74B. To extend these studies, we have shown that T1-transduced AAD T cells (CD4+) are also stimulated by murine MART-1 (EAAGIGILIV) and that transfer of T1-transduced T cells into AAD mice did not result in overt toxicity. Thus, the AAD mouse could be used to study efficacy and toxicity of T1 TCR in syngeneic models. We are currently developing a B16/AAD tumor model to compare recognition of B16 melanoma by MART-1 directed TCRs and CARs. In summary, the convergence of specificity of T1 and RD1 suggests that TCRs against a cancer peptide antigen are focused on the same exposed features of the peptide. Furthermore, the results with RD1 suggest that it is possible to engineer TCRs completely in vitro, and that these TCRs can recapitulate the fine-specificities of conventional TCRs isolated from T cell clones.

#3582

Inclusion of a Dap10 costimulatory domain enhances anti-tumor efficacy of chimeric PD1-expressing T cells in multiple types of solid tumors.

Amorette E. Barber, Geoffrey Parriott, Shane Crean, Hailey Kintz. _Longwood Univ., Farmville, VA_.

Adoptive transfer of tumor-reactive T cells is a promising anti-tumor therapy for many cancers. To enhance tumor recognition by T cells, chimeric antigen receptors (CAR) consisting of signaling domains fused to receptors that recognize tumor antigens can be created and expressed in T cells. One receptor that is a prospective target for a new chimeric antigen receptor is PD1 because the ligands for the PD1 receptor are expressed on many cancer types. Therefore, we developed a murine chimeric PD1 receptor (chPD1) consisting of the PD1 receptor extracellular domain and the activation domain of CD3 zeta. In addition, current chimeric antigen receptor therapies utilize various costimulatory domains to enhance anti-tumor efficacy. Therefore, we also compared the inclusion of CD28, Dap10, 41BB, GITR, ICOS, or OX40 costimulatory domains in our chPD1 receptor to determine which costimulatory domain induced optimal anti-tumor immunity. To determine if this novel CAR could potentially target a wide variety of tumors, the anti-tumor efficacy of chPD1 T cells against murine lymphoma, melanoma, kidney, pancreatic, liver, colon, breast, ovarian, prostate, and bladder cancer cell lines was measured. Of the eighteen cell lines tested, all expressed PD1 ligands on their cell surface, making them potential targets for chPD1 T cells. Regardless of the costimulatory domain in the CAR, all of the chPD1 T cells induced similar levels of T cell proliferation and tumor cell lysis. However, differences were observed in the cytokine secretion profiles depending on which costimulatory receptor was included in the CAR. While most of the chPD1 T cell receptor combinations secreted both pro-inflammatory (IFNγ, TNFα, IL-2, GM-CSF, IL-17, and IL-21) and anti-inflammatory cytokines (IL-10 and IL-5) as determined by ELISA and LegendPlex analysis, chPD1 T cells containing a Dap10 costimulatory domain secreted high levels of proinflammatory cytokines, but did not secrete a significant amount of anti-inflammatory cytokines. Furthermore, T cells expressing chPD1 receptors with a Dap10 domain also had the strongest anti-tumor efficacy in vivo. ChPD1 T cells did not survive for longer than 14 days in vivo, however treatment with chPD1 T cells induced long-lived protective host-anti-tumor immune responses in tumor-bearing mice. Therefore, adoptive transfer of chPD1 T cells could be a novel therapeutic strategy to treat multiple types of cancer and inclusion of the Dap10 costimulatory domain in chimeric antigen receptors may induce a preferential cytokine profile for anti-tumor therapies.

#3583

NKG2D as a chimeric antigen receptor - DAP 10 provides optimal co stimulation for NKG2D based CARs.

Jennifer BOLSEE, Eytan BREMAN, Fanny HUBERTY, Benjamin VIOLLE, Jerome MARIJSSE, Céline JACQUES-HESPEL, Celine MARCHAND, Nancy RAMELOT, Thuy NGUYEN, Julien HOUSSA, David E. GILHAM, Sophie AGAUGUE. _Celyad SA, Mont Saint Guibert, Belgium_.

Chimeric antigen receptor (CAR) protein are artificial protein created by the fusion of an extracellular domain targeting one or several cell surface antigens, a transmembrane domain and an intracellular domain responsible for signal initiation and transmission. First-generation CARs consist of an intracellular signaling domain, often a cytoplasmic tail of the CD3ζ chain. The second and third generation include the addition of one or two co-stimulation domains (e.g. cytoplasmic domain of CD28 and/or 4-1BB).

NKG2D is a receptor present on the cell surface of NK cells, NK T cells and subgroups of CD8+ T cells. The receptor exists as a dimer that interacts with a co-adaptor protein called DAP10. Upon interaction of NKG2D with one of its ligand (MICA, MICB, and ULBP1-ULBP6), the DAP10 cytoplasmic tail will induce downstream signaling. DAP10 has a similar signaling motif to CD28 and similarly gets phosphorylated and recruits kinases. NKG2D ligands are expressed on a vast majority of solid and liquid tumors and are thus a very attractive target for CAR T cells.

In this study, multiple NKG2D constructs were created and compared to one another. The first CAR construct tested (chNKG2D.1) consists of a fusion of full-length wild-type NKG2D and the cytoplasmic domain of CD3ζ. This construct is currently tested in clinical settings. This CAR benefits from the co-stimulatory signal provided by DAP10 and can therefore be considered as a second generation CAR. Next to this, a new chNKG2D was constructed by combining DAP10 overexpression (chNKG2D.1.DAP10). Secondly classic second-generation CARs were created by adding CD28 (chNKG2D.2.1) or 4-1BB (chNKG2D.2.2) co-stimulatory domains to the chNKG2D.

Co-expression of DAP10 increased chNKG2D expression at the cell surface of both CD4 and CD8 T cells (although the increase was more pronounced in CD4 T cells). Furthermore, co-cultures of T cells expressing this construct with K562 cells led to similar IFN-γ levels irrelevant of the CD4/CD8 ratio. Other CD4 specific cytokines were highly increased when the ratio was in favor of CD4 positive T cells. Interestingly, the addition of the co-stimulatory domains to the construct (chNKG2D.2.1 and .2.2) did not lead to higher levels of IFN-γ or any of the CD4 cytokines. Next to cytokine release, cytolytic activity was also assessed which interestingly showed no difference in any of the conditions even when the CD4/CD8 cell ratio was different.

In conclusion NKG2D CAR T cells only showed increased cytokine release when DAP10 was overexpressed, all other conditions did not lead to any changes when compared to the 'NKG2D CAR T cells currently tested in the clinic. This implies that NKG2D is optimally costimulated through the DAP 10 co-signaling, and that this co-signaling is a least as potent as traditional CD28 or 4-1BB based co stimulation.

#3584

**Characterization of a novel class of engineered (TCR) fusion constructs (TRuC** TM **s) aimed to treat solid tumors.**

Ekta Patel,1 Jian Ding,1 Nikolaus Thorausch,2 Janani Krishnamurthy,1 Rashmi Choudhary,1 Solly Weiler,1 Bonnie Le,1 Patrick Tavares,1 Adam Zieba,1 Justin Quinn,1 Yan Wang,1 Wolfgang Schamel,2 Irene Scarfo,3 Marcela Maus,3 Patrick Baeuerle,4 Dainel Getts,1 Robert Hofmeister1. 1 _TCR2, Cambridge, MA;_ 2 _University of Freiburg, Freiburg, Germany;_ 3 _Massachusetts General Hospital, Cambridge, MA;_ 4 _MPM, Cambridge, MA_.

T cells expressing chimeric antigen receptors (CARs) have demonstrated remarkable clinical benefit in certain hematological malignancies albeit with side effect such as cytokine release syndrome, on Target off tumor response and limited efficacy in solid tumors. Here, we present the preclinical evaluation of a novel T cell engineering platform designed to overcome potential hurdles of CARs and other T cell receptor modalities. Specifically, we have generated novel T Cell Receptor Fusion Constructs (TRuC™s) that fuse binder domains to subunits of the T cell receptor. These constructs when introduced using lentiviral technology, integrate into endogenous TCR, reprograming T cells to target tumor antigen in a non-major histocompatibility complex (MHC) restricted fashion and harnessing the full spectrum of TCR signaling. TRuC™ variants were constructed by recombinant fusion of an scFv or sdAb to various TCR subunits via a flexible linker sequence. Likewise, CD28ζ and 41BBζ CARs were generated using the same binders for side-by-side comparison. We demonstrated that TRuC™ variants can effectively reprogram T cells to recognize tumor surface antigens in a non-MHC-restricted fashion. TRuC™s are distinct from CARs in their ability to activate T cells through the entire TCR without additional costimulatory domains. In vitro, TRuC™ T cells were equally potent as CAR T cells in eliminating tumor cells. Compared to CAR-T cells, TRuC-T cells produced lower cytokine levels and proliferated less. Despite these difference, TRuC™ T cells were more efficient in clearing tumors in subcutaneous Raji and MSTO-211H mesothelioma models. Our findings support the development of TRuC-T cells for the treatment of solid tumors.

#3585

T cell receptor-stabilized regenerated CD8ab cytotoxic T lymphocytes for cancer immunotherapy.

Atsutaka Minagawa,1 Akitsu Hotta,1 Yohei Kawai,1 Yutaka Yasui,1 Yasushi Uemura,2 Masaki Yasukawa,3 Tetsuya Nakatsura,2 Shin Kaneko1. 1 _Center for iPSC Research and Application, Kyoto, Japan;_ 2 _National Cancer Center, Tokyo, Japan;_ 3 _Ehime University, Ehime, Japan_.

The use of antigen-specific T cell derived induced iPSCs as a cell source for regeneration of cytotoxic T lymphocytes (CTLs) have advantages in rejuvenation profile, and reproducible number of CTLs. However for the safe and efficient regenerated T cell immunotherapy, strict antigen specificity is essential. Here we report CD8αβ T cells differentiated from T cell-derived iPSCs underwent additional rearrangement of the T cell receptor (TCR) α-chain gene at the CD4/CD8 double positive stage during in vitro differentiation and lost antigen specificity. CRISPR knockout of a recombinase gene in the T-iPSCs successfully prevented this additional TCR rearrangement. Moreover, when CD8αβ T cells were differentiated from non-T cell-derived but antigen-specific TCR-transduced iPSCs, they showed monoclonal expression of the transduced TCR. TCR stabilized regenerated CD8αβ T cells effectively inhibit tumor growth in xenograft cancer models. These approaches could contribute to safe and effective regenerative T cell immunotherapies.

#3586

A universal killer T-cell for adoptive cell therapy of cancer.

Pierre Dillard,1 Nadia Mensali,1 June Helen Myklebust,1 Mickael Hebeisen,2 Gjertrud Skorstad,1 Marit Renee Myrhe,1 Anne Fåne,1 Gustav Gaudernack,1 Gunnar Kvalheim,1 Else Marit Inderberg,1 Sebastien Wälchli1. 1 _Oslo University Hospital, Oslo, Norway;_ 2 _Lausanne University Hospital, Lausanne, Switzerland_.

T cell-mediated immunotherapy of cancer has achieved remarkable results in hard-to-beat cancers. The main challenge of adoptive T-cell transfer (ACT) is its labor intensive and costly production and logistics as well as its dependency on the quality of the patient's T cells. To overcome these hurdles we have designed a universal cell line for TCR expression by modifying the FDA-approved NK cell line, NK-92. Advantages of using this cell line is that it is easy to expand and can readily be genetically engineered. However, tumor cell recognition and killing by NK-92 is not antigen specific. This can be controlled by introducing an antigen receptor, such as a chimeric antigen receptor (CAR) or, as in the current work, a TCR. We herein present evidence that NK-92 can be modified to become a T cell-like lymphocyte which we named UK-92 cell (Universal Killer). UK-92 expressing a therapeutic TCR showed conserved binding capacity to the cognate pMHC. Phosphoflow cytometry results indicated that the introduced TCR was able to mediate intracellular signaling upon either crosslinking or cognate pMHC binding. Our data showed that both early and late TCR signalling players were activated in a TCR-specific manner (anti-CD3/anti-CD28 stimulation) and further in a pMHC specific manner. In vitro functional assays using TCRs isolated from both CD8 and CD4 T cells demonstrated that UK-92-TCR could be stimulated in a pMHC-specific manner and, importantly, could kill tumor cells specifically even in a tumor-like microenvironment such as spheroids. We have now shown in vitro that UK-92 cells are as specific and potent as redirected T cells to kill target cells. Finally, encouraging in vivo data showed that mice receiving UK92 cells expressing a therapeutic TCR experienced reduction in tumor load and enhanced survival compared with control mice. If confirmed, the use of UK92 as a universal cell line might pave the way to truly off-the-shelf therapeutic effector cells for cancer immunotherapy and leading to drastic reduction of cell production time, logistic and cost.

#3587

TCR-transduced T cells targeting a truncal mutation caused by a nsSNV destroy large solid tumors despite intratumoral genetic heterogeneity.

Matthias Leisegang,1 Karin Schreiber,2 Steven P. Wolf,2 Vasiliki Anastosopolou,1 Kimberley Borutta,1 PohYin Yew,2 Kazuma Kiyotani,2 Yusuke Nakamura,2 Hans Schreiber2. 1 _Max Delbrück Center, Berlin, Germany;_ 2 _Univ. of Chicago, Chicago, IL_.

Most if not all cancers have individually specific, so-called unique antigens that we discovered in 1995 to be caused by somatic tumor-specific, non-synonymous single nucleotide substitutions, now usually referred to as nsSNVs, and we showed already then that adoptive transfer of nsSNV-specific T cells has specific anti-cancer effects (Monach P. et al. Immunity 2:45-59, 1995). With the rapid advances in sequencing technologies, these antigens, now generally also called "mutant neoantigens", have been discovered on all primary cancers examined in the past decade, human or experimental, (unless a cancer was the result of artificial genetic engineering). The number of somatic mutations, mostly nsSNVs, differs widely between cancers and tumor types. Importantly however, even patients bearing cancers with a low mutational burden usually have 12 different HLA alleles to possibly present a given mutant peptide as antigen to T cells. Since most mutations in cancers are individually patient-specific, a truly personalized, i.e., patient-specific therapy is required. Encouragement for investing into developing such an approach comes from our recent studies in which we show that a single type of T cell receptor (TCR) targeting a single cancer-specific point mutation, i.e. nsSNV-encoded mutant peptide, can eradicate large solid tumors when the mutation-specific TCR is transduced into peripheral T cells and adoptively transferred (Leisegang, M. et al. Clin. Cancer Res. 22: 2734-43 2016). Here, we examine the relative influence of mutant antigen expression level, intratumoral heterogeneity as well as competence of the transferred mutation-specific T cells on escape of the cancer from therapy using adoptive transfer of T cells specific for a given mutation. The importance and mechanisms of bystander killing in cancer escape will also be discussed and the importance of cross-presentation of the neoantigen by the tumor stroma will be examined. Together, our current studies are consistent with the notion that adoptive transfer of T cells transduced with TCRs recognizing nsSNV-encoded peptides may provide a novel and potentially extremely powerful completely cancer-specific approach in patients with established solid tumors. In addition, the concept should be applicable to most cancer patients, if efforts are made to truly individualize cancer therapy. This requires focus on identifying the set of patient-specific nsSNV and the set of 12 patient-specific MHCI and MHCII that are available to potentially present the mutant peptides of a given patient's cancer as targets for tumor destruction. Supported by NIH grants R01-CA22677 and R01-CA37156, the Cancer Research Foundation, a collaborative research grant by the Berlin Institute of Health and the Einstein-Stiftung Berlin.

#3588

ACTallo: A novel approach using gamma-delta T cells to allogeneic cellular therapy to treat cancer.

Mo Dao,1 Melinda Mata,1 Leonie Alten,2 Aleksandra Nowicka,1 Sabrina Kuttruff,2 Sarah Missel,2 Ali Mohamed,1 Toni Weinschenk,2 Yannick Bulliard,1 Steffen Walter1. 1 _Immatics US, Inc, Houston, TX;_ 2 _Immatics Biotechnologies GmbH, Houston, Germany_.

Adoptive cellular therapy (ACT) has dramatically changed the landscape of cancer immunotherapy; however, the complex manufacture of individualized products from late-stage, heavily pre-treated cancer patients, together with high cost-of-goods, encourages the development of simpler 'off-the-shelf' alternatives. The unique properties of γδ (gamma-delta) T cells make them a seductive candidate for effective allogeneic ACT. In contrast to αβ T cells, γδ T cells are endowed with intrinsic antitumor activity while unable to induce graft-versus-host disease. γδ T cells have a high proliferative potential before exhibiting exhaustion. Moreover, in the context of αβ TCR-engineered ACT, γδ T cells pose a lesser risk of off-target toxicity, due to the absence of expression of endogenous αβ TCR and hence, risk of mispairing with the engineered αβ TCR.

Here, we will present the ACTallo® platform, which combines the unique properties of γδ T cells with Immatics' XPRESIDENT® target/TCR discovery platform. The ACTallo® process selectively expands Vγ9Vδ2 T cells that are retrovirally-transduced to co-express CD8 together with a tumor-specific αβ TCR. Over a period of 45 days or less, ACTallo® γδ T cells can expand >30,000 fold. γδ T cells obtained this way recognize tumor cells, express cytokines, degranulate and kill tumor cells specifically. In addition, ACTallo® γδ T cells show no signs of exhaustion after expansion, as evidenced by the lack of expression of PD1, TIM3 or LAG3. ACTallo® is intended to overcome the limitation of autologous cellular therapies towards off-the-shelf approach, by combining the large-scale expansion potential of allogeneic γδ T cells with novel TCRs against XPRESIDENT® mass-spectrometry guided tumor-specific targets.

#3589

Preclinical evaluation of mesothelin-specific T cell receptor (TCR) fusion constructs (TRuC™s) utilizing the signaling power of the complete TCR complex: A new opportunity for solid tumor therapy.

Jian Ding, Ekta Patel, Patrick Tavares, Justin Quinn, Rashmi Choudhary, Bonnie Le, Olga Kiner, Solly Weiler, Daniel R. Getts, Patrick Baeuerle, Robert Hofmeister. _TCR2 Thearpeutics, Cambridge, MA_.

Despite success in treating hematological malignancies, T cells expressing chimeric antigen receptors (CARs) have shown poor efficacy in solid tumor indications. The poor utility of CARs is thought to be due to a number of factors including T cell exhaustion and a lack of persistence. Utilizing only the CD3ζ chain of six distinct T cell receptor subunits in combination with a costimulatory domain, the CARs bypass the natural TCR signaling complex. The failure to initiate and harness a complete TCR response is arguably a primary underlying factor preventing CAR-T cell success in solid tumor indications. Here, we present a novel T cell engineering platform: T Cell Receptor Fusion Constructs (TRuC™s), which target tumors in a MHC non-restricted fashion. Unlike CARs, the constructs integrate into the TCR complex, harnessing the full potential of natural T cell activation, effector function and regulation. Here we demonstrate preclinical evidence underscoring the efficacy of TRuC™-T cells re-programmed to target the solid tumor antigen mesothelin (TC-210). TC-210 showed robust anti-tumor activity in vitro and in vivo in mesothelioma, lung and ovarian tumor models. Surprisingly, this activity was associated with less proinflammatory cytokine production compared to CAR-T cells with the same binder and was supported by strong TRuC™-T cell persistence. Taken together, mesothelin targeting TRuC™-T cells have potent anti-tumor activity and present a promising approach for treating mesothelin-expressing solid tumors.

#3590

Preclinical evaluation of NKG2D-specific T cell receptor (TCR) fusion constructs (TRuCs): A broad spectrum engineered T cell treatment for solid tumors.

Yan Wang, Janani Krishnamurthy, Jian Ding, Ekta Patel, Patrick Tavares, Justin Quinn, Olga Kiner, Solly Weiler, Patrick Baeuerle, Robert Hofmeister, Daniel R Getts. _TCR2 Thearpeutics, Cambridge, MA_.

Chimeric Antigen Receptor (CAR) T cells targeting solid tumor antigens have only yielded marginal results in the clinic. It is believed that poor efficacy is the combinatorial result of suboptimal CAR-T cell trafficking, expansion and and/or persistence as well as tumor antigen heterogeneity. One potential mechanism to alleviate this issue is to generate engineered T cells that target a family of ligands expressed on tumor cells and signal through the T cell receptor (TCR), thereby harnessing the full potential of T cell activation and effector functions. To overcome these barriers, T cells expressing monomeric or dimeric extracellular domain of Natural Killer Group 2D (NKG2D) were fused to the CD3ε subunit. Upon lentiviral transduction of T cells, the T cell receptor fusion construct (TRuC™) is integrated into the TCR to reprogram the T cell specificity to recognize NKG2D ligands. T cells expressing the NKG2D TRuC™ variants (NKG2DL TRuC™-T cells) potently killed tumor cell lines that express one or more NKG2D ligands including ULBP1, ULBP2/5/6, ULBP3, ULBP4, and MICA/B. Furthermore, T cells transduced with the NKG2DL TRuC™ variants shrank tumors in a mesothelioma mouse model. Taken together, NKG2DL TRuC™-T cells show potent preclinical efficacy and support the development of a broad-spectrum TRuC-T cells for cancer therapy to prevent tumor escape due to loss of antigen.

#3591

CSPG4 as innovative target for CAR redirected CIK lymphocytes against soft tissue sarcomas.

Valeria Leuci,1 Ramona Rotolo,1 Chiara Donini,1 Giulia Mesiano,2 Erika Fiorino,1 Loretta Gammaitoni,2 Giovanni Grignani,2 Lorenzo D'Ambrosio,1 Soldano Ferrone,3 Gianpietro Dotti,4 Massimo Aglietta,1 Dario Sangiolo1. 1 _University of Torino, Candiolo Cancer Institute IRCCS, Candiolo, Italy;_ 2 _Candiolo Cancer Institute IRCCS, Candiolo, Italy;_ 3 _Massachusetts General Hospital, Harvard Medical School, Boston, MA;_ 4 _Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC_.

Purpose of our study is to explore the anti-sarcoma activity of cytokine-induced killer cells (CIK) engineered with a chimeric antigen receptor (CAR) against the Chondroitin sulfate proteoglycan 4 (CSPG4). CIK are ex vivo expanded T lymphocytes endowed with MHC-independent tumor killing potential. The underlying hypothesis is to combine and potentiate the innate antitumor activity of CIK with a new anti-sarcoma specificity by CAR redirection against CSPG4. Soft tissue sarcomas (STS) include multiple histotypes and, in advanced stages, remain mostly incurable. A promising approach in the field is represented by adoptive immunotherapy with genetically redirected lymphocytes that require the identification of relevant tumor targets. CSPG4 may be an innovative candidate in such perspective, as it is highly expressed by several malignancies with restricted presence in normal tissues.

Experimental procedures and results. We exploited a patient derived-platform with CIK and tumor cultures derived from patients. CSPG4 was expressed by 8/8 patient-derived STS cell lines (undifferentiated pleomorphic sarcoma n=3; Liposarcoma n=1; GIST n=4). CAR-CIK were generated starting from PBMC (n=3) engineered with a 2nd-generation anti-CSPG4 CAR, including the 4-1BB co-stimulatory domain. CAR expression ranged between 70%-95%, the expansion rate (ranging from 50 to 100 fold) and phenotype (mean CD3CD56=45%; CD8=76%; NKG2D=83%) of CAR-CIK were comparable with parallel unmodified controls. CAR-CIK included higher rates of differentiated central memory and effector memory cells (36% vs 13% and 36% vs 29%, respectively). Anti-CSPG4 CAR-CIK efficiently killed STS in vitro, without differences among STS histotypes. Mean tumor-specific killing was significantly higher compared with unmodified CIK and notably relevant at low effetor/target (E/T) ratios: 95% vs 63% (10:1), 69% vs 31% (1:1), 54% vs 11% (1:4), 38% vs 8% (1:16), 31% vs 5% (1:32), 24% vs 4% (1:64). CAR-CIK activation, upon recognition of STS, led to intense IFNγ release (12,4 ng/ml), 1 log higher compared with unmodified CIK . CAR-CIK, but not unmodified controls, efficiently killed CSPG4-positive STS lacking NKG2D ligands that are naturally resistant to unmodified CIK (n=2). CAR-CIK presented a low cytotoxicity against normal keratinocytes (20% killing at E/T 3:1) that disappeared at lower but therapeutically effective concentrations (2% E/T 1:1, and 0% E/T 1:2).

Conclusions. Our findings support CSPG4 as a new target for adoptive immunotherapy against advanced sarcomas. CAR redirection highly improves the innate tumor killing ability of CIK that may be a favorable platform for the translation of CAR-based strategies into the field of solid tumors, contrasting the possible heterogeneous expression of CAR-targets. The intense sarcoma killing at very low E/T ratios is representative of realistic scenarios and promising in clinical perspective.

#3592

Pharmacological intervention to temporally stimulate or inhibit ICAM-1 targeting CAR-T cells.

Yogindra Vedvyas, Jaclyn McCloskey, Yanping Yang, Irene M. Min, Moonsoo M. Jin. _Weill Cornell Medical College, New York, NY_.

Gene-engineered adoptive T cell therapies have recently been approved by the FDA. We have previously published data utilizing this technology to target intercellular adhesion molecule-1 (ICAM-1), a broad tumor biomarker. Using an affinity modified version of the physiological ligand of ICAM-1,leukocyte function-associated antigen-1 (LFA-1) I-domain, we have shown efficient clearance of a human metastatic anaplastic thyroid cancer (ATC). We were able to visualize and quantify the bio-distribution of CAR-T cells with a PET gene reporter, somatostatin receptor 2 (SSTR2) (Park et. al, Scientific Reports 7, Article number: 14366, 2017). To expand on this platform, we now want to control the activity of our CAR-T cells by exploiting these features using two FDA approved drugs. First, a somatostain analogue, Lanreotide, has been approved for treating Acromegaly and we have recently observed that Lanreotide can activate SSTR2-transduced T cells via calcium release. Second, Lovastatin, a drug clinically used for lowering cholesterol levels, was found to bind the LFA-1 I-domain and inhibit LFA-1 binding to ICAM-1. We hypothesized that in addition to our I-domain CAR-T cell, systemic use of Lanreotide will stimulate CAR-mediated tumor killing while systemic use of Lovastatin will inhibit CAR-mediated tumor killing.

In-vitro testing involved effector to target (E:T) assays performed in the presence and absence of Lovastatin or Lanreotide. ICAM-1(+) ATC target cells or ICAM-1(-) 293T off-target cells were utilized, both of which were transduced with GFP-Firefly Luciferase (GFP/Fluc) for quantification. Effector T-cells derived from healthy donor PBMCs were either transduced with our I-domain CAR-T cell construct that contains SSTR2, or were left nontransduced. Similar assays were translated in-vivo using a previously established solid tumor model with GFP/Fluc ATC ICAM-1(+) cells. Luminescence was used to track tumor progression and regression throughout both in-vivo and in-vitro experiments. [Ga68] DOTA-TOC was administered via IV injections to visualize the bio-distribution of the CAR-T cells using SSTR2 via PET/CT.

Both in-vitro and in-vivo experiments showed Lovastatins ability to compromise I-domain CAR-mediated tumor killing while Lanreotide expedited the rate of I-domain CAR-mediated killing. We report here that Lovastatin inhibits the interaction of our I-domain CAR with ICAM-1 that is present on our target tumor cells and compromises tumor killing. In addition, we have shown a unique phenomena where we can activate SSTR2-transduced CAR-T cells using Lanreotide. The ability to control CAR-T cell activity is an essential tool when addressing adverse effects and/or less than robust treatment outcomes. This will be crucial when translating our adoptive T cell therapy to the clinic.

### Biomarker Discovery 4

#3593

Noncoding RNAs in early detection of radiation-induced late pulmonary effects.

Marshleen Yadav, Feifei Song, Zahida Qamri, Arnab Chakravarti, Naduparambil K. Jacob. _Comprehensive Cancer Center, The Ohio State University, Columbus, OH_.

Normal tissue toxicity is an undesired outcome in patients receiving radiation therapy, manifested as acute radiation syndromes (ARS) and as late effects in survivors. The purpose of the study is to develop biomarkers for assessing the extent of lung inflammation and early detection of delayed and late radiation toxicities such as pneumonitis and pulmonary fibrosis. Circulating miRNAs offer great promise as diagnostic predictive biomarkers due to their high abundance, high stability and sensitivity. A longitudinal analysis of evolutionarily conserved noncoding RNAs was done using NanoString platform in serum samples from leukemia patients and pneumonitis-prone BALB/c mice exposed to clinically relevant doses of whole-body, organ-targeted/protected irradiation to compare the treatment-induced alterations. The associated immune response was evaluated by cytokine multiplexing and gene expression analysis and imaging with micro-CT. Bleomycin and targeted miRNA knockout mice models were used to evaluate functional significance of the identified biomarkers. Quantification of time-dependent alterations in serum and plasma miRNAs from rodents samples collected after thoracic, gut versus whole-body exposure have identified a panel of five miRNAs that are capable of providing an early readout of delayed lung toxicities. The specificity of the candidate miRNAs as potential early indicators of lung injury was confirmed in a bleomycin model of acute lung injury where an overlapping miRNA signature was observed in serum. Among these conserved miRNAs, serum miR-21 and miR-29a were found significantly elevated in two weeks in lung irradiated animals with concomitant increase in proinflammatory cytokines. The significance of the markers was further validated in serum miRNA profile of leukemia patients who exhibited pneumonitis symptoms post radiation therapy. The functional role of miR-21 in promoting radiation-induced inflammation was evident from compromised response of genes associated with inflammation in irradiated miR-21-/- mice. Our study has identified a panel of evolutionarily and functionally conserved sensitive miRNA biomarkers that facilitate rapid and early detection of late pulmonary toxicities in leukemia and lung cancer patients who receive therapeutic radiation. Analysis of circulating biomarkers will potentially assist in making therapeutic and follow-up decision based on individual's own physiologic response and developing strategies for timely mitigation and treatment.

#3594

DEK overexpression in breast epithelial tissue creates a pro-oncogenic environment.

Megan Johnstone, Jon Cheek, Lisa M. Privette Vinnedge. _Cincinnati Children's Hospital Medical Center, Cincinnati, OH_.

Dense epithelial breast tissue, as detected by mammography, is correlated with a four- to six-fold increased risk for developing breast cancer (BC). It is currently not possible to distinguish dense breast tissue from that which will develop into cancer. Therefore, the need to identify novel markers will improve breast cancer screening and reduce expensive overdiagnosis and overtreatment. Previous research has failed to define reliable diagnostic biomarkers that specify dense breast tissue from that which has the possibility to transform into malignant tissue. DEK, a chromatin binding and DNA repair protein, is upregulated in the majority of solid cancers including breast cancer. Previous publications from our lab demonstrate, in vitro, that DEK overexpression promotes proliferation, increased motility, and epithelial polarization in part through canonical WNT/β-catenin signaling. In vivo experiments with a DEK knockout murine model display a mitigated tumor development response as compared to wild-type mice. Given that DEK is highly expressed in over 60% of BC, we generated a conditional mammary epithelial specific doxycycline-regulated Dek overexpressing (DEK OE) murine model. We find that DEK OE leads to dense epithelial breast tissue, largely due to lobuloalveolar hyperplasia. Longitudinal findings demonstrate that DEK OE results in transformation of mammary epithelial tissue with microtumor development. DEK OE tissue sections display increased transcription of Wnt genes, with upregulation of downstream WNT signaling targets such as AXIN and β-catenin. Conversely, longitudinal repression of DEK OE with doxycycline displays normal tissue morphology and organization. Our data suggest that DEK OE stimulates hyperplasia of mammary epithelium, due to increased proliferation, similar to dense breast tissue as detected by mammography, and may increase risk for spontaneous transformation and BC development later in life.

#3595

A panel of serum proteins, metabolite and lipid for prognosing prostate cancer progression.

Michael Kiebish,1 Jennifer Cullen,2 Amina Ali,3 Leonardo O. Rodrigues,1 Emily Y. Chen,1 Eric Milliman,1 Lixia Xang,1 Vladimir Tolstikov,1 Fei Gao,1 Kiki Panagopoulos,1 Jeonifer Garren,1 Yongmei Chen,3 Gyorgy Petrovics,2 Inger L. Rosner,2 Isabell A. Sesterhenn,4 David McLeod,3 Elder Granger,1 Rangaprasad Sarangarajan,1 Alagarsamy Srinivasan,3 Viatcheslav Akmaev,1 Albert Dobi,2 Niven Narain,1 Shiv Srivastava5. 1 _BERG, Framingham, MA;_ 2 _Center for Prostate Disease Research, Uniformed Services University and the Walter Reed National Military Medical Center and John P Murtha Cancer Center, Bethesda, MD;_ 3 _Center for Prostate Disease Research, Uniformed Services University and the Walter Reed National Military Medical Center, Bethesda, MD;_ 4 _Walter Reed National Military Medical Center and Joint Pathology Center, Silver Spring, MA;_ 5 _Center for Prostate Disease Research, Uniformed Services University and the Walter Reed National Military Medical Center and John P Murtha Cancer Center, Framingham, MA_.

Background: Predicting the clinical course of prostate cancer is challenging due to the wide biological spectrum of the disease. The limited prognostic value of pretreatment PSA, grade, and clinical stage prompted discoveries of early biomarkers for predicting the clinical course of the disease. Along these lines new biomarkers have been developed including pre-diagnostic urine-based tests, serum-based assays for PSA derivatives, and diagnostic biopsy tissue-based assays. Although these advancements continue to improve early diagnosis and prognosis there is a need for further developments to complement current prognostic approaches. While most of the prostate cancer detection and prognostic approaches have been developed by using one type of analyte, e.g. mRNA/DNA or proteins, or metabolites, some also include combination of multiple types of analytes. Indeed, development of prognostic panels that include multiple type of analytes requires sensitive and reproducible detection methods and advanced bioinformatic platforms. The objective of our study was to discover prostate cancer prognostic markers employing an advanced multi-analyte discovery platform. Methods: Pre-surgery serum samples were evaluated among a longitudinally followed (median 10 years), racially diverse prostate cancer patient group (N=385) by mass spectrometry, integrating proteomic, metabolomic and lipidomic (multi-omics) data to differentiate disease progression-free patients (N=310) from patients with disease progression (N=75) by using the Bayesian computational approach. Results: Integrated disease progression data with multi-omics profiles have identified the combined predictive performance of two proteins, a metabolite and a phospholipid molecular species with a cumulative performance of AUC= 0.782 for differentiating patient groups with disease progression-free survival from the group of patients with progression. This panel demonstrated a 0.94 negative predictive value with a modest positive predictive value of 0.34. The odds ratio of disease progression was 6.56 (2.98, 14.40) with the panel of markers. Conclusions: We have identified a panel of multi-analytes with promising performance in predicting disease progression-free survival of prostate cancer patients by evaluating pre-surgery serum samples. This panel offers new opportunities complementing current prognostic markers with potential impact on primary treatment and follow up strategies.

#3596

Biomarkers of response to CDK4/6 inhibitor (CDK4/6i) in hormone receptor (HR) positive and HER2-positive breast cancer (BC) patient-derived xenografts (PDX).

Marta Palafox,1 María Teresa Herrera-Abreu,2 Meritxell Bellet,1 Mafalda Oliveira,1 Alejandra Bruna,3 Olga Rodriguez,1 Marta Guzmán,1 Judit Grueso,1 Cristina Vilaplana,1 Joaquín Arribas,1 Emmanuelle di Tomaso,4 Faye Su,4 Carlos Caldas,3 Nicholas C. Turner,2 Rodrigo Dienstmann,5 José Baselga,6 Maurizio Scaltriti,6 Javier Cortés,1 Cristina Saura,1 Violeta Serra1. 1 _Vall D´Hebron Institute of Oncology, Barcelona, Spain;_ 2 _The Institute of Cancer Research, London, United Kingdom;_ 3 _CRUK Cambridge institute, Cambridge, United Kingdom;_ 4 _Novartis Pharmaceutical Corporation, Cambridge, United Kingdom;_ 5 _Vall D´Hebron University Hospital, Barcelona, Spain;_ 6 _Memorial Sloan-Kettering Cancer Center, New York, NY_.

The cell cycle G1-restriction point is frequently deregulated in HR+ BC by alterations of cyclin D1 (CCND1), p16 (CDKN2A) or pRb (RB1). CDK4/6i (ribociclib, abemaciclib and palbociclib) have shown clinical activity in metastatic HR+ BC, both as single agents and in combination with endocrine therapy. Currently, no biomarkers of response to CDK4/6i have been identified beyond HR expression and little is known about mechanisms of acquired resistance.

Twenty-one PDXs were established from HR+, HER2+ or HR+/HER2+ BC patient biopsies and their response to ribociclib was evaluated in vivo and ex vivo in matrigel cultures. Acquired-resistance was generated in vivo by isolating tumors that escaped therapy overtime. In order to identify response biomarkers, genetic and proteomic analysis of PDXs were performed and correlated with ribociclib antitumor activity. Candidates were validated in a cohort of 8 tumor samples from patients treated with abemaciclib monotherapy and in vitro. Combination with the PI3K-alpha inhibitor (PI3Ki) BYL719 was explored in vivo.

In vivo, ribociclib exhibited antitumor activity in five out of 21 PDXs (24%), two of which acquired resistance after continuous dosage (75mg/kg, 6IW). Ex vivo matrigel cultures recapitulated the in vivo response with 75% sensitivity and 92% specificity (p=0.01), providing a novel approach for high throughput screening. Baseline levels of ER, PR and Ki67 protein or PIK3CA/ESR1 mutations did not discriminate between ribociclib-resistant/sensitive PDXs, whereas CCND1/D2-amplification/overexpression were only found in ribociclib-resistant models. Importantly, sensitive PDXs exhibited significant Ki67 reduction upon ribociclib treatment, higher baseline pRb- and lower p16-staining compared to ribociclib-resistant PDXs (p=0.004, 0.02 and 0.03, respectively). Three out of 8 acquired-resistant tumors (37.5%) exhibited pRb loss. In vitro, RB1 knockdown and cyclin D1/D2-overexpression resulted in higher BrdU incorporation and higher IC50 than control cells upon ribociclib treatment. p16 expression was significantly lower in samples of patients exhibiting clinical benefit with abemaciclib monotherapy (p=0.04). Remarkably, combination of ribociclib with a PI3Ki resulted in appreciable antitumor activity in 18 out of 20 PDXs (90%), including two models resistant to fulvestrant given in combination with ribociclib.

In conclusion, HR+, HER2+ and HR+/HER2+ BC PDXs expressing both high Rb- and low p16-protein levels are sensitive to CDK4/6i whereas deregulation of the G1-restriction point due to low pRb or high cyclin D1/D2 protein levels is associated with resistance to ribociclib monotherapy. Addition of a PI3Ki markedly improves the antitumor response of ribociclib in most of PDXs, suggesting that the PI3K pathway may play a pivotal role in limiting the efficacy of CDK4/6 inhibition.

#3597

Xenobiotic detoxification enzymes and cancer risk: A multiplex immunofluorescence detection of AE1/AE3, GSTA1 and GSTA3 in the tissue microarrays of gastrointestinal tract.

Victor Wan-San Ma, Goretti Hoi-Yan Cheung, Eunice Yuen-Ting Lau, William Chi-Shing Cho. _Queen Elizabeth Hospital, Kowloon, Hong Kong_.

Background: Glutathione S-transferases (GSTs) are an enzyme family responsible for the metabolism of toxins and carcinogens in the phase II detoxification pathway. Among the 5 alpha class isoforms, GSTA1 and GSTA3 are involved in metabolizing several active carcinogenic substrates generated from benzo-[a]-pyrene mainly found in grilled meats and burnt coal. The expression of GSTA1 and GSTA3 in gastrointestinal (GI) tract may be implicated with cancer development associated with the intake of xenobiotic substances.

Methods: Formalin-fixed and paraffin-embedded tissue cores of esophagus (190), stomach (180), small intestine (372), colon (120) and rectum (142) were stained with a multiplex tyramide-immunofluorescence (T-IMF) technique for the simultaneous demonstration of cytokeratin AE1/AE3, GSTA1 and GSTA3. The tissue types range from normal to cancer progression spectrum. All tissue cores were screened for AE1/AE3 expression to confirm the epithelial cell lineage before scoring for GSTA1 and GSTA3 positivity. A p-value <0.05 by Chi-squared test is considered significant.

Results: T-IMF is useful in studying the interaction of multi-markers in a single slide. This technique is superior to chromogenic immunohistochemical application in multi-color presentation. This highly sensitive and handy technique will save multiple sectionings and precious tissues. We found a significant down-regulation of two alpha class GSTs in the malignancies of GI tract (Table 1). | |  | |  | |  | |  | |

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

Table 1. Positive percentages of glutathione S-transferases markers

Marker | Esophagus | Stomach | Small intestine | Colon | Rectum

N | M | N | M | N | M | N | M | N | M

GSTA1 | 41% | 24% | 99% | 40% | 90% | 15% | 22% | 3% | 43% | 21%

p-value | 0.0603 | <0.0001 | <0.0001 | 0.001 | 0.0194

GSTA3 | 78% | 35% | 99% | 54% | 96% | 64% | 100% | 39% | 88% | 40%

p-value | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001

Abbreviation: N= normal; M= malignant

Conclusion: The differential expression of these detoxification enzymes in normal epithelium vs GI cancer may shed some light on their role in protecting normal tissue from cancer development. This finding may provide a potential target for the development of intervention strategies against GI cancer. Further investigations will be conducted to decipher the mechanism underlying this observation.

#3598

Classification of the collagen ECM in normal human tissue as a biomarker for future breast cancer incidence.

Ryan J. Gigstad, Tianjie Wang, Yifei Liu, Menggang Yu, Yuming Liu, Adib Keikhosravi, Kevin W. Eliceiri, Patricia J. Keely, Matthew W. Conklin. _University of Wisconsin, Madison, WI_.

Because we do not know who will develop breast cancer, or who will have a relapse of cancer following surgery, there is an urgent need for the development of biomarkers that can be detected specifically in normal, nondiseased tissue. For 30 years there has been a search for the genetic markers that will predict risk of developing breast cancer, but even while family history is a known risk factor, only 10% of breast cancer risk can be specifically linked to a genetic mechanism (BRCA-1, BRCA-2). Furthermore, individuals who are at high risk due to genetics or family history do not always develop breast cancer. Breast density is another indicator of future incidence, as high-density tissue is correlated with a 4-6 fold increased risk of developing cancer. We hypothesized that a population analysis of the properties of the individual collagen fibers that comprise the extracellular matrix (ECM) in nondiseased breast tissue is a biomarker for the onset of disease. We used multiphoton second-harmonic generation (SHG) imaging to generate high-contrast images of the collagen ECM that can be gathered from common histologic slides that require no additional staining. Our goal was to determine the range of heterogeneity in the properties of the collagen matrix in normal samples. We imaged samples from 141 normal patient donors to comprise 4 different cohorts that define this ground truth state of the matrix. The cohorts were from donors who had never been diagnosed with breast cancer; who had previously been diagnosed with breast cancer but now were disease free; who had never been diagnosed with breast cancer at the time of tissue donation but later went on to develop breast cancer; and those who had donated tissue while in remission from an initial breast cancer diagnosis but later went on to have a recurrence of breast cancer. The SHG images of each patient sample were then analyzed using the curvelet transform-based ctFIRE and CurveAlign software platforms to generate data (fiber length, width, straightness, density, angle with respect to a boundary of epithelial ducts/lobules, and the relative alignment of fibers to each other) on each individual collagen fiber. Advanced statistical measurements and principal component analysis were used to classify the nature of each of the cohorts. Based on our measurements of the structure and organization of collagen fibers, we found that while measurements within a cohort were consistent, there were unique attributes of the collagen matrix that defined each cohort. Furthermore, we performed regression analysis of our measurements against the standard clinical features (age, race, BMI, etc.). Because our various cohorts delineate the nondiseased, involuted, precancer, and prerecurrence normal matrix, respectively, we feel that these data serve as a highly useful, novel classifier, one that describes the clinical impact of the timbre of the matrix in nondiseased women.

#3599

Novel and established acute myeloid leukemia (AML) subsets correlate with ex vivo sensitivity to BCL-2 and PI3K inhibition.

Teiko Sumiyoshi,1 Chris Bolen,1 Jeff Tyner,2 Steve Kurtz,2 Uma Borate,2 Shannon McWeeney,2 Beth Wilmot,2 Dan Bottomly,2 Erik Segerdell,2 Monique Dail,1 Timothy R. Wilson,1 Wan-Jen Hong,1 Jeffrey M. Venstrom1. 1 _Genentech, Inc., South San Francisco, CA;_ 2 _Oregon Health & Science University, Portland, OR_.

Anti-apoptotic BCL-2 family proteins contribute to AML pathogenesis and chemotherapy resistance. Venetoclax (VEN) monotherapy has limited activity in relapsed/refractory AML with a 19% overall response rate. PI3K pathway is constitutively active in 50-80% of AML pts and is tightly linked to the apoptotic pathway. Using clinical, genomic and ex vivo drug sensitivity (IC50) data from 802 AML pts from the multi-institutional Beat AML 1.0 collaboration, we tested the hypothesis that aberrant activation of the PI3K/FLT3 pathway is a mechanism of VEN resistance, with potential utility for identifying pt subsets likely to respond to novel drug combinations. IC50 values were determined by ex vivo drug sensitivity assay, and RNAseq analysis was performed on Illumina HiSeq 2500. To identify pts with activated PI3K signaling, we derived a novel 24-gene expression signature from genes enriched among PIK3CA-mutant tumors, and calculated a PI3K signaling score by applying singular value decomposition to the expression value matrix. Drug response biomarkers were evaluated using a student's t-test without adjustments for multiple comparisons. 45.2% (161/356) of AML pt samples were sensitive to VEN (median IC50, 1.64μM), 70% (190/272) to taselisib (tas), a selective PI3Kα and δ but β-sparing inhibitor (median IC50, 0.06μM) and 95.8% (115/120) to VEN-tas combination (median IC50, 0.02μM). Response to PI3K inhibition varied; 23% (150/659) of AML samples were sensitive to PI3Kδ inhibitor idelalisib (idela; median IC50, 7.23μM) and 63% (337/533) to pan-PI3K inhibitor pictilisib (pic; median IC50, 0.45μM; p=0.03 vs tas). 92% (47/51) of VEN-resistant AML samples tested were sensitive to VEN-tas. VEN sensitivity correlated with high BCL-2 expression, and was greatest among pts with both high BCL-2 and low MCL-1 expression or low PI3K signature. Low expression of PIK3CD (but not PIK3CA/B/G) and the PI3K signature were associated with VEN sensitivity. High BCL-2 expression was associated with resistance to pic, idela and FLT3 inhibitor quizartinib (quiz), with a weaker trend for tas that did not reach statistical significance. Activities of tas, pic and quiz were significantly greater among AML samples with FLT3-ITD mutations, a previously reported VEN resistant subgroup. Expression of activated PI3K signature was higher among AML samples sensitive to pic and quiz. Among 30 AML samples refractory to tas, 18 (60%) were sensitive to VEN and 28 (93%) to VEN-tas. In summary, we identified novel AML subsets characterized by either high gene expression of an activated PI3K gene signature that are refractory to BCL-2 inhibitors or by high BCL-2 expression associated with resistance to PI3K/FLT3 inhibitors. 95.8% of AML pt samples were sensitive to combined inhibition of PI3K and BCL-2. Dual inhibition of PI3K and BCL-2 apoptotic pathways may be a promising combination and should be evaluated in clinical trials.

#3600

Early detection of esophageal adenocarcinoma using near-infrared lectin-based imaging.

Andre A. Neves,1 Massimiliano Di Pietro,2 Maria O'Donovan,3 Dale J. Waterhouse,1 Sarah E. Bohndiek,1 Kevin M. Brindle,1 Rebecca C. Fitzgerald2. 1 _University of Cambridge Cancer Research UK Cambridge Research Inst., Cambridge, United Kingdom;_ 2 _Medical Research Council Cancer Cell Unit, Cambridge, United Kingdom;_ 3 _Cambridge University Hospitals, Cambridge, United Kingdom_.

Background: Long procedure times and sampling error frequently limit the efficacy of endoscopic surveillance for Barrett's esophagus (BE). Near-infrared (NIR) fluorescence imaging minimizes optical scattering and tissue autofluorescence. We assessed, in an ex vivo setting, the feasibility of using a topically applied NIR dye-labeled lectin for the detection of early neoplasia in BE.

Methods: We recruited consecutive patients undergoing endoscopic mucosal resection (EMR) for BE-related early neoplasia. EMR specimens were freshly collected and sprayed at the bedside with a fluorescent lectin and then imaged ex vivo. A maximum of two punch biopsies were collected from each EMR under NIR light guidance. Fluorescence intensity from dysplastic and nondysplastic areas within EMRs were compared and from punch biopsies with different histological grades.

Results: We analyzed 29 EMR specimens from 17 patients. Dysplastic regions showed significantly lower fluorescence across whole EMR specimens (P < 0.001). A 41% reduction in fluorescence was found for dysplastic compared to nondysplastic punch biopsies (P < 0.001), with a sensitivity and specificity for dysplasia detection of 80% and 82.9%, respectively.

Conclusion: NIR imaging using fluorescently-labeled lectins can distinguish dysplastic from nondysplastic Barrett's mucosa ex vivo. We are currently planning a first-in-human study to validate this imaging platform.

#3601

Targeting USP7/PTEN axis regulates the metastatic competency of bone marrow-resident melanoma cells.

Monika Vishnoi,1 Debasish Boral,1 Haowen N. Liu,1 Marc L. Sprouse,1 Wei Yin,1 Michael A. Davies,2 Isabella C. Glitza Oliva,2 Dario Marchetti1. 1 _Houston Methodist Research Inst., Houston, TX;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Recurrence is the major cause of melanoma death due to cell dissemination from primary/metastatic tumor (CTCs). During asymptomatic periods, these cells reside in bone marrow (bone-marrow resident tumor [BMRT cells]) and remain quiescent. We hypothesized that disseminated tumor cells survive in bone marrow (BM) during these periods and evolve to metastatic potency during disease progression. First, we isolated CTC-enriched, Lin-neg population from clinically advanced melanoma patients and performed biomarker expression and mutational profiling to confirm presence of putative CTCs. Lin-neg CTC population contained unique transcriptomics signature with elevated melanoma markers expression (BAGE, MAGEA1, B4GALNT1, S100A3). Further, downstream IPA analysis demonstrated that an upregulation of transcripts for genes involved in cell survival and pro-development functions with concomitant decrease in cell proliferative and inflammation properties. Second, we implanted Lin-neg population in murine xenograft models and isolated HLA+/ Melan-A+ cells population from blood and BM at endpoint. Expression of human (HLA-ABC) and melanoma markers (Melan-A, S100, NG2, CD146) on BMRTCs and CTCs was confirmed by employing: (a) immunofluorescence staining, (b) single-cell DEPArrayTM and CellSearchTM capture, (c) genomic profiling, and (d) organ-site colonization of CTCs with concomitant to BMRTCs in BM. Third, we discovered distinct transcriptomic signature of BMRTCs vs CTCs by differential gene expression profiling. Subsequent pathway analysis showed top five altered canonical pathways in BMRTCs: protein ubiquitination pathway, EIF2, actin cytoskeleton, systemic lupus erythematosus and hypoxia signaling. Of note, a strikingly elevated expression of PTEN in BMRTCs was detected. Fourth, because PTEN binds to USP7, a key component of protein ubiquitination pathway, we evaluated USP7/PTEN axis in CTC-driven BMRTCs modulated metastatic competency. The use of two clinically approved USP7 inhibitors to study their effects on the proliferative capacity of BMRTCs led to a significant reduction of CTCs at metastatic sites. This study provides critical insights to identify biomarkers of melanoma recurrence during the asymptomatic periods of the disease, fostering application of USP7 inhibitors for innovative melanoma therapies for patients with metastasis undetectable disease and/or yet to develop metastasis.

#3602

Validation and implementation of next-generation sequencing in liquid biopsies by a novel NGS platform: Focus on non-small cell lung cancer.

Atil Bisgin,1 Ozge Sonmezler,2 Ibrahim Boga2. 1 _Cukurova University Faculty of Medicine Department of Medical Genetics, Adana, Turkey;_ 2 _Cukurova University AGENTEM (Adana Genetic Diseases Diagnosis and Treatment Center), Adana, Turkey_.

Introduction: Liquid biopsy as a novel and non-invasive test for the surveillance of cancer is rapidly growing, by next-generation sequencing (NGS) technologies with multi-gene panels.

Objective: The aim of this study was to describe the validation and implementation of a cell free tumor DNA (cfDNA) from the patients with non-small cell lung cancer (NSCLC), to sequence for 19 genes and copy number variation analyses for 5 genes via a novel NGS platform; GeneReader NGS System.

Materials and Methods: We performed the complete NGS workflow with modifications, and sequenced the cfDNA extracted from 100 NSCLC patients. The multi-gene panel includes 19 lung cancer related genes (AKT1, ALK, BRAF, DDR, ERBB2, ESR1, KIT, KRAS, MAP2K1, NRAS, NTRK, PDGFRA, PIK3CA, PTEN and ROS) to sequence and additional copy number variations of 5 genes (RICTOR, EGFR, MET, FGFR1 and ERBB2) that all were selected to represent the most actionable genes for the clinical oncology and molecular diagnostics. The sequencing was performed using a novel NGS platform (GeneReader NGS System, Qiagen).

Results: The validation process and the implementation of genetic testing from the cfDNA by this novel NGS system have been utilized for the clinical use. After quality assessment, the data from this NGS system was bioinformatically processed and a tertiary analysis was performed using QCI-Analyze and QCI-Interpret bioinformatics tools. The data was then reviewed on alterations and those reported in different mutation databases. The clinically relevant alterations were identified and reported due to the testing indications. Overall, actionable variants in EGFR, ALK, KRAS, PIK3CA, MET, FGFR1 and ERBB2 genes were detected and reported in 62 % of all NSCLC patients while the majority were in EGFR.

Conclusions: For each of the 100 samples tested thus far, at least one variant was detected showing the high sensitivity and specificity of both the multi-gene panel and this novel NGS platform. Our evaluation yielded good results for the actionable multi-gene panel selected and provides a comprehensive NGS analysis and workflow for this new platform.

#3603

Metabolic profiles distinguish breast cancer progression in African American women.

Delisha A. Stewart,1 Wimal W. Pathmasiri,1 Susan L. McRitchie,1 Lance Buckley,2 Tammey J. Naab,2 Robert L. DeWitty,3 Vikisha T. Fripp,3 Estelle Cooke-Sampson,2 Desta A. Beyene,2 Luisel Ricks-Santi,4 Robert L. Copeland,2 Susan J. Sumner,1 Yasmine M. Kanaan2. 1 _UNC-Chapel Hill, Kannapolis, NC;_ 2 _Howard University Cancer Center, Washington, DC;_ 3 _Providence Hospital, Washington, DC;_ 4 _Hampton University, Hampton, VA_.

Breast cancer (BCa) is one of the most common malignancies in women and the incidence, distribution, clinical outcome and mortality rates vary widely among women of different ethnic backgrounds. Because tissue from African American (AA) women is difficult to obtain for biomarker studies, new population-relevant biomarkers that enable earlier detection and novel therapeutic intervention development are critical. To identify new biomarkers and targets that have the potential to be leveraged for earlier detection, classification of disease progression and development of improved therapeutics for AA patients, plasma and tissue samples were selected from two AA BCa case and control cohorts at the Tissue, Plasma and Clinical Bank at the Howard University Cancer Center (HUCC). Samples were from women either diagnosed with BCa, screened for potential BCa lesions or undergoing reduction mammoplasty surgery at both Howard University and Providence Hospitals. Samples were analyzed by untargeted metabolomics using 1H nuclear magnetic resonance (NMR) spectroscopy. Multivariate and statistical analyses determined bins important to differentiating BCa by progressive stage and grade from control reduction mammoplasty tissues and fibrocystic fibroadenoma. Significant bins were library-matched to identify corresponding metabolites and distinguish common and unique metabolites between tissue groups and compare tissue profiles to plasma samples. Metabolites from each study group were also correlated with other known clinicopathologic BCa risk factors, including age, BMI, and smoking status, to determine their influence on disease progression. Several metabolites were found to distinguish nonmalignant reduction mammoplasty tissues from fibrocystic fibroadenomas, and from Grade (G) I-II estrogen receptor (ER)-positive or GI-II ER-negative tumors and GIII ER-positive or GIII ER-negative tumors. For example, in three comparisons using orthogonal partial least squares discriminant analyses (OPLS-DA) between reduction mammoplasty, fibrocystic fibroadenomas and the malignant tissues, we found 8 unique metabolites when comparing reduction mammoplasty versus fibrocystic fibroadenomas (4-hydroxybenzoate, dimethylamine, formate, glutamine, glutathione, histidine, methionine and UDP-N-acetylglucosamine); 2 unique metabolites comparing reduction mammoplasty versus GI-II (ER-positive and -negative) tumors (2-phenylpropionate and succinate); and 6 unique metabolites comparing reduction mammoplasty versus GIII (ethanolamine, glycine, hypoxanthine, maltose, sucrose and uridine). Our results demonstrate the continued usefulness of metabolomics-based research and the potential for these findings to identify early detection or disease staging biomarkers in a population that experiences a disparate burden of this disease.

#3604

Centrosome amplification in epcam-captured circulating tumor cells from metastatic cancer patients.

Ashok Singh, Ryan A. Denu, Jamie M. Sperger, Beth A. Weaver, Mark E. Burkard, Joshua M. Lang. _Univ. of Wisconsin-Madison, Madison, WI_.

Background: Genomic instability and evolution of cancers is linked to chemotherapy sensitivity and resistance in patients with metastatic cancer. Taxane-based chemotherapies operate by promoting chromosomal instability (CIN) by increasing multipolar mitoses. Breast tumors with CIN are more sensitive to paclitaxel compared to tumors without CIN. There is a critical need for new biomarkers based on biological evaluation of mechanisms of response and resistance in metastatic cancer. Although CIN is difficult to detect directly, centrosome amplification (CA) is a common mechanism of CIN. In this study, we develop an assay to detect CA in circulating tumor cells (CTCs) as a minimally invasive assay to evaluate sensitivity to taxane-based chemotherapies.

Methods: Blood samples from patients with metastatic breast and prostate cancer were collected and processed for nucleated cells using a Ficoll gradient and magnetic depletion of CD45-positive cells. EpCAM-positive CTCs were captured using an exclusion-based sample preparation technology known as the VERSA (Versatile Exclusion-based Rare Sample Analysis) platform. CTCs were defined as cells positive for Hoechst, cytokeratin and negative for multiple immune and endothelial cell markers (CD45/CD11b/CD14 and CD34). EpCAM-captured CTCs were also stained with pericentrin and centrin to identify CA and evaluated for pericentrin (whole centrosome marker) and centrin (centriole marker) protein-staining intensity, number and size.

Results: CA was detected in a subset of CTCs from patients with metastatic breast and prostate cancer patients. As a control, centrosomes were also analyzed in matched peripheral blood mononuclear cells from the same patients. CA was heterogeneous with 100% of patients demonstrating some degree of CA (>4 centrioles), ranging from 15-48% of CTCs.

Conclusion: In conclusion, we report the presence of CA in EpCAM-captured CTCs from metastatic cancer patients. Evaluation of CA in CTCs using pericentrin and centrin staining may serve as a predictive biomarker of taxane based chemotherapy response and resistance. We are recruiting taxane-naïve metastatic cancer patients to test the hypothesis that CA and/or CIN predict taxane sensitivity.

#3605

Next generation sequencing from cerebral spine fluid yields actionable targets in leptomeningeal carcinomatosis.

Louisa von Baumgarten,1 Anna Reischer,1 Jörg Kumbrink,2 Andreas Jung,2 Sibylle Liebmann,2 Steffen Ormanns,2 Klaus Metzeler,1 Julian Holch,1 Volker Heinemann,1 Thomas Kirchner,2 Benedikt Westphalen3. 1 _University Hospital LMU Munich, Munich, Germany;_ 2 _LMU Munich, Munich, Germany;_ 3 _Comprehensive Cancer Center Munich, Munich, Germany_.

Background and Rationale: Leptomeningeal carcinomatosis is a cause of major morbidity and mortality in many solid malignancies. Therapeutic options are limited and include radiation therapy as well as intrathecal administration of cytotoxic agents. Accordingly, novel diagnostic and therapeutic modalities are needed in order to improve the prognosis of patients diagnosed with leptomeningeal carcinomatosis. Liquid biopsies are safe and offer great potential to define actionable targets in a variety of cancers. However, a role for liquid biopsies from cerebral spine fluid (CSF) has not been defined to date.

Patients and Methods: CSF samples from ten consecutive patients diagnosed with leptomeningeal carcinomatosis were collected as part of clinical routine and evaluated by next generation sequencing. All cases were discussed in the Molecular Tumorboard (MTB) as part of the "Molecular Diagnostics and Therapy" unit at the Comprehensive Cancer Center Munich.

Results: Samples from patients diagnosed with cholangiocarcinoma (1), ewing's sarcoma (1), breast (5), lung (2) and gastric cancer (1) were submitted for panel sequencing. Sequencing was technically successful in nine out of ten patients and circulating tumor DNA (ctDNA) was found in all of these nine patients. Of note, ctDNA was also detectable in cases in which microscopy did not reveal malignant cells in the CSF sample (4/10 cases). Tumorigenic alterations were found in seven out of these nine patients. Importantly, NGS of cerebral spine fluid detected previously unknown and therapeutically relevant alterations in this cohort. Case discussion in the Molecular Tumorboard led to therapeutic recommendations in three cases and targeted therapy was ultimately initiated in one case.

Conclusions: Next generation sequencing from cerebral spine fluid is feasible in clinical practice and yields tumorigenic alterations in a large fraction of patients. Importantly, our approach demonstrated the therapeutic relevance of this approach in patients with leptomeningeal carcinomatosis.

#3606

Circulating tumor cells and tumor-derived extracellular vesicles as a possible marker for tumor response and survival in patients with non-small cell lung cancer treated with immunotherapy.

Menno Tamminga,1 Sanne de Wit,2 Joost F. Swennenhuis,3 T. Jeroen N. Hiltermann,1 Ed M. Schuuring,1 Leon W.M. Terstappen,3 Harry J. Groen1. 1 _University Medical Centre Groningen, Groningen, Netherlands;_ 2 _University of Twente, Enschede, Netherlands;_ 3 _University Twente, Enschede, Netherlands_.

Background: Lung cancer remains the number one cause of cancer related death worldwide. Immune modulating therapy have shown long lasting survival, however, they are only effective in about 20% of patients. Although PD-L1 expression predicts tumor response, it is not a robust predictor. Circulating tumor cells (CTCs) or tumor derived extracellular vesicles (tdEV) may be another tumor response predictor. CTCs have been associated with survival in NSCLC. Our hypothesis is that CTC or tdEV changes predict early tumor response and it is an early marker of treatment efficacy.

Methods: Patients with proven advanced NSCLC referred to our center for immunotherapy by PD-L1 or PD-1 antagonists were eligible for inclusion. CellSave tubes were drawn at start of therapy (T0) and after 4 weeks of treatment (T1). EpCAM expressing CTCs were detected by the CellSearch system. EpCAM expressing tdEV which are present in the blood during CTC enumeration, were counted in the images from the CellTracks Analyzer, which were analyzed with the open source program ACCEPT. Response to therapy was measured by the response evaluation criteria in solid tumors (RECIST) and according to clinical evaluation of the treating physician. Patients were divided in groups based on response to therapy and presence of CTCs. Cox regression analyses were used to study the association between CTCs and tdEVS with both OS and PFS.

Results: 114 patients have been included (104 T0 samples, 71 T1 samples, 52 both a T0 and T1 sample). 53 patients have progressive disease (PD), 24 stable disease (SD), 23 partial response (PR) and 2 a complete response; 12 could not be evaluated yet. CTCs were present in 33 T0 samples (32%), and 20 T1 samples (28%). When CTCs were present at T0, patients had no response to therapy in 83% of cases, while patients without CTCs had no response in 68% of cases. Patients with increased CTC counts at T1 responded in 9% of the cases, versus 33% in patients with decreased CTC counts. CTCs were significantly correlated to survival at both time points independently (T0, HR=1.06, p=0.042; T1, HR=1.04, p=0.009). Comparing patients with a decrease of CTCs with those with an increase, Cox regressions showed a HR of 8.9 (p=0.041), while using the difference of CTCs between T1 and T0 as a continuous variable in the cox regression gave a HR of 1.41 (p=0.02).tdEVS were present in 90% of cases (median 7, range 0-1752)at T0 and in 94% at T1 (median 5, range 0-1975). Cox regression showed a significant correlation with survival of tdEVs at T0 (HR=1.0, p=0.004) and T1 (HR=1.0, p=0.009) and the change in tdEVs counts between T0 and T1 (HR=1.01, p=0.04)

Conclusion: CTC and tdEV decrease in the first weeks of immunotherapy was associated with tumor response, whereas an increase in CTC was associated with no response to immunotherapy, indicating a possible use as an early marker.

#3607

Quantitative dimensions of the PAM50 in breast tumors are prognostic and predict paclitaxel response.

Nicola J. Camp,1 Michael J. Madsen,1 Jesus H. Valera,2 Eva Carrasco,2 Rosalia Caballero,2 Miguel Martin,3 Philip S. Bernard1. 1 _University of Utah, Salt Lake City, UT;_ 2 _GEICAM, Madrid, Spain;_ 3 _Hospital General Universitario Gregorio Marañón, Universidad Complutense, Madrid, Spain_.

Standard of care for women with locally advanced breast cancer is aggressive chemotherapy with a combined anthracycline-taxane regimen. Multiple randomized clinical trials have shown survival benefit to adding a taxane, e.g., paclitaxel; however, this can cause serious toxicities such as peripheral neuropathy. Identifying patients who benefit from taxane would improve survival outcomes, reduce toxicities and allow future randomization to more effective agents. So far, gene expression signatures have proven prognostic in the adjuvant chemotherapy setting but not predictive to particular regimens. We recently described 5 breast tumor dimensions using principal components (PC1-5): orthogonal quantitative tumor traits capturing the majority of expression variance in the PAM50 genes. Here we evaluate these dimensions and disease-free survival (DFS) in 819 patients in the Grupo Español para la Investigación del Cáncer de Mama (GEICAM) trial 9906, which evaluated fluorouracil, epirubicin and cyclophosphamide (FEC) vs. FEC followed by weekly paclitaxel (FEC-P). To investigate prognosis, we first ignored trial arm. PC1 was significantly associated with DFS in a Cox regression analysis (p=6.7x10-7), outperforming other predictors including nodal status, tumor size, grade, ER/PR/HER2/ki67 status, age at diagnosis and intrinsic subtype. PC1 remained significant in multivariate Cox regression adjusted for nodal status, tumor size and grade (p=8.7 x 10-4). Modeled as quintiles in a Kaplan-Meier (KM) analysis, PC1 was not only significant (p=3.7 x 10-6) but quantitatively prognostic, with the proportion surviving to 4 years decreasing for each quintile: 91%, 88%, 79%, 71%, 66% for low to high PC1 quintiles, respectively. To investigate response we examined interactions between dimensions and trial arm. Interactions were seen for PC3* arm (p=0.05) and PC4* arm (p=0.05). KM curves showed women with low-PC3 tumors responded to the addition of paclitaxel (p=0.006, 85% survival on FEC-P vs 72% FEC at 4 years), but women with high-PC3 tumors did not (p=0.94, 81% FEC-P, 82% FEC). Survival rates suggest that cases with high-PC3 were FEC super-responders, gaining no benefit from paclitaxel. Response to paclitaxel was seen for low-PC4 tumors (p=0.003, 89% FEC-P, 75% FEC), but not high-PC4 tumors (p=0.92, 70% FEC-P, 71% FEC); cases with high-PC4 tumors were poor responders to either treatment. Combining orthogonal traits PC3 and PC4 defined 44% of women (high-PC3 or high-PC4) with no benefit from paclitaxel (both arms 76% at 4 years), and 56% with lower PC3/PC4 tumors and substantial survival gain (p=0.002, 90% FEC-P, 73% FEC). In conclusion, breast tumor dimension PC1 shows great promise for quantitative prognostics. Dimensions PC3 and PC4 predict paclitaxel response, in particular identifying a large proportion of women (44%) who may be overtreated with paclitaxel, a drug class currently part of standard of care.

#3608

Molecular fingerprint sequencing for minimal residual disease detection in breast cancer.

Iñaki Comino-Mendez,1 Ros Cutts,1 Isaac García-Murillas,1 Neha Chopra,1 Maria Afentakis,1 Abigail Evans,2 Duncan Wheatley,3 Anthony Skene,4 Simon Russell,5 Mohini Varughese,6 Mitch Dowsett,1 Ian E. Smith,7 Nick Turner1. 1 _The Institute of Cancer Research, London, United Kingdom;_ 2 _Poole General Hospital, Dorset, United Kingdom;_ 3 _Department of Oncology, Royal Cornwall Hospitals NHS Trust, Truro, United Kingdom;_ 4 _Royal Bournemouth Hospital, Bournemouth, United Kingdom;_ 5 _Hinchingbrooke Hospital, Hinchingbrooke Park, Huntingdon, United Kingdom;_ 6 _The Beacon Centre, Musgrove Park Hospital, Taunton and Somerset NHS Foundation Trust, Taunton, United Kingdom;_ 7 _Royal Marsden Hospital, London, United Kingdom_.

Introduction: The substantial majority of breast cancers present with early-stage disease, although micro-metastatic disease may be established at the time of diagnosis and may ultimately result in metastatic disease recurrence. Prior studies have shown that analysis of circulating tumour DNA (ctDNA) can detect micro-scopic minimal residual disease (MRD) serving as biomarker to anticipate future cancer recurrence, although sensitivity of current assays is limited. We present a novel molecular fingerprint ctDNA assay that exploits tumour whole genome sequencing (WGS) to develop highly personalised assays.

Methods: WGS was conducted on paired tumor and germline DNA from 11 early breast cancer patients to identify tumour specific small indels (SIs) to track in plasma as individual molecular fingerprints. Patient-specific amplicon-based panels were designed and validated to identify somatic reporters with no sequencing background. DNA was extracted from plasma samples taken at pre-surgery (baseline), post-surgery, and every 3 months until 1 year and every 6 months thereafter, and sequenced to 100,000X.

Results: Molecular fingerprint assays were demonstrated to detect less than a single cancer genome, with undetectable background in control samples. Using plasma samples from patients with early stage breast cancer, we detected ctDNA in 80% of the baseline plasma time-points with 100% specificity (95% confidence limits (CI) 98.7%-100%). ctDNA was detected in all patients prior to relapse, with an observed lead-time of 15.5 months, substantially improved compared with previous techniques, detecting residual disease that was not detectable by tracking mutations using digital PCR.

Conclusions: This proof-of-principle study demonstrates that tumor-specific molecular fingerprints combined with ultra-deep plasma DNA sequencing have the potential to transform MRD detection. Identifying those patients who have undetectable recurrence would pave the way for identifying who is cured by surgery alone and who requires further therapy.

#3609

Differential gene expression is associated with response to chemoradiation and relapse-free and overall survival in rectal adenocarcinoma.

Michael S. Lee,1 Cheryl Carlson,1 Benjamin F. Calvo,1 Bert H. O'Neil,2 Dante S. Bortone,1 Benjamin G. Vincent1. 1 _University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 2 _Indiana University, Indianapolis, IN_.

Background: Neoadjuvant chemoradiation is a standard therapy for stage II-III rectal adenocarcinoma, and the degree of pathologic response observed upon resection informs prognosis. However, there is a need to identify novel biomarkers of response to chemoradiation and survival after chemoradiation, particularly using modern next-generation sequencing methods.

Methods: We prospectively collected pretreatment endoscopic tumor biopsies from 43 patients with stage II-IV rectal adenocarcinoma prior to neoadjuvant chemoradiation with concurrent fluoropyrimidine. Tumor samples were fresh frozen, and subsequently RNA was extracted, paired end libraries for mRNA sequencing (RNASeq) were prepared using TruSeq RNA Access library prep kits (Illumina), and samples were sequenced on Illumina HiSeq. Differentially expressed genes were determined using DESeq2 and Ingenuity pathway analysis (Qiagen) was performed. Additionally, the association between "claudin-low"-like gene sets established in breast and bladder cancers and clinical outcomes was determined.

Results: Among the 36 patients with adequate RNA quality, 7 had a pathologic complete response (pCR) and 29 did not, with 22 differentially expressed genes with false discovery rate (FDR) <0.1 between the two cohorts, including EME2 (2.0-fold change, FDR p-value 0.00627). At 5 years of follow-up, 11 patients had known relapse and 11 were known to be relapse-free, with 33 differentially expressed genes with FDR<0.1 between the two cohorts. Pathway analysis demonstrated that p53 signaling and Wnt/β-catenin signaling pathways were associated with relapse, while dendritic cell maturation and Toll-like receptor signaling pathways were associated with relapse-free survival. Rectal cancers with greater expression of a "claudin-low"-like signature had significantly inferior relapse-free survival (HR 1.79, 95% CI 1.07-3.00) and overall survival (HR 1.91, 95% CI 1.17-3.12) on univariate Cox proportional hazards model.

Conclusions: There are multiple differentially expressed genes associated with response to neoadjuvant chemoradiation in rectal adenocarcinoma. EME2, which forms an endonuclease that cleaves stalled replication forks, was one of the most differentially expressed genes overexpressed in patients with pathologic complete response and thus is a rational target for further investigation. Extension of "claudin-low" gene expression signatures to rectal cancers may serve as a new prognostic biomarker. Further investigation into the association of gene expression subtypes and responses to neoadjuvant chemoradiation is warranted.

#3610

CANCER ID and the identification of circulating tumor cells (CTCs) in NSCLC.

Linda Scarrott,1 Hanny Musa,1 Harriet Wikman,2 Sonja Loges,2 Juergen Braunger,1 Klaus Pantel,2 Ralph Graeser1. 1 _Boehringer Ingelheim, Biberach, Germany;_ 2 _Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany_.

The IMI-funded CANCER-ID consortium (https://www.cancer-id.eu/) was created in order to facilitate the use of Circulating Tumor Cells (CTCs) and other liquid biopsies (ctDNA, miRNA, exosomes) in the monitoring and treatment of cancer, via establishing and validating protocols for their detection and characterization. CTCs are tumor cells that have passively or actively migrated from the bulk of the primary or metastatic tumor(s) into the peripheral blood stream. While some of these cells may establish new metastases and mediate outgrowth of the disease, they can also serve as diagnostic, prognostic or potentially predictive biomarkers, providing non-invasive access to tumor material and information on tumor evolution. However, a major challenge remains in the identification of these very rare cells (1:106-8 WBCs). In the past years, a plethora of methods have been developed to isolate CTCs, via antibody enrichment/depletion strategies, size exclusion and/or based on density and dielectric properties. Yet despite this progress, the detection rate of CTCs in NSCLC (non-small cell lung cancer) is still very low (only ca. 20-30% of metastatic NSCLC patients have detectable CTCs). In stark contrast, SCLC (small cell lung cancer) patients have usually hundreds of detectable CTCs. A potential reason for the lack of detection of NSCLC CTCs may be the choice of surface markers used to detect the CTCs. Using NSCLC cell lines spiked into healthy donor blood, novel antibody cocktails, including positive and negative selection markers, and also markers for cells undergoing epithelial-mesenchymal transition were tested for their sensitivity and specificity to detect tumor cells in blood. Blood samples from NSCLC and SCLC patients are currently being analyzed using the Cell Search system (an EpCAM-based, FDA approved device - the current 'gold standard' for CTC detection and enumeration), in parallel with the Parsortix device (a micro-fluidic platform that isolates CTCs according to size and deformability). This direct comparison permits an understanding of whether the combination of the Parsortix device with downstream novel antibody cocktails for CTC identification improves sensitivity, over Cell Search, while still retaining good selectivity. Results from this ongoing study will be presented. The goal of this project is to generate and clinically validate a protocol that would enable the use of CTCs as a liquid biopsy for NSCLC patients. Such an approach would be of particular benefit to these patients, since tumor biopsies cannot always being readily attained and the use of invasive intervention carries some risk of bleeding and infection. This work is supported by IMI JU & EFPIA (grand no. 115749).

#3611

Serum DCLK1 levels are elevated in melanoma patients and it is a novel predictive marker for survival and response.

Dongfeng Qu, Nathaniel Weygant, Parthasarathy Chandrakesan, Kamille Pitts, Randal May, Sripathi Sureban, Adam Asch, Alexandra Ikeguchi, Courtney Houchen. _Univ. of Oklahoma Health Sciences Ctr., Oklahoma City, OK_.

Melanoma is the deadliest form of skin cancer, and the incidence of melanoma has been rising for the last 40 years. There is a paucity of readily available tools to predict for melanoma progression and response to therapy, leaving an unmet medical need to identify predictive and prognostic biomarkers. Doublecortin-like kinase 1 (DCLK1) is overexpressed and marks a population of tumor-initiating cells in colonic and pancreatic cancers. It regulates key oncogenes, pluripotency factors, angiogenic factors, and epithelial mesenchymal transition (EMT) related transcription factors. In this study, we evaluated whether DCLK1 can be detected in the bloodstream and if its levels in serum samples could be quantitatively assessed in melanoma patients. ELISA assay was performed to detect DCLK1 levels in the serum samples of melanoma patients before and after treatment with either targeted agents or immune checkpoint inhibitors. Western blotting was also performed to detect DCLK1 protein in the serum samples of melanoma patients. Immunohistochemistry (IHC) analysis was performed to detect DCLK1 in the same patients' tumor tissues. Additionally, analysis of DCLK1 and correlative gene expression profiles was performed using TCGA Melanoma dataset. Here we report that the intensity of DCLK1 staining in melanoma tumor tissues is increased compared to normal tissues. DCLK1 levels in the serum were elevated in melanoma patients (n=20) compared to healthy volunteers. Next, we compared DCLK1 levels in 10 patients for whom pre- and post- treatment samples were available. Among these patients, seven had elevated DCLK1 before treatment and had a significant decrease (about 30-50%) in serum DCLK after treatment. Three patients had no change in DCLK1 levels after treatment. The patients with a decrease in their level of DCLK1 post-treatment demonstrated radiologic response to therapy as well, while the patients with no change in DCLK1 level post therapy showed either no improvement or progression (p<0.002). These data suggest that reduction in serum DCLK1 levels following therapy for melanoma was associated with clinical response, and the absence of change in DCLK1 after therapy was associated with poor response. Analysis of the TCGA dataset demonstrated that melanoma patients with high levels of DCLK1 had worse overall survival (p<0.05) compared to the patients with low levels of DCLK1, suggesting that DCLK1 RNA levels could be used as a prognostic biomarker. These data taken together suggest that DCLK1 has potential utility as a novel predictive and on-treatment biomarker for melanoma prognosis and therapeutic response.

#3612

Elevated TGFa levels in chronic mylogenous leukemia (CML) patient plasma measured by a highly sensitive single molecule array immunoassay.

Ahmed Chenna,1 Li Cai,2 Christos Petropoulos,1 John W. Winslow1. 1 _Monogram Biosciences_Laboratory Corporation of America Holdings, South San Francisco, CA;_ 2 _Center for Molecular Biology and Pathology_Laboratory Corporation of America Holdings, Research Triangle Park, NC_.

Introduction: Recent reports suggest that cytokine and growth factor profiling are prognostic in myelodisplasias and CML. TGF-α and IL-6 plasma levels selectively identified CML patients failing to achieve an early molecular response (EMR), or progressed on imatinib therapy (Nievergall et al, 2016). In patients failing to achieve EMR, the median TGFα concentrations were in the low pg/mL range while IL-6 levels were sub-pg/mL or undetectable. Thus, to accurately and reproducibly measure TGFα and IL-6 levels in blood as prognostic or predictive markers of imatinib response, there is a need for highly sensitive immunoassays. Here we demonstrate the utility of using a digital immunoassay to measure sub-pg/mL levels of TGFα and IL-6 in CML plasma samples assessed for BCR-ABL fusion transcripts.

Methods: Single molecule array immunoassays (Simoa) for TGFα and IL-6 were performed on the HD-1 platform (Quanterix), qualified based on precision, reproducibility, linearity, sensitivity according to kit specifications. CML blood samples were obtained from CMBP, LabCorp, Inc, following BCR-ABL e1b2, b2b2, and b3b2 transcript fusion testing by RT-PCR.

Results: TGFα and IL-6 concentrations in the plasma of 77 CML patients were determined and compared to levels in healthy controls. TGFα in CML plasma was elevated and ranged ~20-fold (6.13-118.6 pg/mL), in contrast to the low levels and narrow range in healthy controls (range = 1.11-3.17 pg/mL). Median TGFα levels were 10-fold higher in CML patient plasma (median = 18.8 pg/mL) relative to healthy controls (median = 1.8 pg/mL; p <0.0001). IL-6 levels ranged broadly in CML plasma (0.12-12.6 pg/mL) relative to healthy controls (0.3-1.04pg/mL), though median levels were not significantly different (CML = 0.61 vs 0.69, p = 0.873). TGFα and IL-6 concentrations were not significantly correlated. TGFα levels appear elevated in CML samples expressing all 3 BCR-ABL transcripts, however, a larger number of samples enriched in fusion transcripts are being assessed to confirm possible correlations.

Conclusions: We have qualified novel highly sensitive single molecule array immunoassays to measure TGFα and IL-6 in the plasma of CML patients and healthy controls, and found significantly elevated levels of TGFα in CML plasma. These results support further investigation of TGFα and IL-6, using sensitive immunoassays, as potential prognostic and predictive markers of CML disease progression and treatment outcome.

#3613

**Lab-on-a-chip-based** in-vitro **functional profiling proves to be effective in predicting therapy outcome in AML patients.**

Laura Rocchi,1 Andrea Faenza,1 Viviana Guadagnuolo,1 Laura Rambelli,1 Giovanni Marconi,2 Maria Chiara Fontana,2 Martina Pazzaglia,2 Cristina Papayannidis,2 Giorgia Simonetti,2 Nicola Pecorari,1 Luca Giulianelli,1 Dario Biscarini,1 Marco Bettelli,1 Michele Federici,1 Rita Ruggiano,1 Giovanni Martinelli,3 Roberto Guerrieri,2 Massimo Bocchi1. 1 _CellPly srl, Bologna, Italy;_ 2 _University of Bologna, Bologna, Italy;_ 3 _Seragnoli Institute of Hematology, Bologna, Italy_.

Functional profiling is an emerging trend in precision medicine. Manual laboratory methods proved to be effective in predicting the response to drug treatments both in patients stratified according to genetic profiling and in patients without any specific molecular aberration. In this work, we validated the predictive power of an innovative functional profiling platform, based on lab-on-a-chip technology, which allows testing the response of live tumor cells exposed to anticancer drugs in a fully-automated process, requiring only a Ficoll stratification as a manual step.

15 AML patients (Age: 40% <60 years, 46% in the 60-70 years range, 14% >70 years; Gender: 40% Female; ELN Classification: 20% Favorable, 26% Intermediate-I, 33% Intermediate-II, 14% Adverse; AML stage: 53% De novo, 7% Relapse, 40% Refractory; Treatment: 20% FLAI-3, 40% FLAI-5, 26% Decitabine; 7% Cytarabine, 7% 5-azacitidine) were treated at the Policlinico Sant'Orsola in Bologna. At the same time, bone marrow samples were challenged with the same drugs at scaled concentrations in the functional profiling platform developed by CellPly and an output score was extracted from time-lapse fluorescence image analysis of patient tumor cells exposed to the drug or drug combination for 24 hours. Clinical follow up resulted in the following classification: complete hematologic response (CR) was found in 6/15 patients (6/6 with a De Novo disease), while the remaining 9/15 patients (2/9 de novo; 1/9 Relapse; 6/9 Refractory) were classified as stable disease (SD).

In-vitro functional profiling of 14 patients provided a correlation with the clinical outcome (p=0.01) irrespective of the stage of the disease. 100% of the patients identified as responders by functional profiling (n = 5), resulted in a CR outcome. 89% of the patients identified as non-responders (n = 9) resulted in an SD outcome.

The same patient samples were also investigated for the molecular assessment of the most relevant genes commonly analyzed in AML patients. FLT3-ITD mutation was found in 2/14 patients, both in SD. Negative FLT3 was not correlated to clinical outcome. FLT3-TKD mutation was found in 3/14 patients, 2/3 resulted in CR, 1/3 in SD. TP53 mutation was found in 1/13 patients, classified as SD. Intronic variant rs1625895 was found in 6/13 patients equally distributed in CR and SD. NPM1 mutation was identified in 4/13 patients, 2/4 resulted in CR, 2/4 in SD indicating no direct correlation. IDH2 was found in 1/11 patients (with R172K mutation) with an SD.

As a conclusion, functional profiling proved to be highly correlated with clinical outcome irrespective of patient stage and demonstrated a superior predictive power compared to molecular profiling.

#3614

Evaluation of a commercial targeted NGS panel for tumor mutation burden assessment in FFPE tissue.

Peng Fang,1 Zhenyu Yan,1 Quyen Vu,2 David Smith,2 Chad Galderisi,2 Cynthia S. Spittle,1 Jin Li1. 1 _MolecularMD Corp., Cambridge, MA;_ 2 _MolecularMD Corp., Portland, OR_.

Checkpoint inhibitors have been approved for the treatment of solid tumor and hematological malignancies. While significant responses have been observed in a subset of patients, outcomes are variable and there is a need to identify additional predictive biomarkers beyond PD-L1 levels as measured by IHC. Tumor mutation burden (TMB) has been correlated with response to checkpoint inhibitors and is emerging as a key biomarker for predicting checkpoint inhibitor response. So far, the methods used to assess tumor mutation burden have included exome sequencing and multiple laboratory-developed targeted NGS panels (e.g., FoundationOne and MSK-IMPACT). In order to fully determine the value of TMB as a predictive biomarker for immunotherapy, a standardized panel, workflow and data analysis pipeline for TMB assessment are needed. In this study we evaluated the performance of a commercially available targeted NGS panel and workflow for TMB analysis.

A set of 30 FFPE tumor samples including colon, renal, gastric, endometrial, and lung tissues was analyzed. DNA and RNA were extracted using the RecoverAll Total Nucleic Acid Isolation Kit. DNA quantity and quality were assessed using Qubit and qPCR, respectively.

Samples were analyzed with the ThermoFisher Oncomine™ Mutation Load Research Assay (TML), which evaluates tumor mutation load (mutations/Mb) by interrogating 409 cancer-related genes, spanning ~1.7 megabases of the genome. TMB was measured by counting somatic single-base substitutions per Mb at ≥10% allele frequency in single, non-matched, tumor DNA samples. The impact of DNA quality on the TMB score was evaluated. Deamination errors (i.e. G>A and C>T) in poor quality FFPE samples was found to cause the overestimation of TMB. Therefore, a delta Ct cutoff was established to qualify DNA samples for TMB analysis. 12 of the 30 samples were also analyzed using a comparator NGS panel covering ~1.25 megabases. The correlation of TMB results between the two panels was 0.87. Overall, TMB was lowest in RCC (9-17/Mb) compared to NSCLC and CRC (16-37/Mb). MSI status was determined using the Promega MSI Analysis System v1.2. A correlation was observed between TMB and MSI status in a subset of samples. Reproducibility of the assay was also evaluated. To identify clinically relevant mutations and genetic alteration associated with high mutation burden, the Oncomine Comprehensive assay v3 (OCAv3) was also used to analyze the sample set. Mutations in genes involved in several DNA repair pathways were found to correlate with TMB.

This study demonstrated the feasibility of utilizing a commercial targeted NGS panel and data analysis pipeline for TMB evaluation in clinical FFPE tumor samples. Standardization of TMB analysis will enable the clinical validation of TMB as a predictive biomarker for therapy selection.

#3615

Analysis of microRNAs in cerebrospinal fluid of brain tumor patients.

Jiri Sana,1 Alena Kopkova,1 Marek Vecera,1 Jaroslav Juracek,1 Tana Machackova,1 Parwez Ahmad,1 Natalia Anna Gablo,1 Pavel Fadrus,2 Marek Svoboda,3 Ondrej Slaby1. 1 _Masaryk Univ., Brno, Czech Republic;_ 2 _University Hospital Brno, Brno, Czech Republic;_ 3 _Masaryk Memorial Cancer Institute, Brno, Czech Republic_.

Introduction: Cerebrospinal fluid (CSF) is a body fluid with many important functions that is in direct contact with the extracellular environment of the central nervous system (CSN). Therefore, CSF serves inter alia as a both communication channel allowing the distribution of various substances among CNS cells as well as a reservoir of waste products that these cells released. For these reasons, CSF is a potential source of diagnostic biomarkers of many neurological diseases including brain tumors. Recent studies have revealed that CSF contains also circulating microRNAs (miRNAs), short non-coding RNAs, which have been described as biofluid diagnostic markers in many cancers. The analysis of CSF miRNAs in patients affected by brain tumors could enable more precise diagnosis and patient stratification.Material and methods: We performed next-generation sequencing analysis of small RNAs (small RNAseq) in 89 CSF samples taken from 35 GBM, 14 low-grade glioma (LGG), 14 meningioma, and 8 brain metastasis patients as well as 18 non-tumor donors. Informed consent approved by the local Ethical Commission was obtained from each patient before the lumbar puncture. cDNA libraries were prepared using CleanTaq Small RNA Library Prep Kit (TriLink BioTechnologies) and, subsequently, purified by Agencourt AMPure XP (Beckman Coulter). The final sequencing analysis was performed by Next 500/550 High Output v2 Kit - 75 cycles using the NextSeq 500 instrument (both Illumina). For miRNA mapping and analysis, an online tool Chimira was used. Obtained data were subsequently statistically evaluated in the environment of statistical language R using the Bioconductor edgeR and DESeq2 package.Results: We observed 211 miRNAs to be expressed in more than 60 CSF samples with 1 395 normalized counts in average (min 3, max 77 284; miR-486-5p was excluded from analysis due to its artificial expression). From these miRNAs, 63 showed different levels in CSF of GBM, 16 in LGG, 24 in meningioma, and 31 in brain metastases patients in comparison with CSF of non-tumor donors. MiR-10a-5p and miR-196a-5p have significantly highest levels in CSF of GBM samples. Moreover, 6 miRNAs (miR-30c-5p, miR-30e-5p, miR-145-5p, miR-320c, miR-3960, and miR-6131) indicated significantly different levels between GBM and LGG (P<0.01).Conclusion: We have demonstrated that CSF of various brain tumors (GBM, LGG, meningioma, and brain metastasis) is characterized by specific miRNA signature. Moreover, we found miRNA signature with ability to differentiate LGG and GBM from CSF. Our results suggest, that after independent validations, CSF miRNAs could serve as promising biomarkers in brain tumors. This work was supported by Ministry of Health of the Czech Republic, grant nr. 15-34553A, 15-33158A, 15-31627A, 15-34678A, 16-31314A, 16-31765A and by grant of Czech Grant Agency nr. 16-18257S, 17-17636S.

#3616

Low-pass whole-genome sequencing of single circulating tumor cells for detection of tumor mutation burden and chromosomal instability across multiple cancer types.

Angel Rodriguez, Jerry Lee, Ramsay Sutton, Rhett Jiles, Gordon Vansant, Yipeng Wang, Mark Landers, Ryan Dittamore. _Epic Sciences, San Diego, CA_.

Background: Recent studies show tumor mutation burden (TMB) is a biomarker for patient responses to immune checkpoint inhibitors whereas chromosomal instability (CIN) is a marker for PARP inhibitors drug response. Assessment of TMB and CIN in bulk tumor samples is well explored but limited by sample accessibility and tumor heterogeneity. Analysis of ctDNA is challenging for TMB and CIN, especially for patients who harbor subclonal genomic alterations, limiting its clinical utility. Epic Sciences' CTC platform employs a non-enrichment based approach to provide insight into subclonal heterogeneity. Here we present a single cell genomics assay for detection of TMB and CIN from individual CTCs by low pass whole genome sequencing.

Methods: Contrived samples were prepared by spiking prostate cancer cell lines LNCaP, PC3 and VCaP into healthy donor blood. Red blood cells were lysed, nucleated cells deposited onto glass slides and immunofluorescently stained (DAPI, CK, CD45 and androgen receptor). Identified cancer cells were individually isolated from the slides, lysed, whole-genome amplified (WGA), shotgun library prepared, and low pass whole genome sequenced to ~ 0.1X coverage. Data were analyzed for TMB and large-scale transitions (LST, a surrogate of CIN). Microsatellite instability (MSI) was measured by the Qiagen Type-It Microsatellite PCR kit as per manufacturer's protocol on the control cell lines. 1,047 CTCs from 108 prostate, breast, colorectal, bladder and lung cancer patients were evaluated for clinical feasibility.

Results: TMB scores from LNCaP (average 181) were significantly higher than PC3 (127), VCaP (132), and healthy donor WBC (94). MSI confirmed LNCaP was MSI-H (high) with INDELs detected in 3 of 4 microsatellite sites, whereas PC3 was MSI-L (low) and VCaP was MSS (stable). Although with lower MSI, PC3 (average 33) and VCaP (33) had higher LST scores vs. LNCaP (11). A wide range of TMB (21-480) and LST scores (0-67) was observed in patient samples. TMB cutoff of 175 was set based on cell line TMB scores, and an LST cutoff of 10 was set based on WBC data. Across CTCs from all cancer types and stages, 117 (11.2%) CTCs were TMB-H/LST-L (H: high; L: low), 479 (45.7%) CTCs were TMB-L/LST-H, 31 (3.0%) CTCs were TMB-H/LST-H, and 420 (40.1%) CTCs were TMB-L/LST-L. A significant lower incidence of TMB-H/LST-H CTCs (3.0%) was observed by Fisher's exact test (p<0.0001).

Conclusions: These data demonstrate the feasibility of detecting TMB and CIN simultaneously at the single cell level using the Epic Sciences CTC Platform. Inter- and intrapatient heterogeneity was observed in this large patient cohort. Although MSI and HRD are were mutually exclusive driver events in single cells, patients often had both subclonal populations. Studies are ongoing to investigate the potential correlations of TMB with checkpoint inhibitor and CIN with PARPi response.

#3617

Prediction of irinotecan and flurouracil efficacy in metastatic colorectal cancer by early measurement of circulating tumor DNA.

Iben Lyskjær,1 Camilla J. Kronborg,2 Mads H. Rasmussen,3 Boe S. Sørensen,4 Mona Rosenkilde,5 Michael Knudsen,1 Søren R. Krag,6 Karen-Lise G. Spindler,7 Claus L. Andersen1. 1 _Department of Molecular Medicine (MOMA), Aarhus University Hospital, Aarhus N, Denmark;_ 2 _Department of Oncology, Aarhus University Hospital, Denmark, Aarhus N, Denmark;_ 3 _Department of Molecular Medicine (MOMA), Aarhus University Hospital, Denmark, Aarhus N, Denmark;_ 4 _Department of Clinical Biochemistry, Aarhus University Hospital, Denmark, Aarhus N, Denmark;_ 5 _Department of Radiology, Aarhus University Hospital, Aarhus N, Denmark;_ 6 _Department of Pathology, Aarhus University Hospital, Aarhus N, Denmark;_ 7 _Department of Experimental Clinical Oncology, Aarhus University Hospital, Aarhus N, Denmark_.

Background: Chemotherapy resistance in metastatic colorectal cancer (mCRC) is a major challenge, and development of biomarkers for determining therapy efficacy is vital to ensure optimal palliative treatment. Recently, quantification of changes in circulating tumor DNA (ctDNA) levels has attracted substantial attention as a predictive marker. Here, we provide evidence that ctDNA has the potential to be used as an early marker of therapeutic efficacy of irinotecan and fluorouracil (FOLFIRI).

Experimental Procedures: Patients diagnosed with mCRC, and with indication for first-line combination chemotherapy, were prospectively enrolled in this phase II study. Blood was drawn at baseline, day 7, 14, 21, 40 and then monthly until progression. Response evaluation was done at eight-week intervals by CT scanning using RECIST criteria. Chemotherapy was administered at day one and two every two weeks. The levels of cell-free DNA (cfDNA) and ctDNA were determined using sensitive digital droplet PCR assays. ctDNA assays were designed either to mutations identified by targeted sequencing or on basis of known tumor mutations detected in primary or metastatic lesions using a panel of mutation-specific qPCR based assays (KRAS, NRAS, BRAF).

Results: Twenty-four patients were enrolled. A KRAS, NRAS or BRAF mutation was observed in tumor tissue of 16 patients. In 12 of these the mutations were confirmed in baseline samples and used for assessing ctDNA. For the remaining 12 patients, a mutation for ctDNA quantification was identified by targeted tumor sequencing. Patients with high baseline ctDNA levels (75th centile) had significantly shorter progression-free survival (PFS) than patients with low baseline ctDNA levels. After first treatment cycle all patients experienced a decline in ctDNA levels demonstrating initial response to treatment; however, the reduction in ctDNA was not associated with prolonged PFS or overall survival (OS). Conversely, change in ctDNA levels from first to second cycle of treatment (three weeks after treatment start) predicted FOLFIRI efficacy. Patients with increasing ctDNA levels at this time point had a significantly shorter PFS and a shorter OS than patients with stable or decreasing ctDNA levels.

Conclusion: In agreement with the literature, we show that baseline tumor DNA levels predicted PFS. The initial observed decline in ctDNA levels upon administration of FOLFIRI indicates that all tumors had cancer cells sensitive to treatment; however, after elimination of these, resistant clones expanded, leading to treatment resistance. Importantly, our study demonstrates that FOLFIRI efficacy can be evaluated as early as three weeks after treatment initiation by measurement of ctDNA levels. This enables early detection of treatment failure and allows for rapid adaptation of the treatment strategy.

#3618

Gene mutation and protein co-expression in melanoma and colorectal cancer by BRAF V600 mutation status.

Matthew A. Carnell,1 Rebecca Feldman,2 Michael B. Atkins,3 Wafik S. El-Deiry,4 Michael J. Pishvaian,3 Mohamed E. Salem,5 Ari M. VanderWalde1. 1 _West Cancer Center, Memphis, TN;_ 2 _Caris Life Sciences, Phoenix, AZ;_ 3 _Georgetown University Hospital/Lombardi Comprehensive Cancer Center, Washington, DC;_ 4 _Fox Chase Cancer Center, Philadelphia, PA;_ 5 _Levine Cancer Institute, Carolinas HealthCare System, Charlotte, NC_.

BRAF V600 oncogenic mutations are targetable in the treatment of melanoma but are more difficult to treat in colorectal cancer (CRC). The difference in the biological profile of BRAF V600 mutant melanoma and CRC compared to BRAF V600 wildtype melanoma and CRC has not been established. 5,139 tumor samples (CRC, 4007 and Mel, 1132) submitted for IHC (protein expression), ISH (gene amplification), and NGS sequencing of between 44 and 592 genes between 2009 and 2015 at a CLIA-certified laboratory (Caris Life Sciences, Phoenix, AZ) were retrospectively studied. Of these, 270 colorectal (6.7%) and 334 melanoma (29.5%) samples had BRAF V600 mutations. Genes or proteins with more than 100 samples in each BRAF wildtype and mutant group were tested for co-expression or co-mutation in each malignancy and correlations with a p-value <0.01 were reported. The rate of co-mutations/expression in BRAF V600 mutant melanoma compared to BRAF wildtype melanoma was lower for 5 genes: KRAS (0% vs 3%), NRAS (0% vs 37 %), HNF1A (1% vs 2%), c-KIT (1% vs 7%), and GNAQ (0% vs 4%), and 3 proteins: MGMT by IHC (23% vs 31%), PTEN by IHC (64% vs 81%), and TOPO1 by IHC (44% vs 53%). The rate of co-mutations/expression in BRAF V600 mutant CRC compared to BRAF wildtype CRC was lower for 3 genes: KRAS (0% vs 52%), NRAS (0% vs 4%), and APC (26% vs 61%), and 2 proteins: MLH1 (66% vs 98%), PMS2 (65% vs 97%); and higher for 10 genes: HNF1A (7% vs 1%), JAK2 (2% vs 0%), MPL (1% vs 0%), AKT1 (3% vs 1%), EGFR (4% vs 1%), SMARCB1 (2% vs 0%), ATM (8% vs 4%), STK11 (3% vs 1%), PTEN (8% vs 3%), BRCA2 (18% vs 9%), and 5 proteins: EGFR (77% vs 60%), PD-1 (61% vs 42%), PD-L1 (10% vs 2%), RRM1 (63% vs 44%), and TS (58% vs 32%), and MSI by fragment analysis (35% vs 4%). In melanoma, all 8 proteins and gene mutations with significantly different expression were expressed less frequently in BRAF V600 mutants compared to wildtype. However, in CRC, 16 of 21 gene mutations and proteins were expressed more frequently among BRAF V600 mutants compared to wildtype. HNF1A was expressed more frequently in BRAF mutant CRC than in wildtype CRC but less frequently in BRAF mutant melanoma compared to BRAF wildtype melanoma. BRAF V600 mutations are associated with a biological profile that is distinct from BRAF V600 wildtype in both melanoma and colorectal cancer. Furthermore, BRAF mutant melanoma tends to have different genes and proteins co-mutated/expressed than colorectal cancer which supports previous findings suggesting differential activation of protective feedback loops. These differences in expression of gene mutations and proteins may play a role in the different responses to therapies between BRAF V600 mutant melanoma and BRAF V600 mutant colorectal cancer.

#3619

The role of CXCR4 in progression of malignant glioma.

Mingli Liu. _Morehouse School of Medicine, Atlanta, GA_.

High grade malignant gliomas are devastating, uniformly fatal cancers for which no effective therapies exist. Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. Recent studies have shown that cytokines/chemokines can regulate many malignant features of tumor cells. CXCL12/CXCR4 axis activates various signaling pathways that promote chemotaxis, adhesion and migration, cell proliferation and survival. PI3 kinase, Ras, stress-activated protein kinase (SAPK)/c-Jun Nterminal kinase (JNK), phospholipase C (PLC)/mitogen-activated protein kinase (MAPK), p38 MAPK and AKT are all downstream effectors of CXCL12/CXCR4 axis, through which tumor cell growth, dissemination and migration are facilitated. To enhance our understanding of the development and progression of glioma which may lead to novel and useful biomarkers and/or therapeutic targets in the context of GBM, we analyzed the role of CXCR4 in glioma using the published data and literature. (1).To determine the prognostic role of CXCR4 in malignant glioma, we analyzed the microarray data from Rembrandt Databases by Affymetrix HG U133 v2.0 Plus on 523 glioma patients. Kaplan-Meier analysis of overall survival showed that glioma patients with high CXCR4 expression (n=329) have short overall survival compared to those with low CXCR4 expression (n=54), logrank test: P=2.2e-8 9. Among GBM, high CXCR4 expression (n=178) have short overall survival compared to those with low CXCR4 expression (n=9), logrank test: P=0.044. Among Astrocytoma, high CXCR4 expression (n=102) have short overall survival compared to those with low CXCR4 expression (n=33), logrank test: P=0.0247 (http://www.betastasis.com/glioma/rembrandt/kaplan-meier_survival_curve/). (2).To determine the relationship between glioma progression and chemokine receptor CXCR4, we made a meta-analysis based on 14 published articles. All 14 eligible studies having 805 patients from China and USA were included. These studies showed that CXCR4 expression in glioma was correlated to high WHO grade glioma (OR=8.26, 95% CI=3.42-19.93, P<0.00001). Our results indicate that CXCR4 is a predictive marker for malignant glioma progression and survival. Suppression of CXCR4 inhibited proliferation, migration, and invasion of malignant human gliomas indicating that CXCR4 may represent a novel and promising target for therapeutic intervention of malignant glioma.

#3620

Histopathological associations with PTEN and ERG status in prostate biopsies from men treated with radiation.

Onur Ertunc, Mark Markowski, Phuoc T. Tran, Amol Narang, Jessica Hicks, Daniel Song, Jiayun Lu, Elizabeth A. Platz, Theodore Deweese, Angelo M. De Marzo. _John Hopkins Univ. School of Medicine, Baltimore, MD_.

Loss of the PTEN tumor suppressor gene in prostate cancer, which usually occurs by genomic deletion, is associated with adverse outcome. Recent work (PMID:23888040) suggested that PTEN activity is also required for maintaining genomic integrity and its loss may be associated with resistance to DNA damaging agents, such as ionizing radiation. We assembled a series of patients with intermediate to high grade clinically localized prostate cancer who underwent external beam radiation as their primary treatment. We obtained and regraded all positive biopsy cores according the the new Grade Grouping system for Gleason scores (PMID: 26492179) and performed analytically and clinically validated IHC assays for PTEN (associated with PTEN genomic alterations) and ERG (associated with the TMPRSS2-ERG gene fusion) on all positive tissue cores from a given biopsy that were contained in up to 5 separate FFPE blocks (n=589 positive cores, n positive cores ranged from 1 to 13, with median of 2 and mean of 3) from 193 patients. PTEN and ERG status were correlated with the new Grade Groups as well as other histopathological features, including the presence of cribriform architecture and intraductal carcinoma. Patients were 66% White, and 27% African American, and median age was 68.5. Grade Group designations (taken as the highest Grade group in the case) were as follows (Group 1 = 27%, Group 2 = 30%, Group 3= 15%, Group 4 = 18%, Group 5 = 11%). Overall there was at least some PTEN loss in 36% of cases. The presence of any PTEN loss in a given case was strongly associated with Grade Group (P<0.001, chi square), in that 17% of patients with Grade Group 1 showed any PTEN loss and 77% of patients with Grade Group 5 showed any PTEN loss. As seen in prior studies, PTEN loss was frequently heterogeneous in that only 11% of cases overall showed complete loss of PTEN in all tumor tissue examined. Homogeneous PTEN loss was also significantly associated with Grade Groups since 3.8% of cases of Grade Group 1 showed such loss and 27% of cases of Grade Group 5 showed such loss (P=0.024, chi square). PTEN loss was also associated with cribriform morphology (P<0.001) and the presence of intraductal carcinoma of the prostate (P<0.001), both known independent prognostic factors. Having any PTEN loss was also associated with ERG gene fusions since 22% of cases showed PTEN loss that were negative for ERG and 52% of cases showed PTEN loss in cases positive for ERG (P<0.000). As also previously found, the rate of PTEN loss was higher in samples from White patients than African Americans (complete PTEN loss = 3.9% in African Americans and 14% in White patients). The presence of any ERG positive tumor was also higher in tissues from White patients (54%) compared to African Americans (29%) (P=0.003). Additional analyses are underway regarding associations between PTEN and ERG status and outcomes after radiation therapy.

#3621

High-plex immune marker spatial profiling quantitation by NanoString Digital Spatial Profiling technology and quantitative immunofluorescence.

Maria I. Toki,1 Pok Fai Wong,1 Harriet Kluger,1 Yuting Liu,1 Chris Merritt,2 Giang Ong,2 Sarah Warren,2 Joseph M. Beechem,2 David L. Rimm1. 1 _Yale Univ. School of Medicine, New Haven, CT;_ 2 _NanoString Technologies, Seattle, WA_.

Background: Quantitative immunofluorescence (QIF) offers the advantage of multiple target measurement on a single slide, but is limited by the number of fluorescence channels. NanoString's Digital Spatial Profiling (DSP)* can detect and quantify immune markers at higher multiplex with spatial resolution within specific regions of interest on FFPE tissue. Here, we compare NanoString DSP to automated QIF (AQUA), for immune marker compartment specific measurement. Additionally, we assess their agreement on the prognostic value of commonly used biomarkers in Non-Small Cell Lung Cancer (NSCLC). Finally, we explore the predictive value of a 30-plex panel of immune markers on two cohorts of treated patients.

Methods: NanoString DSP technology uses a cocktail of primary antibodies conjugated to indexing DNA oligos. Regions of interest (ROI) on the tissue are selected with fluorescently labeled antibodies, and oligos from that region are UV cleaved and quantified on the nCounter platform. The comparator for this technology was the AQUA method of QIF. We retrospectively examined a NSCLC cohort of over 100 patients for prognostic markers and two treated patient cohorts for associations between immune markers expression and response to treatment, all in tissue microarray format. The treated cohorts included an Epidermal Growth Factor Receptor Tyrosine kinase inhibitor (EGFR TKI) treated NSCLC cohort and a melanoma immunotherapy (ITx) treated cohort.

Results: Multiple immune markers (CD3, CD4, CD8, CD20, PD-L1) were assessed and a high correlation was found between NanoString DSP counts and QIF scores, when the measurements were performed in the same ROIs (tumor or stroma). The prognostic value of the immune markers tested was concordant between the two assays with high expression of CD3 assessed by either assay showing a statistically significantly better overall survival (OS). For the EGFR TKI treated NSCLC cohort none of the immune markers was correlated with response to treatment or favorable outcome. However, in the ITx treated melanoma patients, PD-L1, PD-1, CD68, CD3, CD8A and b2-microglobulin tumor expression predicted response to treatment. Each marker also predicted better outcome, but only CD8A was an independent predictive marker of prolonged survival.

Conclusion: NanoString DSP offers the capacity of highly multiplexed immune marker measurements on selected compartments. It shows high concordance with AQUA, which was further validated by comparing prognostic significance. The pilot study of the Melanoma ITx cohort also illustrates the potential to simultaneously evaluate a range of markers and possibly construct new predictive signatures based on a cohort represented by very small tissue samples. *FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.

#3622

Identify prognostic biomarkers for colorectal cancer by human villi development model.

Botao Zhang, Lin Feng, Ting Xiao, Shujun Cheng. _National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical, Beijing, China_.

Background Tumorigenesis and embryogenesis have many similar biological characteristics, such as proliferation, invasion, metabolism and immunity. The development of villi is a physiological process, it will stop after the fetus is born. Trophoblast have been defined as "physiological metastasis" or "pseudo-malignant". However, tumorigenesis is a pathological process that will go on indefinitely. We hypothesize that if the interactions between gene and gene in tumors deviate from the interactions in villi, it is more likely to develop into malignant tumors and have worse prognosis.

Methods We collected tissue samples including human chorionic villus samples at 6 to 10 weeks of gestation (n = 15) and leaf chorionic samples from postpartum placental tissue (n = 6), then extracted RNA and carried out RNA sequencing. We processed bioinformatics analysis in combination with multi-stage colorectal cancer (CRC) transcriptome data, including normal (n = 12), low-grade adenoma (n = 21), high-grade adenoma (n = 30), cancer (n = 25).

Results We found that CRC progress and villi development involve many genes with similar biological behaviors, such as proliferation, immunity, metabolism and invasion, but the genes of the relevant biological behavior are different between CRC and villi. Then we filtered those genes, of which the interactions in CRC are far from that in villi. Twenty-four genes, such as CBL, COL1A1, ATP5C1, CDC42 etc were finally selected. Abnormal expression of the 24 genes was significantly associated with poor survival in six independent CRC cohorts (The Cancer Genome Atlas, P<0.001; GSE39084, P= 0.002; GSE14333, P<0.001; GSE17536, P=0.003; GSE39582, P<0.001; GSE29621, P=0.001).

Conclusions Villi development is a reliable and strictly regulated model that can illuminate the trajectory of human cancer development. Our study indicated that the interactions between gene and gene in CRC development deviate from that in villi, the deviation interactions of gene may have some substantial impacts on CRC development and reveal novel prognostic biomarkers. 

### Immune Checkpoints 3

#3623

The CTLA-4 x OX40 bispecific antibody ATOR-1015 induces anti-tumor effects through tumor-directed immune activation.

Niina Veitonmäki, Mia Thagesson, Doreen Werchau, Karin Hägerbrand, Kristine Smedenfors, Anne Månsson-Kvarnhammar, Anna Rosén, Maria Johansson, Christina Furebring, Per Norlén, Peter Ellmark. _Alligator Bioscience, Lund, Sweden_.

ATOR-1015 is a CTLA-4 x OX40 bispecific immune activating antibody developed for tumor-directed immunotherapy. ATOR-1015 binds both targets simultaneously, promoting cell-cell interactions expected to enhance the immuno-stimulating effect of the compound. The mode of action of ATOR-1015 is thought to be a combination of regulatory T cell (Treg) depletion and effector T cell activation. It can be seen as a next generation CTLA-4 antibody with tumor-directed activity and augmented Treg depletion. The ability to induce ADCC of human Treg was investigated using an FcγR expressing reporter assay demonstrating superior effect of ATOR-1015 compared to the monospecific antibody counterparts. Further, ATOR-1015 has been shown to induce activation of T cells in the presence of CTLA-4 expressing cells, ability that is not observed when combining the monoclonal counterparts. Syngeneic tumor models in vivo using human transgenic mice cross-reacting with both targets demonstrate that ATOR-1015 reduces tumor growth and prolongs survival. Further, ATOR-1015 treatment demonstrates superior increase in the intratumoral CD8+ T cell/Treg ratio compared to the monospecific counterparts, without affecting systemic T cells. This tumor directed immune activation is demonstrated to be due to the tumor localization abilities of ATOR-1015. In conclusion, ATOR-1015 is a next generation CTLA-4 antibody with tumor directed activity with augmented T-reg depletion. It is currently in GLP manufacturing of clinical material and will start clinical trials in the second half of 2018.

#3624

Tanshinone IIA could decrease programmed death ligand 1 expression in human breast cancer BT-20 cells.

Chin Cheng Su. _Changhua Christian Hospital, Changhua City, Taiwan_.

Breast cancer is the leading cause of cancer-related death in women. Many prescriptions for chemotherapy have been derived from plants. Danshen (Salviae miltiorrhizae radix) has been used in traditional Chinese medicine and appeared in the Chinese book, Shennong Bencao Jing (ca. 100 A.D.). Tanshinone IIA (Tan-IIA) is extracted from danshen and first described by researchers in 1968. Tan-IIA is well known to have antioxidant and anti-inflammatory properties. Tan-IIA could inhibit many human breast cancer cell lines through different molecular mechanisms in vitro and in vivo have been well documented. Our previous study showed Tan-IIA could inhibit human breast cancer cells through increased Bax to Bcl-xL ratios. Tan-IIA inhibits BT-20 (estrogen-, progesterone-, and HER2-negative subtype (TNBC)) human breast cancer cell proliferation through increasing caspase 12, GADD153 and phospho-p38 protein expression. Tan-IIA also inhibits human breast cancer MDA-MB-231 cells (estrogen-, progesterone-, and HER2-negative subtype (TNBC)) by decreasing LC3-II, Erb-B2 and NF-κBp65 in vivo. However, the effects and molecular mechanisms of Tan-IIA in human breast cancer have not been elucidated clearly. Programmed cell death-ligand 1 (PD-L1) is expressed on many cancer cells, which played a protective role against the cytotoxicity. PD-L1 interacts with programmed cell death-1 receptor (PD-1) to inhibit the T cells and block the antitumor immune response. PD-L1 antibodies, has demonstrated the possibility of dismiss immune suppression in many cancer cells. PD-L1 expression is a favorable biomarker for the prognosis of breast cancer; therefore, immune checkpoint blockade agents may be offering the opportunity to be the future treatment for breast cancer. The present study focused to evaluate the efficacy of Tan-IIA to be one of the immune checkpoint blockade agents for human breast cancer cells. Material and Method: In the present study, the human breast cancer BT-20 cells were treated with SJKJT in vitro. The effects of Tan-IIA on the protein expressions of PD-1, PD-L1, Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), B7-1 (CD80) and B7-2 (CD86) were measured by Western blotting. Results: The results showed that Tan-IIA can inhibit the protein expressions of PD-L1, in breast cancer BT-20 cells significantly. Conclusion: Breast cancer BT-20 cells were treated with Tan-IIA could inhibit the activity of PD-L1, CTLA-4, B7-1 (CD80) and B7-2 (CD86) significantly. These results suggest that one of the molecular mechanisms for Tan-IIA to inhibit breast cancer BT-20 cells maybe through inhibiting the protein expression of PD-L1. The use of Tan-IIA may become a feasible novel therapy option. Further studies are warranted to fully elucidate its mechanisms of action.

#3625

MET signaling upregulates PD-L1 expression in human lung and gastric cancer.

Hyunkyung Ahn,1 Sehui Kim,1 Ji Young Jang,1 Dohee Kwon,1 Jaemoon Koh,1 Young A Kim,2 Doo Hyung Chung,1 Yoon Kyung Jeon1. 1 _Seoul National University, Seoul, Republic of Korea;_ 2 _Seoul Metropolitan Government Seoul National University Boramae Medical Center, Seoul, Republic of Korea_.

Background: Tyrosine kinase receptor, MET, plays an important role for the progression of tumor through diverse mechanisms. Cancer immunotherapy targeting PD-1/PD-L1 pathway shows clinical benefit in patient with PD-L1 expressing solid tumor. In our previous study on non-small cell lung cancer (NSCLC) using immunohistochemistry, MET overexpression was significantly associated with programmed cell death-ligand 1 (PD-L1) overexpression. Thus, we investigated if MET signaling pathway is involved in PD-L1 expression in tumor cells.

Method and Result: Lung adenocarcinoma cell lines including H596 (harboring MET exon 14 skipping mutation), H1993 (harboring MET gene amplification), H23 and H522 (MET wild-type), and a gastric carcinoma cell line, Hs746T (harboring MET exon 14 skipping mutation and gene amplification) were used. Hs746T cells were treated with MET inhibitor or transfected with MET siRNA and subject to microarray analysis. MET inhibition or knockdown led to changes in expression level of variable immune-related molecules, of which PD-L1 was one of the most down-regulated molecules. Stimulation of H596, H23 and H522 cells with HGF, a MET ligand, increased PD-L1 expression at the mRNA and protein level. Treatment of H1993 and Hs746T cells with MET inhibitor or MET knockdown using siRNA resulted in down-regulation of PD-L1 at the mRNA, total protein, and surface expression levels. PI3K/Akt and MAPK/Erk signaling pathways were involved in MET-mediated PD-L1 up-regulation in H1993 and Hs746T cells. Finally, the Cancer Genome Atlas (TCGA) analysis showed that MET and PD-L1 expression are significantly positively correlates with each other in patients with lung cancer (in NSCLC, rho=0.219 [p<0.001]; in adenocarcinoma, r=0.401 [p<0.001]; in squamous cell carcinoma, r=0.144 [p=0.001]) and in patients with gastric cancer (r=0.137 [p=0.005]).

Conclusion: This study demonstrates that MET signaling increases PD-L1 transcription and protein overexpression in lung and gastric cancer cells, which might contribute to immune escape of tumor via PD-1/PD-L1 pathway. MET-targeted therapy may be considered as combination therapy with PD-1/PD-L1 blockades.

#3626

A genome scale screen to identify regulators of PD-L1 in HNSCC.

Jacqueline Mann, Megan Ludwig, Aditi Kulkarni, Judy Kafelghazal, Julia Eisenberg, Rebecca Hoesli, Alexey Nesvizhskii, Chad Brenner. _Univ. of Michigan, Ann Arbor, MI_.

The immune checkpoint inhibitor Pembrolizumab was recently approved for the treatment of metastatic and recurrent HNSCC, but only 18% of patients in the initial trial responded to therapy, and it remains difficult to predict patients likely to experience benefit. We expect a broader understanding of checkpoint regulation in HNSCC to lead to improved response rates and identification of potential responders. To this end, we sought to identify targetable genetic factors modulating PD-L1, a molecule that serves to dampen the anti-tumor immune response. We developed a high throughput screen employing a Genome-scale CRISPR Knock-Out (GeCKO) library in HNSCC cell lines to identify targetable genes and pathways that may regulate PD-L1 expression. In UM-SCC-49, stable knockout pools with representation of approximately 300 gRNAs for each of over 20,000 target genes were expanded and serially sorted to create stable sub-populations with enhanced or diminished PD-L1 expression. These sub-populations were sequenced to identify gRNAs that increased or reduced PD-L1 expression, indicating that their genetic targets may function to regulate PD-L1. We observed 70% library coverage (44,900 gRNAs) in the initial population and 15% and 11% coverage in the sub-populations selected for high and low PD-L1 expression, respectively. We prioritized for validation 61 gRNAs that were enriched at least 2 fold in High or Low PDL1 pool over the control pool. Future experiments focus on validation of these targets in UM-SCC-49 and other cell lines. As we expand this screen and validate hits across multiple genetic backgrounds, we expect to reveal signaling networks promoting immune evasion and potential targets for combination immunotherapeutic protocols.

#3627

Organ-specific differential responses to immune checkpoint inhibitors in patients with advanced hepatocellular carcinoma.

Li-Chun Lu,1 Chiun Hsu,1 Yu-Yun Shao,1 Yee Chao,2 Chia-Jui Yen,3 I-Lun Shih,1 Yi-Ping Hung,2 Chun-Jung Chang,4 Ying-Chun Shen,1 Jhe-Cyuan Guo,1 Tsung-hao Liu,5 Chih-Hung Hsu,1 Ann-Lii Cheng1. 1 _National Taiwan Univ. Hospital, Taipei, Taiwan;_ 2 _Taipei Veterans General Hospital, Taipei, Taiwan;_ 3 _National Cheng Kung University Hospital, Tainan, Taiwan;_ 4 _National Taiwan University College of Medicine, Taipei, Taiwan;_ 5 _National Taiwan University Hospital Hsin-Chu Branch, Hsinchu, Taiwan_.

Background: Immune checkpoints inhibitors (ICIs) such as anti-PD-1/PD-L1 monoclonal antibodies exhibit significant clinical activity in patients with advanced hepatocellular carcinoma (HCC). Immune microenvironments of different organs are often different and may affect the efficacy of immunotherapy. The study explores whether tumor response to ICIs of HCC may vary among different organs.

Patients and Methods: We reviewed patients with advanced HCC who had received anti-PD-1, anti-PD-L1, or anti-CTLA-4 therapy, alone or in combination, in 3 medical centers in Taiwan. Patients with measurable diseases were enrolled. An organ-specific response criteria, modified from RECIST 1.1, allowing a maximum of 5 measurable lesions per organ system, was used to evaluate the responses to ICIs for tumors of individual organ system. The organ-specific complete responses (CR), partial responses (PR), stable diseases (SD), or progressive diseases (PD) were determined for individual organ system.

Results: A total of 76 patients (Male: Female= 62: 14, median age of 59.1 years) with advanced HCC were enrolled. Among them, 51 (67.1%) and 12 (15.8%) patients had chronic hepatitis B virus or chronic hepatitis C infection. The ICI treatments included anti-PD-1 or anti-PD-L1 alone, anti-CTLA4 alone, and anti-PD-1/PD-L1 plus anti-CTLA4 combination in 59, 1, and 16 patients; 20 and 56 patients had received ICIs as first-line or ≥ 2nd-line therapy. The overall response rate (RR) by RECIST 1.1 was 28.9%. Fifty-nine, 34, 18, and 18 patients who had measurable hepatic tumors, lung, lymph node, and intra-abdomen metastases were subject to organ-specific response evaluation. Organ-specific RRs of hepatic tumors, lung, lymph node, and intra-abdomen metastases, were 23.7, 41.2, 27.8, and 38.9%, respectively. Among 39 patients who had both hepatic and extrahepatic tumors, 16 had significant differential responses between hepatic and extrahepatic tumors: 12 of them had disease control (CR/PR/SD) in extrahepatic tumors and PD in hepatic tumors, and 4 exhibited disease control in hepatic tumors and PD in extrahepatic tumors (p=0.046). Moreover, in 16 patients with only evaluable hepatic tumors and lung metastases, 8 had disease control in lung metastases and PD in hepatic tumors, and none experienced disease control in liver tumors and PD in lung metastases (p=0.005).

Conclusions: The hepatic tumors of HCC are less responsive to ICIs than the extrahepatic lesions. Lung metastases show best response to ICIs. The underlying mechanism warrants further investigation. (This work is supported by grants from the Ministry of Science and Technology, Taiwan, MOST 106-2314-B-002-210).

#3628

PD-1 checkpoint blockade therapy enhances adoptive immunotherapy by human Vγ2Vδ2 T cells against prostate tumors in a preclinical model.

Mohanad H. Nada, Hong Wang, Craig Morita. _University of Iowa, Iowa City, IA_.

Cancer immunotherapy is an effective treatment against a variety of malignancies. Checkpoint blockade with anti-PD-1 or anti-CTLA-4 antibodies has resulted in durable clinical responses. However, targeting PD-1 or PD-L1 provides clinical benefits for only a minority of patients. Moreover, the effectiveness of checkpoint blockade correlates with the numbers of nonsynonymous mutations present in the tumor explaining the poor responsiveness of certain tumors such as prostate cancers. Therefore, additional therapies are needed for patients not currently responding.

Unlike αβT cells, the anti-tumor activity of Vγ2Vδ2 T cells does not require MHC and is independent of the number of tumor mutations. Instead, Vγ2Vδ2 T cells use their TCRs to surveil tumor cells for alterations in isoprenoids metabolism induced by bisphosphonate treatment in a process requiring butyrophilin 3A1. This allows Vγ2Vδ2 T cells to recognize and kill all types of tumors. In clinical trials involving more than 200 patients, adoptive immunotherapy with Vγ2Vδ2 T cells has been proven to be safe. However, there have been few partial or complete responses with the most common beneficial response being stable disease. To find ways to increase its effectiveness, we have now assessed the benefits of combining PD-1 checkpoint blockade with the adoptive transfer of human Vγ2Vδ2 T cells. Vγ2Vδ2 T cells were found to express the major inhibitory receptors, PD-1, CTLA-4, LAG3, and TIM3 at varying times during the fourteen day ex vivo expansion period. To assess the effect of checkpoint blockade in vivo, immunodeficient NSG mice were inoculated with human PC-3 prostate cancer cells that naturally express the PD-L1 ligand. After 2 weeks, weekly treatments were started with intravenous pamidronate followed a day later by purified Vγ2Vδ2 T cells combined with either anti-PD-1 (clone J116) or control IgG1 mAbs. The addition of anti-PD-1 mAb treatment, markedly enhanced Vγ2Vδ2 T cell immunity to PC-3 tumors. Combination treatment was able to reduce tumor volume almost to zero after 5 weeks. These results demonstrate that PD-1 checkpoint blockade can greatly enhance adoptive immunotherapy with γδ T cells in a preclinical tumor model and suggests that combination therapy with anti-PD1 and Vγ2Vδ2T cells could be an effective strategy to treat patients with a variety of cancers.

#3629

Repeated abscopal effect with radiation and anti-PD-1 blockade in MMR deficient endometrial cancer.

Michael S. Oh, Young Chae. _Northwestern University Feinberg School of Medicine, Chicago, IL_.

The combination of radiation therapy and immune checkpoint inhibitors has raised interest as possibly having synergistic antitumor effects. Radiation can induce both a local response as well as a systemic one that stimulates immune cells, which then can be further potentiated by immunotherapy. This abscopal effect has been validated in preclinical studies and is currently being studied in clinical trials, though it has yet to be reported in gynecologic cancers. A 64-year-old woman initially presented with vaginal bleeding and was subsequently diagnosed with stage IIIC endometrial adenocarcinoma with pelvic and para-aortic lymphadenopathy. She underwent surgical resection with adjuvant carboplatin and paclitaxel. Follow-up CT scans showed progression of disease, and over the next 18 months, the patient was sequentially treated with doxorubicin, topotecan, melphalan, docetaxel, and capecitabine. Analysis of a tumor biopsy demonstrated an MMR deficient phenotype with absent MLH1 and PMS2. Based on the recently-published data on the efficacy of PD-1 inhibitors in MMR deficient solid tumors, the patient was started on nivolumab. Meanwhile, she was given palliative radiation therapy to the following areas: painful cutaneous metastases on her left thigh; an L3 spinal metastasis; lymph nodes in the retroperitoneum, external iliacs, and left inguinal chain; and a symptomatic bladder mass. After radiation, the patient exhibited a strong partial response not only in the targeted lesions but also throughout her tumor burden, consistent with an abscopal effect. This response was sustained over many months and repeatable with each radiation session. The synergistic use of radiation therapy and immune checkpoint blockade may thus represent a powerful treatment modality in endometrial cancer and warrants further clinical investigation.

#3630

Deep and durable response with dual anti-CTLA-4 and PD-1 blockade in mismatch repair (MMR) proficient endometrial cancer.

Michael S. Oh, Young Chae. _Northwestern University Feinberg School of Medicine, Chicago, IL_.

Immune checkpoint inhibitors have shown promising efficacy in multiple cancer types, but have yet to be extensively validated in endometrial cancer. However, the recent FDA approval of PD-1 inhibitors for mismatch repair (MMR) deficient tumors has extended use of these treatments to all cancer types. PD-L1 positivity in tumor tissue has also been shown to predict susceptibility to immunotherapy and has been used to guide use of these treatments. Despite these advances, the response to immunotherapy in endometrial cancer remains poorly understood. Here, we describe the case of a patient with MMR proficient, PD-L1-negative stage IV endometrial cancer with positive response to combination PD-1 and CTLA-4 inhibition. The patient initially presented with ascites and elevated tumor markers, and cytology from peritoneal fluid showed endometrial adenocarcinoma with squamous and mucinous differentiation and clear cell features. She underwent surgical debulking with both neoadjuvant and adjuvant cycles of docetaxel and carboplatin, but unfortunately had progressive metastatic disease within 4 months. The patient declined further chemotherapeutic agents, and thus was instead trialed on combination nivolumab and ipilimumab despite her cancer exhibiting MMR proficiency and negative PD-L1. She demonstrated deep and durable on-going partial response to this treatment that has persisted still after 10 months. Her course has notably been complicated by hypophysitis that manifested after 5 months of treatment as worsening fatigue, but she has been able to continue with her treatment regimen. This case indicates the potential existence of an endometrial cancer subtype that is sensitive to immune checkpoint blockade, but based on mechanisms other than those driven by MMR deficiency or PD-L1 positivity. Improved understanding of immunotherapy in advanced endometrial cancer is clearly needed and offers the potential to significantly enhance patient outcomes.

#3631

Passenger fusion genes are novel predictive biomarker of anti-PD-1 antibody nivolumab for ovarian cancer.

Junzo Hamanishi, Ryusuke Murakami, John B. Brown, Tsukasa Baba, Kaoru Abiko, Eiji Suzuki, Masaki Mandai. _Kyoto University, Kyoto, Japan_.

Background: Immune checkpoint PD-1 signal blockade provides significant clinical efficacy and durable response for several cancer patients. However it is crucial to identify a biomarker to predict the response to PD-1 signal blockade. Herein we analyzed the somatic mutanomes and transcriptomes of tumor samples from ovarian cancer patients treated with nivolumab to identify potential biomarkers of antitumor response.

Methods: We previously reported a phase II clinical trial for resistant ovarian cancer with nivolumab (UMIN000005714) and showed disease control rate was 45% including 2 complete responders. To estimate the somatic mutation burden (TMB) of tumor samples, the number of non-synonymous single nucleotide variants (nsSNVs), insertions and deletions of genes was analyzed by whole exome sequencing of formalin fixed paraffin embedded tumor tissues. Next, to evaluate whole transcriptome of these samples, we performed an RNAseq analysis and detected several fusion genes in some patients. We evaluated the correlation between clinical anti-tumor response for nivolumab treatment with the numbers of somatic mutations or transcriptomic alignments containing gene fusions.

Results: 16 tumor samples (7 responders, 9 non-responders and 1 loss) for whole exome sequencing and 17 samples (7 responders and 10 non-responders) for RNA sequencing were available. Contrary to expectations generated by previously reported papers in melanoma or lung cancers, the levels of TMB including nsSNVs, insertions and deletions of genes were not significantly correlated to antitumor response. However, more than 2 fusion genes were detected (mean=6.3, range 0-15) in 6 of 7 responders, while 8 of 10 non-responders did not have any fusion genes and the remaining non-responders had only 1 fusion gene each (mean=0.2, range, 0-1). Therefore an antitumor response to nivolumab was strongly correlated to the number of fusion genes in ovarian cancer (p=0.0003, sensitivity: 86% [6/7] specificity: 100% [10/10]).

Conclusions: This study, to our knowledge, is the first to describe passenger fusion genes of tumors, and might provide a clinically useful and novel predictive biomarker of antitumor response to anti-PD-1/PD-L1 antibody therapy for ovarian cancer.

#3632

Combination of EGFR antibody with PD-1 pathway inhibitors improves anti-tumor efficacy and enhances intra-tumor immune response in preclinical mouse tumor models.

Veena Kandaswamy,1 Amelie Forest,1 Marianne Deroose,1 David A. Schaer,1 Ting Chen,2 Shengwu Liu,2 David Surguladze,1 Yung-mae Yao,1 Thompson Doman,3 Gerald Hall,1 Kwok-Kin Wong,2 Michael Kalos,1 Ruslan D. Novosiadly1. 1 _Eli Lilly and Company, New York, NY;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _Eli Lilly and Company, Indianapolis, IN_.

Necitumumab (EGFR inhibitor) in combination with chemotherapy provides a modest, yet a significant improvement in overall survival over chemotherapy alone in patients with advanced squamous non-small cell lung carcinoma (NSCLC). However, combination therapies targeting EGFR and PD-1 pathway blockers may represent a better way to extend clinical benefit to more cancer patients given that PD-(L)1 antibodies have emerged as a standard of care in NSCLC. Antibodies targeting EGFR have the potential to promote an inflamed tumor microenvironment through engagement of Fc-gamma receptors (FcγR) on innate immune cells resulting in an improved antigen presentation and T cell priming. Therefore, the present study was initiated to understand the combinatorial effect of immune checkpoint (PD-(L)1) inhibitors with necitumumab. Preclinical modeling of EGFR/PD-(L)1 mAb combination in mice is challenging due to the lack of cross-reactivity of necitumumab with mouse EGFR. Syngeneic mouse tumor models widely used to study effects of immunomodulatory agents express low or no EGFR. To overcome this limitation, we used two immunocompetent model systems to study the combination effect of EGFR mAb with PD-1 (RMP1-14) or PD-L1 (178G7) mAbs: 1) genetically engineered mouse model of lung adenocarcinoma (TD model) driven by mutant forms of human EGFR (exon 19 deletion and T790M mutation) and 2) CT26 syngeneic mouse tumor model with ectopic expression of human EGFR (CT26-hEGFR). To engage mouse immune cells more efficiently, a murinized version of necitumumab was generated through antibody engineering, with human EGFR binding Fabs and a mouse Fc backbone. Intratumor immune response was evaluated by immunohistochemistry and a custom-made immune profiling Quantigene Plex (QGP) gene expression panel. In both models, targeting EGFR and PD-1 pathway resulted in the combinatorial antitumor efficacy exemplified by decreased tumor burden compared to the monotherapy groups. QGP analysis of CT26-hEGFR tumor tissue revealed that the combination treatment enhanced intra-tumor immune response exemplified by an upregulation of immune-related genes indicative of T cell infiltration (Cd3e, Cd4, Cd8b1), T cell activation (Ifng, Cd274, Pdcd1lg2, Icos, Tnfrsf4, Tnfrsf18, Cd69, Ido1, Havcr2, Lag3), myeloid cell infiltration (Cd86, Timd4, Vista, Cd68, Mpo, Nos2). Histopathological analysis confirmed an increase in T cell infiltration indicating an improved immune response in the combination therapy group. Taken together, these results provide a rationale for further evaluation of EGFR and PD-(L)1 mAbs in clinical setting.

#3633

Immunofluorescence profiling of co-expression of multiple immune checkpoints in malignant and tumor infiltrated lymphocytes in non-small cell lung carcinomas using image analysis system.

Edwin Roger Parra, Mei Jiang, Jiexin Zhang, Carmen Behrens, Jack Lee, John Victor Heymach, Marie-Andrée Forget, Cara Haymaker, Chantale Bernatchez, Cesar Moran, Jianjun Zhang, Don Gibbons, Ignacio Wistuba. _UT MD Anderson Cancer Center., Houston, TX_.

Background: The immunocheckpoint represents a group of membrane proteins expressing on immune cells, composed of multiple co-inhibitory and co-stimulatory pathways. They are involved in eliminating unwanted substances while maintaining self-tolerance, which play a crucial role in immunomodulation. Malignant cells (MCs), on the other hand, often tends to take advantage of immunocheckpoints, for instance, by activating inhibitory checkpoint pathways in situ, to evade immune responses. Therefore, cancer immunotherapies, which target key immune checkpoints to restore effective anti-tumor immune function, emerge as a revolutionary strategy during recent years. The aim of this study was analyze a full range of checkpoint available to investigate their role in the microenvironment and their frequent co-expression in MCs and tumor infiltrated lymphocytes (TILs) in non-small cell lung carcinomas. Material and Methods: 4 µm-thick histology sections obtained from formalin-fixed paraffin-embedded (FFPE) tissues from a TMA containing 256 NSCLCs (adenocarcinomas, ADC=156; squamous cell carcinomas, SCC=100); were used for multiplex immunofluorescence (mIF) analyses. Nine immune markers including PD-L1, B7-H3, B7-H4, IDO-1, ICOS, OX-40, LAG3, TIM3, VISTA, CD3 and CD20 was optimized for immunofluorescence. Two mIF immunocheckpoints panels were composed included seven antibodies as follows: Panel 1: AE1/AE3, PD-L1, B7-H3, B7-H4, IDO1, VISTA, and CD3; and Panel 2: AE1/AE3, ICOS, LAG3, TIM3, OX40, CD3, and CD20. For quantitative image analysis of mIF markers, the stained TMA slides were scanned using the Polaris™ multispectral microscope (Perkin Elmer) and analyzed using the inForm™ 2.3.1 software. Results: In this preliminary report using the median density in MCs and TILs expressing those markers, we identified overall higher expression of immunocheckpoints in SCC than ADC. The higher density co-expression by MCs (AE/AE3+) was B7-H3 + IDO-1+ following by B7-H3+PD-L1+ expression when compared with the other markers (P~0.001) in the panel 1. Furthermore, we observed higher densities of TILs (CD3+) expressing IDO-1+ as well as B7-H3+ and high densities of TILs (CD3+) co-expressing B7-H3+PD-L1+ when compared the other (P~0.001) markers in the same panel. In panel 2, we observed higher densities of TILs (CD3+) expression ICOS+ than the other markers (P~0.001). Conclusions: Here, we describe a mIF platform with computational image processing workflows, enabling simultaneous evaluation of 12 biomarkers to FFPE tissue section. These data highlight digital image processing pipelines to the community for examining immune complexity of immunocheckpoints expression in MCs and TILs to improve biomarker discovery and assessment.

#3634

PET/CT imaging prediction of response to checkpoint blockade in advanced non-small cell lung cancer patients.

Wei Mu, Jin Qi, Hong Lu, Mathew Schabath, Yoganand Balagurunathan, Ilke Tunali, Shari Pilon-Thomas, Robert J. Gillies. _Moffitt Cancer Center & Research Institute, Tampa, FL_.

Immune checkpoint blockade with monoclonal antibodies directed at the inhibitory immune receptors PD-1 and PD-L1 has emerged as a one of the most successful treatment approaches in advanced Non-small Cell Lung Cancer (NSCLC) patients. However, given its adverse effects, high cost and only approximately 20% response rate, accurate response predication is import to identify patients most likely to respond. In previous work, we and others have shown that tumor acidosis, generated from high rates of glucose metabolism, was a potent inhibitor of anti-tumor immunity and that neutralization of acid improved response to checkpoint blockade. In this study, we investigated the value of pre-therapy PET/CT images on response prediction to test the hypothesis that the most metabolically active tumors would have the poorest response to checkpoint blockade. 64 patients were retrospectively curated with pathology verified NSCLC and who did not receive intervening treatments between the PET/CT imaging and initiation of anti-PD1 therapy. Responders (long-term and ongoing responders, n=22) and non-responders (non-responders and transient responders, n=42) were classified according to RECIST criteria. These patients were randomly divided into two groups (n=32 each) for training and testing, respectively. From their images, we constructed 5 different fusion images comprised of PET and CT data. We then extracted 455 quantitative features from these fusion images, as well as the original PET/CT images. We then generated an additional 975 fusion features calculated from the above 455 features, as well as some clinical features (location, age, sex, and histologic type). From these, the most informative features were selected with Receiver Operating Characteristic Curve (ROC) analysis, and these were analyzed with a combined 2-layer cascade support vector machine (cSVM) classification model to relate the features to prediction of objective response. From this we determined that the most informative features were fusion features. With the cSVM, the training set achieved accuracy and area under the ROC curve (AUROC) for classifying response of 100% and 1.00, respectively. Using this model on the test set achieved accuracy and AUROC of 89.66% and 0.86, respectively. We conclude from that PET/CT fusion images may be useful prior to initiation of therapy to predict response to checkpoint blockade in advanced non-small cell lung cancer patients.

#3635

CTC and ctDNA profiling to detect 6 NCCN-guideline recommended classes of alterations for immunotherapy and targeted therapy selection using sample from a single blood draw.

Huangpin B. Hsieh,1 Jen-chia Wu,1 Feng-Ming Lin,1 Julian Lucas,1 Alex Atkins,1 Pratyush Gupta,1 Hung-Jen Shao,1 Yen-Lin Chen,2 Wen-Jie Huang,3 Chia-Hsun Hsieh,4 Ruey Kuen Hsieh,5 Kuo-Wei Chen,6 Ming-Hong Yen,7 Mana Javey,1 Shih-En Chang,1 Twinkal Marfatia,1 Drew Watson,8 Mahul Amin,9 Ashish Nimgaonkar,10 Oscar Segurado,11 Rui Mei1. 1 _CellMax Life, Sunnyvale, CA;_ 2 _Cardinal Tien Hospital, New Taipei, Taiwan;_ 3 _Mackay Memorial Hospital, Taipei, CA;_ 4 _Chang Gung Memorial Hospital, Taoyuan, Taiwan;_ 5 _Mackay Memorial Hospital, Taipei, Taiwan;_ 6 _Cheng Hsin General Hospital, Taipei, Taiwan;_ 7 _Cathay General Hospital, Taipei, Taiwan;_ 8 _Kit Bio Inc, Los Altos, CA;_ 9 _University of Tennessee Health Science Center, Memphis, TN;_ 10 _Johns Hopkins Hospital, Baltimore, MD;_ 11 _MedicAffairs Consulting, San Jose, CA_.

Introduction The availability of targeted and immunotherapies has provided NSCLC patients with more effective treatment options. However, this has resulted in an increase in the number and modality of tests required for treatment selection. Given 30-50% of advanced lung cancer patients have insufficient or unavailable tissue for comprehensive genomic profiling, there is a need for a non-invasive assay that can accurately detect all guideline-recommended markers for NSCLC treatment selection. To meet this need, we have developed a blood test that detects six classes of alterations (SNV, Indels, Rearrangements, CNA, Microsatellite Instability and PD-L1 expression) for therapy selection.

Methods & Results Three tubes of blood from a routine blood draw were sent to our CLIA-certified and/or CAP accredited lab for analysis. PD-L1 expression was evaluated in circulating tumor cells (CTCs) utilizing two different assays; (i) Immunofluorescence (IF) antibody staining, (ii) mRNA qPCR. CTCs were captured on the CMxTM CTC Platform coated with lipid bilayer and antibodies to EpCAM. PD-L1 expression results were highly correlated between IF and qPCR assays in ten solid tumor cell lines (lung, breast, prostate and colorectal cancer) spiked into whole blood to mimic the actual patient CTC capture process. In an ongoing study on clinical samples from NSCLC patients (N=20), we observed greater than 90% concordance between tissue (IHC by 22C3 PD-L1 clone) and blood (CTC IF and mRNA assays). A proprietary Single Molecule Sequencing (SMSEQTM) NGS assay was performed on plasma in order to detect 5 classes of genomic alterations (SNV, Indels, Rearrangements, CNA, MSI) from ctDNA. This assay was validated in accordance with the latest ACMG and AMP guidelines to accurately detect variants at low mutant allele fraction (.1% for SNVs and Indels, 1% for rearrangements and 5 copies for CNA) with high sensitivity and specificity. MSI status was determined by assessing nucleotide repeat sequences in five standard markers (BAT-25, BAT-26, MONO-27, NR-21, NR-24), and was detectable down to a MAF of 1%. In an ongoing study on clinical samples from NSCLC patients (N=20), we observed high concordance of MSI status between tissue (immunohistochemistry for dMMR/MSI status) and blood (ctDNA SMSEQ assay).

Conclusion Tissue insufficiency and procurement challenges are the primary reasons why ~90% of patients diagnosed with advanced NSCLC are not comprehensively tested per NCCN-guidelines in the community setting where most cancer is treated, leading to suboptimal treatment selection. An accurate blood test that detects all 6 NCCN-recommended markers for immunotherapy and targeted therapy selection has the potential to significantly improve adherence to NCCN testing guidelines and enable optimal treatment selection.

#3636

The immune checkpoint, HVEM, is associated with PDL1 expression in lung cancer cell lines.

Shengxiang Ren,1 Nadav Amar,2 Hui YU,3 Christopher Rivard,2 Kim Ellison,2 Leslie Rozeboom,2 Fred R. Hirsch2. 1 _Shanghai Pulmonary Hospital, Shanghai, China;_ 2 _University of Colorado Anschutz Medical Campus, Aurora, CO;_ 3 _Departments of Medicine, Medical Oncology University of Colorado Anschutz Medical Campus, Aurora, CO_.

Background: Herpes Virus Entry Mediator (HVEM) is an important immune checkpoint in cancer recognition. HVEM expressed on tumor cell membranes activates immune cell signaling pathways leading to either inhibition of activity (BTLA) or activation of immune activity (LIGHT). This study is to investigate the prevalence of HVEM expression and its association with PDL1 expression in cell lines and patient tumor tissue.Methods: A TMA of 53 characterized NSCLC, 48 SCLC cell lines and 35 malignant tumors were evaluated for HVEM and PD-L1 expression. The IHC assay for HVEM was Dako Link48 autostainer using a polyclonal antibody from R&D Systems(AF356). PD-L1 IHC was performed on the Dako Link48 autostainer using the PD-L1 22C3 pharmDx kit. Scoring HVEM employed the H-score system while for PD-L1 the tumor proportion score (TPS) was used.Results: IHC HVEM staining was optimized and demonstrated membranous staining with little or no cytoplasmic or nuclear staining. HVEM expression in the NSCLC and SCLC cell lines and malignant tumor TMA revealed a positive H-score more than 1 was 45.3%, 58.3% and 82.9% respectively. In comparison, PDL1 TPS expression of more than 1% was in 59.2%, 16.67% and 45.71% in NSCLC, SCLC cell lines and malignant tumor respectively. A significant correlation of HVEM with PDL1 expression was found in the NSCLC cell lines (r=0.518, p<0.001), while there was no significant association of HVEM with PDL1 expression in the small cell line cohort (r=0.059, p=0.688). Conclusion: HVEM expression was found to be higher in SCLC cell lines as compared to NSCLC cell lines and substantially greater in the general malignant tumor tissues. HVEM expression is associated with PD-L1 expression in NSCLC cell lines but not in SCLC cell lines. This data suggests a potential benefit of combined PD-L1 and HVEM blockade in patients with NSCLC to improve the efficacy of immune-therapy.

Table 1. HVEM Expression in the study | H score

---|---

|

0 | 1-100 | 101-200 | 201-300

NSCLC Cell lines(n=53) | 29 (54.72%) | 23 (43.4%) | 1 (1.8%) | 0

SCLC Cell lines(n=48) | 20 (41.67%) | 26 (54.17%) | 2 (4.17%) | 0

Malignant TMA n=35 | 6 (17.14%) | 16 (45.71%) | 9 (25.71%) | 4 (11.43%)

#3637

Expression of an immune checkpoint- Poliovirus Receptor (PVR) in small cell lung cancer.

Hui Yu,1 Camilla Koczara,1 Andrzej Badzio,2 Dexiang Gao,1 Christopher J. Rivard,3 Kim Ellison,1 Kenichi Suda,1 Shengxiang Ren,1 Charles Caldwell,1 Kristine A. Brovsky,1 Leslie Rozeboom,1 Fred R. Hirsch1. 1 _University of Colorado Anschutz Medical Campus, Aurora, CO;_ 2 _NU-Med, Elblag, Poland;_ 3 _University of Colorado Anschutz Medical Campus, AURORA, CO_.

Background: Although immunotherapy has renewed hopes for the improved treatment of SCLC, the "classical" PD-1/PD-L1 pathway is not commonly expressed in SCLC. PVR (CD155) is expressed at low levels in a number of cell types of epithelial origin and is overexpressed in various carcinomas with epithelial and neurological origins, including colorectal carcinoma, breast carcinoma, neuroblastoma and glioblastoma. In this study, we evaluated the expression of PVR in SCLC cell lines and a cohort of patient tumors with association to clinical characteristics.

Methods: Immunohistochemistry (IHC) was performed to evaluate PVR protein expression in 39 SCLC cell lines as well as a cohort of 77 resected tumors from SCLC patients with clinical data using a monoclonal PVR antibody (D8A5G,Cell Signaling, MA). Scoring data was generated using both the H-score and tumor proportion score (TPS) systems. PVR expression was correlated with characteristics of both the cell lines and the clinical cohort.

Results: The SCLC cell lines were evaluated by the H-score system only. A total of 39 SCLC cell lines in a TMA were evaluated in which 37 cell lines (95%, 37/39) demonstrated PVR staining > 0 and 85% of the cell lines showed positive staining with an arbitrary H-score cutoff of 50. PVR expression was higher in cell lines established from pre-treated SCLC patients as compared to those established from treatment-naïve patients (P=0.037). There was no correlation between PVR expression and cell line growth as suspension versus adherent cells. In the SCLC patient cohort, PVR expression was found to be predominantly on the membrane of tumor cells, with minimal expression observed on immune cells in the tumor microenvironment. PVR expression in the SCLC patient cohort was 82% with H-score cutoff of ≥ 50. SCLC patients with positive PVR expression demonstrated a poorer prognosis, however the difference was not statistically significant (p=0.05). Positive PVR expression was also correlated with advanced stage (P=0.0073) and higher in male patients (P=0.04). With a TPS score system (cutoff of ≥ 50%) the prevalence of PVR expression is 79%. Using the TPS system, higher PVR expression was associated with poor prognosis and advanced stage (P=0.046,

and P=0.045). PVR expression also as evaluated with the TPS score system was correlated with large tumor diameter (P=0.043) and older age of patients (P=0.046).

Conclusion: PVR was broadly expressed in SCLC cell lines and tumor tissues. High PVR expression was associated with poor prognosis, advanced tumor stage, large tumor diameter, and advanced age of patients. We also found that PVR expression may correlate with chemotherapy treatment history in SCLC cell lines. These data suggest that the PVR-TIGIT pathway may be a promising target for immunotherapy in SCLC.

#3638

Quantitative assessment of tumor-infiltrating lymphocytes and immunotherapy outcome in metastatic melanoma.

Pok Fai Wong, James W. Smithy, Kim R. Blenman, Harriet M. Kluger, David L. Rimm. _Yale School of Medicine, New Haven, CT_.

Background: Durable responses to immune checkpoint blockade against programmed cell death 1 (PD-1) are limited to a subset of metastatic melanoma patients, so there is a need for predictive biomarkers. Tumor-infiltrating lymphocytes (TILs) are the major cellular target of anti-PD-1 therapy, so we hypothesized that pretreatment TIL profiles would be associated with response.

Methods: Pretreatment whole-tissue sections from 94 melanoma patients treated with anti-PD-1 therapy (pembrolizumab, nivolumab, or ipilimumab plus nivolumab) from 2011-17 were collected from Yale Pathology archives, and stained to visualize nuclei (DAPI) and melanoma cells (S100 & HMB45) in combination with two multiplex immunofluorescence panels to: (1) perform TIL quantitation of helper T cells by CD4 (SP35, Spring), cytotoxic T cells by CD8 (C8/144B, Dako), and B cells by CD20 (L26, Dako); and (2) assess TIL activation by identifying T cells by CD3 (SP7, Novus), cytolytic activity by GZMB (4E6, Abcam), and proliferation by Ki67 (MIB-1, Dako). Cell phenotyping and counting were performed using inForm software (PerkinElmer) and protein expression was measured by the AQUA method of quantitative immunofluorescence (QIF). Response Evaluation Criteria In Solid Tumors (RECIST) 1.1 were used to classify best overall response as complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD). Objective response rate (ORR; CR/PR), disease control rate (DCR; CR/PR/SD), and progression-free survival (PFS) were correlated with TIL parameters measured by both methodologies.

Results: Pretreatment lymphocytic infiltration, by cell counts or QIF, was significantly higher in CR/PR than in SD/PD, particularly for CD3 and CD8 (P < 0.0001). Neither TIL activation (CD3 high, Ki67 and/or GZMB high) nor TIL dormancy (CD3 high, Ki67 and GZMB low) was significantly associated with outcome. Multivariable analyses revealed significant CD8 associations (HR > 3) with PFS independent of age, sex, mutation, stage, treatment, and prior immune checkpoint blockade, which accounted for similar CD3 PFS associations as expected. The favorable predictive performance of CD8 cell count (and QIF) had an area under the receiver operating characteristic (ROC) curve of 0.75 for ORR and 0.78 for DCR, which reached 0.83 (ORR or DCR) for dual therapy (ipilimumab plus nivolumab). Interestingly, in contrast to previous hypothetical classifications, there were a number of responders (CR/PR) in the CD3 or CD8 low (immune desert) category.

Conclusions: Pretreatment lymphocytic infiltration, by cell counts or QIF, is associated with anti-PD-1 response in metastatic melanoma. Multiplex analysis of the tumor microenvironment has the potential to be used as a companion diagnostic test for precision immunotherapy.

#3639

**Matching-adjusted indirect comparison of nivolumab + ipilimumab and BRAF+MEK inhibitors for the treatment of** BRAF **-mutant treatment-naive advanced melanoma.**

Michael B. Atkins,1 David McDermott,2 Ahmad Tarhini,3 Michael Rael,4 Komal Gupte-Singh,5 Elliot O'Brien,6 Corey Ritchings,5 Sumati Rao5. 1 _Georgetown Lombardi Comprehensive Cancer Center, Washington, DC;_ 2 _Beth Israel Deaconess Medical Center, MA;_ 3 _Center for Immuno-Oncology Research, Cleveland Clinic, OH;_ 4 _Evidera, Inc., CA;_ 5 _Bristol-Myers Squibb, NJ;_ 6 _Evidera Inc., CA_.

OBJECTIVES: Assess the relative overall survival (OS) and progression-free survival (PFS) of nivolumab + ipilimumab (NIVO+IPI) versus dabrafenib + trametinib (DAB+TRAM) and vemurafenib + cobimetinib (VEM+COBI) in treatment-naive patients with BRAF-mutant advanced melanoma. METHODS: In the absence of a direct head-to-head comparison, matching-adjusted indirect treatment comparisons (MAIC) were conducted using patient-level data for NIVO+IPI patients with BRAF-mutant tumors from the CheckMate 067 and 069 trials and published summary level results from BRAF+MEK pivotal trials (COMBI-d, COMBI-v, and coBRIM). NIVO+IPI baseline characteristics were matched to the DAB+TRAM or VEM+COBI patient characteristics by reweighting the NIVO+IPI patients by their odds of enrollment in the studies. Additional potential biases such as use of subsequent therapy were not controlled for. Cox proportional hazard models were fit to the weighted OS and PFS data for NIVO+IPI and virtual event and censor times derived from the published DAB+TRAM or VEM+COBI curves. To capture the non-proportionality observed in the OS and PFS curves, Cox models with separate hazard ratios (HRs) for <12 months and ≥12 months were also fit. RESULTS: The effective sample size of the NIVO+IPI patients was 106 after matching to DAB+TRAM and 59 after matching to VEM+COBI. The overall OS Cox models showed that NIVO+IPI had superior OS with HRs (95% confidence intervals) of 0.64 (0.46, 0.89) versus DAB+TRAM and 0.56 (0.36, 0.89) versus VEM+COBI. This benefit emerged with longer follow-up. At 12 months, the OS HRs were similar for NIVO+IPI versus DAB+TRAM (1.0; 0.67, 1.49) and VEM+COBI (1.01; 0.57, 1.80). However, beyond 12 months, NIVO+IPI had a significantly greater OS benefit versus DAB+TRAM (0.33; 0.18, 0.60) and versus VEM+COBI (0.29; 0.14, 0.63). PFS results were similar with insignificant differences observed at 12 months and superior PFS for NIVO+IPI beyond 12 months; HRs for ≥12 months were 0.29 (0.14, 0.62) for NIVO+IPI versus DAB+TRAM and 0.15 (0.04, 0.54) for NIVO+IPI versus VEM+COBI. CONCLUSION: After adjusting for patient population differences, NIVO+IPI had a significant OS benefit over DAB+TRAM and VEM+COBI. For both OS and PFS, the NIVO+IPI benefit emerges after 12 months of treatment. In the future, these results can be validated by ongoing randomized trials directly comparing sequential combination approaches. Disclosure: This research was funded by Bristol-Myers Squibb.

#3640

Treatment strategies using anti-PD1/PD-L1 (anti-PD) and BRAF/MEK inhibitor (BRAFi) therapy: a retrospective study comparing sequential vs. concurrent administration in BRAF-mutated metastatic melanoma (BMMM).

Rodabe N. Amaria, Van A. Trinh, Jun Gu, Susan McIntyre, Carlos Torres Cabala, Rinata Simien, Adi Diab, Hussein A. Tawbi, Michael A. Davies, Michael K. Wong, Patrick Hwu, Isabella C. Glitza, Sapna P. Patel, Wen Jen Hwu. _MD Anderson Cancer Center, Houston, TX_.

Background: It is unclear whether anti-PD and BRAFi therapy should be administered sequentially or concurrently in patients (pts) with BMMM, although clinical trials using varying strategies are ongoing. We performed a retrospective, single-center analysis of BMMM pts who received anti-PD therapy either sequentially or concurrent with BRAFi therapy. Methods: A total of 457 metastatic melanoma pts (excluding uveal) were treated with anti-PD therapy at MD Anderson Cancer Center from 11/2009-12/2015. Of those, 148 pts had BMMM and were categorized into 4 treatment groups: BRAFi-naïve treated with anti-PD alone (group 1) N=39 (26%); BRAFi-naive treated with combination therapy (group 2) N=30 (20%); BRAFi-refractory treated with anti-PD alone (group 3) N=45 (31%); and BRAFi-refractory treated with combination therapy (group 4) N=34 (23%). Clinical outcomes were analyzed and tabulated for each group. Pretreatment tissue samples were obtained for correlative biomarker immunohistochemistry (IHC) analyses. Results: BMMM pts with elevated LDH are more likely to receive upfront combination therapy. BRAFi-refractory pts have a higher incidence of brain metastases than BRAFi-naïve pts. The clinical outcomes of overall responses, median PFS and OS are provided in the table. Preliminary biomarker IHC analyses in 11 pts showed PD-L1 expression on the tumor cells correlated with median PFS and OS : 15 mo and 22 mo, respectively in 7 pts with PD-L1 >1%, vs. 6.5 mo and 12 mo in 4 pts with PD-L1 <1%. Conclusions: Single-agent anti-PD therapy is an effective strategy in BMMM pts who are either BRAFi-naïve or refractory. Combination therapy of anti-PD and BRAFi in either BRAFi-naïve or refractory BMMM did not appear to improve clinical outcomes. Confirmation data are needed from ongoing prospective randomized clinical trials.

Group | LDH > upper limit normal (%) | Prior CNS metastases (%) | Prior anti CLTA-4 exposure (%) | Number of anti PD-1 doses (#) | Overall Responses CRs + PRs (%) | Median PFS (mo) | Median OS (mo)

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

Group 1 BRAFi-naïve; anti-PD alone | 23.7 | 23 | 17.9 | 12.5 | 30.8 + 25.6= 56.4 | 10 | 21

Group 2 BRAFi-naïve; combination | 37.9 | 20 | 6.7 | 10.5 | 23.3 + 43.3 = 66.6  | 8 | 12.5

Group 3 BRAFi- refractory; anti-PD alone | 25 | 40 | 73.3 | 16 | 37.8 + 17.8= 55.6 | 9 | 25

Group 4 BRAFi- refractory; combination | 17.6 | 44.1 | 72.7 | 11.5 | 29.4 + 5.9 = 35.3 | 5 | 19

#3641

Use of adjuvant interferon alfa-2b or ipilimumab 10 mg/kg for high-risk patients with melanoma, and associated adverse events and duration of therapy.

Ahmad Tarhini,1 Komal Gupte-Singh,2 Min You,2 Amit Gupta,2 Le Trong Kim,2 Sumati Rao2. 1 _Center for Immuno-Oncology Research, Cleveland Clinic, Cleveland, OH;_ 2 _Bristol-Myers Squibb, Princeton, NJ_.

OBJECTIVE: Adjuvant systemic therapy with interferon alfa-2b (IFN) or ipilimumab 10 mg/kg (IPI10) has been the standard for high-risk patients with melanoma who have undergone surgery; however, a high proportion of patients remain untreated. This analysis examines the adverse events (AEs) and duration of therapy (DoT) associated with IFN or IPI10. METHODS: A retrospective cohort study was conducted among each of the four US healthcare databases: OptumInsight, IMS PharMetrics, Truven Commercial, and Truven Medicare Supplemental. Patients receiving melanoma-related surgery (lymph node dissection) from July 1, 2006, to September 30, 2016, were identified. The study estimated the incidence rate (IR) per patient per year of AEs that commonly occur with IFN or IPI10 across different DoT levels among patients with melanoma following surgery. RESULTS: Of 46,401 eligible patients with melanoma surgery claims, 2423 (5%) were treated: 2316 with IFN and 107 with IPI10. Mean age was 51.7 years (60.5% male), and mean (standard deviation) Charlson comorbidity index score was 6.6 (2.8) and 7.5 (2.5) for patients prescribed IFN or IPI10, respectively. The DoT (median, mean (SD)) for IFN was 142, 182 (162) days and for IPI10 was 84, 73 (26) days. Similarly, the number of prescriptions (median, mean (SD)) for IFN were 20, 26 (28) and for IPI10 were 4, 3 (1). The overall AE IR per year was 4.4 in patients receiving IFN and 4.9 for those receiving IPI10. Among patients receiving IFN, the most common AEs were hepatitis, nausea, and fatigue (IRs: 0.92, 0.67, and 0.66, respectively). The most common AEs among IPI10-treated patients were fatigue, rash, nausea, and diarrhea (IRs of 0.95, 0.71, 0.66, and 0.63, respectively). AE rates were greater in patients with low DoT compared with those with a high DoT for most AEs in both IFN and IPI10 cohorts. CONCLUSION: Only 5% of high-risk patients with melanoma following lymph node dissection surgery were treated with adjuvant IPI10 or IFN, while the remaining were untreated. Patients treated with IPI10 or IFN had high AE rates impacting their ability to remain on therapy. Newer adjuvant treatments with more favorable AEs and DoT profiles are urgently needed for high-risk patients with melanoma.

Disclosure: This research was funded by Bristol-Myers Squibb.

#3642

Relationship of argininosuccinate synthetase (ASS) and PD-L1 expression in arginine enriched or depleted environment in melanoma.

Chunjing Wu,1 Ying-Ying Li,2 Medhi Wangpaichitr,2 Lynn G. Feun,3 Macus T. Kuo,4 Niramol Savaraj2. 1 _VA Medical Ctr., Miami, FL;_ 2 _Univ. of Miami/VA Medical Ctr., Miami, FL;_ 3 _Univ. of Miami, Miami, FL;_ 4 _University of Texas M. D. Anderson Cancer Center, Miami, FL_.

While treatment with BRAF/MEK inhibitors represents a breakthrough for melanoma with 80% response rate, recurrences often occur. Salvage treatment with check point inhibitors yield 40% response rate, but usually time to response is long and not suitable for aggressive disease. In this report, we investigate a new modality to improve treatment outcome in melanoma. We have previously shown that BRAF inhibitor resistant (BR) and combined BRAF and MEK resistant (BMR ) melanomas express low levels of argininosuccinate synthetase (ASS1-) compared to their parental cells (Oncotarget 7:17665 ,2016) . Treatment with pegylated arginine deiminase (ADI-PEG20 from Polaris Inc), which degrades arginine (Arg) to citrulline results in growth inhibition and cell death. However, its effect on check point inhibitor ligand PD-L-1 is not known. In this report, we studied PD-L1 gene expression upon arginine deprivation in parental, and their respective BR and BMR cells as well as BRAF wild type cell. We have analyzed PD-L1 expression by flow cytometry as well as immunoblot in 4 pairs of melanoma cells (A2058 ,A375, Mel1220 and UACC62) and their BR and BMR counterpart . Our results showed a 5-20% increase in PD-L1 expression in both BMR and BR cells. BMR cells showed higher levels ( 5-10%) more compared to BR cells. The highest levels of increase in BR and BMR cells are found in A2058. Interestingly, this cell line is known to have inducible ASS upon arginine deprivation. The induction can be found at 72 hr. post ADI-PEG20 treatment. Next, we treated this panel of cell lines with ADI-PEG20 for three days and assessed the PD-L1 expression. The results showed that PD-L1 expression increased slightly (<5% ) in parental cells but more in BR and BMR cells (10-20% ). Interestingly, A-2058 R, a cell line made resistant to ADI-PEG20 with high levels of ASS expression, expressed the highest amount of PD-L1 (approximately 50 times higher). In contrast, the ASS transfected cell lines (A2058ass, A375ass , Mel1220ass) showed no changes in PD-L1 compared to their parental cells. This indicates that ASS does not influence PD-L1 expression. However, Arg deprivation in (ASS-) cells is the factor influencing PD-L1 expression. Interestingly, we also found increase in PD-L1 expression in tumor samples obtained from patients who failed BRAF and MEK inhibitor. On the other hand, melanoma cells with wild type BRAF and low ASS expression (one cell line and 5 primary cultures ) showed variable amounts of PD-L1 expression with minor increase upon ADI-PEG 20 treatment. There is no relationship with mTOR activity or pERK with PD-L1 expression in these panels of cell lines. The mechanism(s) involving HIF1 alpha is being investigated. Overall, our data suggest that combination with ADI-PEG20 and PD-L1 inhibitor, sequentially or concomitantly, should be beneficial for BR and BMR patients. Supported by VA Merit Review Award (1BX003328).

### Liquid Biopsy 3

#3643

Utility of peritoneal and pleural lavages for detection of somatic alterations: A proof-of-concept study.

Núria Jordana Ariza,1 Mónica Garzón Ibañez,1 Alejandro Martinez-Bueno,2 Sonia Gatius Caldero,3 Clara Mayo de las casas,1 Ana Velasco,3 Maria Ruiz,3 Berta Roman Canal,3 Jordi Bertran Alamillo,1 Sonia Rodríguez Muñoz,1 Raquel Campos Fuentes,1 Beatriz García,1 Ariadna Balada Bel,1 Sergi Villatoro,1 Miguel Ángel Molina-Vila,1 Xavier Matias-Guiu3. 1 _Pangaea Oncology, Barcelona, Spain;_ 2 _Hospital Quirón Dexeus, Barcelona, Spain;_ 3 _Hospital Universitario Arnau de Vilanova (HUAV), Barcelona, Spain_.

Background: Surgical resection remains the best treatment option for early-stage endometrial (CE), colorectal (CRC) and lung cancer (LC) patients. However, a proportion of patients (p) develop tumor recurrence, even after curative resection. Peritoneal or pleural lavages may be performed during surgery but little data is available about their potential as liquid biopsies with predictive value for recurrence. This proof-of concept study aimed to determine if somatic mutations can be detected in these lavages.

Methods: We analyzed mutations in paired surgical biopsies, blood and peritoneal or pleural lavages supernatants in an early-stage cohort of 25 EC, 12 CRC and 19 LC p. Molecular characterization of surgical biopsies was performed using the GeneReader NGS platform (Qiagen) and the Actionable Insight Tumor Panel, which includes 12 genes (KRAS, NRAS, KIT, BRAF, PDGFRA, ALK, EGFR, ERBB2, PIK3CA, ERBB3, ESR1, and RAF1). cfDNA was purified from blood and lavages using an automatic extractor and the hotspot mutations identified in tumor tissue were tested using a sensitive Taqman assay for codons 12, 13, and 61 in KRAS, codons 542, 545 and 1047 in PIK3CA and codon 600 in BRAF.

Results: NGS of tumor biopsies revealed hotspot KRAS, PIK3CA or BRAF mutations in a total of 11/25 (44%) early-stage EC p, 7/12 (58%) early-stage CRC p and 6/19 (32%) early-stage LC p. In peritoneal lavages, hotspot mutations were found in 6/11 (55%) EC p and 2/7 (29%) CRC p and, in pleural lavages, hotspot mutations appeared in 2/6 (33%) LC p. Furthermore, 2/11 (18%) blood samples from EC p, 1/7 (14%) blood samples from CRC p and 1/6 (17%) blood samples from LC p were positive.

Conclusions: Our study reveals that mutational analysis in peritoneal or pleural lavages from early stage tumors is feasible and that mutations are more frequently detected in lavages than in blood. Further research will determine if positivity in blood or lavages is associated with early relapse.

#3644

Blood tumor mutational burden (bTMB) from circulating tumor DNA (ctDNA) as a biomarker for both mutational status and tumor burden in non-small cell lung cancer (NSCLC).

Andrew A. Davis, Carlos Galvez, Alan Pan, Nisha Mohindra, Victoria Villaflor, Young Kwang Chae. _Northwestern Univ., Chicago, IL_.

Background:

Tissue tumor mutational burden (tTMB) has emerged as a viable biomarker for response to immune checkpoint blockade (ICB) in multiple histologies including NSCLC. Blood TMB (bTMB) is a novel summative measure of ctDNA genomic alterations in the peripheral blood. Few studies have examined the clinical utility and correlation of bTMB with tumor burden and survival.

Methods:

We identified 53 patients, retrospectively, who had paired blood (Guardant Health) and tissue (Foundation Medicine) next-generation sequencing testing. bTMB was determined by taking the number of reported mutations over the sequencing length of ctDNA. bTMB was calculated for research purposes and has not been approved for clinical use. From the cohort, 36 patients had baseline CT scans with measurable scores by RECIST version 1.1. Survival analyses by quartile were performed for the subset of patients (N=18 PFS, N=19 OS) treated with ICB.

Results:

bTMB was significantly correlated with baseline RECIST tumor burden estimations (p=0.010, Pearson Correlation, r=0.42, Table 1). There was a trend toward mutant allele frequency (MAF) of the highest clone in blood correlating with RECIST score (p=0.061). No significant correlation was found when comparing blood and tissue TMB scores in paired patients (p=0.70). In the subset of patients treated with ICB, higher bTMB was significantly associated with shorter PFS (HR 1.81 [1.03-3.18], p=0.038) and OS (HR 2.23 [1.09-4.58], p=0.029).

Conclusions:

This study demonstrated the feasibility and clinical utility of bTMB. Our findings may indicate that bTMB reflects tumor shedding into the blood as a surrogate for tumor burden and heterogeneity given the correlation with RECIST scores. In survival analyses, higher bTMB predicted shorter survival, which also reflects bTMB as a biomarker for tumor burden. Prospective studies are necessary to study bTMB in larger cohorts of patients treated with ICB. | |

---|---|---

|

|

|

Correlation Coefficient | P

RECIST-Blood TMB  | r = 0.424 | P = 0.010

RECIST-Tissue TMB  | r = 0.238 | P = 0.198

RECIST-MAF | r = 0.316 | P = 0.061

MAF-Blood TMB  | r = 0.182 | P = 0.289

MAF-Tissue TMB  | r = 0.336 | P = 0.065

Tissue TMB-Blood TMB  | r = 0.073 | P = 0.695

#3645

Quantifying cell populations in CTC enriched samples with the open-source imaging program ACCEPT.

Sanne De Wit,1 Leonie L. Zeune,1 T. Jeroen N. Hiltermann,2 Harry J. Groen,2 Leon W.M. Terstappen1. 1 _Univ. of Twente, Enschede, Netherlands;_ 2 _University Medical Center Groningen, Groningen, Netherlands_.

Introduction.

Composition of cell suspensions enriched for Circulating Tumor Cells (CTC) by the CellSearch system has not yet been explored as the system only presents DAPI+/Cytokeratin+ objects to the operator to identify CTC. We used ACCEPT (http://github.com/LeonieZ/ACCEPT) to identify and characterize all DAPI+ nuclei in EpCAM enriched samples and explored avenues to increase the number of nuclei that can be assigned to a cell lineage. Addition of CD16, expressed on granulocytes and NK-cells, the detection of a cellular plasma membrane and the use of LED light, as opposed to the mercury arc lamp, to improve the excitation of CD45-APC were tested.

Methods.

In 127 controls and 300 samples from 192 metastatic lung cancer patients, CD16-PerCP was added to the CellSearch CTC identification antibody cocktail. A CellTracks Analyzer equipped with six filter cubes was used to acquire the images. For exploration, after the CellTracks image acquisition 5 cartridges were scanned again on a microscope that contains a LED light source to determine if this would improve the detection of CD45-APC. Also, wheat germ agglutinin (wga) conjugated to AlexaFluor488 stains the cellular plasma membrane and was added to the CellSearch antibody cocktail to discriminate between bare nuclei and unstained cells in 4 samples. The stack of fluorescence images was analyzed with the open source imaging program ACCEPT to define and identify various cell populations.

Results.

In 427 samples 26,285 ±26,590 [603-156,519] (mean, SD, [range]) nuclei were identified with ACCEPT in the CellSearch images of which only 14% ±15 were identified as leukocytes or CTC, remaining 86% ±15 cells unidentified. Adding CD16 increased the leukocyte population by 47% ±17, thereby reducing the unidentified population to 39% ±19 of the total number of nuclei. CD16-staining was found on 13% of the CTC. Using a LED-light source improved the identification of APC+ cells, and reduced the amount of unidentified nuclei in these samples from 29% (±5) to 14% (±10). Exploration with wga indicated that 43% (±11) (mean 987 cells, range 331-2,638 cells) of the unstained cells contained a cellular membrane.

Conclusion.

In 427 blood samples run on CellSearch, only 14% of the nuclei obtained after EpCAM enrichment could be assigned as a leukocyte or a CTC. By adding CD16 to the antibody cocktail, the unidentified population is reduced from 86% to 39%. Also, 13% of nuclei scored as CTC express CD16, suggesting these are granulocytes that are incorrectly designated as tumor cells. Using a LED light source improves the identification of dimly stained CD45+ cells. Exploration with wga indicates that 57% of the unidentified nuclei are simply bare nuclei and the origin of 43% still remains unknown. Further studies are needed to determine whether EpCAM+/Cytokeratin- CTC are present among the nucleated cells of which the origin is not yet known.

#3646

Investigation of origin of circulating free DNA: Is exosomal DNA the carrier.

Chiho Nakashima, Tomonori Abe, Akemi Sato, Tomomi Nakamura, Kazutoshi Komiya, Eisaburo Sueoka, Shinya Kimura, Naoko Sueoka-Aragane. _Saga University, Saga city, Japan_.

The usefulness of circulating free DNA (cfDNA) for analysis of genetic alterations is largely accepted. We accomplished multicenter prospective study to investigate sequential change of cfDNA in lung cancer patients who acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors. The detection frequency was elevated as cancer progressed, and the prognosis of the patients in which cfDNA was detected was poorer than those not detected. These results suggest that appearance of cfDNA was associated with tumor progression. To verify that, animal experiment using immunodeficient mice, NOJ mice, was performed. After transplantation with human lung cancer cell line, H1975 carrying EGFR L858R, and T790M, into dorsal flanks of these mice, systemic metastasis occurred. cfDNA was sequentially analyzed, resulting that the amount of cfDNA was correlated with tumor burden and metastatic status. In spite of these results, origin, kinetics or possible function on tumor progression of cfDNA has not been elucidated. We have reported there were large sized DNA fragments, around 5 Kb, which is longer than 170 bp of cfDNA conventionally detected in peripheral blood of advanced cancer patients. Exosomes, extracellular vesicles detected in peripheral blood has been reported to be involved in tumor progression through vesicle-mediated communication. In general, exosomes deliver protein, lipid and RNA, and few DNA was contained. However, some researchers reported a large proportion of cfDNA was localized in exosomes. To investigate the origin and localization of cfDNA in peripheral blood, we analyzed relationship between cfDNA and exosomal DNA. We isolated both of cfDNA and exosomal DNA simultaneously, from plasma of healthy individuals and advanced non-small cell lung cancer patients, and compared the DNA yield, DNA size distribution and EGFR mutation detection rate. Localization of DNA in exosome is also investigated using fluorescent dye. We used Total Exosome Isolation Kit® from plasma with/without proteinase K for isolation of exosome from 200 uL plasma, and extracted DNA using Maxwell RSC® circulating cell free DNA cartridge. The proportion of exosomal DNA /cfDNA (e/c DNA ratio) varied individually, but e/c DNA ratio was lower in plasma from cancer patients than healthy individuals. However, large sized fragments of cfDNA were observed in exosomal DNA in lung cancer patients. These results suggest that exosomal DNA might have some interaction with large sized cfDNA fragments observed in plasma isolated from advanced cancer patients.

#3647

Prior DNA repair improves the read quality in next-generation sequencing of cell-free tumor DNA.

Hong Yin, Adam H. Greer, Glenn Mills. _Louisiana State Univ. Health Sciences Ctr., Shreveport, LA_.

The cell-free tumor DNA (ctDNA) in patient's plasma is a noninvasive tumor biomarker. The detection of ctDNA is helpful to evaluate tumor progression, tumor response to the therapy, and subtyping of tumor cells. Targeting next-generation sequencing (NGS) technology provides a sensitive and specific technique for ctDNA detection. It can detect multiple tumor-related gene mutations. However, collection of high quality of cell-free DNA (cfDNA) may not be available in some cases. The quality of cfDNA in plasma is impacted by multiple factors, such as plasma collection and storage conditions, DNase activity in the blood, and the apoptotic and necrotic status of the tumor. These factors affect the integrity of cfDNA, leading to full or partial degradation of cfDNA, including double-strand DNA breaks and single strand DNA nicking. Lesions on cfDNA decrease the usability of ctDNA by digital PCR and targeted NGS. Due to amount limitation of cfDNA in some cases, the repair of damaged cfDNA could be a necessary rescue step in producing viable libraries for NGS.

CfDNA was extracted from the plasma with Zymo Research's Quick-cfDNA Serum and Plasma kit and was quantified by realtime PCR with Alu primer sets. The size of cfDNA was about 160 base pairs, as estimated by Agilent Tapestation 2200 with the High Sensitivity DNA kit. The DNA repair of cfDNA was conducted with PreCR Repair Mix of New England BioLabs. The sequencing library was prepared with Accel-Amplicon 56G Oncology Panel of Swift Biosciences and sequenced in Illumina's MiSeq. The quality of sequencing data was determined with FastQC. Compared to PreCR Repair Mix treated cfDNA samples, fastQ data from some of untreated cfDNA samples showed failed per base sequencing quality, about 50% of sequencing cycles with the mean quality score below 30. The per sequencing quality score of these cfDNA samples failed also to pass the quality filter. The read size distribution analysis indicated that about 60 to 70% of read sized between 30 to 45 bases and about 75 to 80% of read was smaller than 75 bases. However, sequencing data from same cfDNA samples treated with PreCR Repair Mix show a greatly improved sequencing quality. Both per base sequencing quality and per sequencing quality score passed quality filter. The above 97% of reads sized over 92 bases, complying with the requirement of 56G Oncology Panel.

In conclusion, the DNA repair process before library preparation is necessary to rescue damaged cfDNA for amplicon-based NGS.

#3648

Performance of the AVENIO ctDNA assays across multiple high-throughput next-generation sequencing platforms.

Jonathan Choi, Richard Dannebaum, Ashla Singh, Rob Foley, Jorge Dinis, Cindy Choi, Bosun Min, Jingchuan Li, Liang Feng, Fergal Casey, Janet Jin. _Roche Sequencing Solutions, Pleasanton, CA_.

Introduction:

Molecular profiling of tumor samples is becoming routine practice in the clinic, particularly for therapy selection. However, in some cases, tumor tissue is not available for testing. Liquid biopsy, which enables the analysis of circulating tumor DNA (ctDNA) shed from the tumor into the blood, can be used as a surrogate for conventional tissue based testing to detect somatic mutations. Furthermore, ctDNA profiling can have potential applications in tumor burden monitoring. Some technical challenges must be overcome to accurately and sensitively detect the often low amount of ctDNA present in plasma. The AVENIO ctDNA Analysis Kits (Targeted, Expanded and Surveillance Kits; Research Use Only) were developed to sensitively detect all 4 major mutation classes: single nucleotide variants, insertions/deletions, fusions, and copy number variations, using a hybrid capture target enrichment workflow, molecular barcoding, and next generation sequencing. We previously presented a comprehensive study of 370+ samples demonstrating analytical performance of the assays on the Illumina NextSeq 500. As ctDNA based analysis becomes more prevalent in the clinical research community, there is great desire for high performing and cost-effective assays that are easy to implement and available across multiple sequencing platforms.

Methods:

We used the commercially available AVENIO ctDNA Analysis Kits (Expanded, Target and Surveillance Kits; Research Use Only) pre-sequencing workflow to process a panel of cell line blends and plasma-derived ctDNA samples in order to compare the performance of the assays on multiple Illumina sequencing platforms: NextSeq 500, HiSeq 4000 and HiSeq 2500. Ninety samples were tested across all platforms. The samples contain SNVs at 0.5% minor allele frequency (MAF), deletions at 1% MAF, selected fusions at 1% MAF, and MET amplification at 2.3 copies. Analysis was done using the AVENIO ctDNA analysis pipeline for all sequencing runs across the sequencing platforms.

Results: On the NextSeq 500, the AVENIO ctDNA Analysis Kits achieve sensitivities of >99% for 0.5% SNVs, >99% for 1% deletions, >96% for 1% fusions, and >99% for 2.3 copies of MET amplification, with 96% to >99% specificity for all mutations. Similar performance is observed across additional Illumina platforms, HighSeq 4000 and HiSeq 2500. Across all platforms, key sequencing metrics are consistent to achieve required sequencing depths, on-target rates, and uniformity. Also, it is noted that in order to achieve required depths, each platform requires different levels of sample multiplexing. We demonstrated that the AVENIO ctDNA Analysis Kits achieve high sensitivity and specificity across multiple high throughput sequencing platforms. We also will highlight the key performance differences and considerations when performing the assay across these sequencing platforms.

#3649

Circulating microRNA-181a monitors response to FOLFIRINOX chemotherapy in pancreatic ductal adenocarcinoma.

Laura L. Meijer,1 Ingrid Garajová,2 Chiara Caparello,3 Tessa Y.S. Le Large,1 Niccola Funel,3 Enrico Vasile,3 Geert Kazemier,1 Elisa Giovannetti1. 1 _VU University Medical Center, Amsterdam, Netherlands;_ 2 _University Hospital of Bologna, Bologna, Italy;_ 3 _University Hospital of Pisa, Pisa, Italy_.

FOLFIRINOX is nowadays the standard combination chemotherapy for patients with advanced pancreatic ductal adenocarcinoma (PDAC). However, not all patients benefit from this intense therapy and biomarkers to monitor response are lacking. MicroRNAs (miRNAs) have been shown to regulate multiple genes involved in cancer and are able to serve as biomarker. Therefore, this study aimed to explore the role of circulating miRNAs as stratifying and monitoring biomarkers to predict response to FOLFIRINOX. Further, we investigated the effects of miR-181a-5p in vitro on cell proliferation, key targets, and drug sensitivity to 5-fluorouracil, oxaliplatin and irinotecan. In a discovery cohort of 11 patients grouped according to their long vs. short progression-free survival (PFS), the profiles of 179 miRNAs were analyzed with a specific microarray for plasma/serum miRNAs in matched plasma samples before and after FOLFIRINOX therapy. Using quantitative RT-PCR, nine selected miRNAs were validated in a second cohort (n=43). Circulating miR-181a-5p emerged as the most promising miRNA, with significant down-regulation in non-progressive compared to progressive patients after FOLFIRINOX (p=0.003; fold change = -1.70 versus 3.84, respectively). This down-regulation correlated with improved PFS and OS in uni- and multi-variate analyses, especially in combination with decrease of CA19.9 (log-rank p<0.001 and p=0.033, respectively). In situ hybridization in tissue-microarrays built with representative cores from 15 patients revealed that miR-181a was mostly expressed in tumor cells and not in stromal tissue. Remarkably, tissue expression displayed similar down-regulation of miR-181a after treatment. In vitro oligonucleotide transfection of miR-181a showed that enhanced expression of miR-181a increased proliferation of PDAC cells and decreased expression of ATM, a key target of miR-181a. Most strikingly, inhibition of miR-181a in combination with oxaliplatin treatment increased DNA damage and decreased cell viability. This study supports the use of miR-181a-5p as monitory biomarker to select patients most likely to respond to FOLFIRINOX and guide therapy. MiR-181a-5p inhibition could enhance response to oxaliplatin by amplifying DNA damage response and cell death. In conclusion, our clinical data, together with our in vitro findings, strongly suggest that miRNA profiling in plasma provides promising tools for prognostic, predictive, and therapeutic purposes in PDAC patients.

#3650

Use of unique molecular identifiers to gain insight about the true positive mutations in cfDNA of breast cancer patients for implementation of personalized treatment.

Corinna Keup,1 Karim Benyaa,2 Peter Hahn,2 Siegfried Hauch,2 Pawel Mach,1 Mitra Tewes,1 Hans-Christian Kolberg,3 Sabine Kasimir-Bauer1. 1 _University Hospital of Essen, Essen, Germany;_ 2 _QIAGEN GmbH, Hilden, Germany;_ 3 _Marienhospital Bottrop gGmbH, Bottrop, Germany_.

Background: Blood analytes, as liquid biopsies, are discussed to be surrogate markers for therapy stratification, as serial sampling enabled by the minimal invasive nature of blood draw facilitates monitoring of clonal evolution. Mutational analysis of cell-free DNA (cfDNA) in plasma of breast cancer (BC) patients (pts) may predict the ideal therapy strategy. In this regard, PIK3CA mutations in BC pts' cfDNA were already shown to correlate with treatment response. However, in Next Generation Sequencing (NGS) workflows, used for cfDNA analysis, artefacts are often induced during library construction. Here we used unique molecular identifiers (UMIs) to verify true positive PIK3CA mutations in cfDNA of BC pts.

Patients and methods: cfDNA was isolated from 54 plasma samples of 38 BC patients by affinity-based binding to magnetic beads (QIAamp MinElute ccfDNA Kit, QIAGEN, Germany). 30ng - 60ng cfDNA was used for library construction with the QIAseq Targeted DNA Panel for Illumina (QIAGEN, Germany) with integrated UMIs. Sequencing was executed on the NextSeq ® 500 platform (Illumina, US). Data analysis was performed by QIAseq Targeted Sequencing Data Analysis Portal and the Biomedical Genomics Workbench. As a reference, the PIK3CA mutational status of matched tumor tissue DNA (analyzed by Sanger sequencing) was consulted.

Results: Library preparation was successful (yield >15ng) in 52/54 cases. Mean coverage was ~20.000x (mean UMI coverage ~2.500x) and >10.000x in 48/52 cases. The minimal allele frequency found for PIK3CA hotspot mutations (P539S, E545K, H1047R) by UMI analysis was 0.72%. In total, 133 mutations of the PIK3CA gene were identified as true positive mutations by UMI analysis in all 52 samples, which is a reduction of 69% (294/427) of all PIK3CA mutations incorrectly called by conventional data analysis. 59% of all different PIK3CA mutations called by UMI analysis appeared in over 5% of all pts. In the cohort of pts with PIK3CA hotspot mutant tumor tissue, 32% (6/19) showed the mentioned mutations also in matched cfDNA, whereas 16% of pts (3/19) without PIK3CA mutant tumor tissue were identified with true positive PIK3CA hotspot mutations in their plasma. Longitudinal analysis across two years during therapy revealed the increase in allele frequency (0%;11%;39%) of the PIK3CA H1047R mutation in one pt, whereas another pt showed a stable allele frequency of the PIK3CA P539S mutation (52%; 51%; 48%). Results will be expanded by consideration of mutations in BC hotspot genes despite PIK3CA and correlation to clinical parameter.

Conclusions: Unique molecular identifiers enable the identification of true positive mutations in cfDNA and can thus, be used in clinical practice to determine molecular drivers of individual cancer progression and to employ personalized therapy.

#3651

Implementation of a new and easy device, the QIAscout, in the selection and molecular characterization of single circulating tumor cells.

Janina Levermann,1 Norbert Hochstein,2 Siegfried Hauch,2 Ruth Klaever,2 Rainer Kimmig,1 Sabine Kasimir-Bauer1. 1 _University Hospital of Essen, Essen, Germany;_ 2 _QIAGEN GmbH, Hilden, Germany_.

Background: The molecular characterization of circulating tumor cells (CTCs) is widely used to identify targets for individualized treatment options. However, analyzing the whole bulk of CTCs does not give information whether a co-expression of more than one CTC-marker is derived from CTCs, actually co-expressing these markers on the same cell, or from separate CTCs. Here we present a new device, the QIAscout (QIAGEN, Germany), offering the possibility of microscopic single cell isolation for subsequent molecular analysis.

Methods: CTCs were isolated from 2x 5ml whole blood of 15 metastatic breast cancer (MBC) patients (pts) using positive immunomagnetic selection (AdnaTest EMT2/StemCell SelectTM) followed by QIAscout single cell isolation in one of the two blood samples. Cells added to the QIAscout array settle down and adhere to microrafts which is supported by pre-treating the array with Corning® Cell-TakTM Adhesive. Using an inverted microscope, single cells can be mechanically isolated by picking out one of the 12000 microrafts. Subsequently, in both samples, mRNA bound to Oligo-dT beads was purified, reverse transcribed (AdnaTest EMT2/StemCell DetectTM) and the cDNA was pre-amplified and analyzed by a multimarker qPCR Panel (AKT2, ALK, AR, AURKA, BRCA1, KIT, MET, EGFR, ERCC1, ERBB2, ERBB3, KRT5, mTOR, NOTCH1, PARP1, PIK3CA, SRC, GAPDH), normalized by data of healthy donors (n=20) with CD45 as leukocyte control. QIAscout further allowed single cells analysis, amplified by whole transcriptome amplification (WTA - REPLI-g WTA Single Cell Kit) followed by qPCR analysis as performed for the bulk of cells. Establishment of the methods was performed using the breast cancer cell line MCF-7 spiked into blood of healthy donors. All consumables were purchased from QIAGEN (Germany).

Results: "Spike-in" experiments revealed CTC recovery rates of about 70% and subsequent CTC characterization in MCF-7 cells and pts` blood samples resulted in a markedly decreased leukocyte contamination implementing the QIAscout into CTC analysis. However, the positivity rate of overexpressed genes was reduced in MBC pts up to 33% using QIAscout as compared to bulk samples. In five pts, gene expression in the bulk of CTCs was compared with at least 2-4 single cells of the same pts, using QIAscout. A good concordance was achieved for CTC-positivity and all single CTCs showed a broad variety in gene expression. At least one of the single cells expressed more genes than the bulk of CTCs and the expression of target genes that define treatment options, e.g. HER2, was often expressed on only 1/4 analyzed cells whereas resistance markers were detected in nearly every CTC of the same pts.

Conclusion: The implementation of the QIAscout in CTC analysis allows the molecular characterization of single tumor cells to get insights into single cell heterogeneity for further therapeutic strategies.

#3652

Analytical validation of a novel circulating tumor DNA detection platform for targeted and immunotherapy selection.

Pratyush Gupta,1 Julian Lucas,1 Alex Atkins,1 Wen-Sy Tsai,2 Twinkal Marfatia,1 Shih-En Chang,1 Oscar Segurado,3 Rui Mei1. 1 _CellMax Life, Sunnyvale, CA;_ 2 _Chang Gung Memorial Hospital, Taoyuan, Taiwan;_ 3 _MedicAffairs Consulting, San Jose, CA_.

Introduction Comprehensive genomic profiling of solid tumors using circulating tumor DNA (ctDNA) has enabled the detection of all NCCN guideline-recommended somatic genomic classes of alterations from a single, non-invasive blood draw. However, current ctDNA tests still face two major challenges: the inability to reliably identify somatic variants at low mutant allele fraction (MAF), and inconsistency in how the tests have been validated. This study shows how the Single Molecule Sequencing (SMSEQ) platform addresses these challenges. The platform integrates innovative ctDNA extraction methodology, highly optimized library preparation and an error-based variant-calling algorithm to drastically improve sensitivity and specificity. The platform analyzes 5 classes of somatic variants: single nucleotide variants (SNVs), insertions and deletions (Indels), copy number variants (CNVs), fusions and microsatellite instability (MSI).

Methods We analyzed a 73 gene panel covering NCCN recommended actionable variants for solid tumors in 60 reference ctDNA samples with known variants to establish the limit of detection, sensitivity, specificity, accuracy and reproducibility of the SMSEQ platform. For clinical validation, we tested 36 patients with metastatic colorectal cancer (mCRC) and 34 healthy controls from the Chang Gung Memorial Hospital, and 227 patients diagnosed with solid tumor from Taiwan. Circulating DNA was extracted from plasma followed by library preparation using a highly optimized NGS workflow. Somatic variants in ctDNA are identified using locus-specific modeling to separate tumor variants from normal errors.

Results Validation according to recently published ACMG/AMP guidelines shows that the SMSEQ platform allows calling of variants with >99.999% analytical specificity for SNVs, Indels and fusions; and >99% analytical specificity for CNVs and MSI. The platform successfully detected variants at low MAF: 0.1% for SNVs and Indels, <1% for fusions, 5 copies for CNVs, and 1% for MSI. Somatic variants were identified in 35 of 36 mCRC patient samples (97.2%). No false positives were observed within the targeted region for all 34 healthy controls tested. In paired samples, the SMSEQ platform showed 89.7% concordance with tissue biopsy. Observed gene mutation profiles from ctDNA were consistent with published tissue biopsy data: the most frequently mutated genes were TP53, APC, and KRAS; KRAS and BRAF variants were mutually exclusive. In addition to mCRC patients, we tested 227 patients diagnosed with various solid tumors from Taiwan. Actionable variants were detected in 170/227 (75%) patients.

Conclusion The CellMax 73-gene liquid biopsy test detects 5 NCCN-guideline recommended variant classes: MSI for immunotherapy and SNVS, Indels, CNVs, and fusions for targeted therapy selection at low variant allele fraction/copy number at high sensitivity and specificity.

#3653

Sequencing of plasma cfDNA from patients with locally advanced bladder cancer for surveillance and therapeutic efficacy monitoring.

Karin Birkenkamp-Demtröder,1 Emil Christensen,1 Shruti Sharma,2 Ann Taber,1 Mads Agerbæk,1 Himanshu Sethi,2 Hsin-Ta Wu,2 Svetlana Shchegrova,2 Philippe Lamy,1 Iver Nordentoft,1 Ryan Swenerton,2 Samantha Navarro,2 Tony Tin,2 Ramya Srinivasan,2 John So,2 Spenser Alexander,2 Solomon Moshkevich,2 Bernhard Zimmermann,2 Cheng-Ho Jimmy Lin,2 Jørgen Bjerggaard Jensen,1 Lars Dyrskjøt1. 1 _Aarhus University Hospital, Aarhus N, Denmark;_ 2 _Natera, inc., San Carlos, CA_.

Minimally invasive methods for assessment of tumor burden, early detection of disease relapse and for monitoring therapeutic efficacy are needed to improve individualized follow-up and treatment for patients diagnosed with bladder cancer. The ability to predict pathologic complete response after neoadjuvant chemotherapy (NAC) through detection of ctDNA in plasma may enable strategies for bladder preservation. The aim of the study was to use patient-specific mutations to identify residual disease, metastatic relapse and to monitor treatment response in longitudinally collected plasma samples. For this, 50 patients diagnosed with locally advanced muscle-invasive bladder cancer (MIBC) and scheduled for chemotherapy were prospectively recruited between 2013 and 2017. In total, 42 patients received four cycles of cisplatin-based NAC prior to cystectomy and 82% showed response (pathologic downstaging). Eight patients received 2-6 cycles of cisplatin-based first-line chemotherapy due to diagnosis of T4b or lymph node metastasis prior to cystectomy (3CR, 2PR, 2PD, 1 ongoing). So far, 8/50 patients (16%) experienced disease relapse and three patients had metastatic progression. The mean follow-up time after radical cystectomy (RC) was 320 days (65-973); the mean follow-up time for disease-free patients was 397 days (119-778). Whole-exome sequencing (106x mean target coverage) of tumor and germline DNA was performed from a time point before systemic treatment. MUTECT2 identified a mean of 33 high (6-121), 340 (67-2838) moderate and 223 (29-2955) low-impact SNVs or insertion-deletions (InDels) per tumor. High-impact mutations in known cancer genes such as TP53 (60%), KDM6A (34%), ARID1A (32%), RB1 (28%), BRCA2 (26%), FGFR3 (22%) and ERCC2 (20%) were identified with no significant difference between responders vs nonresponders. Signatures 2, 13 (APOBEC), and 3 (BRCA) were frequently identified, with signature 13 being more prevalent in nonresponders (p=0.05). Personalized, multiplex-PCR assay-panels were individually designed, targeting each patient's tumor-specific mutations in plasma. Targeted sequencing was performed using cfDNA from 4-16 longitudinally collected plasma from each patient taken pre- and post-systemic therapy and at scheduled control visits after RC. In a blinded fashion, ctDNA was analyzed and results were compared to pathologic response and radiographic imaging data for each patient. Conclusion: Preliminary results indicated that analysis of plasma cfDNA is an appropriate method to detect metastatic relapse after RC and to monitor treatment efficacy. Here we perform a larger ctDNA study, applying direct sequencing of plasma cfDNA using highly sensitive NGS methods to determine clinical applicability of the method. Data collection is ongoing and all details will be presented at the AACR 2018 meeting.

#3654

**Identification of resistance mutations to crizotinib through circulating tumor cell (CTC) analysis in** ALK **-rearranged non-small-cell lung cancer (NSCLC).**

Emma Pailler,1 Vincent Faugeroux,1 Marianne Oulhen,2 Claudio Forcato,3 Mélanie Laporte,4 Yann Lecluse,4 Ludovic Lacroix,4 Maud NgoCamus,4 Claudio Nicotra,4 Jordi Remon,4 Laura Mezquita,4 David Planchard,4 Jean-Charles Soria,4 Nicolò Manaresi,3 Benjamin Besse,4 Françoise Farace1. 1 _Gustave Roussy / INSERM U981 / Univ Paris-Saclay, Villejuif, France;_ 2 _Gustave Roussy / INSERM U981, Villejuif, France;_ 3 _Menarini Silicon Biosystems, Castel Maggiore, Italy;_ 4 _Gustave Roussy, Villejuif, France_.

Background

Non-invasive methods including CTCs are crucial to develop for the implementation of precision medicine in the treatment of NSCLC. We recently reported how CTCs with abnormal ALK FISH-patterns monitored on treatment can stratify patients at risk of early-resistance to crizotinib (Pailler et al., Cancer Res, 2017). ALK-rearranged NSCLC patients treated with crizotinib develop resistance which manifest by the upregulation of bypass signaling pathways in two-thirds of cases. Here, we evaluated whether individual CTCs could identify resistance mutations to crizotinib and represent tumor diversity compared to single-site tumor-biopsies.

Methods

Individual CTCs were isolated from seven ALK-rearranged patients at resistance to crizotinib. Matched tumor-biopsies were available for three. Two isolation strategies were used: (i) ISET-filtration, immunofluorescence (Hoescht/CD45/epithelial-markers/mesenchymal-marker), scanning, laser-microdissection; (ii) RosetteSep, immunofluorescence (Hoescht/CD45/ALK/cytokeratins), cell-sorting. A process including Ampli1 whole-genome amplification, quality controls, multiplex PCR with two panels (Ampli1 CHPCustomBeta cancer panel developed by Silicon Biosystems and a home-made panel targeting all ALK mutations) and Ion Torrent next-generation sequencing (NGS) was established. Ten to 17 pools with 1-5 CTCs and one CD45+ cell pool were analyzed for each patient. A bioinformatic workflow was developed including determination of allele drop-out (ADO), false-positive rate (FPR) and positive predicted value (PPV).

Results

By comparing germline variants present in constitutional and CD45+ pool DNA, we calculated for each sample the statistics of mean ADO, FPR and PPV which were respectively 19%, 2.6x10-4 and 69%. When variants were present into two pools, the PPV and FPR were 96% and 2.39 10-5 respectively. In the three patients with an available tumor-biopsy, a limited number of shared mutations between CTC and matched tumor-biopsies were identified. In contrast, CTC-private mutations (exclusively present in CTCs and not in matched biopsies) present in at least two CTC pools were identified including in FLT3, SMAD4, KIT, PI3KCA, EGFR, KRAS, PDGFRA genes among which several were COSMIC mutations. A higher degree of mutational diversity was observed in CTCs compared to tumor-biopsies.

Conclusion

Using a rigorously qualified workflow, we reported for the first time the identification of resistance mutations in individually isolated CTCs of ALK-rearranged patients. In accordance with published series from tumor-biopsies, our CTC results highlighted the role of bypass signaling pathways in resistance to crizotinib. Our study provides important perspectives on using CTCs for precision medicine and informing on tumor heterogeneity in NSCLC patients.

#3655

DNA methylation analysis in liquid biopsy: A detailed study on quality control, sample storage and whole genome amplification procedures for downstream applications.

Sofia Mastoraki, Maria Chimonidou, Eleni Tzanikou, Evi S. Lianidou. _Analysis of Circulating Tumor Cells Lab, Univ. of Athens, Athens, Greece_.

Background: Studies on epigenetic alterations and especially DNA methylation in liquid biopsy samples like plasma circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) are of growing interest nowadays. To ensure the reliability of DNA methylation results prior to any clinical applications in liquid biopsy, there is an urgent need to standardize a plethora of parameters. The aim of our study was to evaluate: a) the stability of DNA methylation in plasma, b) the stability of sodium bisulfite (SB) converted DNA samples under different storage temperatures, and c) the accuracy and precision of whole genome amplification (WGA) protocols for downstream DNA methylation analysis. Methods: a) Stability of DNA methylation in plasma: Plasma from healthy donors was pooled and aliquoted in six tubes and spiked with genomic DNA (gDNA) isolated from 106 MCF-7 cells. Five aliquots were kept at -700C, while one was immediately processed for ctDNA isolation and SB treatment. Stored aliquots were processed in the same way after storage for a period of 3 months. All SB-treated DNA samples were further evaluated by real time methylation specific PCR (MSP) for ACTB and SOX17. b) Stability of SB-treated DNA: gDNA was isolated from 106 SKBR3 cells and processed for downstream SB-treatment. SB-treated DNA was split into aliquots and stored at -20°C and -70°C. One aliquot, used as control, was immediately processed for SB treatment followed by real time MSP for ACTB and BRMS1 amplification. The remaining aliquots were processed in exactly the same way after storage for different time points with a maximum period of one year. Additionally, a sample that was subjected to repeated freeze-thaw cycles was added in each run. c) Evaluation of WGA for SB-treated DNA: we evaluated two different SB conversion kits, EZ DNA Methylation-Gold (Zymo), and Epitect Fast Bisulfite, (Qiagen) in combination with downstream WGA (EpiTect Whole Bisulfitome Kit, Qiagen), using 0%, 100% methylated DNA standards and MCF-7, SKBR3 cell lines. Results: a) DNA methylation in plasma is stable at -700C for at least 3 months, since the average Cqs detected, varied from 1 to 2 cycles for ACTB and SOX17, respectively, b) Quality of SB-treated DNA is not affected by repeated freezing-thawing cycles; SB-treated DNA samples are stable when kept for up to one year both at -200C and -700C. c) Identical results by real time MSP were obtained before and after WGA amplification (Qiagen), after using either the Zymo or Qiagen kits for SB conversion. Conclusions: Plasma and SB-treated DNA samples are stable and can be safely used for multiple real time MSP assays over time when kept at -70°C. Downstream WGA of SB-treated DNA could bypass the limited amount of available sample for liquid biopsy applications. In any case, quality control checking should be included in each step of analysis.

#3656

Detecting MSI in plasma: Implications for early detection of lynch associated tumors.

Preethi Srinivasan, Christina Tran, Jonathan Reichel, Juber Ahamad Patel, Maysun Hasan, Fanli Meng, Xiaohong Jing, Sumit Middha, Ahmet Zehir, Rona D. Yaeger, Diane Reidy, Dana Tsui, Zsofia Stadler, Michael F. Berger. _Memorial Sloan Kettering Cancer Center, New York, NY_.

Lynch Syndrome (LS) is characterized by germline mutations in mismatch repair pathway genes. The increased cancer susceptibility risk in these patients and the association of these tumors with microsatellite instability (MSI) provides a transformative opportunity for early detection of cancer in these patients. The presence of microsatellite unstable DNA fragments or zygosity changes of the MMR mutations in the plasma of LS patients is predictive of the presence of cancer. Here we describe an approach to detect MSI from plasma cell-free DNA (cfDNA) of LS associated cancer patients. First, to determine the background rate of MSI in cfDNA of microsatellite stable patients, we screened plasma samples collected from a large number of advanced cancer patients using MSK-IMPACT, a custom sequencing assay targeting 468 cancer genes encompassing ~1.5 megabases. MSK-IMPACT is approved by the NYS Department of Health and authorized by the FDA for clinical testing, including MSI assessment based on more than 1,000 microsatellite regions covered by the assay, and has been used to profile more than 20,000 patients at our institution. We were also able to confirm the ability to detect MSI in cfDNA in a small cohort of plasma samples collected from patients with MSI High tumors, and determine thresholds to delineate MSI from MSS cases from plasma. Using MSK-IMPACT, we detected MSI in the plasma of four patients with advanced MSI-H cancer, including one with confirmed LS. Three of these were patients with confirmed MSI signature from tumor tissue (one prostate cancer and two colorectal cancers. The fourth patient with prostate cancer had no tissue available for sequencing, though MSI was independently confirmed. To improve the sensitivity to detect MSI in patients with earlier stage disease where the fraction of tumor-derived cfDNA is lower, we have developed a novel targeted panel with optimized sequencing process and informatics, incorporating a set of highly informative microsatellite regions as well as SNPs to assess tumor-specific zygosity changes in mismatch repairs pathway genes. Through this work, we demonstrate the ability of our assay to detect MSI in plasma cfDNA with high sensitivity.

#3657

Multi-laboratory assessment of a new reference material for quality assurance of circulating tumor DNA measurements.

Erica Stein,1 Hua-Jun He,1 Kenneth D. Cole,1 Russell K. Garlick,2 Yves Konigshofer,3 Tony E. Godfrey,4 Michael G. Goggins,5 Michael Borges,5 Margaret Gulley,6 Mickey Williams,7 Chris Karlovich,7 Corinne Camalier,7 Lynn Sorbara,8 Matthew R. Young,8 Sudhir Srivastava8. 1 _National Institute of Standards and Technology, Gaithersburg, MD;_ 2 _SeraCare Life Sciences, Milford, MA;_ 3 _SeraCare Life Sciences, Gaithersburg, MD;_ 4 _Boston University School of Medicine, Boston, MA;_ 5 _John Hopkins University, Baltimore, MD;_ 6 _University of North Carolina School of Medicine, Chapel Hill, NC;_ 7 _Frederick National Laboratory for Cancer Research, Frederick, MD;_ 8 _National Cancer Institute, Bethesda, MD_.

The accurate diagnosis and monitoring of cancer, using circulating tumor DNA (ctDNA), is a major challenge, given the low concentration and complexity of the target molecules. Suitable reference materials are required for clinical laboratories to achieve the high levels of measurement assurance and reproducibility. The methods and instruments for ctDNA assays are rapidly evolving to improve the sensitivity and specificity, resulting in a challenge to compare these different assays for the measurement of the diverse classes of ctDNA cancer biomarkers. To address this goal, a new reference material was developed consisting of 40 cancer mutations in a background of wild-type DNA at six different allele fractions (2%, 1%, 0.5%, 0.25%, 0.125, and 0 %). The cancer mutations include single nucleotide variants, insertions, deletions, and two structural variations that were selected for their clinical importance and challenges for next generation sequencing (NGS) methods. The reference material is formulated in a synthetic plasma matrix at a concentration of 25 ng/mL, levels that are consistent with patient samples. The DNA has been processed to accurately simulate the size of patient ctDNA, resulting in a similar conversion rate of the DNA for next generation sequencing methods. The samples were sent to five different laboratories involved in ctDNA measurements. Digital PCR assays for the variants were developed to confirm the allele fractions by several laboratories. When absorbance, fluorescent dye binding, and PCR methods were used to quantify the extracted ctDNA reference material, the results were significantly different, indicating the need for standard methods and reference materials to ensure reproducible results of this critical step. Different NGS methods (including targeted amplicons, hybrid selection, and SiMSen-seq) and instruments were used by the labs to analyze the refence materials at the different allele fractions. The results showed that the assays had significant differences in the ability to detect classes of variants at different concentrations. This new reference material was shown to be useful to benchmark the sensitivity and selectivity of different NGS methods and bioinformatic pipelines. This multi-laboratory assessment clearly demonstrates that the new reference material is highly valuable tool to ensure the reproducibility of ctDNA measurements including the sample extraction, analytical, and bioinformatic steps.

#3658

DNA methylation of circulating tumor educated leukocytes predicts IDH1/2 mutation status in adult patients with diffuse gliomas.

Andreas Kloetgen,1 Jonathan Serrano,1 Seema Patel,1 Christopher Bowman,1 Guomiao Shen,1 David Zagzag,1 Matthias A. Karajannis,2 John G. Golfinos,1 Dimitris Placantonakis,1 Aristotelis Tsirigos,1 Andrew S. Chi,1 Matija Snuderl1. 1 _New York Univ. Langone Medical Ctr., New York, NY;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY_.

INTRODUCTION: Identification of IDH1/2 mutations and association with better outcome revolutionized diagnosis and management of patients with gliomas. Liquid biopsy can detect driver mutation from blood, however, detection of circulating tumor DNA or circulating tumor cells from patients with brain tumors is difficult due to the low amount of circulating tumor material in the peripheral blood. We hypothesized that brain tumors alter the epigenetic profiles of circulating leukocytes and sought to identify specific methylation patterns in peripheral blood that would identify molecular subtypes of gliomas.

METHODS: We analyzed brain tumors and matched whole peripheral blood collected in EDTA tubes from 63 patients with primary gliomas. Tumor IDH1/2 mutation status was analyzed using next-generation sequencing and DNA methylation was analyzed using Illumina Human EPIC array. A complete blood count with differential (CBC) was analyzed to control for variations in CBC profiles. Peripheral blood methylation profile was compared to blood of 40 individuals with no known history of brain tumors. DNA methylation of Acute Myeloid Leukemia (AML) samples (source: TCGA) was used to identify overlapping DNA methylation changes induced by IDH1/2 mutations. Methylation data were analyzed with the R Bioconductor package minfi, including quality control, data normalization and differentially methylated CpG site analysis. Subsequent filtering was performed using a p-value cutoff = 0.01 and a minimal mean difference of the Beta-value of 0.1.

RESULTS: Peripheral blood methylation profiles of patients with brain tumors are distinctly different from normal controls. DNA methylation of IDH1/2 mutated brain tumors and AML showed significant overlap (enrichment = 3, p-value 2.2x10-16) and DNA methylation of IDH1/2 mutated AML cells showed significant overlap with circulating leukocytes from patients with IDH1/2 mutated brain tumors (enrichment = 6.5, p-value 2.2x10-8 ). Using this classifier supported by AML data, DNA methylation of circulating leukocytes was able to predict IDH1/2 mutation status of diffuse gliomas with 98% accuracy, nearly perfectly separating IDH status by clustering.

CONCLUSIONS: Diffuse gliomas induce epigenetic changes in the methylome of the leukocytes, which resembles methylation changes of IDH1/2 mutated AML. IDH wildtype and mutant gliomas can be distinguished with high accuracy by profiling the epigenome of leukocytes. Our study demonstrates the potential of peripheral blood DNA methylation assessment for non-invasive diagnosis of brain tumors.

#3659

Detection and validation of cancer immunotherapy biomarkers in blood.

Feng Xie, Jianjun Yu, Shidong Jia. _Predicine Inc., Hayward, CA_.

Immunotherapy response varies widely, making it difficult for physicians to know whether immunotherapy will be effective for a given patient. Indeed, ~80% or more patients with cancer fail to respond to checkpoint inhibitor immunotherapy. In addition to PD-L1 IHC staining, recent studies reported that patients with deleterious mutations in mismatch repair (MMR) genes, high tumor mutation burden (TMB) or microsatellite instability (MSI) are also associated with better clinical response. As tissue biopsy represents a practical challenge due to its insufficient quantity or lack of access, non-invasive molecular has emerged as an efficient complementary test and attracted increasing attention in clinical development of cancer immunotherapy.

With Predicine's gene RARDAR technology, we developed a blood-based PrediSeq NGS panel to capture genomic alterations in 180 cancer genes including tumor mutation burden (TMB) and microsatellite instability (MSI) and a Predi-CI (Cancer Immunotherapy) panel to measure dynamic expression change of 212 genes related to immune cells or immune responses.

Technical validation was performed to evaluate assay sensitivity, specificity and accuracy using reference samples with known genetic profiling. The panels have been further tested using tissue biopsy and plasma samples from cancer patients. The development of PrediSeq Panel and Predi-CI RNA gene expression tests offer a comprehensive solution to stratify and monitor cancer patients who may benefit from cancer immunotherapy.

#3660

Can the presence of fusion genes in circulating cell-free DNA serve as potential biomarkers for Ewing's sarcoma.

Shintaro Iwata,1 Hajime Kageyama,2 Tsukasa Yonemoto,2 Makiko Itami2. 1 _National Cancer Center Hospital, Tokyo, Japan;_ 2 _Chiba Cancer Center, Chiba, Japan_.

Background: Circulating cell-free DNA (cfDNA) is fragmented DNA derived from tumors that circulates in the blood of patients. With liquid biopsy, cfDNA has the potential to be used to monitor tumor burden in real time. Most reports have described that tumor-specific alterations in cfDNA are mutations, although little has been reported for the detection of tumor specific gene fusions. The objective of this study was to identify whether cfDNA can be used to detect tumor specific gene fusions and whether this would reflect the tumor burden of patients with Ewing's sarcoma.

Patients and Methods: Multiplex long-range genomic PCR was performed to detect patient-specific genomic breakpoints in eight patients. Probes for digital PCR were designed based on the genomic breakpoint of each patient. cfDNA was extracted from the serum of patients, which was drawn at multiple time-points (pretreatment, completion of the treatment, and/or relapse). cfDNA quantification was performed by digital PCR (BioRad Qx-200).

Results: Gene fusions in the cfDNA were detected in six (75%) of eight patients with high specificity. The relative gene fusion copy ratio (gene fusion copy number/wild KRAS copy number) was higher in the patients with M1b (extrapulmonary metastases at diagnosis) compared to the patients with M1a (pulmonary metastases at diagnosis) or M0 (no metastasis), although no significant relationship was observed between the relative gene fusion copy ratio and chemosensitivity. In each patient, the relative gene fusion copy ratio was the highest in pretreatment samples and decreased as the treatment progressed. Patients with relapse displayed an elevated relative gene fusion copy ratio in advance of clinical manifestation or elevation of lactase dehydrogenase.

Conclusion: The amount of cfDNA in the serum strongly correlated with the treatment stage and would be useful as an early biomarker for Ewing's sarcoma patients.

#3661

Comparison of ctDNA markers in matched urine and plasma from patients with liver cancer.

Tai-Jung Lee,1 Surbhi Jain,2 Jamin D. Steffen,2 Adam M. Zhang,2 Chi-Tan Hu,3 James P. Hamilton,4 Ying-Hsiu Su1. 1 _The Baruch S. Blumberg Institute, Doylestown, PA;_ 2 _JBS Science, Inc, Doylestown, PA;_ 3 _Hualien Tzu-Chi Hospital, Hualien, Taiwan;_ 4 _The Johns Hopkins University School of Medicine, Baltimore, MD_.

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer worldwide and a leading cause of global cancer mortality. Lack of efficient early detection and limited therapeutic options are two key contributing factors for the dismal prognosis of this disease. We have previously shown that urine contains fragmented, circulation-derived cell-free DNA that can be used for detection of circulating tumor DNA (ctDNA) if a tumor is present, suggesting urine as an alternative body fluid to plasma for cancer liquid biopsy. This study compared the detectability of five known HCC-associated DNA modifications, including genetic mutations (TERT -124 G>A, TP53 249 G>T and CTNNB1 hotspot at codon 32-45) and epigenetic methylation (mGSTP1, mRASSF1A), in matched urine and plasma from patients with liver cancer. In order to increase the sensitivity of detecting DNA modifications from the short, fragmented DNA templates, specialized short amplicon PCR assays were developed. To compare the detectability of HCC ctDNA markers in urine and plasma, 26 pairs of urine and plasma samples from HCC patients were identified and tested for five HCC ctDNA markers. In this cohort, 17 of 26 (65%) HCC samples had serum AFP above 20 ng/mL and were considered AFP-positive HCC. By using our HCC DNA biomarker panel composed of 5 assays, 22/26 (84.6%) urine and 23/26 (88.5%) plasma samples were found to have at least one DNA marker detected. In the combination of our HCC DNA biomarker panel with serum-AFP, the sensitivity of HCC detection in a liquid biopsy of urine and plasma as compared to serum-AFP alone (the current most used screening marker) increased from 65% to 92.3% for both urine plus AFP and plasma plus AFP. Further studies to include non-HCC samples (such as cirrhosis) to determine the specificity of the HCC DNA biomarker panel for liquid biopsy are in progress. In conclusion, our research provides the promising potential for HCC liquid biopsy screening in at-risk populations and precision medicine by using plasma, urine, or both as body fluids.

#3662

Cell-free DNA sequencing demonstrates persistence of nucleic acid-based therapy—opportunities for detection and monitoring.

Marcin Sikora,1 Alexander Artyomenko,1 James Strauss,2 Richard B. Lanman,1 Victoria M. Raymond,1 Justin Odegaard1. 1 _Guardant Health, Redwood City, CA;_ 2 _Mary Crowley Cancer Research, Dallas, TX_.

Introduction: Cell-free circulating tumor DNA (cfDNA) is utilized for genomic alteration detection to inform therapy selection and is an emerging noninvasive disease monitoring tool. Nucleic acid-based therapies (NATs) directly or indirectly manipulate DNA or mRNA transcript levels for therapeutic effect. cfDNA is in routine clinical use and NATs are in an expanding number of clinical trials, yet little is known about potential interactions of these technologies including the need for cfDNA next-generation sequencing (NGS) to recognize this source of exogenous DNA.

Methods: A 62-year-old male with weight loss and abdominal pain was found to have a tumor in the head of the pancreas and a metastatic liver lesion. Fine-needle aspirate of the tumor confirmed mucinous adenocarcinoma of the pancreas but sample was insufficient for molecular testing. cfDNA NGS was performed (Guardant Health, CA) to identify genomic alterations (e.g., BRCA, EGFR alterations, ERBB2 amplification).

Results: cfDNA NGS revealed dramatic over-representation (41% of all reads) of TP53-derived sequences with atypical insert sizes and distribution, read start/stop positions, and post-clipping read lengths. Atypical sequences comprised only TP53 coding sequence, without introns. Alignment and reconstruction assembled the atypical reads into a 6003 bp circular construct with the complete TP53 coding sequence inserted into a synthetic expression vector. Consultation with the treating physician revealed that patient progressed on FOLFIRINOX and was subsequently enrolled on a clinical trial utilizing a liposome-encapsulated TP53 expression vector with gemcitabine and nab-paclitaxel. His first experimental treatment was four days prior to cfDNA sample collection. Importantly, over-representation of this synthetic construct obscured native TP53 sequences, resulting in a 47% increase in predicted assay limit of detection despite standard performance metrics being within normal ranges. The atypical features of the expression vector reads make it possible to automate detection and quantification, even at significantly lower concentration than described here. The expression vector bears no human genome homology, allowing read identification via mapping. TP53 intron "deletions" were detected by a novel deletion detection algorithm. Finally, insert length and read start-stop distributions are markedly dissimilar between native and synthetic sequences

Conclusions: NATs generate unique and previously unappreciated interference with cfDNA sequencing which can be identified and filtered using automatable and generalizable bioinformatics techniques. cfDNA sequencing identified an NAT construct, four days after administration, demonstrating that the ability to identify and quantify NATs may enable noninvasive assessment of NAT delivery efficacy and/or pharmacologic persistence.

#3663

Non-invasive detection of fusion, splicing and mutation events from circulating cell-free RNA in cancers.

Zhixin Zhao. _Predicine Inc, CA_.

Circulating cell-free DNA (cfDNA) and cell-free RNA (cfRNA) in cancer patients are valuable resource of biomarkers for early detection, diagnosis, treatment and monitoring of disease progression. Compared to cfDNA, cfRNA is more labile to degrade and fluctuate, and thus more challenging to be detected in blood. In the present study, we reported PrediSeq, a validated NGS-based liquid biopsy assay to detect fusions, splicing and mutation events using cfRNA. By using Predicine proprietary ultra-sensitive DeepSeaTM (Deterministic, Efficient, Precise, Sensitive Algorithm) variant detection algorithm, pre-analytical study demonstrated RNA-based detection of fusion, RNA splicing, and mutations using reference materials. In a LOD study, PrediSeq NGS assay is more sensitive than the digital PCR based assay in terms of mutation detection. In clinical validation study, tissue and plasma samples from various types of cancer patients (lung, breast, prostate etc) were used, where concordant fusion events with tissue were observed and RNA mutations confirmed variants detected at DNA level. In conclusion, a highly sensitive and accurate RNA-based liquid biopsy test has been developed to complement the use of cfDNA for detection of fusion, splicing and mutations.

#3664

A workflow to evaluate PD-L1 protein expression on circulating tumor cells (CTCs) from non-small cell lung cancer (NSCLC).

Corinne Renier,1 Charles L. Wilkerson,1 SJ Claire Hur,2 Da Eun Rachel Park,2 Clementine A. Lemaire,1 Melissa Matsumoto,2 James Carroll,3 Steve Crouse,1 Jonathan W. Goldman,3 Edward B. Garon,3 Dino Di Carlo,2 Elodie Christen-Sollier1. 1 _Vortex Biosciences, Menlo Park, CA;_ 2 _University of California Los Angeles, Los Angeles, CA;_ 3 _David Geffen School of Medicine, UCLA, Los Angeles, CA_.

Introduction

Many tumors evade immune surveillance by deploying immunosuppressive mechanisms and co-opting immune checkpoint pathways, such as PD-1/PD-L1. PD-1/PD-L1 pathway blockade is a highly promising therapy and has elicited durable antitumor responses in a variety of cancers. Challenges in advancing immunotherapies lie in patient stratification and monitoring therapy. Evaluation of PD-L1 expression is generally regarded as an inclusion criterion for clinical trials. Current methods rely on the analysis of excisional biopsies, which are invasive and carry associated medical risks. Isolation and analysis of CTCs is a noninvasive alternative that may provide, in real-time, more representative information on tumor biomarkers, and allow the monitoring of treatment effectiveness over time. Here, we present an integrated workflow for the analysis of PD-L1 protein expression on CTCs from metastatic NSCLC patients isolated with the Vortex Biosciences VTX-1 liquid biopsy system.

Methods

Cell lines expressing various level of PD-L1 and white blood cells prepared from healthy donors were used to optimize the PD-L1 immunostaining assay. Several PD-L1 antibodies were titrated, and their specificity estimated with isotype controls. PD-L1 expression quantification was evaluated using two independent methods i) Zen 2 imaging (Zeiss) and ii) CellProfiler software. Clinical validation was performed on blood samples collected from a cohort of metastatic NSCLC patients (stage IV) with known PD-L1 tumor status. CTCs were isolated using the VTX-1 Liquid Biopsy System, stained (PD-L1, CK, CD45, CD11b, DAPI) and classified, then PD-L1 expression was quantified.

Results

We have developed a PD-L1 immunofluorescent assay that shows specificity, sensitivity, and very good intra-assay repeatability. The two methods evaluated for the quantification of PD-L1 expression provided equivalent data and could be used interchangeably. The PD-L1 assay was further validated on CTCs isolated from a small cohort of metastatic NSCLC patients. Preliminary data shows heterogeneity in PD-L1 expression levels across CTCs from the same patient. Interestingly, among PD-L1 positive CTCs, both traditional CK+/PD-L1+ and CK-/PD-L1+ cells were observed. To move toward a more automated workflow for immunostaining, imaging, and fluorescence signal analysis, methods to collect CTC on glass slides were evaluated, with cell recovery ranging from 33 to 98%.

Conclusion

We demonstrated the feasibility of evaluating PD-L1 protein expression on NSCLC's CTCs isolated with the VTX-1 Liquid Biopsy System. While further clinical validation is warranted, this test could represent a simple, non-invasive method to help identify patients most likely to respond to PD-1/PD-L1 immunotherapies. Future work will focus on compatibility of the assay with autostainers and fully automated imaging and analysis platforms.

#3665

Longitudinal monitoring of prostate cancer evolution by plasma genome sequencing.

Naveen Ramesh, Emi Sei, Ruli Gao, Pei Ching Tsai, Christopher Logothetis, Amado J. Zurita, Nicholas E. Navin. _MD Anderson Cancer Center, Houston, TX_.

Castration-resistant prostate cancer (CRPC) is associated with poor survival and frequent metastasis to the bone. Disease progression and evolution of therapy resistance have been challenging to study, in part due to the difficulty in collecting invasive core biopsy samples longitudinally over time. While liquid biopsy methods hold great potential for longitudinal monitoring, they are currently limited to targeted measurements of single genes, or small cancer gene panels. To address this challenge, we developed an unbiased whole-genome sequencing method called PEGASUS (Plasma Exome and Genome Analysis by Size-Selection and Unbiased Sequencing) that enables the detection of copy number aberrations and exome mutations from circulating-tumor DNA (ctDNA). We applied PEGASUS to 15 CRPC patients, which identified common driver mutations and copy number aberrations in plasma DNA including AR, MYC, RB1 and PTEN. We compared matched ctDNA and metastatic tumor samples in 8 CRPC patients, which showed a high correlation (r=0.79) of copy number profiles and moderate concordance of exome mutations (~56%), but also revealed a number of ctDNA and metastasis-specific mutations. We further applied PEGASUS to analyze longitudinal samples from 10 prostate cancer patients that were treated with chemotherapy or androgen inhibition to delineate clonal evolution in response to therapy. Clonal subpopulations and dynamics were inferred from mutation frequencies, revealing both chemoresistant and chemosensitive clones. These data showed that while a subset of point mutations were under positive and negative selection; most copy number aberrations remained stable and did not change in response to therapy. Our data show the technical feasibility of performing whole-genome and exome profiling of plasma DNA and pave the way for future clinical applications in biomarker discovery and informing clinical treatment decisions in prostate cancer patients.

#3666

Diagnostic application of novel ACE technology: Treatment response monitoring via quantification of cell-free DNA (cfDNA) in plasma from late-stage cancer patients.

Robert Kovelman,1 Juan Pablo Hinestrosa,1 Delia Ye,1 Patrick Desmond,1 Michelle Nguyen,1 Robert Turner,1 David Searson,1 Suren Uswatta,1 James Madsen,1 David Bodkin,2 Lyudmila Bazhenova,3 Afshin Bahador,4 Rajaram Krishnan1. 1 _Biological Dynamics, San Diego, CA;_ 2 _Cancer Center Medical Oncology Group, La Mesa, CA;_ 3 _UCSD Moores Cancer Center, La Jolla, CA;_ 4 _South Coast Gynecologic Oncology, San Diego, CA_.

We have developed a novel microfluidic technology that makes use of Alternating Current Electrokinetics (ACE) to isolate macromolecules such as high-molecular-weight DNA and protein complexes as well as nanoparticles, including cell-derived exosomes. As one of the first applications of this novel technology, we have developed procedures and assays for the isolation and quantification of cell-free DNA (cfDNA) in plasma. We show that this methodology allows for the rapid, robust, and highly quantitative assay of cfDNA in undiluted plasma. Analytical studies indicate that these ACE assays are sensitive, have a very broad dynamic range, and allow for highly effective calibration strategies, thereby enabling comparison of results over time and between laboratories. Importantly, we show correspondence between the results of ACE assays and those that employ traditional technologies such as PCR. We have applied this technology to the quantification of cfDNA in plasma from cancer patients, with an initial focus on stage III and IV non-small cell lung cancer (NSCLC). With a goal of enabling less invasive, more frequent, and lower-cost treatment response monitoring in cancer patients, we have conducted clinical studies showing that the results generated from ACE assays provide informative and useful findings related to disease progression and therapeutic alternatives. We are conducting laboratory and clinical studies oriented toward the development of this technology for in vitro diagnostic use.

#3667

Technical advances in plasma genomic biomarkers for mutation detection and monitoring in cancer patients.

Sandra Fitzgerald, Annette Lasham, Cherie Blenkiron, Paula Shields, Ben Lawrence, Cristin Print. _University of Auckland, Auckland, New Zealand_.

A sea-change is imminent for cancer medicine, due to the use of non-invasive genomic biomarkers in blood to inform screening, diagnosis and the selection of treatment. This technology may be used routinely in oncology within five years. Although numerous studies, including work in our laboratory, have shown that genomic analysis of blood can detect the presence and even the type of cancer, researchers have only scratched the surface of what this technology can do. In our laboratory, we are generating new methods to improve the sensitivity and accuracy of such tests. Cell-free DNA from a metastatic colorectal cancer patient has been used to directly compare the sensitivity of mutation detection across three platforms - a custom QiaSeq Targeted amplicon sequencing panel, droplet digital PCR (ddPCR) and UltraSEEK lung panel. UltraSEEK and ddPCR platforms show better sensitivity for detection of the KRAS G12A mutation, compared to the QiaSeq Targeted DNA sequencing panel. This pilot study shows that when looking to specifically detect a small number of known mutations, UltraSEEK or ddPCR gives the highest sensitivity, but when mutations are not limited to a few or are completely unknown, the QiaSeq Targeted DNA panel allows identification of the entire repertoire of mutations in specific cancer-associated genes.

#3668

ctDNA and TCR dynamics predict response toimmune checkpoint blockade in non-small cell lung cancer.

Valsamo Anagnostou, Patrick Forde, Jarushka Naidoo, Kristen Marrone, Vilmos Adleff, James White, Jillian Phallen, Alessandro Leal, Carolyn Hruban, Ashok Sivakumar, Franco Verde, Rachel Karchin, Julie Brahmer, Victor Velculescu. _Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD_.

The dynamic nature of the cancer-immune system crosstalk has rendered single biomarker approaches insufficient to predict outcome from immune-targeted agents. These therapies also pose a challenge to radiographic response assessments that may underestimate the therapeutic benefit. There is therefore an urgent clinical need to develop molecular assays of response and resistance.In order to capture the dynamic nature of the anti-tumor immune response under checkpoint blockade, we devised an integrative non-invasive approach that captures the evolving neoantigen landscape and the associated T-cell receptor (TCR) repertoire. We employed whole exome sequencing to determine the genomic landscape of tumors and identify tumor-derived alterations in subsequent analyses of circulating cell-free tumor DNA (ctDNA), for 14 patients with metastatic NSCLC treated with immune checkpoint blockade. Liquid biopsies were obtained prior to treatment, at week 4-6 and at additional timepoints until disease progression. We used the ultrasensitive targeted error correction sequencing approach to analyze 58 cancer driver genes in the circulation of these patients and assessed the value of longitudinal ctDNA monitoring as a surrogate for response. In parallel, we evaluated dynamics changes in the TCR repertoire by TCR next generation sequencing of serially collected peripheral T cells and tumor infiltrating lymphocytes for each patient. Radiographic response assessments were performed using the RECIST 1.1 criteria. These analyses revealed that ctDNA dynamics predict outcome significantly earlier than imaging. Patients that responded to therapy had a significant drop in ctDNA early during the treatment course whereas non-responders had either limited changes or significant increase in ctDNA. For patients with acquired resistance, ctDNA kinetics revealed clearing of ctDNA at the time of response followed by a new peak at the time of progression. ctDNA detection early during the treatment course was a significant prognostic factor for progression-free and overall survival (log rank p=.004 and .002 respectively) and ctDNA elimination was superior to tumor mutation burden as a predictor of response to therapy. In parallel, we identified changes in the TCR repertoire that are predictive of outcome: peripheral T cell expansion of a subset of intra-tumoral clones was noted at the time of response whereas there was no evidence of T cell expansion in non-responders. Peripheral T cell expansion of a subset of intra-tumoral clones was noted to peak at the time of response and decrease to baseline levels at the time of resistance for patients with acquired resistance. Our findings suggest that a dynamic assay able to capture the tumor-immune system equilibrium would be superior to conventional analyses of static time points. Such an integrated approach would be highly relevant to tailored cancer immunotherapy strategies.

#3669

Assessing the genetic heterogeneity of localized, multifocal prostate cancer via cell-free DNA.

Emmalyn Chen, Clinton L. Cario, Lancelote Leong, Karen Lopez, Jeffry P. Simko, Peter R. Carroll, Caroline Tai, John S. Witte. _University of California - San Francisco, San Francisco, CA_.

At the time of diagnosis, it is common to find multiple spatially distinct foci within a single prostate gland in men with prostate cancer. Recent studies evaluating the genetic heterogeneity of localized, multifocal prostate cancers through whole-genome sequencing and whole-exome sequencing have only recently been described. These tumors were highly heterogeneous for single-nucleotide variants, copy number alterations, and genomic rearrangements. One exciting use of cfDNA is its potential ability to simultaneously capture all genetically distinct tumor subclones. Currently, the degree to which a cfDNA sample is representative of the entire genetic landscape of localized prostate cancer is unknown. The objective of this study is to determine if somatic mutations identified in tumor tissue are detectable in cfDNA. Tumor tissue and blood samples have been collected from twenty-nine patients undergoing radical prostatectomy. Samples from fourteen patients have already been subjected to whole-exome sequencing with a target sequencing depth of 200X (HiSeq 4000). Preliminary analyses indicate that tumor foci within a single prostate gland in an individual are genetically heterogeneous. Further analysis of mutational concordance between tumor tissue DNA and cfDNA along with clinical data will be performed.

#3670

Urine- and blood-based DNA+RNA liquid biopsy tests in prostate, bladder and kidney cancers.

Amy Wang, Zhixin Zhao, Cheng Xie, Tak Cheung, Carlos Montesinos, Phoebe Zhang, Pan Du, Jianjun Yu, Shidong Jia. _Predicine, Inc, Palo Alto, CA_.

The estimated incidence of genitourinary (GU) cancers (prostate, bladder, and kidney) will be over 2 million worldwide and responsible for ∼800 000 deaths by 2020. Current diagnosis and monitoring methods of GU cancer patients are often invasive and/or lack sensitivity and specificity. The recent tremendous success in cell-free DNA (cfDNA) blood test has laid the groundwork for using body fluids such as blood and urine as minimally invasive "liquid biopsies" to identify RNA- and DNA-based molecular biomarkers in urologic malignancies.

Here we report the development and clinical application of PrediSeq panel, a urine- and blood-based next generation sequencing-based diagnostics assay that offers integrated genomic cfDNA and cell-free RNA (cfRNA) profiling of genitourinary cancers to detect RNA-based splicing variants (e.g. AR-V7) and rearrangement (e.g. TMPRSS2-ERG), DNA/RNA-based point mutation (e.g. p53, PIK3CA, FGFRs), DNA-based copy number change (e.g. AR) as well as tumor mutation burden (TMB) and microsatellite instability (MSI) using a single tube of blood and/or urine sample in prostate, bladder, and kidney cancers, respectively. In addition to NGS-based multiplex test, we also developed a Bio-Rad digital PCR assay measuring drug response and resistance biomarkers including AR-V7 and PD-L1.

Collectively, PrediSeq NGS and ddPCR assays offer comprehensive genomic profiling of both cfDNA and cfRNA using blood and/or urine samples in patients with genitourinary cancers. The successful development and clinical validation of these tests has potential to enable early diagnosis, cancer screening, and precision medicine in genitourinary cancers. 

### Molecular Classification of Tumors 1: Epigenetic Therapy, Functional and Molecular Imaging, and Tumor Heterogeneity

#3671

**Phenotypic heterogeneity within prostate cancer bone metastases measured by** 18 **F-DCFBC PET/CT and** 18 **F-NaF PET/CT.**

Stephanie A. Harmon,1 Esther Mena,2 Joanna Shih,2 Ethan Bergvall,2 Stephen Adler,1 Sherif Mehralivand,2 Ravi A. Madan,2 James L. Gulley,2 William L. Dahut,2 Baris Turkbey,2 Peter L. Choyke,2 M Liza Lindenberg2. 1 _Leidos Biomedical Research, Bethesda, MD;_ 2 _National Cancer Institute, Bethesda, MD_.

Purpose: The purpose of this study was to compare the spatial colocalization of prostate-specific membrane antigen (PSMA) targeting agent 18F-DCFBC PET/CT, a marker which demonstrates correlation with tumor aggressiveness, and 18F-NaF PET/CT, a marker of osteoblastic activity, within bone lesions of patients with metastatic prostate cancer. Methods: Twenty-eight metastatic prostate cancer patients received 18F-DCFBC PET/CT and 18F-NaF PET/CT scans (median interval 7 days, range 1-57). Tracer-specific regions of interest (ROI) were derived from threshold containing 80% of maximum SUV (SUV80%). Lesion detection and uptake was compared across the two PET/CT agents using non-parametric testing (Spearman correlation, Wilcoxon rank sum) accounting for intra-patient correlation. Lesion-specific registration was completed by global alignment of skeletal segments followed by local neighborhood optimization of area surrounding lesion. Spatial concordance was evaluated using overlap volume (OV), calculated as the volume of ROI intersection (shared volume) relative to the minimum volume of NaF-ROI or PSMA-ROI to account for differences in ROI volumes. Differences in uptake and CT Hounsfield units (HU) were compared within concordant (overlapping) and discordant (NaF-only, PSMA-only) regions of the same lesion using Wilcoxon signed rank test accounting for intra-patient correlation. Results: Twenty-six patients had positive NaF or DCFBC PET/CT scans, with a total of 241 metastatic sites identified. Lesion-level NaF SUV80% and DCFBC-2hr SUV80% were not significantly correlated at baseline (ρ=0.41, p>0.1). Lesion-level NaF-SUV80% was lower in bone lesions detected only by NaF (N=96) compared to NaF-SUV80% in bone lesions detected by both tracers (N=82), nearing significance (p=0.065). This pattern was not observed in DCFBC-SUV80% (p=0.5). To avoid bias from motion artifacts, only lesions in pelvis and spine were considered for voxel-based analysis (N=42). NaF and PSMA ROIs showing varying levels of spatial concordance (OV mean=0.26, range=0-1), which was modestly associated with higher sclerosis (ρ=0.4, p=0.01) and weakly associated with lower PSMA activity (ρ=0.3, p=0.07). Intra-lesion PSMA-only regions were significantly less sclerotic compared to concordant (p=0.04) and NaF-only (p=0.03) regions, indicating regions of active tumor seeding extend beyond regions of active bone remodeling within the tumor. Conclusions: Metastatic seeding and growth of prostate cancer in bone is biologically complex and dependent on bone microenvironment. We find regions of aggressive cellular activity (PSMA-targeting PET/CT) show heterogeneous colocalization with regions of rapid bone remodeling (NaF PET/CT) within sites of metastatic bone disease. These preliminary findings are important for consideration of targeted radionuclide therapies in metastatic prostate cancer.

#3672

The three-dimensional (3D) spatial lamin A/C organization in normal lymphocytes, and in Hodgkin and Reed-Sternberg cells.

Fabio Contu,1 Aleksander Szczurek,1 Roberta Vanni,2 Hans Knecht,3 Sabine Mai1. 1 _University of Manitoba, Winnipeg, Manitoba, Canada;_ 2 _University of Cagliari, Cagliari, Italy;_ 3 _Jewish General Hospital, McGill University, Montréal, Quebec, Canada_.

Classical Hodgkin Lymphoma (HL) is a B-Cell lymphoma comprising mononucleated Hodgkin cells (H) and diagnostic bi- to multi-nucleated Reed Sternberg cells (RS). Genomic instability in HL is characterized by telomere dysfunction caused by critically short telomeres and telomere uncapping. Super resolution microscopy showed significant increases in the amount of DNA-poor nuclear spaces in HL compared to normal primary lymphocytes. A direct 3D interaction between telomeres and shelterin protein TRF2 is progressively disrupted from H to RS cells by either massive upregulation or downregulation of TRF2. TRF2 interacts with nuclear matrix protein lamin A/C in the maintenance of the 3D genome organization, which is disturbed in H and RS cells. Therefore we hypothesized that the TRF2- telomere-lamin A/C interaction is also disturbed in H and RS cells and leads to organizational alterations of the genome in HL. To this end, we analyzed lamin A/C expression levels of lymphocytes, H and RS cells and found that lamin A/C is upregulated in cancer cells compared to both activated and resting lymphocytes. The regular homogeneous and round shaped pattern found in lymphocytes was replaced in H and RS cells by a more irregular one, characterized by the presence of lamin A/C invaginations and septa that subdivide the nuclei into multiple smaller compartments. Our data show no co-localization between telomeres and lamin A/C in the DNA-poor spaces. Lamin A/C bordered the DNA-poor spaces but was never found inside them or in association with inner telomere fragments. 3D TRF-2 staining showed upregulation of the protein compared to normal lymphocytes, in correlation with the observation made in EBV-negative patients with clinically aggressive disease. The analysis of pre-treatment Hodgkin's patients' samples will allow to determine whether the TRF2-telomere-lamin A/C binding is different in patients with recurrent versus non-recurrent disease.

#3673

Targeting the epigenome of small cell hypercalcemic carcinoma of the ovary, hypercalcemic type (SCCOHT).

Yemin Wang,1 Shary Yu-ting Chen,1 Shane Colborne,1 Krystal Orlando,2 Jessica Lang,3 Anthony Karnezis,1 William Hendricks,3 Gregg Morin,1 Bernard Weissman,2 Jeffrey Trent,3 David Huntsman1. 1 _University of British Columbia, Vancouver, British Columbia, Canada;_ 2 _University of North Carolina, Chapel Hill, NC;_ 3 _Translational Genomics Research Institute, Phoenix, AZ_.

Small cell carcinoma of the ovary hypercalcemic type (SCCOHT) is a rare and poorly differentiated cancer that impacts young women with an average 2-year survival of less than 35%. We and others have discovered the inactivating mutations of SMARCA4 as the only consistent genetic alternation in SCCOHT genomes. SMARCA4 is the ATPase of the SWI/SNF chromatin-remodeling complex, which modulates chromatin accessibility to regulate transcription and plays critical roles in many biologic processes such as cell cycle control, apoptosis, and differentiation. SCCOHT also lacks the expression of SMARCA2, the alternative ATPase in the complex, contrasting the requirement of SMARCA2 for survival of most SMARCA4-deficient cancer cells. This suggest that the complete loss of chromatin-remodeling activity may rewire the epigenome and create opportunities for synthetic lethal targeting. A rational epigenetic drug screen identified EZH2 inhibitors and HDAC inhibitors as promising therapeutic agents in SCCOHT cells. We confirm that catalytic inhibition of EZH2 selectively suppressed the growth of SCCOHT cells in vitro and in xenograft models through activation of apoptosis and differentiation. Pan-HDAC inhibitors also displayed a more robust anticancer effect in SCCOHT cells than other in other ovarian cancer cell lines. Furthermore, combined treatment of EZH2 inhibitors and pan-HDAC inhibitors increased the global acetylation level at histone H3K27 site and synergistically suppressed the growth of SCCOHT cell lines and xenografts through robust induction of apoptosis. Although EZH2 or HDAC inhibitor treatment led to re-expression of SMARCA2, depletion of SMARCA2 had only minimal effect on drug response. Proteomic analysis identified key signaling pathways underlying the efficacy of epigenetic therapy that are under investigation. Therefore, inactivation of SWI/SNF chromatin remodeling complex may drive SCCOHT development through PRC2-dependent rewiring of the epigenome. Targeting these oncogenic events can be the feasible strategies for treatment of SCCOHT that warrant clinical investigation.

#3674

Pharmacodynamic assessment in whole blood for the BET bromodomain inhibitor CPI-0610 of target engagement in patients with progressive lymphoma.

Jennifer A. Mertz,1 Kristie A. Blum,2 Anas Younes,3 Jeremy S. Abramson,4 Michael B. Maris,5 Ian W. Flinn,6 Andre Goy,7 Darrell R. Borger,4 Michael R. Cooper,1 Robert J. Sims1. 1 _Constellation Pharmaceuticals, Cambridge, MA;_ 2 _Ohio State University, Columbus, OH;_ 3 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 4 _Massachusetts General Hospital, Boston, MA;_ 5 _Colorado Blood Cancer Institute, Denver, CO;_ 6 _Sarah Cannon Research Institute, Nashville, TN;_ 7 _Hackensack University Medical Center, Hackensack, NJ_.

Defining the relationship between small molecule exposure, clinical efficacy and target engagement via pharmacodynamic (PD) biomarkers is a key element of phase 1 clinical development. CPI-0610 is an orally bioavailable inhibitor of the bromodomain and extra-terminal (BET) domain family of proteins currently being evaluated in a number of phase I trials. To support the clinical development of CPI-0610, we sought to identify BET target genes in whole blood that function as PD markers for target engagement in patients. The utilization of whole blood rather than tumor samples for PD assessments allows for a high level of compliance for sample collection and assessment of serial samples to understand the kinetics of optimal gene regulation. This is important for the mechanism of action of BET bromodomain inhibitors, as it has been shown that target gene suppression is rapidly reversible upon removal of drug. A preliminary list of BET target genes was first identified after ex vivo treatment of healthy donor blood samples with a BET inhibitor followed by transcriptional profiling. Further characterization was carried out using blood samples recovered from in vivo studies in mice, as well as rat and dog toxicology studies for CPI-0610. Blood samples collected from patients with progressive lymphoma participating in a Phase I clinical study (NCT01949883) were analyzed for changes in the BET target genes and correlated with CPI-0610 dose, pharmacokinetic (PK) exposure and clinical activity. This enabled further refinement of the BET target gene signature to identify the most robust gene expression changes in patient samples. IL8 and CCR1 were the most strongly regulated genes, showing an exposure-dependent downregulation in patient samples. Using these target genes, we were able to identify a minimum threshold of exposure required for BET target engagement as well as show that BET target engagement can be achieved at doses below the maximum tolerated dose. Furthermore, ≥50% downregulation of IL8 was consistent with anti-lymphoma activity in patients from our trial. CCR2, FN1, CSF1R and THBS1 were identified and validated preclinically but in patient samples were difficult to interpret due to lack of robust regulation or low basal levels of expression. In summary, a BET target gene signature assay using whole blood samples was established and implemented in a Phase I lymphoma study which demonstrated that CPI-0610 regulates direct BET target genes in blood cells in a robust and dose-dependent manner.

#3675

Loss of alveolar type II epithelial and endothelial cell lineages in invasive mucinous adenocarcinoma of the lung (IMA).

Koichi Tomoshige,1 Minzhe Guo,1 Tomoshi Tsuchiya,2 Iris Fink-Baldauf,1 Takeshi Nagayasu,2 Kelsey Dillehay McKillip,3 Yutaka Maeda1. 1 _Cincinnati Children's Hospital Medical Ctr., Cincinnati, OH;_ 2 _Nagasaki University Graduate School of Biomedical Sciences, Japan;_ 3 _University of Cincinnati College of Medicine, Cincinnati, OH_.

Background: Invasive mucinous adenocarcinoma of the lung (IMA) is pathologically characterized as lung tumor cells with goblet cell morphology containing abundant intracytoplasmic mucin. IMA has been reported to account for 2-10% of lung adenocarcinomas. Genetically, the majority of IMA harbor KRAS mutations. Recently, by analyzing RNA-seq data using human IMA cases, we reported mucus-related genes specifically expressed in IMA, which includes MUC5AC, MUC5B, MUC3, SPDEF, FOXA3 and HNF4 (Guo, Tomoshige et al., 2017). Notably, immunohistochemical analyses have determined that NKX2-1 (also known as TTF-1) is reduced in IMA (Travis et al., 2011); however, other than NKX2-1, genes reduced in IMA compared to normal lungs have not been determined.

Methods: In order to further understand the demographic occurrence of IMA, we surveyed the clinical records at the University of Cincinnati College of Medicine in the U.S. and Nagasaki University of School of Medicine in Japan. Genes reduced in IMA compared to normal lungs were identified using the RNA-seq datasets as we have previously reported (Guo, Tomoshige et al., 2017). Using single-cell and sorted RNA-seq datasets (LGEA; Du et al., 2017), genes were further categorized into groups that define different cell types that exist in normal lungs.

Results: IMA occurred in 4.6% (3/65 cases in 2017) and 1.9% (2/105 cases in 2017) of lung adenocarcinomas at the University of Cincinnati College of Medicine and Nagasaki University of School of Medicine, respectively. Importantly, analysis using normal lung cell type specification markers obtained from the single-cell and sorted RNA-seq datasets identified that genes that define alveolar type II epithelial cells (e.g., SFTPC and ABCA3) and endothelial cells (e.g., FOXF1 and SOX17) were significantly reduced in IMA compared to normal lungs.

Conclusion: IMA comprises mucus-producing goblet cell lineage populations devoid of alveolar type II epithelial and endothelial cell-lineage populations, suggesting a unique tumor microenvironment distinct from other lung adenocarcinomas.

#3676

CMS classification of colorectal adenomas.

Malgorzata A. Komor,1 Linda J. Bosch,1 Gergana Bounova,1 Anne S. Bolijn,1 Pien Delis-van Diemen,1 Christian Rausch,1 Youri Hoogstrate,1 Andrew Stubbs,1 Mark de Jong,2 Guido Jenster,3 Nicole C. an Grieken,4 Beatriz Carvalho,1 Lodewyk Wessels,1 Connie R. Jimenez,4 Remond J. Fijneman,1 Gerrit A. Meijer,1 NGS-ProToCol Consortium. 1 _Netherlands Cancer Institute, Amsterdam, Netherlands;_ 2 _GenomeScan, Leiden, Netherlands;_ 3 _Erasmus Medical Centre Rotterdam, Rotterdam, Netherlands;_ 4 _VU University Medical Center, Amsterdam, Netherlands_.

Background Consensus molecular subtyping (CMS) is an RNA-expression-based classification of colorectal cancers (CRC). Genomic alterations, resulting in specific RNA-expression patterns, accumulate during CRC pathogenesis, including the premalignant adenoma stage.

Aim This study aimed to investigate whether differentiation of colorectal neoplasia into CMS classes can already be recognized at the adenoma stage, and whether specific CMS classes could be associated with DNA copy number aberrations that mark adenomas at high-risk of progressing to CRC.

Materials and Methods RNA-sequencing was performed on 62 advanced adenomas and 59 CRCs. DNA copy number analysis in adenomas was performed by low-coverage DNA-sequencing (n=30) or array-Comparative Genomic Hybridization (n=32). Microsatellite instability (MSI) status was determined by PCR methods. Adenomas and CRCs were classified into CMS subtypes together with CRCs (n=556) from The Cancer Genome Atlas, using the Random Forest CMS classifier.

Results The majority of the adenomas were classified as CMS3 (n=45; 72%), the 'metabolic subtype', recognized as least common among CRCs. No adenomas were classified as the 'mesenchymal' CMS4 subtype. One adenoma was classified as the 'MSI immune' CMS1 (2%), and 8 adenomas as the 'canonical' CMS2 (13%) type. The remaining 8 (13%) could not be classified. The CMS3 class was enriched with adenomas at low-risk of progression.

Conclusion Most adenomas were successfully classified. The lack of CMS4 adenomas is consistent with the fact that adenomas lack invasion-associated stroma. Adenomas showing cancer-associated chromosomal instability (CIN) or MSI (24%) were mostly classified as CMS2 and CMS1, respectively. The CMS3 subtype appeared to be the predominant adenoma signature.

#3677

Scalable assembly of individual patient profiles for clinical trials accrual and research.

Ryan Duren,1 Ryan Smith,1 Nick Tackes,1 Shane Neeley,1 James Welsh,2 Xuan Shirley Li1. 1 _MolecularMatch, Inc, Houston, TX;_ 2 _MD Anderson, Houston, TX_.

As clinical data are digitized in electronic medical records (EMR), the amount of historical data becomes a challenge for utilization and outcomes studies. An automated approach for structuring clinical data into machine-readable format is essential due to scale. We previously developed MMPower, an ElasticSearch-based technology platform for named entity extraction defined by SNOMED conditions, >2,000 Entrez Genes, >900,000 cancer alterations, and ~7,000 FDA-approved and experimental therapeutics/progression that enables clinical trials search. In a pilot study, MMPower was applied to characterize and index oncology EMR for elucidating clinical histories and identifying clinical trial participants. 52,509 individual EMR were collated by Medical Record Number (MRN) across facets including Demographics, Providers/Sites, Diagnosis, Medication, Imaging, Lab results, Pathology, Performance status, and Encounter with Physician Notes. Applying MMPower yielded a total cohort of 43,987 (83.8% of EMR received) EMR with searchable, structured metadata profiles. We found that 39,317 (89.4%) of the total cohort had a cancer diagnosis as defined by SNOMED cancer types. We also extracted an Entrez gene from 36,152 (82.2%) EMR, while cancer alterations or stratification biomarkers such as ER/PR status, ALK fusions, IGH-BCL1, EGFR exon 19 deletion, CDKN2A loss, etc., were identified in 25,208 (57.3%) EMR. Furthermore, we found that the majority of EMR (69.7%) with identifiable genes also contained a cancer alteration. Interestingly, although most EMR (39,615 or 90% of total cohort) held identifiable cancer therapeutics, only 1.7% of the total cohort were flagged as cases of progression. We then determined the feasibility of matching EMR to clinical trials and found that, strikingly, 16.7% of the total cohort were potentially eligible for institution-specific clinical trials. Finally, we sought to enrich individual EMR with molecular alterations data from external lab reports, so we developed a PDF to JSON transform that enables mapping of report results by MRN to our cancer alterations model, yielding a single searchable record from multiple data sources. These results suggest that the ability to structure machine-readable clinical and molecular data for individuals from EMR at scale would accelerate translational research by promoting more efficient clinical trials accrual and could be extended to prevalence calculations, evidence-backed treatment guidance through molecular assertions, biomarkers discovery, and outcomes studies of therapeutic efficacy.

#3678

DNA methylation profiling of colorectal cancer identifies molecular subtypes and subtype-specific biomarkers for improved prediction of patient prognosis.

Trine Block Mattesen, Mads Heilskov Rasmussen, Jesper Bramsen, Claus Lindbjerg Andersen. _Department of Molecular Medicine, Aarhus University Hospital, Denmark, Aarhus N, Denmark_.

By analysis of RNA profiles from >1000 fresh-frozen (FF) colorectal cancer (CRC) samples we recently identified distinct molecular subtypes of CRC with different etiology, prognosis and prognostic biomarkers. The clinical validation and routine utilization of this exiting knowledge is, however, complicated by the unavailability of high quality RNA from clinical collected formalin-fixed paraffin-embedded (FFPE) samples and the high cost/complexity of RNA sequencing. As part of a solution we have established a novel CRC subtyping approach "methCORR", which only require DNA from either FF or FFPE tissue to classify CRC into subtypes based on their genome-wide DNA methylation profile. Using methCORR we were able to stratify CRC in independent datasets into two major cancer epithelial subtypes, "CRC1" and "CRC2", which are distinguished by their chromosomal/microsatellite stability status, the activity of the BMP-, WNT- and NOTCH signaling pathways and cellular composition of their tumor microenvironment (TME). Very notably, we also found evidence of subtype-specific prognostic biomarkers reflecting inter-tumor TME heterogeneity: aggressive CRC1 tumors were associated with an absence of infiltrating immune cells, whereas aggressive CRC2 tumors were instead associated with immune cell infiltration and stromal fibroblast activation. To derive cost-efficient prognostic biomarkers from our genome-wide methCORR analysis we next exploited that CRC1- and CRC2 cancer epithelial cells as well as TME cell types, such as T-lymphocytes and fibroblasts, have unique and robust DNA methylation patterns that can be interrogated by quantitative methylation-specific PCR (QMSP). Indeed, QMSP biomarker assays specific for CRC1 and CRC2 cancer epithelial cells successfully classified our discovery cohort (n=174) according to methCORR CRC1 and CRC2 epithelial cancer subtypes. Furthermore, additional quantification using T-lymphocytes and fibroblast-specific QMSP biomarker assays confirmed our methCORR estimations: CRC1 aggressive tumors were associated with a high content of cancer epithelial cells and absence of T-lymphocytes whereas aggressive CRC2 tumors were, in contrast, associated with a high content of T-lymphocytes and fibroblasts. Finally, we applied our top 5 QMSP biomarker panel to an independent validation cohort of 192 FFPE samples, which confirmed our observations from the discovery cohort. In conclusion: we have developed and validated novel QMSP-based biomarkers for CRC molecular subtyping and subtype-specific prediction of patient prognosis. Our assays are cost-efficient and compatible with FFPE samples hereby making them potentially relevant for clinical applications.

#3679

PD-L1 driven excluded phenotype in melanoma.

Carl Morrison,1 Jeffrey Conroy,1 Mary Nesline,1 Sean Glenn,1 Blake Burgher,1 Maochun Qin,1 Jonathan Andreas,1 Vincent Giamo,1 Felicia L. Lenzo,1 Angela Omilian,2 Wiam Bshara,2 Antonios Papanicolau-Sengos,1 Yirong Wang,1 Marc Ernstoff,2 Mark Gardner,1 Sarabjot Pabla1. 1 _OmniSeq, Inc., Buffalo, NY;_ 2 _Roswell Park Cancer Institute, Buffalo, NY_.

Background: The excluded phenotype has been previously described by the presence of abundant immune cells that do not penetrate the parenchyma of these tumors but instead are retained in the stroma that surrounds nests of tumor cells. These features support a pre-existing anti-tumor immune-related response, but the details of this mechanism are not well elucidated.

Methods: 300 formalin-fixed, paraffin-embedded (FFPE) metastatic cutaneous melanoma samples were evaluated by the RNA-seq component of a comprehensive immune profile panel to measure transcript levels of immune-related genes. Resultant data was QC filtered, normalized and ranked based on an assorted reference population of various tumor types. The expression of PD-L1 on the surface of tumor cells was assessed in tumor samples by means of an automated IHC assay (28-8, Dako). A tumor was considered PD-L1 positive if ≥1% of viable tumor cells exhibited complete circumferential or partial linear plasma membrane staining at any intensity. Excluded pattern of T-cell infiltration was defined by CD8 immunohistochemistry by the following definition: Restriction of more than 95% of all CD8+ T-cells present in a tumor to the periphery or interstitial stromal areas and not actively invading nest or groups of neoplastic cells.

Results: The excluded phenotype was identified in 34 of 300 (11%). PD-L1 by IHC was positive (≥1%) in the neoplastic cases in 16 of 34 (47%) cases. Membranous staining in immune cells was identified in only 3 of 34 (9%) cases, but was present in >90% of all cases in a non-membranous pattern. In more than one-half of all cases the non-membranous pattern of PD-L1 IHC staining was restricted to the excluded TILs. Higher expression of PD-L1 by RNA-seq was associated with this excluded PD-L1 pattern of staining.

Conclusion: In 300 metastatic cutaneous melanoma cases we demonstrated that the excluded phenotype is frequent and represents ~10% of all cases. Moreover, in a significant number of excluded cases expression of PD-L1 by IHC was distinctly limited to the excluded TILs. This finding would support a unique mechanism of PD-L1 anti-tumor immune-related response that needs further investigation.

#3680

Comparative mutational evaluation for multiple lung cancer by multiplex oncogene mutation analysis.

Yuta Takahashi, Kazuhiko Shien, Shuta Tomida, Eisuke Kurihara, Yusuke Ogoshi, Kei Namba, Takahiro Yoshioka, Hidejiro Trigoe, Hiroki Sato, Hiromasa Yamamoto, Junichi Soh, Shinichi Toyooka. _Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan_.

[Background] In the treatment of synchronous or metachronous multiple lung cancer (MLC), determination whether multiple primary lung cancer (MP) or intrapulmonary metastases (IM) is very important to make an appropriate management. Clinical or pathological diagnoses have been adopted to distinguish whether MLC were MP or IM, however, the accuracy of these approaches seemed to be insufficient. On the other hand, recent evolution of high-throughput sequencing made it possible to perform comprehensive gene mutation analysis in cancer cells. The aims of this study were to investigate mutational profiles of synchronous or metachronous MLC, and to compare multiplex gene mutation analysis of MP or IM among paired tumors with clinical or pathological evaluations. [Methods] We performed targeted sequencing for 20 lung cancer related oncogenes using next-generation sequencing technology in 82 tumors from 37 patients (18 patients with synchronous MLC and 19 patients with metachronous MLC) who underwent surgical resection in our department from July 2002 to April 2013. Then, classification of MP or IM was made by clinical, pathological, and gene mutational evaluation. [Results] Among paired tumors, matching of mutation was observed in 20 (54%) cases (nine cases with completely matched and 11 cases with partially matched), which were diagnosed as IM by mutational evaluation. In pathologically suggested IM cases (n=7), six (86%) patients were interpreted as IM by mutational evaluation, and most of them (n=5) had multiple matched mutations, which suggested the clonality between paired tumors strongly. In pathologically suggested MP cases (n=17), the mutational diagnosis was discordant in eight (47%) patients. Among these cases, four paired tumors had multiple matched mutation, suggesting the pathological diagnosis in these cases might be incorrect. In addition, careful interpretation was required when the paired tumors harboring frequent mutation including TP53 or EGFR because such mutation may match accidentally. [Conclusion] Our findings suggest that multiplex mutational analysis of synchronous or metachronous MLC could complement the pathological diagnosis in differentiation whether MP or IM. In the cases with pathologically equivocal or those with discordant between pathological diagnosis and mutational evaluation, the frequency and the number of matched mutation may be helpful for the differentiation.

#3681

A biologic basis for locoregional failure in patients with oral cavity cancers.

Elif I. Sarihan, Brian B. Burkey, Joseph Scharpf, Robert Lorenz, Eric D. Lamarre, Brandon Prendes, Jessica L. Geiger, David J. Adelstein, Shlomo A. Koyfman, Mohamed E. Abazeed. _Cleveland Clinic, Cleveland, OH_.

BACKGROUND: The standard treatment for patients with oral cavity cancer (OCC) with intermediate risk pathologic variables after surgery is adjuvant radiotherapy. Despite this, one-third of patients experience locoregional failure (LRF). Clinicopathologic prognostic models have not been able to identify subsets of patients at higher risk of failure in whom treatment intensification with the addition of systemic chemotherapy should be considered. We posited that gene expression-derived tumor taxonomies can predict treatment failures and therefore guide more nuanced clinical decision making. Herein, we report on a score model based on OCC gene expression characteristics that can be incorporated into risk stratification and treatment decisions.

METHODS: Formalin-fixed paraffin-embedded (FFPE) tissue samples from patients with intermediate risk OCC treated with surgery followed by radiation alone were subjected to quantitative nuclease protection and next-generation sequencing to measure gene expression (HTG Molecular EdgeSeq™). A subset of samples that had corresponding frozen tumor samples were profiled by RNAseq to validate the FFPE results. Patients were divided into two groups based on LRF. Differentially expressed genes were identified using the R limma package. 98 genes were selected on the basis of unadjusted P values and predicted biological impact, as measured by gene set enrichment results (GSEA) and resultant biological pathway scores. The Cancer Genome Atlas (TCGA) HNSCC dataset (n=521) was used to validate the prognostic performance of our gene set.

RESULTS: Of the 78 patients included in the study, 35% of patients had LRF. GSEA of the 98 genes demonstrated a role for DNA repair, oxidative phosphorylation, hypoxia and p53 pathways, indicating radiobiologic plausibility for a significant subset of the genes that constitute the score. The mean composite score was 0.42 for patients with LRF, and -0.19 for patients without LRF (P = 0.0002). The Kaplan-Meier estimates of progression free survival at 3 years for the 1st (high risk) and 4th quartile (low risk) groups were 0.65 (0.47 to 0.89; 95% CI) and 0.93 (0.82 to 1; 95% CI), respectively. On multivariate analysis, the composite score was the strongest predictor of LRF (P = 0.0073). Composite scores also strongly predicted for overall survival in the TCGA HNSCC dataset (P < 0.01) and the Kaplan-Meier estimates of overall survival at 2 years for the 1st and 4th quartile groups were 0.55 (0.45 to 0.68; 95% CI) and 0.73 (0.63 to 1; 84% CI), respectively. Composite scores performed the best in patients with OCC (P = 0.033).

CONCLUSIONS: We developed a gene signature that predicts LRF in patients with intermediate risk OCC treated with surgery and adjuvant radiotherapy. Further validation on larger datasets are needed. This biomarker can potentially identify higher risk patients who should be considered for intensification strategies with the addition of systemic therapy.

#3682

Expression levels of genes in primary melanoma associated with clinically meaningful characteristics.

Marianne Berwick,1 Ivan P. Gorlov,2 Irene Orlow,3 Carol Ringelberg,2 Marc Ernstoff,4 Joel Parker,5 Meg Gerstenblith,6 Cheryl Thompson,7 Eva Hernando,8 Klaus Busam,9 Cecilia Lezcano,3 Sergio Corrales,3 Siok Leong,3 Nancy E. Thomas,5 Christopher I. Amos2. 1 _Univ. of New Mexico Health Sciences Ctr. - Albuquerque, Albuquerque, NM;_ 2 _Dartmouth College, Lebanon, NH;_ 3 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 4 _Roswell Park Cancer Institute, Buffalo, NY;_ 5 _University of North Carolina, Chapel Hill, NC;_ 6 _Case Western University, Cleveland, OH;_ 7 _Case Western Reserve University, Cleveland, OH;_ 8 _New York University Medical Center, New Yrok, NY;_ 9 _Memorial Sloan Kettering Cancer Center, New Yrok, NY_.

Melanoma survival appears to be improving; however, between 13-68% of individuals with melanoma diagnosed with Stages II-III ultimately die from their melanoma. Factors influencing melanoma survival include sex, age, clinical stage, lymph node involvement and several primary tumor characteristics, such as Breslow thickness, presence of infiltrating lymphocytes (TILs), mitotic rate and ulceration. Identification of genes whose expression in primary tumors is associated with these key tumor/patient characteristics can shed light on molecular mechanisms of melanoma survival. Here we show results from a gene expression analysis of 32 formalin-fixed, paraffin embedded (FFPE) primary melanomas with extensive clinical annotation. We extracted RNA, and measured the expression of 760 genes selected based on published evidence of association with melanoma initiation or outcome using NanoString. The Cancer Genome Atlas (TCGA) data on primary melanomas was used for validation of nominally significant associations. We identified 5 genes that were significantly associated with the presence of TILs in the combined analysis of both datasets after adjustment for multiple testing using the Benjamini-Hochberg false discovery rate method: IL1R2, PPL, PLA2G3, RASAL1, and SGK2. We also identified two genes significantly associated with lymph node status (PIK3CG and IL2RA), and two genes significantly associated with sex (KDM5C and KDM6A). We found that LEF1 was significantly associated with Breslow thickness and CCNA2 with mitosis. These results demonstrate that genetic expression in primary tumors is associated with clinically meaningful characteristics of cutaneous melanoma.

#3683

Reciprocal expressions of TFF-1 and TTF-1 in lung adenocarcinomas.

Daisuke Matsubara,1 Manabu Soda,2 Taichiro Yoshimoto,1 Yusuke Amano,1 Atsushi Kihara,1 Yuji Sakuma,1 Shunsuke Endo,1 Koichi Hagiwara,1 Masashi Fukayama,2 Hiroyuki Mano,2 Toshiro Niki1. 1 _Jichi Medical University, Tochigi, Japan;_ 2 _The University of Tokyo, Tokyo, Japan_.

The molecular targeted therapies against EGFR, ALK etc. have improved progression-free survival of patients with lung adenocarcinomas. However, targetable driver mutations have mainly been found in TTF-1-positive terminal respiratory unit (TRU)-type, but have yet been undiscovered in non-TRU type despite the advances in genomics technologies. In order to find the molecular candidate characterizing major subtype of non-TRU type adenocarcinomas, we compared TTF-1-positive/CDH1-positive adenocarcinoma cell lines ("TRU-type" cell lines) (n=11) and TTF-1-negative/CDH1-positive adenocarcinoma cell lines ("non-TRU-type" cell lines) (n=8), and found that TFF-1 (trefoil factor family-1), a gastric mucosal protective factor, was characteristically highly expressed in "non-TRU-type" cell lines. Immunohistochemical analysis of primary lung adenocarcinomas (n=70) revealed that TFF-1 expression was inversely correlated with TTF-1 expression (p=0.0116) and was very rare in primary lung adenocarcinoma cases (5.7%:4/70). So we focused on the non-TRU type adenocarcinomas on the basis of histologic pattern (acinar, solid or mucinous morphology predominant tumors without non-mucinous lepidic growth components) (n=43). Among them, TFF-1 was frequently expressed (62%:27/43), and significantly correlated with the expressions of MUC5AC, HNF4A and CK20 (p<0.0001, p=0.0001, and p=0.0007, respectively), and inversely correlated with TTF-1 expression (p=00039). Next generation sequencing analysis of the 43 cases revealed that KRAS gene mutations and Inactivating mutations and hypermethylation of the TTF-1 gene were frequent in TFF-1 positive cases. The analysis of publicly available data of 442 lung adenocarcinoma cases revealed that high expression of TFF-1 correlated with low expression of TTF-1 and worse prognosis. We confirmed that shRNA against TFF-1 inhibited the cell proliferation and the colony formation, and induced the apoptosis, in the TFF-1 positive "non-TRU-type" cell lines. These results indicate that TFF-1 is not only the marker but also can possibly be the molecular target of a subset of non-TRU-type lung adenocarcinomas.

#3684

HDAC inhibitors restore BRAF inhibitor sensitivity by altering PI3K and survival signaling in melanoma.

Peter Hersey, Stuart J. Gallagher. _Univ. of Sydney, Sydney, Australia_.

Mutations in BRAF activate oncogenic MAPK signaling in almost half of cutaneous melanomas. Inhibitors of BRAF (BRAFi) and its target MEK are widely used to treat melanoma patients with BRAF mutations but unfortunately acquired resistance occurs in the majority of patients. Resistance results from mutations or non-genomic changes that either reactivate MAPK signalling or activate other pathways that provide alternate survival and growth signaling.Here we show the histone deacetylase inhibitor (HDACi) panobinostat overcomes BRAFi resistance in melanoma, but this is dependent on the resistant cells showing a partial response to BRAFi treatment. Using patient- and in vivo- derived melanoma cell lines with acquired BRAFi resistance, we show that combined treatment with the BRAFi encorafenib and HDACi panobinostat synergistically induced caspase-dependent apoptotic cell death. HDAC inhibition decreased PI3K pathway activity associated with a reduction in the protein level of a number of receptor tyrosine kinases including AXL, YAP, ERBB2 and ERBB3 but not EGFR or ERBB4. Both NOXA and BIM proteins were implicated as causing cell death, but in a cell line specific manner. Independent of these changes, panobinostat reduced c-Myc and pre-treatment of cells with siRNA against c-Myc reduced BRAFi/HDACi drug-induced cell death. These results suggest that a combination of HDAC and MAPK inhibitors may play a role in treatment of melanoma where the resistance is due to activation of MAPK-independent pathways. This work identifies patients that may respond well to this drug combination and demonstrates the necessity of disrupting multiple cell survival pathways in melanoma.

#3685

The BET inhibitor INCB057643 suppresses ALDH activity by targeting the ALDH1A1 super-enhancer in high-grade serous ovarian cancer.

Sergey Karakashev. _Wistar Institute, Philadelphia, PA_.

The emergence of tumor cells with certain stem-like characteristics such as high aldehyde dehydrogenase (ALDH) activity due to ALDH1A1 expression contributes to chemotherapy resistance and tumor relapse. However, clinically applicable inhibitors of ALDH activity have not been reported. There is evidence to suggest that epigenetic regulation of stem-related genes contributes to the efficacy of chemotherapy. Here we show that the bromodomain and extra-terminal (BET) inhibitor INCB57643 suppresses ALDH activity by abrogating BRD4-mediated ALDH1A1 expression through a super-enhancer element and its associated enhancer RNA. INCB57643 suppressed the outgrowth of platinum-treated ovarian cancer cells in a concentration-dependent manner. Consistently, INCB57643 synergizes with carboplatin in suppressing the growth of ovarian cancer cells with BRD4 expression, which was accompanied by induction of apoptosis. These phenotypes correlate with inhibition of ALDH1A1 expression through a super-enhancer element and additional stem-related genes, including LIF, HES1 and WNT5a in promoter regions bound by BRD4. Thus, targeting the BET protein BRD4 using small molecule BET inhibitor INCB057643 is a promising strategy for targeting ALDH activity in epithelial ovarian cancer.

#3686

RAIDs: Future prospects for innovative targeted treatments in cervical cancer.

Maud Kamal,1 Els Berns,2 Windy Luscap Rondof,1 Leanne De koning,1 Gemma Kenter,3 Attila Kerezst,4 Marina Popovic,5 Choumouss Kamoun,1 Balazs Balint,6 Suzy Scholl,1 RAIDs Consortium1. 1 _Institut Curie, Paris, France;_ 2 _ERASMUS, Rotterdam, Netherlands;_ 3 _NKI, Amsterdam, Netherlands;_ 4 _SeqOmics, Szeged, Hungary;_ 5 _IOV, Novisad, Serbia;_ 6 _Seqomics, Szeged, Hungary_.

Routine molecular diagnostic tests have yet to be introduced to guide personalized cervical cancer treatment in contrast to other solid cancers. To assess cancer functional events (CFEs), the RAIDs consortium (Rational Molecular Assessment and Innovative Drug Selection, www.raids-fp7.eu) accrued consecutive tumour tissues, whole blood and sera from 419 cervical cancer patients in 18 centers from 7 European Union countries, between 2013-2016 (BioRAIDs: NCT02428842). Whole exome sequencing (WES) is presently available for the first 98 patients, for 20 CC cell lines (list available in supplementary data) and Reverse Phase Protein Array (RPPA) data for 154 patients with a common core set of 91 patients.

By comparing the proportion of mutated tumours for each gene and after correction for multiple testing, we detected no significant difference in the frequency of significantly mutated genes (SMG) between the TCGA and RAIDs datasets. However, alterations in epigenetic regulator genes, which are of potential high clinical interest, are not discussed in the TCGA paper. Mutations, frameshift deletions, insertions or stop codons in MLL2 and MLL3 genes, are present in 17% and 19 % respectively (total: 30%) of the first 98 RAIDs tumours, as well as in all 20 cervical cancer cell lines analyzed. Similar to the TCGA results, frequent mutations in genes coding for acetyltransferases such as EP300/CREBBP (9%) were detected in RAIDs tumours and cell lines (15%/25%). With the exception of SHKBP1, all other novel and previously confirmed genes by TCGA were equally detected in the RAIDs dataset, such as PIK3CA (33%), FBXW7 (14%), NFE2L2 (7%), KRAS (4%), ERBB2 (4%), PTEN (5%), CASP8 (3%).

Similarly to TCGA, RAIDs RPPA data defined 3 stable clusters. TCGA and RAIDs cluster 1 regrouped proteins frequently associated with EMT (epithelial mesenchymal transition). Significant features of RAIDs cluster 1 were high phospho-YAP (p= 3.11e-06) and phospho-Met (p=4.74e-03), high Met (p=2.42e-11), high Notch (p=4.14e-17), low E-cadherin (p=2.41e-06) as well as significant levels of the phosphorylated forms of EGFR, HER2-3 and AKT and high PD-L1 and B7-H4 expression. While TCGA cluster 2 had been associated with "Hormone" signaling, RAIDs cluster 2 was associated with "DNA damage" signaling. Antibody selection was not identical between TCGA and RAIDs and further comparisons are warranted. In RAIDs, pre-treatment activation of DNA repair proteins (Rad50, Phospho-Chk2, Phospho-DNA-PK, Phospho-FANCD2, Phospho-53BP1) together with low levels of Ki67 (proliferation) appeared predictive for a complete response to standard radio-chemotherapy. This needs validation on an independent population. RAIDs and TCGA cluster 3 were associated with p38 MAPK and PI3K signaling.

The RAIDs consortium's future objectives are to better define actionable CFE in relation to outcome and patient's quality of life, allowing the focus on relevant CFEs for patients at high risk.

#3687

Dot1l inhibition selectively impairs androgen receptor dependent prostate cancer growth through loss of telomere integrity.

Rajita Vatapalli, Yara Rodriguez, Changsheng Zhao, Vinay Sagar, Jonathan Anker, Sahithi Pamarthi, Kenji Unno, Benedito Carneiro, Debabrata Chakravarti, Sarki Abdulkadir. _Northwestern University, Chicago, IL_.

The histone methyltransferase DOT1L methylates lysine 79 (K79) on histone H3 and this histone mark is associated with activation of gene expression. DOT1L has a well-known role in MLL fusion leukemogenesis, however, its role in prostate cancer is yet to be elucidated. Here we show that DOT1L is overexpressed in many solid tumors including prostate cancer and is associated with a poor outcome. Genetic and chemical targeting of DOT1L showed a preferential loss in cell viability and colony formation in androgen receptor (AR)-signaling competent prostate cancer cells, including castration-resistant and enzalutamide-resistant cells. DOT1L inhibition reduced AR levels and upregulated enzymes involved in testosterone catabolism but only affected the expression of a subset of AR target genes. More importantly, loss of DOT1L selectively impaired telomere integrity by promoting telomere uncapping and shortening in AR-signaling competent cells. DOT1L inhibition also reduced the expression of telomerase and Shelterin complex genes including TRF2 and POT1. Recruitment of AR to telomeres and K79 methylation at sub-telomeric regions was also diminished. Consequently, loss of DOT1L leads to induction of Telomere dysfunction induced foci (TIF) and activates a cellular senescence program in these cells. Hence, these results provide compelling evidence for DOT1L inhibition as a novel therapeutic approach in advanced prostate cancers.

#3688

A novel biomarker for pancreatic cancer, sTRA, and CA19-9 define separate subpopulations of PDAC cells with distinctions in behavior and molecular characteristics.

Daniel Barnett,1 Ying Liu,1 ChongFeng Gao,1 Luke Wisniewski,1 Katie Partyka,1 Anna Barry,1 Galen Hostetter,1 Aatur Singhi,2 Randall E. Brand,2 Richard R. Drake,3 Brian B. Haab1. 1 _Van Andel Research Inst., Grand Rapids, MI;_ 2 _University of Pittsburgh Medical Center, Pittsburgh, PA;_ 3 _Medical University of South Carolina, Charleston, SC_.

Cancer cells in pancreatic ductal adenocarcinomas (PDACs) exhibit tremendous diversity in behavior and phenotype, but no biomarkers are available to identify subtypes with clinically significant differences in behavior. We recently identified a new marker of pancreatic cancer, called sTRA, that is expressed on cancer cells with different morphologies and molecular characteristics than cancer cells expressing the biomarker CA19-9. Here we tested the hypothesis that the two types of cells have differences between them in behavior, gene expression, and associations with outcome. In cell line models, sTRA-expressing cancer cells were more resistant to drugs, more invasive, and slower-growing than the CA19-9-expressing cells. Patient-derived xenograft tumors produced either CA19-9 or sTRA, and RNA-seq analysis revealed significant, nonrandom differences between the sTRA-expressing and CA19-9-expressing tumors. Each subpopulation was found in lymph nodes or livers with levels that significantly correlated with their matched primary tumors. In an analysis of two tissue microarrays covering 45 and 41 patients, the tumors containing high levels of sTRA-expressing cancer cells had the shortest survival times. These data support the existence of two distinct subtypes of PDAC cells, respectively marked by CA19-9 and sTRA. Together the markers could have value for predicting the behavior of tumors and for isolating, characterizing, and targeting specific subtypes of cancer cells.

#3689

Identification of candidate therapeutic targets in BCG unresponsive bladder cancer- inflammatory subtypes of BCG unresponsive bladder cancer.

Woonyoung Choi,1 Roger Li,2 Chinedu Mmeje,3 I-ling Lee,2 Shanna Pretzsch,2 Jolanta Bondaruk,2 Max Kates,1 Trinity Bivalacqua,1 Bogdan Czerniak,2 Ashish M. Kamat,2 Colin Dinney,2 Peter Black,4 David J. McConkey1. 1 _Johns Hopkins University, School of Medicine, Baltimore, MD;_ 2 _MD Anderson Cancer Center, Houston, TX;_ 3 _Banner MD Anderson Cancer Center Clinic, Gilbert, AZ;_ 4 _University of British Columbia, Vancouver, British Columbia, Canada_.

Intravesical immunotherapy with Bacillus Calmette-Guérin (BCG) is used for the first line treatment of high risk non-muscle invasive bladder cancer. Despite high initial response rates (70%), recurrence is a major problem and many patients develop BCG unresponsive disease, for which the primary treatment option is definitive surgery (cystectomy). In order to define the biological properties of BCG unresponsive disease, we performed whole transcriptome RNAseq on 29 matched tumors obtained from patients before and after the development of BCG resistance. Unsupervised cluster analysis revealed the presence of two clusters - BCG cluster 1, containing 8 pre- and 19-post BCG treatment tumors, and BCG cluster 2, containing 21 pre- and 10- post BCG tumors (Fisher's exact test, p<0.01). To characterize the biological properties of the two clusters, we extracted the significantly differentially expressed genes and analyzed them by Ingenuity Pathway Analysis (Sigma). Interestingly, multiple immune response pathways (T cell receptor signaling, IL8 signaling, IL12-mediated signaling, IFN-gamma pathways, immune cell trafficking, etc) were significantly enriched (p<0.01) in the BCG cluster 1 that was enriched for BCG unresponsive tumors. To determine the potential relationship between BCG clusters 1 and 2 and the previously defined basal and luminal molecular subtypes, we generated a batch-corrected bladder cancer meta-dataset combining two publically available datasets (GSE48075 and GSE32894) consisting of mixtures of non-muscle invasive and muscle-invasive tumors. We performed consensus cluster analysis using the meta-data and identified 3 distinct molecular clusters. The results indicated that the gene expression signature that characterized BCG cluster 1 was also present in tumors assigned to the basal/SCC-like and p53-like/infiltrated tumors defined previously. Together, the results indicate that the tumors in BCG cluster 1 may be enriched with T cells and therefore may have increased sensitivity to immune checkpoint blockade. However, it is likely that alternative therapeutic targets will need to be identified for BCG unresponsive tumors that display the 'immune desert' phenotype characteristic of BCG cluster 2.

#3690

Measuring functional signal transduction pathway activity on breast cancer tissue samples to determine intra-tumor heterogeneity and heterogeneity between primary and metastatic tumors.

Anja Van De Stolpe,1 Anne van Brussel,1 Cathy Moelans,2 Marcia A. Inda,1 Wim Verhaegh,1 Eveline den Biezen,1 Paul van Diest2. 1 _Philips Research, Eindhoven, Netherlands;_ 2 _UMCU, Utrecht, Netherlands_.

Background

Treatment with targeted drugs is increasingly used in breast cancer aiming to block the tumor driving pathway(s). Drug choice is often based on a single primary tumor biopsy. It is important to ascertain that biopsied tumor material is representative for the tumor or metastases to treat with respect to tumor driving signaling pathway. Little is known about pathway heterogeneity within primary breast cancer, and between primary and metastatic tumors. A novel analysis method was developed to identify and quantify activity of signal transduction pathways in cancer tissue, based on Bayesian models which infer a pathway activity score from transcription factor target gene mRNA levels (Cancer Res 2014 Jun 1;74(11):2936-45).

Methods

Originally for AffymetrixU133Plus2.0, the pathway analysis was adapted for RT-qPCR enabling use on FFPE tissue. Calibration was performed using tissue samples with known pathway activity. Functional pathway activity of ER, AR, PI3K-FOXO, Hedgehog (HH), TGFbeta, and Wnt pathways was analyzed to assess (1) intra-tumor heterogeneity: 2-5 FFPE blocks of 17 primary breast cancers (9 luminal A, 4 luminal B, 1 HER2, 3 triple negative (TN), as defined by surrogate immunohistochemistry); (2) heterogeneity between primary and associated metastases: 9 patients with FFPE blocks from primary and 12 metastases.

Results

Intra-tumor heterogeneity: In 11 out of 13 Luminal A- and B-like tumors the ER pathway was active in all samples, 1 showed ER pathway heterogeneity; 11 had an active TGFbeta pathway, heterogeneic in 3; in one ER inactive tumor one (out of 5) samples was AR active. In 2 TN tumors TGFbeta and PI3K pathways were combined active, in one TN tumor heterogeneic TGFbeta activity was observed. PI3K pathway activity increased with malignancy grade and showed most intra-tumor variation. Overall, pathway heterogeneity was detected in over one third of tumors, least in Luminal A-like (2/9) compared to Luminal B-like (3/4) and triple negative (1/3) tumors.

Extensive heterogeneity was found between primary breast cancer and metastases, and between metastatic locations of the same patient. ER pathway activity decreased in 5/9 patients and increased in 4/9 patients in one or more metastatic tumors; PI3K became active in metastasis of 2 patients and inactive in 1; the Wnt pathway became active in 3 patients in bone and ileum metastases; TGFbeta was lost in 5 patients, and became active in 1 bone metastasis; HH became active in 1 ovarian and AR in 1 bone metastasis.

Conclusion

Intra-tumor heterogeneity was lower in ER active compared to TN breast cancer, suggesting a need for multiple biopsies to adequately characterize TN for neoadjuvant therapy. Between primary tumor and metastases, heterogeneity was extensive indicating the need for pathway analysis on metastatic tumors prior to targeted treatment.

#3691

Collagen 1 fiber content may predict for recurrence in non-small cell lung cancer.

Samata Kakkad, Peng Huang, Malcolm Brock, Balaji Krishnamachary, Zaver M. Bhujwalla. _Johns Hopkins Univ., Baltimore, MD_.

Non-small cell lung carcinoma (NSCLC) comprises 85% of all lung cancers. Approximately 30-50% of NSCLC recur despite curative resection. There is a major unmet need to identify NSCLC that are likely to recur so that these patients may be treated with more aggressive therapies. Collagen 1 (Col1) fibers are a major structural component of the extracellular matrix (ECM) of tumors. While increased Col1 fibers have been associated with increased metastasis in breast cancer, the role of Col1 in NSCLC and its relationship to recurrence has not been previously examined. We performed a blinded second harmonic generation (SHG) microscopy study on tumor tissue obtained from 5 patients with nonrecurrent NSCLC and 5 patients with recurrent NSCLC. Col1 fibers were detected with SHG microscopy that detects an intrinsic signal derived from the noncentrosymmetric molecular structure of Col1 fibers. SHG microscopy of tumor regions was performed on the H&E sections to quantify Col1 fiber volume and inter fiber distance in the tumors. At least 10 fields of view were analyzed from 1-2 sections obtained from each tumor. We additionally performed Haralick feature analyses to further analyze Col1 fiber patterns. Adjacent sections were immunostained for CD44 and α-SMA to identify cancer associated fibroblasts (CAFs) and understand their role in these tumors. We observed a significant increase of Col1 fiber volume and a significant decrease of inter fiber distance in recurrent NSCLC. Four Haralick features (orientation, contrast, energy and homogeneity) were also significantly higher in the recurrent tumors compared to the non recurrent tumors. We are currently analyzing the CAFs in the tumor sections to understand their role in the significant differences observed in Col1 fibers between recurrent and nonrecurrent tumors. If these preliminary data are confirmed in an expanded sample set, Col1 fiber analysis may provide a companion diagnostic test that can be performed rapidly on H&E tumor sections, using a confocal microscope, to evaluate the likelihood of tumor recurrence. These studies provide new insights into the role of the tumor microenvironment in NSCLC recurrence, and identify new targets for future therapeutic strategies. This work was supported in part by NIH R35CA209960.

#3692

PRECISION (Profiling Early Breast Cancer for radiotherapy Omission): An ongoing phase II study of breast-conserving surgery without adjuvant radiotherapy for favorable-risk breast cancer.

Lior Zvi Braunstein,1 Ashley Iannone,2 Alphonse G. Taghian,3 Julia Wong,2 Jennifer Bellon,2 Jay R. Harris2. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Dana Farber Cancer Institute, MA;_ 3 _Massachusetts General Hospital, Boston, MA_.

Background: Radiation therapy (RT) following breast conserving surgery (BCS) is well-known to improve outcomes for invasive breast cancer. However, RT can be resource-intensive, disruptive, and confers a small risk of long-term adverse effects. In addition, reports have suggested that not all patients stand to benefit equally from RT, with certain features portending an excellent prognosis regardless of the adjuvant treatment approach. Specifically, favorable luminal A cancers exhibit a 10-year locoregional recurrence (LRR) rate of 2% following BCS and RT. However, distinguishing luminal A cancers from their less-favorable luminal B counterparts remains a challenge. This trial employs molecular profiling to identify favorable-risk, early-stage breast cancer patients who may safely omit RT following BCS.

Methods: To identify a low-risk, luminal A cohort, we are employing well-characterized clinicopathologic parameters in addition to molecular profiling. Eligible patients are 50-75 years of age and must have invasive breast cancer ≤2 cm (T1), that is estrogen or progesterone receptor positive, low- or intermediate-grade, without HER2 amplification, and with negative margins (no ink on tumor) following BCS. Following resection, tumor samples undergo prediction analysis of microarray 50 (PAM50) profiling via the NanoString Prosigna assay to characterize intrinsic biologic subtype. Patients with tumors classified as luminal A (low risk of recurrence score) are eligible for the omission of RT. The trial is a single-arm prospective cohort study designed to assess the viability of RT omission in this favorable population. The primary endpoint is LRR at 5 years; a rate of <5% will be considered of interest. Projected accrual is 345 patients over 4 years. Using this design, the study has 90% power to distinguish a 5-year LRR rate of 1% from 5% using a one-sample exponential test with one-sided type I error of 0.025. Secondary endpoints include 5- and 10-year recurrence rates of any type (local, regional and/or distant), disease-free survival, invasive-disease-free survival and overall survival, in addition to rates of salvage mastectomy and radiotherapy.

Results: PRECISION opened to accrual in April 2016 and, as of October 2017 has registered 57 patients to undergo molecular risk profiling. Of these, 2 (3.5%) withdrew consent prior to receiving their PAM50 results and 11 (19.3%) were ineligible based on their risk-of-recurrence score, leaving 43 patients eligible for the omission of RT. Of these, 4 (7.0%) ultimately chose to undergo RT, while 39 (68.4%) opted to omit RT on the investigational arm of the study.

Conclusions: The omission of RT is of interest to low-risk breast cancer patients. The safety and feasibility of this approach is currently being studied.

#3693

**Immunologic and clinical implications of polymerase δ (** POLD **) and Polymerase ε (** POLE **) gene mutations in non-small cell lung cancer (NSCLC).**

Young Kwang Chae, Kyunghoon Rhee, Lee Chun Park, Anderson Cho, Sangmin Chang, Taeyeong Ko, Andrew Davis, Manali Bhave, Marcello Cruz, Wadw Iams. _Northwestern University Feinberg School of Medicine, Chicago, IL_.

POLD1 plays a role in proofreading the polymerase δ complex while POLE encodes the major catalytic and proofreading subunit of the polymerase ε complex. Mutations in POLD1 and POLE increase spontaneous somatic mutation rates which have been linked with favorable response to PD-1 inhibitor in NSCLC. However, little is known about its impact on the immune landscape and prognosis.

We analyzed genomic, transcriptomic and clinical data from The Cancer Genome Atlas (TCGA) database using cBioPortal of patients with lung adenocarcinoma (ADC, 522 samples) and squamous cell carcinoma (SQCC, 504 samples). We compared patient samples with at least one mutation (M group) in POLD/E family genes (POLD1, POLD2, POLD3, POLD4, POLE, POLE2, POLE3, POLE4) with patient samples without any mutations in above mentioned gene set (N group). The tumor immune landscape was analyzed from RNA-seq z-scores of 812 'immune metagene' signatures (Angelova, M. et al, 2015) which were used to predict immune infiltration of 31 distinct immune cells for each tumor sample.

POLD family gene mutation frequency was 0.5% (5/1026 samples) while POLE family gene mutation frequency was 2.0% (21/1026 samples). M group showed significantly higher tumor mutational burden (TMB) in both ADC (median mutation count M:475 N:149, t-test p=0.003) and SQCC (median mutation count M: 318 N:218, t-test p=0.01). In ADC, infiltration of activated CD8 T-cells (M:46% N:26%, chi square test p=0.11) and activated CD4 T-cells (M:46% N:29%, chi square test p=0.19) were not significant. In SQCC, M group showed significantly higher infiltration of activated CD8 T-cells (M:54% N:27%, chi square test p=0.03) while infiltration of activated CD4 T-cells (M:46% N:36%, chi square test p=0.44) was not significant. In terms of survival, in ADC, there was significantly worse survival in M group compared to N group (median M:21 N:50 months, log-rank test p<0.001), while in SQCC, there was no significant difference (median M:66 N:62 months, log-rank test p=0.70).

This is the first comprehensive report describing the immunologic and clinical implication of POLD/E family gene mutation status in NSCLC. In summary, POLD/E family gene mutation was linked with higher TMB in NSCLC. In adenocarcinoma, mutational status of POLD/E was associated with an unfavorable survival outcome suggesting potential prognostic implications.

#3694

Measuring oncogene signaling with transferrin-based PET: From bench to bedside.

Michael J. Evans,1 Charles Truillet,1 Davide Ruggero,1 Jason S. Lewis,2 Rahul Aggarwal,1 Spencer C. Behr1. 1 _University of California San Francisco, San Francisco, CA;_ 2 _Memorial Sloan Kettering Cancer Center, New York, MD_.

Background: Non-invasive tools that measure the activity of central oncogenes could be broadly useful for cancer detection and management. We have shown that radiolabeled transferrin molecules can be used to measure mTORC1 and MYC signaling in prostate cancer models, because the transferrin receptor is a MYC target gene. The purpose of this study was to complete the preclinical assessment of 89Zr-transferrin, as well as demonstrate that clinical disease harbors avidity for transferrin using 68Ga-citrate. Methods: Human prostate cancer cell lines have been manipulated pharmacologically and genetically to alter mTORC1 and/or MYC signaling, and radiolabeled transferrin has been used to study the impact of aberrant oncogene signaling on transferrin biology. A first in man study with 68Ga-transferrin PET was also performed at UCSF using a GE SIGNA PET/MR. A dose escalation study was performed in eight patients with castration resistant prostate cancer to identify the optimal signal to noise ratio. Results: Activation of mTORC1 or MYC resulted in higher transferrin receptor expression and higher transferrin uptake into cells, as expected. Pharmacological inhibition of either oncogene suppressed transferrin uptake in vitro and in vivo. 68Ga-transferrin PET/MR resolved ~75% of lesions that were detectable by CT or bone scan. Approximately 4 mCi and >3.5 hours post injection was required to visualize tumor lesions. Conclusions: These experiments establish that like MYC, mTOR activity can be quantified in prostate cancer models with 89Zr-transferrin. Moreover, our first in human study with 68Ga-transferrin shows that human disease is generally avid for the radiotracer, but the uptake is variable among lesions, consistent with a molecularly diverse disease.

#3695

Mapping of molecular landscape underlying drug resistance and recurrence in glioblastoma: Paired analysis of primary and recurrent tumors.

Magdalena Houdova Megova, Zuzana Sporikova, Radek Trojanec, Ondrej Kalita, Jana Vrbkova, Jiri Drabek, Vladimira Koudelakova, Marian Hajduch. _Faculty of Medicine and Denistry, Palacky University Olomouc, Olomouc, Czech Republic_.

Background: Glioblastoma (GBM) is the most frequent and the most malignant brain tumor in adults. Despite aggressive therapy, overall survival of GBM patients remains poor due to intrinsic or acquired resistance to combined radiation and chemotherapy. Underlying biology behind GBM progression after first line therapy is mostly unknown and thus, there are only limited targeted therapeutic options for the 2nd line treatment. The aim of this study was to investigate genetic landscape in paired samples of primary versus recurrent tumors and to validate prognostic and predictive value of the most significant differences in independent cohort of GBMs.

Patients and methods: 43 paired tumor samples were obtained during the 1st surgery followed by radiochemotherapy and after tumor recurrence during the 2nd palliative surgery. 24/43 paired samples were analyzed for whole genome copy number variations (CNV), 23/43 by fluorescent in situ hybridization for determination of EGFR, p53, RB1, MDM2, CDKN2A, 1p, 19q and 10p statuses. Methylation-specific PCR was used for analysis of MGMT promoter methylation and competitive amplification of differentially melting amplicons PCR for IDH1/2 mutations detection. The impact of most relevant alterations identified in paired GBMs was then validated in independent cohort of unpaired 104 tumors.

Results: The most significant differences induced by chemoradiotherapy and/or GBM progression in primary versus recurrent tumors were EGFR amplification (19/24 primary tumors, 16/24 recurrent tumors), CDKN2A loss (18/24 primary tumors, 16/24 recurrent tumors), MDM2 gain (4/24 primary tumors), PDGFRA gain (3/24 recurrent tumors) and GSTT1 loss in primary tumors (7/24) and GSTM1 gain in recurrent tumors (7/24). Univariate analysis of overall survival for significant copy number alterations shown significantly worse prognosis for patients with GSTT1 deletion (HR = 5.356, CI = 1.161-24.701, p = 0.031) in primary tumor and trend for better prognosis in patients with CDKN2A deletion (HR = 0.391, CI = 0.124-1.229, p = 0.108) and GSTM1 amplification (HR = 0.341, CI = 0.090-1.288, p = 0.112) in recurrent tumors. Significantly worse prognosis for GBM patients with aberrations of GSTT1 gene was also confirmed in independent validation cohort of unpaired GBMs in multivariate analysis adjusted for age and Karnofsky score (gain: HR = 1.95, CI = 1.116-3.260, p = 0.011; loss: HR = 2.15, CI = 1.185-3.900, p = 0.012).

Conclusion: Molecular genetic profiling of 24 recurrent GBM showed association between recurrence and status of glutathione S-transferases genes. Significance of GSTT1 gene aberration was validated in independent group of glioblastomas and indicates involvement of redox enzymes in GBM chemoradiation response and tumor progression.

Acknowledgment: The financial support of grants TE02000058, IGA UP LF_2017_013 is gratefully acknowledged.

#3696

RNAseq of patient-derived cancer stem-like cells and exosomes provides new insights into Ewing's sarcoma.

Mariona Chicón Bosch,1 Elizabeth A. Roundhill,1 Alastair P. Droop,2 Michael Parry,3 Lee Jeys,3 Susan A. Burchill1. 1 _University of Leeds, Leeds, United Kingdom;_ 2 _MRC Medical Bioinformatics Centre, Leeds, United Kingdom;_ 3 _The Royal Orthopaedic Hospital, Birmingham, United Kingdom_.

Background

Cancer stem-like cells (CSCs) are responsible for disease progression and relapse (Schatton et al, Bioessays 2009, 31:1038-49). Therefore, the eradication of Ewing's sarcoma (ES)-CSCs is expected to improve outcome for patients. Since the transfer of exosome cargo between cells drives ES progression (Ventura et al, Oncogene, 2016, 35:3944-54) or can be exploited as biomarker (Miller et al, Biology of the Cell 2013, 105:289-303; Tsugita et al, PLoS ONE 2013, 8:e77416) we have characterized primary patient-derived ES, paired ES-CSCs, and the exosomal cargo from these cultures to identify potential prognostic biomarkers of risk and targets for the development of new treatments.

Methods

ES taken at diagnosis and resection were collected following patient consent (GenoEwing; IRAS-167880). ES-CSCs were isolated using a functional self-renewing assay. EWSR1 translocations were confirmed by FISH using a break-apart probe and RT-PCR. Exosomes were collected after incubation of cultures in serum-free media for 48h, isolated using exoEasy Maxi kit (Qiagen) and characterized by Nanoparticle tracking, Western blot and flow cytometry. The expression of the ES-marker CD99 was investigated by immunocytochemistry, Western blot and flow cytometry. RNA was extracted from ES cells and paired exosomes using the miRNeasy Micro kit (Qiagen) and RNA quality confirmed employing the Agilent Bioanalyser. Total RNA libraries were prepared and sequenced using the HiSeq3000 (Illumina®). Reads were processed using fqtools (Droop, Bioinformatics 2016, 32:1883-4), aligned by STAR (Dobin et al, Bioinformatics 2013, 29:15-21) and differential expression was determined by DESeq2 (Anders et al, Genome Biology 2010, 11:R106). Genes were ranked on adjusted p value and log2 fold change; candidates were validated by RTqPCR.

Results

ES patient-derived cells expressed CD99 and contained an EWSR1 gene translocation (23/23). The median size of isolated ES exosomes was 84nm (range 32-132nm). Exosomes were enriched for small RNA (53±5% in exosomes vs 12±3% in cells). The optimal protein panel to identify ES exosomes is CD81, CD63 and TSG-101; CD9 was not detected. Consistent with the exosomal cell of origin, expression of CD99 was detected in 71-79% of isolated vesicles.

The RNA profile of parental cultures and daughter ES-CSCs was compared and two genes with increased expression in ES-CSCs were validated by RTqPCR (p<0.0001). Using a novel experimental approach in ES, we have sequenced the total RNA profile of paired exosomes from patient-derived ES cultures and confirmed the expression of CD99 and one putative ES-CSC biomarker in exosomes.

Conclusions

For the first time, we have sequenced paired exosomes and patient-derived ES cells to identify a potential prognostic biomarker and novel target for therapy.

Work funded by University of Leeds, Ewing's Sarcoma Research Trust (ESRT) and Bone Cancer Research Trust (BCRT).

#3697

High PD-L1 expression and targetable mutations occur independently in NSCLC and are not influenced by smoking or race.

Noura J. Choudhury, Mansooreh Eghtesad, John Cursio, Sabah Kadri, Lauren Ritterhouse, Jeremy Segal, Aliya Husain, Jyoti D. Patel. _Univ. of Chicago, Chicago, IL_.

Background: Targeted therapies such as EGFR tyrosine kinase inhibitors (TKIs), and immunotherapy (IO) with PD-L1 inhibitors have revolutionized first-line treatment options for advanced non-small cell lung cancer (NSCLC). Several small studies suggest that high PD-L1 expression rarely overlaps with classic driver mutations, yet it remains unclear how smoking history, race, and stage may influence the interaction between PD-L1 expression and targetable driver mutations. In our racially diverse population with a high proportion of African American patients, we investigated whether high PD-L1 expression occurs concurrently with targetable mutations in EGFR, BRAF, and MET and whether smoking history, stage, or race alters this distribution.

Methods: In this retrospective study, we selected all NSCLC patients of any stage at our institution whose tumor samples had undergone both in-house next-generation sequencing (NGS) and PD-L1 staining with the Dako Clone 28.8 antibody between March 2016 to July 2017. Our validated NGS assay, the OncoPlus, detects mutations, deletions, and insertions in 147 clinically reported genes, including all classic driver mutations in lung cancer. Patient demographics, treatments and response data were extracted from the electronic medical record through October 1, 2017. We then used Wilson proportion test and chi-squared analysis to analyze whether PD-L1 expression and actionable mutations varied by race, smoking, age and stage.

Results: 114 cases had both PD-L1 staining and NGS analysis available. 71 (62.2%) patients had ≥10 pack year smoking history while 21 (18.4%) patients were never smokers. 41 (35.9%) patients were African American, 62 (54.4%) Caucasian and 7 (6.1%) were Asian. 26 (22.8%) had high expression of PD-L1 (≥50% cells staining positive), and 15 (13.2%), 1 (0.8%), 4 (3.5%), and 6 (5.2%) patients, respectively, had an EGFR, BRAF (V600E), ERBB2, and MET exon 14 skip mutation. 4/26 patients with high PD-L1 expression had a targetable mutation, compared to 22/88 patients with low PD-L1 expression (p<0.001, Wilson test). This distribution did not vary significantly when stratified by race, stage, or smoking history.

Conclusions: We present here our institution's novel workflow of obtaining both in-house NGS and PD-L1 staining at time of initial biopsy, in a diverse population with 1/3 African American patients. We demonstrate that high PD-L1 expression and actionable driver mutations infrequently coexist in the same tumor, and this relationship is not driven by smoking, race, or stage. Our results strongly suggest the necessity of obtaining both PD-L1 staining and NGS at time of initial biopsy to identify optimal therapies for patients with advanced disease. Our results imply that patients with actionable driver mutations are unlikely to benefit from later treatment with IO, which we hope to test explicitly in future studies.

#3698

Unraveling triple-negative breast cancer molecular heterogeneity using an integrative multiomic analysis.

Yacine Bareche, David Venet, Philippe Aftimos, Michail Ignatiadis, Martine Piccart, Francoise Rothe, Christos Sotiriou. _Jules Bordet Institute, Brussels, Belgium_.

Introduction : Triple negative breast cancers (TNBCs) represent about 10-20% of all breast cancers and show worse prognosis compared to other molecular subtypes with increased likelihood of early distant recurrence and death. Recent efforts of genome-wide gene expression profiling analyses have improved our understanding of the biological complexity and diversity of TNBCs reporting, at least 6 different molecular subtypes of TNBC namely Basal-like 1 (BL1), basal-like 2 (BL2), immunomodulatory (IM), mesenchymal (M), mesenchymal stem-like (MSL) and luminal androgen receptor (LAR). However, little is known regarding the potential driving molecular events within each subtype, their difference in survival and response to therapy. Further insight into the underlying genomic alterations is therefore needed.

Aims : Here, we aimed to study the genomic aberrations that drive each of these TNBC subtypes by applying an integrated analysis of somatic mutation, copy number and gene expression of 550 TNBC derived from Molecular Taxonomy of Breast Cancer International (METABRIC) and The Cancer Genome Atlas (TCGA) consortia.

Methods : The METABRIC & the TCGA datasets were used, with available data for copy-number aberrations, somatic mutations and gene expression. ER, HER2 and PR status were inferred based on the bimodality of their mRNA expression level. TNBC samples (n=550) were classified according to Lehmann's molecular subtypes using the TNBCtype online subtyping tool (http://cbc.mc.vanderbilt.edu/tnbc).

Results : We were able to globally reproduce Lehmann's classification with Basal-like 1 (BL1), Immunomodulatory (IM), Androgen-Receptor-like (LAR), Mesenchymal-like (M) and Mesenchymal Stem Cell-like (MSL) being the most stable TNBC subtypes. Each subtype showed significant clinic-pathological characteristic differences. Using a multivariate model, the IM and LAR subtypes showed to be associated with a better prognosis (HR=0.68; CI=0.46-0.99; p=0.043) and a worst prognosis (HR=1.47; CI=1.0-2.14; p=0.046), respectively. BL1 subtype was found to be most genomically instable subtype with high TP53 mutation (92%) and copy-number deletion in genes involved in DNA repair mechanism (BRCA2, MDM2, PTEN, RB1 & TP53). LAR tumours were associated with higher mutational burden with significantly enriched mutations in PI3KCA (55%), AKT1 (13%) and CDH1 (13%) genes. M and MSL subtypes were associated with higher signature score for angiogenesis. IM showed high expression levels of immune signatures and check-point inhibitor genes such as PD1, PDL1 and CTLA4.

Conclusions : Our findings highlight for the first time the substantial genomic heterogeneity that characterize TNBC molecular subtypes, allowing for a better understanding of the disease biology as well as the identification of several candidate targets paving novel approaches for the development of anti-cancer therapeutics for TNBC.

#3699

Histone deacetylase inhibitor ITF2357 induces apoptosis and increases doxorubicin cytotoxicity in preclinical models of human sarcoma.

Maria Grazia Tupone,1 Marta Di Martile,1 Marianna Desideri,1 Simonetta Buglioni,1 Barbara Antoniani,1 Carlotta Mastroiorio,1 Rita Falcioni,1 Virginia Ferraresi,1 Nicola Baldini,2 Roberto Biagini,1 Michele Milella,1 Daniela Trisciuoglio,3 Donatella Del Bufalo1. 1 _Regina Elena National Cancer Institute, Rome, Italy;_ 2 _Istituto Ortopedico Rizzoli, Bologna, Italy;_ 3 _National Research Council, Rome, Italy_.

Sarcomas are rare malignancies with generally poor prognosis, for which current therapies have shown limited efficacy. In order to improve the prognosis of patients, there is an urgent medical need to identify new molecular targets that can be exploited for the development of innovative treatment approaches. Inhibition of histone deacetylases (HDACs) has emerged as a potential strategy to reverse aberrant epigenetic changes associated with cancer, and HDAC inhibitors (HDACi) have been reported as potent and specific anticancer molecules in preclinical and clinical studies. Here we investigated the molecular and functional effects of a new generation HDACi, ITF2357, in preclinical models of soft tissue and bone sarcomas. A wide panel of sarcomas cells, both established and patient derived cell lines, were used. For in vitro experiments Western Blot, RT-PCR, flow cytometry and conservative isobologram analysis have been performed. In vivo efficacy of ITF2357/doxorubucin combination was evaluated in nude mice bearing sarcoma xenografts. Using in vitro and in vivo sarcoma models, we demonstrate that ITF2357 decreases cell viability, activates apoptosis and increases the growth inhibitory efficacy of doxorubicin. The molecular mechanisms of action of ITF2357-mediated cell death implied the activation of mitochondrial apoptosis, as attested by the increase of pro-apoptotic BH3-only proteins, and both caspase and bcl-2 dependent cell death. We also found that ITF2357 transcriptionally upregulated the pro-apoptotic BH3-only genes Bax, Puma, Noxa and BIM in cells harbouring mutant or wild type p53, thus indicating a p53 independent mechanism. Both bcl-2 overexpression and caspases inhibition strongly reduced apoptosis, corroborating the evidence that activation of mitochondrial pathway occurs upstream and before caspases activation. We also found that ITF2357 induces a canonical autophagic process, and that inhibition of autophagy increased apoptosis induced by ITF2357, indicating that in our models autophagy shows a prosurvival effect. We also reported that ITF2357 induced FOXO1/3a up-regulation, FOXO proteins nuclear accumulation, and transcriptional activation of FOXO-target genes, such as BH3-only proteins Puma, Noxa and Bim. Knockdown experiments demonstrated that reduction of both FOXO1 and 3a did not inhibit the induction of Bim expression in response to ITF2357, and consequently did not protect sarcoma cells against ITF2357-induced apoptosis, independently from p53 status. Notably, ITF2357 synergized with doxorubicin to induce in vitro cell death and to reduce in vivo tumor growth. Therefore ITF2357 may represent an important therapeutic agent against human sarcoma regardless p53 status, and the pharmacological combination of ITF2357 with doxorubicin has the potential to enhance sensitization in different preclinical models of sarcoma.

#3700

A window into human tumor progenitor cell subsets: Functionalizing a novel platform, the micropallet array, for molecular evaluation of single adherent cells with defined cell surface phenotype.

Edward L. Nelson, Trisha Westerhof, Giulia Giammo, Guann-Pyng Li, Mark Bachman. _University of California, Irvine, Irvine, CA_.

The recognition of unique cellular elements such as cancer stem cells (CSCs) and processes such as epithelial to mesenchymal transition (EMT) raises predictions that are biologically provocative and clinically impactful. To better understand the interplay between individual cellular elements, and improve current molecular and cellular characterizations of an individual's cancer, the development of a technology to rapidly identify, isolate, and evaluate a patient's tumor biopsy at single cell resolution is required. The micropallet array is a photoresist-based technology that provides a means to isolate and recover single adherent cells on individual pedestals, termed "micropallets". We have functionalized this platform to identify cells of defined surface phenotype in heterogeneous mixtures, such as would be obtained from a tumor tissue biopsy, by incorporating multicolor immunofluorescent confocal imaging to interrogate the expression pattern of up to 6 different cell surface molecules on heterogeneous cell mixtures distributed over a micropallet array. The ability to interrogate the expression of multiple cell surface molecules provides a critical addition to the existing micropallet array platform, in that it enables the identification, enumeration, and along with the previously demonstrated capacity to release and retrieve micropallets carrying single adhered cells, the study of single cells of defined cell surface phenotype. Herein, we report functionalization of the platform and its application to human breast tumors as a proof of principle for its application. We demonstrate the capacity to identify, retrieve, and study single adherent cells that are characterized as; putative CSCs, endothelial progenitor cells, bulk malignant epithelium, leukocytes, and cells with hybrid cell surface phenotype suggestive of cells undergoing EMT, using a panel of antibodies to detect select surface molecules (CD24, CD44, CD45, CD133, CD309, and CD326 [Epithelial Surface Antigen or ESA]). Furthermore, we have applied single cell nanoString analyses to characterize their respective molecular profiles. This provides a unique and unmatched platform for the study of minimally perturbed, primary human tumor cellular subsets, obtained from standard small volume tumor biopsies, and to address biological and cancer biology questions, at single cell resolution, that have been heretofore beyond reach.

## CANCER CHEMISTRY:

### Drug Delivery

#3701

Targeted Bcl2 siRNA delivery using DNA nanoparticles in cancer therapy.

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

Background: Short interfering RNA (siRNA), highly capable of knockdown of specific genes, has emerged as a promising molecular therapeutic tool in targeted cancer treatment. Nonetheless, only limited success has been achieved in the systematic administration of siRNA. Therefore, the lack of a robust and versatile siRNA delivery system is a critical issue in translating this therapeutic tool for cancer treatment. Recent development in DNA nanotechnology has made programmable DNA nanoparticles (DNPs) a potent drug delivery platform. Rationally designed DNPs have emerged as facile delivery vehicles since their physicochemical properties can be precisely controlled compared to other delivery systems. This study focuses on the development of tumor targeted novel DNP-based siRNA delivery system to knockdown Bcl2 gene, as a targeted cancer therapeutic.

Methods: Structural DNA technology was applied to design a library of DNPs with different sizes and shapes. Flow cytometry, confocal imaging, and transmission electron microscopy (TEM) were utilized to study the cellular internalization of DNPs. The efficacy of Bcl2 knockdown by DNP-siBcl2 and the resulting influence on cell growth and progression were assessed in cancer cells (in vitro) and in mice bearing corresponding tumor xenografts (in vivo).

Results: The correlation between DNPs design (size, shape, tumor-targeting) and internalization efficiency in different cancer cell lines remains elusive. We investigated the cellular uptake of different sizes and shapes of DNPs with or without aptamer (nucleolin targeting molecule) in multiple human cancer cell lines. The cellular uptake efficiency of DNPs was influenced by targeting ligand, size, shape, and cell line. We observed distinct stages of the internalization process of a gold nanoparticle (AuNP)-tagged DNP, using high-resolution TEM. This study provides detailed understanding of cellular uptake and intracellular trafficking of DNPs in cancer cells. Anti-cancer therapeutic potential of siBcl2 delivered by DNPs were studied in mice bearing DMS53 tumor xenografts. Significant tumor growth inhibition was observed in mice treated with DNPs-siBcl2 (5 mg/kg or 10 mg/kg, IV) relative to vehicle alone and naked siBcl2. No toxicity was observed by pathologic examination of lung, liver, kidney, heart, brain, or spleen.

Conclusions: Our study offers new insights for future optimization of DNP-based drug delivery systems for cancer therapy. Our novel aptamer conjugated DNP formulations demonstrated substantial cellular internalization. Targeting Bcl2 and its downstream signaling intermediaries reduced cellular growth. We validated the strategy of silencing of Bcl2 using DNPs in order to inhibit cancer progression in vivo. We believe that the DNPs and methodologies developed in this project will be applicable to knockdown of Bcl2 and other gene targets and may be an excellent platform to apply in future anti-cancer therapy.

#3702

A novel nanoparticle-based theranostic agent targeting low-density lipoprotein receptor-related protein-1 enhances the efficacy of neoadjuvant radiotherapy in colorectal cancer.

Kyoung Jin Lee,1 Serk In Park,1 Seong-Yun Jeong,2 Eun Kyung Choi2. 1 _Korea University College of Medicine, Seoul, Republic of Korea;_ 2 _Asan Medical Center, Seoul, Republic of Korea_.

Neoadjuvant radiotherapy is becoming an important therapeutic option for colorectal cancer (CRC) patients, although complete tumor response is observed only in 20 to 30% patients. However, precise biologic mechanisms underlying resistance to radiotherapy remain to be elucidated. In this study, we established a radiotherapy-resistant CRC patient-derived xenograft (PDX) mouse model, and we discovered that low-density lipoprotein receptor-related protein-1 (LRP-1) is increased in radiotherapy-resistant tumors compared with radiotherapy-sensitive tumors. Immunohistochemistry, mRNA and protein expression analyses of radiotherapy-sensitive vs. -refractory CRC patient samples confirmed a statistically significant association between LRP-1 levels and radiotherapy resistance of CRC. We also found that LRP-1 expression is significantly associated with the prognosis of CRC patients by analyzing gene expression data of CRC patients in Gene Expression Omnibus public database. Subsequently, aiming to develop radio-sensitizing nanoparticles targeting LRP-1 for chemotherapeutic drug delivery and in vivo imaging, we screened and identified an LRP-1-binding peptide in a radiotherapy-resistant CRC PDX mouse model using M13 phage display. We next engineered human serum albumin nanoparticles (HSA NPs) containing the LRP-1-binding peptide (for tumor localization), 5-FU (for cytotoxic chemotherapy) and Cy7 fluorophore (for in vivo imaging). When administered in CRC PDX-bearing mice, the LRP-1-targeting HSA NPs accumulated specifically in the CRC tumor tissue and dramatically increased the efficacy of radiotherapy. In addition, whole-body fluorescence imaging of PDX CRC-bearing mice showed that the HSA-NP accumulated only in radiation-treated tumors with high ROI values. In summary, we identified that LRP-1 is a novel signature protein of radiotherapy-resistant CRC, and validated that LRP-1 is closely associated with the progression of CRC. We developed a novel HSA nanoparticle-based theranostic agent loaded with LRP-1-binding peptide, 5-FU and a fluorophore that is applicable to both diagnostic imaging and radio-sensitizing neoadjuvant therapy of CRC. This approach is clinically applicable to improve the effectiveness of neoadjuvant radiotherapy and increase the ratio of complete tumor response in radio-resistant CRC.

#3703

Superior efficacy of novel albumin-binding auristatin E-based prodrugs compared to auristatin E in a panel of human xenograft models in mice.

Stephan D. Koester, Lara Pes, Johannes P. Magnusson, Serghei Chercheja, Federico Medda, Friederike I. Nollmann, Patricia Perez Galan, Javier Garcia Fernandez, Heidi-Kristin Walter, Khalid Abu Ajaj, Anna Warnecke, Felix Kratz. _CytRx Corporation, Freiburg, Germany_.

Auristatins are highly cytotoxic antimitotic tubulin-binding peptides. Of this family, only Adcetris®, an antibody drug conjugate (ADC) derived from monomethyl auristatin E (MMAE), is approved and marketed. Other auristatins such as dolastatin 10, dolastatin 15, and auristatin PE reached phase 1 and 2 clinical trials, but due to systemic toxicity and low antitumor activity they were discontinued.1 Because of the high cytotoxicity, it is important to deliver these drugs selectively to the tumor avoiding premature release in the blood circulation.

We developed an acid-sensitive drug delivery system that uses circulating endogenous serum albumin as a macromolecular carrier. The drug is rapidly bound covalently to albumin upon i.v. injection and, following accumulation in the tumor due to enhanced uptake and retention mechanisms, the highly cytotoxic agent is released in a pH-dependent manner.2 Herein we present in-vivo data of the considerably improved efficacy of two auristatin E derived prodrugs, AE-Keto-Sulf07 and AE-Ester-Sulf07, compared to the parent compound auristatin E. The acid-sensitive hydrazone prodrugs were prepared from the respective carbonyl-containing auristatin E derivatives and the water-solubilizing maleimide-bearing linker Sulf07.

Female NMRI nu/nu mice were inoculated subcutaneously with patient- or cell-derived human tumor xenografts (A375, A2780, RXF631, LXFA737) and randomized (n=7-8 per group) with starting tumor volumes in the range of 140-350 mm3. AE-Keto-Sulf07 showed excellent antitumor response over a wide dose range (3.0-6.5 mg/kg twice per week over 3-4 weeks), with optimal dosage at 4.5 mg/kg twice per week over 4 weeks. AE-Ester-Sulf07 was highly efficacious between 1.9 and 2.4 mg/kg dosed twice per week over 3-4 weeks or at 3.8 mg/kg dosed once per week over 4 weeks, but caused cumulative skin lesions due to scratching and biting. In contrast, auristatin E was dosed at 0.3 mg/kg twice per week over 3-4 weeks and was only marginally active.

In summary, we have shown for the first time that auristatin E derivatives, namely AE-Keto-Sulf07 and AE-Ester-Sulf07, bound to circulating albumin demonstrate promising antitumor efficacy and induce statistically significant long-term partial or complete remission in a panel of human xenograft models in mice.

#3704

Efficacy of DR5-targeted immunoliposomes in pancreatic cancer models primed with SMO inhibitors.

Darren Chan,1 Charlene Minx,1 Jun Wang,1 Arindam Sen,2 Michael Johnston,3 Wen Wee Ma,4 Christopher J. Scott,3 Robert M. Straubinger1. 1 _University at Buffalo, Buffalo, NY;_ 2 _Roswell Park Cancer Institute, Buffalo, NY;_ 3 _Queen's University of Belfast, Belfast, United Kingdom;_ 4 _Mayo Clinic, Rochester, MN_.

Background: Pancreatic cancer (PaCA) is a deadly disease with 5-year survival of 6%. Poor prognosis is attributed to low tumor microvessel density, poor perfusion, and amplified stroma, constituting a drug delivery barrier. Tumor priming using NVP-LDE225, an inhibitor of sonic hedgehog (sHH) signaling, can compromise the stromal drug delivery barrier by mediating stromal thinning, and discharge of tumor interstitial pressure (IFP) and decompression of tumor vasculature, resulting in increased perfusion and deposition of drug-containing nanoparticles into tumors. However, convective fluid efflux may increase as interstitial tumor pressure declines during priming, increasing drug washout. We hypothesize that a targeting nanoparticle delivery system will increase drug retention during priming, thereby increasing efficacy.

Approach: Patient-derived xenograft (PDX) PaCA tumors possessing varied stromal architecture and degrees of desmoplasia were implanted s.c. in SCID mice. They ranged from low- to high desmoplasia and varied in microvessel density. The sHH inhibitor (sHHI) NVP-LDE225 was administered p.o. for 10 days, and fluorescent 80nm sterically-stabilized liposomes (SSL) were administered at intervals to probe tumor vascular perfusion/permeability. Efficacy was investigated with SSL liposomes containing doxorubicin (SSL-DXR), optionally coupled covalently with Death Receptor 5 monoclonal antibody AMG655 (SSL-DXR-DR5), which binds the pro-apoptotic DR5 receptor and initiates the extrinsic apoptotic pathway.

Results: Quantitative fluorescence microscopy showed that sHH inhibitor treatment mediated a temporal window of enhanced permeability/perfusion and deposition of SSL that varied with the amount of desmoplasia in PDX models, which are otherwise highly impermeant to SSL. Quantitative IFP measurements using the "wick and needle" technique showed reduction of IFP paralleled establishment of the priming window. In the absence of sHH inhibitor priming, SSL-DXR (2 mg/kg), SSL-DXR-DR5 (2 mg/kg of DXR), and free DR5 (dose-matched to SSL-DXR-DR5 protein) were administered to evaluate the effect of tumor priming on therapeutic efficacy. Any DR5-containing treatment was curative for MiaPaCA-2 tumors (high DR5 expression). The response of Panc-1 tumors (low DR5 expression) was greatest with SSL-DXR-DR5. Tumor responses with the PDX model #12424, (high DR5 expression) were greater in sHHI-treated animals compared to controls. SSL-DXR + free DR5 mediated a transient inhibition of tumor progression, whereas a durable inhibition of tumor progression was observed with SSL-DXR-DR5 immunoliposomes.

Conclusions: Efficacy of SSL-DXR-DR5 immunoliposomes was superior to other treatments in two cell line-based tumors, and tumor response varied with DR5 receptor density. Efficacy in low permeability/perfusion PDX tumors was greatest following tumor priming by sHH inhibitors.

#3705

Targeted anticancer drug delivery to Ewing's sarcoma using human anti-CD99 targeted hybrid polymerization liposomal nanoparticles.

Hyunggyoo Kang,1 Jon Nagy,2 Timothy Triche1. 1 _USC/Children's Hospital Los Angeles, Los Angeles, CA;_ 2 _NanoValent Pharmaceuticals, Inc., Bozeman, MT_.

Most anticancer therapies are unspecific and therefore inevitably lead to off-target toxicity. To overcome these shortcomings, targetable hybrid polymerization liposomal nanoparticles (HPLNs) have been invented. Multiple innovative and proprietary technologies have been developed to enable creation of this unique form of nanoparticle therapy, including the use of cross linked lipids to improve both stability and bioavailability, modified polyethylene glycol (PEG) for both 'stealthiness' (to prevent immune response to the nanoparticle) and for covalent attachment of targeting molecules such as tumor specific antibodies or peptides. Our present study showed the superior efficacy of human anti-CD99 targeted HPLNs in the treatment of Ewing sarcoma. Human monoclonal anti-CD99 antibody-targeted HPLNs encapsulating irinotecan (CD99-HPLN/Ir) can efficiently reach implanted Ewing sarcoma tumors in xenograft mice and dramatically reduce and/or eliminate the tumor burden. Complete tumor ablation has been observed at doses as low as 1 mg irinotecan/kg, treated twice per week. In other cases, animals who failed untargeted (HPLN/Ir) treatment showed complete tumor ablation after 'salvage' treatment with CD99-HPLN/Ir. Our CD99 targeted HPLN/irinotecan formulation, termed NV103 showed much better efficacy than the commercial untargeted liposomal irinotecan (Onivyde™) and doxorubicin (Doxil™). Toxic side effects, normally associated with systemic administration of free irinotecan, were minimized or undetectable. Our nanoparticles showed excellent bioavailability. Even the untargeted HPLN/Ir improve drug bioavailability six-fold in a comparison experiment with the drug in liposome form (Onivyde™), with no discernable systemic toxicity. Antibody targeting of the HPLN irinotecan formulation (NV103) improves the efficacy 12-fold, over the untargeted liposome form. In conclusion, this formulation may be effective for treatment of other CD99-expressing tumors like certain leukemias, lymphomas and glioma. Ultimately, after development of other tumor-specific targeting agents and varied encapsulated therapeutics, this technology can be usefully adapted for the treatment of other cancers as well.

#3706

Novel therapeutic strategy for pancreatic cancer with lectin drug conjugate (LDC): The efficacy and pilot safety test.

Osamu Shimomura,1 Tatsuya Oda,1 Hiroaki Tateno,2 Yusuke Ozawa,1 Sota Kimura,1 Jun Hirabayashi,2 Nobuhiro Ohkohchi1. 1University of Tsukuba, Tsukuba-city, Japan; 2National Institute of Advanced Industrial Science and Technology, Tsukuba-city, Japan.

Background: We recently developed lectin-drug conjugate (LDC) for targeting pancreatic cancer specific glycan and showed potent anti-cancer effect both in vitro and in vivo (Osamu Shimomura et al., Molecular Cancer Therapeutics 2017). The additional evaluation of anti-cancer effects and the toxicity studies of LDC need to be clarified.

Methods: LDC, rBC2LC-N lectin fused with 38 kDa of pseudomonas exotoxin (PE) A, was used in this study. We confirmed of the anti-cancer effect of LDC in orthotopic mouse models and developed dissemination mouse models to make sure of the survival benefits in LDC by both intra-peritoneally and intra-venously injection by log-rank test. We checked the damage of all organs pathologically after injection of the minimal or maximal tolerance doses and performed the blood sample analyses.

Results: We have already validated the cytotoxic effect of LDC for PDAC cell lines in vitro i.e., IC50= 1.04 pico g/ml. Intraperitoneal and intravenous injection of LDC reduced tumor weight from 390 mg in normal saline group to 130.8 mg and 203 mg, respectively in the orthotopic models (P=0.001 and P=0.007, respectively), and improved median survival duration from 62 days to 105 and 90 days in the peritoneal dissemination models, respectively (P<0.0001 and P<0.0001, respectively). Intravenous injection of LDC conferred comparable therapeutic effects to intraperitoneal injection. Inconsistent with the apprehension that all lectins mediate harmful hemagglutination, the rBC2LC-N never caused hemagglutination with human erythrocytes in every blood types. The laboratory test after injection of therapeutic dose (1µg/mouse) of LDC showed no obvious abnormality except slightly elevation of aspartate and alanine aminotransferase. Surprisingly, no adverse event were observed after administration of rBC2LC-N lectins without PE up to 15 µg/mouse in wild type mice. Although some epithelial desquamations were revealed in the mucosa of the gut after fatal dose of LDC injection, we could not figure out the organ damage after the therapeutic dose of LDC injection pathologically.

Conclusions: We show the successful in vivo application of a lectin for cancer treatment. Lectin itself did not cause severe adverse events and could be a potential candidate of a drug carrier, providing a new insight into anti-cancer drug development.

#3707

PD-L1 antibody drug conjugate for cancer immune-chemo combination therapy.

Samaresh Sau, Alex Petrovici, Hashem Alsaab, Arun K. Iyer. _Wayne State University, Detroit, MI_.

Antibody drug conjugates (ADCs) are a new form of targeted therapy to cancer, consisting of an antibody, a conditionally stable linker, and a cytotoxic drug. Immune checkpoint molecule, such as programmed death ligand-1 (PD-L1) overexpressed in various solid tumors including triple negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC) and it has a crucial role in tumor immune evasion. Blocking the interaction between PD-L1 and PD-1 (programmed death-1 receptor) resurrects tumor immune surveillance, resulting CD8+ cytotoxic T cells activation to attack the tumor cells. This is an emerging anticancer mechanism and more than four antibody inhibitors have been recently FDA approved against PD-1 or PD-L1 biomarker. Targeting PD-L1 in TNBC and NSCLC is an excellent approach for tumor immunotherapy. However, the major challenges of anti-PD-L1 therapy (Atezolizumab) are (i) the transient expression of PD-L1 in tumor microenvironment, (ii) development of anti-PD-L1 resistance, (iii) difficulty of tumor stroma penetration that retard its clinical outcome. To overcome these challenges, we chemically conjugated Atezolizumab (ATZ) with potent chemotherapeutic payload, such as Doxorubicin (Dox) through polyethylene glycol (PEG) linkers. The role of Dox is to break the tumor stroma so that PD-L1 antibody can penetrate to the core of the tumor. Introduction of PEG-linker with high molecular weight ATZ (155 kD) has drastically improved the solubility of ADC and successful chemical conjugation of between ATZ and PEG-Dox was evaluated by MALDI-TOF analysis. Our data has revealed significantly tumor cell killing effect and induction of apoptosis in PD-L1-Dox treated MDA-MB-231 cells. The near infrared imaging of PD-L1 dye conjugate in TNBC and NSCLC patient derived xenograft (PDx) supports the selective tumor targetability. Based on the synergistic anticancer effect and selective tumor targetability demonstrated in this study build a strong rational of using PD-L1 ADC approach for cancer immune-chemo therapy and diagnosis.

#3708

Targeted delivery of doxorubicin to glioblastomas by thermally sensitive polypeptides.

Sonja Dragojevic,1 Rebecca B. Mackey,1 Felix Kratz,2 Drazen Raucher1. 1 _Univ. of Mississippi Medical Ctr., Jackson, MS;_ 2 _CytRx Corporation, Freiburg, Germany_.

Glioblastoma (GBM) ranks among the most common, aggressive, and least curable cancers due to a strong tendency for intracranial dissemination, high proliferation potential and inherent tumor resistance to radiation or chemotherapy. Current treatments face the critical challenge of adverse treatment effects in normal tissue within the brain itself and also when the agent cannot adequately penetrate the blood brain barrier (BBB) to reach the tumor microenvironment. We have developed a system to selectively deliver chemotherapeutic doxorubicin to GBM tumors. This carrier is based on elastin-like polypeptide (ELP), which is soluble at physiological temperatures but undergoes a phase transition and accumulates at tumor sites with externally applied, mild (40-41°C) hyperthermia. The CPP-ELP-Dox conjugate consists of a cell penetrating peptide (CPP), which facilitates transcytosis through the BBB and cell entry, and the 6-maleimidocaproyl hydrazone derivative of doxorubicin (Dox) at the C-terminus of ELP. The acid-sensitive hydrazone linker ensures release of Dox in the lysosomes/endosomes after cellular uptake of the drug conjugate. We have shown that CPP-ELP-Dox effectively inhibits cell proliferation in three GBM cell lines; GBM6, D54, and U251 with IC50 values of 250 nM, 60nM, and 30nM, respectively. Both the free drug and CPP-ELP-Dox conjugate exhibited similar in vitro cytotoxicity, although their subcellular localization was considerably different. The Dox conjugate was mainly dispersed in the cytoplasm, while free drug was largely dispersed in the cytoplasm but also had partial nuclear accumulation. The accumulation of free Dox in GBM cells was measured by flow cytometry to determine whether the cytotoxic activity of free Dox or CPP- ELP-Dox is related to intracellular Dox levels. Intracellular Dox concentration was increased in the CPP-ELP-Dox cells compared to that in the free Dox cells, which positively correlates with cytotoxic activity. Flow cytometry was also used to quantitatively investigate apoptosis of GBM cells. After incubation for 24 h, the total apoptosis percentage of free Dox was 1.5% in GBM6, 6% in D54, and 7% in U251 cells. However, the total apoptosis rates of CPP-ELP-Dox treated cells were 5%, 12% and 5% in GBM6, D54, and U251 cells respectively, which confirms increased apoptosis compared to free Dox treated cells. Cell cycle analysis of GBM cells treated with free Dox showed enhanced arrest of the cells in G1 phase, while there was increased percentage arrest of cells in G2 phase in cells treated with CPP-ELP-Dox.In summary, our findings demonstrate that CPP-ELP-Dox effectively kills GBM cells. Development of such a drug carrier has the potential to greatly improve current therapeutic approaches for GBM by increasing the specificity and efficacy of treatment and reducing cytotoxicity in normal tissues.

#3709

Characterization, in vitro and in vivo efficacy studies of docetaxel trastuzumab stealth immunoliposome in human breast cancer models.

Anne Rodallec,1 Jean Michel Brunel,2 Stephane Robert,3 Sarah Giacometti,1 Ariel Savina,4 Fanny Bouquet,4 Bruno Lacarelle,1 Joseph Ciccolini,1 Raphaelle Fanciullino1. 1 _SMARTc - Aix Marseille University, Marseille, France;_ 2 _CRCM, Marseille, France;_ 3 _AMUTICYT - Aix Marseille University, Marseille, France;_ 4 _Institut Roche, Boulogne Billancourt, France_.

In HER2-positive breast cancer, the Trastuzumab + Docetaxel doublet has a significant efficacy but is hampered by frequent toxicities that could be addressed with nanoparticles. We have developed an Antibody-Nano Conjugate (ANC), combining docetaxel encapsulated in a stealth liposome engrafted with trastuzumab. This entity is expected to improve the efficacy/toxicity balance of this doublet, by improving trafficking and delivery to the tumors. ANCs were synthesized using thin-film method and trastuzumab was grafted via sulfydryl groups on maleimide polyethylene glycol. Two compositions (ANC-1 and ANC-2) were tested and compared (i.e., size, morphology, encapsulation rate, engrafting rate, and stability). Antiproliferative activity was tested on a panel of human breast cancer models ranging fromHER2-negative (MDA-MB-231) to HER2-positive (MDA-MB-453) or -overexpressing (SKBR3), both on 2D and spheroid models. Biodistribution, efficacy and survival were tested in MDAMB-231 and MDA-MB-453 bearing nude mice. ANC-1 presented a greater size (140 ± 3 nm), docetaxel encapsulation rate (73 ± 6 %) and stability as compared with ANC-2. In vitro, both ANCs were found to be more efficient than standard docetaxel + trastuzumab, with a better performance for ANC-1. In vivo, ANC-1 accumulated better in tumors, resulting in a higher reduction in tumor growth (i.e. >+36%) and prolonged survival (i.e. >+33%) as compared with standard treatment. Both in vitro and in vivo results suggest that higher antiproliferative efficacy and efficient drug delivery can be achieved in breast cancer models. Of note, although modest, even Her2- model (i.e., MDA231) was found to benefit from the nanoparticle.

#3710

Nanoparticle-mediated concomitant radiation dose amplification and PARP inhibition in lung cancer.

Ana G. Vazquez-Pagan,1 Paige Baldwin,1 Ravina M. Ashtaputre,2 Sijumon Kunjachan,3 Srinivas Sridhar,1 Rajiv Kumar,1 Ross Berbeco4. 1 _Northeastern University, Boston, MA;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _Brigham and Women's Hospital, Boston, MA;_ 4 _Brigham and Women's Hospital, Dana-Farber Cancer Institute, Boston, MA_.

Introduction: More than 50% of cancer patients receive radiation therapy at some point during their care. Gold nanoparticles (GNPs) can amplify the radiation dose by facilitating the ejection of low-energy photoelectrons, resulting in increased DNA damage. One of the main challenges of radiation therapy in cancer is to sustain this damage for longer durations. DNA single-strand breaks (SSBs) are repaired by base excision repair, which utilizes Poly(ADP-ribose) polymerase (PARP). PARP inhibition during radiotherapy provides an attractive alternative in maximizing treatment outcomes. Here, we explore a strategy to combine the radiosensitizing effect of GNPs with the DNA-repair inhibiting ability of NanoTalazoparib (nTLZ), a liposomal formulation of the PARP inhibitor, talazoparib (TLZ).

Methods: GNPs were synthesized by reducing AuCl4 using Tetrakis(hydroxymethyl)phosphonium chloride and further PEGylating using heterobifunctional PEGs. A liposomal formulation of TLZ was synthesized using the Nanoassemblr, a microfluidics-based device. Physicochemical characterization of GNPs and nTLZ was carried out using TEM, DLS and release kinetics studies. In vitro studies were carried out to assess the toxicity of the GNPs using MTT assay in non-small cell lung cancer (NSCLC) cell line Calu-6. The therapeutic efficacy of combination treatment using GNPs, nTLZ and radiation was done using clonogenic survival assays. Clonogenic assay was carried out using Calu 6 cells, which were sequentially treated with GNPs (1mg/mL), TLZ (0.5 uM) and nTLZ (0.5 uM) with and without radiation. The radiation doses varied from 0-10Gy for each set of treatments.

Results: PEGylated GNP's showed a hydrodynamic diameter of ~10-12 nm with a spherical morphology whereas nTLZ size was 70 nm encapsulating TLZ at a concentration of ~200 ug/ml. MTT assay showed no toxicity for PEGylated GNPs treated upto a concentration of 3.0 mg/mL. Cells treated with either GNPs, TLZ or nTLZ did not show significant reduction in colony formation, but were reduced with increasing doses of radiation. The survival plots showed a highly additive antiproliferative effect for the GNP + nTLZ combination at all radiation doses, while the free TLZ + GNPs combination was not as effective at inhibiting colony formation. In vivo experiments assessing the combination therapy in a subcutaneous xenograft mice model using Calu 6 are currently under way.

Conclusions: The preliminary in vitro results indicate that the combination of radiosensitizing GNPs with a potent DNA repair enzyme inhibitor, TLZ, has an immense potential as a complimentary combination therapy in conjunction with radiation therapy in treatment of lung cancer.

This work is supported by the CaNCURE program (grant #1CA174650-02), American Lung Association, Dana-Farber Cancer Institute and Brigham and Women's Hospital.

#3711

**Smart therapy: Using** CCND1- **targeted siRNA-loaded liposomes to sensitize mantle cell lymphoma cells to cisplatin.**

Tanvi N. Gandhi, Chioniso P. Masamha, Sudip K. Das, Nandita G. Das. _College of Pharmacy and Health Sciences, Butler University, Indianapolis, IN_.

Mantle cell lymphoma (MCL) is the most aggressive subtype of B-cell non-Hodgkin's lymphoma, due to its refractory nature and high relapse rate. It is characterized by the aberrant overexpression of the cell cycle regulatory oncogene, cyclin D1 (CCND1), which occurs as a result of a t (11; 14) chromosomal translocation event, the initiating lesion for MCL tumorigenesis. Current therapy includes a series of different chemotherapy regimens often involving a combination of at least four drugs, but there is no gold standard combination treatment regimen. One of the most commonly used drugs in combination treatment is the chemotherapeutic agent, cisplatin. High concentrations of cisplatin in the body cause nausea, vomiting, loss of taste and, more severely, nephrotoxicity. A commonly used approach to reduce the concentration of a chemotherapeutic agent is to use it in combination with a molecular targeting agent. Since cyclin D1 is overexpressed in MCL, we hypothesize that knocking down CCND1 by using siRNA will sensitize MCL cells to cisplatin. We developed CCND1 primers to detect reduction in CCND1 mRNA levels after RNAi using PCR. The greatest challenge against siRNA delivery is its poor stability in vivo and low efficacy of transfection into tumor cells. Our objective is to formulate a dosage form that could be practically adapted in a clinical setting in the future. We developed a liposome formulation that uses a balanced ratio of neutral and cationic lipids to serve as a carrier for the siRNA and improves delivery to tumor cells. Our CCND1 siRNA and control siRNA-loaded liposomes have an average particle size below 150 nm and an overall cationic charge less than 30V. These characteristics are important for the trafficking of the liposome carrier in vivo and its eventual uptake into tumor tissue. Fluorescence assays were used to detect and quantify the amounts of siRNA bound to the liposome. We determined the effect of the liposomes in combination with cisplatin on MCL cell viability using a colorimetric MTT assay. The novel CCND1 siRNA-loaded liposome combined with cisplatin was found to show a dose-dependent decrease in cell viability and was more potent than either CCND1 siRNA liposomes or cisplatin alone (p<0.05). In conclusion, our preliminary studies indicate a potential advantage of using smart therapy by co-administering cisplatin with CCND1 siRNA-loaded liposomes.

#3712

Optimizing design parameters of a VLA-4-targeted liposomal nanoparticle in a multiple myeloma disease model.

Basar Bilgicer, Tanyel Kiziltepe, David Omstead, Peter Deak. _Univ. of Notre Dame, Notre Dame, IN_.

Ligand-targeted liposomes have garnered interest as drug delivery vehicles for cancer therapy, however they have not consistently produced successful outcomes. These inconsistencies likely arise from differences in disease models and target receptors, as well as from nanoparticle design parameters which can significantly influence therapeutic efficacy. We systematically evaluated the role that peptide-linker length, peptide hydrophilicity, peptide density, and nanoparticle size play on tumor targeting using a multifaceted synthetic strategy to prepare highly controlled and consistent peptide-targeted liposomes. We analyzed these parameters in a VLA-4-expressing multiple myeloma model system where liposomes were functionalized with a VLA-4-antagonist peptide to evaluate in vitro targeting efficiency and in vivo biodistribution and tumor cell uptake. Our studies demonstrated that by increasing the hydrophilicity of the targeting peptide sequence via addition of an oligolysine chain and simultaneously optimizing the EG peptide-linker length, cellular uptake of targeted liposomes was significantly enhanced in vitro. Specifically, the cellular uptake was greatly enhanced for 50-100 nm size liposomes with shorter peptide-linker lengths of EG6 when compared to the industry standard EG45 linker. For in vivo applications, although targeted formulations did not enhance tumor accumulation directly relative to non-targeted formulations, liposomes designed with EG6 linker and an oligolysine chain provided a significant advantage by demonstrating significantly enhanced tumor cell uptake relative to non-targeted liposomes. These results highlighted the importance of creating a comprehensive understanding of the effect of each liposome design parameter on multifactorial biological endpoints in determining the therapeutic potential of peptide-targeted liposomes.

#3713

Targeting β-catenin pathway by nanoparticle-mediated delivery of norcantharidin impairs the stemness of triple negative breast cancer cells.

Yunfei Li, Zhishan Wang, Yajuan Xiao, Chengfeng Yang. _University of Kentucky, Lexington, KY_.

Background Norcantharidin (NCTD) is known as an inhibitor of serine/threonine protein phosphatase types 1 and 2A (PP1/PP2A) and exhibits potent anticancer efficacies. Our preliminary study identified NCTD interfering with the interaction between phospho-β-catenin and PP1/PP2A could downregulate the levels of non-phospho β-catenin in the cytoplasm in Triple Negative Breast Cancer (TNBC) cells. As β-catenin pathway plays an important role in cancer stemness, we hypothesize that NCTD could prevent β-catenin nuclear translocation, impair the stemness and reduce the metastasis of TNBC. However, the inherent features of short half-life and nephrotoxicity hinder NCTD's further in vivo application. The goal of this study was to test the therapeutic effect of targeting β-catenin pathway by nanoparticle-mediated delivery of NCTD against TNBC.

Experimental Design NCTD was loaded into an integrin α5 targeting nanoparticle (NP) (RGD-LPH) via complexing with polyethyleneimine. The targeting efficiency of RGD-LPH was tested by the in vivo imaging of RGD-LPH labeled by cyanine 7.5 (cy7.5). The effects of NP-mediated delivery NCTD on TNBC cell malignant behaviors and tumor metastasis were tested in various in vitro and in vivo models.

Results We found that RGD-LPH NP-mediated delivery of NCTD significantly reduced the active and total β-catenin levels as evidenced by Western Blot. The self-renewal capacity was remarkably blocked by RGD-LPH, as examined by sphere formation. RGD-LPH also suppressed features of cancer cells related to proliferation, as examined by MTT, clonogenic assay and soft agar colony formation. In particular, compared to free NCTD, RGD-LPH demonstrated extended-release profile and long-last effect in the

long-term experiments such as clonogenic assay and soft agar colony formation assay. In vivo, systemic delivery of RGD-LPH(cy7.5) indicated a preferential uptake in primary tumor and lung of the tumor-bearing mice. Efficacy studies indicated reduced metastasis of mice from RGD-LPH treatment.

Conclusion: Collectively, our results strongly suggest that NCTD being encapsulated in RGD-LPH, which warrants further development for preclinical and clinical studies as a novel TNBC therapeutic approach, is a robust modulator of Wnt signaling with anti-metastasis activity.

#3714

Stealth liposomes encapsulating plasmid DNA of telomerase specific oncolytic adenovirus.

Yoshihiko Kakiuchi. _Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan_.

Background: Oncolytic virotherapy has the potential to become the next breakthrough in cancer therapy. We previously established a telomerase-specific oncolytic adenovirus (Telomelysin), which is controlled to replicate and induce oncolytic cell death selectively in tumor cells, and its safety for humans was proven in the phase I clinical trial performed in the USA. While oncolytic adenoviruses have promising antitumor effects, the application is basically limited to local administration because of non-specific trapping in the liver and immune elimination after systemic delivery. Liposomes are among the most common nanoparticles used as drug carriers, and liposome-encapsulated therapeutic agents enhance the efficacy by increasing their stability in the bloodstream. We investigated the potential of liposome-encapsulated plasmid DNA of telomerase-specific oncolytic adenovirus as an oncolytic adenoviral agent suitable for systemic delivery.

Methods: TelomeScan, a modified Telomelysin expressing the green fluorescence protein (GFP), was used in this study. Cytotoxic effects of TelomeScan plasmid DNA-lipid complexes (Lipo-pTS) and control GFP plasmid DNA-lipid complexes (Lipo-pGFP) against HCT116 colon carcinoma cells were evaluated in vitro and in vivo. Coxsackie and adenovirus receptor (CAR) blocking assay using anti-CAR antibodies and neutralizing assay using adenovirus type 5-specific antibodies (AdNAbs) were performed to assess the CAR-independency of Lipo-pTS in infectivity and the influence of AdNAbs on Lipo-pTS cytotoxicity, respectively. Immunogenecity of Lipo-pTS was evaluated by assessing the productivity of AdNAbs after systemic administration in immune-competent BALB/c mice.

Results: Lipo-pTS, which has a diameter of 40-50 nm, showed potent antitumor effects on HCT116 cells compared to Lipo-pGFP in vitro and in vivo, by producing actual viable adenoviruses after Lipo-pTS treatment, leading to tumor-specific oncolytic cell death. Infectivity of Lipo-pTS was not affected by pretreatment of anti-CAR antibodies, while infectivity of TelomeScan was reduced to less than half by anti-CAR antibodies, which suggested that tumor selectivity of Lipo-pTS was independent of CAR expression. Importantly, Lipo-pTS reduced production of AdNAbs significantly after intravenous administration into BALB/c mice compared to TelomeScan, and even in the presence of AdNAbs, Lipo-pTS maintained strong cytotoxicity in contrast to TelomeScan whose cytotoxicity was almost nullified by AdNAbs. These findings proved that Lipo-pTS possesses a stealth effect on the immune system in vivo environment, meaning that systemic delivery of Lipo-pTS may be possible.

Conclusion: Lipo-pTS has the potential to become an oncolytic adenoviral agent suitable for systemic delivery with the characteristics of CAR-independent antitumor

activity and a stealth effect on the immune system.

#3715

Targeted nanotherapy using the PARP inhibitor talazoparib for breast cancer treatment.

Paige Baldwin, Rajiv Kumar, Srinivas Sridhar. _Northeastern University, Boston, MA_.

Introduction: PARP inhibitors exploit defects in DNA repair pathways to selectively target cancerous cells. These drugs are currently delivered orally, but Talazoparib, the most potent PARP inhibitor, exhibits greater toxicity than the others. Systemic administration of nanoparticles bypasses the first-pass metabolism of oral drugs and nanoparticles preferentially accumulate in tumors due to the leaky tumor vasculature. Additionally, nanoparticles can be actively targeted to tumors by conjugating different moieties such as antibodies that recognize overexpressed markers on the tumor cells. NanoTalazoparib (NanoTLZ) has been previously formulated and extensively characterized in breast, ovarian, and lung cancer models. Here we describe further characterization of NanoTLZ and the development of a next generation fluorescently labeled EPCAM targeted formulation of NanoTLZ for the treatment of triple negative breast cancer (TNBC).

Methods: Animals with orthotopic TNBC xenografts were injected with NanoTLZ and at various time points tumor biopsies were taken to assay tumor PAR levels. Blood was collected at various time points and plasma separated for drug extraction and quantification. Pharmacokinetic modeling is underway. Fluorescently labeled NanoTLZ was developed with the addition of Cy5, followed by further improvement via conjugation of anti-EPCAM antibodies. Targeting capability was assessed via laser scanning confocal microscopy after treatment of the TNBC cell line MDA-MB-231 with either non-targeted or targeted NanoTLZ. Therapeutic efficacy studies with targeted NanoTLZ alone and in combination with radiation are underway in an MDA-MB-231 xenograft model.

Results: Pharmacodynamics indicated PAR suppression in tumors within 30 minutes of NanoTLZ treatment. Tumor PAR levels began to increase 24 hours after a single dose but remained significantly lower than control levels up to 72 hours (P<0.005). Characterization of NanoTLZ after the addition of Cy5 and after anti-EPCAM targeting indicated that the formulation remained under 100 nm in diameter with a charge of ~10 mV and equivalent drug loading and release. Cy 5 labeling allowed for visualization of nanoparticle uptake and intracellular fluorescence was 70% greater in cells when treated with EPCAM targeted NanoTLZ compared to non-targeted.

Conclusions: The sustained release of Talazoparib from the nanoformulation decreases tumor PAR levels for up to 72 hours after a single dose, allowing for less frequent administration than the current daily regime used for oral Talazoparib. The targeted formulation of NanoTLZ shares the same physicochemical properties as the untargeted formulation, but is taken up much faster in vitro, suggesting it will allow for greater accumulation at the tumor site making it more effective than the previously tested untargeted NanoTLZ. Supported by ARMY/W81XWH-16-1-0731 and Rivkin Foundation.

#3716

Tumor multicomponent targeting nano-micelles with synergistic combination to overcome drug resistance and reprogramming of macrophages in renal cell carcinoma.

Hashem O. Alsaab,1 Samaresh Sau,1 Vino T. Cheriyan,2 Rami Alzhrani,1 Ulka Vaishampayan,3 Arun K. Rishi,2 Arun K. Iyer1. 1 _Use-inspired Biomaterials & Integrated Nano Delivery (U-BiND) Systems Laboratory Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI; _2 _John D. Dingell VA Medical Center, Wayne State University, Detroit, MI;_ 3 _Karmanos Cancer Institute, Wayne State University, Detroit, MI_.

Renal Cell Carcinoma (RCC) contributes >90% of the most common form of kidney tumor and remains one of the ten leading causes of cancer death in the US. Tyrosine kinase inhibitors (TKIs) and mammalian target of rapamycin (mTOR) inhibitors have increased therapeutic possibilities for treating RCC. Although the impact on disease progression is encouraging, a substantial proportion of patients do not respond adequately, and therapy resistance almost inevitably occurs. Eventually, new strategies have emerged that include immunotherapy such as PD-1 inhibitor (Nivolumab) and the combination of chemo-immune therapy. Possibly, combination treatment aimed at different, non-related pathways may be advantageous. In this regard, we would like to come up with synergistic therapeutic strategies for nonresponsive, highly aggressive tumor types to tackle the current clinical challenges. We have worked on different RCCs tumor models that in general have a poor prognosis with conventional therapies. We also pursued different combination regimens, including drugs that work on the mTOR inhibition (everolimus), inhibit RTK-inhibitor or VEGFR (cabozantinib or sorafenib). Also, there is a critical need to develop safe and effective delivery vehicles that can carry the payload to the right target tissue and cell. Thus, different types of nanoparticles have been designed to deliver a variety of therapeutic agents to target hypoxic tumor microenvironment by using carbonic anhydrase-9 (CA9); FRα for targeting cancer epithelial cells; and FRβ for targeting tumor-associated macrophages (TAM) and combination delivery of RTK-inhibitor Cabozantinib (CB) and sorafenib with our own apoptosis inducer/CARP-1 protein activator CFM-4.16 (C4.16) for overcoming drug resistance and reprograming TAM for RCC therapy. The current work also focused on multimodal approaches, including (a) Optimization of hypoxia marker conjugated targeted polymer-lipid nano-formulation (PLNP) using copper-free click chemistry; (b) In vitro and in vivo pre-clinical testing of targeted -PLNP loaded with polypharmacy in inhibiting RCCs using mice bearing resistant RCCs and patient-derived xenografts (PDX). The results of efficacy and biodistribution of targeted PLNPs in animals bearing RCC xenografts and PDX model showed higher accumulation of drugs at tumor sites with higher tumor growth inhibition. Also, the targeted formulation showed high binding affinity and specific tumor uptake, faster normal tissue clearance, and less non-target organ uptake. These findings portent promising therapeutic potential of hypoxia-targeted -PLNPs for treating RCCs.

#3717

**α** 3 **β** 1 **-Integrin targeted DM1 micellar prodrug for targeted therapy against ovarian cancer.**

Xiaocen Li,1 Wenwu Xiao,1 Yayu Wang,2 Xiaojia Chen,2 Ruiwu Liu,1 Kit S. Lam1. 1 _UCDavis, Sacramento, CA;_ 2 _Jinan University, Guangzhou, China_.

Ovarian cancer ranks fifth in cancer deaths among women in America. The standard treatment for advanced stages ovarian cancer consists of primary debulking surgery followed by i.v. and i.p. chemotherapy. DM1, or mertansine, is a thiol-containing maytansinoid, that inhibits microtubule assembly and kills various cancer cells at low nM concentration. However, its application in the clinic has been hampered by severe toxic side effects1. Its trastuzumab conjugate (T-DM1), which was approved by the FDA in 2013, is a promising therapeutic agent for patients harboring ovarian cancers with high Her-2 expression2. However, the general limitation for antibody drug conjugates (ADCs) is low drug content, high clearance by the reticuloendothelial, and high manufacturing cost. To overcome this, we designed and synthesized a novel PEG-based, cholic acid/ DM1 hybrid telodendrimer (PEG5k-CA4-DM1) comprised of one DM1 molecule and three molecules of maleimide per telodendrimer, which self-assembled into 25nm nanomicelles for targeted therapy against ovarian cancer. To enhance the stability of this nanoparticle, PEG5k-Cys4-CA8, which contain 4 thiol groups per telodendrimer is formulated together with PEG5k-CA4-DM1 at a ratio of 2:5, to generate thiol/maleimide crosslinked DM1 micellar prodrug (DM1-nano). To further enhance its tumor-targeting ability, the surface of DM1-nano was decorated with LXY30 via a hydrophilic "stealth" linker (EK)3, using copper-free 'Click' chemistry. LXY30 is a potent and specific ligand against α3β1 integrin which is overexpressed on the surface of many epithelial cancer cells including a panel of ovarian cancer cell lines (e.g. SKOV-3). Compared with free DM1 and non-targeted DM1-nano, LXY30-EK-DM1-nano not only prevented premature drug release, but also further enhanced tumor targeting ability, drug solubility and in vivo circulation time in blood. When loaded with a fluorescent dye (DiD), LXY30-EK-DM1-nano was found to (i) internalize inside SKOV-3 cells in vitro, (ii) significantly accumulated at the tumor site of SKOV-3 xenograft mice, and (iii) exhibit potent anti-cancer activity in vitro (IC50=26nM with SKOV-3 cells) and in vivo (SKOV-3 xenograft model). In addition, LXY30-EK-DM1-nano was found to be significantly more efficacious and less toxic than free DM1 and non-targeted DM1-nano. In conclusion, the LXY30-DM1-nano offers a promising novel nanotherapeutic agent against ovarian cancer.

#3718

An injectable nanoparticle formulation of valrubicin.

Tapas De,1 Steve Miller,2 Zachary Yim,1 Vuong Trieu2. 1 _Lipomedics Inc, Fort Worth, TX;_ 2 _Autotelic Inc, City of Industry, CA_.

Introduction: Valrubicin [AD-32 (N-trifluoroacetyladriamycin-14-valerate)], a cancer therapeutic agent, is used for treating bacillus Calmette Guerin (BCG)-resistant bladder cancer. Although valrubicin has been found to be less toxic to normal tissues than doxorubicin during preclinical studies, its therapeutic applications have been restricted to nonsystemic administration due to its poor aqueous solubility. Its proven antineoplastic properties make it a promising candidate to serve as an effective chemotherapeutic agent for systemic use as a soluble nanoparticle formulation with injectable excipients. The current study was undertaken to formulate valrubicin as nanoparticle formulation using our proprietary Quanticle™ Technology. Here we report the use of biocompatible and injectable phospholipid and protein to produce a stable nanoparticle formulation of valrubicin for systemic use.

Methods: We developed Quanticle manufacturing process, which subjected the nanoparticle to a minimum number of fixed passes to avoid the deterioration of excipients by microfluidization process. This was possible through judicious combination of proprietary ternary solvent mixture and excipients. A Quality by Design (QbD) approach was utilized to design the experimental study and understand the effect of process variables on critical quality attributes (CQAs) during our proprietary medicinization of valrubicin. Drug loading, nanoparticle size (before and after filtration), particle size distribution, ease of filtration, and physical stability of particle during storage at room temperature were evaluated as CQA. The process variables being evaluated were aqueous to organic phase ratio, ratios of solvents constituting the total organic phase, amount of drug, microfluidizer pressure, number of passes, and evaporation temperature. Valrubicin was also examined for its incorporation into whole human plasma or human plasma components (HDL, LDL and serum albumin (HSA).

Results: Quanticle formulation with human serum albumin as excipients produced nanoparticle formulation of very small size of ~100 nm. The formulation was stable for 24 h at RT. This formulation was also lyophilizable and reconstitutionable to its original size and the reconstituted formulation is stable for more than 24 at RT. The formulation was reconstitutionable in less than 3 minutes with 0.9% (w/v) saline; the final conc. of the formulation was ~2 mg/mL and the drug recovery was very high. Valrubicin distributes itself in the particles of HDL, LDL and HSA, with the maximum being in HSA.

Conclusions: A new nanoparticle valrubicin (LM-401) was formulated using injectable excipients. The formulation is sterile filterable, lyophilizable and stable at RT for 24h before and after lyophilization. We are planning for in vitro and in vivo application of this new LM-401 formulation in ovarian (SKOV-3) and prostate (PC-3) cancer cell lines.

#3719

Fabrication of chemotherapy drug temozolomide-loaded poly(lactic-co-glycolic acid) microparticles by electrospraying for the treatment of glioma.

Daniel A. Rodriguez De Anda, Sue Anne Chew. _University Of Texas Rio Grande Valley, Brownsville, TX_.

Glioblastoma (GBM) is a form of brain tumor with a low median survival rate of 16 months after diagnosis despite the many efforts to treat it. The main approach to treat GBM consists of surgery followed by radiation and chemotherapy drugs, such as Temozolomide (TMZ). However, TMZ tends to degrade fast making it hard to deliver enough amounts of the drug to the site of the tumor. The overall goal of our project is to develop a biodegradable composite system to locally deliver both an anti-angiogenic and chemotherapy agent for the treatment of glioma. The objective of this particular work was to successfully fabricate TMZ loaded biodegradable poly (lactic-co-glycolic acid) (PLGA) microparticles as carriers of the drug to protect TMZ from rapid degradation, and providing a method for controlling the release of the drug. In this study, three different methods of fabricating the microparticles were investigated and compared: single-emulsion solvent evaporation, double-emulsion solvent evaporation, and electrospraying to determine which method will result in high drug loading and sustained release of the drug. For the drug release study, 5 mg of each type of microparticle were added to 1 ml of phosphate-buffered saline (PBS) and stored and shaken in a non-CO2 incubator set at 37°C. At each time point (1, 3, 7, 10, and 14 days), the microparticles were frozen down and at the end of the study, the microparticles were dissolved in dimethyl sulfoxide (DMSO) and the drug concentration was determined by absorbance using a spectrophotometer at a wavelength of 328 nm. Compared to the two emulsion solvent evaporation methods investigated, electrospraying provides a way to fabricate TMZ loaded microparticles with a high drug loading efficiency (60% to 97% of the drug used for fabrication was loaded into the particles compared to 0.05% for the emulsion solvent evaporation methods). The lower drug loading efficiency of the emulsion solvent-evaporation technique resulted from the loss of the drug into the PVA solution during the microparticle hardening process which is avoided with the electrospraying method. The electrospraying method showed a constant controlled release of the drug over 14 days and resulted in a drug cumulative release of 90% from the microparticles at the end of the study. However, the amount of drug loaded is still low (0.72% to 1.2% of the total weight of the particles) due to the low solubility of TMZ in the solvent (i.e. dichloromethane) used for electrospraying. Thus, our future work consists of investigating other solvents to increase the amount of drug loaded. In conclusion, electrospraying is a promising method to fabricate microparticles with high drug loading efficiency and sustained release of the drug, however optimization of the solvent used to prepare the electrospraying solution is needed to increase the amount of drug loaded.

#3720

Augmented loading efficiency of doxorubicin into glioma-derived exosomes by an integrated microfluidic device.

Abhimanyu Thakur, Heng Zou, Mengsu Yang, Youngjin Lee. _City University of Hong Kong, Kowloon Tong, Hong Kong_.

Glioma is a lethal brain cancer in the central nervous system (CNS). The therapeutic usage of potent anticancer drugs for glioma is restricted by their poor penetration across the blood-brain barrier (BBB). Current reports have revealed that cell-derived exosomes can cross the BBB, suggesting their prospective use as drug delivery vehicles for targeting glioma as well as neurological disorders. However, conventional isolation of intact, specific, and optimal exosomes for drug delivery from various donor cells is laborious and time-consuming. Particularly, increasing loading efficiency of non-hydrophobic anticancer drugs, such as doxorubicin, into exosomes has been challenged. Therefore, a single integrated microfluidic device has been developed, based on the principles of immunocapture and density-gradient-free centrifugation, using soft lithography for efficient exosome isolation and drug loading consecutively. The isolated glioma-exosomes were characterized by transmission electron microscopy (TEM), immunogold-EM, and nano tracking analyzer. The loading efficiency of doxorubicin into glioma-exosomes was evaluated by UV-visible spectrophotometer as well high performance liquid chromatography (HPLC). Furthermore, the doxorubicin loaded glioma-exosomes were treated to autologous and heterologous glioma cells, followed by evaluation with confocal microscopy. The present study has shown that our integrated microfluidic device isolates pure exosomes from glioma cells, and it dramatically increases loading efficiency of doxorubicin into the exosomes, supporting its usefulness to study the exosome-mediated drug delivery for glioma therapy.

#3721

MMP9 responsive molecular beacon-loaded nanovesicles for imaging and therapy for triple negative breast cancer.

Amalendu P. Ranjan, Rafal Fudala, Sunil Shah, Andrew Gdowski, Piyush Kumar, Ignacy Grycznski, Zygmunt Grycznski, Jamboor K. Vishwanatha. _University of North Texas Health Science Center, Fort Worth, TX_.

African American women are disproportionately affected by triple negative breast cancer (TNBC). Combination of tumor chemotherapy and imaging in an 'all-in-one system' provides a useful multi-modal approach in the battle against metastatic breast cancer. A promising imaging technique, time gated molecular beacon probes, is a noninvasive and painless method for detection and monitoring. Molecular beacon probe-based theranostic nanoparticles is a novel concept in next-generation medicine that combines simultaneous diagnostics and therapeutics to radically change the way we diagnose, image and treat breast cancer. In this study, we formulated pH sensitive nanovesicles containing molecular beacons specific to MMP-9 for imaging tumor microenvironment along with an anticancer drug, thereby enabling visualization of the tumor together with therapy at the tumor site. This theranostic nanosystem will provide an exciting new tool for breast cancer diagnosis and therapy. Formulation of novel pH responsive nanovesicles were carried out using DMPC and DSPE-PG-8CH. Molecular beacons specific to MMP-9, along with cabazitaxel were encapsulated within the nanovesicles. Characterization of the formulated nanovesicles along with intracellular uptake studies were carried out in breast cancer cell lines. The hydrodynamic diameters of the vesicles were measured using a dynamic light scattering (DLS) instrument (Malvern Zetasizer Nano-ZS) and found to be ~65nm. The zeta potential varied between -55 to -60 mV. Validation of molecular beacon loading in liposome and its stability were done using spectroscopy. Results indicated a molecular beacon loading of ≈29 % in the liposomes and the liposomes were stable for atleast 2 weeks. Comparison of cleavage sequence with time MB and MB-loaded liposome showed that even after 72 hours, molecular beacon released from the liposomes were able to cleave MMP-9 and elicit an effect. This unique and innovative combinatorial theranostic strategy can improve the accuracy of imaging and therapy, provide better assessment of the severity of disease and even monitor the response to drug therapy. This innovative proposal of combining an imaging technique together with delivery of anticancer drug at the tumor site will enhance their clinical potential for diagnosis and therapy in metastatic breast cancer therapy.

#3722

Efficacy of irreversible electroporation in combination with liposome-nvp-bez235 for head and neck cancer.

Li Tian,1 Lucas Wang,2 Yang Qiao,1 Saisree Ravi,3 Ashley Chang,4 Thomas Alexander Rogers,5 Linfeng Lu,1 Adam D. Melancon,1 Marites Pasuelo Melancon1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _The University of Texas at Austin Dell Medical School, Austin, TX;_ 3 _Rice University, Houston, TX;_ 4 _McGovern Medical School, Houston, TX;_ 5 _Mississippi State University, Starkville, MS_.

Introduction: Irreversible electroporation (IRE) is an emerging, minimally invasive ablation technique with the advantage of not being affected by heat sink effect and the ability of ablation in close vicinity of major blood vessels. One limitation is local recurrence at the original ablated site. We hypothesize that combining IRE with a chemotherapeutic drug could prevent recurrence. NVP-BEZ235 (BEZ) is a dual PI3K/mTOR inhibitor that has shown promise for treating advanced solid malignancies, including sensitizing head and neck squamous cell carcinoma to radiotherapy. However, clinical trials revealed low bioavailability and high toxicity due to high dose oral administration over a long period of time. Thus, we formulated a liposomal BEZ (L-BEZ) and studied its antitumor effect in combination with IRE and compared the results with orally delivered BEZ (oral-BEZ).

Methods: IRE was performed using ECM 830 (BTX Harvard Apparatus) at various field strengths. BEZ was loaded into liposome (L-BEZ) by hydration-sonication method. The in vitro and in vivo efficacy was tested against head and neck cancer cell line, HN5 cells and in nude mice bearing HN5 xenografts (n=4/group). Mice were divided into control (no treatment), IRE (2500 V/cm for 99 pulses), IRE + L-BEZ (IRE at 2500 V/cm for 99 pulses and 7 doses of L-BEZ) and IRE + oral-BEZ (IRE at 2500 V/cm for 99 pulses and 21 doses of oral-BEZ) groups. Tumor size was monitored for 60 days.

Results: The hydrodynamic volume of NVP-BEZ ranged from 100-500nm. Maximum drug loading was achieved at 2.7mg/mL of BEZ. Electroporation disrupted nanoparticle's integrity even at the lowest tested field strength (250 V/cm) and released BEZ. At the highest field strength (2500 V/cm), approximately 5% of cells survived with IRE, while cells electroporated at 500 V/cm increased cell viability (114%) as compared to the untreated group (100 %). Combination of electroporation and L-BEZ significantly decreased cell viability at 500 V/cm (p<0.05). In vivo, IRE + L-BEZ suppressed tumor growth the longest, as compared with control (no treatment), IRE, and IRE + oral-BEZ. It was also the only group that resulted in no palpable tumor from day 30 to day 60. Survival studies show a significant difference between IRE + L-BEZ and the other groups. IRE + L-BEZ group had 100% animal survival on day 60. IRE + oral-BEZ had 50%, while IRE and control groups had 0% animal survival, on day 60. Also, the total administered BEZ amount in IRE + L-BEZ group was only 6.6% of that in IRE + oral-BEZ group.

Conclusion: Incomplete electroporation increases the viability of surviving cells both in vitro and in vivo. Co-delivery of L-BEZ enhances the antitumor efficacy of IRE alone. L-BEZ also decreases BEZ exposure compared to oral-BEZ. Thus, L-BEZ in combination with IRE potentially ensures complete eradication of surviving cells left after IRE.

#3723

Nanoengineered mesenchymal stem cells successfully inhibit tumor progression in a syngeneic mouse model.

Buddhadev Layek, Jayanth Panyam, Swayam Prabha. _University of Minnesota, Minneapolis, MN_.

Active tumor-targeted drug delivery holds great promise for enhancing therapeutic efficacy and mitigating toxic side effects resulting from nonspecific distribution of chemotherapeutic agents to healthy tissues. However, the majority of current drug delivery strategies depend on inefficient passive accumulation of the drug carrier in the tumor. We have developed a novel and truly active tumor-targeting approach that relies on the tumor tropism of mesenchymal stem cells (MSCs). Our work emphases nanoengineering of MSCs with paclitaxel-loaded nanoparticles to create cellular drug depots capable of selectively homing to tumor sites and releasing the drug over an extended duration. Paclitaxel-loaded PLGA nanoparticles (18.1 ± 1.9 % w/w paclitaxel; diameter of 282.5 ± 5.2 nm; and zeta potential of −18.4 ± 2.5 mV) were formulated by emulsion-solvent evaporation method. Nanoengineered MSCs were generated by incubating MSCs with 100 µg/mL paclitaxel-loaded nanoparticles for 4 hours at 37°C without altering their viability and migration potential. Anticancer efficacy of nanoengineered MSCs was evaluated in C57BL/6 albino female mice bearing orthotopic Lewis Lung Carcinoma (LL/2-luc) tumors. Despite significantly lower doses of paclitaxel (~2.5 mg/kg total dose for nanoengineered MSCs vs. 120 mg/kg for free- and nanoparticle-encapsulated drug), nanoengineered MSCs resulted in significant greater tumor inhibition and superior survival. Furthermore, administration of human MSCs did not elicit any overt immunologic responses in mice. In summary, our results demonstrate that nanoengineered MSCs can serve as an efficient carrier for tumor-specific delivery of anticancer drugs, resulting in greatly improved therapeutic efficacy.

#3724

5-FU-loaded Nucleoside Nanoparticles for Therapeutic Efficiency of Oral Cancer by various administrations.

HUI FENG, Hang ZHAO, Ning Ji, Jing Li, Zhiyong Wang, lu jiang, Qianming Chen. _sichun university, chengdu, China_.

Drug delivery systems are designed to improve bioavailability, reduce degradation, and alleviate the side effect of the loaded drugs. Here, we reported the development of a novel monomeric self-assembled nucleoside nanoparticle (SNNP) into an efficient drug delivery system in such field. Initially, we used a L-configurational pyrimido[4,5-d]pyrimidine nucleoside building nano-bundles and then found its high biocompatibility and low toxicity in vitro and in vivo. Afterwards, the stability in blood circulation and the distribution of 5-FU in OSCC tissues was remarkably increased by the loading with SNNP. Most importantly, 5-FU-SNNP markedly retarded the in vivo growth of OSCC xenografts in mouse models compared with free 5-FU and SNNP by intratumoral, intraperitoneal and intravenous injection, respectively. It was worth mentioning that, SNNP can even reduce the toxic side effects of 5-FU using intraperitoneal injection on OSCC xenograft mouse model. Furthermore, the superior in vivo antitumor efficacy of 5-FU-SNNP compared with free 5-FU and SNNP was related to inhibit cell proliferation and promote cell apoptosis by TUNEL and immunohistochemistry analyses. These findings indicated that when loaded with 5-FU loaded with SNNP has better antineoplastic efficacy using various administrations and lower side-effects, suggesting that this new type of SNNP has a high hope to be a promising candidate nanoparticle-delivery strategy for cancer therapy clinically in the future.

References

Hang Zhao, Hui Feng, Dongjuan Liu, Jiang Liu, Ning Ji, Fangman Chen, Xiaobo Luo, Yu Zhou, Hongxia Dan, Xin Zeng, Jing Li, Congkui Sun, Jinyu Meng, Xiaojie Ju, Min Zhou, Hanshuo Yang, Longjiang Li, Xinhua Liang, Liangyin Chu, Lu Jiang, Yang He, and Qianming Chen. Self-Assembling Monomeric Nucleoside Molecular Nanoparticles Loaded with 5-FU Enhancing Therapeutic Efficacy against Oral Cancer. ACS nano. 2015; 9(10): 9638 - 9651.

The authors gratefully acknowledge support from the National Natural Science Foundation of China (81520108009, 81500860,81700988).

#3725

Pre-clinical evaluation of prostate specific membrane antigen (PSMA) nanobioconjugate encapsulating green tea polyphenol EGCG for prostate cancer.

Imtiaz A. Siddiqui, Vaqar M. Adhami, Islam Rady, Hasan Mukhtar. _Univ. of Wisconsin-Madison, Madison, WI_.

For prostate cancer green tea constituent (-)-epigallocatechin-3-gallate (EGCG) has shown promise in preclinical, geographical and epidemiological studies and more importantly in many clinical trials. However, concerns related to stability, poor intestinal absorption and extensive systemic and enteric metabolism have hampered its use in the clinic. We introduced the idea of nanotechnology to enhance the outcome of chemoprevention with EGCG and coined the term 'Nanochemoprevention' and later reported the efficacy of chitosan nanoparticles encapsulating EGCG for PCa prevention and therapy. Here, we evaluated an aptamer conjugated prostate targeting nanobioconjugate encapsulating EGCG in two relevant pre-clinical mouse models of human PCa. We developed chitosan nanobioconjugates encapsulating EGCG, surface functionalized with the A10 2′-fluoropyrimidine RNA aptamers that recognize the extracellular domain of the prostate specific membrane antigen (PSMA), henceforth called chit-EGCG-Apt. Mice implanted with androgen sensitive CWR22Rν1 cells and treated with Chit-EGCG-Apt showed significant inhibition in tumor growth that was also associated with decreased serum PSA levels as compared to native EGCG. Tumor tissues from mice treated with Chit-EGCG-Apt exhibited significant (i) induction of PARP cleavage, (ii) increase in the protein expression of Bax with concomitant decrease in Bcl-2, (iii) activation of caspases and (iv) reduction in Ki-67 and PCNA. We further observed inhibition of markers of angiogenesis, invasion and metastasis in tumor tissues in these mice. We next evaluated the efficacy of Chit-EGCG-Apt in the prostate specific Pten knockout mouse model that recapitulates disease progression as seen in humans. Longitudinal MRI analysis suggested significant inhibition of PCa development and tumor growth in these mice. We observed a significant inhibition in tumor growth as evaluated by genitourinary and prostate tissue weight. We further observed a significant decrease in serum IGF-I levels with concomitant increase in serum IGFBP-3 levels in these mice. We analyzed tumor tissues isolated from the mice and observed significant (i) induction of apoptosis, (ii) modulation of Pten/PI3K/Akt, MAPK pathway, IGF-1 signaling and iii) inhibition of markers associated with angiogenesis, invasion and metastasis. These data support our hypothesis that target-specific nanobioconjugates enhance efficacy through boosting bioavailability, thus leading to a significant potential for possible clinical outcome. Targeted nano-encapsulation approach could be viewed as "precision chemoprevention" for PCa patients that are considered low-risk and put on active surveillance.

#3726

Design of personalised polymer based combination therapeutics for advanced stage breast cancer patients.

Ana Armiñán, Zoraida Andreu, Juan J Arroyo-Crespo, David Charbonnier, Esther Masiá, Fernanda Rodriguez-Otomin, Aroa Duro-Castano, Vicent J Nebot, Maria J Vicent. _Centro de Investigación Príncipe Felipe, Valencia, Spain_.

Our objective is to engineer tumour-targeted polymer-based combination therapies specifically designed to treat metastatic breast cancer (BC) in a personalised manner. Our strategy is to develop novel multicomponent polymer conjugates and assess structure activity relationships in clinically relevant models to understand mechanisms of action. Furthermore, we are searching for novel drug combinations, including tumour-derived exosome release pathway inhibitors due to their association with metastasis and tumour drug resistance mechanisms. NCA polymerization techniques have allowed us to precisely control the synthesis of well-defined star-based (STP) and linear (LTP) polypeptidic architectures. After fluorescence labelling, we studied our systems in vitro demonstrating a much more rapid uptake for STP through clathrin-coated mediated endocytosis. pK and biodistribution in healthy mice revealed renal excretion profiles and greater terminal and accumulation half-lives for STP architectures when compared to the LTP. Remarkably, we also observed clear accumulation in immune system-related organs including the spleen and lymph nodes (LN) (up to 40% ID/g tissue accumulation in the LN after i.v. administration). This is possibly due to their inherent structural and morphological features, such as their size (≈100nm), highly negative z-potential values, and/or hydrophilic surfaces. These data highlight the great potential of our stabilized self-assemblies as carriers to target LN metastasis, cancer immunotherapy, or immune system-related approaches, such as vaccination. In parallel, we have performed a High Throughput Screening to select synergistic drug combinations to be used in polymer-based combination approaches through rationally designed linkers that confer adequate drug release kinetics. To perform this approach we selected four metastatic human BC cell lines representing clinical BC subtypes. All cell models have been fully characterized regarding their Cathepsin B activity, intracellular pH, as well as oestrogen, progesterone, Her2 receptors, GSH and exosomes levels; all representing patient-specific biomarkers. Cell viability and exosomes release modulation have been studied following treatments and several drug combinations have been selected for each specific BC subtype. With selected drug combinations different linking chemistry has been designed. We have studied a combination conjugate with the chemotherapeutic agent conjugated to PGA through two different length pH-labile hydrazone linkers. This provided different therapeutic outputs in cells and in a metastatic immunocompetent orthotopic breast cancer model, not only for the primary tumor but also for metastasis progression. The results obtained so far open up a wide range of opportunities for the currently unsuccessful clinical approaches to target LN metastasis and cancer immunotherapy.

#3727

Dual acidic pH targeted mesoporous silica nanoparticles for noninvasive detection of triple negative breast cancer.

Joshua T. Hayes,1 Abhilash Samykutty,1 Molly McNally,1 Alexandra Thomas,2 Akiko Chiba,1 William Grizzle,3 Karen M. Winkfield,2 Lacey R. McNally1. 1 _Wake Forest Univ. Comp. Cancer Ctr., Winston Salem, NC;_ 2 _Wake Forest Univ. Comp. Cancer Ctr., Winston-Salem, NC;_ 3 _University of Alabama Birmingham, Birmingham, AL_.

Purpose: Current screening methods for triple negative breast cancer suggest the presence and size of tumor, but do not identify the tumor microenvironment. The inability to determine precise tumor location and position following diagnosis impedes medical confidence in surgical resection and radiation therapy. Theranostic nanoparticles can be targeted for dye and drug delivery to facilitate non-invasive characterization and treatment of malignancies. Combinational use of nanomedicine and Multispectral Optoacoustic Tomography (MSOT) renders high resolution images through ultrasonic detection of thermoelastic dye expansion (the optoacoustic effect). Approaches in nanomedicine are steadily growing, but clinical evaluation relies on minimizing off-target accumulation and controlling cargo release. We evaluated a mesoporous silica nanoparticles with pH-sensitive chitosan gatekeeper and tumor targeting V3 pH low-insertion peptide (V3 pHLIP) conjugation (V3-CMSN-780) for dual acidic pH targeted delivery to triple negative breast cancer. Methods: Mesoporous silica nanoparticles were synthesized around a structural skeleton of hexadecyltrimethylammonium bromide (CTAB). CTAB was removed from the particles through repeated dialysis. Subsequent particles were characterized with digital light scattering (DLS) and zetasizer to confirm particle size and zeta potential. Mesoporous nanoparticles were then coated with chitosan for extracellular acidic pH selectivity and functionalized with variant 3 pH low-insertion peptide (V3 pHLIP) to promote anchoring of the particle to tumor cells. Functionalized nanoparticles (V3-CMSN) were loaded with NIR 780 (5 mg/mL) and delivered to MDA-MB-231 and MDA-MB-468 malignant cell lines at pH 7.4, 6.8, and 6.6 to confirm pH-sensitive dye release into the cells with Near Infrared fluorescent imaging. V3-CMSN treated cells were inserted into tissue mimicking phantoms which were evaluated using MSOT. Results: The size of V3-CMSN-780 was 35 d. nm with a zeta potential of 25 mV. V3-CMSN were loaded with 780 dye with a dye loading efficiency of 77%. Cellular uptake of V3-CMSN-780 within MDA-MB-231 cells was 2152 a.u., 5242 a.u., and 3639 a.u. at pHs 7.4, 6.8, and 6.6, respectively, based NIR fluorescent imaging. Treatment of MDA-MB-231 cells with V3-CMSN-780 within tissue mimicking phantoms also confirmed acidic pH selectivity of the particles with a 6X and 4X accumulation of particles at pH 6.8 and 6.6 as compared to pH 7.4. While similar results were observed in MDA-MB-468 cells with NIR fluorescent imaging, MSOT imaging indicated that MDA-MB-468 cells treated at pH 6.8 and 6.6 had increased 780 signal, 110X and 16X, incomparison to pH 7.4. Conclusion: Successful dual targeting of breast tumor cells provides a foundation for later in vivo and ex vivo studies using V3-780-CMSNs to deliver diagnostic and therapeutic cargo to malignant breast tissue.

#3728

α-Mangostin-encapsulated PLGA nanoparticles inhibit pancreatic carcinogenesis by targeting cancer stem cells in KC and KPC mice.

Raj K. Verma,1 Wei Yu,1 Anju Shrivastava,2 Rakesh K. Srivastava,3 Sharmila Shankar3. 1 _Kansas City VA Medical Center, Kansas City, MO;_ 2 _St. Joseph's Hospital and Medical Center, Phoenix, AZ;_ 3 _Louisian State University Health Sciences Center, New Orleans, LA_.

Activation of sonic Hedgehog (Shh) in cancer stem cell (CSC) has been demonstrated with aggressiveness of pancreatic cancer. In order to enhance the biologic activity of α-mangostin, we formulated mangostin-encapsulated PLGA nanoparticles (Mang-NPs) and examined the molecular mechanisms by which they inhibit human and KC mice (PdxCre;LSL-KrasG12D) pancreatic CSC characteristics in vitro, and pancreatic carcinogenesis in KPC (PdxCre;LSLKrasG12D;LSL-Trp53R172H) mice. Mang-NPs inhibited human and KrasG12D mice pancreatic CSC characteristics in vitro. Mang-NPs also inhibited EMT by upregulating E-cadherin and inhibiting N-cadherin and transcription factors Slug, and pluripotency maintaining factors Nanog, c-Myc, and Oct4. Furthermore, Mang-NPs inhibited the components of Shh pathway and Gli targets. In vivo, Mang-NPs inhibited the progression of pancreatic intraneoplasia to pancreatic ductal adenocarcinoma and liver metastasis in KPC mice. The inhibitory effects of Mang-NPs on carcinogenesis in KPC mice were associated with downregulation of pluripotency-maintaining factors (c-Myc, Nanog and Oct4), stem cell markers (CD24 and CD133), components of Shh pathway (Gli1, Gli2, Patched1/2, and Smoothened), Gli targets (Bcl-2, XIAP and Cyclin D1), and EMT markers and transcription factors (N-cadherin, Slug, Snail and Zeb1), and upregulation of E-cadherin. Overall, our data suggest that Mang-NPs can inhibit pancreatic cancer growth, development and metastasis by targeting Shh pathway.

#3729

Developing cetuximab-staurosporine conjugate as the therapeutic medicine in KRAS/BRAF mutated colon cancer cells.

Wei-Ting Chao,1 Wei-Ting Sun,2 Hsiang-Ling Chiu,1 Wan-Chen Wei,1 Yi-Che Wu,1 Ling-Yi Kao,1 Yuan-Chiang Chung,3 Shih-Hsien Chuang2. 1 _Tunghai Univ., Taichung, Taiwan;_ 2 _Development Center For Biotechnology, Taipei, Taiwan;_ 3 _Cheng-Ching General Hospital, Taichung, Taiwan_.

Epidermal growth factor receptor (EGFR) is the key receptor for cancer cell growing, cetuximab is the monoclonal antibody that targets EGFR and was approved for treatment of colorectal, head and neck cancer. However, patients with KRAS /BRAF mutation are screened for the resistance. Kinases activities in the signal axes have been demonstrated the role in drug resistance in colon cancer cells. In this study, cetuximab was conjugated with kinases inhibitor staurosporine and the efficacy was examined in KRAS/BRAF mutated colon cancer cells. The cetuximab and staurosprone conjugate was prepared based on the amide bond formation or the thioether bond formation. Both cleavable and non-cleavable linkers were applied to the conjugate. KRAS and BRAF mutated colon cancer cell lines SW480 and HT-29 were treated with cetuximab, staurosporine, cetuximab and Staurosporine combination, cetuximab-staurosporine conjugate, cell viability and the molecular expression were monitored. In the result, staurosporine was demonstrated to suppress cetuximab induced Src activation which may contribute to drug resistance. Wild type cell (SW48) is sensitive (63% viability) to cetuximab treatment compared to BRAF and KRAS mutated cells. Cetuximab-staurosporine conjugate showed twice efficacy than cetuximab on wild type cell, and also resulted in greater toxicity (50% viability) in BRAF/KRAS mutated cells which are resistant to cetuximab treatment. Taken together, cetuximab-staurosporine conjugate has therapeutic potential in BRAF/KRAS mutated colon cancer cells.

#3730

Structure determination, mode of action and safe delivery of an improved K6L9-type peptide.

Tej B. Shrestha, Jing Yu, Stefan Bossmann, Deryl Troyer. _Kansas State Univ., Manhattan, KS_.

There is a significantly increased interest in using antimicrobial peptides for anticancer therapy. D-K6L9, LKLLKKLLKKLLKLL is an antimicrobial peptide that has been explored. It kills cancer cells by depolarizing their cell membranes, thus inducing apoptosis. Further modification of the peptide for increased toxicity and safe packaging into a delivery vehicle for targeted delivery is highly desired for the in vivo applications. Here, the peptide was modified with two extra amino acids (serine and alanine) at both C and N terminals, resulting in SA-LKLLKKLLKKLLKLL-AS. The sequence and structure of the modified peptide were determined by means of 2D 1H-1H -COSY, NOESY, and TOCSY-NMR spectroscopy. The 3D structure of the peptide in the solution phase was generated by CNS software using a distance constraint generated by NOE spectroscopy. The 3-D structure of SA-D-K6L9-AS is maintained, as well as the amphiphilicity required for insertion into cell membranes. This peptide was tested on the following mouse cancer cell lines: GL 26 (glioma), B16-F10 (melanoma lung metastases) and 4T1 (metastasizing breast cancer). The LC50 values of the modified peptide were found to be 0.707 µM for B16-F10, 6.237 µM for 4T1 and 1.707 µM for GL 26, which is 5- 10 fold more active than the original D-K6L9. To explore the findings using the modified peptide, SA-D-K6L9-AS tagged with a rhodamine dye was incubated with GL 26 cancer cells. Sequential confocal imaging (every 30 seconds) revealed that the peptide gets internalized into the cytoplasm in less than 5 min and then localizes in the mitochondria. This peptide is found to be toxic to neuronal stem cells and monocytes as well. To avoid the non-specific toxicity of the peptide for in vivo applications, highly mesoporous silica nanoparticles (MSN) were synthesized. The peptide was loaded into the MSN. MSN were further coated with a polysilazane as a "gift wrap" after loading the peptide. This gift wrap can hold the contents inside the MSN, showing no toxicity at 24 hours and subsequent slow release of the peptide payload into the cytoplasm within 72 hours. This gift wrapping of the highly toxic peptide into the MSN could be very useful for in vivo cancer therapy by means of targeted delivery to the cancer site with appropriate surface modification of MSN.

## ENDOCRINOLOGY:

### Steroid Receptors and Preclinical Studies of Endocrine-Related Cancers

#3731

Deciphering the transcriptional role of estrogen receptor alpha in ovarian cancer.

Irene Lee, Myles Brown. _Dana Farber Cancer Institute, Boston, MA_.

Ovarian cancer is the leading cause of death among gynecologic malignancies. Despite years of research, the 5-year survival rate for ovarian cancer patients has remained poor and the standard of care is largely the same as it was decades ago. Epidemiological evidence and previous studies have suggested a role for steroid hormones in the pathogenesis of ovarian cancer, however, the role of steroid hormones in ovarian cancer remains understudied. We hypothesized that Estrogen Receptor α (ERα) is transcriptionally active and drives a transcriptional program that is sufficient to promote ovarian cancer cell proliferation and survival in a subset of ER+ ovarian cancers. Preliminary data generated in our laboratory has suggested that estradiol (E2) treatment increases cell proliferation in PEO1 cells, an ERα+ ovarian cancer cell line. This increase in cell proliferation can be inhibited by co-administration with the selective estrogen receptor modulator, Tamoxifen, and the selective estrogen receptor degrader, Fulvestrant. To further interrogate the mechanism of action of ERα in ovarian cancer, we performed RNA-seq on PEO1 cells treated with Vehicle, 10 nM E2, 100 nM Fulvestrant, and E2+Fulvestrant for 24 hours. We identified 659 significantly differentially expressed genes following E2 treatment. Fulvestrant inhibited the majority of E2-induced differentially expressed genes, confirming that these genes are dependent upon ERα. Gene Set Enrichment Analysis (GSEA) indicated that the Hallmark early and late estrogen responses are enriched in the E2 dataset, confirming that classical ER activity is intact in these cells. Furthermore, we identified G2/M checkpoint as positively enriched in our dataset, indicating that proliferation genes are upregulated by ERα in these cells. Additionally, we found that apoptosis was negatively enriched in our dataset, indicating that apoptosis pathway genes are suppressed by E2 treatment. Future investigation in this project will center on exploring the mechanisms of ERα transcriptional activity through examining the ERα-dependent cistrome, regulation of target genes, and its interactions with other cofactors. These data will give us insight into how ER is regulated and what pathways and processes ER is driving. Additionally, we will use CRISPR/Cas9 screening to uncover the key genes downstream of ER that are executing the estrogen-dependent effects on proliferation and survival. We believe that these studies will provide additional drug targets that may suggest a combinatorial therapeutic approach in conjunction with endocrine therapies for the treatment of ovarian cancer.

#3732

Insights into the non-coding genome of parathyroid tumors.

Annamaria Morotti,1 Irene Forno,1 Valentina Andrè,2 Vito Guarnieri,3 Andrea Terrasi,1 Rosa Maria Silipigni,4 Silvana Guerneri,4 Chiara Verdelli,2 Alfredo Scillitani,3 Leonardo Vicentini,4 Filomena Cetani,5 Edoardo Beretta,6 Sabrina Corbetta,2 Valentina Vaira1. 1 _University of study of Milan, Milano, Italy;_ 2 _IRCCS Istituto Ortopedico Galeazzi, Milano, Italy;_ 3 _IRCCS Casa Sollievo della Sofferenza, Foggia, Italy;_ 4 _IRCCS Cà Granda, Ospedale Maggiore Policlinico, Milano, Italy;_ 5 _University Hospital of Pisa, Italy;_ 6 _Ospedale San Raffaele, Milano, Italy_.

Background. Parathyroid tumors are characterized by genetic and epigenetic alterations resulting in aberrant expression of protein coding genes and non-coding RNAs. Although long non-coding RNAs (lncRNAs) play a regulatory role in endocrine cancer pathogenesis, a lncRNAs signature in human parathyroid tumors is still missing. Here we investigated the lncRNAs alterations, both at genomic and transcriptional level, in human non-familial parathyroid tumors.

Methods. The expression of 90 lncRNAs was investigated in 4 parathyroid carcinomas (PCas), 12 adenomas (PAds) and 2 normal glands (PaNs) using a commercial array. Both unsupervised (hierarchical clustering-HCL and Principal Component Analysis-PCA) and supervised (Significance Analysis of Microarray, SAM) analyses were performed to identify differences in lncRNAs expression between the 3 tissue types. Significant lncRNAs were validated in a second set of parathyroid tissues including 7 PCas, 26 PAds, 6 atypical PAds (aPAds) and 4 PaNs. Genomic characterization of 21 PAds was performed by array Comparative Genomic Hybridization (aCGH). CDC73 and Multiple Endocrine Neoplasia 1 (MEN1) genes mutations were detected by Sanger sequencing.

Results. HCL analysis of lncRNAs expression identified 2 major groups in which PaNs and PCas were distinguished. Nine lncRNAs were differentially expressed in parathyroid tissues. Specifically, KCNQ1OT1 and SNHG6 were enriched in PaNs, HAR1B, MEG3, HOXA3as and NEAT1 expression characterized PAds, whereas BC200, HOXA6as and WT1-AS were significantly upregulated in PCas. Besides confirming previous data, validation analysis highlighted a different lncRNAs expression pattern in PCas and aPAds according to CDC73 mutation status, with mutated tumors overexpressing the majority of the lncRNAs. Interestingly, BACE1-AS, KCNQ1OT1, NEAT1 and SNHG6 levels in PAds were significantly correlated with MEN1 levels while HAR1B upregulation was associated with chromosome 11 loss of heterozygosity (LOH).

Conclusions. Overall these findings shed light on lncRNAs deregulation in parathyroid pathobiology. Parathyroid tumors histotypes are characterized by different lncRNAs signatures that are related to chromosome 11 derangements and to MEN1 inactivation. Finally, MEN1 may play an epigenetic role in lncRNAs regulation, supporting the important role of chromosome 11 in parathyroid tumorigenesis.

#3733

Combined targeting of estrogen receptor alpha and nuclear transport pathways remodel metabolic pathways to induce autophagy and overcome endocrine resistance.

Zeynep Madak Erdogan,1 Eylem Cotul-Kulkoyluoglu,1 Kinga Wrobel,1 Sunati Sahoo,2 Barbara Haley,2 Yosef Landesman3. 1 _Univ. of Illinois at Urbana-Champaign, Urbana, IL;_ 2 _University of Texas Southwestern, Dallas, TX;_ 3 _Karyopham Therapeutics, MA_.

Majority of breast cancer specific deaths in women with ERα (+) tumor occur due to metastases that are resistant to endocrine therapy. There is a critical need for novel therapeutic approaches to resensitize recurrent ERα (+) tumors to endocrine therapies. The objective of this study was to elucidate mechanisms of improved effectiveness of combined targeting of ERα and XPO1, a nuclear transport protein in overcoming endocrine resistance. Selinexor (SXR), an XPO1 antagonist, has been evaluated in multiple later stage clinical trials in patients with relapsed and /or refractory hematological and solid tumor malignancies. Using Cignalfinder to profile kinase signaling pathways, we found that 4-OH-Tam, SXR or their combination induced differential Akt phosphorylation profiles, changing the localization and activity of the kinase. Since we observed dramatic changes in Akt activity we hypothesized that metabolic profile of breast cancer cells would change in the presence of 4-OH-Tam and SXR. Using Seahorse metabolic profiler and cell viability experiments in limited media conditions we showed that tamoxifen resistant cells were more dependent on mitochondria for energy production. Their glucose and fatty acid dependency decreased in the presence of SXR and cells were more dependent on glutamine as the mitochondrial fuel source. In order to examine metabolic pathways that might result in the observed phenotype we performed transcriptomics and GC/MS whole metabolite profiling and identified aminoacid metabolism pathways to be upregulated when cells were treated with SXR+4-OH-Tam. We demonstrated that combined targeting of XPO1 and ERα rewires metabolic pathways and shuts down both glycolytic and mitochondrial pathways that would eventually lead to autophagy. Remodelling metabolic pathways to regenerate new vulnerabilities in endocrine resistant breast tumors is novel, and given the need for better strategies for improving therapy response of relapsed ERα(+) tumors, our findings show great promise for uncovering the role ERα-XPO1 crosstalk plays in reducing cancer recurrences.

#3734

Estrogen receptor β agonists suppress the growth and progression of mammary tumors in immune-competent mouse models.

Kumaraguruparan Ramasamy, Cathy Samayoa, Naveen K. Krishnegowda, Shaorong Chen, Ratna K. Vadlamudi, Rajeshwar R. Tekmal. _UT Health Science Ctr. at San Antonio, San Antonio, TX_.

Background: Despite medical advances in early detection and treatment, breast cancer still has a relatively high mortality rate in women due to recurrence and metastasis. Many human cancers are able to suppress the activity of the immune system. With the emerging importance of the immune system in tumor surveillance, the need to employ immunocompetent in vivo models to study breast cancer progression is evident. Syngeneic tumor mouse models are a useful tool to study drug development and therapeutic utility of novel drugs. Emerging evidence suggest that Estrogen receptor (ER) β functions as a tumor suppressor in many cancers including breast cancer. Therefore, targeting ERβ with selective agonists may provide therapeutic benefit in the treatment of breast cancer. Herein, we examined the therapeutic efficacy of ERβ agonists on the growth of syngeneic mouse mammary tumors. Experimental design: To test the effects of ERβ agonists on growth, we carried out cell proliferation, invasion and migration, and clonogenic assays in all three-mouse mammary tumor models with different genetic background. Cell cycle was analyzed using FACS analysis. D2A1 (BALB/c) and MM51 (FVB) syngeneic models and ex-vivo culture of E0771 (C57/B6) cells were used to evaluate the antitumor effects of ERβ agonists. Results: First, we assessed the effects of ERβ agonists on cell proliferation of these three mouse mammary tumor cells. Cells were treated with different concentrations of LY500307 (100nM-10µM) and S-Equol (1µM-100µM) for 72 and 96 hrs. D2A1 cells and E0771 cells showed IC50 of 2.5µM for LY500307 and 50 µM for S-Equol and MM51 cells had an IC50 of 2 µM for LY500307 and 40 µM for S-Equol. Our results also showed that ERβ agonists reduce the colony formation ability of D2A1 and MM51 cells. In D2A1 and MM51 cells, LY500307 treatment decreased colonies by 32% and 50%, and S-Equol reduced colonies to 18% and 40% as compared to control respectively. Cell cycle analysis showed that LY500307 and S-equol treatment in D2A1 and E0771 cells resulted in a significant accumulation of cells in S phase. Further, we analyzed the therapeutic efficacy of LY500307 in two syngeneic mouse tumor models from D2A1 and MM51 cells. Our results demonstrated that LY500307 inhibited the tumor growth and the effect was more pronounce in combination with aromatase inhibitor letrozole. Further, using ex-vivo model of tumor explants from E0771 cells, we showed that ERβ agonists inhibited the mammary tumor growth. Conclusions: Our results suggested that ERβ agonists have potential to prevent the progression mammary tumors in immunocompetent hosts.

#3735

Somatostatin receptor-based imaging and treatment of murine pancreatic neuroendocrine tumors induced by MEN1-loss.

Janet W. Li,1 Hanwen Zhang,1 Sean D. Carlin,2 Nitya Raj,1 David S. Klimstra,1 Steven K. Libutti,3 Wolfgang A. Weber,1 Diane Reidy-Lagunes,1 Brian R. Untch1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _University of Pennsylvania, Philadelphia, PA;_ 3 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ_.

Background: Somatostatin receptor ligands are used for the detection and treatment of neuroendocrine tumors. Mutations of MEN1 are frequently observed in human pancreatic neuroendocrine tumors (PanNETs). We utilized a previously described murine model of PanNET that harbors a floxed Men1 allele to study the feasibility of somatostatin receptor type 2 (SSTR2) imaging and therapy in mice. Methods: Mice with pancreatic-specific expression of Cre recombinase and floxed Men1 (Pdx1-cre, Men1f/f) were aged to 9-12 months to allow for the development of PanNETs. Mice were injected with either the SSTR2 agonist 68Ga-DOTA-TATE or antagonist 177Lu-DOTA-JR11 via tail vein. Imaging with 68Ga-DOTA-TATE was performed using PET/CT. Percent uptake of injected activity was calculated for visualized tumors. 177Lu-DOTA-JR11 binding was visualized with autoradiography. Immunofluorescence (IF) detected SSTR2 expression and a marker of DNA damage (γ-H2AX). Results: IF staining for SSTR2 showed membranous localization of the receptor limited to areas of neoplasia with high and low levels of SSTR2 expression variable by individual animal and tumor. Tumors were successfully detected in mice injected with 68Ga-DOTA-TATE using PET/CT. The mean maximum percent uptake of injected activity for visualized tumors was 2.8% +/- 0.34. Injection of 177Lu-DOTA-JR11 and subsequent autoradiography confirmed localization of radionuclide to areas of histology-confirmed neoplasia. Treatment effect (DNA damage) was observed with co-localization of γ-H2AX and SSTR2 staining in neoplastic areas as compared to hyperplastic islets and normal pancreatic parenchyma that were both SSTR2 and γ-H2AX negative. Conclusion: PanNETs from Pdx1-cre, Men1f/f mice can be successfully imaged and treated using somatostatin-based radionuclides. This genetically accurate model can be employed to study peptide receptor radionuclide therapy efficacy and toxicity either alone or in combination with other treatment strategies.

#3736

Role of RAI2 protein in progression of breast and prostate cancer to hormone independent disease.

Katharina Besler, Aleksandra Weglarz, Christina Zill, Jana Jensen, Harriet Wikman, Klaus Pantel, Stefan Werner. _University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany_.

In breast cancer, low retinoic acid-2 (RAI2) gene expression is correlated with presence of disseminated tumor cells in the bone marrow of early stage patients and with poor outcome. Furthermore, the RAI2 protein acts as a transcriptional co-regulator involved in hormonal response and differentiation of hormone dependent breast cancer cells. Here, we evaluate the role of RAI2 in prostate and breast cancer, both steroid hormone driven cancers, focusing on its impact on progression to a hormone independent disease.

First protein expression of RAI2 and hormone receptors (HR) were analyzed in hormone dependent breast and prostate cancer cell lines after treatment with RAI2- or HRspecific shRNAs. Quantitative PCR was applied to examine expression of HR regulated genes in RAI2-depleted VCaP prostate cancer cells and KPL-1 breast cancer cells after hormone stimulation. The effect of a somatic RAI2 knockout in LNCaP prostate cancer cells and KPL-1 breast cancer cells on hormone independent growth was analyzed under hormone deprivation or HR inhibition. Anchorage-independent growth as well as cellular motility of RAI2 knockout LNCaP cells were also tested. Protein expression of the MAPK/ERK and PI3K/AKT signaling axis was analyzed in RAI2 knockout KPL-1 cells, cultured under hormone deprivation or fulvestrant treatment.

Silencing of RAI2 decreased the expression of AR in prostate cancer and ER in breast cancer cell lines, respectively. Moreover, silencing of hormone receptors increased RAI2 expression in cell lines of both tumor entities, revealing an interdependent regulation of RAI2 and tested hormone receptors. Furthermore, after hormone stimulation RAI2 depleted VCaP cells showed increased induction of androgen target genes like STEAP4, MME and SGK1. Similarly, loss of RAI2 expression in KPL-1 cells increased the induction of several estrogen target genes like EGR3, PGR and SNAI1 after hormone stimulation. In LNCaP prostate cancer cells complete loss of RAI2 significantly increased cell viability, colony formation, anchorage independent growth and cell migration. Additionally, those cells were more resistant to chemical castration. Similarly, KPL-1 cells were less sensitive to anti-estrogens and showed an additional growth advantage in hormone free media after RAI2 loss. Analysis of differentially expressed proteins revealed an upregulation of HER2 in RAI2 knockout KPL-1 cells under hormone deprivation.

Our results show that RAI2 acts as a corepressor of steroid regulated genes in prostate and breast cancer cells. Moreover, we revealed a functional association between RAI2 and hormone dependent tumor growth. RAI2 loss could thus contribute to resistance to endocrine therapy in both cancer entities by the induction of receptor tyrosine kinases, like HER2.

#3737

**Apalutamide (ARN-509) demonstrates therapeutic efficacy in genetically engineered mouse models of** Pten **-deficient prostate cancer.**

Marco A. De Velasco,1 Masahiro Nozawa,1 Yurie Kura,1 Naomi Ando,1 Noriko Sato,1 Kazuhiro Yoshimura,1 Kazuko Sakai,1 Kazuhiro Yoshikawa,2 Kazuto Nishio,1 Hirotsugu Uemura1. 1 _Kindai University Faculty of Medicine, Osaka-Sayama, Japan;_ 2 _Aichi Medical University, Japan_.

Apalutamide (ARN-509) is an oral nonsteroidal androgen receptor (AR) antagonist that is currently undergoing late-stage clinical development for the management of castration-resistant prostate cancer (CRPC). In this study, we use genetically engineered mouse prostate cancer (GEM-PCa) models to examine the preclinical activity of apalutamide in the setting of castration-naïve and castration-resistant disease and delve into the molecular mechanisms underlying CRPC progression. In an early-stage efficacy model of Pten-deficient prostate cancer, four weeks of treatment with apalutamide (30 mg/kg/d) significantly reduced tumor burden by 33.5% (P=0.002) in castration-naive mice but did not alter tumor burden in orchidectomized mice bearing castration-resistant tumors. Interim analyses from long-term survival studies, using models of advanced prostate cancer driven by the conditional inactivation of Pten and Trp53, have indicated improved median overall survival (OS) times in both castration-naïve (17 days, 95% CI 11.6-38.9 in control mice vs. 36 days, 95% CI 21.8-56.9 in apalutamide treated-mice, P=0.280) and CRPC settings (21 days, 95% CI 15.1-25.8 in control mice vs. 40 days, 95% CI 31.2-50.1 in apalutamide treated-mice, P=0.001). In the CRPC model, time to tumor progression was significantly longer for mice treated with apalutamide, median time 20 days 95% CI 9.8-20.6, in apalutamide treated-mice vs. 7 days, 95% CI 5.1-10.8, in control mice, P=0.029. Molecular characterization indicated increased PI3K-AKT signaling in response to treatment with apalutamide in castration-naïve prostate tumors. In vitro studies using four mouse prostate cancer cell lines revealed synergistic responses with apalutamide and AKT inhibition (GSK-690693), the mean combination index at 50% effective dose ranged from 0.585-0.791. In vivo efficacy studies of combination therapy in Pten-KO mice showed tumor burden reductions of 33.4%, 17.7% and 40.9% in apalutamide, GSK-690693 and combination treatments, respectively, P=0.002 in the castration-naïve setting, and reductions of 1.0%, 12.7% and 15.5% in apalutamide, GSK-690693 and combination treatments, respectively, P=0.101 in the CRPC setting. Studies to further evaluate the therapeutic benefit of apalutamide plus PI3K/AKT signal blockade in models of advanced prostate cancer are underway. In conclusion, our findings show that apalutamide is active in GEM-PCa models and support further investigation for developing rational treatment combinations for the management of advanced prostate cancer.

#3738

**Three-dimensional treatment-resistant breast cancer spheroids as a predictive model of** in vivo **response to endocrine therapy.**

Carlo I. Rosales, Jiong Zhao, Lauren M. Gutgesell, Rui Xiong, Debra A. Tonetti, Gregory R. Thatcher. _University of Illinois at Chicago, Chicago, IL_.

1 out of every 8 U.S. women will develop invasive breast cancer during her lifetime, making it the second most common form of cancer affecting women. Breast cancer is also a leading cause of cancer related deaths for women in the U.S., second only to lung cancer. Activation of estrogen receptor alpha (ERα) is the primary proliferative mechanism of breast cancer cells, making it a logical target for therapy. ER ligands with antiestrogenic activity, such as the selective estrogen receptor modulator (SERM), tamoxifen, and selective estrogen receptor degrader (SERD), fulvestrant, have proven clinically successful as treatments for breast cancer; however, resistance in up to 50% of patients provides a therapeutic challenge. Once resistant, breast cancer cells become endocrine-independent, because of this, there is an urgent need for both novel therapy and improved models of resistant breast cancer. Our lab has created a panel of various endocrine-independent cell lines to mimic SERM and SERD resistance. Along with traditional 2D cell culturing, 3D spheroids have also been utilized to gain a better understanding of resistance. Importantly, the response to therapeutic agents, of cell lines in 2D versus 3D cultures is not identical. We observe that 3D cultures better replicate observations in mouse xenograft models, demonstrating that elements of the spheroid microenvironment, such as cell-cell interactions and the presence of extracellular matrix (EM), mimic aspects of the tumor microenvironment in vivo. Cells cultured as spheroids are therefore a suitable in vitro model for drug discovery, predictive of response in preclinical animal models, in contrast to 2D monolayer cell cultures.

#3739

Nuclear hormone receptors modulate imatinib resistance in chronic myeloid leukemia (CML).

Bharathi M. Rajamani, Sreeja Karathedath, Raveen Stephen Illangeswaran, Esther Sathya Bama Benjamin, Vikram Mathews, Aby Abraham, Poonkuzhali Balasubramanian. _Christian Medical College, Vellore, India_.

Despite the impressive success of molecular targeted therapy in chronic myeloid leukemia (CML) with tyrosine kinase inhibitors (TKI) such as imatinib (IM), a proportion of patients exhibit suboptimal response and intolerance to the drug. While the mechanism by which resistance or intolerance develops is not completely clear, it is possible that several secondary modifiers could determine disease progression and drug response in CML. Nuclear Hormone Receptors-NHRs are attractive but less explored therapeutic targets in myeloid leukemia. The impressive success of treatment with ATRA, a RAR ligand, in the treatment of APL has not been translated to other myeloid leukemia subtypes. Recently, Prost et al., 2015 showed that Peroxisome proliferator-activated receptor-γ agonist Pioglitazone eliminates the residual CML stem cell pool and these patients remained in complete molecular response even after withdrawal of IM. The role of other NHRs in CML TKI resistance has not been explored extensively.

Based on the in-vitro drug sensitivity data, CML cell lines KU812, EM2 were grouped as sensitive (IC50-0.15 & 2.29uM) and Lama84, KCL22 were grouped as resistant (IC50-54.45 & 66uM) to IM. The NHRs and coregulators that are differentially expressed between IM sensitive and resistant cell lines were identified using NHR RT2 PCR profiler array (SAbiosciences). Based on +2 fold cutoff, the differentially expressed NHRs between test and control group were identified. Eight NHRs (AHR, AR, ESR1, ESRRG, PPARγ, RXRA, RXRB & THRA) were upregulated in the sensitive compared to the resistant cell lines, which was further validated by qRT-PCR.

To understand the effect of these NHRs in sensitizing resistant CML cells to Imatinib, KCL22 and Lama84 cells were pretreated with NHR ligands, followed by increasing concentrations of Imatinib (5µM-100µM). Ligands of RXR (9-cis retinoic acid), AR (testosterone) and ESRRG1 (GSK4716) significantly improved IM sensitivity by increasing the RNA expression of IM influx transporter hOCT1, which was also reported by Wang et al., 2012; Austin et al., 2014 using ligands for RXR, PXR and PPARγ. Pretreatment with these ligands did not have significant effect on the mRNA expression of BCR-ABL, but there was increased inhibition of the BCR-ABL downstream targets such as pCRKL, pSTAT5 and pAKT at the protein level in combination with IM. The AR, RXR & ESRRG ligands significantly decreased the IC50 to IM (p<0.0001) in primary CML cells (n=22) in combination with IM. We also observed significant sensitization of primary samples (n=7) to IM when used in combination with clinically available RXR and AR ligands isotretinoin (p=0.0011) & stanozolol (p=0.0054). Our finding suggests that NHR ligands, especially RXR, AR & ESRRG when used in combination with IM could overcome resistance. Further studies are warranted to confirm the mechanistic effects of these ligands in modulating drug resistance in CML.

#3740

Functional cross-talk between nuclear receptor LRH-1 and androgen receptor signalling in prostate cancer.

Wenxing You, Yuliang Wang, Leung Chan. _The Chinese University of Hong Kong, Hong Kong, China_.

Liver Receptor Homolog 1 (LRH-1, NR5A2) is an orphan nuclear receptor that is over-expressed in cancers in tissues such as the breast, colon and pancreas. In prostate cancer, LRH-1 is reported as a potent transcription factor to facilitate the formation of CRPC (castration-resistant prostate cancer cells) which is resistant to androgen deprivation therapy (ADT). However, it is still elusive how LRH-1 responses to ADT and facilitates the growth of CRPC. After deprivation of androgen in mouse prostate cancer xenograft model, LRH-1 mRNA was upregulated dramatically at a very low androgen level. After CRPC formation, LRH-1 mRNA was downregulated when androgen level recovered. KLK3/PSA is an important downstream target of activated AR (Androgen receptor), whose expression level at 4 days after castration is downregulated, while upregulated after relapse. Since PSA level/AR signaling activity showed an opposite expression pattern to LRH-1, we hypothesized that LRH-1 was suppressed by androgen in a negative-feedback manner. With androgen treatment of an androgen-sensitive prostate cancer cell line LNCaP, LRH-1 was downregulated in both dose- and time-dependent manner, while PSA shows an opposite expression pattern. And knocking down of AR blocked the suppression of androgen on LRH-1, indicating that androgen suppresses LRH-1 through AR. Furthermore, AR antagonist Enzalutamide can block the binding of androgen to AR thus upregulate LRH-1. According to ChIP-seq data, AR binds to the promoter and enhancer of LRH-1,and the binding intensity increased with androgen treatment. Through ChIP-qPCR, several binding sites of AR on LRH-1 promoter and enhancer were validated. The transcription activity of luciferase reporter with these AR binding sites was also suppressed by treatment of androgen. In the future work, to find out the epigenetics modifiers in the AR complexes when suppressing LRH-1 will be critical for the understanding of the detailed mechanism.

#3741

Deciphering the regulome of androgen receptor variants in prostate cancer.

Mark L. Daniel,1 Theresa E. Hickey,2 Jason S. Carroll,3 Wayne D. Tilley,2 Luke A. Selth,2 Scott M. Dehm1. 1 _University of Minnesota, Minneapolis, MN;_ 2 _University of Adelaide, Adelaide, Australia;_ 3 _University of Cambridge, Cambridge, United Kingdom_.

Prostate cancer is an androgen-dependent disease. Androgen function is controlled by the androgen receptor (AR), and treatments targeting AR are effective at preventing disease progression. Despite this, resistance and emergence of castration resistant prostate cancer (CRPC) is responsible for virtually all prostate cancer-related death. Constitutively active AR variants (AR-Vs) have been implicated as drivers of CRPC. AR-Vs consist of the unstructured AR NH2-terminal domain (NTD), and the AR DNA binding domain, but lack the ligand binding domain (LBD), which is targeted by all current therapies. While our understanding of proteins that regulate the AR LBD is mature, there is less known about the molecular regulation of the NTD. This represents a gap in knowledge given that the AR NTD is essential for AR/AR-V transcriptional activity. To address this, we used a technique termed Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins (RIME) with a pair of isogenic prostate cancer cell lines that were manipulated via genome engineering to express either full-length AR or AR-V. RIME identified several candidate proteins that interact with the AR NTD including BCL9, a β-catenin transcriptional coactivator. Interrogation of public databases revealed that BCL9 mRNA is overexpressed in metastatic prostate cancer compared with localized disease. Additionally, knockdown of BCL9 in cell lines expressing full-length AR inhibited expression of AR target genes as well as β-catenin target genes. Interestingly, knockdown of BCL9 did not affect these same genes in cell lines expressing AR-Vs, indicating that AR truncation could represent a mechanism of escape from BCL9 regulation. Collectively, these findings identify a new mechanism of AR regulation by BCL9 in prostate cancer.

#3742

Vitamin D inhibits hyaluronan synthesis and extracellular matrix gene expression in triple negative breast cancer.

Carmen J. Narvaez, Seamus Balinth, JoEllen Welsh. _University at Albany-Cancer Research Center, Albany, NY_.

Human breast tumors with abundant hyaluronan (HA) and high expression of the HA synthesizing enzyme HAS2 are aggressive and exhibit poorer survival than tumors with low HA/HAS2. The vitamin D receptor (VDR) and its ligand 1,25(OH)2D3 (1,25D) exert anti-tumor effects in cancer cells and inhibit breast cancer growth in animals, but considerable heterogeneity has been reported in different model systems. Genomic profiling has demonstrated that, while a few genes are commonly regulated by 1,25D in all VDR positive cell lines, the majority of VDR regulated genes are cell line specific. In general, cell lines derived from the more aggressive tumor subtypes such as Triple Negative Breast Cancer (TNBC) express low levels of VDR and are less sensitive to 1,25D than cell lines derived from less aggressive subtypes. Through genomic profiling of TNBC cells derived from wild-type (WT) and VDR knockout (VDRKO) mice, we identified HAS2 as a 1,25D repressed gene. To understand the mechanistic impact of vitamin D and Has2 on HA synthesis and tumorigenic potential in TNBC cells, we utilized the human TNBC cell line Hs578T, which is representative of the mesenchymal/stem-like (MSL) subtype of TNBC. 1,25D inhibited HAS2 expression and HA synthesis in Hs578T cells, and also down-regulated CD44, a breast cancer stem cell marker which acts as the receptor for HA, the product of HAS2. Further studies determined that Hs578T cells are heterogeneous with respect to morphology and HAS2 expression. We therefore sorted parental Hs578T cells into HAhigh and HAlow populations. HAhigh populations exhibited elevated HA production, smaller size, increased proliferation and higher motility than HAlow populations. Through genomic profiling, HAhigh populations demonstrated higher levels of expression of genes involved in the enzymatic production and degradation of HA (HAS2, HAS2-AS, HYAL1), expression of extracellular matrix (ECM) proteins involved in HA binding and motility (PTX3, CEMIP, ACAN) and epithelial-mesenchymal transition (SNAI1) compared to HAlow populations. Despite their more aggressive phenotype, HAhigh populations retained expression of VDR protein at levels comparable to that of parental Hs578T cells and HAlow subclones. Treatment with 1,25D decreased production of HA in both HAhigh and HAlow populations, but significantly reduced expression of genes involved in the production of HA (HAS2, HAS2-AS) and HA binding and motility (ACAN, CEMIP) only in the HAhigh populations. This study suggests that 1,25D/VDR can suppress HAS2 and HA production and signaling in the ECM, and thus may reduce the aggressive phenotype of TNBC.

#3743

Translational regulation by ERα in hormone-dependent cancers.

Julie Lorent,1 Vincent van Hoef,1 Richard Rebello,2 Mitchell Lawrence,2 Ivan Topisirovic,3 Ola Larsson,1 Luc Furic2. 1 _Karolinska Institutet, Stockholm, Sweden;_ 2 _Peter MacCallum Cancer Centre, Melbourne, Australia;_ 3 _McGill University, Montreal, Quebec, Canada_.

The estrogen receptor α (ERα) activities are complex: in the cytoplasm ERα can directly stimulate survival signalling at the cell membrane, while in the nucleus ERα activates and represses the transcription of target genes. We recently showed that in prostate cancer ERα expression is associated with increased proliferation and higher clinical grade.

Here we explore the role of ERα in coordinating transcription and mRNA translation. Unexpectedly, loss of ERα expression leads to decoupling of transcription and translation events. Namely, mRNAs whose levels are induced by ERα loss exhibit reduced translation efficiency and, vice versa, mRNAs whose levels are reduced by ERα loss exhibit enhanced translation efficiency. Such regulation is manifested at the protein level and targets a range of key cellular functions including translation and metabolism.

Our detailed mechanistic assessment reveal that while ERα-regulated microRNA levels contribute to global changes in mRNA levels, translational buffering is explained by changes in ribosome processivity and elongation rate.

Overall, we have recently identified a process by which ERα drastically impacts the translation of a subset of mRNAs in cancer cells. We propose that this new regulatory pathway plays a major role in mediating biological effects of ERα in neoplastic tissues. Moreover, our findings have important implications in understanding alterations in gene expression programs following treatment with ERα antagonists.

#3744

Multiple endocrine-resistant breast cancer cell lines retain ER and sensitivity to endocrine therapy.

Lauren M. Gutgesell, Rui Xiong, Jiong Zhao, Huiping Zhao, Debra A. Tonetti, Gregory R. Thatcher. _Univ. of Illinois at Chicago, Chicago, IL_.

Endocrine therapy is the standard of care for breast cancer expressing estrogen receptor (ER),

which occurs in 70% of patients. Unfortunately, acquired or de novo resistance to endocrine

therapy is observed in up to 50% of patients, leaving a significant portion of patients with

insufficient treatment options. Endocrine-resistance, usually defined as resistance to tamoxifen

and aromatase inhibitor (AI) therapy, can also include resistance to selective estrogen receptor

degraders (SERDs), since these also target ER. Since multiple mechanisms contribute to

resistance, development of multiple resistant cell lines is needed for drug discovery and to identify

characteristics that may suggest susceptibility to alternative and combination therapies. We have

developed 5 stable, endocrine-resistant cell lines from a parent MCF-7 cell line, which all retain

ER: one of these cell lines, MCF-7:CFR is resistant to the SERD fulvestrant. Clinical metastatic

breast cancers that have gained endocrine resistance are overwhelmingly ER+. In addition to ER,

progesterone receptor (PR) and glucocorticoid receptor (GR) status of these lines was assessed,

and correlated with the response of these cells in culture to four classes of endocrine therapeutics:

SERDs, selective ER modulators (SERMs), and selective human ER partial agonists (ShERPAs).

Growth of all 5 cell lines was endocrine independent, indicating resistance to AI therapy. Two of

the ER+, PR- cell lines were most resistant to the spectrum of endocrine therapies, but these cell

lines both showed sensitivity to ShERPAs, especially in combination with non-endocrine targeted

therapies, such as the PI3K inhibitor, alpelisib. Paradoxically, all endocrine-resistant cell lines

responded to at least one of the endocrine therapies tested, demonstrating that if ER is not lost

in the metastatic state, it remains a vulnerability suitable for therapeutic targeting.

#3745

Inhibition of Protein Kinase N1 prevents control of AR over SRF action in advanced prostate cancer.

Varadha Balaji Venkadarkrishnan,1 Adam DePriest,2 Yixue Su,1 Giridhar Mudduluru,1 Salma Ben-Salem,1 Sangeeta Kumari,1 Qiang Hu,2 Eduardo Cortes,2 Scott Dehm,3 Cristina Magi-Galluzzi,1 Eric Klein,1 Nima Sharifi,1 Song Liu,2 Hannelore Heemers1. 1 _Cleveland Clinic, Cleveland, OH;_ 2 _Roswell Park Cancer Institute, Buffalo, NY;_ 3 _University of Minnesota, Minneapolis, MN_.

A major limitation in the management of castration-recurrent prostate cancer (CR-CaP) is the lack of treatments to inhibit androgen receptor (AR) action that is driving CaP growth when androgen-deprivation therapy (ADT) has failed. AR action breaks down in fractions that are regulated differently at the molecular level, control diverse aspects of CaP biology and contribute differentially to CaP progression. Blocking a specific fraction(s) of AR action that mediates CaP progression may serve as alternative treatment strategy, yet evidence for such an approach and drugs that render this approach feasible are not known. Our laboratory has identified a mechanism of AR action in which AR conveys androgen-responsiveness to Serum Response Factor (SRF) target genes via androgen activation of RhoA. Androgen-responsive SRF action mediates cell migration and is associated with aggressive CaP behavior and recurrence. Importantly, control of AR over RhoA-SRF action is maintained in CR-CaP, which renders this signaling a potentially attractive novel therapeutic target. Using an siRNA screen, we identified Protein Kinase N1 (PKN1) as the Rho effector that transduces androgen-responsiveness from RhoA to SRF. In promoter-reporter, qRT-PCR and oligoarray assays, knockdown of PKN1 severely impaired the androgen-regulation of SRF target genes, but affected androgen response of only a few (12 of 452) direct AR target genes. In contrast, transient overexpression of PKN1 preferentially stimulated androgen-responsive SRF target gene expression. Stable overexpression of PKN1 hastened growth and shortened survival in CaP orthotopic xenografts. Site-directed mutagenesis showed that the effect of PKN1 on SRF target genes relied on its kinase activity. Treatment with lestaurtinib, a multikinase inhibitor that is also a potent inhibitor of PKN1, mimicked the effects of PKN1 knockdown on expression of SRF and AR target genes in several CaP cell lines and ex vivo CaP explants. Lestaurtinib also inhibited the proliferation of CaP cells that are either ADT-naïve or -resistant, express only the constitutively active AR variant ARv567es, or are growth-stimulated by the AR-activating ADT drug metabolite 5α-abiraterone. RNA-Seq followed by MSigDB analyses confirmed that lestaurtinib impairs androgen-dependent PKN1 activity and revealed that 100 of the 127 MgSigDB gene sets isolated after PKN1 silencing overlapped with 123 gene sets derived after lestaurtinib treatment. Overlapping gene sets included several related to hormonal carcinogenesis and cancer progression, prostate development, signaling cascades that have been implicated in SRF and RhoA function and/or are relevant to CaP. PKN1 inhibition via lestaurtinib, which is used already to treat human hematologic malignancies and is well tolerated by carcinoma patients, may thus serve as novel alternative treatment strategy to target AR-dependent SRF action in CR-CaP.

#3746

Targeting adrenal androgen to treat prostate cancer.

Yue Wu, Jianmin Wang, Li Tang, Elena Pop, John J. Stocking, Kristopher M. Attwood, Gary J. Smith. _Roswell Park Cancer Inst., Buffalo, NY_.

Background: Adrenal androgens are the most abundant androgens in humans, with dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEA-S) being the major adrenal androgens in circulation. Significantly, adrenal androgens may serve as substrates for intratumoral (intracrine) production of testosterone (T) and/or dihydrotestosterone (DHT), the ligands for androgen receptor (AR). However, adrenal androgens also have functions that are independent of AR. The present study addressed the capability of prostate cancer tissue to use adrenal androgens for T/DHT production, delineated signaling pathways activated by adrenal androgens, and determined whether adrenal androgens sustained tumor growth after androgen-deprivation therapy (ADT).

Experimental Procedures: Metabolism of adrenal androgen was evaluated ex vivo using fresh clinical prostate tissue specimens. The production of DHT or DHEA was determined using ELISA. Human prostate cancer cells lines were used for in vitro experiments. The effects of adrenal androgens on tumor growth were evaluated in vivo using xenografts of the human prostate cancer cell line VCaP in castrated or intact mice.

Results: 1) Human prostate tissue was able to produce DHT using DHEA-S at a physiologic concentration (3.5 μM). The production of DHT from DHEA-S was absolutely dependent on the activity of steroid sulfatase (STS). DHEA at a physiologic concentration (10 nM) was not a suitable substrate for DHT production, but was used by prostate tissue when available at a supra-physiologic concentration (3.5 μM) to produce DHT. 2) DHEA-S was able to stimulate growth of prostate cancer cells in vitro. The growth stimulatory effect of DHEA-S was mediated by AR-dependent and AR-independent signaling pathways. A subset of the genes modulated specifically by DHEA-S were distinctive from the T-modulated genes. 3) DHEA sustained tumor growth after castration. DHEA-stimulated growth was attributed to activation of the insulin-like growth factor 1 receptor (IGF1R) signaling pathway.

Conclusions: Prostate tissue can use adrenal androgens for DHT production. Adrenal androgens may stimulate prostate cancer growth using AR-dependent pathways that are mediated via intratumoral T/DHT production, or using AR-independent signaling pathways such as the IGF1R signaling pathway. Identification of targets and therapeutics to block the adrenal androgen-stimulated pathways may help develop novel strategies to treat prostate cancer.

#3747

Translating the functional interactions of checkpoint kinase 2 and the androgen receptor into more effective therapies for the treatment of prostate cancer.

Huy Q. Ta, Natalia Dworak, Rosalie Sleppy, Jeffery A. Allende, Daniel Gioeli. _University of Virginia, Charlottesville, VA_.

Prostate cancer remains the most diagnosed cancer among men in the United States behind skin cancer, and advanced prostate cancer is the third leading cause of cancer-related deaths, with a 5-year survival rate of 26%. Radiation is the standard of care for the treatment of prostate cancer at the early and late stages. Checkpoint kinase 2 (CHK2) is a serine/threonine protein kinase whose main function is regulating the DNA damage response (DDR) induced by ionizing radiation. The androgen receptor (AR) is a major driver of prostate cancer, even at the castration-resistant stage of the disease. The development of the second-generation anti-androgen enzalutamide, which is a selective AR antagonist, highlights the enduring importance of the AR. We have previously demonstrated that CHK2 is a critical negative regulator of prostate cancer cell growth, androgen sensitivity, and AR transcriptional activity. We have now uncovered novel molecular interactions between CHK2 and AR that provide mechanistic insight into our observation that CHK2 regulates prostate cancer growth. The AR directly interacts with CHK2, and that interaction increases with radiation. We found that the interaction of CHK2 and AR occurs at sites of DNA damage. The binding of CHK2 with AR can be disrupted with CHK2 kinase inhibitors suggesting that the kinase activity of CHK2 is required. This was verified using kinase-impaired CHK2 variants, including the K373E variant associated with 4.2% of prostate cancer. Furthermore, the radiation-induced increase in CHK2-AR interactions requires AR phosphorylation on both serine 81 and serine 308. Interestingly, CHK2-depletion in LNCaP cells increases ionizing radiation induced AR expression and DNA damage. Together, these data provide the rationale for targeting the CHK2-AR signaling axis to improve the effectiveness of prostate cancer therapies. The combination of CHK2 or CDK1 inhibitors with androgen deprivation therapy (ADT) and radiation shows an additive effect on the repression of tumor cell growth. Nearly every patient with disseminated prostate cancer will relapse following ADT and develop incurable castration-resistant prostate cancer. We have uncovered the molecular details of a signaling axis involving CHK2 and AR that, when perturbed in combination with ADT and/or ionizing radiation, effectively inhibits prostate cancer cell growth. This may enable resensitization of castration-resistant prostate cancer to the currently approved treatment options.

#3748

p53 status as a determinant of functional duality of estrogen receptor beta in breast cancer: Therapeutic implications.

Gokul M. Das,1 Utpal K. Mukhopadhyay,1 Christina Adams,1 Nadi Wickramasekera,1 Rajesh Medisetty,1 Sanjay Bansal,1 Laxmi Silwal-Pandit,2 Anne-Lise Borresen-Dale,2 Austin Miller,1 Wendy M. Sweizig,1 Angela Omilian,1 Wiam Bshara,1 Alka Mukhopadhyay1. 1 _Roswell Park Cancer Inst., Buffalo, NY;_ 2 _Oslo University Hospital Radium Hospital, Montebello, Oslo, Norway_.

Whether estrogen receptor beta (ERβ) is a pro- or anti-oncogenic protein in breast cancer has been controversial. ERβ levels are high in ERα negative cancers including triple-negative breast cancer (TNBCs). Recent reports including the Cancer Genome Atlas (TCGA) show that about 80% of TNBC express mutant p53 (mut-p53) and it is the most predominant driver in these cancers. We tested the hypothesis that p53 status in breast cancer will have an important role in determining the duality of ERβ functions. We have shown that ERβ directly binds to p53 in human breast cancer cells. Using glutathione-S-transferase (GST)-pull down and co-imunoprecipitation assays, we have delineated the domains of both proteins that are required for the ERβ-p53 interaction. The DNA binding domain (DBD) along with the hinge domain of ERβ and the C-terminal regulatory domain of p53 are essential for the interaction. Using the highly sensitive proximity ligation assay (PLA), we show ERβ-p53 interaction in situ in breast cancer cells expressing either wild type (wt)- or mut-p53. ERβ and p53 antibodies validated for specificity were used. In multiple cell lines, a combination of proliferation and apoptosis assays, RNAi technology, quantitative chromatin immunoprecipitation (qChIP), and quantitative real-time PCR (qRT-PCR) showed that ERβ is pro-proliferative in the context of wt-p53, whereas it is anti-proliferative in the context of mut-p53. The results were recapitulated in isogenic MDA-MB-231 TNBC cells (generated by CRISPR technology) that differ only in the presence of wt-versus mut-p53. ERβ binds and sequesters mut-p53 from mut-p53−p73 complex leading to reactivation of tumor suppressor p73. Consistent with these data, combination of immunohistochemistry (IHC) and PLA in TNBC patient tumor tissue microarray (TMA) showed that patients with tumors expressing wt-p53/ high ERβ had worse prognosis, both in terms of overall survival (OS) and progression-free survival (PFS). On the contrary, tumors expressing mut-p53/ high ERβ were of smaller size and stage. These findings were complemented by data from our analysis of the mut-p53 subgroup of the basal-like tumors in the METABRIC dataset which showed that patients with tumors expressing higher levels of ERβ RNA (ESR2) had better prognosis. Surprisingly, 4-Hyroxy Tamoxifen (Tam) increased ERβ-mut-p53 interaction in TNBC cells and combination of doxorubicin (Adriamycin) and Tam decreased the IC50 of doxorubicin by 10 fold leading to increased apoptosis. These data have significant clinical implications in targeting ERβ and mutant p53 signaling pathways for therapeutic purposes especially in ERα negative cancers such as TNBC. Importantly, although at present Tam is not standard of care for TNBC, our data suggest a possibility for repurposing Tam therapy alone or in combination with chemotherapy to treat TNBC stratified based on p53 status.

#3749

Negative regulation of CPSF6/paraspeckles /A-to-I RNA editing by prolactin hormone in aggressive breast cancer.

Najat Binothman, suhad Ali. _McGill University, Montreal, Quebec, Canada_.

Prolactin (PRL) hormone plays a fundamental role in mammary gland development and terminal differentiation of mammary epithelial cells. However, The role of PRL in breast tumorigenesis is not fully elucidated. Recently, we described the Cleavage and Polyadenylation Factor-6 (CPSF6), a PRL-down-regulated target gene, to play a critical pro-oncogenic role in aggressive breast cancer. Importantly, we found aggressive breast cancer cells of luminal B, HER2-overexpressing and triple negative subtypes to require CPSF6 for viability and tumorigenic capacity. Mechanistically, we found CPSF6 to interact with components of the pro-oncogenic A-to-I RNA editing machinery, paraspeckles and ADAR1 enzyme and to be required for their physical integrity. Moreover, we found PRL to suppress mRNA and protein expression levels of CPSF6 and core paraspeckles proteins (PSPC1, P54 and SFPQ) as well as NEAT1 long non-coding RNA level and to modulate their intracellular localization. Finally, we found prolactin to suppress A-to-I RNA editing activity in aggressive breast cancer cell. Together, this study revealed a novel mechanism contributing to the anti-tumorigenic role of PRL in breast cancer.

#3750

Xenoestrogens cause estrogen receptor-dependent R-loop formation and DNA damage.

Karen A. Dunphy, Prabin Dhangada Majhi, Aman Sharma, Amy L. Roberts, Elizabeth A. Daniele, Sallie S. Schneider, D Joseph Jerry. _Univ. of Massachusetts Amherst, Amherst, MA_.

Background: The hormone 17β-estradiol (E2) plays an important role in breast cell proliferation and development but prolonged exposures and higher levels of E2 have been linked to genomic instability and breast cancer. Recent studies show E2 induced transactivations lead to DNA damage. A variety of environmental chemicals mimic the activities of estrogen, referred to as xenoestrogens. Thus, our hypothesis is to determine whether xenoestrogens stimulate transcriptional response and induce DNA damage similarly like E2.

Methods: The T-47D breast cancer cell line is used as a model because it expresses both estrogen receptors (ERα & ERβ) as found in normal breast tissue. Cells were exposed a physiologic range of E2 (17β-estradiol) [0.5-100nM] and the xenoestrogens, e.g., benzophenone-3 (BP-3) [0.5-50µM] and propyl paraben (PP) [0.5-10µM] with or without ER-antagonist ICI (1µM) for 24 hours. Transcriptional responses were quantified using an integrated ERE-luciferase reporter and qPCR for endogenous genes. Proliferation was monitored using the Alamar Blue assay. DNA damage was determined using γ-H2AX. R-loops were detected using the S9.6 antibody against DNA-RNA hybrids in the genome.

Results: Exposure of T-47D cells to physiologic levels of E2, PP and BP3 causes significant increase in DNA double-strand breaks (DSBs) as determined by γ-H2AX staining. We demonstrate a concomitant increase in R-loop formation with increase in γH2AX intensity. Treatment with RNaseH depletes R-loop intensity, corroborating the result. E2 and PP induce transactivation as determined by Luciferase reporter assay as well as expression of ER-response genes (PGR and AREG), whereas BP3 has modest effect on transactivation of PGR. ER transactivation as well as R-loop formation by E2, PP and BP3 were abrogated with ICI, demonstrating ER-dependency of R-loop formation. Consistent with the transcriptional response, proliferation studies determined that E2 showed highest proliferation followed by PP. However, BP3 showed no increase in proliferation.

Conclusions: Our results show that xenoestrogens have strikingly different activities in transactivation and proliferation. However, both xenoestrogens appear to stimulate R-loop accumulation that leads to DSB in an ER-dependent manner.

## IMMUNOLOGY:

### Immunomodulatory Agents and Interventions 1

#3751

**Inhibition of A** 2A **R by AZD4635 induces anti-tumor immunity alone and in combination with anti-PD-L1 in preclinical models.**

Alexandra Borodovsky,1 Yanjun Wang,1 Minwei Ye,1 Joseph C. Shaw,1 Kris Sachsenmeier,1 Nanhua Deng,1 Kelly Goodwin,1 James D. Clarke,1 Richard Goodwin,1 Nicole Strittmatter,1 Carl Hay,2 Vasu Sah,1 Lawson Deborah,1 Corinne Reimer,1 Miles Congreve,3 Jonathan Mason,3 Fiona Marshall,3 Paul Lyne,1 Richard Woessner1. 1 _AstraZeneca, Boston, MA;_ 2 _MedImmune, Gaithersburg, MD;_ 3 _Heptares Therapeutics, Hertfordshire, United Kingdom_.

Adenosine signaling through the high affinity adenosine 2A receptor (A2AR) on immune cells elicits a range of immunosuppressive effects which can promote tumor growth and limit the efficacy of immune checkpoint inhibitors. AZD4635 (HTL-1071) is a potent and selective oral A2AR antagonist, currently in a Phase 1 clinical trial as a single agent and in combination with durvalumab (anti-PD-L1 Ab) in patients with solid malignancies. In functional in vitro assays, the IC50 of AZD4635 for inhibition of A2AR signaling is dependent on adenosine concentrations and ranges from 1, 10.0, 143 nM in the presence of 0.1, 1, 10 μM adenosine, respectively. However, full understanding of the impact of adenosine on AZD4635 mediated anti-tumor responses requires characterization of intratumoral adenosine concentration and spatial heterogeneity. Using a novel LC/MS based method, measurement of intratumoral adenosine concentrations in a panel of syngeneic tumor models demonstrated a wide range of adenosine levels (5-122 μM). Additionally, measurement of intratumoral adenosine by desorption electrospray ionisation - mass spectroscopy (DESI-MS) demonstrated that adenosine levels are spatially heterogeneous, with levels varying up to 50-fold among regions of a single tumor. High intratumoral adenosine concentrations were correlated with decreased intratumoral CD8 infiltration in vivo. The therapeutic benefit of A2AR blockade alone and in combination with anti-PD-L1 was evaluated in a panel of syngeneic mouse tumor models with varying adenosine concentrations. In sensitive models, inhibition of A2AR signaling with AZD4635 led to a dose dependent reduction in tumor growth alone and in combination with checkpoint inhibitors. Tumors harvested from the treated mice exhibited increases in the functional activity of T cell and myeloid subsets. These results demonstrate that AZD4635 is a potent and selective A2AR inhibitor, that blockade of A2AR signaling with an inhibitor such as AZD4635 can reduce tumor burden and enhance antitumor immunity.

#3752

Preclinical pharmacology of MP0310: A 4-1BB/FAP bispecific DARPin drug candidate promoting tumor-restricted T-cell costimulation.

Alexander Link, Julia Hepp, Christian Reichen, Patricia Schildknecht, Ivana Tosevski, Joanna Taylor, Laurent Juglair, Alexander Titz, Mirela Matzner, Ralph Bessey, Christof Zitt, Guy Lemaillet, Joerg Herbst, Keith M. Dawson, Hong Ji, Victor Levitsky, Dan Snell, Michael T. Stumpp, Andreas Harstrick, Elmar vom Baur. _Molecular Partners AG, Schlieren, Switzerland_.

Agonistic antibodies against the T cell costimulatory receptor 4-1BB (CD137) have proved to be very efficacious anti-tumor agents in preclinical animal models. However, clinical development of 4-1BB agonistic antibodies has met with limited success thus far. Anti-4-1BB monoclonal antibodies have either been reported to cause significant dose-limiting hepatotoxicity or demonstrated limited efficacy as single agent therapeutics. Here we describe the generation of a tumor-targeted 4-1BB agonist aimed at inducing more effective triggering of 4-1BB without associated systemic toxicity. Tumor targeting is achieved via fibroblast activation protein (FAP) which is abundantly expressed by cancer associated fibroblasts present in many solid tumors. Drug candidate MP0310 comprises DARPin domains binding to 4-1BB and FAP and is devoid of an antibody Fc domain. Compared to first generation monoclonal antibodies targeting 4-1BB, MP0310 shows high potency in vitro and less systemic activation in vivo. In vitro reporter and T cell assays indicate that MP0310 is a potent T cell co-stimulator whose activity is restricted to the presence of FAP-expressing cells. In humanized mouse xenograft studies, FAP-targeted 4-1BB activation induced potent co-stimulation of CD8 T cells leading to tumor growth inhibition. On the other hand, the DARPin molecule did not induce effects associated with strong systemic activation such as hepatotoxicity or exacerbation of graft versus host disease observed in such models, unlike the first generation FcγR-dependent 4-1BB antibodies. In addition, no systemic activation of T cell proliferation was observed in the absence of FAP-positive tumors. In healthy cynomolgus monkeys, administration of MP0310 did not induce systemic stimulation of memory T cell proliferation in contrast to an anti-4-1BB antibody despite MP0310 being fully cross-reactive to cyno 4-1BB and binding effectively to cyno FAP. Therefore, we conclude that the tumor-restricted co-stimulation of 4-1BB may prevent toxicities caused by systemic 4-1BB activation and provide a safe and effective way to boost anti-tumor T cell responses. This could allow more effective dosing and better combination therapies with checkpoint inhibitors and other immune stimulating drugs. MP0310 is in preparation to enter clinical development.

#3753

Indoximod modulates AhR-driven transcription of genes that control immune function.

Erik L. Brincks, James Adams, Michael Essmann, Benjamin A. Turner, Lifu Wang, Jiyuan Ke, Agnieszka Marcinowicz, Nicholas Vahanian, Charles J. Link, Mario R. Mautino. _NewLink Genetics, Ames, IA_.

The IDO pathway mediates immunosuppressive effects by metabolizing tryptophan (Trp) into kynurenine (Kyn). The depletion of Trp stimulates downstream signaling through nutrient sensors GCN2 and mTOR, while the production of Kyn stimulates signaling through the aryl-hydrocarbon receptor (AHR) transcription factor. The activation of these signaling pathways has pleiotropic effects on immune cells, including influencing the differentiation of dendritic cells (DCs), helper T cells, and regulatory T cells (Treg) as well as enhancing the proliferation of effector T cells and Treg. Indoximod has been demonstrated to relieve IDO-mediated immunosuppression in vitro and in vivo, by creating an artificial Trp-sufficiency signal that bypasses activation of GCN2 and inhibition of mTOR in conditions of Trp deprivation. We hypothesized that indoximod's activity could also include the disruption of AHR activation by Kyn and other Trp catabolites, causing differential AHR signaling and transcriptional activity, resulting in an augmented antitumor immune response. We observed that indoximod activates AHR-dependent transcriptional activity in HepG2 cells as evidenced by increased AHR-driven luciferase and endogenous CYP1A1 activity. AHR activation by indoximod was also observed in primary human T cells, as measured by the induction of CYP1A1 mRNA. In a Kyn-driven Treg differentiation assay, indoximod altered transcription of genes associated with T helper and Treg phenotypes. Indoximod induced upregulation of Rorc expression, a TH17-associated transcription factor, while concurrently downregulating transcription of Foxp3, the master transcription factor of Treg cells. These effects were reverted by an AHR inhibitor. Consistent with the gene expression profiles, indoximod shifted the cellular phenotype from Foxp3+ Treg toward Th17-producing CD4 helper T cells. Transcription of Ido1 is controlled by IFNγ and AHR-response elements in its promoter. Consistent with the role of indoximod in blocking the IDO pathway, we observed that indoximod downregulated the expression and function of IDO in in vitro derived plasmacytoid DC that normally would express IDO under the same differentiation conditions. When these in vitro differentiated human pDCs were used in an MLR culture, the indoximod-induced downregulation of IDO resulted in decreased Kyn production and increased T cell proliferation. Moreover, indoximod treatment of tumor-bearing mice decreased expression of IDO in the pDC in tumor-draining LN. Together, these data suggest that indoximod modulates AHR signaling to exert multiple immunomodulatory effects, including a shift from suppressive Foxp3+ Treg toward TH17 helper T cells as well as the downregulation of IDO expression in pDC, contributing to enhanced antitumor immunity.

#3754

The novel hexavalent human CD137/4-1BB agonist HERA-CD137L promotes anti-cancer immunity by activating CD8 T cells while regulatory T cells are not affected.

Meinolf Thiemann, Jaromir Sykora, David M. Richards, Christian Merz, Viola Marschall, Mauricio Redondo Mueller, Julian P. Sefrin, Karl Heinonen, Harald Fricke, Christian Gieffers, Oliver Hill. _Apogenix AG, Heidelberg, Germany_.

CD137/4-1BB is an inducible costimulatory receptor mainly expressed on immune cells following activation. Binding of the cognate ligand CD137L/4-1BBL leads to receptor trimerization and activation of signaling cascades involved in expansion and survival of T cells and myeloid cells as well as memory formation and protection against autoimmunity. Apogenix´ HERA technology is based on trivalent single-chain molecular mimics of the TNF-SF receptor binding domains fused to a silenced human IgG1-Fc-domain which serves exclusively as a dimerization scaffold. This results in hexavalent agonists replicating the natural receptor binding mode. Due to their inherent high receptor clustering capacity, HERA compounds are true agonists and their biological activity is, in contrast to bivalent anti-TNFR-SF antibodies, independent of FcR-mediated crosslinking events. HERA-CD137L was produced in CHO-S cells and purified resulting in highly pure non-aggregating protein lots. PK studies in mice demonstrated a terminal half-life of 75 hours indicating excellent in vivo stability. To study the effects on immune cells in vitro, T cells were isolated from healthy-donor buffy coats and stimulated with anti-CD3 antibody alone or in combination with HERA-CD137L. Using multicolor flow cytometry, we confirmed that expression of CD137 increased on CD8+ T cells following stimulation with anti-CD3 antibody. In accord with upregulation of the activation markers CD25 and CD69 and the memory marker CD45RO, treatment with HERA-CD137L enhanced proliferation of both CD4+ and CD8+ T cells, as determined by CFSE analysis. Intracellular accumulation of IFN-γ, TNF-α, Granzyme B and Perforin upon CD137 ligation was observed in CD8+ but not CD4+ T cells. HERA-CD137L treatment of THP-1 monocytes co-cultured with primary T cells also increased their cytotoxic activity against multiple tumor cell lines, including colorectal and mammary, as shown in a real-time live cell analysis (RTCA) assay. Primary human monocytes express low levels of CD137 and differentiation to macrophages in vitro did not increase expression. However, pro-inflammatory cytokines such as TNF-α and the chemokine CCL4 were secreted after stimulation with HERA-CD137L. RTCA assays further demonstrated enhanced antigen-specific killing of MDA-MB231 tumor cells by HERA-CD137L treated T cells. HERA-CD137L conveys its activity through effector T cell proliferation while regulatory T (Treg) cell proliferation or production of anti-inflammatory cytokines are not altered in Treg cell cultures. In contrast, HERA-CD137L prevented Treg-mediated suppression of effector T cells. Based on the in vitro data presented, HERA-CD137L is a promising candidate to promote anti-tumor immune responses either as single agent or in combination with other IO-compounds.

#3755

Comprehensive antitumor immune activation by a novel TLR7/8 targeting agent NKTR-262 combined with CD122-biased immunostimulatory cytokine NKTR-214.

Saul Kivimae, Marlene Hennessy, Rhoneil Pena, Yolanda Kirksey, Wildaliz Nieves, Phi Quatch, Janet Cetz, Zhongxu Ren, Haiying Cai, BoLiang Deng, Wen Zhang, John L. Langowski, Christie Fanton, Neel K. Anand, Werner Rubas, Steve Doberstein, Jonathan Zalevsky. _Nektar Therapeutics, San Francisco, CA_.

Background: NKTR-262 is a novel therapeutic, which delivers intratumoral engagement of the TLR 7/8 pathway, promoting an immune stimulatory environment and local injection site tumor antigen release. When NKTR-262 is administered in combination with NKTR-214, a CD122-biased cytokine agonist currently in clinical trials as a monotherapy and in combination with nivolumab, the combined effect of innate immune stimulation and enhanced antigen presentation with sustained T cell activation leads to systemic tumor immunity. Materials and methods: Ten subcutaneous syngeneic mouse tumor models with diverse histologies were assessed for NKTR-262 and NKTR-214 combination treatment efficacy. Once established, tumors were treated with a single peritumoral dose of NKTR-262, while NKTR-214 was administered i.v. on q9dx3 schedule. A subset of tumor models were inoculated bilaterally to assess abscopal effect of the combination treatment. Regression of injected tumors and the abscopal effect in contralateral untreated tumors was assessed by tumor size measurements. In bilateral models immune cell activation in both tumors was assessed by flow cytometry. Cytokine induction was measured in plasma and tumors in select models. Results: Combination treatment with NKTR-262 and NKTR-214 showed efficacy in all tested tumor models. Efficacy varied from significant tumor growth inhibition to up to 100% complete responses in multiple models. Synergistic efficacy was demonstrated in select models where single agent NKTR-262 or NKTR-214 activity was compared to the combination treatment. Immune cell phenotyping showed that combining NKTR-262 with NKTR-214 induced a two-step immune response in treated and abscopal tumors. At early timepoints, accumulation of activated granulocytes correlated with tumor cell death and dendritic cell activation. The innate response was followed by selective tumor infiltration by CD8 T cells and a reduction of immunorepressive cells. Single agent treatment showed only a subset of the cellular changes observed in the combination. Immune cell activation was shown to correlate with immune stimulatory cytokine release in NKTR-262 treated tumors. Conclusions: We present a designed combination therapy that mimics a natural immune response by activating a broad immune cell network in multiple nonclinical tumor models independent of tissue origin. Combining NKTR-262 and NKTR-214 engages the entire immune activation cascade required for systemic tumor clearance from local tumor antigen production to a sustained systemic T cell response. Unlike treatments that stimulate downstream components of select immune pathways without eliciting systemic tumor immunity, a comprehensive anti-tumor immune activation by coordinated engagement of innate and adaptive immune cells may increase the success of immune therapy for patients.

#3756

Small synthetic, multivalent bicyclic peptides that activate T cell costimulatory protein CD137.

Peter U. Park,1 Rachid Lani,2 Gemma Mudd,2 Julia Kristensson,2 Katerine Van Rietschoten,2 W. Frank An,1 Gavin Bennett,2 Kevin McDonnell,1 Nicholas Keen1. 1 _Bicycle Therapeutics, Lexington, MA;_ 2 _Bicycle Therapeutics, Cambridge, United Kingdom_.

CD137 (4-1BB/TNFRSF9) belongs to the TNF receptor superfamily and provides costimulatory signaling for T cells and NK cells. Agonistic anti-CD137 antibodies have shown potent, often curative anti-tumor activity in preclinical models. These effects are mainly mediated by cytotoxic T cells and generate long lasting, memory responses. Two human anti-CD137 antibodies urelumab and utomilumab are currently undergoing clinical testing. Urelumab has shown several single-agent, partial responses, but its use has been hampered by hepatoxicity, whilst utomilumab lacks hepatotoxicity, but it has shown little or no single agent activity.

Bicycles® are a new class of drugs - fully synthetic, constrained bicyclic peptides that have antibody-like, high affinity and exquisite target specificity unachievable with conventional small molecule approaches. The Bicycle platform uses phage display and chemical optimization to rapidly identify and mature binders for affinity and physicochemical properties. Their small size (1.5-2 kDa) delivers advantages in tumor penetration, and rapid renal elimination may avoid the liver and GI toxicity often associated with other drug modalities, including certain antibodies. We hypothesized that a fully synthetic Bicycle CD137 agonist with rapid renal clearance, minimal liver interaction and no Fc receptor interaction may induce CD137 mediated anti-tumor activity while avoiding liver toxicity.

1015 Bicycles were screened on phage against recombinant CD137. After phage and chemical optimization, a high affinity lead BCY3814 (KD ~30 nM) was selected. BCY3814 competes for binding with the CD137 ligand and utomilumab (known to bind to CD137 ligand binding site) but does not compete with urelumab which binds an alternative epitope. In common with many TNF receptors, CD137 activation requires receptor crosslinking, thus multivalent binders would be expected to recapitulate the action of its natural trimeric ligand. Uniquely, the versatility of the Bicycle format allowed us to rapidly generate more than 50 different bi-, tri- and tetra-valent variants of BCY3814 with chemical linkers and hinges of various lengths and rigidity using different sites of attachments, while maintaining a compact size (<15 kDa). We developed molecules exhibiting a wide range of potency and efficacy in cell-based CD137 activation assays. Several of these synthetic Bicycle CD137 agonists were 10-fold more potent than the clinical antibodies or the natural ligand. These potent Bicycles are being tested in a humanized CD137 mouse model to demonstrate anti-tumour activity with reduced liver toxicity. We hypothesise that such molecules will be promising, novel cancer immunotherapy candidates and pave the way for development of synthetic agonists of other TNF receptors.

#3757

Targeting the IDO/TDO pathway through degradation of the immunosuppressive metabolite kynurenine.

Silvia Coma,1 Jill Cavanaugh,1 James Nolan,1 Jeremy Tchaicha,1 Karen McGovern,1 Everett Stone,2 Candice Lamb,2 Christos Karamitros,2 John Blazek,2 Kendra Garrison,2 George Georgiou,2 Mark Manfredi,1 Xiaoyan Michelle Zhang1. 1 _Kyn Therapeutics, Cambridge, MA;_ 2 _University of Texas Austin, Austin, TX_.

The tryptophan/kynurenine pathway has been clinically validated in several tumor types with small-molecule IDO1 inhibitors in combination with checkpoint inhibition. Indoleamine-pyrrole 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase 2 (TDO2) are upregulated in a number of tumor types, metabolizing tryptophan to form immunosuppressive kynurenine. We are developing Kynureninase (Kynase), a kynurenine depleting enzyme, to treat IDO1 and TDO2 positive tumors. The human Kynase has been successfully engineered to vastly increase catalytic activity and stability toward kynurenine over the wild type enzyme. In mice, Kynase achieved prolonged Kynurenine degradation (≥5 days) in plasma and tumor draining lymph node (TDLN), leading to anti-tumor activity as a single agent and in combination with check point inhibitors in mouse syngeneic tumor models. Kynase demonstrated superior tumor growth inhibition and survival benefit relative to a leading IDO1 inhibitor epacadostat in these models. The effects of Kynase on a number of immune cell types, both in vitro and in vivo, are being investigated. Human Kynase has also shown a favorable PK profile and kynurenine degradation in non-human primates, and Kynase variants are now moving toward development candidate selection for treatment of cancers where both IDO/TDO pathways play a significant immunosuppressive role.

#3758

TLR9 agonist SNA-induced innate and adaptive immune responses in tumor microenvironment enhance checkpoint inhibitor antitumor activity in mouse tumor models.

SubbaRao Nallagatla, Bart R. Anderson, Richard Kang, Ekambar R. Kandimalla. _Exicure, Inc., Skokie, IL_.

Spherical Nucleic Acids (SNAs) are a novel class of therapeutic agents with oligonucleotides densely packed and radially oriented in 3D format around liposomal nanoparticles. As a result of the 3D-architecture, SNAs exhibit increased cellular uptake and nuclease stability compared with linear oligonucleotides.

Checkpoint inhibitors (CPI) have shown antitumor efficacy in a variety of solid and liquid tumors. However, tumors with low immunogenicity and lacking pre-existing T lymphocyte infiltrates (TILs) in the tumor microenvironment (TME) are less responsive to CPI. TLR9 agonists induce potent innate and adaptive immune responses and have been shown to increase TIL and activate APC to secrete IFNs leading to activation and expansion of tumor-specific CTLs in TME increasing effectiveness of CPIs.

In the present study, we have evaluated antitumor effects of SNA following intratumoral delivery in A20 lymphoma and CT26 colon cancer models. SNA was also studied in combination with anti-PD-1 or anti-PD-L1 in A20 and with IDO-1 inhibitor in CT26 models. We have also evaluated biomarkers in TME to elucidate the mechanism of SNA-mediated effects in enhancing anti-PD-1 efficacy.

Monotherapy of SNA or oral dosing of IDO-1 inhibitor in CT26 tumor model resulted in a tumor growth inhibition (TGI) of up to 80% or 20%, respectively, compared with vehicle. The combination of SNA and IDO-1 inhibitor enhanced TGI up to 90% compared with either monotherapy. Mice bearing A20 tumors were treated with SNA, intraperitoneal dosing of anti-PD-1 or anti-PD-L1 or the combination of SNA/anti-PD-1 or SNA/anti-PD-L1. SNA alone showed potent dose-dependent TGI. Anti-PD-1 or anti-PD-L1 monotherapy resulted in modest TGI compared with vehicle. The combination of SNA with anti-PD-1 or anti-PD-L1 enhanced TGI compared with either monotherapy. FACS analysis of A20 tumor tissue following treatments revealed reduced T-regulatory cells and increased T-effector cells. Moreover, SNA treatment led to increased expression of IFN-inducible genes, ISG15, IRF7, MX1, and IP10 and a CD8 T cell marker CD8A in TME and maintained these gene expression levels when combined with anti-PD-1. These results establish the mode of action of TLR9 agonist SNA in TME in potentiating antitumor effects of anti-PD-1.

These data demonstrate that the TLR9 agonist SNA showed potent antitumor activity as a result of increased IFN gene expression coupled with increased CD8+ T cells and decreased T-regulatory cells locally in the tumor and thereby enhancing efficacy of checkpoint inhibitors. A TLR9 agonist SNA, AST-008, is currently in clinical development.

#3759

Characterization of INCB081776, a potent and selective dual AXL/MER kinase inhibitor.

Margaret Favata, Kerri Lasky, Yvonne Lo, Patricia Feldman, Jun Li, Yaoyu Chen, Christina Stevens, Min Ye, Hui Wang, Ke Liu, Richard Wynn, Yanlong Li, Jennifer Harris, Robert Landman, Yu Li, Xiaozhao Wang, Chunhong He, Yun-Long Li, Chu-Biao Xue, Wenqing Yao, Jonathan Rios-Doria, Zhenhai Gao, Maryanne Covington, Xuesong M. Liu, Holly Koblish, Peggy Scherle. _Incyte, Wilmington, DE_.

Tyro-3, Axl, and Mer constitute the TAM family of receptor tyrosine kinases (RTKs), which are amplified, translocated, or over-expressed in numerous types of human cancer. These RTKs play important roles in tumor growth, survival, cell adhesion and migration as well as drug resistance. In addition, it has been shown that both AXL and MER are critical regulators of innate immunity, phagocytosis, and immune-suppressive activity. Therefore targeting both AXL and MER kinases may not only impact the growth, survival and malignant progression of neoplastic cells directly, but also has the potential to restore and enhance host immunity against cancers. INCB081776 is a potent inhibitor of AXL and MER that exhibits selective pharmacological activity and enhanced anti-tumor immune activity. In biochemical assays, INCB081776 potently inhibited the kinase activity of recombinant AXL/MER enzymes and was highly selective against a panel of 192 kinases (IC50 = 0.61±0.31 nM and 3.17±1.97 nM against AXL and MER, respectively). INCB081776 is greater than 30 fold selective against TYRO3. Selectivity against TYRO3 is important as retinal toxicity associated with loss of the Mer gene appears to be modulated by TYRO3 in mice. In cellular assays, INCB081776 effectively blocked auto-phosphorylation of AXL or MER including BAF3 cells transfected with constitutively active AXL or MER, AXL in H1299 tumor cells, or MER kinase in G361 tumor cells, with low nanomolar IC50 values. In addition, INCB081776 inhibited activation of MER kinase in primary human macrophages with low nanomolar IC50 potency. More importantly, in an in vitro functional assay, INCB081776 partially reversed M2 macrophage-mediated suppression of T cell proliferation, and increased IFN-γ in co-cultured macrophages and T cells. In vivo, INCB081776 administration to H1299 tumor-bearing mice dose-dependently inhibited the phosphorylation in tumors. Consistent with the proposed mechanism of action, INCB081776 potently inhibited tumor growth in immunocompetent mice, but not in immunodeficient mice, demonstrating that a functional immune system is important for activity. Treatment was associated with dose-related increases in the percent of tumor-infiltrating effector CD4+ and CD8+ T cells, as well as macrophages with the M1 phenotype. In addition, INCB081776 decreased the percentage of intratumoral M2 macrophages and monocytic myeloid-derived suppressor cell (M-MDSC) immune cell populations. In the 4T1 model, combining INCB081776 with anti-PD-L1 resulted in synergistic anti-tumor effects compared to either single agent. Collectively, these preclinical data support the hypothesis and potential therapeutic utility of INCB081776 as an immunotherapeutic agent capable of enhancing tumor immune surveillance mechanisms in cancer patients as a single agent and when combined with therapies mediating immune PD-L1 checkpoint blockade.

#3760

Preclinical antitumor activity of a CC chemokine receptor (CCR) 2/5 dual antagonist as monotherapy and in combination with immune checkpoint blockade.

Qihong Zhao, Anwar Murtaza, Adam Bata, Wendy Sun, Ching-Ping Ho, Ragini Vuppugalla, Robert Cherney, Kevin Stefanski, Z Alexander Cao, Ashwin Sama, Arvin Yang, Mary Struthers, Miguel Sanjuan, John T. Hunt, Percy Carter, Luisa Salter-Cid. _Bristol-Myers Squibb, Princeton, NJ_.

Background: Different immune cell subsets are recruited to the tumor microenvironment (TME) via interactions between chemokines and chemokine receptors (CKRs). The recruitment of immune suppressive cells of both myeloid and lymphoid origin contributes to tumor growth and metastasis. CCR2 and CCR5 are 2 CKRs that are expressed on myeloid and T-cell infiltrates in the TME. CCR2 mediates the migration of monocyte-derived suppressor cells (M-MDSCs) from bone marrow to blood and to the TME. CCR5 directs migration of polymorphonuclear MDSCs (PMN-MDSCs) and regulatory T cells to the TME. In addition, CCR5 potentiates PMN-MDSC immune-suppressive function via arginase-1 and promotes polarization of tumor-associated macrophages into an immune-suppressive phenotype. Each receptor has been shown to play an important role in multiple tumor models. CCR2- and CCR5-selective antagonists, in combination with chemotherapy, have shown positive proof of mechanism in patients with pancreatic and colorectal cancers, respectively. Thus, targeting both receptors with a small-molecule dual antagonist represents a potential novel treatment for cancer. Here we evaluate the antitumor activities of BMS-687681, a small-molecule CCR2/5 dual antagonist, as monotherapy and in combination with immune cell checkpoint blockers (ICBs) in mouse tumor models.

Methods: Three syngeneic mouse tumor models (MC38, CT26, and 4T1) were used to determine the antitumor activities of BMS-687681 ± ICBs (anti-PD-1 and/or anti-CTLA-4). BMS-687681 activity was assessed by comparing its effect on tumor growth inhibition (TGI) and on the number of tumor-free (TF) mice to vehicle control following oral dosing alone or in combination with ICBs. Phenotyping of peripheral and tumor-associated immune cell subsets was also conducted to gain mechanistic insight.

Results: Modest to significant TGI was observed with BMS-687681 alone. Combination of BMS-687681 with ICBs demonstrated significantly improved antitumor activity relative to either monotherapy, as measured by both TGI and the number of TF mice. TF mice were monitored for > 50 days following the last drug treatment, and the majority of mice showed no tumor recurrences. These mice were then rechallenged with tumor cells, and tumor growth was monitored for > 40 days. Although tumor growth initially increased following rechallenge, these tumors regressed after a week, and mice remained TF thereafter. The mechanistic basis for the antitumor activity of BMS-687681 ± ICBs will be presented.

Conclusions: BMS-687681, a potent, selective, and orally active CCR2/5 dual antagonist, demonstrated improved antitumor activity in combination with ICBs relative to monotherapy treatment. These findings support the potential utility of CCR2/5 dual antagonism in combination with ICBs as a novel immunotherapy for cancer.

#3761

Histone deacetylase inhibitor, Entinostat enhances the tumor specific immune response by generating T-cell central memory in the TME.

Pankaj Gaur,1 Vivek Verma,1 Rahul Nandre,1 Pooja Vir,1 Hua Wang,1 Baolin Kang,1 Peter Ordentlich,2 Lei Wang,2 Seema Gupta,1 Samir N. Khleif1. 1 _Georgia Cancer Center, Augusta University, Augusta, GA;_ 2 _Syndax Pharmaceuticals, Inc., Waltham, MA_.

The epigenetic deregulation of T-cells and enhanced numbers of immunosuppressive cells in the tumor microenvironment (TME) are associated with decreased anti-tumor effects. Hence, targeting the epigenetic modifications using modulators such as histone deacetylase inhibitors (HDACi) provides the basis for a potential role for these agents in cancer-immunotherapy. In this context, Entinostat, an oral HDACi has been shown to reprogram the TME by impacting the numbers and activity of the immunosuppressive cell populations resulting in enhanced anti-tumor activity when combined with immune checkpoint blockade. However, the effect of this inhibitor on anti-tumor activity of effector T-cells is not well understood. Here, using animal tumor-models that require antigen-vaccine for T cell priming, we showed that Entinostat significantly synergized with tumor antigen-specific vaccine in delaying the tumor growth and prolonging mice survival. Further, we found that administering Entinostat in combination with antigen-priming resulted in sustainable long-term anti-tumor effects. Immunologically, Entinostat significantly enhanced the total CD8+, antigen-specific CD8+, granzyme B+ (GB+) CD8+, and antigen-specific GB+CD8+ T-cells in the TME when combined with the vaccine. Interestingly, we found that Entinostat led to a significant increase in the number of central memory T-cells (TCM; CD62L+CD44+) and decrease in the effector memory cells (TEM; CD62L-CD44+) in the TME. Notably, we did not find significant immune modulation in the periphery following the combination treatment indicating that the immune modulatory effects of Entinostat were localized to TME and hence may be associated with less systemic toxicity. Taken together these results highlight the ability of Entinostat to significantly enhance anti-tumor responses partly through the generation of central-memory which may translate to durable responses. These data provide further support on the potential of utilizing Entinostat in combinatorial immunotherapeutic strategies.

#3762

**Novel RORγt agonist induces antitumor immune effect through enhancement of tumor antigen-specific CD8** + **T-cell infiltration into the TME.**

Pankaj Gaur,1 Vivek Verma,1 Rahul Nandre,1 Pooja Vir,1 Hua Wang,1 Baolin Kang,1 Laura Carter,2 Xiao Hu,2 Xikui Liu,2 Seema Gupta,1 Samir Khleif1. 1 _Georgia Cancer Center, Augusta University, Augusta, GA;_ 2 _Lycera Corp., Ann Arbor, MI_.

RORγt is a master transcription factor, which regulates the proliferation and functionality of Type 17 T-cells (TH17 and Tc17). Recent nonclinical studies have shown that in addition to promoting effector differentiation, stemness and plasticity, RORγt also inhibits Treg differentiation, an important component of suppressive tumor microenvironment (TME). Therefore, stimulation of RORγt by synthetic, small-molecule agonists holds promise as an immunotherapy. We tested the effects of a RORγ agonist (LYC-54143; 100 mg/kg BID given continuously till the end of study) on the tumor growth and survival in mice in combination with a tumor antigen-specific vaccine (3 doses, one week apart), which is required for proper priming in this model. We found that the RORγ agonist resulted in a highly significant enhancement of the antitumor effect delaying the tumor growth (p≤0.001 at day 21 compared to vaccine alone treatment) and prolonging survival. At day 36 after tumor implantation, 80% of mice survived in the RORγ agonist + vaccine treatment group compared to 0% survival following RORγ agonist or vaccine alone treatments. Immunologically, we found that RORγ agonist treatment led to a significant decrease in the numbers of Tregs and a significant increase in IL-17+ IFNγ+ T cells in the TME. Moreover, RORγ agonist treatment led to a significant increase in the numbers of T cells including increased antigen-specific CD8+ T-cells in the TME. Interestingly, scheduling RORγ agonist before or with the vaccine demonstrated comparable antitumor activity, suggesting that the scheduling of RORγ agonist treatment does not affect the therapeutic outcomes. These results show that an RORγ agonist can enhance the antitumor immune response by enhancing effector functions and by decreasing immune suppression in the TME by increasing cytokine production and switching the differentiation of immune-suppressive Tregs to effector Th17 cells and support that RORγ agonists are promising immune-modulatory agents in cancer.

#3763

IDO vaccine enhances antigen-specific anti-tumor effects by reducing IDO-expressing antigen presenting cells and MDSCs.

Rahul Nandre,1 Vivek Verma,1 Pankaj Gaur,1 Pooja Vir,1 Hua Wang,1 Baolin Kang,1 Ayako Wakatsuki Pedersen,2 Mai-Britt Zocca,2 Mads Hald Andersen,2 Seema Gupta,1 Samir Khleif1. 1 _Georgia Cancer Center, Augusta University, Augusta, GA;_ 2 _IO Biotech ApS, Denmark_.

Tumors evade immune surveillance using several mechanisms such as induction of regulatory T-cells (Tregs) and upregulation of indolemine-2,3-dioxygenase (IDO) enzyme. IDO is an intracellular enzyme that mediates its immunosuppressive effects by depleting tryptophan leading to accumulation of kynurenine and in turn reducing the proliferation of effector cells while activating Tregs. IDO is expressed by several suppressive immune cells including Tregs, suppressive antigen presenting cells (APCs), as well as by tumor cells. These immunosuppressive effects of IDO make it an attractive target for cancer immunotherapy, including a target for vaccine therapy. To test the anti-tumor immune effect of IDO vaccine, we evaluated the anti-tumor effect of an IDO-derived MHC I restricted peptide (IDO-pep) vaccine in the low IDO-expressing (TC-1) and high IDO-expressing B16 (melanoma) mouse tumor models. For this, mice were treated with three doses of IDO-peptides in combination with the respective tumor-specific vaccines (E7 for TC-1 and gp100 for B16) at an interval of seven days, followed by assessment of tumor growth and mice survival. We observed a significant delay in the tumor growth and prolonged survival in both tumor models when IDO vaccine was combined with tumor-specific antigen vaccine. We found that tumor-infiltration of antigen-specific CD8+ T-cells was significantly enhanced following the addition of IDO vaccine. Furthermore, while CD4+ T-cells increased in the tumor microenvironment (TME), there was a significant reduction in the numbers of Treg cells. We also found a significant reduction in the numbers of IDO-expressing myeloid-derived suppressor cells (MDSCs), dendritic cells as well as macrophages out of the total CD45+CD11b+ myeloid cells in the TME after the administration of IDO-pep in combination with tumor antigen-specific vaccine. In conclusion, our findings suggest that IDO-pep vaccine enhances the specific anti-tumor immune response by increasing the tumor antigen-specific effector T-cell infiltration in the TME through inhibition of immune suppressive cells. These results also suggest that MHC I restricted IDO peptide treatment is a promising strategy to improve cancer immunotherapy.

#3764

Porcupine inhibitor RXC004 enhances immune response in pre-clinical models of cancer.

Inder Bhamra, Richard Armer, Matilda Bingham, Catherine Eagle, Alicia Edmenson Cook, Caroline Phillips, Simon Woodcock. _Redx Pharma Ltd, Alderley Edge, United Kingdom_.

Background: RXC004, a potent and selective porcupine (PORCN) inhibitor, is being investigated in a safety and tolerability study in cancer patients with solid tumours (CT 2017-000720-98). In addition to the tumour targeting role of RXC004 and other Wnt pathway inhibitors, we present pre-clinical data which suggests further potential for RXC004 in modulating the immune system of the tumour microenvironment. Materials and Methods: To evaluate the potential of a novel porcupine inhibitor, RXC004, as an immunomodulatory anti-cancer agent, sub cutaneous B16F10 melanoma (C57BL/6 mice) and CT26 colorectal (BALB/c mice) murine tumour models were utilised. Mice in both models were treated with RXC004 alone or in combination with mouse anti-PD-1 antibody. Flow cytometry analysis was utilised to measure key immune cell populations in the tumour microenvironment. To probe the underlying mechanism of immune modulation in these models and to provide a link to the emerging clinical data suggesting a role for Wnt pathway activation in immune escape, human monocytic cells were isolated from PBMCs and human dendritic cells were derived in vitro. The Wnt pathway was induced in derived Dendritic cells and expression of IDO was measured. Results: In the murine CT26 model, RXC004 treatment reduced tumour size when dosed in combination with anti-PD-1 antibody, causing regression and cures in some animals. Furthermore, flow cytometry showed RXC004 in combination with anti-PD-1 antibody increased the proportion of CD8+ cytotoxic T cells as well as decreasing FoxP3+ regulatory T cells when compared to the monotherapy anti-PD-1 arm. In a syngeneic murine melanoma B16F10 model, RXC004 monotherapy at a dose of 5mg/kg QD orally significantly inhibited tumour growth, as did RXC004 combined with anti-PD-1. RXC004 had no effect on the proliferation of B16F10 cells in vitro, suggesting this was not caused by the compound directly affecting B16 cell proliferation. Flow cytometry analysis of the B16 tumours showed significant immune modulatory effects in the tumour microenvironment. In addition to mouse model data, Wnt pathway activation in human dendritic cells was shown to increase IDO expression. Conclusion: Taken together, data from murine syngeneic mouse models corroborate literature data suggesting that inhibiting the Wnt pathway may promote the immune response against human cancers.

#3765

An anti-CD137 antibody can show greater efficacy in syngeneic mouse tumor models when combined with other immuno-oncology (IO) therapies.

Matthew J. Robinson,1 Ines Osma-Garcia,1 Jane Coates-Ulrichisen,1 Amanda Watkins,1 Suzanne Mosely,1 Chrisopher Lloyd,1 Geoff Williams,1 Michelle Morrow,1 Simon Dovedi,1 Ronald Herbst,2 Robert Wilkinson1. 1 _MedImmune, Cambridge, United Kingdom;_ 2 _MedImmune, Gaithersburg, MD_.

CD137 is a costimulatory molecule associated with tumour infiltrating lymphocytes and cytotoxic T lymphocytes in particular. Murine anti-CD137 monoclonal antibodies (mAbs) have shown potent anti-tumour effects in syngeneic tumour models, which can be further enhanced by combination with checkpoint inhibitor (CPI) mAbs to PD-1, or CTLA-4 and OX40. Two anti-CD137 monoclonal antibodies (mAbs), Urelumab and Utomilumab, are in development for both haematological and solid cancers, and studies include combinations with other IO therapies such as anti-PD-1, anti-PD-L1 and anti-OX40 mabs. We studied the impact of combining anti-CD137 with CPIs anti-PD-L1, anti-CTLA4, and IO agonist GITRL fusion protein (GITRL-FP) and anti-OX40 on pharmacodynamic responses, tumour growth and survival in mouse syngeneic models. In a responsive model, CT26, combining anti-CD137 with anti-PD-L1 had a greater impact on tumour growth inhibition, which correlated with an enhanced proliferation of CD8 T cells, as determined Ki67 expression using flow cytometry. In MC-38, a model that is less sensitive to both monotherapies, the combination was synergistic and resulted in enhanced tumour growth inhibition. Next, we compared the anti-CTLA4, anti-OX40 and GITRL-FP as monotherapies and in combination with anti-CD137 in the CT26 model. As before, anti-CD137 alone induced CD8 T cell proliferation, whereas anti-OX40 and GITRL-FP stimulated expansion of CD8 and CD4 T cells. Interestingly, in all combinations anti-CD137 appeared to enhance both CD8 and CD4 T cell proliferation. Anti-CTLA4 and GITR-FP had a greater impact on tumour growth inhibition when combined with anti-CD137. In summary, in vivo models indicate that anti-CD137 mAbs are a promising therapy in cancer, but the most benefit may be obtained in combination with other IO therapies.

#3766

Radiation enhances anti-tumor activity of immune checkpoint blockade in syngeneic tumor models.

Sumithra Urs, Mary Anne Meade, Kevin Guley, Sarah Krueger, Alden Wong, Scott Wise, Maryland Franklin. _MI Bioresearch, Ann Arbor, MI_.

More than 50% of cancer patients receive some sort of radiation therapy during the course of their illness. While radiation treatment is a mainstay in clinical oncology, there is limited preclinical data in syngeneic models. The advent of image-guided small animal irradiators such as the Small Animal Radiation Research Platform (SARRP; Xstrahl) allow for use of targeted focal irradiation (RT) in a broad range of models. Here we evaluated the use of RT to broaden efficacy and response duration of immunomodulatory therapies.

As a first step, subcutaneous syngeneic tumor models were tested with single doses of RT. The A20 B cell lymphoma, the CT26 colon carcinoma, and the Pan02 pancreatic carcinoma all demonstrated dose response anti-tumor activity with increasing dose (5, 10 or 20Gy) of RT. In the A20 model, doses of 10 and 20Gy RT resulted in 2 tumor free survivors (TFS) each. CT26 had 1, 2 and 2 TFS each in RT groups of 5, 10 and 20Gy, respectively. In these 2 models, tumor free mice were rechallenged with appropriate tumor cell implants on the contralateral flank and, in all cases, tumors failed to develop suggesting a memory immune response was elicited. In Pan02 we found 1 and 5 TFS in the 10 and 20Gy groups, respectively.

In follow-on work, we used the CT26 model to compare 5Gy RT, bi-weekly dosing of anti-PD-1 (RMP1-14; Bio X Cell) or the combination. Treatments were initiated at approximately 100mm3 tumor volumes. Anti-PD-1 resulted in a slight tumor growth delay (TGD) of 5 days compared to isotype control, whereas RT resulted in a 19 day TGD and more than doubled the time to evaluation size (TES) from 16 days in the control to 35 days. In combination, the TGD was further improved to 40 days and TES to 56 days.

An additional colon model, MC38, was tested with 10Gy RT alone or in combination with anti-PD-1, or anti-PD-L1. Either antibody resulted in modest single agent activity with anti-PD-1 being slightly more efficacious than anti-PD-L1 with 15 days TGD compared to 7 days, respectively. Mice in the RT group demonstrated 20 days TGD. RT in combination with anti-PD-1 resulted in 37 days TGD, however, in combination with anti-PD-L1 TGD was not improved over RT alone. Effects of radiation and anti-PD-1 therapy on tumor infiltrating lymphocytes is being investigated in the MC38 model, and data will be shown.

GL261-luc, an orthotopic model of murine GBM, was also tested. We found that anti-PD-1 improved anti-tumor activity from control, a single dose of 7.5Gy RT further improved activity against control, and the combination was 100% curative. Evaluation of tumor infiltrating lymphocytes showed increased CD8+ T cells along with increased regulatory T cells (Tregs) as the model progressed. RT reduced the number of Tregs and a further reduction was observed following combination therapy. In summary, the

data presented here provides rationale for further evaluation of immunomodulatory agents in combination with RT.

#3767

Imprime PGG, a soluble yeast b-glucan PAMP, enhancement of anti-tumor responses in combination with tumor targeting antibody is highly dependent on NK cell killing.

Kathryn A. Fraser, Takashi Kangas, Ross B. Fulton, Steven M. Leonardo, Ben Harrison, Yumi Yokoyama, Nandita Bose, Jeremy R. Graff, Mark Uhlik, Keith B. Gorden. _Biothera, Inc., Eagan, MN_.

Cancer therapy has been reshaped by checkpoint inhibitors (CPIs), making it possible for durable responses against cancers with traditionally low cure rates. Current efforts are focused on combination therapies in the hopes of evading resistance to CPIs and improving overall response. One escape mechanism attributed to acquired resistance to CPIs includes defective antigen presentation, namely a loss in MHC class I expression. This leads to loss of CD8 T cell-mediated tumor killing and disease relapse. This recent revelation has stimulated a need for therapies that activate other cytotoxic effector cells such as NK cells to kill tumors. Imprime PGG (Imprime) is a soluble, systemically delivered yeast 1,3/1,6 β-glucan PAMP (pathogen-associated molecular pattern) capable of triggering innate immune cell function leading to a cascade of immune activation and enhanced tumor killing. Imprime activates the innate immune system via dectin-1, eliciting production of a variety of chemokines and cytokines, including type I IFN, leading to the mobilization and stimulation of innate cell types including dendritic cells and monocytes. Unlike other PAMPs which systemic administration often leads to toxic side effects, Imprime has been administered safely by intravenous infusion to >400 human subjects. Currently, Imprime PGG is being evaluated in combination with αPD1 therapy in multiple clinical trials.

Previously we have shown that combination therapy of anti-Trp1 antibody and Imprime leads to a significant reduction in both number and size of lung metastases in the B16F10 metastatic melanoma tumor model over anti-Trp1 antibody alone. This reduction of metastases is highly dependent on NK cells but not CD8 T cells. To explore the impact of Imprime on NK-mediated cytotoxicity, we further evaluated in vivo killing of MHC class I deficient TapKO cells after intravenous administration of Imprime. In these experiments Imprime was able to enhance the NK cytotoxic killing of the targets. All NK cell killing observed was dependent on type I IFN, phagocytic cells and dectin 1. Imprime treatment increased cytokines that drive enhanced NK activation and effector phenotype. Significant increases were observed in the cytokines IL15/IL15rα, IL18, IL12p70 in lymph node lysates as well as increases in the effector molecules CD69, GrB, and CD107a on splenic NK cells. The upregulation of all of these molecules during Imprime treatment was dependent on dectin 1. Additionally, IL15/IL15rα production was also dependent on type I IFN, and phagocytic cells. Interestingly, Ly6c hi monocytes, which are increased after Imprime treatment, also show enhancement of IL15rα expression.

Collectively, these data demonstrate that Imprime contributes to enhanced NK functionality and killing which may provide a unique immunotherapeutic approach to complement existing therapies.

#3768

**Identification of a novel non-brain penetrant A** 2A **R inhibitor and proof-of-concept of CD73 and A** 2A **R/CD73 small-molecule inhibitors for cancer immunotherapy.**

Pierre Fons,1 Andy Bell,2 Michael Esquerre,1 Stephanie Versluys,1 Florie Bertrand,1 Celine Poussereau-Pomie,1 Adrian Schreyer,2 Richard Cox,2 Jeremy Besnard,2 Michael Paillasse,1 Mark Swindells,2 Joanna Lisztwan,1 Craig Johnstone,1 Mark Whittaker,1 Andrew Hopkins2. 1 _Evotec, Toulouse, France;_ 2 _Exscientia, Dundee, United Kingdom_.

Adenosine, generated by CD73 is a key driver of immunosuppression in the hypoxic tumour microenvironment (TME). In immune-inflamed tumours, with CD8+ T-cell infiltrates, adenosine signalling is a cause of resistance to immune checkpoint therapies (ICTs) through the inhibition of T-cells, NK cells and more largely of the overall antitumor immunity. Evotec and Exscientia are collaborating to develop a drug discovery platform for accelerating small molecule development in Immuno-Oncology targeting this adenosine pathway. The platform has integrated a unique biophysical screening approach to the adenosine receptors and CD73 to drive automated medicinal chemistry design with a translational-focused screening cascade. We have firstly sought to generate a novel non-brain penetrant A2AR antagonist. We have selected EVOEX21546 as our lead candidate and demonstrated its in vitro potency on primary human CD3+ T lymphocytes for inducing the recovery of IL-2 production after CADO-mediated inhibition of T-cell activation. In addition, we have demonstrated the compounds effect on other key biological features of T-cell activation including IFN-γ production and T-cell proliferation. ADME/DMPK data show that EVOEX21546 has a favourable pharmacological profile consistent with its evaluation in in vivo models resistant to ICTs before its entry into preclinical development. In parallel of this first drug discovery program, we have initiated research of CD73 specific inhibitory molecules and two series binding at orthogonal sites on CD73 have been identified. In vitro functional potency has been demonstrated for the CD73 inhibitors for inducing recovery of AMP-induced inhibition of T-cells activation as measured through IL-2 production. The series are under optimization to improve activity, solubility and ADME/DMPK parameters. Finally, from the perspective of developing dual pharmacological profile for A2AR and CD73 inhibition, we have assessed in a primary human CD3+ T-lymphocyte assay the synergy between the two targets in order to optimize the recovery of T-cell activation. Fragment screening has been performed for this approach to further aid design new molecules with a dual pharmacological profile. These results, have paved the way to an optimized process for identifying, improving and accelerating drug discovery in Immuno-Oncology in the frame of the adenosinergic immunosuppressive pathway.

#3769

**Pharmacokinetic-pharmacodynamic relationship for AB928, a dual antagonist of the A** 2a **R and A** 2b **R adenosine receptors.**

Lisa Seitz, Devika Ashok, Manmohan R. Leleti, Jay P. Powers, Brandon Rosen, Dillon Miles, Lixia Jin, Adam Park, Tim Park, Steve Young, Ferdie Soriano, Aimee Rieger, Ulrike Schindler, Joyson Karakunnel, Matt J. Walters. _Arcus Biosciences, Hayward, CA_.

INTRODUCTION: CD73 converts extracellular adenosine monophosphate (AMP), derived from adenosine triphosphate, into adenosine (ADO). ADO suppresses immune responses, including those of T cells, natural killer (NK) cells and dendritic cells, via A2aR and A2bR receptors. Activation of these receptors results in increased intracellular levels of cyclic AMP (cAMP) and phosphorylation of the cAMP response element-binding protein (CREB). Monitoring the degree of pCREB induced by activation of the Aa2R and A2bR receptors in peripheral blood allows for a specific measurement of target engagement by AB928. In mice, the pCREB assay, coupled with changes in immune cell infiltrate and tumor growth rates, allows for a fuller understanding of the drug's PK/PD relationship.

METHODS: Mouse and human whole blood was stimulated with the adenosine agonist NECA (5'-N-Ethylcarboxamidoadenosine) following in vitro spike in or in vivo dosing with AB928. Multi-color phospho-flow cytometry was used to assess levels of pCREB on immune cells. AB928 levels in plasma samples were determined using LC-MS-MS after protein precipitation.

RESULTS: A2aR mRNA was the most prevalent adenosine receptor in CD4+ and CD8+ T cells. NK cells also express primarily A2aR, although A2bR was also detected. Dendritic cells and CD14+ monocytes expressed both A2aR and A2bR. NECA stimulation of mouse whole blood resulted in a significant increase in the levels of pCREB within CD8+ T cells with an EC50 of 100 nM, while in human whole blood the EC50 was higher at 700 nM. AB928 (100 nM) exhibited comparable shifts in the NECA dose response between human and murine whole blood, 15 and 16-fold, respectively. Due to the high level of adenosine in the tumor microenvironment, doses of AB928 associated with plasma levels that significantly inhibited 5 μM NECA in the whole-blood pCREB assay were selected for mouse efficacy studies. At these doses, significant reductions in tumor growth were noted. In human whole blood, approximately 90 nM AB928 was required to inhibit 50% of the 5 μM NECA-induced pCREB signal. Inhibition was observed on both CD4+ and CD8+ T cells. NECA-induced pCREB elevations were observed in CD14+ cells less frequently than T cells; however, when the signal was observed, AB928 inhibited it. The human pCREB assay is being utilized to monitor the PD responses in an ongoing clinical trial. Preliminary PK/PD results from this study will be presented.

CONCLUSIONS: Systemic extent of receptor (A2aR and A2bR) occupancy by the novel dual antagonist AB928 can be assessed in humans and mice based on the extent of receptor-mediated CREB phosphorylation in blood lymphocytes. AB928 retains much of its inherent potency when competing against high concentrations of adenosine under physiologically relevant conditions (i.e., whole blood).

#3770

**Characterization of novel dual A** 2A **/A** 2B **adenosine receptor antagonists for cancer immunotherapy.**

Michal Galezowski, Paulina Wegrzyn, Aneta Bobowska, Claude Commandeur, Katarzyna Dziedzic, Marcin Nowogrodzki, Alicja Obara, Joanna Szeremeta-Spisak, Anna Dzielak, Iwona Lozinska, Marcelina Dudek, Anita Janiga, Jacek Reus, Marek Wronowski, Mateusz Swirski, Adam Radzimierski, Magdalena Ziembik, Joanna Mierzwicka, Katarzyna Wojcik-Jaszczynska, Elzbieta Gocek, Karolina Grycuk, Aniela Golas, Olga Pierzchala, Julian Zachmann, Mateusz Nowak. _Selvita S.A., Krakow, Poland_.

Adenosine is the key immunometabolite responsible for immune tolerance in tumors. It is present in normal tissue in low concentrations, having various physiological functions. In the tumor, its concentration increases rapidly, as a result of overexpression of enzymes producing adenosine, additionally enhanced by hypoxia and inflammation. Adenosine inhibits the biological functions of T lymphocytes infiltrating the cancer tissue by binding to the A2A receptor. The affinity to A2B receptor is believed to attenuate the response of dendritic cells and other parts of innate system. Thus blocking simultaneously the effects mediated by both receptor subtypes with dual inhibitor seems to be a viable approach to a single agent cancer immunotherapy or a combination with current immunotherapeutic agents.

We have discovered a novel series of potent and selective A2A/A2B inhibitors. Best compounds present subnanomolar activity in both in vitro recombinant cellular model and in primary human cells. Our antagonists dose-dependently restore the adenosine agonist-impaired functional activity of CD4+ and CD8+ human T-lymphocytes (cytokine release assays). We observe also the relieve of adenosine-related suppression in NK cells cytotoxicity. Most importantly tested compounds present improved primary pharmacological profile in comparison to A2A inhibitors currently tested in clinical trials. Further validation in in vivo models showed efficacy standalone and in combination with checkpoint inhibitors.

#3771

Mechanistic and pharmacodynamic characterization of the immuno-oncological activity of RXDX-106, a novel TYRO3, AXL, and MER (TAM) inhibitor in clinical development.

Amanda Albert, Erin D. Lew, Kristen Smith, Elizabeth A. Tindall, Yumi Yokoyama, Amy Diliberto, Heather Ely, Jack Lee, Robin Nevarez, Joanne Oh, Colin Walsh, Jason Christiansen, Gary Li, Robert Shoemaker. _Ignyta, Inc., San Diego, CA_.

The TAM family of receptor tyrosine kinases (RTKs) includes TYRO3, AXL, and MER, and is a unique set of molecular targets for cancer immunotherapy. TAM RTKs play a key role in the tumor microenvironment (TME) as negative regulators of innate immune responses and contribute to the evasion of cancer cells from immunosurveillance. RXDX-106 is a novel small molecule TAM inhibitor with single-digit nanomolar kinase inhibition and slow dissociation binding kinetics resulting in potent and durable target inhibition. In preclinical studies, RXDX-106 has demonstrated immune-mediated single agent in vivo activity in multiple syngeneic tumor models and potentiated anti-tumor activity in combination with immune checkpoint inhibitors. To characterize the mechanism of action of and pharmacodynamic responses to RXDX-106 treatment, an integrated approach was employed to explore RXDX-106-mediated immune modulatory effects at the cellular, protein, and transcriptional levels. In syngeneic tumor models, RXDX-106 treatment resulted in the recruitment of both innate and adaptive immune cell subtypes to the TME. Specifically, RXDX-106 treatment resulted in increased immune cell infiltration, macrophage polarization towards an antitumor M1 phenotype, activation of T cell and NK cell function, and modulation of immune markers at both the protein and RNA level, indicative of a pro-inflammatory and anti-tumorigenic effect. Importantly, the immune modulatory effects of RXDX-106 were concordant across platforms. Immune modulation was further characterized in a dose-dependent manner, with increasing immune cell infiltration observed with escalating doses of RXDX-106. In summary, RXDX-106 has the potential to restore and enhance immune function by modulating the local immunosuppressive TME. The unique mechanism of activating both innate and adaptive immunity, and regulating cross-talk between immune cells and tumor cells, supports clinical evaluation of RXDX-106 as an immunomodulatory agent for the treatment of a variety of cancers.

#3772

Preclinical study of a novel TLR8 selective agonist for cancer immunotherapy.

Yuxun Wang, Heping Yang, Xingzhong Zhang, Shuwen Ren, Huanping Li, Shuda Zhao, Longjun Gu, Yin Zhang, Yikun Zeng, Longsheng Wang, Guangliang Fu, Fang Bao, Fang Liu, Zhiheng Wu, Panhu Zhu, Hui Xu, Yaqiao Gao, Jian Zhang, Pei Wang, Shoujun Chen, Daxin Gao. _Denovo Pharmatech, Shanghai, China_.

The innate immune system has emerged as one of the major pathways for drug discovery focusing on cancer immunotherapy. TLR8 plays a crucial role in activating innate immunity and facilitates adaptive immunity thereby exerting potent immune-mediated anticancer activity. We previously presented preliminary characterization of a novel TLR8 agonist DN-A1 (Abstract 2995, AACR Annual Meeting 2017). In the present study, we carried out further comprehensive comparative preclinical study of DN-A1 and motolimod, the only TLR8 selective drug candidate in clinical trials. The result indicated that DN-A1 clearly differentiated from motolimod. DN-A1 was highly selective for TLR8 while motolimod exhibited moderate activity over TLR7 in addition to its primary agonistic activity over TLR8. DN-A1 had superior in vitro ADMET and in vivo PK profiles. Moreover, DN-A1 displayed more potent in vitro and in vivo biological activity and produced stronger anticancer efficacy than motolimod. These findings were further supported by DN-A1's stronger effect than motolimod in activating innate immunity in both monkey in vivo and human PBMC ex vivo systems. Furthermore, DN-A1 significantly suppressed tumor growth as a single agent and combination with the chemotherapeutic agent enhanced efficacy of either agent alone in leukemia tumor model. GLP toxicity study in rats and preliminary toxicity study in monkeys revealed acceptable safety profile of DN-A1. Importantly, DN-A1 consistently caused skin injection site reaction (ISR) when administered subcutaneously in both rats and monkeys. In contrast, motolimod failed to induce ISR under the same condition where DN-A1 caused ISR. Recent reports of motolimod's clinical data indicated that ISR was strongly correlated with substantial survival benefit in cancer patients whereas there was no survival benefit when motolimod failed to induce ISR. Therefore, DN-A1 holds great potential as the best-in-class TLR8 selective immunotherapeutic drug candidate poised for human clinical trials.

#3773

RORγ agonist enhances antitumor effects of agonist anti-OX40 antibody.

Pankaj Gaur,1 Vivek Verma,1 Rahul Nandre,1 Pooja Vir,1 Hua Wang,1 Baolin Kang,1 Laura Carter,2 Xiao Hu,2 Xikui Liu,2 Seema Gupta,1 Samir Khleif1. 1 _Georgia Cancer Center, Augusta University, Augusta, GA;_ 2 _Lycera Corp., Ann Arbor, MI_.

Combination immunotherapy strategies are being developed to improve the clinical outcome in cancer patients. T-cell costimulation through OX40 has been shown to promote expansion and proliferation of effector T-cells leading to enhanced effector functions, memory generation and immune inflammatory antitumor responses. However, treatment with anti-OX40 as a single agent has not led to major positive clinical outcomes. Therefore, anti-OX40 is an ideal candidate for combination immunotherapy. In preclinical models, we have recently shown that combining anti-PD-1 concurrently with anti-OX40 negates the effects of agonist anti-OX40, making identification of combination partners crucial for antitumor therapy. RORγt, a master transcription factor, is known to drive Type 17 T-cell differentiation. Synthetic, small-molecule RORγ agonists have been shown to enhance Type 17 T-cell effector functions and survival, decrease immune suppressive mechanisms and modulate expression of a number of costimulatory and coinhibitory molecules. We hypothesized that combining RORγ agonist could enhance the antitumor effects of anti-OX40. Using a murine model where vaccine is used to prime the immune system, we assessed the effects of anti-OX40 antibody combined with a RORγ agonist (LYC-54143) on growth of established tumors and survival. We found that the combined treatment resulted in a significantly delayed tumor growth and prolonged mice survival. Analysis of the tumor microenvironment revealed that combination therapy significantly increased the numbers of total CD4+ T-cells including RORγt+ and highly activated INFγ+ cells, decreasing Treg numbers. Moreover, we found that RORγ agonist resulted in an increase in the numbers of total, antigen-specific, granzyme B+, and IFNγ+ CD8+ T-cells in the TME. These results show that inducing Th17 cells within the TME produces better anti-OX40 effect, suggesting that agonist anti-OX40 and RORγ agonist treatment is a novel potent combination for cancer immunotherapy.

#3774

BGB324, a selective small-molecule inhibitor of receptor tyrosine kinase AXL, targets tumor immune suppression and enhances immune checkpoint inhibitor efficacy.

Kjersti Davidsen,1 Katarzyna Wnuk-Lipinska,2 Wenting Du,3 Magnus Blø,2 Agnete Engelsen,1 Stephane Terry,4 Stacey D´mello,1 Maria Lie,1 Jing Kang,1 Linn Hodneland,2 Sebastien Bougnaud,1 Kristina Aguilera,2 Oddbjørn Straume,5 Salem Chouaib,4 Rolf A. Brekken,3 Gro Gausdal,2 James B. Lorens1. 1 _University of Bergen, Bergen, Norway;_ 2 _BerGenBio ASA, Bergen, Norway;_ 3 _Hamon Center for Therapeutic Oncology Research, UT Southwestern Medical Center, Dallas, TX;_ 4 _Université Paris-Sud, Paris, France;_ 5 _Haukeland University Hospital, Bergen, Norway_.

The AXL receptor tyrosine kinase is associated with poor overall survival in a wide spectrum of cancers. AXL signaling is important for tumor cell plasticity related to epithelial-to-mesenchymal transition (EMT), immune escape and intrinsic resistance to cytotoxic lymphocytes. AXL is expressed on several cells associated with the tumor immune microenvironment, including natural killer (NK) cells, dendritic cells and a subset of tumor-associated myeloid cells. AXL signaling enhances secretion of immune-suppressive cytokines from innate immune cells that limit antitumor immunity. Hence AXL resides uniquely at the nexus between tumor and microenvironmental antitumor immune suppression mechanisms. BGB324, a selective clinical-stage small-molecule Axl kinase inhibitor, is currently being evaluated in combination with pembrolizumab in three phase II clinical trials in patients with TNBC (NCT03184558), NSCLC (NCT03184571) and melanoma (NCT02872259). We show that BGB324 targets immune suppression mechanisms in the tumor microenvironment that improve immunotherapy in different murine tumor models. BGB324 treatment reduces myeloid-derived suppressor cells and tumor-associated macrophages, and lowers CCL11, IL-7, IL-1β, and IL-6 in murine pancreatic cancer models. This altered immune landscape is associated with increased tumor infiltration of NK and CD8+ T cells and enhanced therapy responses. Further, BGB324 targets tumor intrinsic immune resistance and enhances human CD8+ T cell and NK-cell mediated NSCLC tumor cell lysis. We are currently using high-dimensional mass cytometry analysis (CyTOF) to map adaptive Axl-dependent immune suppression during immune checkpoint blockade. Collectively these results highlight a prominent function for AXL in resistance to immune therapy and support continued clinical translation of combining BGB324 with immune checkpoint inhibitors to improve cancer treatment.

#3775

Use of RLI-15 a clinical grade fusion protein with IL-15 superagonistic activity for the activation of anti-tumor immune response.

Irena Adkins,1 Lenka Sadilkova,1 Nada Hradilova,1 Jakub Tomala,2 Barbora Tomalova,2 Marek Kovar,2 Romana Mikyskova,3 Milan Reinis,3 Guy de Martynoff,4 David Bechard,4 Ulrich Moebius,1 Radek Spisek1. 1 _SOTIO a.s., Prague, Czech Republic;_ 2 _Institute of Microbiology of the ASCR v.v.i., Prague, Czech Republic;_ 3 _Czech Centre for Phenogenomics, BIOCEV and Department of Transgenic Models of Diseases, Institute of Molecular Genetics of the ASCR, v.v.i., Prague, Czech Republic;_ 4 _Cytune Pharma, Nantes, France_.

RLI-15, a superagonist fusion protein of interleukin (IL)-15 and the IL-15 receptor α (IL-15Rα) sushi+ domain represents a promising candidate for the induction of anti-tumor immunity. RLI-15 was designed to bypass the need of endogenous IL-15Rα, thereby leveraging the activity of IL-15 in vivo on target immune cells. RLI-15 stimulates the proliferation and the cytotoxic activity of natural killer (NK) cells and memory CD8+ T cells with no significant expansion and activation of regulatory T cell compartment. RLI-15 was previously shown to exhibit a potent anti-metastatic activity in B16F10 melanoma and Renca renal cell carcinoma mouse models. RLI-15 also significantly delayed tumor growth and prolonged survival when combined with anti-PD1 therapy in CT26 and MC38 colon carcinoma models. Here, we report that the combination treatment with clinical-grade RLI-15 and an anti-PD1 antibody leads to a significant anti-tumor efficacy in a TRAMP-C2 prostate cancer mouse model with 70 % of mice remaining tumor free after the treatment. We evaluated the optimal schedule of such combination therapy to set the basis for the design of upcoming clinical trials. We further tested how the administration schedule affects the pharmacodynamics properties of clinical-grade RLI-15 and translates into the anti-tumor efficacy in metastatic Renca and CT26 mouse models. In cynomolgous monkeys, various schedules of administration of RLI-15 showed a dose-dependent expansion of peripheral blood lymphocytes, predominantly of NK cell and memory CD8+ T cell compartments. The toxicity in mice and cynomolgous monkeys was evaluated to determine the maximal tolerated dose of RLI-15. Furthermore, the activity of clinical-grade RLI-15 was tested in vitro on human PBMCs and the superiority over IL-2 and IL-15 stimulatory capacity has been confirmed. The complex analysis of RLI-15 behavior and of the induced anti-tumor immune response will be explored in the design of a planned Phase I clinical study in patients with both solid tumors and hematological malignancies.

#3776

Generation and formatting of an Affimer® biotherapeutic for the inhibition of the PD-L1/PD-1 pathway: Proof of concept in mouse.

Amrik Basran. _Avacta Life Sciences, Cambridge, United Kingdom_.

We have generated a range of potent Affimer antagonists to both human and mouse programmed death-ligand 1 (PD-L1) and have demonstrated that when formatted, possess the necessary affinity, pharmacokinetics (PK) and tissue penetration to modulate the immune system and demonstrate efficacy in mouse models. Affimer biotherapeutics are a new protein scaffold with great potential for the generation of biotherapeutics. Based on the human protease inhibitor Stefin A, the scaffold is small (12kDa), lacks any post translational modifications such as disulphide bonds and glycosylation. Large diverse phage display libraries have been created by engineering in two 9 amino acid peptide loops into the scaffold backbone. Using phage display, we have identified competitive binders to both mouse and human PD-L1 which are low single digit nM in affinity as determined by Biacore. By ELISA we have shown that the Affimer molecules identified compete against PD-1 and CD80 for the binding site on PD-L1 and bind to the target on a range of human cancer cell lines (e.g. lung adenocarcinoma cells), as demonstrated by FACs. Following formatting of the lead Affimer protein to the N-terminal Fc region of an IgG, we showed that we could further improve the affinity to give a KD of <40 pM as determined by Biacore. The PK properties of the Affimer-Fc fusion were determined in mouse dosed at 10 mg/kg and shown to have a terminal half-life of over 100 hours. When dosed in a tumor mouse model the Affimer-Fc fusion protein showed a statistically significant reduction in tumor volume when compared to a control antibody. We have demonstrated that the Affimer scaffold has all the necessary attributes to be developed as a therapeutic platform. The Affimer scaffold was well tolerated in vivo, demonstrated a potency and PK profile that ensured sufficient exposure in the animal to modulate the immune system to slow tumor progression in a relevant mouse model.

#3777

RORγ agonist enhances antitumor effects of immune checkpoint anti-PD1.

Pankaj Gaur,1 Vivek Verma,1 Rahul Nandre,1 Pooja Vir,1 Hua Wang,1 Baolin Kang,1 Laura Carter,2 Xiao Hu,2 Xikui Liu,2 Seema Gupta,1 Samir Khleif1. 1 _Georgia Cancer Center, Augusta University, Augusta, GA;_ 2 _Lycera Corp., Ann Arbor, MI_.

Anti-PD1/PDL1 therapies have demonstrated great clinical benefit in some cancers, albeit in a limited percentage of patients. RORγt is a master transcription factor, which promotes Type 17 T cell differentiation and is known to enhance the stemness and plasticity of effector cells and to modulate PD1 expression. Since one of the potential failure mechanism of anti-PD1 is the lack of sustained CD8 infiltration within the TME, we assessed whether combining a synthetic, small-molecule RORγ agonist could enhance the antitumor effects of anti-PD1 treatment. To assess this combination treatment, we tested the effects of anti-PD1 antibody (1 mg/kg twice weekly) combined with a RORγ-agonist (LYC-54143; 100 mg/kg PO, BID given continuously till the end of study) in tumor-bearing murine mode lthat utilizes a vaccine for proper priming. We found that RORγ agonist combined with PD1 antibody significantly enhanced the anti-PD1 antitumor effect. This result was manifested by further delaying the tumor growth and prolonging mice survival. At day 35 after tumor implantation, 66% of mice survived in the group treated with anti-PD1 and RORγ agonist and vaccine when compared to 0% survival following anti-PD1 + vaccine or vaccine- alone treatments. This enhanced antitumor activity was observed/confirmed in several tumor models with the combination of RORγ agonist + anti-PD1 treatment. These results show that RORγ agonists may enhance the effectiveness of anti-PD1 therapy and therefore this combination is a promising therapeutic strategy for cancer.

#3778

Combination immunotherapy intercepts murine pancreatic cancer progression.

Aleksandra Popovic,1 Nina J. Chu,2 Todd Armstrong,2 Elizabeth M. Jaffee2. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _Johns Hopkins University School of Medicine, Baltimore, MD_.

Immunotherapy agents that activate T cells are changing clinical practice for some metastatic cancer patients. Vaccines targeting viral oncogenes prevent the development of hepatocellular and cervical cancers. We therefore tested the hypothesis that a vaccine targeting the oncogene that initiates pancreatic cancer (PDA) given in combination with immunotherapy agents that reprogram the earliest procarcinogenic inflammatory changes will slow (intercept) PDA development. The KRAS proto-oncogene is a driver mutation expressed in >90% of pancreatic intraepithelial neoplasms (PanINs) and an ideal candidate target for early vaccination against PDA. However, even early PanINs have immunosuppressive microenvironments highly expressing inhibitory immune checkpoints like CTLA-4 and infiltrating immunosuppressive cells such as regulatory T cells (Tregs). The overall goal of this research is to reprogram the immunosuppressive environment while specifically activating immunity against early premalignancies to prevent PDA development. We previously reported that early dosing of KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) mice, which endogenously recapitulate human PDA tumorigenesis, with the Treg depleting and inactivating agents cyclophosphamide (cy) and PC61 antibody in combination with an attenuated Listeria monocytogenes vaccine expressing mutant KrasG12D (LM-Kras) can slow tumorigenesis in 40% of mice. In this study we attempted to improve the efficacy of a vaccine approach by specifically inhibiting the immunosuppressive effects of the CTLA-4 immune checkpoint on Tregs with a CTLA-4 blocking antibody. KPC mice at 4-5 weeks with early PanINs received 2 doses of LM-Kras and cy/PC61 and 8 doses of anti-CTLA-4. Control mice received isotype antibody with LM-Kras and cy/PC61 or no treatment. At 10-11 weeks, pancreata were pathologically evaluated and assigned grades normal, low (1) and high-grade (2 and 3) PanINs, and PDA. Mice receiving LM-Kras + anti-CTLA-4 had a significantly lower incidence of low- and high-grade PanINs and PDA compared to controls. Furthermore, mice receiving LM-Kras, cy/PC61, and anti-CTLA-4 did not develop lesions beyond PanIN 1, indicating that the combination therapy may halt PDA development at early stages.

To investigate the immune profiles of mice receiving treatment, flow cytometry was performed on tumor-infiltrating lymphocytes (TILs) isolated from pancreata to assess abundance of Tregs, infiltration of effector and memory T cell populations, and expression of inhibitory immune checkpoint molecules. Mice receiving combination therapy had decreased numbers of Tregs, increased numbers of effector and memory T cells, and increased CD8+ T cells to Tregs ratio in the microenvironment compared to controls. Further studies using immunohistochemistry and RNA expression profiles are under way to further characterize the immune changes in the microenvironment following treatment.

#3779

The TLR7/8 agonist R848 induces antitumor responses and attenuates cachexia in a murine model of pancreatic ductal adenocarcinoma.

Katherine A. Michaelis, Mason A. Norgard, Xinxia Zhu, Peter R. Levasseur, Katherine R. Pelz, Kevin G. Burfeind, Terry K. Morgan, Daniel L. Marks. _Oregon Health & Science University, Portland, OR_.

PURPOSE: With recent advances in immunotherapy, many novel cancer treatments are rapidly entering the clinical arena. However, immunotherapies may differ from traditional chemotherapies in their effects on cachexia and treatment-associated sickness. Cachexia is a common comorbidity of cancer that limits therapeutic options, decreases quality of life, and increases mortality risk. Many chemotherapy agents induce or worsen cachexia by independently causing anorexia, weight loss, muscle wasting, and fatigue. Acute systemic inflammation, an effect of many immunotherapies, results in sickness responses that are typically self-limited in healthy individuals. Whether immunotherapy-associated sickness is self-limited or chronic in the context of cancer, and how cachexia is impacted by immunotherapy, remains unknown.

METHODS: We recently found that the TLR7/8 agonist R848 reduces tumor size in mice implanted with epithelial cells from a syngeneic KRASG12D/+ P53R172H/+ Pdx-Cre (KPC) pancreatic tumor. To assess cachexia outcomes and R848-induced sickness, mice were orthotopically implanted with KPC cells or saline, and 2 days later were randomized to daily IP R848 (10µg) or vehicle until sacrifice (n=5-7/group/experiment, 3 experiments). Mice were tracked for weight, food intake, body composition, and locomotor activity (LMA), with end-stage analysis of tissue mass and gene expression.

RESULTS: Initially, KPC-bearing mice treated with R848 developed significant weight gain and ascites, but had a similar degree of anorexia and decreased LMA as vehicle-treated KPC-bearing mice. However, ongoing R848 resulted in subsequent tumor regression, decreased ascites, increased appetite, and increased LMA. At necropsy, KPC-bearing mice treated with R848 had a 50-70% reduction in tumor mass, histologically characterized by lymphocytic infiltrate and germinal centers. Furthermore, R848-treated KPC-bearing mice had improved total lean mass and heart mass; decreased expression of genes related to skeletal and cardiac muscle catabolism (Mafbx, Murf1, Foxo1, Bnip3, Gabarapl, Ctsl) and hepatic acute phase reactants (Orm1, Apcs); a trend toward decreased CNS inflammatory gene expression (Selp, Il1r1); and unchanged brown adipose tissue thermogenic gene expression (Ucp1). In sham-operated mice, R848 resulted in self-limited anorexia and weight loss, without muscle wasting or decreased LMA. Current work is underway to elucidate tumor intrinsic and tumor extrinsic mechanisms of R848 in this model.

SUMMARY: These studies show that R848 does not independently cause sustained sickness, and in a murine model of pancreatic cancer, can induce antitumor responses and improve cachexia outcomes. This represents a key difference from many cytotoxic chemotherapies and suggests immunotherapy approaches may be useful in the treatment of cachexia-associated malignancies.

#3780

Preclinical pharmacokinetics, pharmacodynamics and safety of BION-1301, a first-in-class antibody targeting APRIL for the treatment of multiple myeloma.

John Dulos,1 Lilian Driessen,1 Peter van Zandvoort,1 Jos van de Crommert,1 Nitya Nair,2 Britta Randlev,2 Eduard de Cock,1 Jeroen Elassaiss Schaap,3 Hans van Eenennaam,1 Andrea van Elsas1. 1 _Aduro Biotech Europe, Oss, Netherlands;_ 2 _Aduro Biotech, Berkeley, IL;_ 3 _PD Value, Houten, Netherlands_.

APRIL critically triggers BCMA to drive proliferation and survival of human multiple myeloma (MM) cells. Importantly, APRIL induces resistance to lenalidomide, bortezomib and other standard-of-care drugs. Furthermore, APRIL drives expression of PD-L1, IL-10, VEGF and TGFβ forcing an immunosuppressive phenotype on BCMA+ cells. MM survival, resistance to treatment and the immunosuppressive phenotype can be blocked by BION-1301, the first-in class humanized antibody neutralizing APRIL. BION-1301 blocks APRIL binding to the MM relevant receptors BCMA and TACI in contrast with anti-BCMA bispecifics, ADC and CAR T-cells which use only BCMA for MM cell killing. In addition, BION-1301 inhibits immune suppressive effects of regulatory T-cells via TACI which differentiates it from BCMA targeting cytotoxic approaches. In vivo, BION-1301 was shown to suppress T cell-independent B cell responses to NP-Ficoll. Furthermore, APRIL blockade demonstrated single agent anti-MM activity in a humanized SCID model confirming its activity in vivo targeting MM cells in the tumor-protective bone marrow microenvironment. Here, we report on the preclinical pharmacokinetics (PK)/ pharmacodynamics (PD) relationship and safety analysis of BION-1301. A single-dose non-human primate (NHP) study administering intravenous BION-1301 at 0.3, 3 and 30 mg/kg, yielded PK parameters typical for IgG4 class antibodies and absence of tolerability issues. PD analysis showed a statistically significant reduction in total IgA and IgM in a dose-dependent fashion. Another measure of target modulation by BION-1301 was provided by co-injection of the hapten TNP-Ficoll (T-cell independent antigen) in different dose groups. Consistent with previous observations in hAPRIL transgenic mice, BION-1301 reduced in a dose-dependent fashion, TNP-specific IgA and IgM. Using a weekly dosing regimen, a 4-week repeat-dose toxicity study was conducted at doses of 10, 30 and 100 mg/kg, demonstrating that BION-1301 had no effect on vital organs and other toxicology related factors examined, at any dose level up to 100 mg/kg. Chronic exposure to BION-1301 led to significantly reduced levels of IgA, IgG and IgM observed at all dose levels. To determine target engagement of BION-1301, quantitative assays were developed to detect free APRIL and BION-1301. BION-1301 reduced free APRIL levels in serum in a dose-dependent manner following single and multiple dose administrations. Using a MABEL approach, PK/PD modeling informed a proposed human starting dose of 50 mg. In summary, BION-1301 showed no toxicity in NHP and binding of APRIL resulted in decreased IgA, IgG and IgM levels. PK and target engagement biomarkers predict the first in human dose using PK/PD pharmacometric modeling. A first-in-human study is ongoing to characterize safety and PK/PD of BION-1301 in heavily pretreated MM patients.

### Innate Immune Responses in Cancer

#3781

A focused dendritic cell CRISPR/Cas9 screen to identify novel regulators of cross-presentation and antitumor immunity.

Jesse T. Davidson, Derek J. Theisen, Carlos G. Briseño, Vivek Durai, William E. Gillanders, Theresa L. Murphy, Kenneth M. Murphy. _Washington University School of Medicine, Saint Louis, MO_.

Introduction: Dendritic cells (DCs) are professional antigen-presenting cells with two major subsets, the classical DC type 1 and 2 (cDC1 and cDC2). The cDC1, also known as the CD8α+ DC, is especially equipped for the process of cross-presentation in which dead cell antigen is presented on MHC-I to prime cytotoxic T cells. This process is relevant to anti-cancer immunity, as mice deficient in the transcription factor BATF3, which specifically lack the cDC1, succumb to highly immunogenic tumors that are normally rejected and cleared in wild-type mice. The mechanism of cross-presentation by the cDC1 is poorly understood. In this study, we developed a focused CRISPR/Cas9 screen to identify novel genes that endow the cDC1 with the ability to cross-prime cytotoxic T cells.

Methods: A curated list of target cDC1 genes was constructed using the Immunological Genome Project Database as the primary resource (www.immgen.org). 120 genes specifically expressed by the cDC1 in comparison to other murine immune cells were selected. Guide RNAs (gRNAs) were designed to early exon sequences using the MIT CRISPR Design Tool to minimize off-target effects (www.crispr.mit.edu), and 2-3 different gRNAs were evaluated in tandem for each candidate. These gRNAs were transduced via retroviral vector into lineage depleted c-Kithi bone marrow cells isolated from C57BL/6J Cas9 knock-in mice. These cells were differentiated in vitro for 8 days in the presence of the cytokine Flt3L. Transduced DCs were sorted by FACS and co-cultured in the presence of a source of dead cell antigen (heat-killed L. monocytogenes expressing ovalbumin, or HKLM-OVA) as well as CFSE-labelled transgenic CD8+ T cells with an ovalbumin peptide-specific T cell receptor (OT-1). The proliferation of OT-1 cells was measured by CFSE dilution.

Results: To validate our CRISPR/Cas9-based screening approach, we targeted the first exon of the beta-2 microglobulin gene (B2M), which is required for cell surface expression of MHC-I. B2M knockout DCs were co-cultured in the presence of HKLM-OVA and CSFE-labelled OT-1 cells for 3 days. Controls included DCs transduced with a scramble gRNA with or without antigen. As expected, targeting B2M in DCs led to near-complete reduction in OT-1 proliferation compared to scramble control (3.7% vs. 20.6%, p=0.003), similar to the absence of antigen (3.7% vs. 0.96%, p=0.99). We then expanded our assay to our curated list. Promising candidate genes have been discovered and are currently being validated in murine knockout models.

Conclusions: The CRISPR/Cas9 system can be adapted to screen the cDC1 for novel genes involved in cross-priming cytotoxic T cells. Validation of candidates in vivo, including tumor assays, will be presented at the meeting. These findings may have important implications for adaptive immunity against cancer.

#3782

Transcriptional regulation of MHC Class II in tumor associated plasmacytoid dendritic cells in glioblastoma.

Sreenivasulu Chintala, Brij N. Tewari, Mario Henriquez, Kaleigh Fetcko, Anna Filley, Mahua Dey. _Indiana University School of Medicine, Indianapolis, IN_.

Background: Glioblastoma (GBM), an aggressive grade IV astrocytoma, is the most common primary brain tumor in adults and carry very poor prognosis. One of the hallmark features of GBM is tumor induced dense immunosuppression that renders immunotherapeutic strategies against GBM ineffective. In order to develop successful immunotherapeutic strategies for GBM, it is critical to understand the mechanism of immunosuppression in the tumor microenvironment (TME). In our previous publication, we have shown that plasmacytoid dendritic cells (pDCs) accumulate in TME and preferentially up-regulate MHC-II expression in comparison to myeloid DCs (mDCs). GBM infiltrating pDCs are immunosuppressive in nature and lack the ability to secrete IFN-alpha, and MHC-II upregulation by immunosuppressive pDCs lead to development of tumor tolerance. MHC-II molecules are transcriptionally regulated by the multiprotein complex enhanceosome consisting of a catalytic Class II transactivator (C2ta) in a cell type-dependent mechanism. There are no studies clearly showing differential enhanceosome and C2ta regulation in GBM associated pDC and mDC. In the present study, we investigated the mechanism of differential expression of enhanceosome multiprotein complex in GBM associated pDC and mDC and identify the upstream regulators and downstream effectors. Methods: GL261 cells were intracranially injected (i.c.) in C57BL6 mice and allowed to establish tumors. Tumor and normal brain (n=5), along with cervical lymph nodes (cLN), were collected and processed for isolation of pDC and mDC using flow sorting. Quantitative RT-PCR analysis was used to determine the enhanceosome components. IHC/IF and Western blot analysis were performed to confirm differential expression of enhanceosome components in mouse and human GBM specimens. Cytokine/chemokine protein array and ELISA methods were used to determine the differential expression of cytokines/chemokines in pDC and mDC. Results: We found differential expression of enhanceosome components in tumor associated pDC compared to mDC. Additionally, we found that GBM derived cytokines regulate differential expression of enhanceosome in tumor associated pDC and mDCs. Conclusions: In conclusion results from our studies show that GBM microenvironment regulates differential expression of enhanceosome transcription factors in pDC compared to mDC in a cytokine dependent process. This in turn helps to maintain an immunosuppressive microenvironment for continued tumor growth.

#3783

Mitochondrial metabolism in tumor-associated macrophages: The role of MIF and tumor-derived lactate.

Jordan Noe, Beatriz Rendon, Eun Jung Kim, Robert Mitchell. _University of Louisville, Louisville, KY_.

Purpose: Within the tumor microenvironment, tumor-associated macrophages (TAMs) polarized to an "M2" phenotype promote angiogenesis, metastasis, and the suppression of anti-tumor immune responses. Previously, we have found that macrophage migration inhibitory factor (MIF) promotes lactate-enhanced M2-polarization, however the underlying mechanisms were not elucidated. As cancer cells that exhibit the Warburg Effect secrete copious amounts of lactate into the tumor microenvironment, highly glycolytic cancers may promote tumor progression by producing lactate to support an immunosuppressive tumor microenvironment. The goal of this study is to investigate the mechanism by which MIF and tumor-derived lactate support pro-tumorigenic M2-TAMs.

Methods: Using primary bone marrow-derived macrophages (BMDMs), we explored the mechanisms of MIF and lactate-enhanced M2 polarization using metabolic inhibitors and gene expression assays. To investigate the role of MIF in mitochondrial metabolism, we conducted extracellular flux analysis to determine mitochondrial OCR and ECAR in macrophages polarized to an M0 and M2 phenotype.

Results: Blocking mitochondrial pyruvate uptake reduces both M2 polarization and HIF1α stabilization. Blocking mitochondrial metabolism and respiration inhibits M2 polarization. MIF deficiency decreases mitochondrial OCR, NRF2 stability, and M2 polarization. Increasing CSN5 activity "rescues" the loss of M2 polarization with MIF deficiency.

Conclusions: Tumor-derived lactate per se does not promote M2 polarization but instead requires subsequent metabolism to pyruvate. Additionally, we have identified that mitochondrial pyruvate metabolism is a previously undetermined requirement for M2 macrophages. Further, we have found a potential link between MIF and metabolism, where MIF likely regulates CSN5 activity and NRF2 stability to increase mitochondrial respiration. As inhibiting mitochondrial respiration blocks M2 polarization, MIF may promote lactate-enhanced M2 polarization by enhancing mitochondrial metabolism. Subsequent studies will be conducted to further investigate the metabolic pathways and mechanisms required for the mitochondrial metabolism of tumor-derived lactate in promoting M2-TAM polarization.

#3784

Intraoperative changes of low density granulocyte (LDG) in gastrointestinal cancer surgery.

Yuko Kumagai. _Jichi Medical University, Tochigiken Shimotsukeshi, Japan_.

Background: Low density granulocytes (LDG) have been shown to be increased in peripheral blood of autoimmune disease, severe sepsis and cancer. In this study, we examined the frequency and function of the LDG in patients who received abdominal surgery for gastrointestinal malignancy. Method: In 57 patients who received laparotomy due to the gastrointestinal cancer ( 5 esophageal, 25 gastric , 27 colorectal cancer), peripheral blood was collected before and after operative procedure in operation room. After Ficoll-Hypaque centrifugation, intermediate layer which consists mainly of mononuclear cells was collected and immunestained with mAbs to CD66b, CD45 as well as 7AAD and their expressions were examined with flow cytometer. CD66b(+) CD45(+) cells were defined as LDG and the ratios of LDG in total CD45(+) leukocytes were calculated in 7AAD(-) live cell fraction. Next, CD66b(+) LDG and CD66b(-) mononuclear cells were separated with MACS method, and LDG were cultured and examined for extracellular traps (NETs) formation in vitro. Finally, CD66b(-) mononuclear cells were cultured on anti-CD3 mAb coated plate with or without autologous LDG, and proliferation of T lymphocytes was examined with CFSE dilution method. Results: The ratio of the LDG is low (median= 1.14%, 0.62 ~1.66%, n=57) at a beginning of surgery. In contrast, the ratio was markedly increased (median= 8.10%, 5.91~10.3%, n=57, p<0.001) at the end of surgery. The ratio of postoperative LDG showed significant correlation with operation time and positive trend with intraoperative bleeding. The LDG showed relatively week expression of CD11b and CD66b, but highly expressed in IL8 receptor and CD62L, suggesting immature LDG. The short-term culture of the LDG produced massive SYTOX-positive web like stricture which was characteristic for NETs. The mixture of tumor cells on the LDG culture demonstrated that many tumor cells were trapped in the NETs within 5 min incubation and vigorously grew thereafter. Finally, addition of the LDG on mononuclear cell culture strongly suppressed the anti-CD3 stimulated T cell division. Conclusion: LDG with immature phenotype increase in circulating blood of the patients who underwent gastrointestinal cancer surgery, probably due to the surgical stress. The LDG produce NETs which may efficiently trap circulating tumor cells, and can inhibit T lymphocyte mediated-immune reaction. Thus, the LDG provide tumor permissive microenvironment in target organ and have supportive roles on the development of postoperative hematogenous metastasis in gastrointestinal cancer.

#3785

Loss of vacuolar ATPase in hematopoietic stem cells promotes breast tumor progression and pathogenesis.

Manoranjan Sahoo, Gajendra K. Katara, Mahamood Y. Bilal, Sara Fleetwood, Arpita Kulshrestha, Safaa A. Ibrahim, Kenneth D. Beaman. _Rosalind Franklin University of Medicine and Science, North Chicago, IL_.

Vacuolar ATPase (V-ATPase) is expressed intracellularly on vesicular membranes, and controls intracellular pH and acidification of various intracellular vesicles. Most cancer cells express V-ATPase on their cell surface where they appear to acidify the extracellular environment and help cancer cells to metastasize. We have previously demonstrated that the a2 isoform of V-ATPase (a2V) of mammary epithelial cells plays a key role during breast tumor progression and metastasis. However, the role of a2V of hematopoietic stem cells during breast tumor pathogenesis is not known. Therefore, to understand the role of a2V in hematopoietic stem cells during breast tumor progression, we conditionally knocked out a2V (cKO) from the hematopoietic stem cells of mice. We observed that deletion of a2V leads to a significant reduction of CD4+ and CD8+ T cells in the periphery and spleen of the a2V cKO mice compared to control mice. We hypothesized that reduction of helper CD4+ cells, or cytotoxic CD8+ T cells would result in larger and faster-growing tumors in the a2V-cKO mice. After implantation of a syngeneic tumor cell line (E0771) in the breast tissue of mice, we observed that deletion of a2V led to larger and faster-growing breast tumors compared to control mice. Further investigation of the tumor microenvironment revealed that there was a significant reduction in percentage of CD4+ and CD8+ T cells in the a2V-cKO mice compared to control mice. Targeted RNAseq of tumor microenvironment demonstrated that out of 499 genes that were analyzed, only 3 genes were significantly upregulated while 144 genes were significantly downregulated in the a2V cKO mice. The 144 downregulated genes included genes for pro-inflammatory cytokines, T cell specific genes, death receptors and proapoptotic genes. Collectively, these results demonstrate that deletion of a2V from the hematopoietic stem cells leads to a decrease in CD4+ and CD8+ T cells in the periphery, which results in a faster-growing and larger breast tumor. Further study is needed to understand the relative contribution of CD4+ or CD8+ T cells during breast tumor pathogenesis.

#3786

ILC-k: Human innate lymphoid cells displaying unique metabolic features and KIR-independent cytotoxicity, impaired in acute myeloid leukemia.

Bérengère Salomé,1 Alejandra Gomez-Cadena,1 Romain Loyon-Bonato,1 Madeleine Suffiotti,1 Valentina Salvestrini,2 Antonio Curti,2 Paolo Tentorio,3 Domenico Mavilio,3 Carsten Riether,4 Adrian Ochsenbein,4 Emanuela Marcenaro,5 David Gfeller,1 Pedro Romero,1 Sara Trabanelli,1 Camilla Jandus1. 1 _University of Lausanne, Epalinges, Switzerland;_ 2 _Institute of Hematology, University of Bologna, Bologna, Italy;_ 3 _Humanitas Clinical and Research Center, Rozzano-Milan, Italy;_ 4 _University of Bern, Bern, Switzerland;_ 5 _University of Genoa, Genoa, Italy_.

Innate Lymphoid Cells (ILCs) are a recently identified family of lymphocytes, divided into 3 different groups that mirror the functional specialization of helper CD4 T cells. However, it is now clear that the spectrum of ILC diversity is much broader than originally appreciated. ILCs have been shown to play important roles in inflammatory and autoimmune diseases, and their involvement in anti-tumor responses has also recently been evidenced by us and others. Here, we report on the identification of a previously undescribed human ILC subset, named hereafter "ILC-killer" (ILC-k), characterized by a Lineage negative, CD127+ cKIT- CRTh2- CD56+ phenotype, thus being distinct from conventional helper ILCs, e.g ILC1 (CD56-), ILC2 (CRTh2) and ILC3 (cKIT+). Transcriptomic profiling of ex-vivo highly pure ILC-k, conventional ILC subsets and conventional Natural Killer (NK) cell subsets showed closeness between ILC-k, ILC3 and CD56bright NK cells. However, ILC-k display a specific metabolism (low glucose uptake, high mitochondrial activity) and cytotoxic gene signature, also confirmed at protein level. By their functional evaluation, ILC-k showed specific ability to kill tumor cell lines in a KIR-independent, but NKp30- and Trail-dependent manner. Their relative frequency is decreased and both their metabolic fitness and cytotoxic potential are impaired in Acute Myeloid Leukemia (AML) patients at diagnosis (N=51). Upon remission, ILC frequencies and functions are restored to normal levels. Overall, we identified cytotoxic ILCs, distinct from conventional NK cells, that are functionally impaired in AML. Given their KIR-independence, ILC-k might represent promising therapeutic candidates to improve NK-cell based immunotherapy, that currently necessitates KIR-HLA mismatch between donor and recipient for full efficiency.

#3787

Exploring the effect of chemotherapies on STING-dependent cytokine release.

Richard D. Wilkinson, David I. Johnston, Eileen E. Parkes, Nuala McCabe, Richard D. Kennedy. _Queen's University Belfast, Belfast, United Kingdom_.

Introduction: Blockade of PD-1/PD-L1 has shown potent antitumor activity but these therapies are limited to certain diseases with higher intrinsic somatic mutation burden. A key research priority is now optimising response to these agents in diseases with limited clinical response. Activation of the cGAS-STING innate immune pathway has been identified as synergistic with anti-PD-1 therapy, and also a means of overcoming resistance to IO (immune-oncology therapy). We have reported activation of the cGAS-STING immune pathway as a result of cytosolic DNA released in response to intrinsic and extrinsic DNA damage, and that upregulation of PD-L1 in response to DNA damage is dependent on STING. Therefore, activating the STING pathway as a combination treatment with IO therapy could result in improved clinical responses. Experimental procedures: We perform qPCR analysis of the STING target genes, CXCL10/CCL5 after treatment with different classes of chemotherapy. We perform immunofluorescence to determine dsDNA translocation to the cytosol and the requirement of STING for this, an siRNA screening approach was used to identify the key nucleases involved in cytosolic dsDNA translocation and chemokines expression. Results: We observed increased expression of CXCL10/CCL5 gene expression following treatment with the topoisomerase I (irinotecan) and II (Doxorubicin and Etoposide) inhibitors. This chemokine expression was associated with increased expression of dsDNA in the cytosol. Using an siRNA approach, depletion of STING and the DNA nuclease MUS81 demonstrated abrogation of CXCL10/CCL5 expression following treatment with Doxorubicin. Conclusions: Here, we show that topoisomerase I and II inhibitors are the most potent chemotherapy at activating the cGAS-STING pathway and associated expression of interferon-dependent chemokines. We propose that combination therapies of topoisomerase I and II inhibitors with IO therapy will be effective in tumours which have had limited clinical response to single agent IO therapy.

#3788

Antigen presentation by MHC-I molecule and immune escape in acute myeloid leukemia with high burden of genomic aberrations.

Giovanni Marconi,1 Maria Chiara Fontana,2 Cristina Papayannidis,2 Mariachiara Abbenante,2 Antonella Padella,2 Giorgia Simonetti,2 Michele Cavo,2 Giovanni Martinelli2. 1 _Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy;_ 2 _University of Bologna, DIMES, Istituto Seragnoli, Bologna, Italy_.

Background: Antigen presentation is crucial for tumor immune response and MHC-I plays a key role. Solid and hematological tumors with high load of newly tumor associated antigens develop evading immune surveillance. A subset of patients with acute myeloid leukemia (AML), precisely ones with complex karyotype and TP53 alterations have a high load of mutation or genomic aberrations. In our study, we screened the entire genome of a large set of newly diagnosed AML to discover if MHC I antigen presentation is altered in the set of AML with a high neo-antigen load.

Methods: We screened 300 samples of AML bone marrow at diagnosis from 3 hematological Institutions. We analyzed 636 single nucleotide polymorphism (SNP) arrays to detect karyotype aberration at genomic level; germline SNP arrays were performed in 114/300 patients, the remaining 186/300 were normalized with a reference pool of 222 healthy donors. Of the 300 patients, we collected clinical data and we tested TP53, IDH1, IDH2, FLT3, NPM1, DNMT3A and RUNX1 mutations according ordinary practice. Genes in MHC class I antigen presentation pathway were selected basing on Reactome. We considered MHC-I antigen presentation altered if pathway's alteration rate (altered genes in a pathway/gene in a pathway ratio) scored in the top 25% between all pathways.

Results: Twenty out of 300 patients (6.7%) had loss of genes in MHC I antigen presentation (group A). Patients had alterations in one or more genes; overall, 53 genes were founded altered in our set. Patients in group A did not have differences in age or in white blood count at diagnosis compared with other patients in our set (group B). Group A was enriched for patients with of complex karyotype (14/20 patients, p<.001), isolated del(5q) (3/20) and for patients harboring TP53 mutation (11/20 patients, p<.001). In 280 patients (20 not tested), 70 patient had complex karyotype; 14/70 (20%, p<.001) patients with complex karyotype were from group A. Group A had a higher median burden of genomic than patients in group B (0.08 vs 0.04 alterations per pathway, p<.001). Patients in group A had a lower median overall survival (OS) than patients in group B (3.0 months vs 15.7 months, p<.001).

Conclusion: We demonstrated that patients with MHCI alterations have a high chance to be TP53 mutated or to have complex karyotype; patients with complex karyotype have a high chance to harbor concomitant alterations in MHC I antigen presentation. In the set of AML with high burden of aberrations, MHC I alterations could be permissive for leukemia development toward immune escape. Our approach is a good candidate for further testing inside clinical trials to identify a cohort of patients that are unlikely to respond to immune check-point inhibitors within patients with larger sets of patients with high burden of alterations. Acknowledgements: ELN, AIL, AIRC, Regione-Università 2010-12, FP7 NGS-PTL, HARMONY, Fondazione del Monte BO e RA.

#3789

Role of NOS2-COX2 crosstalk in tumor microenvironment of estrogen receptor-negative breast cancer and its therapeutic implications.

Debashree Basudhar,1 Sharon Glynn,2 Madison Greer,1 Veena Somasundaram,1 Jae H. No,1 David A. Scheiblin,1 Pablo Garrido,2 William F. Heinz,1 Aideen E. Ryan,2 Jonathan M. Weiss,1 Robert Y. Cheng,1 Lisa A. Ridnour,1 Stephen J. Lockett,1 Daniel W. McVicar,1 Stefan Ambs,1 David A. Wink1. 1 _National Cancer Institute, Frederick, MD;_ 2 _National University of Ireland Galway, Galway, Ireland_.

Tumor is often described as a wound that never heals. This leads to a chronic inflammatory tumor microenvironment characterized by infiltration of M2 macrophages and Th2 cells causing dysregulated release of multiple cytokines, chemokines and growth factors, thus creating a conducive environment for tumor growth and metastasis. In spite of significant progress in breast cancer treatment, metastatic breast cancer still remains a major health hazard with a high mortality rate among women. Moreover, there is cellular heterogeneity within and among different breast tumors, which poses a significant challenge in developing effective therapeutics, thus making it important to understand subtype-specific mechanisms. Our laboratory and other groups have previously shown that inducible nitric oxide synthase (NOS2), an enzyme involved in production and regulation of endogenous nitric oxide (NO), is a predictor of poor survival among highly metastatic ER-negative (ER-) breast cancer patients. Another proinflammatory enzyme, cyclooxygenase-2 (COX2,) responsible for conversion of arachidonic acid to prostaglandin E2 (PGE2), is also highly expressed in breast cancer and is detectable in ductal carcinoma in situ, invasive breast carcinoma, and metastatic lesions. We investigated the role of inflammation associated enzymes, NOS2 and COX2, and established that their simultaneous elevated expression significantly reduced patient survival (33%) when compared to greater than 95% survival of ER- patients with low NOS2/COX2 tumor expression. We further investigated their tumor subtype specific novel signaling mechanism in vitro and showed TNFα and/or endoplasmic reticulum stress as key players. Proinflammatory cytokines present in tumor microenvironment play a key role in regulation of this pathway and effectiveness of chemotherapeutics. Moreover, the ability of NOS2 and COX2 to regulate different cytokines in the tumor microenvironment further emphasizes the importance of their crosstalk in tumor progression, metastasis and ability of cancer cells to escape immune surveillance. Last, we demonstrated that simultaneous inhibition of COX2 and NOS2 using commercially available inhibitors significantly reduced tumor growth in murine models of ER- breast cancer, thus suggesting the beneficial effects of dual NOS2/COX2 therapy.

#3790

Human tumor-infiltrating monocytes/macrophages do not predominantly inhibit tumor-specific effector T cell responses in early-stage lung cancer: The role of macrophage versus tumor PD-L1.

Jason Stadanlick,1 Abhishek Rao,1 Sunil Singhal,1 Michael Annunziata,1 Pratick Bhojnagarwala,2 Shaun Obrien,2 Edmund Moon,2 Edward Cantu,2 Gwenn Danet-Desnoyers,1 Hyun-Jeong Ra,1 Leslie Litzky,1 Wayne W. Hancock,1 Steven M. Albelda,2 Evgeniy Eruslanov1. 1 _Abramson Cancer Ctr. of Univ. of Penn., Philadelphia, PA;_ 2 _University of Pennsylvania, Philadelphia, PA_.

Our current understanding of tumor-associated monocyte/macrophage lineage cells (MMLC) as being immunosuppressive is based largely on murine tumor models that generated a widespread interest in targeting these cells therapeutically. Unfortunately, this approach has not yet been successful in many clinical trials. One potential explanation is that there are fundamental differences between mice and humans in tumor evolution and immunity that might impact the function of human MMLC, limiting the success of myeloid cell-related therapies. Moreover, despite recent successes with checkpoint blockade therapy, the contribution of PD-L1 expressed on MMLC to T cell suppression in humans remains unclear. Thus, the overall purpose of this study was to determine the phenotypic composition of monocyte-macrophage cell lineage in a large cohort of early stage (resected) human lung tumors and delineate how different cell populations of this lineage regulate the effector phase of tumor-specific T cell responses. In addition we also explored the role of MMLC-expressed PD-L1 by interrogating the tripartite functional interactions between tumor-specific effector T cells, PD-L1\+ or PD-L1\- tumor cells, and TAM that possess a varied surface PD-L1 expression. Phenotypically, we find that (i) MMLC are not the predominate cell population of infiltrating leukocytes within the early-stage lung tumors, (ii) tumor MMLC consist primarily of tumor-associated macrophages (TAM), (iii) the TAM phenotype is complex and does not fit the conventional M1 and M2 phenotype denominations, (iv) TAM are able to co-express T cell co-inhibitory and co-stimulatory receptors. Functionally, MMLC are not primarily suppressive, but have diverse effects, including stimulation of the effector phase of tumor-specific T cell responses. Mechanistically, TAM-expressed PD-L1 (in contrast to tumor-expressed PD-L1), does not inhibit the interaction between tumor-specific effector T cells and tumor cells, but does protect TAMs expressing a tumor antigenic peptide/MHC class I complex from being killed by effector T cells recognizing the cognate peptide on the tumor. These data provide new insights into the phenotype of tumor MMLC and functional cross-talk between TAM, tumor-specific T cells, and tumor cells. Our results show that the prevailing premise that TAMs are predominantly immunosuppressive does not apply to early-stage human lung cancer and thus the current MMLC-targeted approaches would not be helpful in majority of early-stage lung cancer patients.

#3791

Triggering receptor expressed in myeloid cells 1 (TREM1) is a predictive marker for breast cancer therapeutics.

Ashok K. Pullikuth,1 Julia Chifman,2 Eric D. Routh,1 Jeff Chou,1 Guangxu Jin,1 Michael A. Black,3 Lance D. Miller1. 1 _Wake Forest University School of Medicine, Winston Salem, NC;_ 2 _American University, Washington, D.C., DC;_ 3 _Otago School of Medical Sciences, University of Otago, Dundein, New Zealand_.

Triggering receptor expressed on myeloid cells-1 (TREM1) is a cell surface immunoglobulin that amplifies pro-inflammatory responses in myeloid cell populations and has been implicated in carcinogen-induced liver cancer. However, its role in breast cancer is unknown. In a recent screen to identify gene expression patterns associated with breast cancer outcomes, we identified a significant and reproducible positive association between TREM1 expression and breast cancer recurrence in two large breast cancer cohorts comprising 1,984 and 616 patients (p<0.001 and p=0.003, respectively). TREM1 expression was significantly associated with recurrence in both basal-like (p<0.001) and luminal tumor subtypes (Luminal A, p=0.047; Luminal B, p=0.022) and remained significant (p<0.001) in a multivariate Cox regression model, independent of histologic grade, tumor size, nodal status, patient age, treatment and molecular subtype. In pre-treatment biopsies of patients treated with neoadjuvant chemotherapy, high TREM1 expression was associated with a significant decrease in pathologic complete response (pCR) rate (p<0.001), especially in those tumors estimated to have high levels of lymphocytic infiltrate that otherwise associated with an increased pCR rate, consistent with an immune regulatory function for TREM1. Bioinformatics analysis of genes reproducibly correlated with TREM1 expression in breast tumors revealed significant enrichment in myeloid immune cell populations (p<0.001) in general, and neutrophils (p<0.001), M0 macrophages (p<0.001) and M2 macrophages (p<0.001), in particular. Immunohistochemical analysis of breast tumor tissues confirmed colocalization of TREM1 protein expression with the pan-macrophage marker, CD68. These findings, together with the established role for TREM1 in promoting inflammation, fit a model where tumor-infiltrating TREM1-positive myeloid cells impact breast cancer outcomes by promoting immune evasion.

#3792

**Intratumoral CD56** bright **natural killer cells are associated with improved survival in bladder cancer.**

Neelam Mukherjee,1 Niannian Ji,1 Maggie E. Tomasini,1 Vincent Hurez,1 Tyler J. Curiel,1 Maureen O. Montgomery,1 Andrew J. Braun,1 Marlo Nicolas,1 Marcela A. Flores,2 Qianqian Liu,1 Jianhua Ruan,2 Robert S. Svatek1. 1 _UT Health Science Ctr. at San Antonio, San Antonio, TX;_ 2 _UTSA, San Antonio, TX_.

Antitumor immune responses are largely mediated by cytotoxic lymphocytes (CD8+ T cells) that recognize tumor-associated antigens presented in the context of a major histocompatibility complex class I molecule (MHC I). However, many tumors downregulate MHC I expression as a means of immune escape. Under these conditions, additional effector lymphocytes, such as natural killer cells (NK cells) which mediate non-MHC restricted cytotoxicity, provide antitumor defenses. Although the presence of intratumoral CD8+ T cells has been associated with an improved survival in muscle-invasive bladder cancer, the role of other effector lymphocytes including NK cells in bladder cancer (BC) is less studied. Here, a comprehensive examination of intratumoral lymphocytes in murine and human urothelial tumors revealed a relatively high proportion of macrophages, γδ T cells and NK cells and whereas macrophages were detrimental, NK cells were protective to BC growth. γδ T cells and NK cells contribute to innate effector immune responses and likely crosstalk as γδ T cells can boost NK cell cytotoxic function. Our findings suggest that γδ T cells could potentially have a role in activating NK cells function marked by increased IFN-γ and TNF-α expression. In patients undergoing cystectomy for muscle-invasive BC, a higher frequency of intratumoral NK cells was associated with an improved survival and this association was maintained in MHC I low but not MHC I high tumors. Higher stage bladder tumors had a higher proportion of intratumoral CD56dim NK cells, which exhibit less cytotoxicity and IFN-γ secretion compared to their CD56bright counterparts. Moreover, bladder infiltrating CD56bright but not CD56dim NK cells predicted improved survival for cystectomy patients. Further, older patients with BC had significantly less intratumoral NK cells compared to younger patients. Interestingly, surface expression of CD56 on NK cells decreased with increasing patient age and the proportion of CD56bright subsets decreased with increasing age whereas the proportion of CD56dim subsets slightly increased with age. Finally, because intravesical Bacillus Calmette-Guérin (BCG) activates NK cells and is commonly used to treat non-muscle invasive BC, we conducted a proof-of-concept trial of administering BCG to patients with muscle-invasive BC prior to cystectomy. While intravesical BCG was well tolerated and increased the percentage of intratumoral NK cells in this neoadjuvant setting, it had no discernable antitumor activity and did not alter CD56 expression or functional status of intratumoral NK cells. We conclude that NK cells are protective in BC and the extent of an intratumoral CD56bright NK cells is a prognostic indicator in BC. While BCG may induce NK cells, alternative approaches to improve NK cell function in BC are warranted.

#3793

Immune cells in gastric cancer.

Carlos Castaneda, Miluska Castillo, Luis Bernabé Monsalve, Joselyn Sanchez Sifuentes, Eduardo Payet Meza, Eloy Ruiz, Fernando Barreda Bolaños, Manuel Chumpitaz La Rosa Sanchez, Brayam Felix, Katherine Tello, Claudio Flores, Carlos Chavez, Ivan Chavez Passiuri. _Instituto Nacional de Enfermedades Neoplasicas, Lima, Peru_.

Introduction: Role of TIL and interaction with H. pylori infection is not understood in Gastric Cancer.

Methods: Gastric tissue samples were retrospectively collected from 50 patients who were refereed to surgery at Instituto Nacional de Enfermedades Neoplasicas between 2011 and 2012. We prospectively evaluated the distribution and density of tumor-infiltrating lymphocytes (TIL) as well as the presence of H. pylori. An evaluation of the role of the density of different subpopulations of infiltrating immune cells in tissue TMA was additionally performed. Clinicopathological data were collected from medical and pathology reports. Kaplan-Meier estimation curves overall survival (OS) was applied. A p-value ≤0.05 was considered statistically significant.

Results: Median age was 52 years and 50% were male, most were HG-3 (83%), Lauren classification (76%) and clinical stage III (50%). Most cases went to subtotal gastrectomy (64%). H. pylori was present in 34% (17/49) and most frequent density was moderate (++) (10/49).

Intratumoral TIL mild (92%) and bandlike infiltration (68%).

Median percentage of intratumoral CD3, CD4, CD8, CD20, CD68 and CD163 TILs in 5 HPF were 12.00±0.108, 1.55±0.017, 7.96±0.093, 1.34±0.057, 12.30±.054 and 8.88±0.069, respectively.

No significant association between immune cell subpopulation density and features was found, however it was a trend to association for: Older age and CD163 (p=0.184), male sex and CD8 (p=0.166) and CD4 (p=0.171), poorly differentiated HG and CD4 (p=0.071), early stage and CD4 (p=0.084), diffuse Lauren classification and CD4 (p=0.13), and H. pylori 0&+/+++ and CD68 (p=0.171).

The median follow-up was 4.7 years (CI95%: 4.5-5.0), median survival was 1.7 years (CI95%. 0.6-2.9). Earlier clinical stage (p=0.003), non-lymphatic vascular invasion (p=0.024) and absence of node involvement (p=0.018), early Bormann classification (p=0.030) and absence of metastasis (p=0.029) were associates to survival. In relation to the types of TILs, CD3 (<27.5, p=0.051), CD4 (<2.6, p=0.015), CD8 (>6.2, p=0.049), and CD4/CD8 (<0.31, p=0.006) were associated with poor survival. However, in the Cox model age (>60yr HR: 5.1) Bormann (IV, HR: 22.8), NLR (<2.13, HR: 29.2), LMR (>3.86, HR: 19.8) and CD4/CD8 (<0.31, HR: 10.3) were associated with poor prognosis.Conclusion: Patients with NLR and CD4/CD8 TIL high had better survival. These results suggest the prognostic implications of biomarkers and type of TIL in patients with AGC that could be used in clinical practice.

Keywords: Gastric cancer, tumor-infiltrating lymphocytes, H. pylori

This study was supported by the CIENCIACTIVA-CONCYTEC, under the contract #197-2015-FONDECYT

#3794

CCL5 from tumor-associated macrophages/microglia (TAMs) regulates glioma migration and invasion via calcium-dependent matrix metalloproteinase-2.

Caren Yu-Ju Wu,1 Chia-Hua Chen,2 Chun-Yen Lin,2 Li-Ying Feng,2 Yung-Chang Lin,2 Kuo-Chen Wei,2 Chiung-Yin Huang,2 Jia-You Fang,1 Pin-Yuan Chen3. 1 _Chang Gung University, Taoyuan, Taiwan;_ 2 _Linkou Chang Gung Memorial Hospital, Taoyuan, Taiwan;_ 3 _Keelung Chang Gung Memorial Hospital, Keelung, Taiwan_.

Tumor-associated macrophages/microglia (TAMs) are either macrophages of peripheral origin or brain-intrinsic microglia, and represent a considerable portion of the glioma mass, which facilitate gliomagenesis. Chemokine C-C motif ligand 5 (CCL5) is an inflammatory mediator produced by immune cells, and involves in tumor growth, and migration in serval cancers including glioma. However, the mechanism of how CCL5 facilitates glioma invasion remains largely unresolved. In this study, we demonstrate that CCL5 modulate human glioma cell lines, A172 and U87, migratory and invasive activities along with MMPs expression. In response to CCL5, glioma synchronized an increase of intracellular calcium levels, and kinase elevations including p-CaMKII, and p-Akt expressions in time and dosage manners. Importantly, glioma invasion along with MMPs elevation are dependent on CCL5-propogated p-CaMKII expression, and inhibition of p-CaMKII effectively reduced CCL5-directed glioma MMPs expression and invasion. Using three-dimensional cultures, we demonstrated that glioma cells tend to homing toward GM-CSF-activated TAM condition media. This homing effect associated with MMPs elevation which can be ameliorated either by controlling the intracellular and extracellular calcium levels or by CCL5 antagonism. Clinical results also reveal association of CCL5 with TAM activation, and patients' overall survival. Our results suggest that modulation of glioma CaMKII may restrict the effect of CCL5 on glioma migration and could be a potential therapeutic strategy for alleviating glioma invasion.

#3795

Novel mechanism underlying tumor immune evasion through tumor endothelial cells.

Kazuhiro Taguchi,1 Takashi Onoe,1 Hideki Ohdan2. 1 _National Hospital Organization, Kure Medical Center/Chugoku Cancer Center, Hiroshima, Japan;_ 2 _Hiroshima University, Hiroshima, Japan_.

Background: There has been a recent interest in how tumor cells escape from immune surveillance while establishing tumor microenvironments. Tumor endothelial cells (TECs), which constitute the lining of the tumor vessels, develop in the tumor microenvironment and come in contact with circulating immune cells. As such, TECs may control the trafficking or the anti-tumor reaction of an immune cell. We hypothesized that TECs may have an immune-suppressive potential in tumor microenvironments; thus, TECs may contribute to tumor immune-evasion.

Objective: We aimed to investigate the immune-suppressive functions of TECs.

Materials and Methods: We established a mouse cancer-model using the B16 mouse melanoma cell line and isolated TECs from the tumors. The immunological character of TECs was assessed by performing flow cytometric phenotyping of TECs and in vitro antigen endocytosis assay using exogenous antigen ovalbumin (OVA). Next, to identify the antigen presentation and immune-suppressive functions of TECs, B16 cells transfected with ovalbumin (B16-OVA) were inoculated in mice and the TECs isolated from the B16-OVA tumor were analyzed. An antibody reacting specifically to complex of OVA-derived antigen presented with mouse MHC class I was used to test antigen presentation. Further, to verify antigen-specific suppressive functions, TECs isolated from B16-OVA tumors were co-cultured with OVA-specific CD8+ T cells (OT-I cells) stimulated by OVA-pulsed bone marrow derived dendritic cells (BMDCs) in vitro. In this suppression assay, we performed a blocking assay using an anti-programmed death-ligand 1 (PD-L1) antibody. We also performed an antigen-specific cytotoxicity assay using B16-OVA cells as targets and OVA-specific CD8+ T cells which had been co-cultured with TECs as effectors.

Results: TEC expressed MHC class I/II and co-stimulating molecules and PD-L1, suggesting that TECs had antigen-presenting and immune-suppressive properties. We found that TECs endocytosed B16-OVA derived protein and presented tumor-derived OVA peptides with their MHC class I molecule. TECs from B16-OVA tumor showed significantly greater suppression of OT-I CD8+ T cell proliferation compared to those from non-transfected B16 tumor. Similarly, in a cytotoxicity assay, TECs from B16-OVA tumor suppressed cytotoxicity of CD8+ T cells in an antigen specific manner. Strikingly, this suppressive function of TECs for the proliferation and cytotoxicity of OVA-specific T cells was abrogated by blocking PD-L1 using an anti-PD-L1 antibody, suggesting that the PD-1/PD-L1 axis contributes towards the immune-evasion mechanism.

Conclusions: TECs have the characteristics of antigen presenting cells and regulate the immune response of tumor antigen specific T cells via the PD-1/PD-L1 pathway. Our data may suggest the novel mechanism of tumor immune-evasion through TECs.

#3796

Substrate specificity of human kallikrein-related peptidase 2 (KLK2) towards beta chain of interleukin-10 receptor.

Luiz J. Neto,1 Maria A. Juliano,2 Juliana R. Oliveira2. 1 _A.C.Camargo Cancer Center, São Paulo, Brazil;_ 2 _Universidade Federal de São Paulo, São Paulo, Brazil_.

The kallikrein-related peptidases (KLKs) comprise a family of 15 homologous secreted trypsin- or chymotrypsin-like serine proteases. KLK2 and KLK3 have the most organ restricted expression profile of all KLKs; specifically, they are abundantly expressed in the luminal epithelium of the prostate. The expression of KLK2 gene is regulated by the androgens and androgen receptor (AR) signaling and like human PSA, serum KLK2 can also function as a biomarker for prostate cancer (PCa). The present work provides biochemical data related to the hydrolytic activities of KLK2 on libraries of fluorescence resonance energy transfer (FRET) peptides in the presence of sodium citrate, a kosmotropic salt that is present in normal prostate tissue and seminal fluid in high concentration. The peptide SYR↓IF (cleaved at↓) was the highly hydrolyzed and best substrate so far reported for KLK2. The sequence YRIF is present in the N-terminal segment (59YRIF62) in the β chain of interleukin-10 receptor. In order to verify the effect of KLK2 in the IL10RB surface expression in the bone marrow-derived macrophages (BMDM) cells, they were incubated with KLK2 at 37ºC for 2 hours. BMDM treated with KLK2 showed a significant reduction in the IL10RB surface expression compared with control group, indicating that KLK2 acts directly on this receptor, decreasing its expression. The effect of this reduction was evaluated trough BMDM activation assay. After incubation of BMDM cells with KLK2, they were washed and incubated with or without LPS, IFN-γ and recombinant IL10. When incubated with recombinant IL10, control macrophage showed a significant reduction of NO, TNF-α and IL-12p40 levels indicating the IL10RB functionality and according to IL10 immunosuppressive effect. However, KLK2 pretreated macrophage maintained NO, TNF-α and IL-12p40 levels, even in the presence of recombinant IL10, leading a proinflammatory status in BMDM culture, while KLK3 doesn't show the same effect. Taken together, the data indicates that KLK2 decreases the surface expression of IL10RB on macrophage cells and contributes to a proinflammatory environment.

#3797

Chromosome instability as a prognostic factor& an immunotherapeutic target in acute myeloid leukemia.

Ning Jin, Mark Burkard. _University of Wiscosin, Madison, WI_.

Chromosome instability (CIN) is an increased rate of chromosome mis-segregation due to errors in mitosis. CIN generates genetic and karyotypic diversity and is common in hematological malignancies. Low to moderate levels of CIN are well tolerated, and moderate levels can promote cancer proliferation by generating genetic diversity for tumor evolution. However, very high levels of CIN are lethal and enhancing CIN can be therapeutic. Therefore, CIN status could be prognostic for clinical outcomes as well as a therapeutic target. Despite its promise, little is known about CIN in AML.

We first determined if CIN is correlated with clinical outcome in patients with acute myeloid leukemia (AML). We quantified the chromosome mis-segregation frequency (lagging chromosomes and chromatin bridges) in dividing AML cells on bone marrow core biopsies. Our study reveals a wide distribution of CIN in AML core biopsy specimens, ranging from 20% to 67%. High CIN correlates with a better overall survival in patients with AML.

Recent data suggest that chromosome mis-segregation promotes senescence and initiates immune clearance. Our hypothesis is that chromosome instability is immunogenic due to DNA damage, proteotoxic/oxidative stress with a high mutagenic potential, which can be recognized by immune system. To test this, we used an inhibitor of Monopolar spindle 1 (Mps1) to enhance CIN and tested if that altered immunogenicity. Mps1 is a key component of the mitotic checkpoint and is also involved with chromosome alignment. Inhibition of Mps1 by AZ3146 results in severe chromosome mis-segregation, micronuclei formation, and apoptosis in AML cell lines. In addition, AZ3146 treatment causes activation of γH2AX due to DNA damage, and calreticulin exposure from endoplasmic reticulum stress. This generates danger signals to activate innate immune system (natural killer cells and dendritic cells) and induces immunogenic cell death (ICD) through priming of cytotoxic T cells. This effect is mainly seen in synchronized mitotic cells, suggesting AZ3146 acts specifically on mitosis. Additionally, we observed the overexpression of programed death ligand-1 (PD-L1) after the treatment of AZ3146 in AML cells. As evidence showed, PD-L1 participates in the immune evasion and protects AML blasts from cytotoxic T cells in mouse models of leukemia.

Taken together, our observations in Mps1 inhibitor treatment indicate the immunogenicity of CIN in AML. Additionally, these data support combination treatment of mitotic checkpoint inhibitor with immunotherapy to improve therapeutic efficacy in AML patients.

#3798

Gut microbiota modulates adoptive cell therapy via CD8α dendritic cells.

Mireia Uribe-Herranz,1 Kyle Bittinger,2 Stavros Rafail,1 Sonia Guedan,1 Stefano Pierini,1 Ceylan Tanes,2 Saar A. Gill,1 Janos Tany,1 Mark A. Morgan,1 Frederic D. Bushman,1 Carl H. June,1 Andrea Facciabene1. 1 _University of Pennsylvania, Philadelphia, PA;_ 2 _Children's Hospital of Philadelphia, Philadelphia, PA_.

Adoptive T-Cell therapy (ACT) is a promising new modality for malignancies. Here, we report that adoptive T-cell efficacy in tumor-bearing mice is significantly affected by differences in the native composition of the gut microbiome, treatment with antibiotics or by heterologous fecal transfer. Depletion of bacteria with vancomycin, decreased the rate of tumor growth in mice receiving ACT, whereas treatment with neomycin and metronidazole had no effect, indicating the role of specific bacteria in host response. Vancomycin treatment induced an increase in systemic CD8α\+ dendritic cells, which mediated a sustained systemic expansion of adoptively transferred antitumor T cells in an IL-12-dependent manner. In subjects undergoing allogeneic hematopoietic cell transplantation, we found that oral vancomycin also increased IL-12 levels. Collectively, our findings demonstrate an important role played by the gut microbiota in the antitumor effectiveness of ACT and suggest new avenues to improve response to ACT by altering the gut microbiota.

#3799

Heparan sulfate modifiers and signaling between tumor-associated macrophages and cancer-associated fibroblasts in a biomimetic hydrogel model of bone-metastatic prostate cancer.

Fabio Henrique Brasil da Costa,1 Nora M. Navone,2 Mary C. Farach-Carson,3 Daniel D. Carson1. 1 _Rice University, Houston, TX;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _The University of Texas Health Science Center at Houston, Houston, TX_.

The establishment of a new tumor niche for most carcinomas is orchestrated primarily by the bidirectional engagement of cancer cells and fibroblasts. Cancer cells reprogram and activate fibroblasts into a wound healing-like state and, in turn, these cancer-associated fibroblasts (CAFs) react by remodeling the extracellular matrix (ECM) and secreting chemokines and growth factors. Macrophages are rapidly recruited to the tumor and represent the primary immune cell in the microenvironment. Tumor-associated macrophages (TAMs) enter a wound healing-like loop, displaying the full spectrum of macrophage phenotypes and behaviors as the disease progresses. Among these activities is the ability to modulate growth factor bioavailability. Deposition of the heparan sulfate (HS) proteoglycan perlecan/HSPG2 in the ECM and secretion of HS modulators are increased as result of the cancer-CAF-TAM trialogue and play critical roles in the availability and signaling of growth factors, cytokines, and chemokines. In this work, we developed a novel, biomimetic 3D hydrogel system composed of hyaluronic acid and collagen I (1:1) to study prostate cancer bone metastases. Our goal was to explore the molecular signaling between TAMs and CAFs to learn how this interaction affects the expression of the enzymes that modulate HS as a growth factor depot. Essential modulators of HS include heparanase (HPSE) and sulfatases 1 and 2 (SULF1, 2), all of which selectively release growth factors to influence signaling in the tumor microenvironment. We found that alternatively activated macrophages (M2) and TAMs (M2-like) increase levels of HPSE and SULFs in CAFs. We next examined the impact of TAM-CAF co-signaling on prostate cancer viability, growth, and dependence on HS modulators. Our data show that as a consequence of the TAM-CAF interaction, the elevated levels of HS-modifying enzymes are sufficient to trigger substantial changes in prostate cancer cell behavior, including viability, proliferation, and response to growth factors. We currently are assessing if these outcomes are seen in a series of patient-derived prostate xenograft lines representing different prostate cancer subtypes. We conclude that our 3D hydrogel model is a physiologically relevant in vitro system to discover fundamental aspects of HS signaling in the tumor microenvironment and thus uncover new ways to reduce the bioavailability of growth factors in the prostate cancer microenvironment and interfere with cancer progression. (Supported by NIH P01 and CAPES.)

#3800

Inhibition of Polycomb Repressive Complex 2 EZH2 lysine methyltransferase improves tumoricidal activity of macrophages towards mesothelioma cells.

Malik Hamaidia, Clotilde Hoyos, Luc L. Willems. _National Fund for Scientific Research, Gembloux, Belgium_.

Rationale: Clinical evidence indicates that tumor infiltration by tumor associated macrophages (TAMs) correlates with poor prognosis in malignant mesothelioma (MM). By attenuating the immune response, TAMs indeed promote survival of MM cells. TAMs share properties with alternative macrophages (M2) that are activated by anti-inflammatory (e.g. IL-10) or Th2-associated (i.e. IL-4, IL-13) cytokines.

Objectives: We hypothesized that macrophage activation is mediated by a transcriptional program tightly regulated by epigenetic modifications. We focused on the Polycomb Repressive Complex 2 (PRC2) EZH2 lysine methyltransferase responsible for trimethylation of histone H3 at lysine 27 (H3K27me3). We investigated the effect of a selective EZH2 inhibitor (EPZ005687) on tumoricidal activity of primary human monocyte-derived macrophages.

Results: Our data show that inhibition of EZH2 reduces CD206 expression and phagocytic activity of macrophages conditioned in MM pleural effusions (PE-macrophages). Moreover, EZH2 inhibition enhances the cytotoxic potential of PE-macrophages towards MM cells. Inhibition of EZH2 also increases the ability of macrophages to support proliferation of autologous T-cells.

Conclusion: Induction of cytotoxic activity of PE-macrophages via PRC2 EZH2 lysine methyltransferase could be of therapeutic value in MM.

#3801

Functional relevance of A-to-I RNA editing on the immune response in breast cancer.

Floriane Dupont,1 Florian Clatot,2 David Venet,1 Véronique Kruys,3 Debora Fumagalli,4 Vincent Detours,5 Françoise Rothé,1 Christos Sotiriou1. 1 _Institut Jules Bordet - ULB, Bruxelles, Belgium;_ 2 _Henri-Becquerel Center and INSERM U1245, Bruxelles, France;_ 3 _Institut de Biologie et de Médecine Moléculaires - ULB, Gosselies, Belgium;_ 4 _Breast International Group (BIG) - AISBL, Bruxelles, Belgium;_ 5 _IRIBHM, Université Libre de Bruxelles (ULB), Bruxelles, Belgium_.

INTRODUCTION: RNA editing is a post-transcriptional modification that changes double stranded RNA sequences (dsRNA). The most common way of editing in humans is the Adenosine-to-Inosine type, which is catalyzed by members of the ADAR enzymes family. A-to-I RNA editing has been shown to be essential for normal tissue development but also plays a crucial role in many cancers. We have notably shown that in breast cancer the editing frequency is increased in tumors as compared to normal tissue and correlates with ADAR expression. We also showed that type I IFN response and ADAR DNA copy number together explain 53% of ADAR expression in breast cancers. Moreover, the implication of ADAR-mediated RNA editing in preventing the sensing of endogenous dsRNA leading to inhibition of the immune response has been recently reported. ADAR-mediated RNA editing may play a crucial role in tumor progression by modulating immune response through the dsRNA sensing pathway. Here, we aim to assess the influence of ADAR-mediated RNA editing in the sensing of endogenous dsRNA and the immune response in breast cancer.

MATERIAL & METHODS: ADAR expression was modulated in MDAMB231 breast cancer cell line using shRNA lentiviral transduction and lentiviral plasmid transduction for ADAR inhibition and ADAR wild-type and editing-deficient mutant overexpression respectively. ADAR expression was assessed by RTqPCR/Western blotting. dsRNA level was evaluated by immunofluorescence using the dsRNA-specific J2 antibody (Scicons).

RESULTS: Poly I:C transfection, mimicking dsRNAs, as well as IFN Type I exposure induced the immune response in BC cell lines, in particular it increases the expression of RIG-I, MDA5 and IRF7 involved in the dsRNA sensing pathway. DNMTi treatment, known as an inducer of dsRNA level, also activated cytosolic dsRNA response in these cells in a dose dependent manner. In order to assess the role of ADAR-mediated RNA editing in the activation of the dsRNA sensing pathway, we exposed MDAMB231 cell lines in which ADAR expression was modulated to DNMTi. A wide range of doses of DNMTi, from 0.3 to 50μM, and time of exposure, from 3h to 72h, were tested. The cytosolic dsRNA pathway was induced in a dose dependent manner after DNMTi exposure. However, no effect of ADAR modulation on the activation of the pathway was observed. No difference in dsRNA levels was observed after activation of the immune pathway by DNMTi or IFN exposure, nor after ADAR modulation.

CONCLUSION & PERSPECTIVES: dsRNA pathway activation by DNMTi, poly I:C transfection or IFN Type I treatment is not dependent of ADAR in MDAMB231 cell lines. Moreover, in contrast to data in colorectal cell lines, our data suggest that DNMTi exposure does not increase cytosolic dsRNA level.

#3802

Human NKG2D ligand regulation of Natural Killer cell function and its implications in cancer and inflammation.

Payal Dhar, Jennifer Wu. _Northwestern University, Chicago, IL_.

The significance of ligand-induced activation of Natural Killer cell activating receptor NKG2D in controlling tumor growth has been well established in various experimental animal models. Amongst different types of NKG2D ligands, MHC I chain related molecule MICA/B is expressed most prevalently in various human malignancies, but has not been found in rodent family. Proteolytic-mediated shed-form of soluble MICA/B (sMIC) has been shown to suppress the immune system and correlate with advanced disease stages in cancer patients. Previous studies in our lab using two humanized bi-transgenic mice, one expressing native ligand (TRAMP/MICB) and alternatively expressing the shedding-resistant mutant (TRAMP/MICB.A2) revealed opposite roles of membrane-bound and soluble form of MIC in regulating tumor immunity. Mice with high levels of sMIC exhibited increased tumor progression, whereas mice expressing membrane-bound MICB.A2 in tumors enjoyed tumor-free survival. These findings raised the intriguing question of why do these structurally similar forms of MIC have vastly different effects on tumor immunity. To address this question, we investigated the signaling events and functional outcomes in NK cells upon stimulation by the two forms of ligands. In vitro co-culture studies of NK cells with tumor cell lines expressing sMICB and MICB.A2 revealed elevated pro-inflammatory cytokine production by NK cells upon stimulation with sMICB. In contrast, NK cells stimulated with MICB.A2 displayed enhanced expression of cytotoxicity mediators and signaling molecules of cytotoxicity pathway. This suggests that signaling through sMICB may polarize the NKG2D signaling pathways with preferential activation of inflammatory cytokine pathways. Our data has uncovered a new potential mechanism whereby sMIC promotes tumor progression and supports the notion that sMIC is a viable target for cancer immunotherapy.

#3803

Immune escape from NK cells and activated CD8 T cells by miR-183 repression of MICA/B expression in human lung cancer.

Nhan Tu, Thu Le Trinh, Sarah S. Donatelli, Melba M. Tejera, Danielle L. Gilvary, Jun-Min Zhou, Erika A. Egsioglu, Sheng Wei, Domenico Coppola, Julie Y. Djeu. _Moffitt Cancer Center, Tampa, FL_.

Immune escape is a hallmark of cancer. In human lung cancer, we have identified a unique microRNA (miR)-based pathway employed by tumor cells to repress detection by immune cells via the NKG2D-MICA/B receptor-ligand system. MICA/B is readily induced by cell transformation and serves as a danger signal and ligand to alert NK and activated CD8+ T cells. However, immunohistochemical analysis indicated that human lung adenocarcinoma and squamous cell carcinoma specimens express little MICA/B while high levels of miR-183 were detected in both tumor types in a TCGA database. Human lung tumor cell lines confirmed the reverse relationship in expression of MICA/B and miR-183. Importantly, a miR-183 binding site was identified on the 3' untranslated region (UTR) of both MICA and MICB, suggesting its role in MICA/B regulation. Luciferase reporter constructs bearing the 3'UTR of MICA or MICB in 293 cells supported the function of miR-183 in repressing MICA/B expression. Additionally, anti-sense miR-183 transfection into H1355 or H1299 tumor cells caused the upregulation of MICA/B. Abundant miR-183 expression in tumor cells was traced to transforming growth factor-beta (TGFβ), as evidenced by anti-sense TGFβ transfection into H1355 or H1299 tumor cells, which subsequently lost miR-183 expression accompanied by MICA/B upregulation. Most significantly, anti-sense miR-183 transfected tumor cells became more sensitive to lysis by activated CD8+ T cells that express high levels of NKG2D. Thus, high miR-183 triggered by TGFβ expressed in lung tumor cells can target MICA/B expression to circumvent detection by NKG2D on immune cells.

#3805

Expression of tryptophan -2, 3-dioxygense (TDO) in metastatic uveal melanoma.

Mizue Terai, Emma Link, Eric Link, Bao Lam, Marlana Orloff, Takami Sato. _Sidney Kimmel Medical Collegen of Thomas Jefferson University, Philadelphia, PA_.

Uveal (eye) melanoma (UM) is the most common primary eye malignancy in adults. Despite effective treatments for primary UM, up to 50% of patients subsequently develop systemic metastasis, predominantly in the liver. Once hepatic metastasis develops, the survival of UM patients is generally short and currently available treatments fail to show meaningful improvement of their survival. Metastatic uveal melanoma (MUM) is highly resistant to traditional systemic therapies, including immunotherapy. Immune checkpoint blockades approved for metastatic cutaneous melanoma (MCM) such as anti-CTLA4 antibody and anti-PD-1 antibody have shown only marginal clinical benefit in MUM (response rates <5%). We have reported that, while PD-1 expression was seen in both MCM and MUM, there was a stark difference in PD‐L1 expression between these two types of melanoma (MCM vs. MUM, 20.8% vs. 0%) (ASCO 2016, A Javed, et al). It is of note that, of 6 MCM specimens obtained from hepatic metastasis, none expressed PD‐L1. These results indicate the presence of unique immune microenvironment in the liver, harboring melanoma metastasis. One of the factors dominant in the liver is tryptophan 2,3-dioxygenase (TDO). TDO and indoleamine 2,3-dioxygenase (IDO) catalyze tryptophan and produce kynurenine, a metabolite that reportedly inhibits anti-tumor T cell immune responses. Recent studies have revealed that TDO is constitutively expressed in a wide variety of cancer cells. Investigation on TCGA data indicates expression of TDO2 in 62% of primary UM. 42% of samples showed increased TDO2 expression (RPKM >= 1 RPKM), while TDO2 message is not present in 1/3 of the samples. Expression of TDO2 in primary UM correlated to poor survival (Cox regression hazard ratio 0.7, p=0.04). Moreover, TDO2 expression correlated to patients with BAP1 mutations (p=0.00071) and differences between patients with monosomy 3 and disomy 3 (p=0.00017). We further investigated whether MUM cell lines and MUM tumor specimens express TDO. We detected TDO protein in all of 4 MUM cell lines by Western blotting. Although inducible by exogenous IFN-gamma, IDO1 protein was not detected in any of these MUM cell lines without stimulation. TDO mRNA was increased 3.5 fold by stimulating MUM cells with recombinant human TNF-alpha. All MUM cell lines possess TNF-alpha receptors. We have also investigated TDO and IDO1 proteins in 10 MUM specimens obtained from hepatic metastasis by immuno-histochemical staining. TDO was positive in both normal hepatocytes as well as tumor themselves in all MUM specimens. Interestingly, the intensity of TDO staining is much stronger in MUM, compared to the surrounding liver tissues. On the other hand, IDO1 protein was not positive in any of MUM tissues obtained from liver metastasis. These results indicate that expression of TDO by MUM cells might be one of the key mechanisms for escape from T-cell immune surveillance in hepatic metastasis from primary UM.

#3806

The pattern of NKG2DL expressions determines immunogenicity and antitumor effect of protein-bound polysaccharide-K in tumor-bearing mouse models.

Tomohiro Takehara,1 Kenzo Soejima,1 Tomoko Betsuyaku,1 Kiyoshi Yoshimura2. 1 _Keio University, Tokyo, Japan;_ 2 _National Cancer Center, Tokyo, Japan_.

Background and Aim: Natural killer group 2, member D ligands (NKG2DLs) have paradoxical immunological characteristics, exerting both immune-stimulatory and immune-suppressive effects on T cell activation. First, we attempted to determine the relationship between the antitumor effect of protein-bound polysaccharide-K (PSK) in a tumor-bearing mouse model and the expression profile of NKG2DLs on a tumor. Next, we investigated whether NKG2DL expression on tumor cells affects T cell activation by examining the expression of NKG2D on and IFN-γ production in CD8+ T cells in mice bearing NKG2DL‒expressing and NKG2DL‒nonexpressing tumors.Materials and Methods: PSK was administered to evaluate its effectiveness against tumor growth. The expression of Rae-1 and H60 were analyzed in the cell lines. Results: PSK showed the highest antitumor effects in mice implanted with cells expressing neither Rae-1 nor H60. PSK had little antitumor effect in mice implanted with cells expressing both Rae-1 and H60. A correlation between the expression of NKG2DLs and the antitumor effect of PSK was observed. After PSK administration, INF-γ production in CD8+ T cells increased in mice with cells expressing neither Rae-1 nor H60, but did not change in mice implanted with cells expressing both Rae-1 and H60.Conclusion: we demonstrated that the expression pattern of NKG2DLs affects tumor immunity and the efficacy of immunotherapy in a tumor-bearing mouse model. In particular, the expression of H60 and Rae-1 family members may be an immunosuppressive factor. Further analysis is required to gain a better understanding of the mechanism of interaction between NKG2D expressed on immune effector cells and H60/Rae-1 expression on tumors. Although NKG2DL expression may represent a marker predicting response to immunotherapy, further studies in humans are required to validate this finding in the clinical setting.

#3808

Interrogation and modulation of the immunosuppressive activity of human TAM-like macrophages using in vitro cultures.

Laura E. Gallagher, Andrew Hall, Lauren A. Patience, Lucia Janicova, Stephen Anderton. _Aquila BioMedical, Edinburgh, United Kingdom_.

Tumor Associated Macrophages (TAMs), characteristic of an M2-like immune-suppressive phenotype, can induce proliferation and survival of tumor cells, facilitate angiogenesis, and suppress anti-tumor immune responses via expression of co-inhibitory molecules (e.g. PD-L1) and cytokines (e.g. IL-10, TGF-β). TAMs are therefore a highly attractive target of innovative cancer immunotherapies. Understanding the ability of pre-clinical candidate compounds to reverse TAM (M2-like)-mediated immune suppression and the potential for reprogramming of M2-like macrophages to a M1-like phenotype is key in the development of effective TAM-targeted cancer immunotherapies. Here we outline development of an assay to assess the capabilities of pre-clinical compounds to reverse M2 macrophage-mediated immune suppression.

Monocytes were isolated from whole blood obtained from healthy volunteers and cultured under M2-polarising conditions. The resulting macrophages were phenotypically characterized and then used in co-culture with autologous PBMC, stimulated through T cell receptor ligation. Resulting cytokine production was assessed, alongside CD4+ and CD8+ T cell viability and cell cycle status (flow cytometry).

M2-like macrophages polarized with M-CSF displayed an immune suppressive phenotype as shown by their production of IL-10 and their inhibition of IFN-γ production by, and cell cycle status of, T cells in co-culture assays. This suppressive activity was only partially reversed by PD-1-blockade. Modifications to the macrophage polarization protocol were seen to alter the resulting macrophage cell surface phenotype (e.g. expression levels of PD-L1, TIM-3 and CD200R) and their suppressive activity in the co-culture assay. These cell surface phenotypes broadly reflected those seen amongst tumor-derived macrophages from renal and ovarian carcinoma patients (CD14+ CD163+ with expression of TIM-3 and LAG-3). Moreover, compound-mediated changes in functionality seen with macrophages polarized from healthy PBMC monocytes could also be seen using patient-derived material.

The assay outlined here therefore provides a M-o-A human in vitro system to test novel compound activity (either singly or in combination) upon TAM-like macrophage generation, phenotype and suppressive function. This allows selection of the most efficacious compounds for further investigation using patient-derived immune cells.

#3809

T-cell activation through the inhibition of tumor-expressed IDO1 activity in tryptophan metabolism pathway.

Chunlan Dong, Ying Wang, Kefeng Gong, Frank Xing, Qian Shi. _CrownBio, Taicang, Jiangsu, China_.

Indoleamine 2,3-dioxygenase-1 (IDO1) has been described as a major mechanism of immunosuppression in tumors. Despite histologic evidence showing that most human tumor tissues have extensive infiltration by various pro-inflammatory and immune cells, expression of IDO1 by tumor cells results in aggressive tumor growth and resistance to T cell-targeting immunotherapies. To de-suppress T-cell function through inhibition of IDO1-mediated tryptophan metabolism pathway, we established an in vitro assay system allowing robust detection of production level of tryptophan metabolism product N-formyl-kynurenine (Kyn) in culture medium, plasma and tumor tissue through ELISA and LC-MS/MS. We screened a panel of human and murine tumor cell lines for IDO1 expression with and without INFg induction. Our data suggest that a few of these models constitutively express IDO1; about half of the tumor cell models in the panel express IDO1 in an IFNg-dependent manner while the others do not respond to the cytokine stimulation at all. The mechanism underlying the lack of response to cytokine induction on IDO1 production is under investigation. We next tested the reduction of kynurenine in culture medium through inhibition of IDO1 enzymatic activity. After 24h stimulation by IFNg, a murine cell model was treated by INCB024360 (Incyte) for 48h and 72h. Cell-free supernatants were then collected, followed by Kyn detection by LC-MS/MS. The results showed that longer compound treatment (72h) offered a more robust detection window (1,500 ng/mL Kyn down to 2,00 ng/mL), compared to 48h results (350 ng/mL Kyn down to 100 ng/mL). This reproducible assay offers a reliable platform for initial IDO compound screening. To correlate effective reduction of kynurenine production to reactivation of T-cell activity, a co-culture system was established in that T-cell proliferation and cytokine production (IL2) were examined upon IDO1 inhibition in a time course. To achieve maximal therapeutic effect, a combination of INCB024360 and aPD1 antibody was applied in a T cell-mediated tumor cell killing assay. Detailed data will be discussed.

#3810

Ovarian tumor-induced changes in macrophage phenotype are associated with tumor metastasis.

Aparna Yellapa, Pincas Bitterman, Sameer Sharma, Sanjib Basu, Animesh Barua. _Rush University Medical Center, Chicago, IL_.

Background: Ovarian cancer (OVCA) differs from other gynecologic malignancies in its dissemination pattern where the tumor migrates in a diffuse intrapelvic and -abdominal manner. OVCA also triggers a variety of cellular and/or humoral immune responses to a variety of tumor-associated proteins. OVCA is mostly diagnosed at late stages where tumor has already metastasized, indicating an escape from immune responses. Despite several immune response factors, ovarian tumor metastasizing to omentum indicates presence of one or more immune evasion mechanisms. Immune system recognizes and eliminates pathogenic products and tumor cells and macrophages play a critical role in this process. Macrophages in tumor condition express anti-inflammatory cytokines that induce immune suppression. Cellular immune response maximizes killing efficacy of macrophages and proliferation of CD8+ T cells. M2 macrophages are tumor-associated macrophages involved in active growth and progression of tumors. However, the mechanism of ovarian tumor-induced changes in M1 macrophages to M2 phenotype is not well understood. Therefore, the goal of our study was to determine the tumor-induced changes in macrophage phenotype and cytotoxic T cells in association with OVCA metastasis.

Materials and Methods: This study was performed in exploratory design using ovarian specimen from normal subjects (60-80 years, n=10) and ovarian malignant tumors at early (n=16, 4 from each histologic type of OVCA) and late stages (n =20) as well as omental tissues from normal (n=3) and OVCA patients (n= 3). Tissue specimen were processed for immunohistochemistry (IHC), Western blotting (WB) and gene expression studies. Expression of M1 and M2 macrophages, CD8 and PI3K was examined by immunohistochemistry (IHC), Western blotting (WB) and qPCR.

Results: Compared with normal ovaries, more M1 and M2 macrophages were localized in tumor tissues. However, compared with early-stage OVCA, the frequency of M2 macrophages was significantly higher in ovarian tumors and omental tissues at late OVCA. Intensity of PI3K was also higher in ovarian tumors and omental tissues at late OVCA compared with normal ovarian tissues. Frequency of CD8+T cells was higher in tumor ovarian samples in comparison with normal ovarian tissues.

Conclusion: Results of this study suggest that the growth and metastasis of ovarian tumors are associated with increased conversion of M1 macrophages to M2 phenotype. This change in macrophage phenotype may be mediated by the increase in PI3K expression.

Support: Swim Across America. 

### Therapeutic Antibodies, Including Engineered Antibodies 3

#3811

Targeted Interleukin 2 and synergy with immune check-point inhibitors.

Cornelia Deborah Hutmacher-Berndt, Dario Neri. _ETH Zurich, Zurich, Switzerland_.

Systemic high-dose Interleukin 2 (IL2) is able to elicit complete responses in patients suffering from metastatic melanoma. However, the majority of patients cannot receive the whole treatment dose due to severe toxicities. Other approved drugs for the treatment of metastatic melanoma include chemotherapeutics (e.g. Dacarbazine), a protein kinase B-raf inhibitor (Vemurafenib) and an immune check-point inhibitor (Ipilimumab, an anti CTLA-4 antibody). These drugs provide a clear benefit to patients, but cancer cures are still rare.

Targeting IL2 to the site of disease represents a promising strategy to circumvent the toxicity and maximize the efficacy of this immunomodulatory payload. Various formats of targeted IL2 fusion proteins are currently in clinical development. Furthermore, recent successful approaches in the field of immunotherapy focus on the inhibition of check-point pathways, aiming to release the immunosuppressive brakes of the immune system. To explore the possibility of improving therapeutic efficacy of the single agents, a targeted version of IL2 (namely F8-IL2, where the F8 antibody recognizes the alternatively-spliced extra-domain A of fibronectin) was investigated in combination with an anti PD-1, anti PD-L1, or anti CTLA-4 antibody in an immunocompetent mouse model of CT26 colon carcinoma. In a first study, the combination of F8-IL2 and CTLA-4 blockade led to significant tumor growth retardation (curing 3/ 5 mice). Increasing the dose of F8-IL2 from 30 to 45 µg led to a 100 % cure rate with no signs of toxicity (body weight loss <5 %). Moreover, all cured mice acquired protective long-term immunity, which prevented tumor growth upon subsequent rechallenge with CT26 colon carcinoma cells. Furthermore, high dose F8-IL2 combined with the anti PD-1 antibody showed better therapeutic results than the respective monotherapies. In contrast, combination of F8-IL2 with the anti PD-L1 antibody did not improve the therapeutic outcome.

Taken together, these data provide a strong rationale for the clinical use of targeted IL2 in combination with Ipilimumab.

#3812

A best in class anti-CD38 antibody with antitumor and immune-modulatory properties.

Nina Eissler,1 Simone Filosto,1 Jake Y. Henry,2 Michael F. Maguire,1 Kristina Witt,3 Andreas Lundqvist,4 Teresa Marafioti,2 Pascal Merchiers,1 Kevin Moulder,1 Beatriz Goyenechea,1 Haw Lu,1 Camilla Fairbairn,1 Sarah Windler,5 JD Aurellano,5 Omar Duramad,5 Dominic Smethurst,1 Sergio A. Quezada,2 Anne Goubier1. 1 _Tusk Therapeutics, Stevenage, United Kingdom;_ 2 _University College London Cancer Institute, London, United Kingdom;_ 3 _Karolinska Insitutet, Stockholm, Sweden;_ 4 _Karolinska Insitutet, Stevenage, United Kingdom;_ 5 _iQ Biosciences, Berkeley, CA_.

Targeting CD38 in multiple myeloma has resulted in outstanding responses. CD38 is widely expressed on myeloma cells and other hematological malignancies. Not much is known about its expression on solid tumors and its role in the immune system. We have analysed a range of solid tumors for CD38 expression and distribution. To optimally target CD38, we have generated a novel antibody that is depleting CD38-high expressing cells, but also has immune modulatory properties. To dissect CD38 expression in solid tumors we exploited mRNA expression libraries, performed immune histochemistry (IHC) on tumor sections, and flow cytometry on patient tumor material. Bioinformatic analysis of the immune cell atlas revealed varying CD38 expression among all cancers analysed, and CD38 expression could be correlated with immune markers, e.g. Foxp3, PD-1/L1. IHC and flow cytometry confirmed CD38 expression across common cancer types, mostly confined to infiltrating lymphocyte and myeloid subsets. Expression on tumor cells was patient dependent. CD38 expression on immune cells was heterogenous and found on NK cells, T cells, suppressive myeloid cells, as well as regulatory T and B cells. Of note, high expression of CD38 was found to be correlated to a subset of exhausted T cells co-expressing PD-1 and other exhaustion markers. To target CD38 in solid tumors, we have screened a panel of CD38-binding antibodies. All antibodies have the potential to deplete CD38 positive tumor cells in vitro and in vivo. Additionally, their ability to influence effector T and NK cell activation has been evaluated. Among a panel of antibodies binding to distinct epitopes of CD38 and exerting unique functional properties, we have identified a fully human antibody, with strong capacity to deplete CD38-high cells in vitro and in vivo by varying killing mechanisms. This antibody was found to increase TCR-mediated signaling and proinflammatory cytokine secretion by human T cells, and further to enhance NK cell activation in vitro. Low dose injection to non-human primates resulted in increased expression of activation markers on both CD4 and CD8 T cells, while no T cell depletion was observed. Other selected antibodies comprise distinct modalities including strong to weak agonistic activity, differential killing properties, modulation of CD38 enzymatic activity, and offer a selection of candidates applicable for different treatment settings. In summary, we found heterogenous expression of CD38 in solid tumors, mostly confined to immune subsets. To target CD38, we present a potent anti-CD38 antibody with depleting effects on CD38-expressing cancer cells, as well as suppressive immune cells, and the capacity to increase the function of immune effector cells. This dual activity might allow to fully exploit the therapeutic potential of targeting CD38, not only in hematological malignancies but also in solid tumors.

#3813

Development of JNJ-64164711, a low fucose anti-GITR antibody for enhanced depletion of tumor regulatory T cells (Tregs).

Cam V. Holland,1 John Kehoe,1 Judith Hailey,2 Rupesh Nanjunda,1 Eilyn Lacy,1 Robin Ernst,1 Di Zhang,1 Sam Wu,1 Alexey Teplyakov,1 Weimin Li,1 Michelle Kinder,1 Edward W. Thompson,3 Patrick Wilkinson,1 Jackson Wong,1 Gerald Chu,1 Linda Okonkwo,1 John D. Alvarez,1 Anhco Nguyen,1 Iqbal S. Grewal,1 Moitreyee Chatterjee-Kishore,1 Mark Salvati,1 Marco Gottardis1. 1 _Janssen Research & Development, LLC, Spring House, PA; _2 _VWR, Radnor, PA;_ 3 _Kelly Services, Troy, MI_.

Background: Checkpoint blockade of PD-1/PDL-1 or CTLA-4 has demonstrated clinical benefit in a subset of cancer patients (pts). To further improve the outcome, approaches to deplete immunosuppressive Tregs are being developed. Emerging evidence supports that the TNF receptor superfamily member 18 (TNFRSF18 or GITR) is highly expressed on intratumoral (i.t.) Tregs with limited expression on effector T cells or circulating immune cells (1,2). This differential expression allows for development of monoclonal antibody (mAb) therapies that eliminate this population while sparing other immune subsets. Herein we characterize JNJ-64164711 (JNJ-711), a fully human anti-GITR mAb being developed to selectively deplete GITR+ Tregs and expand the population of pts that may respond to immunotherapy.

Methods: Treg frequency and GITR expression on human cancer specimens was performed by IHC and multicolor flow cytometric analysis (FC). JNJ-711 was assessed for binding to GITR (human and cyno) and human Fcγ receptors, and inhibition of hGITRL binding to hGITR. The JNJ-711 binding epitope was mapped by hydrogen-deuterium exchange. JNJ-711 functional activity was determined by 1) an NF-kB reporter assay, 2) antibody dependent cellular cytotoxicity (ADCC) on GITR+ cell lines, and 3) JNJ-711 mediated Treg depletion in primary pts tumor samples.

Results: GITR expression prevalence was evaluated by IHC in multiple solid tumors. Immune cell GITR expression was observed in head and neck (HN), esophageal (ESO), non-small cell lung carcinoma (NSCLC), colorectal (CRC) and prostate cancers. GITR was moderately expressed on HN and ESO tumor cells. GITR expression (by FC) was higher on tumor Tregs versus other T cell populations in the tumor and periphery across NSCLC, CRC, ovarian and renal cell carcinoma samples. GITR was differentially higher on i.t. effector Tregs (eTreg) whereas CCR4 was highest on CD4+ T cells in circulation. JNJ-711 binds to hGITR on cells with high affinity [KD= 180 pM], has a log greater affinity to FcγRIIIa [KD= 37 nM on 158V; KD= 180 nM on 158F], a slower dissociation rate than most GITR agonist (Ag) mAb tested [Koff= 5.47 x 10-4 s-1 for Fab], and binds to the CRD1 of GITR. Unlike other GITR Ag mAbs tested, JNJ-711 does not block GITR interaction with GITRL. JNJ-711 demonstrates higher ADCC mediated cell depletion than other GITR Ag mAbs in clinical development on 1) GITR+ hematologic tumor cell lines (HuT102, KMS-12-BM, RPMI 8226, JJN-3), 2) primary activated GITR+ T cells, and 3) in vitro expanded Tregs. Using dissociated primary pts samples, JNJ-711 selectively depleted i.t. eTregs.

Conclusion: JNJ-64164711 selectively depletes hGITR+ cells including Tregs and has potential to enhance antitumor immune response. This antibody is being evaluated for clinical development.

References:

1. Asma et al. Tumor Biol 2015;36:3727-34.

2. Pedroza‐Gonzalez et al. OncoImmunology 2015;4:12, e1051297.

#3814

TriTACs are novel T cell-engaging therapeutic proteins optimized for the treatment of solid tumors and for long serum half-life.

Holger Wesche,1 Wade Aaron,1 Richard J. Austin,1 Patrick A. Baeuerle,2 Adrie Jones,1 Bryan Lemon,1 Kenneth Sexton,1 Timothy Yu1. 1 _Harpoon Therapeutics, South San Francisco, CA;_ 2 _MPM Capital, Cambridge, MA_.

T cell engagers are antibody-based therapeutics that transiently tether T cells via the T cell receptor complex (TCR) to surface antigens on tumor cells. This leads to activation of T cells and redirected lysis of the attached target cell. The therapeutic potential of this modality was demonstrated by blinatumomab, a CD19/CD3-bispecific T cell engager approved for the treatment of adult patients with relapsed/refractory acute lymphoblastic leukemia. Despite success of this T cell-engaging therapy in a hematologic malignancy, clinical studies in solid tumors with other T cell engagers have been less encouraging so far. The TriTAC (Tri-specific T cell Activating Construct) platform was developed to address shortcomings of existing T cell engagers, including short serum half-life, limited tissue penetration, and suboptimal activity. TriTAC constructs are made of a single polypeptide designed to bind to a cancer surface antigen, the CD3 epsilon subunit of the TCR, and to human serum albumin. CD3 is bound by a single-chain variable fragment (scFv) while both tumor targeting and albumin binding are achieved by single domain antibodies. The latter allow TriTACs to be very small, stable, and easily produced and purified. Noncovalent binding to serum albumin has been validated as an effective way to extend the serum half-life of other proteins up to several weeks. Even though TriTACs have three binding domains, their overall size is only ~50 kDa, one third of the size of a monoclonal antibody. This is expected to allow for faster diffusion into human tumor tissues than is possible with antibodies given the high interstitial pressure and dense extracellular matrix in solid tumors. TriTACs can induce T cell to kill tumor cells in vitro at single-digit picomolar to femtomolar concentrations with concomitant induction of inflammatory cytokine release and T cell proliferation. TriTACs can diffuse much faster across an extracellular matrix than antibodies, and eradicate tumors in mouse xenograft models supplemented with human T cells. In nonhuman primates, TriTAC molecules have serum half-lives of approximately 4 days, and appear well tolerated.

#3815

Decomposition of parameters contributing to the improved therapeutic index of ADCs obtained by GlycoConnect™ and HydraSpace™ Technologies.

Floris van Delft, Brian Janssen, Remon van Geel, Marloes Wijdeven, Jorge Verkade, Sander van Berkel. _Synaffix BV, Oss, Netherlands_.

We have found that conjugation of toxic payloads to the native glycan of a monoclonal antibody by chemoenzymatic remodeling (GlycoConnect™ technology) consistently provides antibody-drug conjugates (ADCs) with enhanced therapeutic index (TI) increases versus those of ADCs prepared by mainstream clinical technologies. For example, head-to-head comparison of GlycoConnect™ ADCs -based on the same antibody and payload components- with the marketed drugs Adcetris and Kadcyla revealed an improvement in both efficacy and safety, which is further enhanced by use of a polar HydraSpace™ technology. Importantly, a similar improvement in TI was noted for comparison of a GlycoConnect™ ADC versus or a site-specific ADC derived from a cysteine-engineered antibody, the most important emerging technology in the clinic. With the aim to better understand the superiority of ADCs based on combined GlycoConnect™ and HydraSpace™ technologies, we have performed in-depth in vitro and in vivo investigation into factors contributing to the overall performance of ADCs prepared by glycan conjugation with a polar spacer. For example, it is found that stability is significantly improved versus mainstream technologies in terms of aggregation and linker stability. Rapid aggregation was observed for both Kadcyla and Adcetris, as well as ADC decomposition due to retro-Michael reaction resulting in free payload (for Adcetris) or albumin-conjugated DM1 (for Kadcyla), as indicated by LC-MS and immunoassay analysis. The latter image is confirmed by in vivo pharmacokinetic analysis, showing a dramatic difference in total antibody versus total conjugated antibody by premature release of payload. Finally, analysis with a solid 3D tumoroid model indicates enhanced penetration of site-specific versus randomly conjugated ADC, which may also contribute to better efficacy in solid tumors. The data generated further underline the importance of conjugation and linker technology as critical quality attributes for next-generation ADCs with higher therapeutic index.

#3816

Efficacy of CDX-1140, an agonist CD40 antibody, in preclinical tumor models.

Lawrence J. Thomas,1 Li-Zhen He,2 James Testa,2 Anna Wasiuk,2 Jeffrey Weidlick,2 Crystal Sisson,2 Laura A. Vitale,2 Thomas O'Neill,2 Eric Forsberg,1 Catherine D. Pilsmaker,1 Lauren E. Gergel,1 Elizabeth Q. Do,1 James Boyer,1 April R. Baronas,1 Mallary Rocheleau,1 Michelle E. Grealish,1 Kathleen M. Borrelli,1 Henry C. Marsh,1 Tibor Keler2. 1 _Celldex Therapeutics Inc., Needham, MA;_ 2 _Celldex Therapeutics Inc., Hampton, NJ_.

CD40 on antigen presenting cells plays a critical role in the induction of effective innate and adaptive immune responses. In contrast, CD40 signaling on certain malignant cells, particularly B cell lymphomas, inhibits proliferation or triggers apoptotic cell death. Thus, two independent mechanisms provide opportunities for the use of agonist anti-CD40 monoclonal antibodies in cancer therapy: enhancement of anti-tumor immunity, and direct inhibition of tumor growth. CDX-1140 is a human IgG2 antibody selected from a panel of fully human mAbs specific for CD40 generated by hybridoma technology from human Ig transgenic mice. We previously demonstrated the potent immune enhancing effects of CDX-1140 using in vitro models and in non-human primates. CDX-1140 was shown to activate dendritic cells and B cells in an Fc receptor independent manner. CDX-1140 does not bind to the CD40L binding site, and synergizes with CD40L in stimulation of the CD40 receptor and subsequent functional activities. Here we further characterized the anti-tumor activity of CDX-1140 on CD40 positive tumors using xenograft models in immunodeficient mice. Using the Ramos and Raji human lymphoblastoma cell lines, CDX-1140 was shown to attenuate tumor growth and prolong survival. Addition of CDX-1140 and human PBMC was highly effective at promoting complete rejection of both Ramos and Raji tumors. Importantly, the epithelial EJ138 bladder carcinoma cell line was also highly sensitive to CDX-1140 treatment. For example, in a study where mice received EJ138 cells subcutaneously, all animals that were treated with 300 µg of CDX-1140 on days 1, 8, and 15, showed suppression of tumor growth through day 60, in comparison to saline-treated animals which developed significant tumors in 7 of 8 animals. These data support the potential of CDX-1140 for direct anti-tumor effects on CD40-positive tumors (including epithelial tumors) that may supplement its activity as an immune activating agent. CDX-1140 is currently in a phase 1 dose-escalation study in patients with advanced solid tumors.

#3817

Improved T cell activation bioassays to facilitate the development of bispecific antibodies and engineered T cell immunotherapies.

Pete Stecha, Denise Garvin, Jim Hartnett, Gopal B. Krishnan, Frank Fan, Mei Cong, Zhi-jie Jey Cheng. _Promega Corp., Madison, WI_.

T cells play a critical role in cell-mediated immunity and can mediate long-term, antigen-specific, effector and memory responses. A variety of immunotherapy strategies have been developed recently that are aimed at inducing, strengthening or engineering T cell responses. These have emerged as promising approaches for the treatment of diseases such as cancer and autoimmunity. Current methods used to measure TCR-mediated T cell proliferation and cytokine production rely on primary PBMCs as a source of T cells, which must be stimulated via co-culture with APCs or anti-TCR/CD3 antibodies. These assays are laborious and highly variable due to their reliance on donor primary cells, complex assay protocols and unqualified assay reagents. As a result, these assays are difficult to establish in quality-controlled drug development settings. To overcome this barrier, we developed two reporter-based bioluminescent T cell activation bioassays that can be used for the development of bispecific antibodies and engineered T cell immunotherapies. The assays consist of Jurkat T cells genetically engineered to express luciferase downstream of either NFAT or IL-2 response elements. The T cell activation bioassays reflect the mechanisms of action of biologics designed to induce TCR and/or CD28-mediated T cell activation, as demonstrated using anti-CD3 and/or anti-CD28 antibodies as well as blinatumomab, a bispecific antibody that simultaneously binds CD3 expressed on T cells and CD19 expressed on malignant B cells. The bioassays are pre-qualified according to ICH guidelines and demonstrate assay specificity, precision, accuracy and linearity required for routine use in potency and stability studies. Finally, our data illustrate the use of reporter-based T cell activation bioassays for characterizing and measuring the activity of engineered chimeric antigen receptor T cells.

#3818

A novel multi-specific antibody targeting PD-L1-overexpressing cancers that redirects and stimulates antigen-committed CD8+ T cells through concomitant engagement of a T cell costimulatory receptor.

Tea Gunde,1 Sebastian Meyer,1 Stefan Warmuth,1 Alexandre Simonin,1 Christian Hess,1 Matthias Brock,1 Timothy Egan,2 David M. Urech3. 1 _Numab Innovation AG, Wädenswil, Switzerland;_ 2 _Numab US, LLC, Cambridge, MA;_ 3 _Numab Therapeutics AG, Pfäffikon, Switzerland_.

Targeting PD-L1-overexpressing cells with therapeutic antibodies is a clinically validated strategy for the treatment of multiple solid tumors. In order to increase efficacy, PD-1/PD-L1 blocking agents are currently being tested in combination with additional immune checkpoint modulators (ICMs). However, such combination therapies are associated with considerable treatment-related adverse events, resulting in a narrow therapeutic window and thereby limiting treatment efficacy. To maximize potency and improve the safety of ICM combination approaches, we sought to design a multi-specific molecule bearing two ICM domains that depletes PD-L1-overexpressing cancer cells via selective recruitment and stimulation of tumor-reactive effector T cells in the tumor microenvironment. The molecule's design consists of three monovalent antibody variable-domain fragments (Fvs) specific for PD-L1, human serum albumin and a T cell costimulatory receptor fused in a single-chain (PD-L1/HSA/costim tri-specific scDb-scFv). The monovalent and Fc-less structure of the molecule ensures that agonism of the costimulatory receptor on effector cells can only arise when the molecule concomitantly binds to PD-L1 on the surface of target cells. The addition of a half-life-extending anti-SA domain, meanwhile, not only enables convenient dosing but also should promote delivery of the molecule to tumor microenvironments. Here, we demonstrate the successful production of a novel multi-specific antibody that potently blocks PD-L1/PD-1 signaling and elicits T cell costimulation solely in the presence of cells that overexpress PD-L1, as confirmed using a transgenic Jurkat reporter T cell-line in co-cultures with HT29 and HCC827 cancer cell-lines. We show that the molecule elicits such costimulation considerably more selectively and potently than a first-generation, clinical-stage, anti-costim IgG. Furthermore, the tri-specific is incapable of stimulating T cells in the absence of primary T cell activation. Finally, in an HCC827 xenograft model in hPBMC-supplemented NOG mice, the tri-specific slowed tumor growth and enhanced intratumoral CD8+ T cell activation to a greater extent than monospecific IgG variants of the anti-PD-L1 and anti-costimulatory receptor domains. These data support the hypothesis that broadening the therapeutic window for a promising, clinically validated ICM-based treatment strategy can be achieved by combining multiple ICM domains in an Fc-less, monovalent multi-specific that selectively forms immunological synapses between cancer cells and activated effector cells.

#3819

INCAGN02385 is an antagonist antibody targeting the co-inhibitory receptor LAG-3 for the treatment of human malignancies.

David Savitsky,1 Rebecca Ward,1 Christina Riordan,1 Cornelia Mundt,2 Shawn Jennings,3 Joe Connolly,1 Mark Findeis,1 Michele Sanicola,4 Dennis Underwood,1 Horacio Nastri,5 Peggy Scherle,5 Gregory Hollis,5 Reid Huber,5 Robert Stein,1 Marc van Dijk,1 Nicholas S. Wilson6. 1 _Agenus Inc, Lexington, MA;_ 2 _Agenus Inc (Current Affiliation - Novartis), Lexington, MA;_ 3 _Agenus Inc (Current Affiliation - Takeda Pharmaceuticals), Lexington, MA;_ 4 _Agenus Inc (Current Affiliation - Sanofi Genzyme), Lexington, MA;_ 5 _Incyte Corporation, Wilmington, DE;_ 6 _Agenus Inc (Current Affiliation - Gilead Sciences), Lexington, MA_.

Lymphocyte activation gene 3 (LAG-3) is a cell surface receptor that negatively regulates antigen-specific T cell responses. LAG-3 expression is generally restricted to populations of recently activated and chronically stimulated exhausted T cells, and is often correlated with general T cell dysfunction across several human malignancies. Accordingly, the LAG-3 pathway has been identified as a potential barrier to productive tumor-specific T cell immunity generated by PD-1/PD-L1 blockade. The antitumor activity from targeting the LAG-3 pathway in preclinical models has provided further rationale for pharmacologic modulation of the LAG-3 axis in cancer patients. INCAGN02385 is an Fc-engineered IgG1κ antibody chosen for development based on its high-affinity binding to human LAG-3, cross-reactivity with cynomolgus monkey LAG-3, and ability to potently block LAG-3 binding with its MHC class II ligand. INCAGN02385 also enhances T cell responsiveness to TCR stimulation alone or in combination with PD-1/PD-L1 axis blockade. Evaluation of INCAGN02385 in cynomolgus monkeys was well-tolerated and demonstrated the expected pharmacokinetic profile. Altogether, these data support assessment of INCAGN02385 in patients with advanced or metastatic solid tumors.

#3820

Novel PD-1/GITRL bispecific fusion protein delivering concurrent T cell co-stimulation and checkpoint pathway inhibition.

Simon J. Dovedi,1 Jonathan Seaman,1 Ines Osma-Garcia,1 Katharina Deschler,1 Edmund Poon,1 Ronald Herbst,2 Robert Wilkinson1. 1 _MedImmune, Cambridge, United Kingdom;_ 2 _MedImmune, Gaithersburg, MD_.

Glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) is a co-stimulatory receptor, and programmed cell death protein 1 (PD-1) is an important co-inhibitory receptor involved in controlling T-cell activation and function. Preclinical studies using murine cross-reactive molecules demonstrate that the combination of an αPD-1 monoclonal antibody (mAb) with a GITR-ligand (GITRL) fusion protein can elicit synergistic antitumor activity and long-term survival benefit in syngeneic mouse tumor models. Here we describe the activity of two bispecific fusion proteins, each comprising two GITR ligand trimers fused to the Fc region of an αPD1 mAb, in an IgG1 (MEDI5771) or IgG4P (MEDI3387) format. We show that these bispecific molecules are able to concurrently engage both targets and are effective in cell-based PD-1 and GITR reporter assays, and in a range of human primary T-cell assays. Furthermore, the PD-1/GITRL FP bispecific constructs elicit T-cell responses in vitro that are at least equivalent to a combination of a) the parental molecules and b) MEDI1873 (a GITR agonist currently undergoing clinical evaluation) and an αPD-1 mAb. Moreover, using surrogate molecules we demonstrate that IV administration leads to dose-dependent pharmacodynamic responses on CD4+ and CD8+ T cells for both isotypes of the bispecific molecule. These data demonstrate that the antitumor activity for these novel molecules is equivalent to the combination of GITRL-FP and PD-1 mAb in the B16 model. In summary, the simultaneous dual targeting of PD-1 and GITR pathways mediated by these two novel bispecific molecules induces robust T-cell activity that is at least equivalent to a combination of molecules targeting these pathways, thereby demonstrating strong potential as a novel anticancer therapy.

#3821

The neoantigen-targeting antibody NEO-201 enhances NK cell-dependent killing of tumor cells through blockade of the inhibitory CEACAM5/CEACAM1 immune checkpoint pathway.

Justin M. David,1 Massimo Fantini,1 Christina M. Annunziata,2 Philip M. Arlen,1 Kwong Y. Tsang1. 1 _Precision Biologics, Inc., Rockville, MD;_ 2 _National Cancer Institute, Bethesda, MD_.

Background: Immunotherapy using checkpoint blockade antibodies that target effector cell inhibitory receptors, like PD-1 and CTLA-4, have elicited some dramatic and durable responses in several tumor types. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a cell-surface protein expressed by immune cells and tumor cells, and it can inhibit T cell function similar to PD-1 and CTLA-4. CEACAM1 is also a potent inhibitor of natural killer (NK) cell function; binding between CEACAM1 on NK cells and CEACAM1 or CEACAM5 on tumor cells inhibits activation signaling by NKG2D, which prevents NK cell cytolysis and permits tumor cells to evade NK killing.

NEO-201 is a novel humanized IgG1 monoclonal antibody (mAb) that was derived from an immunogenic preparation of tumor-associated antigens (TAAs) from pooled allogeneic colon tumor tissue extracts. It reacts against a wide variety of human carcinoma cell lines and tumor tissues, but is largely non-reactive against normal tissues. NEO-201 binds to members of the CEACAM family, and can activate innate immune mechanisms such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) to kill tumor cells. This investigation was designed determine whether NEO-201 blocks the CEACAM1 inhibitory pathway to restore antitumor functionality to NK cells.

Methodology: In vitro assays using human tumor cell lines were conducted to identify CEACAM family members bound by NEO-201. Functional assays were conducted to assess the ability of NEO-201 to potentiate the in vitro killing of tumor cells by the NK cell line NK-92, which expresses CEACAM1 and lacks CD16 and the ability to mediate ADCC.

Results: NEO-201 was found to react with distinct variants of CEACAM5 and CEACAM6, but not with CEACAM1 or CEACAM8. Expression profiling revealed that various NEO-201+ cell lines expressed differing levels of the native forms of CEACAM5/6 vs. the NEO-201-reactive variant forms of these molecules. Functionally, NEO-201 treatment augmented the cytolytic activity of NK-92 cells against NEO-201+ tumor cells in proportion to their level of CEACAM5 expression (average increase of 2-fold), but not against NEO-201+ cells that only expressed CEACAM6.

Conclusions: This study demonstrates that NEO-201 is reactive against a tumor-associated variant of CEACAM5/6, and provides evidence that this antibody can block the interaction between tumor cell CEACAM5 and NK cell CEACAM1 to reverse CEACAM1-dependent inhibition of NK cytotoxicity. Experiments are in progress to determine the involvement of NK cell CEACAM1 and/or other checkpoint pathways in this mechanism of action. These results suggest that NEO-201 may potentially reverse CEACAM1-dependent immunosuppression of NK cells in patients whose tumors express the NEO-201-reactive variant of CEACAM5.

#3822

Characterization of the first chicken-derived anti-PD-1 clinical stage antibody with a unique epitope and promising anticancer activity.

Torben Gjetting, Monika Gad, Camilla Fröhlich, Maria C. Melander, Gunther Galler, Johan Lantto, Thomas Bouquin, Ivan D. Horak, Michael Kragh, Mikkel W. Pedersen, Klaus Koefoed. _Symphogen, Denmark_.

Inhibition of immunologic checkpoints like Programmed Cell Death 1 (PD-1) has shown clinical efficacy in a broad range of cancers by improving or restoring T-cell activity. Anti-PD-1 antibodies show great promise in treating cancer malignances when administered alone or in combination with other immune activating approaches. However, high protein sequence identity between human and mammalian species used for antibody generation often disfavor generation of antibodies against functionally conserved epitopes, or prevents isolating antibodies cross reacting with ortholog species used for evaluating potential toxicity. Chickens are phylogenetically distant from mammals and are better at generating antibodies against epitopes that are conserved in mammals. Because chickens generate antibodies from a very restricted set of V-gene germline genes that are diversified by "gene conversion", we envisioned that high throughput humanization of antibody frameworks was achievable by "mass CDR grafting" after recovering antibodies by immunization and B-cell cloning. Wild type chickens were immunized with PD-1 antigen, and a repertoire of 120 antibodies was generated with Symplex™ technology, by combining single B-cell FACS sorting and high throughput RT-PCR cloning of cognate VH and VL chains. The isolated PD-1 repertoire was cloned with an inert Fc backbone and humanized by a combination of in silico CDR grafting and gene synthesis. Humanized antibodies were expressed and screened for retained binding affinity and functionality in T-cell based assays. We successfully generated a humanized PD-1 antibody repertoire and found that most antibodies retained affinity and functionality similar to that of parental chicken antibodies. Furthermore, the antibody repertoire displayed broad binding epitope coverage on PD-1, often with strong pM affinity, and showed biophysical properties acceptable for drug development. Our lead antibody Sym021 blocked PD-L1 and PD-L2 ligand binding and downstream PD-1 signaling, resulting in elevated T-cell cytokine production in vitro. Moreover, Sym021 bound human PD-1 with much stronger affinity of 30 pM compared to clinical PD-1 mAbs nivolumab and pembrolizumab, while also cross reacting to cynomolgous and mouse PD-1. This enabled direct testing of Sym021 in syngenic mouse in vivo models and evaluation of preclinical toxicology in cynomolgus monkeys. Detailed epitope mapping showed that the epitope recognized by Sym021 was unique compared to clinical antibodies pembrolizumab and nivolumab. These results supported entry of PD-1 targeting Sym021 into clinical trials.

#3823

Bat4306f, an anti-CD20 antibody devoid of fucose modification, demonstrates enhanced ADCC effect and potent in vivo efficacy.

Jin-Chen Yu, Chao Qin, Shengfeng Li. _Bio-Thera Solutions, Guanzhou, China_.

Depletion of malignant B cells using anti-CD20 antibodies is one of the most effective approaches for CD20-positive non-Hodgkin lymphoma therapy. Anti-CD20 antibodies can activate several pathways including antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), or programmed cell death to kill tumor cells. ADCC is mediated via Fc region of antibody binding to FcγRIIIa receptor on effector cells, such as NK cells. Previous studies have shown that non-fucosylated antibody binds to FcγRIIIa with increased affinity and can thus trigger FcγRIIIa-mediated effector functions more efficiently than native, fucosylated antibody. Obinutuzumab, an FDA-approved anti-CD20 antibody with approximate 30% fucose level of a normal antibody, has demonstrated better efficacy than fucosylated rituximab. We generated BAT4306F, which has the same sequence as obinutuzumab yet is completely devoid of fucose modification. BAT4306F induced higher level of ADCC effect compared with rituximab and obinutuzumab in vitro. BAT4306F also showed significantly superior activity on depleting B cells in whole blood compared to rituximab, and superior activity compared to obinutuzumab. In monkeys dosed once with these antibodies, BAT4306F demonstrated much stronger B-cell depletion activity than that of obinutuzumab and rituximab, whether detected in serum, spleen, or lymph node. The residual B cells detected in spleen of monkey treated with BAT4306F were 8-fold less than that of obinutuzumab, and 10-fold less than that of rituximab. Furthermore, BAT4306F demonstrated stronger tumor-inhibition activity than rituximab in a DLBCL tumor cell xenograft mouse model. Preclinical acute and long-term toxicity studies in monkey reveal that BAT4306F is well tolerated. Together these data demonstrate that BAT4306F is more potent than obinutuzumab and rituximab due to a complete defucosylation. The result warrants further clinical development of BAT4306F.

#3824

Antitumor activity of REGN4659, a fully human anti-CTLA-4 monoclonal antibody, against MC38.Ova tumors grown in immunocompetent human CTLA-4 knock-in mice.

Elena Burova, Omaira Allbritton, Susannah Brydges, William Poueymirou, Robert Durso, Douglas MacDonald, Ella Ioffe, Markus Mohr, William Olson, Gavin Thurston. _Regeneron Pharmaceuticals, Inc., Tarrytown, NY_.

Cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) is an inhibitory checkpoint receptor that is expressed by activated conventional T cells and regulatory T cells and negatively regulates T cell activation. CTLA-4 plays a key role in restraining the adaptive immune response of T cells against a variety of antigens, including self-antigens. However, in the tumor microenvironment, the co-inhibitory CTLA-4 receptor contributes to tumor immune escape. CTLA-4 shares its two ligands, CD80 and CD86, which are largely expressed on antigen-presenting cells, with CD28, a co-activator of T cells; however, CTLA-4 binds CD80 and CD86 with higher affinity than CD28. Blockade of CTLA-4 elicits an effective antitumor immunity in preclinical animal models and has demonstrated favorable clinical results in several solid malignancies. REGN4659 is a human IgG1 antibody that binds to the extracellular domain of human and monkey CTLA-4 with high affinity and specificity. REGN4659 blocks CTLA-4 interaction with CD80 and CD86 ligands and enhances the cytokine release of activated primary human T cells in vitro. The effects of treatment with REGN4659 on the subcutaneous growth of a mouse adenocarcinoma tumors expressing chicken ovalbumin (MC38.Ova) were evaluated in immunocompetent mice genetically engineered to express a human CTLA-4 chimeric protein from the endogenous mouse locus (CTLA-4 hum/hum knock-in). Prophylactic treatment with REGN4659 significantly suppressed MC38.Ova tumor growth in CTLA-4 knock-in mice in a dose dependent manner, and improved mouse survival. Because patients treated systemically with CTLA-4 blockade have suffered from immune-related adverse events, we investigated the efficacy of a peritumoral low-dose administration of a mouse-reactive surrogate anti-CTLA-4 antibody in an attempt to minimize systemic exposure. Peritumoral injection of anti-CTLA-4 antibody was as efficacious as intraperitoneal delivery at inducing anti-tumor immunity. Peritumoral anti-CTLA-4 antibody delivery was associated with lower circulating antibody levels but nonetheless caused systemic antitumor immunity as revealed by growth retardation of concomitantly implanted distant tumors. The mechanism of action and robust anti-tumor efficacy of REGN4659 supports its clinical development for the treatment of human cancers.

#3825

INCAGN02390, a novel antagonist antibody that targets the co-inhibitory receptor TIM-3.

Jeremy Waight,1 Priyadarshini Iyer,1 Ekaterina Breous-Nystrom,1 Christina Riordan,1 Mark Findeis,1 Dennis Underwood,1 Joseph Connolly,1 Michele Sanicola-Nadel,2 Horacio Nastri,3 Peggy Scherle,3 Gregory Hollis,3 Reid Huber,3 Robert Stein,1 Mark van Dijk,1 Nicholas S. Wilson4. 1 _Agenus Inc, Lexington, MA;_ 2 _Agenus Inc (Current Affiliation - Sanofi Genzyme), Lexington, MA;_ 3 _Incyte Corporation, Wilmington, DE;_ 4 _Agenus Inc (Current Affiliation - Gilead Sciences), Lexington, MA_.

Unprecedented rates of durable clinical responses have been observed for antibody-based therapeutics targeting immune checkpoint proteins such as cytotoxic T lymphocyte antigen-4 (CTLA-4) or programmed death receptor-1 (PD-1). Nonetheless, a significant number of patients experience de novo resistance or relapse due to adaptive resistance mechanisms. T-cell immunoglobulin and mucin domain containing-3 (TIM-3) is an inhibitory receptor involved in immune tolerance often co-opted by tumors to prevent successful antitumor responses. Accordingly, TIM-3 is frequently expressed on myeloid and so-called exhausted T and NK cells within the tumor microenvironment. Targeting the TIM-3 pathway in preclinical models has provided additional rationale for pharmacologic modulation of this axis in cancer patients. INCAGN02390 is a novel and fully human Fc-engineered IgG1κ antibody developed to antagonize the TIM-3 pathway for the treatment of human malignancies. INCAGN02390 forms a high-affinity interaction with TIM-3, occluding access to the CC'-FG binding cleft and blocking phosphatidylserine binding. In addition, INCAGN02390 elicits rapid receptor internalization, potentially obviating interactions with other described or undescribed ligands. INCAGN02390 also enhances IFN-γ production from T cells undergoing tonic TCR stimulation when combined with PD-1 blockade. Finally, to demonstrate combinatorial potential, we show potent antitumor activity of an anti-mouse TIM-3 antibody in concert with other checkpoint antibodies in vivo. In summary, these data support assessment of INCAGN02390 in patients with advanced or metastatic solid tumors.

#3826

GITRL-Fc biomarker and mechanism study: GITRL-Fc reduces Treg frequency in tumors and requires effector function for inhibition of tumor growth.

Gretchen M. Argast, Belinda Cancilla, Fiore Cattaruzza, Pete Yeung, Reyhaneh Lahmy, Erwan Le Scolan, Rose Harris, Alayne Brunner, Min Wang, Fumiko Axelrod, Jorge Monteon, Jennifer Elechko, Andrew Lam, MingHong Xie, Earth Light Lowe, Gilbert O'Young, Austin Gurney, Ann M. Kapoun. _OncoMed Pharmaceuticals, Inc, Redwood City, CA_.

Activation of the co-stimulatory receptor GITR (Glucocorticoid-Induced Tumor Necrosis Factor Receptor) by GITR-Ligand (GITRL) promotes proliferation and activation of effector T cells (T eff) and inhibits suppressive activity of regulatory T cells (Treg). Here, we have further characterized the mechanism of action of a single-gene GITRL trimer fused to an immunoglobulin Fc domain (GITRL-Fc, 336B3) by examining pharmacodynamic (PD) biomarkers in time course studies. Mice bearing CT26.WT colon tumors were treated with weekly GITRL-Fc and sacrificed 24 hours, 7 and 14 days after the first dose. Immuno-phenotyping of tumor-associated immune cells revealed a reduction in Treg frequency in tumor by 24 hours post-dose that was maintained at 7 and 14 days. Furthermore, GITRL-Fc treatment increased activation markers on tumor-associated CD4+ and CD8+ T cells, suggesting an increased cytotoxic environment within the tumor. This was supported by significant and sustained increase in CD8+ T cell:Treg ratio in the tumor after GITRL-Fc treatment. To determine whether intratumoral (IT) injection of GITRL-Fc is an effective route of administration, we compared efficacy, pharmacodynamic (PD) markers and pharmacokinetics in IT- and intraperitoneal (IP)-injected mice bearing bilateral CT26.WT tumors. Both routes of administration showed similar tumor growth inhibition (TGI) and PD markers in both the treated and the abscopal tumors, but IT injection resulted in a significantly lower serum GITRL-Fc concentration, suggesting that IT administration may be an alternative route of administration to IP with similar efficacy. The GITRL-Fc molecule 336B3 is effector function competent and able to induce cell-mediated cytotoxicity upon binding. To determine whether this effector function is required for GITRL-Fc-induced TGI, we treated CT26.WT tumor-bearing mice with 336B3 and 336B22, a GITRL-Fc molecule deficient in effector function. The effector function-competent 336B3 induced significant TGI and a more robust activation of Teff cells and reduction in Treg frequency, when compared to 336B22, suggesting that effector function is important for efficacy. To identify biomarkers for GITRL-Fc, we performed microarray analyses on multiple syngeneic mouse models treated with GITRL-Fc and developed GITRL gene signatures from blood and from tumors. We also developed multiplexed immunohistochemistry panels designed to quantify frequency of GITR and GITRL expression (GITR+CD8, GITR+FOXP3) in tumors. In conclusion, we have examined the effects of GITRL-Fc on preclinical mouse models. Biomarker analysis showed that loss of Tregs, activation of T cells and Fc-mediated effector function are key elements in the mechanism of action of the molecule. We have identified potential biomarkers to be used for PD and potential predictive analysis in clinical trial patient samples.

#3827

Bispecific antibodies with an anti-PD-1 backbone for cancer therapy generate enhanced immune activity.

Baiyong Li, Zhaoliang Huang, Xinghua Pang, Tingting Zhong, Na Chen, Max Wang, Xiaoping Jin, Dennis Xia, Peng Zhang, Michelle Yu Xia. _Akeso Biopharma, Inc., Zhongshan Guangdong, China_.

PD-1 antibody combination therapy shows great promise to further enhance clinical efficacy of PD-1 antibody. Bispecific antibody represents an emerging platform that allows simultaneous targeting of multiple pathways employing a single agent. Here we present a series of bispecific antibodies using anti-PD-1 antibody as backbone, including PD-1/CTLA-4 and PD-1/VEGF bispecific antibodies, which target additional immune suppressive pathways to further enhance cancer immunity. These include complementary check point pathway and suppression of dendritic cell maturation and antigen presentation. Clinical candidates for these projects have been selected demonstrating superior immune enhancing activity in preclinical study. These bispecific antibodes are designed based on Akeso Tetrabody platform, which has demonstrated robust developability properties. Akeso PD-1/CTLA-4 bispecific antibody AK104 has entered clinical trial for advanced solid tumors (ClinicalTrials.gov Identifier: NCT03261011).

#3828

Pritumumab mAb binds cell surface expressed vimentin on pancreatic cancer cells and inhibits tumor growth.

Ivan Babic,1 Natsuko Nomura,1 Eric Glassy,2 Elmar Nurmemmedov,1 Venkata Yenugonda,1 Mark Glassy,3 Santosh Kesari1. 1 _John Wayne Cancer Institute, Santa Monica, CA;_ 2 _Nascent Biotech, Inc, San Diego, CA;_ 3 _Nascent Biotech, Inc., San Diego, CA_.

Monoclonal antibodies (mAbs) as therapeutics for cancer have shown some success in the clinic. These biologics are useful if targeting a cancer specific epitope. Cancer patients can generate tumor-specific B lymphocytes which can be isolated to develop human mAbs against tumor-associated antigens. The best source of anti-tumor antibodies is from sentinel lymph nodes. Pritumumab (also referred to as CLNH-11, CLN-IgG, or ACA-11) is a classic example of a natural human anti-cancer antibody. It is a natural human IgG1 kappa antibody developed by the human hybridoma technology, using B lymphocytes isolated from a regional draining lymph node of a patient with cervical carcinoma. In the original patient from whom the B lymphocyte was isolated, the bioavailability of the target antigen induced a natural immune response resulting in the generation of the pritumumab IgG. The target antigen recognized by pritumumab is cell surface expressed vimentin, also referred to as ecto-domain vimentin (EDV). Vimentin is an intracellular cytoskeletal protein overexpressed during epithelial-to-mesenchymal transition (EMT), a process integral to cancer cell metastasis. Pancreatic cancer is a deadly disease with poor prognosis. No more than 6% of pancreatic cancer patients will survive beyond five years after diagnosis. There is a clear unmet need to help improve outcomes for patients. (A) Purpose of the study: to examine if pritumumab mAb can target pancreatic cancer cells and inhibit pancreatic tumor growth. (B) Experimental Procedures: Immunohistochemical analysis with horseradish peroxidase (HRP)-conjugated pritumumab on pancreatic cancer patient tissue sections along with immunofluorescence, flow cytometry, and In-Cell ELISA to demonstrate antibody binding. Antibody-dependent cell-mediated cytoxicity (ADCC) was performed to determine mechanism of action. In vivo subcutaneous xenograft mouse model treated with pritumumab to assess efficacy of the antibody for tumor inhibition. (C) Results: In this study we demonstrate pritumumab binds to the cell surface of patient pancreatic cancer tissue and binds the surface of established pancreatic cancer cells in culture. We show antibody binding to these cells induces antibody-dependent cell-mediated cytoxicity (ADCC). Furthermore, pritumumab effectively inhibits pancreatic tumor growth in a xenograft mouse model. (D) Statement of Conclusions: overall, these data provide pre-clinical validation of pritumumab mAb as a therapeutic for pancreatic cancer.

#3829

Generation and characterization of novel antibodies against the β subunit of MUC4.

Catherine Orzechowski, Abhijit Aithal, Wade Junker, Prakash Kshirsagar, Surinder Batra, Maneesh Jain. _University of Nebraska Medical Center, Omaha, NE_.

Mucins are high molecular weight glycoproteins that are primarily expressed by the apical surfaces of respiratory, gastrointestinal and reproductive tracts. They are involved in protection, growth and repair of epithelial surfaces. Apart from its protective function, mucins also regulate diverse functions in normal cells and have a crucial role in oncogenesis. MUC4 is a type-1 transmembrane mucin, which is overexpressed in several cancers and has been implicated in oncogenic transformation, proliferation, invasion, inhibition of apoptosis, and chemoresistance properties associated with these cancer cells. It is a 930 kD glycoprotein, consisting of a large high molecular weight subunit, MUC4α, containing the identical 16 amino acid tandem repeat region and MUC4β, a membrane-bound subunit, containing three EGF (Epidermal Growth Factor)-like domains. The latter subunit is responsible for the oncogenic functions of this molecule and can potentially be targeted. The monoclonal antibody 8G7 has been used extensively to study MUC4 expression. Since 8G7 binds an epitope in the tandem repeat (TR) region on the MUC4α subunit it cannot detect the cleaved cell surface associated form of MUC4 or isoforms that lack the TR region or heavily glycosylated TR regions. The MUC4β subunit however, is attached to the surface of the cell making it an ideal candidate for studying and targeting MUC4. The aim of this study is to generate and characterize monoclonal antibodies (mAbs) against the β subunit of MUC4. Thirty-two mAbs against MUC4β subunit were initially isolated using ELISA screening. Flow cytometry was used to test high affinity binders in MUC4 transfected cells. It was then tested whether these mAbs were able to immunoprecipitate transfected MUC4 from detergent extracted lysates. Finally, four high affinity mAbs (E9 (IgG2b), 6E8 (IgG2b), 1C7 (IgG1), and 3B4 (IgA)) were selected from these analyses. All the four mAbs immunoprecipitated full length MUC4 from native cancer cell lines. All the four mAbs bound to native human MUC4 expressed on cancer cell surface of different cell lines albeit with varying affinities. 3B4 recognized a conformational epitope and exhibited cell surface binding. The mAb 3B4 was internalized into MUC4 expressing cells in a concentration time-dependent manner. 6E8 showed partial internalization while E9 mAb did not show any internalization. Both 3B4 and 8G7 were able to cap MUC4 at the cell surface of cancer cell lines. Our results suggested that these antibodies can potentially block MUC4 function. The mAbs, 3B4, 1C7 and 6E8 inhibited the growth of MUC4 expressing cells in MTT and colony forming assays. Studies are currently underway to evaluate the utility of these novel MUC4β-domain specific mAbs to target MUC4-expressing tumors in vivo. Overall these mAbs can be used as reagents to study MUC4 cleavage and can also be developed into MUC4 specific diagnostic and therapeutic agents.

#3830

Monoclonal and bispecific antibodies against human insulin-like growth factors in oncology.

Qi Zhao. _University of Macau, Taipa, Macao_.

The insulin-like growth factor (IGF) axis plays an important role in cell growth and differentiation. During the past decade, IGF type 1 receptor (IGF-1R) was deemed a promising target for cancer therapy. The major challenge is that abnormal activation of IGF-1R ligands (IGF-I and IGF-II) exists in series of cancers and contributes to the resistance in IGF-1R-targeted therapies. The IGF-I/II actions are mediated through the IGF receptor type 1 (IGF-1R) and the insulin receptor (IR), which are overexpressed in multiple types of tumors. Up to date, we have developed different antibody formats against IGF-II or IGF-I/II, including one fully human IgG m708.5 with picomolar affinity[1], two bispecific antibodies (BsAbs) m660 [2, 3] and m67 [4], and one small-size nanobody s7g1 (15 kDa) (unpublished data). BsAb m67 has exhibited in cynomolgus macaques and high stability in serum. Compared with large-size antibodies, nanobody s7g1 has distinct advantages because of greater and more rapid tissue accumulation. All antibodies can eliminate the efflux of IGFs from tumor tissues and circulating IGFs in the serum. The m708.5 shows dual-specific for IGF-I/IGF-II with the highest affinities. The m708.5 has shown satisfying preclinical activities against a broad spectrum of human cell lines. When tested in neuroblastoma cell lines, m708.5 showed strong antitumor activity alone and in combination with chemotherapeutic agents [5]. Therefore, the anti-IGF-I/II antibodies offers an alternative approach to target both the IGF-1R and IR-A signaling pathways through the inhibition of IGF-I/II. These results support the clinical development of anti-IGF antibodies for solid tumors with potential for synergy with chemotherapy. Acknowledgements: This work was supported by the Science and Technology Development Fund of Macau (FDCT/131/2016/A3). References: 1. Zhao, Q., et al.. Mol Cancer Ther, 2011. 10(9): p. 1677-85. 2. Chen, W., et al.. Mol Cancer Ther, 2012. 11(7): p. 1400-10. 3. Feng, Y., et al.. Mol Cancer Ther, 2009. 8(5): p. 1113-8. 4. Feng, Y., et al.. Exp Mol Pathol, 2014. 97(3): p. 359-67. 5. Zhao, Q., et al.. Int J Cancer, 2015. 137(9): p. 2243-52.

#3831

Development of near-infrared photoimmunotherapy targeting GD2-positive neuroblastoma.

Hiroshi Nouso. _Okayama University, Okayama, Japan_.

Background: Neuroblastoma (NB) is a primary malignant tumor of the peripheral sympathetic nervous system. Despite the recent advances in the treatment of NB, patients with high-risk NB still have poor prognosis. Immunotherapy using anti-GD2 monoclonal antibody (mAb), which binds to disialoganglioside GD2 on the surface of NB cells, has recently become a standard treatment for high-risk NB. However, the therapeutic efficacy of anti-GD2 mAb is insufficient in many clinical trials. Therefore, the enhancement of therapeutic potential of anti-GD2 mAb is needed to improve the clinical outcome of patients with high-risk NB. Near-infrared photoimmunotherapy (NIR-PIT) is a promising antitumor strategy to enhance the therapeutic potential of immunotherapy using an antibody-photoabsorber conjugate (APC). In this study, we investigated the therapeutic efficacy of anti-GD2-based NIR-PIT using anti-GD2 mAb conjugated to the photoabsorber IR700 (anti-GD2-IR700) in human NB cells.

Methods: We used 3 human NB cell lines, including IMR-32, CHP-134 and SK-N-SH. The expression of GD2 on the cell surface of NB cells was analyzed by flow cytometric analysis. Anti-GD2-IR700 was synthesized by incubating anti-GD2 mAb and IR700. The mixture was purified with a Sephadex G50 column. The antitumor effect of anti-GD2-IR700 was evaluated using XTT assay. NB cells were incubated with anti-GD2-IR700, anti-GD2 mAb or PBS and NIR light was irradiated at 2.0J/cm2.

Results: IMR-32 and CHP-134 cells, but not SK-N-SH cells, expressed GD2 protein on the cell surface. In GD2-positive IMR-32 and CHP-134 cells, administration of anti-GD2-IR700 significantly suppressed the cell viability compared to anti-GD2 mAb or PBS when combined with NIR light irradiation. In contrast, in GD2-negative SK-N-SH cells, anti-GD2-IR700 showed no significant inhibition of cell viability in combination with NIR light irradiation.

Conclusion: These results suggest that NIR-PIT using an anti-GD2-IR700 is a promising antitumor strategy to promote the therapeutic efficacy of anti-GD2 immunotherapy for high-risk NB.

#3832

A proliferation-inducing ligand (APRIL) directly impacts immune regulatory subsets to regulate suppressive multiple myeloma bone marrow microenvironment.

Yu-Tzu Tai, Liang Lin, Liji Xing, Liji Xing, Kenneth Wen, Nikhil Munshi, Paul Richardson, Kenneth Anderson. _Dana-Farber Cancer Inst., Boston, MA_.

A proliferation inducing ligand (APRIL), abundant in serum samples of multiple myeloma (MM) patients, promotes MM cell progression in vivo predominantly via MM-specific receptor B cell maturation antigen (BCMA) (Blood. 2016; 127:3225). Here, we study whether APRIL directly regulate non-MM subpopulations to influence immunosuppressive bone marrow (BM) milieu and further define molecular mechanisms regulating these processes. First, transmembrane activator and calcium modulator (TACI) protein, the other APRIL receptor expressed at lower levels than BCMA on MM cell membrane, is consistently expressed at higher levels on CD4+CD25highFoxP3high T regulatory (Treg) vs autologous CD4+CD25- conventional T cells (Tcon) from MM patients (n=10, p<0.02). In contrast, BCMA is confirmed undetectable in T cells. TACI levels are even higher in the IL10+Foxp3+ subset within Foxp3+ Tregs of paired BM and PB samples from MM patients (n=17). TACI mRNA is also increased by >10-fold in purified Tregs vs autologous Tcon (n=12, p<0.01), which is strongly associated with upregulated immune inhibitory genes including Foxp3, CTLA4, IL-10, and CD38. APRIL preferentially induces growth and survival genes in Treg than paired Tcon, confirming elevated TACI levels in Treg vs autologous Tcon (n=4). Importantly, APRIL stimulates proliferation of Treg more potently than Tcon (n=3). In the presence of Treg, APRIL further suppresses proliferation of autologous Tcon (n=3) stimulated by anti-CD3/CD28 beads in a dose-dependent manner. MM-induced Treg (iTreg) can be generated in ex vivo co-cultures of MM cells with PB mononuclear cells or Tcon, mimicking increased Tregs linked to disease progression. Importantly, APRIL further augments generation of iTreg. Osteoclasts, a major physiological source of APRIL in the BM, significantly enhance iTreg generation that blocks Tcon proliferation. APRIL, without MM cells, cannot convert Tcon into iTreg, confirming a lack of direct impact due to the absence of TACI in paired Tcon. APRIL preferentially induces IL-10 and PD-L1 in TACIhigh Treg vs paired TACIlow/-Tcon. Finally, APRIL upregulates CD19+CD24highCD38high B regulatory cells (Breg) which interact with MM cells in the BM to confer an immunosuppressive microenvironment. Importantly, APRIL-induced Breg has higher IL-10, which is blocked by a blocking APRIL monoclonal antibody. Taken together, we are the first to characterize significantly high TACI expression that is functional in immune checkpoint Treg and Breg subsets which barely express BCMA. Importantly, we identify additional biological functions of APRIL in regulating immunosuppression via specific TACI- but not BCMA-mediated signaling cascades in the MM BM milieu, further supporting targeting this mechanism-based immunotherapeutic pathway to simultaneously target MM cells and revert immunosuppression.

#3833

SEA-BCMA: A highly active enhanced antibody for multiple myeloma.

Heather Van Epps, Martha Anderson, Changpu Yu, Kerry Klussman, Lori Westendorf, Chris Carosino, Luke Manlove, Julia Cochran, Jason Neale, Dennis Benjamin, Maureen Ryan. _Seattle Genetics, Bothell, WA_.

Despite a number of new therapies for multiple myeloma (MM) most patients relapse, require multiple lines of therapy, and ultimately succumb to disease. Therapies that are well tolerated and active remain an unmet need. Currently, successful MM therapies combine agents with different mechanisms of action and safety profiles. Therapeutic antibodies have recently altered the MM treatment paradigm, allowing patients to achieve deeper responses with minimal added toxicity. SEA-BCMA is a humanized afucosylated IgG1 antibody targeting BCMA, which shows preclinical evidence of encouraging activity and tolerability. BCMA is expressed at the surface of plasma cells, and induces proliferative signals through the binding of its ligands APRIL and BAFF. SEA-BCMA acts through three mechanisms of action. One, it engages in increased binding to FcγRIII through SEA technology leading to enhanced antibody dependent cellular cytotoxicity. Two, it mediates antibody dependent cellular phagocytosis. Three, it blocks the proliferative signals from BCMA ligand binding. This antibody shows activity in all seven tumor xenograft models tested, inducing tumor delays at doses as low as 0.1mg/kg, and generating prolonged survival and durable regressions with repeat dosing. SEA-BCMA is active on tumor xenografts expressing as few as 2000 surface copies of BCMA antigen. Both effector function and ligand blocking contribute to overall in vivo activity. In the absence of effector cell recruitment, ligand blocking alone can induce prolonged durable regressions. In addition, SEA-BCMA can target MM cells in the presence of soluble BCMA. SEA-BCMA is tolerated up to 100mg/kg in cynomolgus monkey, reflecting lack of toxicity from cellular interactions with the Fc portion of the Ab, since SEA-BCMA does not bind to the cynomolgus monkey antigen. SEA-BCMA displays a 12-day half-life in these animals. In vitro testing with human PBMCs or bone marrow mononuclear cells induced minimal cytokine production in the presence of BCMA target. In summary, SEA-BCMA is highly active and well tolerated in preclinical models and is a strong candidate for treatment of MM patients.

#3834

Antibodies targeting Resokine, a soluble immune modulator, inhibit tumor growth in syngeneic mouse models.

Kathy Ogilvie, Cherie Ng, Leslie Nangle, Jeanette Ampudia, Joon Chang, Andrea Cubitt, David J. King, John Mendlein. _aTyr Pharma, San Diego, CA_.

A number of non-canonical functions have been established for proteins generated from the tRNA synthetase gene family. One of these, termed Resokine, is derived from histidyl tRNA synthetase and plays an important role in controlling immune cell activation. Circulating levels are sufficient to down-regulate the extent of T cell activation that can be achieved in vitro. A panel of specific monoclonal antibodies have been generated and tested for their anti-tumor activity in mouse syngeneic tumor models. Antibodies to Resokine demonstrated anti-tumor activity across three different tumor models. Treatment of subcutaneous CT26 tumors resulted in improved efficacy compared to treatment with antibodies that block the PD-1/PD-L1 interaction. Significant efficacy was also observed in the difficult to treat subcutaneous B16F10 melanoma and 4T1 breast tumor models. In addition anti-Resokine demonstrated significant activity in a tumor seeding model using B16F10 melanoma which resulted in inhibition of tumor nodules in the lung, and was more efficacious than a combination of antibodies to PD-L1 and CTLA-4. Combinations of anti-Resokine antibody with either anti-PD-1 or anti-PD-L1 demonstrated at least additive, and potentially synergistic activity in these models. Animals with long-term tumor regressions were re-implanted with viable tumor cells, and demonstrated long-term immune memory with rejection of the newly implanted tumors. To understand the mechanism of anti-Resokine antibody therapy, cell depletion studies were carried out in the B16F10 tumor model. In these experiments activity of anti-Resokine antibodies was demonstrated to be dependent upon the presence of CD8 T cells and also NK cells, but independent of CD4 T cells. The immune based mechanism of antibodies to Resokine was further demonstrated by re-challenge of mice that had regressed tumors upon treatment. Tumor re-growth was not observed even in the absence of further treatment whereas control mice grew tumors at the normal rate, suggesting that immune memory had been induced. Antibodies to Resokine offer an exciting new potential option for immunotherapy of cancer, which has significant activity as monotherapy and is compatible with more established modalities. Anti-Resokine antibodies are currently being developed to initiate clinical evaluation.

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

### Novel and Canonical Targets

#3835

Targeting loss of MTAP expression in glioblastoma multiforme with a combination of 2-fluoroadenine and methylthioadenosine.

Baiqing Tang. _Fox Chase Cancer Center, Philadelphia, PA_.

Methylthioadenosine Phosphorylase (MTAP) is a key enzyme in the purine and methionine salvage pathways that convert the polyamine byproduct 5-deoxy-5-methylthioadenosine (MTA) into adenine and methylthioribose-1-phosphate. Homozygous deletion of the MTAP gene is observed in 55% of all glioblastoma multiforme (GBM) tumors. Since MTAP is a house-keeping enzyme found in all normal tissues, the lack of MTAP in GBM makes MTAP-loss a potential therapeutic target. In this study, we explore a novel strategy to target MTAP-loss in GBM by combining 2-fluoroadenine (2FA), a highly toxic adenine analogue, with MTA. Because MTAP is expressed in all normal tissues, MTA will be converted to adenine, which will protect against 2FA toxicity. However, in tumor cells, the effects of 2FA will not be ameliorated because they lack MTAP. Here we show that there is undetectable expression of MTAP in A172 and U87 GBM cell lines by both western blot and MTAP enzyme activity assays. In vitro toxicity studies in which cells were cultured with either 2FA alone or 2FA plus MTA revealed that both cell lines were sensitive to 2FA with or without MTA. The LD50 of A172 cells was 110 nM (2FA) and 127 nM (2FA + 5 μM MTA), while for U87 cells it was 397 nM (2FA) and 323 nM (2FA + 5μM MTA). This is in sharp contrast to MTAP+ NIH3T3 cells in which we observed a 44 fold shift in the ratio of the LD50 concentration in the presence and absence of MTA (160 nM versus 7000 nM). Addition of MTA protected normal tissues against 2FA toxicity in SCID mice. SCID mice subcutaneously injected with either A172 or U87 cells and then treated with 2FA(10 mg/kg) + MTA(50 mg/kg) showed significantly reduced tumor growth

compared to vehicle treated controls. Our results indicated that 2FA+MTA might be effective in treating GBM.

#3836

Imbalanced nucleotide metabolism sensitizes breast cancer cells to anthracyclines.

Craig Davison, Olivier P. Chevallier, Catherine Knowlson, Melanie McKechnie, Robbie Carson, Jaime Esteve, Richard Wilson, Robert D. Ladner, Melissa J. LaBonte (Wilson). _Queen's University Belfast, Belfast, United Kingdom_.

Introduction. Triple negative breast cancer (TNBC) makes up 15% of breast cancers and is associated with poor prognosis. TNBC treatment remains hampered by early visceral metastasis and lymph node involvement at the time of diagnosis and limited effective therapeutic options. Advances in treatment that translate to significant improvements in outcome have been painstakingly incremental, despite advances in research technologies and TNBC subtyping.

We have identified deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) as a critical gatekeeper that protects tumor DNA from the genotoxic misincorporation of uracil during treatment with anthracyclines that are commonly used in the FEC chemotherapy regimen. dUTPase catalyses the hydrolytic dephosphorylation of deoxyuridine triphosphate (dUTP) to deoxyuridine monophosphate (dUMP). This reaction has the dual function of providing dUMP for thymidylate synthase (TS) as part of the thymidylate biosynthesis pathway and of maintaining low intracellular dUTP pools. This is crucial as DNA polymerase cannot distinguish between dUTP and deoxythymidylate triphosphate (dTTP), leading to dUTP misincorporated into DNA. dUTPase is being evaluated as a target in cancer therapy alongside TS-targeted therapies and uracil misincorporation is believed to be a potential mechanism of cytotoxicity. Hypothesis: Targeting dUTPase and inducing uracil misincorporation during the repair of DNA damage induced by anthracyclines represents a novel strategy to induce TNBC cell lethality.

Methods. Inhibition of dUTPase (DUTi) was carried out using SMARTpool siRNA or small molecule inhibition. The effect of DUTi in combination with anthracyclines was determined by growth inhibition and clonogenics assays. DNA damage was assessed by Western blot, immune-fluorescent foci detection, and flow cytometry. Nucleotide metabolites were quantified by LC/MS.

Results. DUTi significantly sensitised TNBC cell lines to doxorubicin and epirubicin. In the MDA-MB-231 cancer cells, loss of dUTPase expression resulted in a synergistic 80% reduction in survival compared to 0.075µM doxorubicin alone at 40%. This decrease in survival correlated with increased activation of proteins involved in DNA damage response, including ɣ-H2AX(Ser139), a marker for dsDNA breaks. We anticipate that quantification of nucleotides will show an imbalance of dUTP:dTTP, subsequently leading to uracil misincorporation and resulting cell death.

Discussion. These results suggest that repair of anthracycline-induced DNA damage requires dUTPase to prevent uracil misincorporation and that inhibition of dUTPase is a potential novel strategy to enhance the efficacy of anthracyclines. This shows the potential advantage of a dUTPase inhibitor being added to current chemotherapy regimens for TNBC.

Future work will elucidate the mechanism of sensitisation and in vivo translation of combination therapy for TNBC.

#3837

Bumped kinase inhibitor 1553 selectively inhibits androgen receptor positive prostate cancer.

Takuma Uo, Shihua Sun, Kathleen Haugk, Kathryn Soriano Epilepsia, Mamatha Damodarasamy, Matthew Hulverson, Wesley VanVoorhis, Kayode K. Ojo, RamaSubbaRao Vidadala, Dustin Maly, Stephen Plymate, Cynthia C. Sprenger. _Univ. of Washington, Seattle, WA_.

BACKGROUND: Prostate cancer (PCa) is the second most common cancer in men and one of the leading causes of cancer death. Resistance to current PCa therapies, including androgen deprivation, occurs in almost all patients leading to development of castration resistant prostate cancer (CRPC). Resistance is associated with expression of splice variants of the androgen receptor (AR-Vs) that are constitutively active. The intrinsically disordered N-terminus of the AR/AR-Vs makes targeting the AR-Vs difficult. Thus, therapies that indirectly target the AR by inhibiting factors important in regulating AR levels and activity may be the most successful against inhibiting the constitutively active AR variants. Kinases have been shown to be important in regulating the AR, thus kinase inhibitors have the potential to inhibit androgen receptor signaling and function. OBJECTIVE: To demonstrate that our lead kinase inhibitor targets and inhibits AR positive CRPC. RESULTS: Bumped kinase inhibitors designed at the University of Washington are based on a pyrazolopyrimidine backbone, with R1 and R2 groups that confer a "bump" thereby preventing the BKIs from binding to most human kinase ATP binding pockets. While the majority of BKIs do not have activity against CRPC, a subset have activity against AR+ PCa lines, including CRPC lines expressing AR-Vs, but with no activity against AR negative PCa lines or a broad range of other human cell lines. We next examined the effects of our lead BKI compound (1553) on AR expression, phosphorylation, and function. BKI-1553 blocked transactivation activity of AR as judged by the reporter assays using an ARE-luciferase construct. Expression of canonical AR target genes was downregulated in LNCaP cells treated with BKI-1553. Westerns demonstrated a decrease in total AR 4 and 24 hours after treatment with BKI-1553. Levels of phospho-Serine81on the AR, which is critical for AR activity, were decreased even further than total AR by 4 hours after treatment. Thus, AR signaling appears to constitute BKI-sensitive components. We then decided to examine the effect of BKI-1553 on in vivo tumor growth. Toxicity studies demonstrated that it was non-toxic in mice at oral doses that achieved therapeutic levels. In vivo treatment with BKI-1553 significantly decreased growth of a castrate-sensitive patient-derived PCa xenograft and a castration-resistant PCa cell line (LuCaP35, p < 0.01 and LNCaP95, p < 0.005, respectively). SUMMARY: Our lead bumped kinase inhibitor, BKI-1553, selectively inhibits the growth of prostate cancer cells that express full-length AR and/or AR-Vs. BKI-1553 reduced not only cell proliferation, but decreased levels of AR and AR variants, decreased pSer81 levels, and decreased activation of AR-regulated promoters. Since the majority of CRPCs are AR driven, our targeted BKIs could be a novel therapy for cancers that are resistant to currently available therapies.

#3838

Inhibition of the spectraplakin protein MACF1 sensitizes glioblastomas to BCNU.

Danielle Borlay, Kala Bonner, Orica Kutten, Katelyn Atkinson, Quincy A. Quick. _Tennessee State Univ., Nashville, TN_.

Glioblastoma heterogeneity and diversity is recognized as a major contributing factor to the resistance of these cancers to clinical chemo- and radiotherapy treatment regimens. This diversity is attributed in part to a plethora of novel unevaluated proteins whose functions participate in glioblastoma etiology, progression, and recurrence. One such protein is the cytoskeletal cross-linker, Microtubule Actin Crosslinking Factor 1 (MACF1), which we have previously shown was expressed at high levels in glioblastoma tissue as compared to its absence in normal brain tissue. In support of these findings immunoblotting expression analysis in this study showed that MACF1 protein expression was upregulated in response to BCNU exposure in glioblastoma cells. Subsequently, experiments were conducted to assess the combinatorial effect of MACF1 inhibition in conjunction with BCNU treatment. A CRISPR-Cas9 genome editing approach was used to inhibit MACF1 function prior to drug exposure. Cell proliferation analysis revealed a cooperative synergistic effect of MACF1 inhibition and BCNU treatment on the reduction of glioblastoma cells as compared to cells treated with either BCNU or CRISPR inhibition of MACF1 alone. In conclusion, upregulation of MACF1 in response to BCNU suggests that this spectraplakin protein may play a role in the mechanistic resistance of glioblastomas to BCNU and the therapeutic prognosis of glioblastomas treated with this drug. Additionally, this study demonstrated that MACF1 inhibition is a novel neoadjuvant approach that enhances the therapeutic efficacy of BCNU.

#3839

A small-molecule inhibitor of the β-catenin-TCF4 interaction suppresses colorectal cancer growth in vitro and in vivo.

Seung Ho Shin,1 Do Young Lim,1 Kanamata Reddy,1 Margarita Malakhova,1 Fangfang Liu,2 Ting Wang,2 Mengqiu Song,2 Hanyong Chen,1 Ki Beom Bae,1 Joohyun Ryu,1 Kangdong Liu,2 Mee-Hyun Lee,2 Ann M. Bode,1 Zigang Dong1. 1 _The Hormel Institute, University of Minnesota, Austin, MN;_ 2 _The China-US (Henan) Hormel Cancer Institute, Zhengzhou, China_.

Colorectal cancer is associated with aberrant activation of the Wnt pathway. β-Catenin plays essential roles in the Wnt pathway by interacting with T-cell factor 4 (TCF4) to transcribe oncogenes. We synthesized a small molecule, referred to as HI-B1, and evaluated signaling changes and biologic consequences induced by the compound. HI-B1 inhibited β-catenin/TCF4 luciferase activity and preferentially caused apoptosis of cancer cells in which the survival is dependent on β-catenin. The formation of the β-catenin/TCF4 complex was disrupted by HI-B1 due to the direct interaction of HI-B1 with β-catenin. Colon cancer patient-derived xenograft (PDX) studies showed that a tumor with higher levels of β-catenin expression was more sensitive to HI-B1 treatment, compared to a tumor with lower expression levels of β-catenin. The different sensitivities of PDX tumors to HI-B1 were dependent on the β-catenin expression level and potentially could be further exploited for biomarker development and therapeutic applications against colon cancer.

#3840

Plocabulin, a tubulin inhibitor, presents antitumor activity in patient-derived xenograft (PDX) models of gastrointestinal stromal tumor (GIST).

Agnieszka Wozniak,1 Yannick Wang,1 Jasmien Wellens,1 Yemarshet K. Gebreyohannes,1 Maria Jose Guillén,2 Carlos M. Galmarini,2 Pablo M. Avilés,2 Maria Debiec-Rychter,1 Raf Sciot,1 Patrick Schöffski1. 1 _KU Leuven and University Hospitals Leuven, Leuven, Belgium;_ 2 _PharmaMar, Madrid, Spain_.

Introduction: Advanced GIST is commonly treated with tyrosine kinase inhibitors (TKI) [e.g. imatinib (IMA)]. With time the vast majority of patients develops TKI-resistance. GIST is generally believed to be resistant to chemotherapy with cytotoxic agents. The aim of our study was to test plocabulin (PLO; PM060184, PharmaMar), a potent cytotoxic tubulin-dynamics modifier, in two PDX models of GIST, characterized by different sensitivity to IMA.

Experimental set-up: NMRI nu/nu mice (n=34) were transplanted bilaterally with human xenografts UZLX-GIST3sens (KIT: exon 11 p.W557_V559delinsF; IMA-sensitive) or -GIST9res (KIT: exon 11+17: p.P577del;W557LfsX5;D820G; IMA-resistant). Xenografted animals were randomly assigned to three treatment groups: control [vehicle, 5ml/kg/QW intravenously (i.v.)], IMA (50mg/kg/BID orally) and PLO (16mg/kg/QW, i.v.). Treatment lasted 22 days and the antitumor activity was assessed by tumor volume measurement, histopathology and KIT signaling pathway by Western blotting. Histological response (HR) was evaluated as previously described by Antonescu et al. 2005. Mann Whitney U test was used for statistical analysis with p <0.05 considered as significant.

Results: PLO treatment resulted in a reduction of tumor volume to 59% in GIST3sens and to 70% of the baseline volume in the GIST9res model. Good HR (grade 3, 4) was observed in 70% (GIST3sens) and 50% (GIST9res) of tumors. HR obtained with PLO was mainly characterized by necrosis, while IMA produced mainly myxoid degeneration in the sensitive model. In addition, in GIST3sens PLO decreased the microvessel area and increased apoptosis as assessed by immunohistochemistry, which was not observed in GIST9res. In the latter model PLO showed better activity than IMA in terms of tumor volume reduction (59% vs. 146%, p<0.01) and HR (grade 3, 4 in 50% vs. 0%). KIT signaling was not affected by PLO. The experimental drug was well tolerated throughout the experiment at the dose administered.

Conclusions: PLO is the first anti-tubulin agent showing antitumor activity in GIST PDX, both in models sensitive or resistant to IMA. The drug causes cytotoxicity in GIST, mainly through necrosis, without affecting KIT signaling. Due to the different modes of action of PLO and established TKI our work provides a scientific rationale to combine PLO and IMA to overcome resistance to small molecule TKI.

#3841

Biologic evaluation of heterocyclic pyrimidines as novel tubulin inhibitors targeting the colchicine binding site.

Kinsie Arnst. _Univ. of Tennessee Health Science Ctr., Memphis, TN_.

Interfering with tubulin dynamics is an attractive strategy for cancer therapy, and tubulin-targeting agents are some of the most widely used chemotherapeutic drugs. Current FDA-approved tubulin inhibitors target either the taxane site (e.g., paclitaxel) or the vinca alkaloid site (e.g., vinblastine). However, their clinical efficacy is often limited by the development of multidrug resistance and a narrow therapeutic index. Tubulin inhibitors interacting with the colchicine site have shown potent anticancer activities, are generally less susceptible to multidrug resistance, and can disrupt tumor vasculature, leading to rapid tumor cell apoptosis. Here, we report on the biologic evaluation of heterocyclic pyrimidines as a new class of tubulin inhibitors targeting the colchicine binding site with a significantly improved therapeutic index. One of the most promising compounds of the series, SB157, was able to strongly inhibit tubulin polymerization and induce cytotoxicity in cancer cells in vitro. We have also obtained the high-resolution X-ray crystal structure for SB157, confirming its direct binding to the colchicine binding site in tubulin, and elucidated the detailed molecular interactions. SB157 significantly inhibited tumor growth in an A375 melanoma xenograft model, and the tumors revealed elevated apoptotic activity and extensive disruption of tumor vasculature. Finally, we demonstrated that SB157 significantly overcame clinically relevant multidrug resistance in a paclitaxel-resistant PC-3/TxR prostate cancer xenograft mouse model. Collectively, these studies provide preclinical proof of concept and structural basis to support the continued development of SB157 and optimization of this scaffold as an improved class of tubulin inhibitors for cancer therapy.

#3842

Potent antitumor activity of the HSP90 inhibitor SNX-2112 in type 1 papillary renal cell carcinoma.

Roma Pahwa,1 Abhigya Giri,1 Carole Sourbier,1 Craig Thomas,2 W Marston Linehan,1 Len Neckers,1 Ramaprasad Srinivasan1. 1 _Urologic Oncology Branch, National Cancer Institute, National Institute of Health, Bethesda, MD;_ 2 _National Center for Advancing Translational Sciences (NCATS),NIH, Bethesda, MD_.

Papillary renal-cell carcinoma (pRCC) is the second most common form of kidney cancer, accounting for 15 to 20% of all cases. There are currently no treatment options of proven benefit for patients with advanced pRCC. The type 1 subset of pRCC tumors are associated with gain of chromosome 7 and activating mutations of MET that could lead to aberrant MET pathway activation resulting in cell proliferation and anti-apoptotic signaling. While MET kinase inhibitors are active in some patients with type 1 pRCC, primary and acquired resistance is common, prompting us to investigate possible therapeutic alternatives. Heat shock protein 90 (Hsp90), which stabilizes numerous oncogenic proteins, including MET and several downstream mediators of MET activity, is an important regulator of cell proliferation and survival, and has attracted wide interest as a novel target for cancer therapy. Hsp90 inhibitors were also identified as molecules of interest based on antitumor activity against patient derived type 1 pRCC cell lines following quantitative high throughput screening (qHTS) with small molecule libraries. We hypothesized that inhibition of Hsp90 might provide a novel therapeutic approach for patients with pRCC tumors with MET pathway activation. SNX-2112 was chosen as a candidate Hsp90 inhibitor for further evaluation. Our data demonstrate that inhibition of the Hsp90 pathway is lethal to pRCC cell lines with MET pathway activation, and that treatment with SNX-2112 leads to the degradation of MET/pMET and its downstream signaling intermediates p-AKT, AKT, p-ERK. Furthermore, SNX-2112 leads to inhibition of critical downstream transcriptional targets of AKT and ERK, including VEGF, c-Myc and BIRC5. SNX-2112 has potent anti-tumor activity in vitro, inhibiting proliferation, clonogenicity and 3D-anchorage independent colony formation of MET activated pRCC cell lines. These data demonstrate that Hsp90 inhibition is a promising therapeutic strategy in Type 1 pRCC and is worthy of further preclinical and clinical evaluation.

#3843

The potential of heat shock proteins 70/90 as molecular therapeutic targets in breast cancer.

Ho Young Kim, Sanghwa Kim, Lee Su Kim. _Hallym Univ., Anyang, Republic of Korea_.

Purpose: Heat shock proteins (hsps) are molecular chaperones that are synthesized by cells in response to various stress conditions. The expression of hsps have been shown to be associated with carcinogenesis and the expression of hsps have been implicated in the biological behavior of tumors. Recently, hsps have emerged as novel molecular targets in anticancer protocols. The objectives of this study were to investigate the significance of hsp 70/90 in breast carcinogenesis and effect of geldanamycin (a blocker of hsp 90) and quercetin (a blocker of hsp 70) on growth inhibition in different breast cancer cell lines. Methods: Breast tissues from 82 patients were obtained between January 2010 and December 2010 at the Division of Breast & Endocrine Surgery, Hallym University Sacred Heart Hospital. Expression of hsp 70/90 was studied by immunohistochemistry (IHC) on tissue sections from 63 breast carcinomas and 19 benign breast tissues. Both cytoplasmic and nuclear expression was measured. Expression of hsp 70/90 was also analyzed by use of a Western blot with the breast cancer cell lines. We next investigated the effects of blockers of hsp 70/90 on cell growth of the human breast cancer cell lines. Results: More prominent hsp 90 expression was observed in malignant tissue than in benign tissue by both cytoplasmic and nuclear IHC staining (p<0.001, p<0.001). Nuclear hsp 90 expression was associated with a positive lymph node status (p=0.003) and the presence of poorly differentiated tumors (p=0.028). Expression of hsp 70 was not different in malignant and benign tissues as determined by both cytoplasmic and nuclear IHC staining. The breast cancer cell lines all expressed hsp 70/90. Geldanamycin markedly inhibited the cell growth of these breast cancer cell lines in a dose dependent manner and induced apoptosis in the cell lines. Quercetin inhibited cell growth of the cell lines less efficiently. Conclusion: The expression of hsp 90 was associated with breast carcinogenesis and the presence of more aggressive tumors. Geldanamycin inhibited cell growth of hsp 90 expressing breast cancer cell lines. We suggest that Hsp 90 may be a possible molecular target against breast cancer.

#3844

TAK-659, a SYK kinase inhibitor, demonstrates preclinical antitumor activity in solid tumor models.

Jessica J. Sappal, Matthew Theisen, Zhongmin Xiang, Stephen Tirrell, Rudy Christmas, Jie Yu, Mengkun Zhang, Karuppiah Kannan. _Takeda Pharmaceuticals, Cambridge, MA_.

TAK-659 is a highly potent, reversible inhibitor of spleen tyrosine kinase (SYK) and fms related tyrosine kinase 3 (FLT3) that is currently being investigated in Phase I and II clinical trials. SYK is expressed ubiquitously in hematopoietic cells, and abnormal function of SYK has been implicated in several types of hematopoietic malignancies, including diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL), and peripheral T-cell lymphoma (PTCL). Although there are considerable data supporting a role for SYK signaling in hematological cancers, data on the relevance of SYK in solid tumors are emerging. The literature suggests that SYK expression plays a role in the growth of breast and ovarian cancers, and the pancreatic adenocarcinoma microenvironment has been shown to be B-cell rich, giving us a basis to explore the activity of TAK-659 in these indications. Furthermore, ovarian cancer patients with recurrent disease have been shown to have an increase in pSYK at the time of relapse versus at diagnosis, suggesting a role for SYK in chemoresistance. The ability of TAK-659 to inhibit ex vivo colony formation of cells with the ability to grow anchorage independently in semi-solid medium was examined in 31 different human tumor xenografts. TAK-659 showed concentration-dependent inhibition of tumor colony growth in 5/31 tumor models tested, including gastric, breast, and ovarian cancers. Pre-clinically, TAK-659 has exhibited significant antitumor activity in a number of mouse xenograft models of solid tumors. We demonstrate here that daily oral administration of TAK-659 at 60mg/kg over 21 days leads to significant antitumor activity in primary human tumor models of gastric, pancreatic, triple negative breast (TNBC), and ovarian cancers, all of which express pSYK. Significant anti-tumor activity was also observed in the HCC70 TNBC xenograft model which does not express total SYK, demonstrating that TAK-659 may have anti-tumor effects beyond the inhibition of pSYK signaling. A decrease in myeloid derived suppressor cells was seen following TAK-659 treatment in this xenograft model, suggesting an immunomodulatory response may be driving the antitumor activity. Taken together, these data along with published information support the rationale for the investigation of TAK-659 in solid tumor cancer indications.

#3845

**24R,25-Dihydroxyvitamin D3 reduces tumor burden and metastasis in ER+ breast cancer** in vivo **.**

Anjali Verma, David Joshua Cohen, Chandana Muktipaty, Barbara D. Boyan, Zvi Schwartz. _Virginia Commonwealth University, Richmond, VA_.

The purpose of this study was to investigate the effects of 24R,25-dihydroxyvitamin D3 (24,25), on tumor growth and metastasis in an in vivo estrogen receptor positive (ER+) orthotopic breast cancer model. 1α,25(OH)2D3 has been shown to have pro-apoptotic effects on breast cancer both in vitro and in vivo. 24,25, a naturally occurring metabolite, is present in human serum at concentrations up to 10 times higher than 1,25. Moreover, 1,25 regulates production of 24,25 by stimulating expression of the 24-hydroxylase.

6-8 week old female nod scid gamma IL2R mice were kept on an ad libitum vitamin D deficient diet. 15 days before surgery, mice were implanted with 1.7mg 0-order release estrogen (E2) pellets. In the first experiment, mice were given a fast-release pellet (45 days). In the second experiment, mice were given a slow-release (90 day) pellet. After 15 days of acclimation (day 0), mice were injected in the T4 mammary fat pad with 1M fluorescently labeled MCF7 cells (an ER+ breast cancer cell line) and cells in 50:50 PBS:Matrigel solution. Tumors were measured once a week starting at day 14 and tracked throughout the course of the study. On day 30, 24,25 treatment was initiated. Each experiment was divided into three groups: 0 ng 24,25, 25 ng 24,25, or 100 ng 24,25 per injection, with n=6-8 mice per group. Treatments were administered three times a week in a 50µL intraperitoneal injection. On day 70, mice were harvested and assessed for tumor burden and metastasis by fluorescence detection microscopy. Fluorescent tissues were marked 'metastasis positive' and harvested for future immunohistochemical analysis.

Mice that were given 24,25 three times weekly showed decreased tumor burden and metastasis as compared to controls. In the first experiment (45 day E2), 25ng and 100ng 24,25 treated mice had significantly lower relative tumor volumes than controls beginning on day 70. These 24,25 treated mice also showed significantly lower rates of left axial lymph node metastasis, and had statistically longer survival rates. The second experiment (90 day E2) had similar results. Mice treated with 25ng and 100ng 24,25 had decreased tumor burden as compared to 0ng controls on day 70. Mice given 24,25 had decreased lung metastasis; however, mice treated with 100ng 24,25 had increased rates of right axial lymph node metastasis. Altogether, mice in both groups that were treated with 100ng of 24,25 had decreased rates of left axial lymph node metastasis and lung metastasis as compared to controls, and statistically longer survival rates. Thus 24,25 could play a role in 1,25's anti-apoptotic effect on breast cancer, potentially via upregulation of 24,25.

#3846

A metabolic pathway targeted inhibitor for precision therapy of castrate-resistant prostate cancer.

Hirak S. Basu,1 Nathaniel L. Wilgonowski,1 Jessica L. Lieblich,1 Grace T. Wu,1 Izabela Fokt,1 Sumankalai Ramachandran,1 Jiaquin Yu,2 Mark Titus,1 Waldemar Priebe,1 David J. Beebe,2 George Wilding1. 1 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _University of Wisconsin, Madison, WI_.

Background: We have previously reported that an AP-1 factor JunD is overexpressed in PCa. It complexes with androgen receptor (AR) to induce spermidine/spermine acetyl transferase (SSAT). SSAT initiates polyamine oxidation that generates copious amounts of reactive oxygen species (ROS) production in polyamine-rich PCa cells. ROS activate NF-κB. NF-κB also induces SSAT and activates AR. This sets-up a feed-forward loop for PCa growth at low androgen. An IHC assay for SSAT can be utilized to identify PCa patients with this loop activated. AR-N-terminal targeted inhibitors that block AR interactions with JunD and NF-κB can be developed as a precision therapy for these patients. Method: We applied Immunocytochemistry (ICC), proteomic and metabolomic techniques to analyze stationary and invading cells that are separated in a novel micro-scale dvice to understand the mechanism of invasion. Gaussia luciferase reconstitution assay in a high throughput screen (HTS) to identify inhibitors of AR-JunD and AR-NF-κB interactions. In vitro cell growth assay along with in vivo pharmacokinetic (PK), maximum tolerated dose (MTD) and enzalutamide-resistant CRPC cell xenograft growth in nude mice are used to optimize the lead. Unpublished data: Immunocytochemistry (ICC) data show that in cultured enzalutamide-resistant C4-2 and -sensitive LNCaP cells as well as in some patient PCa cells, more SSAT positive cells are in the migratory than in the stationary section of the microscale device. Screening of 27,000+ compounds from 2 different chemical libraries detected a single compound that specifically inhibits both AR-JunD and AR-NF-κB2 (p52) interactions. We have synthesized several of its analogs. Lead analogs inhibit growth of AR-positive C4-2 and LNCaP cells at nanomolar concentration, but have little effect on the growth of AR-negative PC-3 and SSAT silent siSSAT cells in culture. Co-immunoprecipitation (co-IP) assay shows its efficacy in inhibiting AR-JunD and AR-p52 interactions in situ. It has been formulated in 25% ethanol:water and administered orally. PCa tumor bearing nude mice tolerated it at 50 mg/kg p.o. daily for more than 28 days and has a serum Cmax of ~ 3 μM within 30 minutes and a plasma t1/2 of ~1 h after a single oral dose of 100 mg/kg. Twenty four hours after dosing it is found in the flank tumor (1.5 ng/mg tissue; ~450 nM) and in the prostate tissues (10 ng/mg tissue; ~3 μM) in addition to liver and kidney and inhibited growth of C4-2 xenografts in nude mice.

#3847

Inhibition of GCN2 sensitizes ASNS-downregulated cancer cells to asparaginase by disrupting amino acid response.

Akito Nakamura, Tadahiro Nambu, Shunsuke Ebara, Yuka Hasegawa, Kosei Toyoshima, Yasuko Tsuchiya, Daisuke Tomita, Jun Fujimoto, Osamu Kurasawa, Chisato Takahara, Ayumi Kawamura, Ryuichi Nishigaki, Yoshinori Satomi, Akito Hata, Takahito Hara. _Takeda Pharmaceutical Company Limited, Japan_.

General control nonderepressible 2 (GCN2) plays a major role in cellular response to amino acid limitation. Although maintenance of amino acid homeostasis is critical for tumor growth, little is known concerning the contribution of GCN2 to cancer cell proliferation and its potential as a therapeutic target. In this study, we demonstrate that inhibition of GCN2 with small-molecule compounds sensitizes cancer cells to anti-leukemic agent asparaginase (ASNase). We first tested acute lymphoblastic leukemia (ALL) cells and showed that treatment with GCN2 inhibitors rendered ALL cells sensitive to ASNase by preventing the induction of asparagine synthetase (ASNS). The prevention of ASNS expression suppresses asparagine synthesis and thus inhibits protein translation. In addition, comprehensive gene expression profiling revealed that the combined treatment induced stress-activated MAPK pathway and thereby triggering apoptosis. Using cell-panel analyses, we also identified a clear synergy between GCN2 inhibitors and ASNase in acute myelogenous leukaemia (AML) and pancreatic cancer cells besides ALL. Notably, basal ASNS expression levels were significantly correlated with sensitivity to combinatorial treatment. Furthermore, the combination displayed robust antitumor activity in mouse xenograft models of ALL, AML, and pancreatic cancer. These results demonstrate a novel mechanistic insight into a role of GCN2 in amino acid response and provide a rationale for further investigating GCN2 inhibitors for the treatment of cancer.

#3848

Functionalized small-molecule thiosemicarbazones as inhibitors of cathepsin K-mediated collagenase activity.

Mary Lynn Trawick, Kevin G. Pinney. _Baylor Univ., Woodway, TX_.

The metastatic spread of malignant cells from a primary tumor remains the major cause of death in cancer patients. Metastasis also contributes significantly to the debilitating side effects and pain associated with the disease. For example, patients with advanced breast cancer have a high rate of skeletal metastases that can result in intractable bone pain, nerve compression syndrome-associated paralysis, pathologic bone fractures and hypercalcemia. Clearly, the prevention and management of bone metastases has significant potential to improve the quality of life and survival of breast cancer patients. Recently, CatK was found to be overexpressed in various types of cancers, including highly metastatic breast, prostate, osteosarcoma melanoma and glioma cancers. In bone remodeling, the degradation of type I collagen, the major component of the organic matrix in bone, as well as other proteins is largely accomplished by cathepsin K (CatK) with a contribution by neutral matrix metalloproteinases. CatK is a powerful proteolytic enzyme that is synthesized as a proenzyme and is abundantly expressed by osteoclasts, multinucleated cells that form a specialized sealing zone within which their ruffled border secretes acid to dissolve the mineral component, and proteinases to degrade the organic component of bone. Procathepsin K (proCatK) is autocatalytically activated under acidic conditions, and can also be cleaved to its active form by other proteolytic enzymes. It is the major cysteine proteinase of osteoclast lysosomes. While CatK quickly loses activity after cleavage of its proenzyme in vitro, additional studies have shown that the enzyme is greatly stabilized by complex formation with the glycosaminoglycan chondroitin sulfate. Small-molecule thiosemicarbazones have received special attention due to their antibacterial, antiviral, antifungal and antineoplastic effects. Recent studies have also confirmed that thiosemicarbazones have the ability to inhibit human and parasitic cathepsin L like-cysteine proteases and the mammalian cathepsin L. This work reports the investigation of a library of thiosemicarbazones synthesized to test their efficiency to inhibit human cathepsin K. Nine thiosemicarbazone (TSC) compounds had IC50 values of less than 50 nM against CatK using a fluorogenic assay with Z-L-Phe-L-Arg-AMC. Kinetics of their inhibition demonstrated that they were time-dependent but slowly reversible enzyme inhibitors. One of these compounds was shown to inhibit the activity of CatK against collagen. This compound also inhibited the cleavage of the proenzyme to its mature form. The activity of CatK was greatly stabilized by the inclusion of chondroitin sulfate in the reaction mixture, but inhibition of the enzyme was not affected. These data indicate that potent, small-molecule TSC CatK inhibitors may be useful to prevent the metastatic spread of cancer to bone.

#3849

Identification and optimization of chemical compounds as potent agonists of human STING with anticancer activity in mice.

Jian Hui Wu. _McGill Univ. Lady Davis Inst., Montréal, Quebec, Canada_.

The host STING pathway plays a critical role in innate immune sensing of cancer, that drives type-I interferons (IFNs) production and promotes aggressive antitumor responses. Compound DMXAA is an agonist of mouse STING (mSTING) and demonstrated potent antitumor activities in several tumor models, including melanoma. However, DMXAA cannot activate human STING (hSTING), which provides a possible rationale for its failure in recent clinical trial. In this project, we aim to identify novel chemical compounds as potent STING agonists. By combining structure-based drug design and in vitro assays, we have discovered two initial hits as STING agonists that belong to two different chemical scaffolds. Direct binding of our compounds with hSTING was confirmed by Surface plasmon resonance (SPR) analysis. Chemical optimization of our initial hits has led to compound #150 and #171. We demonstrated that #150 and #171 at 10 uM have substantially activated STING pathway in HEK293 cells that were transiently transfected with plasmid expressing hSTING. Compound #171 at 20 uM potently activated STING signaling in human THP-1 cells that harbor hSTINGHAQ as well as PBMC cells from a panel of human donors. Importantly, with DMXAA as the control, we demonstrated that #171 at 20 mg/kg via i.p. has significant antitumor effect against TRAMP-c1 tumor in C57/BL6 mice.

#3850

FOXP3 dimerization by stapled alpha-helical peptides alters regulatory T cell function.

Rachel Eclov, Marie Fefferman, Ravand Samaeekia, James L. LaBelle. _University of Chicago, Chicago, IL_.

Regulatory T cells (Tregs) are critical for the maintenance of immune tolerance and control of immune responses to foreign agents. Tregs depend upon the transcriptional protein FOXP3 for their ontogeny and genetic orchestration of their suppressive mechanisms. While T cells have the ability to target tumor-associated antigens from many cancer subtypes, these T cells are often suppressed in patients with immunologically "silenced" tumors. The tolerizing function of Tregs can limit effective anti-tumor T cell-mediated killing, even in the presence of immunotherapies, including checkpoint inhibition, and their presence predicts reduced survival in patients with a myriad of cancers. Currently, there exists no means of depleting Tregs in patients without also targeting activated effector T cells. In an effort to overcome this, we have specifically inhibited Tregs by targeting FOXP3 through molecular mimicry using peptides designed to inhibit FOXP3 homodimerization. Homodimerization of FOXP3 is mediated by an antiparallel coiled-coil interaction between the leucine-zipper domains of two FOXP3 proteins. A series of single and double hydrocarbon-stapled alpha-helical (SAH) peptides were synthesized using the leucine zipper dimerization domain (DD) interface of FOXP3 (SAH-FOXP3DDs) as a molecular template. SAH-FOXP3DDs, but not native peptides, specifically bind FOXP3's leucine zipper domain with nanomolar affinity. Unlike stapled peptide point mutant controls, lead SAH-FOXP3DDs also showed dose-dependent inhibition of FOXP3 binding to cognate DNA. Lead SAH-FOXP3DDs are cell permeable and nontoxic to T cells as measured by absence of non-specific LDH-release and apoptosis induction following treatment. Flow cytometric analysis of treated Tregs measured changes in expression of a number of Treg/T cell - associated proteins including increases in FOXP3 and CD127 and decreases in CD25, while no changes were observed in treated conventional CD4+ T cells (Tcons) or with cells treated with point-mutant control peptides. In addition, treated Tregs demonstrated mRNA expression changes of FOXP3-regulated genes including Foxp3, CD127, IL2 and Ctla. Treatment of freshly isolated and in vitro expanded Tregs with lead SAH-FOXP3DD compounds inhibited Treg-mediated suppression of Tcon proliferation in response to CD3/CD28 activation, while no effects were measured with a point-mutant control. We believe that SAH-FOXP3DDs will be powerful molecular probes to further our understanding of FOXP3 transcriptional control. Additionally, they may be a foundation for prototype therapeutics to target Tregs in cancer patients in an effort to amplify the endogenous anti-tumor immune response alone or in combination with other cancer immunotherapies.

#3851

Novel small-molecule inhibitor of PD1/PDL1 pathway demonstrated single agent and drug combo effectiveness in cancer immunotherapy.

Yuguang Wang,1 He Zhou,2 Nong Zhang,1 Feilan Wang,1 Qiang Zhao,2 Tianzhi Wu,1 Haiyan Zhu,2 Yuzhi Liu2. 1 _Maxinovel Pharmaceuticals, Inc., Shanghai, China;_ 2 _Shanghai Chempartner Co., Inc., Shanghai, China_.

With 5 antibodies approved for cancer treatments, PD-1/PD-L1 pathway has been the most successful target in cancer Immuno-therapy. Small molecules that inhibit PD-1/PD-L1 interaction are also attractive because their better tissue penetration may lead to stronger and broader anti-tumor efficacy in addition to more convenient dosing regimen. A series of such small molecule inhibitors have been developed by Maxinovel. Among them, Max 10043 and its 2nd generation compound MAX-10129 potently inhibited PD-1/PD-L1 interaction, reversed PD-L1 suppression of anti-CD3 mediated activation of human T cells, and was well tolerated in animal studies. MAX-10043 also demonstrated anti-tumor efficacy in CT26 murine colorectal carcinoma model. MAX-10129, with improved oral bioavailability, displayed significant inhibition of tumor progression with dose dependency in murine colorectal carcinoma MC-38 model. MAX-10129 also demonstrated synergistic anti-tumor efficacy in different combinations with an anti-CTLA4 antibody, an IDO inhibitor Epacadostat, a COX-2 inhibitor Celebrex and a chemo drug Cisplatin, respectively. The p value of the combo treatment group relative to the vehicle group is less than 0.01 for the Epacadostat combo, the Celebrex Combo and the Cisplatin combo while it is less than 0.05 for the anti-CTLA4 combo. More studies are on-going to illustrate mechanism of action as well as the possibility of all oral triple and quadruple combinations . These data suggest the MAX-10129, a small molecule inhibitor of PD-1/PDL-1 interaction, may represent a novel, orally administrated, safe and effective immunotherapy agent.

#3852

Combination efficacy and safety profile of an orally bioavailable small molecule agent targeting CD47/SIRPα axis.

Girish Daginakatte, Sasikumar Pottayil, Gundala Chennakrishna, Wesley Roy Balasubramanian, Sudarshan Naremaddepalli, Archana Bhumireddy, Sandeep Patil, Kavitha Nellore, Priyabrata Chand, Kiran Aithal, Amit Dhudashiya, Samiulla DS, Rajesh Eswarappa, Murali Ramachandra. _Aurigene Discovery Technologies, Bengaluru, India_.

Introduction: Most of the immunotherapies currently approved in the clinic target immune checkpoint proteins that suppress T-cell responses. There is growing evidence that the innate immune system also plays an important role in the initiation and propagation of enduring antitumor responses. Targeting CD47-SIRPα axis is emerging as one of the promising new immunotherapy approaches that targets innate immune response. A number of clinical trials are in progress to evaluate CD47/SIRPα blocking therapies. Most of these molecules are either anti-CD47 antibodies or SIRPα-Fc recombinant proteins. We are developing a novel small molecule CD47 antagonist, AUR-104, as therapeutic agent for solid and hematalogical cancers. AUR-104 is a CD47 antagonist that disrupts CD47- SIRPα interaction and enhances phagocytosis of tumor cells. AUR-104 exhibits good drug-like properties and demonstrates antitumor activity in several pre-clinical tumor models. Here, we report the anti-tumor efficacy of AUR-104 in combination with tumor specific antibodies in pre-clinical models of cancer and also present the safety profile of AUR-104 in rodents.

Materials and Methods: Syngeneic murine tumor models: MC38 colon carcinoma cells were subcutaneously implanted in C57BL/6J mice while A20 B-cell lymphoma cells were implanted in BALB/c mice. Tumor bearing mice were treated with AUR-104 (30 mg/kg, b.i.d, and po) as a single agent or in combination with anti-PD1 antibody (100 µg/animal) or anti-PDL1 antibody (200 µg/animal). Tumor volumes were recorded with calliper's measurement over period of treatment.

A single dose maximum tolerated dose (MTD) study in BALB/c mouse followed by a 14-day repeat dose toxicity study in BALB/c mouse: Adult male and female BALB/c, are dosed with AUR-104 at ascending doses up to the limit dose. End points monitored include clinical observations, toxicokinetic parameters, body weights, food consumption, hematology, clinical pathology investigations, organ weights and histopathology of selected tissues.

Results: AUR-104 combination treatment with anti-PD1 antibody significantly enhanced anti-tumor efficacy in MC38 colon carcinoma model. Combination study with anti-PDL1 antibody in A20 tumor model is in progress. Preliminary observations from efficacy studies indicate that AUR-104 combination treatments with antibodies are well tolerated without any signs of toxicity. Advance in vitro safety evaluation and in vivo 14 day repeat day toxicity study in mice are being initiated. In summary, AUR-104 plus anti-PD1 antibody was a well-tolerated drug combination that exhibited a much greater in vivo antitumor response as compared to the single agent treatments. These results demonstrate the therapeutic potential of CD47 antagonist AUR-104 in combination with other tumor specific antibodies for the treatment of cancer.

#3853

AXL inhibitor promotes anti-tumor immunity through modulation of macrophage polarization.

Shenshen Lai, Qin Xu, Jun Yan, Hong Zhang, Zaihui Zhang. _SignalChem Lifesciences Corp., Richmond, British Columbia, Canada_.

Polarization of tumor-associated macrophages (TAMs) to classic pro-inflammatory M1 or alternatively activated M2 types plays an important role in establishing tumor microenvironment and determining therapeutic responses. It has become increasingly clear that M2 macrophages contribute to tumor progression by producing anti-inflammatory cytokines and suppressing anti-tumor immunity. AXL receptor tyrosine kinase has recently emerged as a dual therapeutic target in oncology, due to its function in tumor growth, survival and metastasis, as well as immunosuppressive activity. AXL and its ligand Gas6 are both involved in attenuating anti-tumor immune response through modulation of TAM polarization. Thus, targeting the AXL signaling pathway is expected to promote a pro-inflammatory tumor microenvironment, in addition to direct inhibition of tumor growth. SLC-391, a selective small molecule inhibitor for AXL, displays high potency against numerous cancer cell lines through inhibition of AXL/PI3K/AKT-dependent cell proliferation and survival in vitro. Additionally, this compound was also found to alter the cytokine profile expressed in THP1-derived M2 macrophages, including upregulating pro-inflammatory CXCL10 and downregulating anti-inflammatory IL-10. Noticeably, the expression level of AXL was significantly upregulated in THP1-derived macrophages in presence of tumor cell-conditioned medium, which indicated the involvement of AXL in M2 polarization, and therefore strengthening the notion of AXL as a dual therapeutic target. In a co-culture system consisting of THP1-derived macrophages and A549 non-small cell lung cancer cells, SLC-391 targeted AXL activity in both cell types for overall tumor cell suppression through direct and macrophage-mediated inhibition. This observation is supported by inhibition of tumor growth and the increased ratio of M1/M2-polarized TAMs from mice treated with SLC-391 in a CT-26 murine colon carcinoma syngeneic model, considering CT26 cells are not sensitive to SLC-391 in cell-based proliferation assay. In summary, in addition to direct inhibition of tumor cells, SLC-391 also appears to promote anti-tumor immunity through modulation of M2 to M1 transition.

#3854

Antizyme inhibitor editing modulates nuclear localization but not antizyme binding: Implications for prostate cancer.

James M. Rice,1 Aram Ghalali,1 Liangzhe Wang,1 Amanda Kusztos,1 Finith Jernigan,2 Chin Lee Wu,3 Bruce Zetter,1 Michael S. Rogers1. 1 _Boston Children's Hospital at Harvard Medical School, Boston, MA;_ 2 _Harvard Medical School, Boston, MA;_ 3 _Massachusetts General Hospital, Harvard Medical School, Boston, MA_.

The endogenous antizyme inhibitor (AZIN) binds to, and is a key regulator of, the cell cycle suppressor antizyme. Antizyme itself is a key regulator of ornithine decarboxylase (ODC), the rate-limiting enzyme in the polyamine synthesis. We have measured the expression and localization of AZIN in 202 prostate cancer specimens, along with 26 adjacent benign samples, and found that nuclear localization of AZIN is dramatically associated with worse outcome. Upregulation and nuclear localization of AZIN have been observed in several cancers, as has editing of the AZIN1 mRNA. Other have hypothesized that the RNA- edited AZIN (edAZIN) may have an increased affinity to antizyme and that could explain the association of edAZIN to the various cancers. We have studied the mechanism behind the nuclear localization of AZIN and found the single base pair substitution caused by RNA editing is sufficient to result in nuclear localization of the protein in all cell types tested. To determine if the nuclear localization might result from increased antizyme-edAZIN affinity, we developed fluorescent protein FRET sensor for protein-protein interaction using Clover-AZIN and antizyme-mRuby2 fusion proteins. Unexpectedly, we found that the editing event decreases edAZIN affinity for antizyme, notwithstanding increased interaction in vivo. Thus, the data indicate that the change in protein localization to the nucleus may be more important to oncogenic function than the actual degree of binding to antizyme. Other functional differences between edAZIN and AZIN might be explained by altered kinetics of binding, by the contribution of an additional adapter protein that modulates the intracellular antizyme:AZIN complex, or by competition for AZIN binding by other partners whose interaction is affected by the editing event. Therefore, we plan to identify AZIN and edAZIN interacting proteins by using proteomic analysis and later study the interaction between AZIN or edAZIN with their binding partners in more detail. Furthermore, our validated FRET assays can be used to identify antagonists of AZIN-antizyme binding (no antagonists are currently known). Given the important role of AZIN in tumor growth, discovery of small-molecule inhibitors of AZIN should lead to antitumor therapies.

#3855

Inhibition of MNK by eFT508 reprograms T-cell signaling to promote an antitumor immune response.

Craig R. Stumpf, Joan Chen, Vikas K. Goel, Gregory S. Parker, Gary G. Chiang, Peggy A. Thompson, Kevin R. Webster. _eFFECTOR Therapeutics, San Diego, CA_.

Mitogen-activated protein (MAP) kinase signaling cascades play a vital role in T-cell activation upon antigen recognition. MNK1 and MNK2 are important downstream effector kinases in the MAPK pathway that largely function in regulating the expression of important signaling molecules, including cytokines and immune checkpoint receptors. MNKs are primarily thought to regulate the expression of select mRNAs, predominantly via post-transcriptional mechanisms involving the phosphorylation of the eukaryotic translation initiation factor eIF4E as well as the RNA binding proteins hnRNPA1 and PSF. eFT508 is a potent and highly selective inhibitor of MNK1 and MNK2 that has been shown to promote antitumor immunity by decreasing the expression of immunosuppressive molecules, such as immune checkpoint receptors. In order to identify key T-cell components that are regulated by MNK phosphorylation and may mediate the effects of eFT508 treatment, we performed an unbiased phosphoproteomic analysis of T cells during the early stages of T-cell receptor-mediated stimulation with and without eFT508 treatment. Primary human T cells were pretreated with eFT508 for two hours prior to stimulation with αCD3/αCD28 for an additional 30 minutes. Protein samples were then prepared for multiplexed phosphoproteomic analysis by mass spectrometry. Consistent with previous proteomic studies using stimulated T cells, a number of phosphopeptides associated with T-cell receptor signaling, among other cellular activities, were detected. Moreover, treatment with eFT508 specifically blocked the phosphorylation of distinct phosphosites on select proteins. The phosphoproteins modulated by eFT508 treatment are involved in important T-cell signaling pathways, cell proliferation and differentiation programs, stress responses, and post-transcriptional and translational gene regulation. Furthermore, there was enrichment for specific sequences surrounding the phosphorylation site in eFT508-sensitive peptides, highlighting a potential mechanism mediating MNK target recognition. Confirmation of MNK-mediated phosphorylation of novel substrates is being conducted in vitro by biochemical analysis of direct phosphorylation of potential substrates by MNK1 or MNK2 and in cellular lysates treated with eFT508 by Western blot analysis using phosphosite-specific antibodies. These findings have significantly expanded our understanding of cell signaling through MNK1 and MNK2 and will help to illuminate potential regulatory programs through which inhibition of MNKs by eFT508 can modulate antitumor immunity.

#3856

**Antitumor activity of ABI-009 (** nab **-rapamycin) in combination with anti-PD1 antibody in a syngeneic mouse model of B16 melanoma.**

Shihe Hou, Berta Grigorian, Anita Schmid, Neil Desai. _Aadi Bioscience, Inc., Pacific Palisades, CA_.

Background: mTOR pathway has been implicated in cell survival and proliferation and is an attractive target for cancer therapy. Loss of PTEN, a negative regulator of mTOR pathway, is frequently observed in multiple cancer types. Recent studies indicate that loss of PTEN promotes resistance to T cell-mediated immunotherapy. Since PTEN loss can result in downstream mTOR activation, we investigated the safety and efficacy of ABI-009 (nab-rapamycin, a novel mTOR inhibitor) in combination with anti-PD1 antibody in a syngeneic mouse model of B16 melanoma. ABI-009 (nab-rapamycin) is a nanoparticle form of human albumin-bound rapamycin with a mean particle size of approximately 100 nm developed with a proprietary nanoparticle albumin-bound (nab®) technology. ABI-009 is currently in phase 1 and 2 clinical studies for the treatment of malignant perivascular epithelioid cell carcinoma (PEComa), severe pulmonary arterial hypertension (PAH), nonmuscle-invasive bladder cancer, soft-tissue sarcomas, and various childhood cancers.

Methods: Syngeneic B16 melanoma tumors were implanted in immunocompetent C57BL/6 mice. ABI-009 was administered IV at 5 mg/kg 3 times weekly. Monoclonal anti-mouse PD-1 antibody (RMP1-14, BioXcell, West Lebanon, NH, USA) was administered IP at 250 μg every 2 days for 3 times. The study drugs were either administered as single agent or in combination with 3 different dosing schedules: concurrent (A), ABI-009 one week before anti-PD1 (B), or anti-PD1 one week before ABI-009 (C).

Results: Overall, all treatments were well tolerated with no significant body weight loss in any group. All treatment groups showed significant antitumor effect and longer survival compared with the saline control. Addition of ABI-009 simultaneously or after anti-PD1 significantly improved tumor growth suppression and survival compared with anti-PD1 alone. Tumor volumes were reduced 35.5% for group A and 44.8% for group C when compared with anti-PD1 alone (p < 0.05). Median survival was 26 days for group A and 26 days for group C vs. 20 days for anti-PD1 alone (p < 0.05). On the other hand, group B failed to significantly improve antitumor effect and survival over anti-PD1 alone. The combination treatment regimen of anti-PD1 given before ABI-009 (group C) was the best schedule among the 3 schedules tested.

Conclusions: The combination of ABI-009 and anti-PD1 antibody was well tolerated. Results from this study support the treatment regimens of anti-PD1 immunotherapy, concurrent or followed by ABI-009. Pretreatment with ABI-009 followed by anti-PD1 is not recommended. A phase 1b investigation of safety/efficacy of nivolumab and ABI-009 in patients with advanced sarcoma has been initiated.

#3857

Selective targeting of dopamine receptor dysregulation in high grade gliomas with imipridone ONC201.

Varun Vijay Prabhu,1 Neel Madhukar,1 C. Leah B. Kline,2 Rohinton Tarapore,1 Wafik S. El-Deiry,2 Joseph Rucker,3 Benjamin Doranz,3 Faye Doherty,4 Alexander VanEngelenburg,4 Jessica Durrant,4 Cyril Benes,5 Sean Deacon,6 Neil Charter,6 R. Benjamin Free,7 Wolfgang Oster,1 David Sibley,7 Isabel Arrillaga,5 Olivier Elemento,8 Joshua E. Allen1. 1 _Oncoceutics, Inc, Philadelphia, PA;_ 2 _Fox Chase Cancer Center, Philadelphia, PA;_ 3 _Integral Molecular, Inc, Philadelphia, PA;_ 4 _HistoTox Labs, Inc, Boulder, CO;_ 5 _Massachusetts General Hospital, Boston, MA;_ 6 _DiscoverX Corporation, Fremont, CA;_ 7 _NINDS/NIH, Bethesda, MD;_ 8 _Weill Cornell Medicine, New York, NY_.

D2-like dopamine receptors (DRD2/3/4) are G protein-coupled receptors (GPCRs) that are overexpressed in glioblastoma (GBM) and their antagonism induces tumor cell apoptosis. We describe the first selective DRD2/3 antagonist for neuro-oncology using computational, receptor pharmacology, biochemical and clinical studies. Consistent with an in-silico prediction and in contrast to antipsychotics that target several dopamine receptors and other GPCRs, β-arrestin recruitment and cAMP assays determined that ONC201 is a selective DRD2/3 antagonist. Schild analyses and radioligand competition assays revealed competitive and non-competitive DRD2 antagonism with a potency (2-3 µM) that is consistent with anticancer activity and driven by an unusually slow association rate. Proof-of-concept studies show that selective DRD2 inhibition induces superior anti-cancer efficacy relative to pan-targeting of the dopamine receptor family. In accordance with superior selectivity, ONC201 also exhibited a wider therapeutic window compared to antipsychotics. Shotgun mutagenesis across 350 amino acids of DRD2 identified 8 residues that are critical for ONC201-mediated DRD2 antagonism. Consistent with competitive inhibition, several mutated residues were within the orthosteric binding site. However, distal residues were identified that were not involved in DRD2 antagonism by antipsychotics and may explain the selectivity and non-competitive antagonism of ONC201. In vitro and in vivo studies have previously demonstrated single agent ONC201 efficacy in GBM models (Allen et al 2013). Analyses of The Cancer Genome Atlas and tissue microarrays revealed high DRD2 expression relative to other dopamine receptors, correlation with poor prognosis and high DRD2 expression in primary rather than secondary GBM. A linear correlation between DRD2 mRNA and ONC201 GI50 was observed among GBM cell lines in the NCI60 panel. Interestingly expression of DRD5, a D1-like dopamine receptor that counteracts DRD2 signaling, was significantly inversely correlated with ONC201 potency in the NCI60 dataset (P <.05). Furthermore, a de novo missense DRD5 mutation was identified in cancer cells with acquired resistance to ONC201, and overexpression of the mutant construct could recapitulate resistance. ONC201 exhibited biological activity in a phase II recurrent GBM study, including tumor regressions (Arrillaga et al, 2017). Among the 15 available archival patient tumor specimens from the first cohort of this trial, all had DRD2 expression and 8 had low DRD5 expression that was associated with superior progression-free and overall survival, with 4/8 DRD5- and 0/7 DRD5+ patients alive after 15 months (P=0.012). Thus, ONC201 possesses unique receptor pharmacology as the first selective DRD2/3 antagonist for clinical neuro-oncology that has exhibited clinical activity in biomarker-defined recurrent high grade glioma patients. 

### Novel Assay Technology and Delivery Systems

#3858

Validation of digital spatial profiling of key immuno-oncology targets for mouse FFPE preclinical models.

Sarah Warren, Heather Metz, Kristi Barker, JingJing Gong, Alison VanSchoiack, Quoc Huynh, Chris Merritt, Lucas Dennis, Yan Liang, Joseph M. Beechem. _NanoString Technologies, Inc., Seattle, WA_.

Mouse models of cancer are an invaluable biomedical research tool in which characterization of the abundance, distribution and co-localization of immuno-oncology targets is critical for the advancement of disease understanding. Nanostring Technologies' Digital Spatial Profiling (DSP) technology allows for the simultaneous analysis of 10s to 100s of proteins from discrete regions of interest (ROI), providing a spatial context to multiplexed data. The purpose of this study is to validate an antibody panel designed to characterize key tumor and immunology markers on the DSP platform. DSP technology utilizes barcoded oligo tags that are covalently linked to detection reagents, in this case, antibodies. The workflow for a typical DSP experiment consists of incubating a formalin-fixed, paraffin-embedded (FFPE) tissue with a cocktail of oligo-labeled primary antibodies and three fluorescently labeled visualization markers. The visualization markers and tissue morphology guide the selection of ROIs. UV light then illuminates each ROI, releasing the oligo tags through a UV photocleavable linker. Tags are collected through microcapillary aspiration and then deposited into a microtiter plate for further analysis on the standard nCounter® platform. In order to validate antibodies for this technology, the specificity and sensitivity of each antibody was evaluated for proper immunohistochemistry (IHC) staining pattern on appropriate tissues as well as signal-to-noise ratios of positive counts above background. Panels will also undergo an evaluation of single vs. multiplex interactions to ensure there are no deleterious effects of multiplexing antibodies. Immunohistochemical analysis of antibodies chosen for panel displays indistinguishable staining patterns on mouse control tissues for unconjugated primary antibodies and oligo-conjugated primary antibodies. Visualization markers allow for an ROI selection strategy that utilizes a range of circular ROIs (e.g., 50-650um) as well as custom masking of areas of interest. Key immuno-oncology targets including B7-H3, beta-catenin, CD19, CD25, CD3, CD4, histone H3, Ly-6G, pan-cytokeratin, PD-1, PD-L1, F4/80, Ki-67, STAT3, etc., have been validated thus far. Ongoing validation efforts will continue to expand this antibody panel. Nanostring Technologies' DSP platform is applicable not only to human tissue but also to mouse preclinical samples. Antibody specificity, sensitivity and interaction within a cocktail have been validated for this application. DSP technology allows for the multiplexing of important mouse immuno-oncology markers so that researchers can evaluate the abundance as well as the spatial distribution of these key targets simultaneously.

#3859

Hormone receptor and HER2/HER3 expression in preclinical breast cancer models of primary tumor and bone metastasis.

Tiina E. Kähkönen,1 Mari I. Suominen,1 Jussi M. Halleen,1 Jenni H. Mäki-Jouppila,1 Azusa Tanaka,2 Michael Seiler,2 Teppo Haapaniemi,3 Jenni Bernoulli1. 1 _Pharmatest Services, Turku, Finland;_ 2 _Taconic Biosciences, Hudson, NY;_ 3 _BioSiteHisto Ltd., Tampere, Finland_.

Breast cancer expressing estrogen receptor (ER) and progesterone receptor (PR) is classified as hormone receptor positive, and triple-positive breast cancer expresses also human epidermal growth factor receptors (HER2). Despite the hormonal and HER2 targeted treatments, breast cancer metastasizes to bone in high frequency and may develop resistance against the used treatment. Prevention and treatment of bone metastases is challenging and moreover, hormones are strong regulators of both bone and immune system. Aim of the present study was to verify and compare ER, PR, HER2 and also HER3 status in preclinical primary and bone metastasis breast cancer models utilizing immunodeficient and human immune system engrafted mice.

BT-474 human breast cancer cells were inoculated orthotopically into mammary fat pad of placebo or 17β-estradiol (E2) supplemented female immunodeficient NOG mice. In a bone tumor study, BT-474 cells were inoculated into the tibia of female NOG or humanized NOG mice (HSCFTL-NOG-F mice, Taconic Biosciences). Tumor growth was followed for 8 weeks and histopathological tumor evaluation and immunohistochemical stainings for ER, PR, HER2 and HER3 were performed.

Orthotopic tumor growth of BT-474 was hormone dependent and only minor growth was observed in the absence of E2. In the presence of E2 supplement, the orthotopic tumor expressed ER, PR and HER2/HER3. However, in the absence of E2 supplement there was reduced PR expression but no major changes in the ER and HER2/HER3 expression. In contrast, when breast cancer cells were inoculated into the tibia, tumor growth was observed also without E2 supplement. In this case, tumor in the bone was positive for ER and HER2/HER3 but negative for PR. No significant changes were observed between immunodeficient and humanized mice regarding intratibial tumor growth or ER, PR and HER2/HER3 expression.

As a summary, estrogen supplementation is needed to support breast cancer BT-474 tumor growth when cancer cells are inoculated orthotopically into mammary fat pad. In contrast, BT-474 tumor growth was observed in bone also in the absence of supplied E2 in immunodeficient and humanized mice. ER and HER2/HER3 expression was observed in primary and bone tumors, but PR expression was significantly reduced if no estrogen supplement was used. Taking together, when developing new therapies against breast cancer, treatment targets in preclinical models should be carefully verified. Focus should be addressed not only on primary tumor but also on bone metastasis where cancer cells are under influence of different tumor microenvironment and may express differently hormone receptors and HER2/HER3. While hormones influence breast cancer progression, they also regulate bone turnover and immune system, and therefore humanized mouse models provide an essential platform for novel therapy development.

#3860

Rodent multispecies multiplex immunohistochemistry using digoxigenin and polymer detection methods in mouse tissues.

Joseph Vargas, David Tacha, Sara Figueroa, Cristin Douglas. _Biocare Medical, Pacheco, CA_.

When mouse, rat, and rabbit antibodies are used, consideration of the target tissue is of critical importance. Endogenous IgGs present in tissues pose significant challenges to interpretation. When a species conflict exists for the antibody and the target tissue, it is likely to generate undesired background staining due to the detection system. In the past, mouse-on-mouse and rat-on-mouse utilized either a biotinylated primary antibody or a biotinylation approach to label the antibody. Tissues rich in endogenous biotin such as kidney or liver were difficult to use in these systems as streptavidin-horseradish peroxidase (HRP) or anti-biotin techniques were used as the detection complement. Attempts to negate endogenous biotin were minimally effective as avidin-biotin blocking steps were unlikely to work acceptably in tissues such as kidney and liver. Using anti-digoxigenin (dig) and polymer technology to generate multi-species multiplex stains may provide a solution.

Materials and Methods: Mouse tissues were fixed for 24 hours and embedded in paraffin. Sections were cut at 5 microns and dried on slides before deparaffinizing and peroxidase blocking. Slides were then heated at 110°C for 15 minutes (spleen, liver, kidney) or 80°C for 60 minutes (brain) in a citrate-based buffer (pH 6.2) prior to application of antibodies. A sequential approach of two applications of dig-labeled mouse-on-mouse (MM) detection was performed with PAX8 (M) and CD10 (M) as well as with Neurofilament (M) and GFAP (M). Anti-rat alkaline phosphatase and anti-rat HRP polymer sequential applications were also executed with rat monoclonals CD4 and CD8. Then, a multispecies sequential multiplex stain using MM anti-dig along with anti-rabbit polymer was achieved with mouse hepatocyte specific antigen and rabbit CD3. Finally, a four-step cocktailed multispecies multiplex was developed using a rat monoclonal F480 combined with a rabbit CD3 in conjunction with a cocktail of anti-rat polymer and anti-rabbit polymer. Visualization in each assay was achieved by the use of two chromogenic end-points. Slides were counterstained in either hematoxylin or methyl green prior to dehydration and mounting.

Results: Rodent multiplex stains were easily achieved with dig-based MM and cocktailed methods that use mouse/rabbit or rat/rabbit approaches. Specific, clean, and background-free staining on spleen, liver, kidney and brain mouse tissues was achieved with excellent signal-to-noise in both sequential and cocktailed formats.

Conclusion: Using mouse-on-mouse anti-digoxigenin technology in multiplex staining is a superior approach to streptavidin or biotin strategies in rodent systems. Additional multiplexing techniques with rat or rabbit antibodies using polymer-based detections generates excellent staining with a significant reduction of background and an exceptional signal-to-noise ratio in mouse tissues.

#3861

3D ex vivo PDX cell model screening to better predict in vivo outcome.

Xiaoxi Xu, Zhongliang Li, Yan Liu, Fanxiu Meng, Yu Lu, Songling Zhang, Chunlan Dong, Frank Xing, Qian Shi. _CrownBio, Taicang, Jiangsu, China_.

Patient-derived xenograft (PDX) models have been highly demanded in preclinical drug discovery and development due to several advantages over conventional 2D cell culture system: 1) reflection of the heterogeneity, molecular and histopathologic signatures of the original tumor than cell lines or genetically engineered mouse models, and 2) certain degree of correlation of their drug-response profiles with clinical response. Certain limitations of using PDX models, however, do exist, including low throughput in candidate drug screening, lack of dose-response curves, high cost and time-consuming studies, and progressive loss of human-derived stromal elements over passages. To overcome these disadvantages, we sought to develop ex vivo 3D assay format on cells isolated from early passage PDX models, and provide genetic mutation information to better interpret results. Moreover, we attempted to incorporate immunotherapy strategy into the 3D system as well. We have recently built up an ex vivo cell bank containing more than 300 frozen ex vivo tumor cells dissociated from freshly isolated PDX tumor tissues. We have collected five panels of such frozen ex vivo cells, each panel representing more than 50 models in a specific tumor type. SOC compounds corresponding to each unique tumor panel were tested, and will be discussed. Moreover, we have established a 3D immune cell and ex vivo somatic tumor cell co-culture system to evaluate drug efficacy of immune checkpoint inhibitors aPD1, aPDL1 and CTLA4 antibodies either as single agent or in combination with small-molecule inhibitors. The data will be presented. In summary, we have developed a robust and reproducible 3D ex vivo assay platform for medium-throughput compound screening with bioinformatics information for data interpretation.

#3862

Empowering research on ubiquitin and ubiquitin-like protein modification cascade using recombinant enzyme systems.

Eric Yao, Shenshen Lai, Jun Yan. _SignalChem Parmaceuticals Inc., Richmond, British Columbia, Canada_.

Protein modifications by the covalent attachment of ubiquitin (Ub) or ubiquitin-like proteins (UBLs) such as SUMO, ISG15 or NEDD8 participate in many critical cellular processes. These include cell-cycle regulation, DNA repair, endocytosis, mitophagy, oncogenesis, post-membrane-protein trafficking, antiviral pathways and most notably, proteasomal degradation of target proteins. Ubiquitination and modification by UBLs share a similar catalytic cascade which requires the sequential action of three classes of enzymes: E1 activating enzymes, E2 conjugating enzymes and E3 ligases. While Ub/UBLs are activated by E1 through an ATP-dependent thioester bond formation and transferred to E2 from E1, the exact mechanism of how they are subsequently attached to a protein substrate depends on the type of E3's containing characteristic catalytic domain - RING, HECT or RBR. It has been generally accepted that E3's mediate substrate specificity and E2's are the main determinants for selection of the lysine to construct the modifier chains, which thereby directly control the cellular fate of the substrate. Recent research has linked dysregulation of the Ub/UBLs modification system to numerous diseases including cancer, immunological disorders and neurodegeneration. The high substrate specificity provided by combinations of over 30 E2's and over 600 E3's in the human genome makes these enzymes emerging drug targets. In response to a growing market demand, SignalChem has developed an extensive array of products encompassing enzymes, Ub/UBL modifiers and substrates in the ubiquitination, SUMOylation, ISGylation and NEDDylation processes. With Promega's AMP-GloTM reagents and an optimized assay protocol, we have identified and validated a variety of functionally competent combinations of these enzyme components. With the established protocol, each enzyme in the catalytic cascade has been assessed for their specific activity towards generation of free AMP. In addition, inhibition profiles of all three enzymes have been obtained using the assay system, further demonstrating their potential to be used in high-throughput screening to identify potential lead compounds for drug discovery and development programs.

#3863

Analytical performance of a novel next generation sequencing assay for myeloid cancers.

Nickolay Khazanov, Wally Zhang, Dinesh Cyanam, Scott P. Myrand, Denis Kaznadzey, Paul D. Williams, Vinay Mittal, Daniel J. Mazur, Sihong Chen, Jason Wustman, Efren Ballesteros-Villagrana, Goutam Nistala, Santhoshi Bandla, Jim Veitch, Jon Sherlock, John Bishop, Seth Sadis. _Thermo Fisher Scientific, Ann Arbor, MI_.

Title: Analytical performance of a novel next generation sequencing assay for myeloid cancers

Nick Khazanov, Wally Zhang, Dinesh Cyanam, Scott P. Myrand, Denis Kaznadzey, Paul Williams, Vinay Mittal, Dan Mazur, Sihong Chen, Jason Wustman, Efren Ballesteros-Villagrana, Goutam Nistala, Jon Sherlock, Michael Hogan, Jim Veitch, John Bishop, Seth Sadis

Introduction: Myeloid malignancies contain a diverse and heterogeneous set of genomic alterations that include recurrent somatic mutations in key driver genes as well as frequent and diagnostic chromosomal rearrangements that generate a wide array of gene fusion products. To support clinical and translational research into precision oncology strategies for myeloid cancers, a next-generation sequencing (NGS) assay was generated to detect common and relevant somatic alterations.

Methods: To define gene targets that were recurrently altered in myeloid cancers and relevant for clinical and translational research, an extensive survey of investigators at hematology oncology research labs was performed. The gene targets identified by researchers were complemented by a comprehensive survey of literature and genomic databases. Clinical guidelines for myeloid diseases in the US and in Europe were reviewed to ensure representation of relevant alterations. A targeted Ion AmpliSeq panel was generated to support the detection of recurrent single-nucleotide variants, insertions/deletions, and gene fusions from blood or bone marrow samples. The panel was developed for manual or automated library preparation and sequencing on the Ion Torrent PGM or Ion S5 instruments.

Results: The Oncomine™ Myeloid Research Assay gene panel included 58 genes and generated an average read depth of >2,000 reads per targeted amplicon with an average uniformity of >95%. Important GC-rich targets such as CEBPA generated sufficient balanced read depth to support variant detection. A cohort of samples positive for FLT3 alterations was analyzed and successful detection of FLT3-internal tandem repeat variants was demonstrated. Several gene fusion transcripts common to myeloid cancers were detected. Comparable results were observed on Ion Torrent PGM and Ion S5 instruments.

Conclusions: A novel myeloid specific NGS assay capable of detecting relevant DNA and RNA alterations from the same sample was developed. The assay is useful for characterizing relevant alterations in a range of myeloid diseases including acute myeloid leukemia, myelodysplastic syndrome, myeloproliferative neoplasms, chronic myelogenous leukemia, chronic myelomonocytic leukemia, and juvenile myelomonocytic leukemia. A review of the analytical studies will be presented.

#3864

Development of high-specific and sensitive reporter assays for the detection of Wnt/β-catenin signaling activity.

Kiyoshi Yamaguchi, Chi Zhu, Tomoyuki Ohsugi, Akari Saku, Tsuneo Ikenoue, Yoichi Furukawa. _Institute of Medical Science, The University of Tokyo, Tokyo, Japan_.

The Wnt/β-catenin signaling pathway is involved in development, tissue homeostasis, and regeneration. Since aberrant activation of this pathway plays a crucial role in human carcinogenesis, inhibition of this pathway has been shown to be a promising therapeutic strategy for cancers with de-regulated Wnt signaling. Although cell-based assays using synthetic TCF/LEF reporters (also known as TOPFLASH) have contributed to the discovery of small molecules that modulate Wnt signaling pathway, these assays often raise a problem of the specificity in the screening. In this study, we aimed to develop a specific reporter for the detection of Wnt signaling activity. We performed integrated transcriptome analysis and found that expression of histidine ammonia-lyase gene (HAL) is negatively regulated by β-catenin/TCF. In addition to TOPFLASH, we leveraged a promoter region of HAL as another transcriptional readout of Wnt signaling. Subsequently, HepG2 cells stably expressing both optimized HAL promoter reporter and TOPFLASH reporter were established. Since inhibition of the β-catenin/TCF transcriptional activity decreased TOPFLASH activity and simultaneously increased HAL reporter activity, we termed this assay as a bidirectional reporter assay. To verify the effectiveness of this assay, we compared the number of hits in the screening of a test library using the bidirectional assay with that using the conventional TOPFLASH/FOPFLASH assay. Among the 361 compounds in the library, the bidirectional reporter assay identified 9 hits, while the TOPFLASH/FOPFLASH assay did 79 hits. Four compounds were overlapped between these assays, suggesting that the bidirectional assay decreased false positives compared with the conventional assay. The concept of bidirectional assay might be useful for the screening of compounds that regulate other transcriptional factors and/or disease-associated signaling pathways.

#3865

ActivSignal platform technology for direct multiplex analysis of signaling pathway activation profiles.

Ilya Alexandrov,1 Irina Brandina,1 Nader Rahimi,2 Michael Sherman,2 Julia Yaglom,2 Rosana Meyer,2 Marwa Ali,2 Esma Ersoy2. 1 _ActivSignal, Natick, MA;_ 2 _Boston University, Boston, MA_.

The multiplex IPAD assay technology from ActivSignal monitors activity of 26 major signaling pathways by examining phosphorylation events or expression of more than 60 most relevant human and mouse protein targets. The technology uses antibodies against established and well known targets that reflect activities of canonical pathways. We utilize paired antibodies for each protein target for high specificity, and these pairs are carefully selected and tested. Only if both antibodies in a pair bind to their specific target molecule will the detection take place during the analysis phase - avoiding false hits. The IPAD assay can be used to profile samples made from cell culture, tissues, blood, etc. The IPAD assay requires only minute amounts of sample, which can be easily taken from your precious sample where available amounts may be limited. Applications include:

  * Direct snapshot of signaling pathway activity rather than downstream effects.
  * Understanding the physiological effects of a drug.

  * What pathways are regulated in response to lead candidate drug?
  * Does the drug of interest cause biologically significant secondary and off target effects?

  * Evaluation of signaling pathway activities in response to shRNA, CRISPR, hormones, stresses and other treatments.
  * Elucidating the signaling proteome in a medium-throughput format (96-well format).
  * Profiling of tissue samples from patients and animals.
  * Signaling profiling of tumor biopsies.

In one application, our collaborators used the IPAD assay to understand signaling effects of a novel protein, TMIGD1, whose expression is markedly downregulated in renal cancer. The Activsignal platform analysis showed changes in activities of several major pathways, e.g. inhibition of cell cycle and cell proliferation after reintroduction of TMIGD1. In another application of our platform our collaborators studied the mechanism of action of a small molecule inhibitor that targets Hsp70. They used ActivSignal platform technology to profile signaling pathways in response to the inhibitor. They identified several pathways whose activities were affected by the inhibitor. ActivSignal is a breakthrough, low-cost tool for the analysis of signaling pathway activation profiles. It enables researchers to rapidly and efficiently gain insight into function of their favorite drug molecules, genes etc.

#3866

A novel assay for rapid identification and isolation of single intact circulating tumor cells.

Lili Huang,1 Le Shen,1 Min Yu,2 Paul Liu1. 1 _Microsensor Labs, Chicago, IL;_ 2 _University of Southern California, Chicago, IL_.

We present a magnetic bead assay for rapid identification and isolation of single intact circulating tumor cells (CTCs). Cell viability is essential for cell culture and downstream molecular analysis. Many CTC isolation technologies, including CellSearch, uses fixation and fluorescent imaging for CTC detection that the captured CTCs are no longer viable. This novel assay utilize magnetic sensing for CTC identification, and therefore preserve CTC integrity and viability. The assay is performed on a microelectronic chip which also generates spatially-patterned microscopic magnetic field to sort single CTCs. We evaluated the performance metrics of the assay with EGFP-expression breast cancer patient derived CTC cells mixed with peripheral blood mononuclear cells (PBMCs) in vitro. Cell suspensions were mixed with magnetic microbeads coated with anti-EpCAM antibodies and then isolated with a magnet. Different amount (20, 50, 100, 500) of CTCs from serial dilution were spiked into 5x10^6 human PBMCs labelled with Alexa Flour 488 anti-human CD45 antibodies. After the assay, no PBMCs are visible which shows high purity in recovered CTCs. Recovery rate, defined by the ratio of the mean of the recovered CTC count to the mean of the spiked CTC count, is in the range of 87% to 92% for the various CTC spiked-in amount. In each spiking experiment, triplicate measurement and controls are used. Total assay time is less than 80 minutes. Conclusion: A rapid assay for isolation and detection of CTCs from PBMCs is demonstrated which shows high recovery rate and viability. This novel assay has great potential for single intact rare cell isolation.

#3867

**Activating** ESR1 **mutations detection by single ddPCR assay.**

Francois-Clement Bidard, Emmanuelle Jeannot, Luc Cabel, Nicolas Epaillard, Radouane El Ayachy, Aurelien Noret, Anne Vincent-Salomon, Jean-Yves Pierga, Ivan Bieche, Charlotte Proudhon, Marc-Henri Stern. _Institut Curie, Paris, France_.

Background: In metastatic breast cancer, most mutations of ESR1 (which encodes the estrogen receptor alpha ERα) occur in exon 5 (E380Q) and/or 8 (L536, Y537, D538). These mutations are responsible for acquired resistance to hormone deprivation but can be targeted by selective estrogen receptor degraders (SERD) such as fulvestrant. Circulating tumor DNA (ctDNA) allows a non-invasive assessment of ESR1 mutations, but implementing its routine use requires a sensitive, specific and fast technique, at a moderate cost.

Methods: We designed a ddPCR assay scanning for all mutations targeting the 536-538 amino acid residues of ERα with a unique pair of probes and multiplexed it with other probes targeting E380Q. Sensitivity and specificity were assessed in vitro; this multiple hotspot scanning (MHS) ddPCR was used to detect ESR1 mutants in cohorts of patients with hormone deprivation-resistant metastatic breast cancer.

Results: All the above-mentioned mutations were detected in a single MHS ddPCR assay with a high specificity and with a limit of detection of ~0.1% in mutant allele frequency, allowing ESR1 mutant detection in circulating tumor DNA. A perfect concordance with targeted next generation sequencing was observed in the first 31 clinical plasma samples tested. This technique also detected ESR1 polyclonal mutations.

Conclusion: This technique allows for a sensitive, large scale and repeated screening of ESR1 mutants with a single ddPCR assay. The clinical utility of ESR1 mutation detection by this technique is now investigated in the randomized phase 3 PADA-1 trial (sponsors: Unicancer/Gineco), in which patients with rising levels of circulating ESR1 mutants during aromatase inhibitor / palbociclib therapy are eventually switched to fulvestrant / palbociclib (NCT03079011).

ESR1 mutants detectable in a single ddPCR assay

---

AA changes | Mutant frequency

(as per Toy et al, Cancer Discov 2017).

E380Q | 19%

L536H | 4%

L536P | 2%

L536R | 1%

Y537S | 11%

Y537C | 6%

Y537N | 5%

Y537D | 1%

D538G | 32%

D538-L539ins | 1%

total % of mutations covered | ~82%

(Many of the 18% remaining mutations occur outside exon 5 & 8 and have no proven functional impact)

#3868

Development of a high-throughput screening co-culture angiogenesis assay system using hTERT immortalized primary cells.

Chaozhong Zou,1 Shuaizhang LI,2 Chia-Wen Hsu,2 Menghang Xia,2 Metewo S. Enuameh1. 1 _ATCC Cell Systems, Gaithersburg, MD;_ 2 _National Institutes of Health, Bethesda, MD_.

Angiogenesis is a multi-step physiological process which is involved in a large number of normal and disease state processes; In vitro angiogenesis models provide useful tools to study these processes, one of which is the analysis of tubule formation. Tubules formed in co-culture assays composed of both endothelial and stroma-producing cells were significantly more heterogeneous and more closely resembled capillaries than mono cell culture models utilizing only endothelial cells to generate tubules in an extracellular matrix. Current co-culture models using primary cells have donor variability and inconsistent results due to lot-to-lot variation. In this study, we established an in vitro co-culture model system consisting of an assay-ready mixture of an aortic endothelial cell line TeloHAEC-GFP and an hTERT immortalized adipose-derived mesenchymal stem cell line in a specially formulated medium containing VEGF supplement. Both cell lines were immortalized by hTERT alone and have been well-characterized; showing that the cells retain the most important characteristic of their primary counterparts. The new co-culture system forms functional tubular structures in less than 7 days, additionally, the hTERT-MSC cells which surround the tubular structures have undergone transformation indicated by elevated positive αSMA staining, indicating that the system has physiological relevance. Notably, our results showed the co-culture system has minimal lot-to-lot variation indicated by the treatment of three lots with the anti-cancer drug, ramucirumab. More importantly, the tubular formation efficiency is reduced or blocked by well-known anti-cancer drugs such as sunitinib and bevacizumab. We also tested four HIF-1 inhibitors identified in previous high-throughput screens and found that those compounds inhibited tubule formation in the co-culture system. These results suggest that the co-culture system can mimic the hypoxic environment in solid tumors. Previously the authors optimized the system for 384-well performance and here we report further optimization of the system into a 1536-well high-throughput format and a shortening of the assay time frame to 3 days. Using this format, we evaluated 2816 drugs from The National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection (NPC), and 35 potent inhibitors (IC50 ≤1 μM) were identified. Moreover, many known angiogenesis inhibitors were identified, such as topotecan, docetaxel, and bortezomib. Several potential novel angiogenesis inhibitors were also identified from this study. Among the inhibitors, some compounds were proven to be involved in the hypoxia-inducible factor-1α and the nuclear factor-kappa B pathways. These results demonstrate that the co-culture model described in this report provides a consistent and robust in vitro system for antiangiogenic drug screening.

#3869

A nanofiber-based biosensor for ultrasensitive detection of serum CRP in melanoma.

Zuan-Tao Lin, Yaxi Li, Tianfu Wu. _Univ. of Houston, Houston, TX_.

Purpose: Early detection of melanoma is important to improve survival; however, the technology for accurate early diagnosis is still challenging. This study is to develop a novel polymeric biosensor that could provide superior sensitivity in the detection of potential early biomarkers (e.g., CRP) of melanoma.

Experimental Procedure: Our strategy is to construct a highly specific molecular recognition core and a highly responsive transducer made of conductive polypyrrole (PPy) nanofibers. Briefly, a polymeric matrix was produced by polymerization of acrylamide (AM), methylenebisacrylamide (MBAA), N-Isopropylacrylamide (NIPAAm) and CRP/CRP-aptamer complex first. Next, taking advantage of the porous structure of this NIPAAm-AM-CRP-aptamer/CRP polymeric matrix, we synthesized a polypyrrole-based conductive nanofiber structure using copper phthalocyanine-3,4',4'',4‴-tetrasulfonic acid tetrasodium salt (CuPT) as a dopant counterion in situ to achieve uniformly dispersed nanofibers within the polymeric network of NIPAAm-AM-CRP-aptamer/CRP. After removing CRP recombinant protein, we were able to obtain a robust CuPT-PPy/NIPAAm-AM polymeric sensor for the detection of CRP with high sensitivity and selectivity. Two-step signal amplification cascades were involved in this CRP-specific polymeric sensor: 1) CRP binding-induced polymeric network shrinkage; 2) shrinkage-triggered conductance change of the polymeric network. Therefore, serum CRP levels could be quantitatively analyzed through monitoring the conductance change caused by polymeric network shrinkage upon aptamer-CRP binding.

Results: The limit of detection (LOD) of the polymeric sensor for detection of human recombinant CRP could reach 10^−19 M. The Fourier transform infrared (FT-IR) spectra confirmed the chemical structure of the polymeric network, and the morphology was observed using scanning electron microscopy (SEM) and atomic force microscopy (AFM). The results of FT-IR reveal that CuPT-PPy nanofiber was successfully synthesized in situ. The nanostructure of the nanofibers was clearly observed using SEM and AFM, indicating that the diameter of nanofiber is about 20 nm. This CRP-specific biosensor and a commercial CRP ELISA kit were used to perform side-by-side measurement of serum CRP in melanoma patients.

Conclusion: Our results indicated that this CRP-specific conductive polymeric senor is highly sensitive and selective in accurately discriminating melanoma patients from healthy controls. Such a flexible conductive polymeric biosensor may hold great promise as a point-of-care device in the diagnostics of melanoma or other cancers.

#3870

Validation of a canine intestinal organoid model as a preclinical screen to assess GI toxicity in novel oncology drug development.

Sarah M. Hoyle, Nicola Tonge, Charlotte Seaman, Mark Drinkwater, Adam Bonas, Vicky Murray-Tait, Aude-Marine Bonavita, Cath Booth. _Epistem Ltd, Manchester, United Kingdom_.

Gastrointestinal (GI) toxicity is a common and often severe dose-limiting side effect of chemotherapy. Symptoms include diarrhea, dehydration and ulceration leading to increased susceptibility to infection, due in part to the damage or loss of crypt and/or villi structures in the small intestine. As improvements in oncology therapeutics are pursued to acquire more efficacious agents, assessment of their potential GI toxicity is therefore crucial. The mouse small intestinal in vitro organoid model was first described by Sato et al. in 2009 and has been used to help understand the intestinal stem cell hierarchy and their regulatory genes. In parallel the pharmaceutical industry has been gaining interest, with the aim of validating organoids as a screening step prior to in vivo studies. We have previously developed and validated the organoid model as a screening tool to predict GI toxicity or mucosal regeneration in three species: mouse, rat and human. These techniques have recently been employed with canine tissue, successfully establishing canine organoids and assessing their suitability in our screening models in comparison to other species. The intestinal organoid culture conditions were designed to mimic the stem cell niche allowing cell differentiation and proliferation to occur. All expected intestinal lineages were present in the canine organoids and the epithelial hierarchy closely resembled that observed in vivo. The response of canine organoids to cytotoxic insult via treatment with common colorectal chemotherapy drugs correlated with their known responses in vivo. The organoid branches, which represent the crypts and contain the proliferative cells, were first lost upon treatment before complete organoid death, presumably due to the loss of stem cells within the branches. The level of toxicity associated with these chemotherapy agents was determined by calculation of IC50 values, which were then compared to published in vivo data, upon which correlation in drug sensitivity within the GI was observed. In summary, we conclude that the canine organoids, alongside other already validated organoid species, are a predictive preclinical model that can be used to identify any potential on-target, off-tissue GI toxicities induced by novel oncology therapeutics. Toxicity and mechanism of action can all be addressed in vitro to potentially reduce in vivo experimentation.

#3871

Monitoring CD73 and CD39 ectonucleotidase activities and their modulation by antibodies and small molecules inhibitors.

Said A. Goueli, Kevin Hsiao. _Promega Corp., Madison, WI_.

Modern immunotherapy approaches focusing on bolstering T cell responses and augmenting cell mediated immunity with final tumor destruction have ushered a new era and a turning point in cancer treatment. This was demonstrated by recent successes of several immunotherapeutic regimes, such as monoclonal antibody blocking of cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD1), which boosted the development of this treatment modality. Although much success have been generated with multiple tumors at different stages, tumors can deploy multiple mechanism to allow their survival by avoiding immunesurveillance. Thus, identification of alternate immunosuppressive pathways that can be targeted to enhance tumor clearance are disparately needed. It is well known that adenosine produced in the tumor microenvironment (TME) hampers the immune reaction towards cancer cells by disabling the cytotoxic anti-tumor immune response while enhancing the proliferation and polarization of immunosuppressive cells and the neovascularization that support tumor growth. It is also known that modulation of adenosine levels in the TME limits tumor growth and improves anti-tumor immune activity. Therefore, targeting the enzymes that produce adenosine and adenosine receptors as potential biomarkers for treatment response and tumor progression can provide a novel therapeutic strategy that synergize with other check point immunoblockade antibodies.

Towards this goal we have developed a novel assay to monitor the activity of the two enzymes responsible for generating extracellular adenosine in the TME, namely, ectonucleotidase triphosphate diphosphohydrolase 1 (CD39) and ecto 5'-nucleotidase (CD73). Since adenosine monophosphate (AMP) is generated from adenosine triphosphate (ATP) and adenosine diphosphate (ADP) by CD 39 followed by generation of adenosine (Ado) from AMP by CD73, we monitored the activity of both enzymes in different tumor cell lines with varied levels of both enzymes. We were able to correlate the activity of the ecto enzymes with their protein level on the cell surface and carried out enzyme characterization using both antibodies and small molecules selective for their targets. Our results clearly show that small molecule inhibitors of CD73 provide a better strategy to combat adenosine levels while small molecules towards CD39 were shown to be promiscuous since they inhibit other enzymes such as kinases. For targeting CD39, a better strategy might be the use of antibodies since they are selective to their cognate enzyme protein. Because the therapeutic potential of targeting CD39 and CD73 is not dependent solely on their nucleotidase enzyme activity but also due to their non-enzymatic role in tumor progression, antibodies might provide an alternative approach for targeting these enzymes.

#3872

**Quantitative measurement of immune-modulatory mediators within tumors of freely moving mice utilizing** in vivo **microdialysis.**

Nadege Morisot, Julien Roeser, Hajo Schiewe, Holden Brown Janssens, Marieke van der Hart, Arash Rassoulpour. _Charles River Laboratories, South San Francisco, CA_.

Monitoring changes in biochemical elements within tumors is crucial to understanding cancer biology and to helping with the development of novel therapies. Yet to date, experimental techniques enabling sensitive and quantitative measurement of the levels of small molecules contributing to tumor development have been limited in preclinical oncology models. In the current set of experiments, we implemented in vivo microdialysis to measure signaling molecules and oncometabolites in the tumor microenvironment of freely moving rodents. To this aim, a small dialysis probe is implanted in syngeneic tumors in mice. Probes are perfused with artificial dialysate fluid and dialysate containing molecules from the tumor microenvironment is collected at regular intervals. Levels of biochemicals in the dialysate are then quantified by liquid chromatography-mass spectrometry (LC-MS), a robust and sensitive quantitative technique. We report here proof-of-concept results supporting the use of in vivo microdialysis coupled to LC-MS in cancer research. First, we tested whether implantation of dialysis probe affects in vivo tumor growth. To do so, tumor volume from probe-implanted and non-implanted MC38 allografts in mice was monitored daily for up to 10 days following probe implantation. We found that the growth curve from probe-bearing MC38 tumors was similar to probe-free tumors, revealing that probe implantation did not influence tumor size progression. Next, the levels of metabolites that are known to be involved in tumor development were measured in tumor allografts of freely-moving mice using in vivo microdialysis. Relevant levels of arginine, ornithine and putrescine were quantified by LC-MS. Levels of key molecules of the adenosine signaling and tryptophan/kynurenine pathway metabolites in MC38 tumors were also quantified. To better understand the homogeneity of this particular allograft model, we measured the concentration of the above-mentioned molecules in different regions of a tumor. To this end, two separate probes were implanted in the center and the peripheral area of the same 300 mm3 MC38 allograft. We found that levels of measured molecules were similar in dialysates collected from both probe locations, suggesting homogenous levels of biochemicals throughout the microenvironment of these non-necrotic tumors. Our proposed experimental approach allows for a quantitative and sensitive measurement of key biochemical mediators of tumor progression. In vivo microdialysis in murine tumor models may be used to elucidate the mechanisms by which therapies, such as chemotherapy and immune checkpoint inhibitors, modulate the tumor microenvironment. Our efforts in preclinical cancer research has the potential to bring new insights on the mechanisms underlying cancer development and help the discovery of next-generation therapies for cancer

#3873

Targeted nanotherapy using a PARP inhibitor for Ewing sarcoma.

Abhik Bandyopadhyay,1 Rostislav Likhotvorik,1 Jodie Cropper,1 Paige Baldwin,2 Srinivas Sridhar,2 Raushan Kurmasheva1. 1 _Greehey Children's Cancer Research Inst., San Antonio, TX;_ 2 _Northeastern University, Boston, MA_.

Ewing family of sarcomas comprises the fourth most common highly malignant childhood cancer. Although dose intensification and compression has improved outcome, for patients with advanced or metastatic disease survival remains poor. Recently, a potential vulnerability of Ewing sarcoma (EwS) cells have been described as being hypersensitive to inhibition of the DNA repair protein PARP. Several groups including ours, identified the combination of a PARP inhibitor talazoparib (TLZ) with temozolomide (TMZ), a DNA damaging agent, as causing regression of ~50% of EwS xenograft models, whereas administration of single agents had little or no antitumor activity. Our preliminary studies were important for developing the current Children's Oncology Group trial (NCT02116777). However, the combination of TLZ with TMZ causes synergistic toxicity and necessitates reducing the dose of TMZ to ~13% of its single agent maximum-tolerated dose in mice and a similar dose reduction in children in the trial (NCT02116777). In this application, we explore the utility of nanoparticle formulation of TLZ (NpTLZ), and an antibody(CD99)-targeted formulation (T-NpTLZ), alone and in combination with TMZ in well-characterized models of EwS in mice. The formulations have been developed by Nanomaterials Synthesis Laboratory at Northeastern University. They are designed to increase tumor drug delivery, reduce normal tissue toxicity (mainly thrombocytopenia), and potentially allow escalation of TMZ dose. All formulations have been well characterized, and our preliminary results show that EwS tumors regress at 1/3 of the free-TLZ dose (1mg/kg of NpTLZ vs 0.33mg/kg free-TLZ), and mice tolerate 3-times the dose of Np-TLZ compared to free drug. In the PPTP study, the MTD for TMZ combined with free-TLZ (0.25 mg/kg PO BID daily x 5) was 12 mg/kg. The efficacy testing of the combination of NpTLZ with TMZ showed maintained complete response (MCR) in TC-71 xenograft at much higher TMZ dose (NpTLZ at 1mg/kg SID days 1-3-5, IV; TMZ at 50mg/kg BID x 5 PO). These data show that in combination with Np-TLZ we are able to increase the dose of TMZ to >75% of the MTD, which can lead to re-sensitization of tumors intrinsically resistant to the combination of free-TLZ and TMZ. This strongly indicates that higher doses of TMZ may be tolerated in combination with Np-TLZ. We will expand the efficacy studies to two sensitive and two resistant to TLZ and TMZ xenograft models, and next approach testing of the antibody-targeted NpTLZ formulation. Translational potential. The use of nano-formulated TLZ may overcome intrinsic resistance to this agent, due to higher drug accumulation in tumor tissue, and because of decreased normal tissue distribution of NpTLZ, allow higher doses of TMZ to be tolerated. NpTLZ formulation has advantages also for young patients (compliance/parenteral administration) and could be rapidly introduced into clinical trials.

#3874

Developing a nanoformulation of the PARP inhibitor talazoparib as a novel delivery for treatment of BRCA-deficient breast cancer.

Di Zhang,1 Paige Baldwin,2 Srinivas Sridhar,2 Karen Liby1. 1 _Michigan State University, East Lansing, MI;_ 2 _Northeastern University, Boston, MA_.

BRCA mutations are the leading cause of hereditary breast cancer. PARP inhibitors have shown promising activities in clinical trials for breast cancer by inducing synthetic lethality, particularly in patients with BRCA deficiency. Moreover, the utility of PARP inhibitors could potentially extend beyond BRCA mutations by targeting defects in homologous recombination, and thus impact up to 33% of breast cancer patients overall. However, conventional oral delivery of PARP inhibitors is hindered by limited bioavailability and significant off-target toxicities, thus compromising the therapeutic benefits and quality of life in patients. Therefore, we developed a new nanoparticle delivery system for PARP inhibitors and hypothesize that nanoformulated Talazoparib can enhance efficacy by increasing drug concentrations in the tumor and reduce off-target toxicities. The nanoparticle formulation includes polymer brushes to prolong the circulation time, enabling tumor accumulation through the enhanced permeability and retention effect. The therapeutic efficacy of Nano-Talazoparib (Nano-TLZ) was assessed after i.v. injection in Brca1Co/Co;MMTV-Cre;p53+/- mice with established tumors and compared to vehicle control (saline, i.v.), empty nanoparticles (i.v.), free Talazoparib (i.v.), and free Talazoparib (gavage). Treatment was started when the tumor was 4 mm in diameter and ended when the tumor size reached defined IACUC endpoints. Nano-TLZ significantly (p<0.05) prolonged the life span of BRCA deficient mice from 11.6±2.6 days with saline injection to 82.2±10.5 days with i.v. Nano-TLZ. Nano-TLZ induced growth arrest in 100% of the tumors and regression (> 50% reduction in tumor volume) in 80% of the tumors. Established tumors regressed more rapidly, and therefore progression free survival significantly improved in the nanoformulated Talazoparib group (p<0.05). Moreover, Nano-TLZ is better tolerated than free Talazoparib with no significant weight lost. In a biomarker study following 10 days of treatment, Nano-TLZ increased double strand DNA breaks (γ-H2AX) and decreased proliferation (PCNA) compared to controls. Interestingly, Nano-TLZ significantly (p<0.05) decreased myeloid derived suppressor cells in both the tumor (41.6±4.7% to 11.2±2.9%) and spleen (10.0±3.2% to 3.7±0.6%) compared to the saline control. Nano-TLZ also significantly (p<0.05) decreased the percentage of tumor-associated macrophages in the mammary gland from 7.4±2.0% in the saline control group to 2.5±0.2% in the Nano-TLZ group. The changes in immune populations suggest potential immunomodulatory effects of Talazoparib. These results demonstrate that the delivery of Talazoparib as a nanoformulation induces superior treatment outcomes with reduced off-target toxicity in BRCA deficient mice, and provides a novel delivery strategy for PARP inhibitors in patients.

#3875

Lymphatically directed MAPK/PI3K/mTOR inhibitor for treatment of cancer growth and metastasis.

Stefanie Galbán, Carlos Espinoza, Kathryn E. Luker, Gary D. Luker, Marcian Van Dort, Brian D. Ross. _University of Michigan, Ann Arbor, MI_.

The Ras-ERK and PI3K/mTOR signaling pathways have profound effects on cancer cell survival, differentiation, proliferation, metabolism and motility. Due to the importance of these pathways, a myriad of compounds has been developed to inhibit key signaling nodes including MEK, PI3K and PI3K/mTOR inhibitors. Evidence has shown that extensive cross-talk and compensation between pathways occurs shifting therapeutic efforts towards strategies to target multiple pathways to improve therapeutic outcomes. However, clinical trials evaluating MAPK and PI3K combination therapies have revealed poor tolerability leading to early discontinuation. Here we show a multifunctional molecular inhibitor (ST-182) capable of simultaneous inhibition of MAPK, PI3K and mTOR pathways. Kinase assays were used to determine IC50s for MEK1, PI3K α, β, δ, γ and mTOR confirming in vitro targeting of these signaling nodes. Phosphorylation changes of ERK and AKT as surrogate markers for kinase inhibition were confirmed in multiple breast cancer cell lines and determined to be independent of their BRCA1 mutational status. Reverse phase protein array performed in MDA-MB-231 cells indicated both efficient MAPK and PI3K/mTOR pathway inhibition and along with differential regulation of epithelial-mesenchymal transition (EMT) pathways compared to combination therapy of single agents (MEK plus PI3K inhibitor). Using excised breast cancer tissue from orthotopic mammary tumor mouse models (MDA-MB-231 and AT-3), ST-182 was found to modulate MEK and PI3K/mTOR activities demonstrating in vivo bioavailability confirming simultaneous multifunctional inhibition of Ras/MEK/ERK and PI3K/AKT/mTOR pathways. Innovative Kinase Translocation Reporters (KTR) were used to confirm in vitro and in vivo inhibition of these pathways. Treatment of breast tumor bearing mice daily with ST-182 (400 mg/kg, PO) achieved a significant reduction in volumetric tumor growth versus control animals with no observed systemic toxicity. Furthermore, we observed a reduction in metastatic tumor load in these mouse models underpinning its regulation of EMT proteins. Pharmacokinetics evaluation of ST-182 following oral administration revealed unique physiochemical properties promoting direct lymphatic system uptake as the primary absorption route at an astonishing >95% level rather than traditional portal vein absorption. Significant implications of lymph-directed uptake include circumventing first pass metabolism, enhanced bioavailability and reduction in systemic toxicities. This paradigm shift in drug development is anticipated to open up new opportunities for delivery of receptor tyrosine kinase (RTK) inhibitors using lymph-directed compounds to improve clinical outcome of breast cancer along with other tumor types.

#3876

**Development and** in vitro **evaluation of cell membrane lipid-extracted nanoliposomes for lung cancer treatment.**

Jasmine King, Aarohi Bhatt, Drew Goodrich, Thomas Preteroti, Hanan Alharbi, Robert Campbell. _MCPHS University- Worcester, WORCESTER, MA_.

Background: Every year more deaths are attributed to lung cancer compared to colon, prostate, and breast cancers combined. Lung cancer is typically diagnosed at late stages, and has demonstrated poor treatment outcomes due to acquired drug resistance. Unfortunately, as one method is introduced to overcome drug resistance a new mechanism can arise. In an effort to overcome drug resistance lipid extracts (LE) were derived from target cells, and the ingredients were used to develop nanoliposomes that would selectively target a desired cell population compared to controls. The early studies were designed to demonstrate the use of CLENs (cellular membrane lipid-extracted nanoliposomes) for the targeting of lung cancer. Experimental Procedures: CLENs were initially prepared using various ratios of LE/DOPC/Chol/PEG by thin film evaporation. Particle size and zeta potential values were determined for all preparations of CLENs (with and without drug). CLENs were evaluated for selectivity for target compared to control cells. Cytotoxicity studies were performed on various doxorubicin hydrochloride-loaded CLENs using a cell model of lung cancer (ChagGo-K-1). For cellular uptake studies ChaGo-K-1 cells were seeded at 10,000 cells/mL in a 48-well plate and allowed to incubate for 24 hours prior to cell studies. A fluorescence microplate reader was used to determine fluorescence intensity values for the two studies. Results: ChaGo-K-1 CLENs demonstrated an average particle size of 206+/-2 nm and zeta-potential of -21+/-3 mV, illustrating favorable characteristics for drug delivery and interstitial drug targeting. The cellular uptake studies suggested that ChaGo-K-1 CLENs were selective for ChaGo-K-1 cells in vitro, when compared to all other control varieties of CLENs. Drug-loaded ChaGo-K-1 CLENs displayed significant cytotoxicity against ChaGo-K-1 cells compared to control cells. Fluorescence microscopy revealed association of ChagGo-K-1 CLENs with target cells. Conclusions: Based on promising early finding CLENs may represent a relatively tumor specific drug delivery system that can be used to overcome drug resistance, improve efficacy, and to limit off-target drug effects. Preclinical studies will investigate whether the relative selectivity and cytotoxicity observed against lung cancer cells in vitro translates to favorable tumor uptake and efficacy in vivo.

#3877

**MPL-5821, a macrophage targeted ESM** TM **p38 MAPK inhibitor, inhibits the production of TLR agonist induced IL-10 whilst sparing T-cell functionality.**

David Moffat,1 Martin Perry,1 A. Marijne Heeren,2 Tanja D. de Gruijl,3 Justyna Rzepecka,4 Lucia Janicova,4 Anastasia Nika,4 Darryl Turner,4 Clare Doris,4 Claire Tebbutt,5 Kathryn Chapman,5 Gary Newton,5 Stephen Anderton4. 1 _Macrophage Pharma, Windsor, United Kingdom;_ 2 _Center Gynecological Oncology Amsterdam (CGOA), Amsterdam, Netherlands;_ 3 _VU University Medical Center, Amsterdam, Netherlands;_ 4 _Aquila BioMedical Ltd, Edinburgh, United Kingdom;_ 5 _Domainex Ltd, Saffron Walden, United Kingdom_.

Compensatory release of immunosuppressive cytokines, such as IL-10, by macrophages present in the tumor microenvironment has been implicated as a mechanism for adaptive resistance to a number of immunotherapies. Our drug discovery effort utilises Esterase Motif TechnologyTM (ESMTM) which selectively targets myelomonocytic cells sparing the concomitant lymphocyte anti-tumor immune response. TLR agonists are known to be stimulators of the immune response, a key component of which is the production of myeloid cell IL-12p70. However, their therapeutic potential has been limited by their accompanying induction of IL-10 and other factors. MPL-5821 is an ESMTM p38 MAPK inhibitor which not only inhibits IL-10 but also enhances LPS stimulated IL-12p70 and in contrast to conventional p38 MAPK inhibitors provides enhancement of lymphocyte IFNγ production.

The present studies contrast MPL-5821 with multiple non-targeted agents, including inhibitors of HDAC, JAK, PI3K, MEK and CSF-1R, in human PBMC assays. These demonstrated the benefit of ESMTM-targeting as applied to p38 MAP kinase inhibition to not only inhibit TLR agonist induced immunosuppression but also enhance IFNγ due to its sparing of the myeloid-lymphocyte axis. None of the other modalities were able to achieve the effects observed for MPL-5821.

Having established the unique ability of MPL-5281 to inhibit LPS induced IL-10 production whilst still maintaining lymphocyte IFNγ production, we extended our studies to human cancer ex vivo models. We chose to evaluate MPL-5821 in combination with TLR agonists in ex vivo assays using tissue and ascites derived from ovarian and cervical cancer patients. A single cell suspension was prepared from cervical cancer tumor draining lymph nodes and incubated with MPL-5821 +/- the TLR 7/8 agonist R848 for 24 and 48 hours. MPL-5821 potently inhibited the R848 induced IL-10 production as measured by Cytometric Bead Array and in contrast to a conventional p38 MAP inhibitor LY2228820 also enhanced IFNγ production.

We studied MPL-5821 in cell suspensions prepared from human ovarian tumor and ascites. For the tumor sample, a single cell homogenate was prepared by mechanical and enzymatic digestion and for the ascites the cells were isolated by centrifugation. The cell populations were then analysed by flow cytometry and the cell preparation cultured for 72 hours with anti-CD3 or TLR agonist in the presence of test compound. Cytokine production was measured after 72 hours by ELISA or Luminex bead array. MPL-5821 again showed potent inhibition of TLR agonist induced IL-10 with concomitant enhancement of IFNγ production. We conclude that application of ESMTM technology to macrophage selective delivery of p38 MAPK inhibitors has the potential to inhibit TLR agonist induction of IL-10, which is implicated in limiting the performance of TLR agonists in the clinic.

#3878

Drug-loaded microparticles as a treatment approach for pancreatic cancer.

Maria Munoz-Sagastibelza,1 Oluwafeyikemi Okome,1 Jenny E. Paredes Sanchez,1 Albert Palileo,1 Catherine Burkhart,2 Laura Martello-Rooney1. 1 _SUNY Downstate Medical Ctr., Brooklyn, NY;_ 2 _Buffalo BioLabs, Buffalo, NY_.

Pancreatic cancer is the fourth leading cause of cancer death in the United States with only 7% of diagnosed patients surviving 5 years. Most pancreatic cancer patients are not surgical candidates due to advanced stage at diagnosis. Also, current systemic chemotherapies have not been very effective at decreasing tumor burden. Poly(lactic-co-glycolic acid)-based (PLGA) microparticles (MPs) are a promising tool for localized drug delivery within the tumor due to their biocompatibility, flexibility in drug encapsulation and extended drug release. The present study investigated whether gemcitabine-loaded microparticles (GMPs), paclitaxel-loaded microparticles (PMPs) or sequential treatment of both, in comparison with blank (no drug) MPs, systemic treatments and no treatment control, are able to promote cancer cell killing effects and modulate drug resistance in vitro and in vivo. We previously showed in vitro studies with PANC-1 cells and we were able to complete the MPs studies with a second human pancreatic cancer cell line, MIAPaCa-2. In both cases, we tested the effect of single and combination treatments on two well-known resistance markers for gemcitabine, ribonucleotide reductase catalytic subunit M1 and cytidine deaminase, as well as on the promotion of cell death measuring cleaved caspase-3 (CC3). When treated with GMPs alone, both markers went up suggesting an increase in resistance against gemcitabine. Interestingly, both cell lines treated with PMPs alone showed an increase in CC3 expression and a significant decrease in the expression of both markers. Subsequently, we tested the in vivo efficacy of MPs by direct injection into established subcutaneous MIAPaCa-2 tumors in nude mice. Following four weeks of treatment, the tumors were excised, biopsied for protein analysis and frozen in OCT to allow visualization of fluorescent MPs and detect apoptosis by immunofluorescence. We also want to evaluate reactive oxygen species production; where we expect to see an increase in the MPs treated groups. This analysis is in progress. In conclusion, we observed a decrease in cell viability and drug resistance proteins in vitro using the drug-loaded microparticles in two grade 3 (or high grade) pancreatic cancer cell lines. The described drug delivery method has the potential to be a more efficient treatment modality than systemic gemcitabine and paclitaxel against early stage and locally advanced pancreatic cancer.

#3879

Enhancement of tumor penetration by drug-loaded nanoparticles: An innovative targeted strategy for neuroblastoma.

Mirco Ponzoni,1 Chiara Brignole,1 Laura Emionite,2 Silvia Bruno,3 Daniela Guarnieri,4 Leopoldo Sitia,4 Matteo Bauckneht,5 Ambra Buschiazzo,5 Andrea Rossi,1 Daniela Di Paolo,1 Patrizia Perri,1 Flavio Curnis,6 Alessandro Gori,7 Angela Rita Sementa,1 Michele Cilli,2 Pier Paolo Pompa,4 Gianmario Sambuceti,5 Angelo Corti,6 Fabio Pastorino1. 1 _IRCCS, Istituto G. Gaslini, Genoa, Italy;_ 2 _IRCSS Ospedale Policlinico San Martino, Genoa, Italy;_ 3 _University of Genoa, Genoa, Italy;_ 4 _Istituto Italiano di Tecnologia (IIT), Genoa, Italy;_ 5 _IRCSS Ospedale Policlinico San Martino, University of Genova, Genoa, Italy;_ 6 _IRCCS San Raffaele Scientific Institute and Vita Salute San Raffaele University, Milan, Italy;_ 7 _Istituto di Chimica del Riconoscimento Molecolare, Consiglio Nazionale delle Ricerche, Italy_.

Background: Anti-cancer drugs-loaded targeted nanocarriers can reduce side-effects and improve therapeutic efficacy in preclinical studies. However, poorly perfused and dysfunctional tumour vessels limit the transport of the payload into the parenchyma of solid tumours. The use of tumour-penetrating nanocarriers might enhance tumour penetration and anti-tumour effects.

Methods: A consensus motif, mediator of tissue penetration (TP) was added to a previously characterized neuroblastoma (NB)-targeting peptide (pep). In vitro NB cell association and internalization of TP-pep, either free or coupled to Stealth Liposomes (SL), were tested by FACS and confocal microscopy. In vitro cytotoxic potential of a novel doxorubicin (DXR)-loaded liposomal (TP-pep-SL[DXR]) was evaluated by MTS assay. Three mouse xenograft models mimicking the growth and spread of NB in humans (injection routes: subcutaneous; adrenal gland; tail vein) were enrolled to examine in vivo penetration, vascular permeability, tumour glucose consumption and sensitivity in response to TP-pep-SL[DXR].

Results: Compared to pep, TP-pep increases its cellular association in vitro on cell lines and cells derived from NB patients. When coupled to SL, TP-pep enhances liposomes penetration and cytotoxic effects in vitro and increases binding and penetration in a mouse model of NB. Moreover, in vivo accumulation of Evans Blue dye within the tumour mass reveals that TP-pep-SL[DXR] increases the tumour vascular permeability into NB tumour mass, but not in non-tumour tissues. Compared to pep-targeted liposomes, TP-pep-SL[DXR] leads to an increased anti-NB effect towards all the animal models tested.

Conclusion: Our findings demonstrate that the enhancement of tumour penetration by drug-loaded nanoparticles might represent an innovative targeted strategy for NB.

#3880

Peptide-targeted chemotherapy for pancreatic cancer.

Chin-Tarng Lin,1 Chen-Der Wu,1 Jen-Chieh Lee,1 Han-Chung Wu,2 Chung-Wei Lee,1 Chin-Feng Lin,1 Ming-Chieh Hsu1. 1 _National Taiwan Univ. College of Medicine, Taipei, Taiwan;_ 2 _Academia Sinica, Taipei, Taiwan_.

The purpose of this abstract was to assess the efficacy of peptide targeted chemotherapy for pancreatic carcinoma. Previously we have constructed four peptides that bind specifically to cancer cell lines, which were derived from three different carcinoma cell lines and their vascular endothelia: L-peptide: (L-P, anti-cancer cell membrane), RLLDTNRPLLPY; SP-94-P (anti-hepato-pancreatic cancer cell membrane), SFSHHTPILP; PC5-52-peptide (anti-tumor endothelia), SVSVGMKPSPRP; and control peptide, RLLDTNRGGGGG. These peptides were linked to pegylated liposomal iron oxide nanoparticles to identify the targeted tumor cells and vascular endothelia using MRI analysis, and were also linked to dextran coated liposomal doxorubicin (L-D) for treatment of non-obese diabetic, severe combined immunodeficiency (NOD-SCID) mice bearing pancreatic cancer cell (PANC-1) xenografts. Our results demonstrated that the tumor intensity of MRI is clearly decreased after SP-94-P-iron oxide was applied, and that in combination of application of L-P linked L-D (L-P-L-D) plus SP-94-P linked L-D (SP-94-P-L-D) and PC5-52-P linked L-D (PC5-52-P-L-D), they could inhibit pancreatic tumor growth with very mild adverse events. The use of the control peptide linked L-D also led to a decrease of the xenograft size, but also induced marked apoptotic change of their visceral organs. It is concluded that the combination of L-P-L-D, SP-94-P-L-D and PC5-52-P-L-D to treat pancreatic cancer xenograft in NOD SCID mice can clearly inhibit pancreatic cancer growth with minimal adverse events.

#3881

Formulating a cell membrane lipid-extracted nanoliposome (CLENS) drug platform for the treatment of prostate cancer.

Abubker Omaer, Ali Alqarni, Carlos Randulfe, Hanan Alharbi, Robert Campbell. _MCPHS University, Worcester, MA_.

Background: Liposome-encapsulated doxorubicin has proven to be an effective drug delivery method to cancer cells. Lipid extracts derived from human prostate cancer cells along with conventional components were employed to develop cell membrane lipid-extracted nanoliposomes (CLENs) for prostate cancer treatment. In an effort to balance efficiency of drug loading with cellular targeting efficiency we report on physicochemical characteristics (i.e., particle size, zeta potential, drug loading efficiency) and cellular uptake studies.

Methods: Five preparations of CLENS were prepared consisting of variable concentrations of DOPC and CRL-1740 lipid extract material varying as a function of optimized fixed ratios of conventional components (Cholesterol and DSPE-PEG5000). The controls used to investigate the effect of the different components on drug incorporation efficiency were preparations of DOPC (100%) and CRL-1740 (100%). Doxorubicin hydrochloride (5 mol%) was employed during the preparation of all formulations. Samples were readily prepared by thin film evaporation. For cellular uptake studies CRL-1740 cells were seeded at 1 x 104 mL in 48-well plate and incubated for 24 hours prior to use. The cells were next exposed to various amounts of rhodamine labeled CLENS for 24 hrs. Cellular uptake was determined using fluorescence intensity values determined by a fluorescence microplate reader. Fluorescence microscopy supplement cellular uptake studies.

Results: The DOPC drug-loaded liposome preparation consistently achieved the lowest percent of drug incorporation. Likewise, 100% CRL-1740 CLENs demonstrated a significant degree of formulation instability, resulting in a relatively low percent of drug incorporation compared to preparations that consisted of lipid extract and conventional components. The finding were supported by values determined for zeta potential and particle size. Cellular uptake studies also favored the preparations that consisted of lipid extract material and conventional components compared to each alone. The results collectively support the importance of balancing lipid extract material derived from prostate cancer target cells with cholesterol and DSPE-PEG5000 for formulation development and cellular uptake.

Conclusion: We formulated seven preparations of CLENS as part of the drug platform. The most promising candidates were determined following completion of the study. Future studies should evaluate the cytotoxicity and safety profile of the most promising formulations vivo.

### Pharmacogenetics and Pharmacogenomics

#3882

Functional validation of a genome-wide association study (GWAS) SNP associated with anthracycline-induced congestive heart failure.

Xi Wu, Gloria Xue, Laura Gardner, Fei Shen, Guanglong Jiang, Santosh Philips, Geneva Cunningham, Bryan P. Schneider. _Indiana University School of Medicine, Indianapolis, IN_.

Anthracyclines are commonly used chemotherapies for the treatment of breast cancer, but they can cause dose-related cardiotoxicities and lead to congestive heart failure (CHF) in ~2% of patients. There are no clinically available predictive biomarkers for this toxicity. Previously we have identified and validated the association of a single nucleotide polymorphism (SNP), rs28714259, with CHF in three large adjuvant phase III breast cancer trials. rs28714259 locates in a putative glucocorticoid receptor (GR) binding site and the risk (minor) allele is predicted to disrupt GR binding. Interestingly, activation of GR-signaling by dexamethasone has been shown to protect cardiomyocytes from doxorubicin-induced apoptosis in rat. To test whether the risk allele affects GR binding efficiency in vitro, we performed electrophoretic mobility shift assay (EMSA) in MCF-7 and A549 cells. 100nM of dexamethasone for 4 hours induced a prominent band shift in either the major- or risk-allele probes. Notably, we observed a ~50% reduction in the shifted band intensity of risk-allele probes compared to major-allele probes, suggesting decreased binding in risk-allele probes. No additional supershift was detected after the binding reaction was co-incubated with a GR antibody. However, the previously shifted band was significantly diminished in both the major- and risk-allele probes, probably due to a conformational change induced by GR antibody and leading to the disruption of GR/DNA binding, which in turn suggests the observed shift was specifically caused by GR. Further validations are ongoing using cardiomyocytes derived from human induced pluripotent stem cells (iPSCs). These studies provide further insight into the function and role of rs28714259 in anthracycline-induced CHF.

#3883

**Gene expression predicts** ex vivo **drug sensitivity in acute myeloid leukemia.**

Brian S. White,1 Suleiman A. Khan,2 Muhammad Ammad-ud-din,2 Swapnil Potdar,2 Mike J. Mason,1 Cristina E. Tognon,3 Brian J. Druker,3 Caroline A. Heckman,2 Olli P. Kallioniemi,2 Stephen E. Kurtz,3 Kimmo Porkka,4 Jeffrey W. Tyner,3 Tero Aittokallio,2 Krister Wennerberg,2 Justin Guinney1. 1 _Sage Bionetworks, Seattle, WA;_ 2 _Institute for Molecular Medicine Finland, Helsinki, Finland;_ 3 _Oregon Health and Science Institute, Portland, OR;_ 4 _University of Helsinki, Helsinki, Finland_.

Introduction: Ex vivo drug sensitivity studies of samples derived from acute myeloid leukemia (AML) patients have been shown to be predictive of in vivo response. These findings are based on a limited number of well-characterized agents for which in vivo patient response data and ex vivo drug sensitivity data—on that same patient—are available. To show the feasibility of scaling such ex vivo studies to large drug screens, we characterized the reproducibility of expression-based models of drug response across two independent data sets—one generated at the Oregon Health and Science University (OHSU) and the second at the Institute for Molecular Medicine Finland (FIMM).

Methods: We harmonized two large-scale AML ex vivo studies screened for drug response and profiled transcriptomically—OHSU (303 AML patient samples and 160 drugs) and FIMM (48 AML samples and 480 drugs). The two panels have 94 drugs in common. Log-logistic curves were fit to the dose-response data and area under the dose-response curves (AUCs) were calculated. Predictive modeling using Ridge regression or an integrative Bayesian approach was performed for each drug AUC independently using 202 highly-variable and/or cancer-associated genes as features.

Results: For each of the 94 drugs in common between the two data sets, we trained a Ridge regression model on the OHSU data set, used the model to predict response in the FIMM data set, and calculated the Pearson correlation between the predicted and observed FIMM responses. 41 of the 94 drug models had a positive and statistically significant correlation [false discovery rate (FDR) < 20%; mean ρ = 0.43; 95% CI = 0.29 – 0.77]. Drugs corresponding to the top decile of these significant models (mean ρ = 0.54; 95% CI = 0.48 – 0.77) clustered into four primary classes: MEK inhibitors (PD184352, Selumetinib, and Trametinib), EGFR/VEGFR inhibitors (Cabozantinib, Erlotinib, Foretinib, and Sorafenib), and singletons Venetoclax and Sirolimus. To confirm these results, we applied a second modeling approach—an integrative Bayesian machine learning method—that allows systematic combination of both data sets. Training and evaluation of this approach using 10-fold cross validation yielded 82 positive and statistically significant correlations (FDR < 20%; mean ρ = 0.35; 95% CI = 0.13 – 0.58). Five of nine drugs (Cabozantinib, Selumetinib, Sirolimus, Sorafenib, and Trametinib) corresponding to the top decile of these significant models (mean ρ = 0.54; 95% CI = 0.49 – 0.60) overlapped with drugs from the top decile of Ridge results (one-sided Fisher p = 2.5 x 10-4)

Conclusions: Our results using independent data sets and two statistical approaches suggest that certain drugs (including MEK and EGFR/VEGFR inhibitors) are amenable to expression-based predictive modeling in AML. Future work will focus on inferring individual biomarkers of response.

#3884

Novel RUNX1 targeted therapy for AML expressing mutant RUNX1.

Christopher P. Mill, Courtney D. DiNardo, Warren Fiskus, Dyana T. Saenz, Agnieszka J. Nowak, Baohua Sun, David N. Saenz, Steven M. Kornblau, Kapil N. Bhalla. _MD Anderson Cancer Center, Houston, TX_.

RUNX1 is a master-regulator transcription factor involved in normal and malignant hematopoiesis. In addition to chromosomal translocations involving the RUNX1 locus, somatic, heterozygous RUNX1 mutations commonly occur in MDS, CMML, as well as in secondary (s) or de novo AML, which confer higher risk, shorter latency to AML transformation and poorer outcome in AML. We demonstrate here for the first time that stable or inducible shRNA-mediated knockdown of RUNX1 induced colony growth inhibition and greater apoptosis of AML cells expressing mutant (mt) RUNX1 versus those expressing wild-type (WT) RUNX1. Knockdown of RUNX1 also inhibited in vivo AML growth and significantly improved survival of immune-depleted mice engrafted with AML cells expressing mtRUNX1. RUNX1 transcription is driven by a BET protein BRD4-occupied super-enhancer in the first intron of the RUNX1 gene. Our findings also demonstrate that shRNA-mediated knockdown of BRD4, or treatment with BET protein (BETP) inhibitor (BETi), or BETP degrader PROTAC, but not their inactive enantiomer or congener, resulted in depletion of RUNX1 and induction of apoptosis in AML cells expressing mtRUNX1. Notably, following CRISPR-mediated editing of the +24 kb enhancer of RUNX1, the surviving clones exhibited markedly attenuated RUNX1 expression and stunted in vitro growth. Another goal of present studies was to find novel agents with an expression signature mimicking the expression signature of RUNX1 knockdown, but without the capacity to exert genotoxic effects on hematopoietic progenitors. To achieve this objective, we performed LINCS (Library of Integrated Network-based Cellular Signatures)-CMap (connectivity mapping) analysis querying with the RNA-seq signature of RUNX1 knockdown by shRNA to RUNX1. The expression-mimickers (EMs) uncovered as top hits included not only agents that have been documented to be BETis, but also agents that engage targets proven to be therapeutically effective when subjected to targeted-inhibition. Importantly, EM hits also included non-genotoxic anticancer agents, such as narciclasine (natural plant alkaloid), fenbendazole (benzimidazole anthelmintic) and cinobufagin (bufanolide steroid). Treatment with these agents depleted RUNX1 levels and dose-dependently induced loss of viability, as well as reduced AML burden and significantly improved survival of mice engrafted with AML cells expressing somatic mtRUNX1. These EMs also induced lethality of HPCs from FPD/MM (familial platelet disorder with myeloid malignancy) due to germline mtRUNX1 with co-occurring leukemogenic somatic mutations, while relatively sparing FPD HPCs without co-occurring leukemogenic mutations or normal HPCs. Collectively, these findings demonstrate BETP antagonists, as well as uncovered novel agents, as targeted, effective agents that deplete RUNX1 levels and induce in vitro and in vivo efficacy against AML expressing somatic or germline mtRUNX1.

#3885

11q13 amplification selects for sensitivity to the ERK inhibitor KO-947 in squamous cell carcinomas.

Francis J. Burrows,1 Linda Kessler,1 Tao Wu,2 Xin Gao,2 Jeffrey Chen,2 Rasmus Hansen,2 Shuangwei Li,2 Carol Thach,2 Shisheng Li,2 Ke Yu,2 Jeff Kucharski,2 Ulf Peters,2 Jun Feng,2 Yi Wang,2 Yvonne Yao,2 Ata Zarieh,2 Matt Janes,2 Jingchuan Zhang,2 Liansheng Li,2 Dana Hu-Lowe,2 Pingda Ren,1 Yi Liu1. 1 _Kura Oncology, San Diego, CA;_ 2 _Wellspring Biosciences, San Diego, CA_.

The MAPK pathway is a major driver of malignant progression, particularly in cancers arising from mutations in pathway components, and BRAF and MEK inhibitors have been approved for treatment of BRAF-mutant melanoma. ERK1/2 kinases are the final node in the MAPK signaling pathway and offer the possibility of clinical benefit in settings where earlier drugs are ineffective. We have previously reported that KO-947, a potent and selective inhibitor of ERK1/2 with extended target residence time and favorable pharmaceutic properties, displays robust single-agent antitumor activity in PDX models of adenocarcinomas with RAS/RAF mutations, and in squamous cell carcinomas (SCCs) that lack mutations in MAPK pathway components. Here we report the discovery of a novel biomarker that is associated with sensitivity to KO-947 in head and neck SCC and esophageal SCC PDX models. Recurring amplification of chromosome 11 between bands q13.3 and q13.4 is a feature of several tumor types, including ESCC and HNSCC. TCGA reports incidence rates of >50% in ESCC and ~20% in HNSCC, although higher frequencies are seen in some subtypes, such as pharyngeal and HPV-negative SCC. The 11q13 amplicon commonly contains about a dozen genes, including several potential oncogenes with functional linkage to the MAPK pathway, such as CCND1, FADD and the recently described calcium-dependent chloride channel ANO1. PDX campaigns were carried out in 24 ESCC and 18 HNSCC models, including 16 and 7 of each with 11q13 amplification, respectively. Groups of three animals were dosed with KO-947 at 300mg/kg QW, tumor growth was monitored for 3-6 weeks and responses were categorized as progressive disease, stable disease (SD, >80% TGI) or partial/complete response (PR/CR, >30% regression). In ESCC tumor-bearing animals, the overall response rates (ORR = PR+CR) were 33% in the overall population, 51% in the 11q13-amplified (11q-AMP) and 3% in 11q13-WT. The disease control rates (DCR = PR/CR+SD) were 54% overall, 77% in 11q-AMP and 21% in 11q-WT. In HNSCC tumor-bearing animals, the ORRs were 24% in the overall population, 56% in 11q-AMP and 9% in 11q-WT, with DCRs of 40%, 69% and 26% in the three subsets, respectively. 11q-AMP was significantly associated with response to KO-947 in both tumor types. ROC analysis defined the minimum effective copy number as 4 and revealed significant associations between expression levels of several 11q13 amplicon genes and response to KO-947. A key role for ANO1 in driving ERK-dependent tumor growth in 11q-AMP cases was further indicated by the observation that ANO1 expression was silenced in some 11q-AMP models and these tumors failed to respond the ERK inhibition. The results suggest that 11q13 amplification in SCCs can drive tumor growth and survival in a MAPK-dependent manner and that 11q-AMP may be a useful biomarker for predicting clinical response to ERK inhibitors.

#3887

Whole exome sequencing to identify genetic markers for trastuzumab-induced cardiotoxicity.

Chihiro Udagawa,1 Hiromi Nakamura,2 Hiroshi Ohnishi,3 Kenji Tamura,4 Tatsunori Shimoi,4 Masayuki Yoshida,4 Teruhiko Yoshida,2 Hiroshi Okamura,1 Yasushi Totoki,2 Tatsuhiro Shibata,2 Hitoshi Zembutsu2. 1 _Toyo kohan, Tokyo, Japan;_ 2 _National Cancer Center Research Institute, Tokyo, Japan;_ 3 _Cancer Institute, Tokyo, Japan;_ 4 _National Cancer Center Hospital, Tokyo, Japan_.

Although trastuzumab-induced cardiotoxicity is an important determinant to limit the use of this drug, the molecular mechanism of risk for this toxicity is not well understood. To identify a genetic marker(s) determining the risk of trastuzumab-induced cardiotoxicity, we performed whole exome sequencing of germline DNA samples from 9 patients with trastuzumab-induced cardiotoxicity, and conducted a case-control association study of 2,258 genetic variants between 9 cases (with trastuzumab-induced cardiotoxicity) and general Japanese population controls registered in Human Genetic Variation Database (HGVD). To further validate the result of the screening study, we carried out a replication study of 10 variants showing Pmin < 0.001 in the screening study using 234 independent patients treated with trastuzumab, including 10 cases and 224 controls (without trastuzumab-induced cardiotoxicity). In the replication study, we observed that three variants had effect in the same direction as in the screening study (SNV1 in exon 2 of HERADR1, SNV2 in exon 2 of HERADR2 and SNV3 in exon 44 of HERADR3). A combined result of the screening and the replication studies suggested an association of a locus on chromosome 6q12 with trastuzumab-induced cardiotoxicity (SNV3 in HERADR3, combined-Pmin = 0.00056, OR = 13.73). This finding provides new insights into personalized trastuzumab therapy for the patients with HER2 positive cancer.

#3888

The NantOmics Pharmacogenomics Test: An integrative panomic approach to pharmacogenomics screening.

Camille Schwartz,1 John Little,1 Charles Vaske,1 Stephen Benz,1 Patrick Soon-Shiong,2 Shahrooz Rabizadeh,2 J. Zachary Sanborn1. 1 _NantOmics, Santa Cruz, CA;_ 2 _NantOmics, Culver City, CA_.

Numerous oncology drugs have pharmacogenomics warnings on their FDA labels, yet pharmacogenomics screening is not routinely applied in clinical practice. Pharmacogenomics testing is used to reduce the chance of drug-induced toxicities, improve patient outcomes, and reduce treatment costs by tailoring therapies to the patient's genotype. Here we present the NantOmics pharmacogenomics test, which uses whole genome (DNA) and whole exome sequencing data from FFPE tumors and matched normal samples to predict and advise physicians of possible drug-induced toxicities. Using FDA labels and Clinical Pharmacogenomics Implementation Consortium (CPIC) guidelines, we developed a clinical pharmacogenomics panel including 31 markers in 11 genes linked to toxicities from 14 cancer therapies, as well as a research panel of 13 gene-drug pairs reported in primary literature. The test screens for both germline and somatic variants to predict how an oncology patient may respond to routinely-used chemotherapies.

Several genes in our panel, including CYP2D6 and other CYP family members, prove challenging to properly genotype due to high levels of polymorphism, multiple similar haplotypes, and structural variations. We have established a kmer-based approach to accurately determine haplotypes for these genes, as well as coverage-based methods to infer hybridizations and copy number variations. For pharmacogenes with ambiguous genotypes comprising multiple heterozygous SNPs, we utilize any allele fraction imbalance observed at these loci in the tumor DNA and/or RNA sequencing data to disambiguate the correct genotype.

The test was run on 1,172 patient samples, 95.8% of which contained a variant in at least one gene from our panel. Furthermore, 7.5% of patients had genomic variants associated with severe or life-threatening drug toxicities. For all alleles in our clinical panel, we observed similar allele frequencies to those reported in the ExAC database. Our test was validated on several cell lines from the CDC GeT-RM, and was able to accurately determine the genotypes for all genes present in our test panels in each of the cell lines tested. To further validate our test, we ran it on a cohort of patients previously genotyped by an independent CLIA-validated PCR-based panel, as well as on a set of synthetic data. In all validation studies, we were able to demonstrate that the test detects each variant in our panel, and correctly determines patient genotype in all studied cases. Given the high percentage of patients with potentially treatment-altering genomic variants, these results underscore the need for more routine pharmacogenomics screening in the oncological setting.

#3889

RPL28 **promoter polymorphism rs4806668 is associated with reduced survival in FOLFIRI-treated metastatic colorectal cancer patients.**

Adrien Labriet,1 Éric Lévesque,2 Elena De Mattia,3 Erika Cecchin,3 Derek Jonker,4 Félix Couture,2 David Simonyan,5 Angela Buonadonna,6 Mario D'Andrea,7 Lyne Villeneuve,1 Giuseppe Toffoli,3 Chantal Guillemette1. 1 _Pharmacogenomics Laboratory, Centre Hospitalier Universitaire de Québec (CHU) Research Center and Faculty of Pharmacy, Laval University, Québec, Quebec, Canada;_ 2 _CHU de Québec Research Center and Faculty of Medicine, Laval University, Québec, Quebec, Canada;_ 3 _Clinical and Experimental Pharmacology, "Centro di Riferimento Oncologico"- National Cancer Institute, Aviano, Italy;_ 4 _Division of Medical Oncology, Department of Medicine, Ottawa Hospital, University of Ottawa, Ottawa, Ontario, Canada;_ 5 _Clinical and Evaluative Research Platform, CHU de Québec Research Center, Québec, Quebec, Canada;_ 6 _Medical Oncology Unit, "Centro di Riferimento Oncologico"- National Cancer Institute, Aviano, Italy;_ 7 _Medical Oncology Unit, "San Filippo Neri Hospital", Rome, Italy_.

Fluorouracil (5-FU), folinic acid, and irinotecan (FOLFIRI) is a standard treatment of metastatic colorectal cancer (mCRC). Our study aimed to investigate genetic variability in candidate genes in relation to patients' outcomes using two independent cohorts of 417 FOLFIRI-treated mCRC cases recruited in Canada and Italy. We used a haplotype-tagging polymorphism (htSNP) approach to maximize the coverage of genetic variability of the selected genes and genotyping was performed using time-of-flight mass spectrometry iPLEX Sequenom Technology. Associations between polymorphisms and clinical outcomes were tested using Cox proportional hazards model adjusted for covariates. Of the genes tested, RPL28 encodes a ribosomal protein and its silencing was shown to enhance sensitivity to 5-FU and irinotecan in vitro.1 RPL28 rs4806668G>T was associated with a shorter progression-free survival (PFS) in the Canadian (hazard ratio (HR) 3.23, P = 0.013), Italian (HR 3.28, P = 0.021) and combined (HR 3.36, P < 0.001) cohorts. This marker was also associated with a reduced overall survival (OS) in the Canadian (HR 3.09, P = 0.032), Italian (HR 3.05, P = 0.030) and combined (HR 3.07, P = 0.002) cohorts. These carriers of the rs4806668 TT genotype associated with reduced survival represent less than 5% of the population of European ancestry and its frequency varies greatly among ethnic groups. This htSNP, located in the promoter region of RPL28, is in strong linkage disequilibrium with six other polymorphisms and may affect RPL28 gene expression. In support, the rs4806668T allele was associated with increased RPL28 expression in colon tissues of healthy individuals of the GTEx cohort, compared to rs4806668G carriers. Functional investigations are required to elucidate the underlying molecular mechanism. These results suggest a role for the ribosomal RPL28 protein in cancer cell response to FOLFIRI treatment.

This work is supported by the Canadian Institutes of Health Research.

1Allen et al. Mol Cancer Ther. 2012 Jan;11(1):119-31

#3890

**Interspecies comparison of in vitro** **endoxifen metabolism.**

Miguel Muzzio,1 Zhihua Huang,1 Elizabeth R. Glaze,2 David L. McCormick1. 1 _IIT Research Institute, Chicago, IL;_ 2 _National Cancer Institute, Bethesda, MD_.

Endoxifen (N-desmethyl-4-hydroxytamoxifen) is an active metabolite of tamoxifen being developed for use in breast cancer prevention and therapy. To optimize model selection for preclinical toxicology and pharmacology studies, the in vitro metabolism of endoxifen was compared using commercially available pools of hepatocytes from humans, rats, dogs, rabbits, minipigs, and non-human primates. Endoxifen (10 μM) was incubated with hepatocytes for 0, 2, or 4 hours. Cells were harvested, extracted, and analyzed by LC-MS/MS to quantitate levels of parent compound and major metabolites. Although substantial differences in patterns of endoxifen metabolism were seen in different species, endoxifen glucuronide and endoxifen sulfate were the primary metabolites identified in all species. Endoxifen was relatively stable when incubated with human hepatocytes: after 2 and 4 hours of incubation, endoxifen levels were > 20-fold greater than levels of endoxifen glucuronide and > 5-fold greater than levels of endoxifen sulfate. Levels of endoxifen glucuronide and endoxifen sulfate in human hepatocytes were similar at both time points. By contrast, endoxifen was extensively glucuronidated (but not sulfated) in both rabbit and minipig hepatocytes: after 4 hours of incubation, levels of endoxifen glucuronide in rabbit and minipig hepatocytes were > 5 times the level of parent drug. Levels of endoxifen sulfate were very low in both rabbits and minipigs. Rats and dogs demonstrated patterns of endoxifen metabolism that were intermediate to those of humans, rabbits, and minipigs. In rat hepatocytes, levels of parent drug were slightly greater than levels of endoxifen glucronide at 4 hours; little endoxifen sulfate was generated in rat hepatocytes. In dog hepatocytes, levels of endoxifen and endoxifen glucuronide were comparable at 4 hours; sulfation of endoxifen was greater in dogs than in other species, as levels of endoxifen sulfate at 4 hours were approximately 2/3 of the levels of parent drug and endoxifen glucuronide. Although endoxifen was somewhat less stable in hepatocytes isolated from non-human primates than in human hepatocytes, endoxifen metabolism in hepatocytes from non-human primates demonstrated the greatest similarity to metabolism in human hepatocyte pools: at 4 hours, levels of parent drug in monkey hepatocytes were approximately 4 times the levels of endoxifen glucuronide and 8 times the levels of endoxifen sulfate. These data demonstrate that endoxifen is quite stable when incubated with hepatocytes from humans and non-human primates. By contrast, endoxifen is rapidly and extensively glucuronidated in hepatocytes from rabbits and minipigs. Rats and dogs, the two species used most commonly in preclinical safety studies, both demonstrate substantially more rapid endoxifen metabolism than do humans. [Supported by HHSN261201500024I from the National Cancer Institute, DHHS.]

#3891

Tolerability and outcomes of trametinib and everolimus combinations in advanced solid tumors.

Jason Patterson, Cory Perry, Joe Windscheffel, Rachel Elsey, Kirstin Williams, Nandini Dey, Pradip De, Brian Leyland-Jones, Casey Williams. _Avera Cancer Institute, Sioux Falls, SD_.

Purpose

Aberrations in the PI3K/AKT/mTOR and MAPK pathways are routinely found in patients with cancer. To date, safe and effective combinations of drugs that hit both pathways concurrently are scarce. Data from Tolcher and colleagues have shown that the combination of trametinib and everolimus is intolerable at the doses and schedules studied in a phase 1 trial for patients with advanced solid tumors. The purpose of this analysis was to determine the tolerability and outcomes for patients prescribed trametinib and everolimus utilized in 2 open trials at our center.

Methods

A retrospective analysis was performed on 31 patients with various tumor types at the Avera Cancer Institute with approval from the IRB. Patients who received at least one dose of trametinib in combination with everolimus in addition to concurrent therapies were included. Fifteen patients (48%) had undergone ≥ three lines of therapy prior to initiation of trametinib and everolimus. Descriptive statistics were used to determine primary and secondary outcomes. Tolerability was defined as number of dose adjustments or holds in therapy for each regimen. Primary outcome was based on comparisons between tolerability of the drug regimens and the schedule of doses, line of therapy, concurrent cytotoxic agents, and primary tumor type. A secondary outcome calculated the PFS of eligible patients (n=5) who had different trametinib and everolimus regimens.

Results

Twenty-three patients (74%) tolerated various combinations of trametinib and everolimus, of which 16 received treatment for more than 3 months. Eight patients (26%) discontinued treatment due to adverse events, of which six patients did so within the first 3 months of treatment. Average time to first hold or reduction in therapy due to toxicities from any trametinib and everolimus regimen was 2.5 months. Most common adverse events were fatigue (n=19), mucositis (n=18), and elevated LFT's (n=17). The most common regimen utilized (n=23) was initiated at trametinib 1 mg daily with everolimus 5 mg every Mon, Wed, Fri. Patients on this regimen experienced dose-limiting toxicities at an average of 1.4 months after initiation. Trametinib and everolimus were most commonly started as ≥ the fourth line of therapy. PFS for evaluable patients was 6.5 months; two patients continue to respond. PIK3CA (n=10) and KRAS (n=9) were the most common mutations identified.

Conclusion

In this analysis, the combination of trametinib and everolimus has shown to be tolerable in many patients with advanced solid tumors, and has also demonstrated clinical efficacy for patients with heavily pre-treated disease. However, the regimens utilized were highly individualized, thus notable variability existed between patient therapies. Therefore, to determine an optimal dose and schedule to evaluate the overall efficacy of trametinib and everolimus combination, an adequately powered and controlled study would be required.

#3892

Upfront DPD screening with adaptive dosing to secure 5-FU administration in head and neck cancer patients.

Joseph Ciccolini,1 Manon Launay,1 Charlotte Dupuis,2 Florence Duffaud,2 Bruno Lacarelle,1 Sébastien Salas2. 1 _Aix-Marseille Univ., Marseille, France;_ 2 _Assistance Publique Hôpitaux de Marseille, Marseille, France_.

Upfront screening for dihydropyrimidine dehydrogenase (DPD) deficiency in patients scheduled for 5-FU-based protocols should reduce the risk of treatment-related toxicities by preventive adaptive dosing. Our group has developed a simple, rapid and cheap functional testing that helps categorizing patients on their DPD status and detecting poor metabolizer (PM) patients, using UH2/U ratio measurement in plasma as a surrogate for DPD activity. 5-FU dosing can be next tailored using a geometric scale, according to the level of DPD deficiency. We report here the performances of this adaptive dosing strategy implemented in routine clinical practice in head and neck cancer patients. A total of 218 evaluable adult patients were scheduled for a 5-FU-regimen (mostly TPF protocol), with prospective search for DPD deficiency and, if required, tailored dosing. Overall, 20 patients (9%) were identified as PM and received subsequently an average 20-50% reduced dose of 5-FU as compared with extensive metabolizer (EM) patients (2102 ± 254 mg VS. 2577 ±353mg, p<0.001 t-test). Gender (Female) was associated with higher risk for being PM (p=0.01, Pearson's Chi-square test), whereas age was not. Early severe toxicities were seen only in 5% of patients, a figure markedly lower than the one usually reported with 5-FU-based regimen in head-and-neck cancer patients. Similarly, all severe toxicities (i.e; including now the delayed or cumulative ones) were observed in 12.8% of patients only. No statistical difference was evidenced in overall toxicities between PM patients with adapted dosing (10%) and EM patients with standard dosing (13%, p=0.7014, Chi-square test), suggesting that being DPD-deficient is not anymore a risk for severe or life-threatening toxicities with 5-FU, provided that corrective action is undertaken. As for early toxicities, incidence of global toxicities was lower than the 30-40% usually described with 5-FU in this setting. Finally, despite an average -20% reduction in 5-FU dosing between PM and EM patients, clinical efficacy was not statistically different between the two groups (CR+PR: 56% VS. 40%, SD: 11% VS. 5%, PD: 22% VS. 43%, p = 0.2774, chi-square test), thus demonstrating how cutting 5-FU dose in PM patients does not hinder treatment efficacy. In conclusion, this study shows that 5-FU-related toxicities can be avoided at low cost by the upfront detection of DPD deficient patients with simple adaptive dosing strategies. Of note, reducing 5-FU dosing in PM patients did not affect negatively treatment efficacy, while limiting the incidence of treatment-related toxicities, thus improving the efficacy/toxicity balance of this drug.

#3893

Quantitative contribution of rs75017182 to dihydropyrimidine dehydrogenase mRNA splicing and enzyme activity.

Qian Nie, Shikshya Shrestha, Erin E. Tapper, Colbren S. Trogstad-Isaacson, Kelly J. Bouchonville, Steven M. Offer, Robert B. Diasio. _Mayo Clinic, Rochester, MN_.

5-Fluorouracil (5-FU) has remained a widely prescribed anti-cancer drug for decades. However, 5-FU–based treatments show inter-individual variability, with up to 34% of 5-FU–treated patients developing severe (grade 3+) adverse toxicity. Dihydropyrimidine dehydrogenase (DPD; DPYD gene) is the rate-limiting enzyme of 5-FU catabolism and converts 80–85% of 5-FU into its inactive metabolites. Three deleterious genetic variations in DPYD are well-established predictors of severe 5-FU toxicity (i.e., *2A, p. I560S, and p. D949V); however, these variants only explain 30–35% of adverse cases. Recently, an intronic variant (rs75017182) has been suggested to contribute to 5-FU–related toxicity by promoting the alternative splicing of DPYD. However, clinical studies are conflicting as to the degree to which rs75017182 affects DPD enzyme function, and the true contribution of the rs75017182 variant towards DPD function and 5-FU toxicity remains unclear and inconclusive. In the present study, we directly examined the effect of the intronic SNP rs75017182 on DPYD mRNA splicing, DPYD expression, and DPD enzyme function. We genotyped 3950 healthy Caucasian volunteers from the Mayo Clinic Biobank to identify the rs75017182, *2A, p.I560S, and p.D949V variants. From the initial cohort, 204 volunteers who were rs75017182 heterozygous carriers were recruited as our study population. Alternative splicing and DPD activity in rs75017182 carriers were measured. The findings were confirmed using a novel mini-gene reporter system in vitro. A moderate, but significant, reduction (30%) was detected in canonically spliced DPYD expression in rs75017182 carriers compared to non-carriers. A correlative reduction (35%) in DPD enzyme activity was observed in those carriers, which was similar to that of D949V carriers (31%). The results demonstrated an association of this deep intronic variant with decreased DPD activity and suggested that rs75017182 may be a predictor of 5-FU toxicity similar to D949V.

#3894

**DPYD-Varifier, a computational model to identify 5-FU toxicity-associated** DPYD **variants.**

Shikshya Shrestha, Cheng Zhang, Calvin R. Jerde, Hu Li, Steven M. Offer, Robert B. Diasio. _Mayo Clinic, Rochester, MN_.

5-Fluorouracil (5-FU) is a cornerstone therapy for the treatment of several aggressive tumors. However, nearly 1/3 of patients receiving 5-FU experience severe (grade 3+) adverse 5-FU toxicity. Genetic variations in dihydropyrimidine dehydrogenase (DPD; DPYD gene) are major contributors of 5-FU toxicity. With continued progress in next-generation sequencing technologies, the number of reported genetic variants is substantially increasing; to date, there are more than 450 reported missense variants in the DPYD gene. Only a handful of those variants, including *2A, I560S, D949V, and rs75017182, have been established as predictors of toxicity. Because most of the remaining variants are unstudied, mainly due to their rare occurrence and unequal racial distribution, it is challenging to conduct statistically powered clinical association studies. Therefore, in vitro and computational methods to identify potential toxicity-associated variants have major appeal. The primary goal of the current study was to develop a highly accurate gene-specific in silico classifier to predict deleterious DPYD variants. For this purpose, using a recombinant cellular model, we characterized 69 missense DPYD variants reported in online databases. We then utilized DPD activity data from the 69 variants in combination with data from 87 previously characterized variants (see Offer, SM et al. Cancer Research 2013 & 2014) to develop the DPYD variant classifier model (DPYD-Varifier). DPD protein-specific biochemical and structural features and amino acid conservation scores were integrated into the model's random forest classifier-based algorithm. DPYD-Varifier showed improved accuracy (85% accurate) compared to other in silico tools. Additionally, the model provided mechanistic insights into alterations of DPD function conferred by DPYD variants. In general, deleterious variants were located in closer proximity to important binding/coordinating sites of the DPD protein. Additionally, DPYD-Varifier allowed us to estimate the cumulative allele frequency of all reported deleterious variants in various geographical/racial groups, which can substantially add to our knowledge of DPD deficiency and 5-FU toxicity observed in these populations. In conclusion, the integration of in vitro and accurate computational data from our study permitted classification of relevant DPYD variants, which could be useful to more effectively individualize 5-FU therapy in the future.

#3895

ABCC11 **gene polymorphism as a potential predictive biomarker for an oral 5-fluorouracil derivative drug S-1 treatment in non-small cell lung cancer.**

Satoshi Fukuda,1 Takehiro Uemura,2 Tetsuya Oguri,1 Akira Takeuchi,1 Kazuki Sone,1 Takamitsu Asano,1 Yoshihiro Kanemitsu,1 Masaya Takemura,1 Hirotsugu Ohkubo,1 Ken Maeno,1 Yutaka Ito,1 Akio Niimi1. 1 _Nagoya City University, Nagoya, Japan;_ 2 _Aichi Cancer Center Hospital, Nagoya, Japan_.

Introduction: ABCC11/MRP8 (ABCC11) is an ATP-binding cassette transporter that is involved in regulating cellular sensitivity and resistance for many anti-cancer drugs including antifolates 5-fluorouracil (5-FU), pemetrexed and methotrexate. ABCC11 has functional genetic variant which regulate the protein expression of ABCC11. However, the relationship between the genetic variant of ABCC11 and the efficacy of anticancer drugs remains to be elucidated. An oral 5-FU derivative S-1 composed of tegafur, 5-chloro-2, 4-dihydroxypyridine (CDHP; a potent inhibitor of dehydropyrimidine dehydrogenase), and potassium oxonate, is used against non-small cell lung cancer (NSCLC) treatment; however the biomarker for S-1 is not fully examined. In this study, we examined whether ABCC11 single nucleotide polymorphism (SNP) which regulate the expression of ABCC11 is related with the efficacy of S-1.

Method and results: We introduced ABCC11 showing 538G or 538A expression vector and parental plasmid into HeLa cells and established HeLa/ABCC-538G, HeLa/ABCC-538A and HeLa/mock cells, respectively. The protein expression of ABCC11 was higher in the HeLa/ABCC-538-G cell than in the other cells. Futhermore, the IC50 for 5-FU with CDHP was 0.94, 9.75 and 0.64 times higher in the HeLa, HeLa/ABCC-538G and HeLa/ABCC-538A cells than in the HeLa/mock cells, respectively. This result indicates that this ABCC11 SNP is related with 5-FU cytotxocity. Then we analyzed 21 human NSCLC cell lines, classifing NSCLC cell lines into two groups based on the genotype of the SNP 538G>A in ABCC11: a combined G/G and G/A genotype group, and an A/A genotype group. The distribution of the cytotoxicity for 5-FU in combination with CDHP was significantly lower in the A/A genotype group than in the combined G/G and G/A genotype group. Next, we examined the clinical usefulness of the ABCC11 SNP in S-1 treatment of 68 NSCLC patients. In these 68 patients, the median progression-free survival (PFS) was 73days, and the disease control rate was 50%. These data was similar to previous reports. Classified these 68 patients into each ABCC11 538G>A genotype group, we found that the disease control rate was significantly better in the A/A genotype group than in the combined G/G and G/A genotype group (A/A:59.2%, G/G and G/A:35.7%, respectively), but the PFS had no significant difference between these two genotype groups.

Conclusion: SNP 538G>A in the ABCC11 gene is a potential determinant for S-1 treatment in NSCLC.

#3896

A big data approach to predicting context specific synergistic drug combinations.

Coryandar M. Gilvary, Neel Madhukar, Olivier Elemento. _Weill Cornell Medical College, New York, NY_.

Combination therapies for various cancers have been shown to increase efficacy, lower toxicity and escape resistance. However, systematically interrogating all possible synergistic therapies is experimentally unfeasible due to the sheer volume of possible combinations. Computational approaches have proven to be an invaluable tool within pharmacogenomics and have helped with prioritizing the development of perspective therapeutics as well as matching the right drugs with the right patients. Here we apply a novel big data approach in the evaluation and ultimately the prediction of drug synergy by using the recently released NCI-ALMANAC, the largest publically available synergistic drug efficacy dataset to date. First, to better understand drug combinations, we distinguished between those that were synergistic and adverse and evaluated various drug similarity metrics for all pairs. We found that certain features showed significant differences between adverse and synergistic drug combinations, such as post-treatment transcriptional effects similarity (D = 0.17, p < 0.001) and chemical structure similarity (D = 0.25, p < 0.001). By exploiting these significant similarities and dissimilarities and incorporating cell line specific data we developed a machine learning model to predict context specific drug synergy and achieved significant predictive performance (AUC = 0.823). We find that our model can be used to both identify novel synergistic drug pairs, as well as find novel indications for known drug combinations by identifying new sensitive cell lines. Moreover, the interpretability of our model allows for the interrogation of features for a deeper understanding of why certain combinations are predicted synergistic. In addition to identifying the cancer types and subtypes a combination therapy would be most synergistic within, we set out to identify the molecular indication for highly synergistic pairs. Specifically, we systematically identified candidate predictive biomarkers which could be used to stratify patient cohorts. Overall, our model and methodology can expedite the development and expansion of combination therapeutics, which can help battle acquired resistance and increase therapeutic efficacy. The thorough understanding of specific combination efficacy dependencies allows for a true precision medicine application of these therapeutics.

#3897

Pharmacogenomic landscape of long non-coding RNAs in human cancers.

Aritro Nath, R. Stephanie Huang. _University of Minnesota, Minneapolis, MN_.

Extensive efforts in recent large-scale drug screens have enabled identification of protein-coding genes (PCGs) as biomarkers for anti-cancer agent sensitivity, but information on the role of long-non coding RNAs (lncRNAs) in drug response is sparse. Lower abundance, difficulty in expression profiling and limited functional knowledge have so far hindered global analysis of lncRNAs in drug screens. However, lncRNAs represent nearly 60% of the human genome (vs. 3% for PCGs) and contain a vast majority of pharmacogenomic SNPs. Thus, it is critically important to evaluate the role of lncRNAs in drug sensitivity. Here, we perform a comprehensive reanalysis of large-scale drug screens using novel computational methods to create a landscape of pharmacogenomic interactions of lncRNAs in human cancers.

We developed an algorithm to accurately impute the lncRNA transcriptome using PCG expression. This approach allowed us to generate lncRNA transcriptome in high throughput drug screening data sets and enabled systematic evaluation of lncRNAs for hundreds of drugs. For example, we applied our method to the Genomics of Drug Sensitivity in Cancer (GDSC) study, where sensitivity to 265 anti-cancer agents were quantified in 963 cancer cell lines. Sparse regression analysis showed lncRNA transcriptome were similar in accuracy at predicting drug response compared to PCGs (P = 0.9), indicating lncRNAs may be equally important biomarkers.

Subsequently, we identified drug-specific lncRNA biomarkers using ANOVA while adjusting for tissue type. Our analysis recapitulated literature-verified lncRNAs associated with drug response, e.g. HOTAIR expression with cisplatin resistance (FDR < 0.05). In addition, we identified several compelling novel lncRNA associations for specific drugs along with previously uncharacterized multi-drug response predictors.

We next identified lncRNAs that may serve as biomarkers for drug response while controlling for known cancer functional events. As an example, we analyzed EGFR-inhibitors (erlotinib, gefitinib, pelitinib) while controlling for EGFR somatic point mutations and amplification status and identified an anti-sense lncRNA overlapping UGT1A locus that may be a potential regulator of erlotinib metabolism. These findings reveal important lncRNAs that predict response independent of known cancer functional events and are currently being evaluated in non-small cell lung cancer patients.

In conclusion, our comprehensive analysis has generated unprecedented insights into the role of lncRNAs in anti-cancer drug response and will be an invaluable resource for researchers studying drug response in various cancer types.

#3898

CDA predicts life-threatening toxicities in AML patients treated with cytarabine.

Raphaelle Fanciullino,1 Laure Farnault,2 Mélanie Donnette,1 Vadim Ivanov,2 Geoffroy Venton,2 Joseph Ciccolini,1 Yael Berda-Haddad,2 Catherine Roche,2 L'Houcine Ouafik,2 Bruno Lacarelle,1 Regis Costello2. 1 _Aix-Marseille Univ., Marseille, France;_ 2 _Assistance Publique Hôpitaux de Marseille, Marseille, France_.

Cytarabine (Ara-C) is the backbone for treating accute myeloid leukemia (AML). As most nucleoside analogs, Ara-C is metabolized in the liver by an exclusive enzymatic step driven by cytidine deaminase (CDA). CDA is a ubiquitous enzyme that proved to be highly polymorphic, with activities in patients ranging from poor metabolizer (PM) to ultra metabolizer (UM). To date, deregulated CDA has been evidenced as a culprit for life-threatening toxicities with gemcitabine. Little evidences have been collected thus far about the possible role erratic CDA could further play with Ara-C. Here, we investigated whether deregulations affecting CDA could be at the origin of unexpectedly severe, and sometimes lethal, toxicities with Ara-C in patients with AML. In this clinical observational study we determined CDA activity levels and searched for CDA genetic polymorphisms in 58 adult patients (25F, 33M, mean age 63 ±15 years) hospitalized for AML and scheduled for induction therapy. All patients were treated following current standard care by Ara-C-containing regimen (i.e., 7+3 protocol) and monitored for toxicity (CTCAEv4 grading) and response. Efficacy was evaluated one-month after starting the induction protocol. Mean CDA activity in the studied population was 3.14 ± 3 U/mg. No relationship was found between CDA phenotype and genotype (i.e., 79A>C polymorphism). A total of 20 patients (43%) displayed severe toxicities (i.e. grade-3 and above) upon administration, including 3 unrecoverable grade-5 toxicities (5%). Relationship between CDA activity and severe toxicity was investigated using ROC analysis. We observed that the incidence of CDA deficiency was much higher in AML patients (40%) than what we previously observed in patients with solid tumors. A cut-off value in CDA ≤ 2 U/mg was associated with increased risk of experiencing severe/lethal toxicities with 74% sensitivity and 65% specificity. Among the 20 patients with severe toxicities, 14 (72 %) were categorized as PM. Importantly, patients who experienced lethal toxicities were all profoundly PM patients. CR was achieved in 35 patients (60%), CRI achieved in 4 patients (7%) whereas 16 patients (27%) failed to respond (PD). No relationship was evidenced between CDA status and response, however regardless of the karyotype we observed that PM patients tended to have longer PFS (278 vs. 517 days) and OS (570 days vs. not reached) as compared with EM patients. Overall this proof-of-concept study strongly suggests that CDA status could be a relevant marker for predicting clinical outcome in patients treated with Ara-C, especially treatment-induced toxicities. A prospective study including full PK and pharmacogenetics support is currently ongoing in our institute to confirm these observations. Should the role CDA plays be confirmed, this marker could be used as a covariate to customize Ara-C dosing in AML patients.

#3899

**Discovery and clinical implementation of individualized therapies in acute myeloid leukemia based on** ex vivo **drug sensitivity testing and multi-omics profiling.**

Disha Malani,1 Ashwini Kumar,1 Bhagwan Yadav,2 Mika Kontro,2 Swapnil Potdar,1 Oscar Brück,2 Sari Kytölä,2 Jani Saarela,1 Samuli Eldfors,1 Poojitha Ojamies,1 Karjalainen Riikka,1 Muntasir Mamun Majumder,1 Imre Västrik,1 Pekka Ellonen,1 Matti Kankainen,1 Minna Suvela,1 Siv Knappila,1 Alun Parson,1 Aino Palva,1 Pirkko Mattila,1 Evgeny Kulesskiy1,1 Laura Turunen,1 Karoliina Laamanen,1 Elina Lehtinen,1 Piia Mikkonen,1 Maria Nurmi,1 Sanna Timonen,1 Astrid Murumägi,1 Bjorn Tore Gjersten,3 Satu Mustjoki,2 Tero Aittokallio,1 Krister Wennerberg,1 Simon Anders,4 Maija Wolf,1 Caroline Heckman,1 Kimmo Porkka,2 Olli Kallioniemi1. 1 _Inst. for Molec. Medicine Finland (FIMM), Helsinki, Finland;_ 2 _Hematology Research Unit Helsinki, Helsinki, Finland;_ 3 _Department of Clinical Science, Bergen, Norway;_ 4 _Center for Molecular Biology of University of Heidelberg (ZMBH), Heidelberg, Germany_.

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by multiple molecular subtypes and lack of effective targeted therapies. Here, we performed extensive molecular profiling and ex vivo drug testing with 515 approved and emerging cancer drugs on 164 AML patient samples. The aim was to i) assign individualized treatment options to advanced AML patients in real time, ii) explore drug response patterns across the molecular subtypes of AML and iii) identify opportunities to repurpose existing and emerging cancer drugs.

Bone marrow samples (n=164) from 129 consecutive AML patients and 17 healthy donors were studied from the Helsinki University Hospital and the Haukeland University Hospital, Bergen. Mononuclear cells were resuspended either in mononuclear cell medium (MCM) or stroma conditioned medium (CM) and tested for drug sensitivity and resistance as previously described (PMID: 24056683) and studied by exome and transcriptome sequencing. The study protocol allowed us to return data to the clinician for consideration of novel treatment options. For the meta-analysis of associations between drug responses and molecular and clinical parameters, Wilcoxon signed ranked test and logistic regression were applied.

Clustering of all patient samples based on ex vivo drug response patterns in both media types identified 7 distinct functional groups of AML. For example, a subgroup of samples was highly resistant to chemotherapeutics and all targeted drugs except BCL-2 inhibitors. The differences in drug responses in the two media types highlighted the importance of assay conditions for ex vivo drug testing. Strong clustering of several drugs in the same drug classes was often observed as well as clustering across different classes, for example between BET (JQ1, I-BET151, birabresib) and MEK (trametinib, cobimetinib) inhibitors. About 24 percent of the FLT3 negative AML patients manifested strong ex vivo sensitivity to glucocorticoids, highlighting a potential drug repositioning opportunity in this subset of AML patients. Overall, we identified 320 significant associations between drugs and mutated driver genes including association between NPM1 mutation and sensitivity to JAK inhibitors.

Altogether, targeted treatment opportunities were clinically tested in 25 occasions in chemorefractory AML patients. The tailored clinical therapy led to transient complete remission or leukemia free state in 36% (9/25) of these cases.

In conclusion, we discovered and clinically implemented individualized therapeutic options for AML patients, which resulted in a 36% clinical responses in a non-randomized proof-of-concept study. The associations identified between ex-vivo drug response and driver mutations provided novel drug repositioning opportunities in specific molecular subtypes.

#3900

Plasma ctDNA multigene mutation analysis for the diagnosis and treatment monitoring of colorectal cancer patients.

Michael J. Powell,1 Elena Peletskaya,1 Qing Sun,1 Jinwei Du,1 Larry Pastor,1 Jyoti Phatak,1 Jenny Wilding,2 Walter F. Bodmer,2 Aiguo Zhang1. 1 _DiaCarta, Inc., Richmond, CA;_ 2 _Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom_.

Introduction

Colorectal cancer is a highly preventable disease as early detection increases rates of patient survival to near 100%. Also during therapy colorectal tumors can develop resistance due to mutations in cellular growth factor receptor signaling pathway genes such as KRAS and BRAF. Herein we report new validation studies of a novel multigene mutation biomarker real-time PCR based assay for qualitative detection of colorectal cancer associated mutations including the tumor suppressor APC, growth factor signaling pathway genes KRAS and BRAF and the essential WNT pathway signaling gene Beta- catenin (CTNNB1) called ColoScapeTM .The assay allows the sensitive detection of the presence or absence of mutations in the targeted regions of the genes interrogated. While tumor-tissue remains the 'gold standard' for genetic analysis in cancer patients, it is challenged with the advent of circulating cell-free tumor DNA (ctDNA) analysis from blood samples; so called 'liquid biopsy'. Here we broaden our previous study on the initial validation of FFPE samples to include plasma DNA in colorectal cancer patients and by evaluating its clinical utility as a method for monitoring tumor ctDNA mutations during and after therapy as well as a potential screening test for early diagnosis of colorectal cancer.

Methodology

he high sensitivity of this multigene biomarker assay is achieved due to xenonucleic acid (XNA) probe technology. XNA probes are novel backbone modified oligomers having natural nucleoside bases (A,T,C and G) that hybridize by Watson-Crick base pairing to natural DNA and RNA with much higher binding affinity than natural deoxyribonucleic acid oligomers of the same sequence. XNA probes are designed that bind to the selected wild-type sequences at the respective genetic loci in the target genes. These XNA probes have a much higher Tm than the primer annealing temperature and suppress amplification of WT DNA templates and only allow amplification of the target mutant DNA templates in the sample. Both single nucleotide polymorphisms (SNP's) and insertion/deletions (indels) mutations can be detected. Performance parameters of the assay were established on DNA of colorectal cancer patients extracted from FFPE as well as reference DNA materials (synthetic and cell line derived DNA). This study presents further validation of the test as a potential landmark clinical test for colorectal cancer initial detection in FIT posiive patients and for recurrence and therapy monitoring

Conclusion:

The ColoScapeTM Colorectal Cancer Mutation Detection assay is shown to be a sensitive tool intended to facilitate research in Colon Cancer development, early detection, disease monitoring and therapeutic interventions. 

### Receptor Targeting and the Tumor Microenvironment

#3901

Characterizing antibody-drug conjugate cytotoxicity using four different real-time assays.

Andrew L. Niles, Kevin Kupcho, Sarah Duellman, Jolanta Vidugiriene, Dan Lazar, James J. Cali. _Promega, Madison, WI_.

Recent clinical successes using antibody drug conjugates (ADC) directed to hematological or solid tumor cancers have re-invigorated immunotherapeutic drug development interest. Unfortunately, efficacious ADC are inherently complex entities comprised of a targeting antibody, a stable but cleavable linker, and a toxic payload, for which the best combinations are often identified through iterative trial and error. Therefore, an efficient and systematic method for rank ordering ADC candidates is sought to evaluate their intrinsic pharmacodynamic properties. We chose SKBR3 (Her2+) and T47D (Her2-) cell models to define the on- and off-target cytotoxic potential of trastuzumab emtansine. We employed a suite of four, newly developed real-time assays to measure exposure-dependent changes in viability, caspase activation, phosphatidylserine (PS) exposure and loss of membrane integrity to define potency and magnitude of response, as well as primary mode of cell death. We discovered that trastuzumab emtansine mediated dose-dependent effects on SKBR3, with cytostasis beginning at 17h near 1μg/ml, caspase induction at 20h, which led to PS exposure at about 21h and secondary necrosis at 30h. During this same time course, full dose-response curves were generated for each biomarker as EC50 values evolved and matured to their respective maximal responses. Conversely, the T47D non-target cell population exhibited only minor cytotoxicity at the latest time points, presumably owing to linker degradation and non-specific payload release. Taken together, these data define the in vitro pharmacological disposition of trastuzumab emtansine, including the kinetic component of functionality related to antigen/antibody engagement, internalization, linker mediated payload release and resulting toxicity. This study may provide a tangible roadmap for the critical evaluation of other ADC under development.

#3902

A novel biologic ADI-TRAIL fusion protein benefits from structural and functional complementarity of its components arginine deiminase and TRAIL, induces cancer cell apoptosis in vitro, and inhibits tumor growth in vivo.

Elena Brin,1 Katherine Wu,1 Yudou He,2 Wei-Jong Shia,1 Mario M. Kuo,1 Li-Chang Chen,1 Eleanor Dagostino,1 Richard Hickey,1 Bob Almassy,1 Richard Showalter,1 Jim Thomson1. 1 _Polaris Pharmaceuticals, San Diego, CA;_ 2 _formerly Polaris Pharmaceuticals, currently UCSD, San Diego, CA_.

TNF-related apoptosis-inducing ligand (TRAIL) receptor agonists have shown good safety profile and efficacy in preclinical cancer models. However, clinical success has been limited due to poor PK and/or development of resistance to death receptor-induced apoptosis. To address these issues we have conceived and produced a fusion protein of arginine deiminase (ADI) and TRAIL. ADI is an enzyme that converts arginine into citrulline. Arginine deprivation can inhibit growth of arginine auxotrophic cancers that lack key enzymes enabling normal cells to produce arginine from citrulline. ADI is currently being evaluated in clinical trials and is well-tolerated. We (and others) observed synergy between ADI and TRAIL in inducing apoptosis in a number of cancer cell lines including those that are otherwise resistant to rhTRAIL. We have explored potential mechanisms of synergy and will present our findings. Using in silico modeling we predicted structural complementarity between ADI and TRAIL with each modality stabilized when part of the fusion protein. We were able to produce multiple ADI-TRAIL fusion protein variants and have tested their activity in enzymatic and cell-based assays. Our experimental data confirmed in silico predictions of the structural advantages for ADI and TRAIL in the fusion protein. ADI enzymatic activity improved when part of the fusion protein and the fusion protein appeared to have higher potency than the combination of the two separate proteins in inducing cancer cell death. In mice ADI-TRAIL fusion protein exhibited extended half-life and was efficacious in HCT116 xenograft model, superior to rhTRAIL administered at the same molar amounts. Serum concentrations of the fusion protein had an inverse correlation with the tumor volume. ADI-TRAIL is a novel biologic that benefits from structural and functional synergies between its components and is showing promise as a cancer therapeutic in preclinical studies.

#3903

Rocaglamide A and synthetic analogues sensitize resistant renal carcinoma cells to TRAIL-induced apoptosis and inhibit cell proliferation.

Ancy D. Nalli,1 Lauren E. Brown,2 Cheryl L. Thomas,1 Thomas J. Sayers,1 John A. Porco,2 Curtis J. Henrich1. 1 _National Cancer Institute, Frederick, MD;_ 2 _Boston University, Boston, MA_.

Induction of cancer cell-specific apoptosis via activation of TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) signaling has been an attractive goal for cancer therapeutics. However, many tumor cells develop resistance to TRAIL. Therefore, the search for enhancers of TRAIL-induced apoptosis has accelerated over the past several years. A high-throughput screening assay was developed that identified several natural product enhancers of TRAIL-induced apoptosis in TRAIL-resistant renal carcinoma cells. Among these natural products was the protein synthesis inhibitor, rocaglamide A, a cyclopenta[b]benzofuran secondary metabolite from the genus Aglaia, with potent antiproliferative and anti-inflammatory properties. Natural and synthetic rocaglates have been reported to have potent anticancer activities in vitro on various human cancer cell lines and in vivo in mouse models. The mechanism of action involved in the anticancer effects of rocaglamide A is generally thought to be inhibition of translation initiation. However, several other cancer-related cellular effects including cell cycle arrest have been reported in various cancer cell types. In this study, rocaglamide A and its synthetic analogs were assessed for their ability to enhance TRAIL-induced apoptosis, inhibit protein synthesis, and regulate cellular processes associated with TRAIL sensitization in TRAIL-resistant ACHN renal carcinoma cells. Rocaglate treatment enhanced TRAIL signaling resulting in caspase-dependent apoptotic cell death upon addition of TRAIL. Rocaglates also inhibited protein synthesis, which correlates with rapid loss of the antiapoptotic FLICE-inhibitory protein (cFLIP) and MCL-1, which are often overexpressed in TRAIL-resistant cancer cells. On average, the rocaglates were ~4-5-fold more potent than TRAIL sensitizers compared to their protein synthesis inhibitory potency. This difference suggests a possible therapeutic window for induction of TRAIL sensitization while minimizing general effects of protein synthesis inhibition. Rocaglamide treatment alone inhibited cell proliferation leading to cell cycle arrest at G2/M phase but not subsequent apoptosis. These studies suggest that the TRAIL-sensitizing activity of rocaglates and their growth-inhibitory effects as single agents are largely dependent on protein synthesis inhibition. The development of a large number of structurally diverse but mechanistically similar enhancers of TRAIL signaling with a wide range of potencies allows for further understanding of structure-activity relationships regarding both protein synthesis inhibition and TRAIL sensitization for this important family of compounds.

Funded (in part) by NCI Contract No. HHSN261200800001E.

#3904

PEP-010, a cell penetrating & interfering peptide as a new therapeutic approach in breast cancer.

Sophie Lebel-Binay,1 Fariba Nemati,2 Leticia Dominguez-Berrocal,1 Justine Fleury,2 Adnan Naguez,2 Didier Decaudin,2 Angelita Rebollo3. 1 _PEP-Therapy, Paris, France;_ 2 _Institut Curie, Paris, France;_ 3 _CIMI-Paris, Paris, France_.

Introduction. The interaction between intracellular caspase-9 and PP2A proteins is critical to apoptosis. We designed PEP-010, a Cell Penetrating & Interfering Peptide, which specifically disrupts the interaction between caspase-9 and PP2A. We evaluated in vitro and in vivo its therapeutic properties to demonstrate its potential as an innovative approach to breast cancers treatment.

Material and Methods. In vitro evaluation was done by apoptosis (Annexin V) and cell viability (MTT) measurements on cancer cell lines from different origins. In vivo efficacy studies were conducted on patient-derived xenograft (PDX) mice models of triple-negative breast cancer (TNBC) and hormone-positive HER2-negative breast adenocarcinoma (BC). Pharmacokinetic and biodistribution studies were conducted on mice after administration by iv and ip route. Preliminary tolerance studies were done on mice and rats after repeated administration of PEP-010.

Results. First, we demonstrated that PEP-010 is able to penetrate into tumor cells and induces caspase-9-dependent apoptosis in several tumor cell types. Moreover, we demonstrated that PEP-010 specifically induces the death of cancer cells (CLL) without harm to healthy cells.After PEP-010 treatment, we observed a significant tumor growth inhibition in PDX mice models of TNBC, compared to the untreated control. In BC PDX mice models, we observed also a significant tumor growth inhibition with complete response after PEP-010 treatment. We also improved the stability and the pharmacokinetic parameters of PEP-010. Point mutations on a protease cleavage site clearly improved peptide stability while keeping the functional activity. Biodistribution studies demonstrated that the optimized peptide is able to reach the targeted tumor and accumulate there at higher concentration than the former peptide. Pharmacokinetic studies in mice administered with PEP-010 by iv and ip route were conducted as well as in vitro study of plasmatic clearance of PEP-010 in different species. We finally performed formulation studies to improve the solubility of PEP-010 and confirmed the efficacy of the drug product in a BC PDX mice model.Preliminary tolerance studies were done on mice and rats after repeated administration of PEP-010 without demonstrating any signs of toxicity. Moreover, no immunogenic response has be observed after repeated administration of PEP-010 in mice.

Conclusion. Using PEP-010, a CP&IP blocking caspase-9/PP2A interaction, we have demonstrated that this peptide has an interesting in vitro and in vivo therapeutic effect. PEP-010 constitutes a new innovative therapeutic approach for the treatment of human breast cancers. PEP-010 will enter GLP toxicity studies shortly in order to prepare the first in human clinical trial.

#3905

Strategic combination of the cyclin-dependent kinase inhibitor CYC065 with venetoclax to target anti-apoptotic proteins in chronic lymphocytic leukemia.

Rong Chen,1 Yuling Chen,1 Sheelagh Frame,2 David Blake,2 William G. Wierda,1 Daniella Zheleva,2 William Plunkett1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Cyclacel Pharmaceuticals, Inc. , Dundee, United Kingdom_.

CYC065 is a cyclin-dependent kinase (Cdk) inhibitor that is highly selective towards Cdk2 and Cdk9. In chronic lymphocytic leukemia (CLL), a disease that is addicted to the over-expression of anti-apoptotic proteins for survival, inhibition of Cdk9 by CYC065 reduced phosphorylation of the C-terminal domain of RNA polymerase II and blocked transcription. These actions depleted the intrinsically short-lived anti-apoptotic protein Mcl-1, but not Bcl-2, and induced apoptosis in CLL cells in vitro. The IC50 for CYC065-induced CLL cell death after a 24-hr incubation was 0.8 μM, a concentration that is achievable in the clinic at tolerated doses. CYC065 killed the CLL cells equally efficiently in the presence or absence of the human stromal cell line, StromaNKtert, and with or without a stimulation condition that mimics the lymphoid tissue microenvironment (anti-IgM, anti-CD-40, IL-4). Venetoclax, which specifically inhibits Bcl-2 function, is approved for treatment of CLL with del(17p); however upregulation of Mcl-1 is associated with resistance to venetoclax in the lymph nodes. Therefore, we hypothesized that the combination of CYC065 with venetoclax would target the parallel mechanisms that promote the survival control in CLL cells, and induce synergistic cell death by apoptosis. A time course study of the single agents showed that under conditions that mimic the lymph node microenvironment, cell death induction by venetoclax required 6-8 hr to reach the plateau of cell killing and maximal killing by CYC065 occurred after 24 hr, consistent with the different mechanisms of action of the two compounds. Following the removal of CYC065 or venetoclax after 4, 8, 12, or 24 hr incubations, there was no evidence for additional cell death after an additional 48 hr in drug-free medium regardless of the duration of drug incubation. Immunoblots showed recovery of RNA pol II phosphorylation, and restored Mcl-1 expression upon washout of CYC065. The reversible action of these compounds has potential implications for clinical scheduling combining these compounds. Median effect analysis indicated that CYC065 and venetoclax combined synergistically in CLL samples with or without 17p deletion. A dose reduction analysis confirmed mutual potentiation of each other when combined. Combination of IC50 concentrations of CYC065 and venetoclax for 24 hr was sufficient to decrease the viability of CLL cells by over 90% in the lymph node mimicking microenvironment. Thus, these data provided rationale for clinical combination of CYC065 and venetoclax in CLL. CYC065 is currently in a Phase I clinical trial in patients with advanced solid tumors (NCT02552953) using an intermittent dosing regimen which causes at least 24 hr Mcl-1 downregulation in patient PBMCs at well tolerated dose levels.

#3906

Inhibition ofNF-kB Inducing Kinase (NIK) to supressNF-kB alternative pathway and cell migration in HNSCC.

Rita Das, Paul E. Clavijo, Clint Allen, Anthony Saleh, Zhong Chen, Carter Van Waes. _NIH-NIDCD, Bethesda, MD_.

Nuclear factor-κB (NF-κB) activation through the alternative pathway has been implicated in cancer progression and survival, however the regulatory signaling and biological significances are not well understood, especially in head and neck squamous cell carcinoma (HNSCC). NF-κB-inducing kinase (NIK) is a key molecule in the alternative NF-κB pathway, which receives signals from Lymphotoxin beta/Lymphotoxin beta receptor (LTβ/LTβR) to activate Inhibitor-κB kinase α (IKKα) and transcription factors RELB/NF-κB2. In a panel of HNSCC cell lines, we observed that dysregulation of the alternative NF-κB pathway occurred primarily through increased LTβ receptor signaling via NIK and the non-canonical NF-κB subunits. We identified a NIK inhibitor 1, 3[2H, 4H]-Iso-Quinoline Dione, which inhibited cell migration at a low dose range, but exhibited minimal anti-proliferative effect in HPV(-) HNSCC cell lines. The biological effects of the inhibitor were through suppression of the alternative NF-κB signaling. Using Western blot and immunofluorescence staining, we showed that the inhibitor reduced the protein expression and/or phosphorylation of NIK, IKKα, and RELB/NF-κB2 (p52) in the cytoplasm, and/or the protein translocation in the nucleus. The NIK inhibitor also blocked LTβ induced translocation of RELB to the nucleus. The effects of NIK inhibitor were validated by knockdown of NIK using siRNA, which inhibited cell migration and LTβ-induced expression of MET. Met is a hepatocyte growth factor receptor implicated in promoting metastasis, and knockdown of MET slowed down HNSCC cell migration. We further extended the study on a murine oral metastasis cell line MOC2, which is a syngeneic mouse model suitable for testing drug effects on tumor growth and migration in vivo. We found that the NIK inhibitor decreased cell proliferation, migration, and invasion through an extracellular matrix barrier at low concentration ranges. Currently, we have identified a potent and selective small molecule NIK inhibitor, which is orally bioavailable and active in vivo. We plan to test the effects of this NIK inhibitor on HNSCC tumor growth and metastasis in the syngeneic MOC2 model. Our study may shed light on how HNSCC tumors migrate and metastasize to local lymph nodes, where the microenvironment promotes alternative NF-κB activation by LTβ through NIK signaling. Targeting NIK mediated alternative NF-κB activation could be a potential therapeutic strategy for preventing tumor migration and metastasis of HNSCC. (Supported by NIDCD intramural projects ZIA-DC-000016, 74)

#3907

Birinapant co-treatments of colon cancer cell lines show consensus molecular subtype-specific synergistic effects.

Michael Fichtner,1 Emir Bozkurt,1 Katherine McAllister,2 Christopher McCann,2 Daniel Longley,2 Jochen H. Prehn1. 1 _Royal College of Surgeons in Ireland, Dublin, Ireland;_ 2 _Queens University Belfast, Belfast, United Kingdom_.

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. Despite recent advances, the treatment success differs strongly between colorectal cancer patients and new treatment options are urgently needed. Since most tumors avoid apoptosis by a dysregulation of Inhibitor of apoptosis proteins (IAPs), IAP inhibitors such as the SMAC mimetic birinapant are promising new drugs for the treatment of CRC. However, it is currently not possible to stratify for patients who might benefit from adjuvant therapy and those who need new treatments. In 2015, an international consortium unified six independent subtyping approaches and defined 4 distinct, so-called consensus molecular subtypes (CMS). The CMSs are the current gold standard for CRC subtyping and have some prognostic values. Nevertheless, whether the CMS classification can be used to estimate the treatment success is currently under investigation. This study aimed to evaluate the efficacy of treatments with the chemotherapeutics oxaliplatin and 5-fluoruracil (5-FU), TNFalpha, the SMAC-mimetic birinapant and a combination of these drugs. We selected 10 cell lines, representing the four CMSs, and treated them for 24h and 48h with the respective drugs. At both time points, we assessed the cell death by staining with Annexin V/PI followed by flow cytometric analysis of the cells. We were able to show that birinapant has very strong synergistic effects if used in combination with either oxaliplatin/5-FU or TNFalpha, but does not induce apoptosis alone. Importantly, the efficacy of the various co-treatments differed between the cell lines. These differences can, to some extent, be linked to the corresponding CMS of the cells. Cells belonging to the CMS1 subtype showed strong synergistic effects of birinapant and oxaliplatin/5-FU. In contrast, birinapant and TNFalpha co-treatments showed synergistic effects in CMS2 cells, but not in CMS1 cells. Cells belonging to the CMS3 subtype, however, showed a mixed but generally weak response to the co-treatments. Our results highlight birinapant as a promising drug for the treatment of CRC. Our data also suggest that CMSs not only have prognostic values but may also be used as patient stratification tools.

#3908

miR-3132 induces TRAIL and cell death in mutant p53-expressing cancer cells.

Amriti R. Lulla, Margret B. Einarson, Yan Zhou, Avital Lev, David T. Dicker, Wafik S. El-Deiry. _Fox Chase Cancer Ctr., Philadelphia, PA_.

Tumor suppressor p53, guardian of the genome, is frequently mutated or functionally dysregulated in more than 50% of human tumors. p53 mutation is a later event in tumorigenesis and a number of p53 mutants have "Gain of Function" (GOF) properties that have been shown to promote invasive and more aggressive phenotypes in cancer cells. Mutant p53 has been an attractive and promising therapeutic target for advanced stages of tumors. Further understanding the GOF properties of mutant p53 may help with design of novel strategies to target mutant p53 and/or key GOF pathways. We developed a novel functional high throughput screening (HTS) assay to identify miRNAs that selectively target GOF in mutant p53-expressing cell lines. Of the 2754 miRNA mimics screened, we identified 56 miRNAs that selectively reduced cell viability in TP53 R175H (mutant) cells. The current study characterizes the role of our lead mimic, miR-3132 in mutant p53 cells. We found that expression of miR-3132 is lost in cancer cells and that miR-3132 expression is correlated to p53 status, with mutant p53 cells expressing lower levels of miR-3132 than those with wild-type p53. Restoring expression of miR-3132 via mimics induces anti-proliferative and pro-apoptotic effects in a broad panel of colorectal (CRC), breast and lung cancer cell lines. In addition, miR-3132 induces TRAIL-dependent cell death in mutant p53-expressing cells. All cell lines show potent upregulation of cleaved caspases -8, -9 and -3, at 72 hrs post transfection with miR-3132. DNA damaging agents and serum starvation endogenously increase mature miR-3132 levels in wild-type p53 cells. However, this increase is blunted in mutant p53 cells, indicating that p53 could have a role in modulating either the transcriptional or post-transcriptional levels of miR-3132. In summary, we have identified miR-3132 as a TRAIL and cell death-inducing miRNA whose regulation is dependent on p53. Our ongoing work aims at finding the key targets of miR-3132 that explain the cellular response, TRAIL induction and subsequent cell death. Our long-term goal is to develop miR-3132 as a novel single or combinatorial anti-cancer agent for mutant p53 cancer cells.

#3909

HDAC inhibition in combination with MEK or BCL-2 inhibition as novel therapeutic strategies in multiple myeloma.

Kevin C. Miller, Jessica Haug, Teresa Kimlinger, Sanjay Kumar, Wilson Gonsalves, S. Vincent Rajkumar, Shaji K. Kumar, Vijay Ramakrishnan. _Mayo Clinic, Rochester, MN_.

Multiple myeloma (MM) is an incurable malignancy of plasma cells. It is the second most common hematologic cancer, affecting nearly 30,000 people in the United States annually. Substantial progress has been made in the past fifteen years in the treatment of MM due to the approval of several new classes of drugs. However, patients inevitably relapse and become refractory to existing therapies. Hence, there is an immediate unmet need to develop novel therapies for MM based on a better understanding of the disease biology. Mutations in RAS have been found to occur in about 40% of newly diagnosed MM patients, with the frequency increasing to around 70% in relapsed/refractory patients. Such mutations are absent in patients with the premalignant conditions monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). Clearly, RAS mutations contribute to both disease progression and relapse. However, targeting the MEK/ERK pathway has been unsuccessful in MM patients to date. Given the high frequency of RAS mutations in MM, we hypothesized that targeting this pathway could still be a promising strategy when combined with existing agents that have multifaceted mechanisms to promote tumor cell death, such as the recently approved histone deacetylase (HDAC) inhibitor LBH589 (panobinostat). Our results clearly demonstrate that low doses of LBH589 in combination with the MEK inhibitor AZD6244 induce BIM-dependent synergistic cell death in several MM cell lines and patient cells. Our studies also suggest that mutations in RAS/RAF could serve as a predictive biomarker for sensitivity to AZD6244/LBH589. RAS/RAF mutations appear to confer Mcl-1 dependence in MM cells, in part by driving up the phosphorylation of Mcl-1. The AZD6244/LBH589 combination is able to decrease the phosphorylation of Mcl-1 at several sites, which dissociates BIM-Mcl-1 complexes, ultimately leading to activation of the intrinsic apoptosis pathway. Additionally, we identified that wild-type RAS/RAF cells have relatively lower levels of phospho-Mcl-1, as well as higher levels of Bcl-2 and phospho-Bcl-2 when compared to mutated RAS/RAF cells. This seems to confer functional Bcl-2 dependence. Consequently, we found that wild-type RAS/RAF cells are sensitive to the BH3-mimetic ABT199 (venetoclax) when combined with LBH589. Through ongoing experiments, we hope to further confirm the mechanism of action of both these combinations, identify the particular HDAC that is required to be inhibited for the observed synergy, and validate RAS/RAF mutational status as a biomarker for predicting sensitivity to either combination. Our findings have broad therapeutic potential given the prevalence of RAS mutations in MM. Moreover, the ABT199/LBH589 combination could emerge as a targeted therapy for wild-type RAS patients, perhaps broadening the scope and capacity of Bcl-2 inhibition in MM.

#3910

Characterization of BNC101 a human specific monoclonal antibody targeting the GPCR LGR5: First-in-human evidence of target engagement.

Daniel J. Inglis, John Licari, Kristen R. Georgiou, Nicole L. Wittwer, Ross W. Hamilton, Donna M. Beaumont, Michaela A. Scherer, Tina C. Lavranos. _Bionomics Ltd., Thebarton, Australia_.

At the base of normal intestinal crypts, stem cells maintain the highly regenerative gut epithelium. These intestinal stem cells are well characterized for their high expression of the G coupled protein receptor LGR5 (also known as GPR49). Together with R-spondins (potent Wnt signaling modulators) and stem cell growth factors, LGR5 forms part of a signaling cascade responsible for the regulation of cellular proliferation. Key mutations in the APC or BRAF pathways of intestinal stem cells lead to lesions and metastatic colorectal cancer if not diagnosed and resected at an early stage. The majority of primary and metastatic tumors arising from these mutations overexpress LGR5. It has been reported that metastatic colorectal cancer (CRC) patients expressing higher levels of LGR5 in tumor biopsies had increased rates of relapse. BNC101 is a first-in-class anti-LGR5 humanized monoclonal antibody. Biacore analysis of BNC101 has demonstrated a high affinity to LGR5 (Kd=16nM) with no cross-reactivity to LGR4 or LGR6 receptors. Immunoprecipitation studies have also shown that BNC101 has no off-target binding. The murine equivalent of BNC101 has demonstrated antitumor activity in multiple CRC patient-derived xenografts. Flow cytometry studies on CRC cell lines have revealed that LGR5 is located intracellularly with a small fraction present on the cell surface. To better understand its mechanism of action, BNC101 was conjugated to Alexa Fluor 647 and incubated with human CRC cell lines. It was shown that BNC101 interacts with membrane-bound LGR5 and is internalized within 5 minutes. Incubation of the cells for 24 hours at clinically relevant concentrations led to accumulation of fluorophore-conjugated BNC101 within the cell. This intracellular accumulation of BNC101 was further demonstrated with receptor recycling kinetic studies whereby only partial receptor recycling occurred, which may be attributed to the large intracellular LGR5 pool. CHO cell lines overexpressing LGR5 with a GFP tag were used to determine co-localization of BNC101 with its LGR5 ligand. BNC101 is currently in a safety and dose escalation phase I clinical trial in patients with recurrent metastatic CRC. We were able to demonstrate BNC101 target engagement with LGR5 for the first time in tumor biopsies following treatment. Tumor biopsies were analyzed by mass spectrometry together with Matrix Assisted Laser Desorption/Ionization; co-localization of BNC101 with LGR5 was observed, providing us with evidence for the first time that BNC101 infiltrates the patient tumor and engages with the overexpressed LGR5 receptor.

#3911

Targeting the EphA2-ligand binding domain for the design of innovative cancer therapeutics.

Parima Udompholkul, Luca Gambini, Ahmed F. Salem, Carlo Baggio, Maurizio Pellecchia. _University of California, Riverside, Riverside, CA_.

Eph receptors are the largest family of receptor tyrosine kinases, and are involved in regulating several cellular processes, including cell migration, and angiogenesis. Among the Eph family, EphA2 has attracted great interest as it is overexpressed in several tumor types such as melanoma, breast, prostate, lung, colon, and ovarian cancers, and it is also implicated in various aspects of oncogenesis (proliferation, tumor angiogenesis, cell adhesion, migration and metastasis). Thus, EphA2 is a promising direct chemotherapeutic target and several trials are ongoing targeting its kinase domain. In addition, due to its ability to endocytose upon activation, EphA2 could also be used as a carrier of cytotoxic agents, particularly in antibody-drug conjugates (ADCs) or peptidomimetic-drug conjugates (PDCs), using agonistic agents that target its extracellular ligand binding domain (LBD). Hence, potent and selective EphA2-LBD targeting ADCs or PDCs may lead to a decrease in off-target cytotoxicity while maximizing the efficacy of chemotherapeutic agents. Recently, a 12-mer agonistic peptide (YSAYPDSVPMMS, termed YSA in short) targeting EphA2-LBD with modest affinity has been identified by a phage display. YSA binding leads to EphA2 activation and internalization. Accordingly, we developed an innovative anti-triazole linker for the synthesis of EphA2-targeting peptide-drug conjugates to avoid the compatibility problems of disulfide and hydrazine linkers typical of ADCs. Using this strategy, our laboratory initially coupled the YSA peptide with an azide linker-derived taxane (paclitaxel, PTX), generating the desired YSA-PTX conjugate. Additional and more recent studies with a mildly improved EphA2-targeting agent, named 123B9, were also reported by our laboratory and tested in cellular and in vivo models of prostate cancer, pancreatic cancer and melanoma. While these studies clearly suggest that YSA and 123B9 are capable of targeting cytotoxic agents to EphA2-expressing tumor cells, the pharmacodynamic and pharmacokinetic properties of the targeting agents need to be improved to translate these studies into potential new therapies. Our central hypotheses are that by using structure-based design strategies, and leveraging on our previous preliminary data, it should be possible to derive novel YSA or 123B9 derivatives with nanomolar potency against the receptor. In addition, we will show that such agents could be used to prevent cancer cell migration in several solid tumors including breast, prostate, pancreatic and melanomas when used as single agents.

#3912

Towards development of next-generation biparatopic ADCs using a novel linker-toxin with expanded therapeutic window.

Rupert H. Davies,1 Stuart D. Barnscher,2 Peter W. Chan,2 Laurence Madera,2 Jamie R. Rich,2 Marylou Vallejo,2 Grant R. Wickman,2 Kevin Yin,2 Vincent Fung,2 Kevin J. Hamblett,1 Patrick G. Kaminker,1 John S. Babcook2. 1 _Zymeworks Biopharmaceuticals, Seattle, WA;_ 2 _Zymeworks, Vancouver, British Columbia, Canada_.

Antibody drug conjugate (ADC) therapies such as Kadcyla® and Adcetris® have significantly improved outcomes for patients. Despite these early advances, many ADCs have failed due to tolerability and efficacy concerns; therefore, there is a need to develop ADCs with a greater therapeutic window. We have previously reported the increased tolerability of a novel N-acyl sulfonamide auristatin payload conjugated to trastuzumab via a protease cleavable linker. In non-human primates (NHPs), the HNSTD for this ADC was 18 mg/kg compared to 3 mg/kg for the MMAE conjugate control. Separately, we have also reported that a biparatopic antibody targeting a tumor associated antigen (e.g. anti-HER2 bispecific antibody ZW25) can lead to enhanced receptor clustering and improved internalization, thereby increasing the efficiency of payload delivery. Our aim is to develop a series of novel biparatopic ADCs with expanded therapeutic windows against multiple targets. Here we present the proof-of-concept in vitro and in vivo characterization of benchmark ADCs against 3 different targets with improved tolerability and equivalent efficacy. Benchmark antibodies against 3 known clinical targets were conjugated to our N-acyl sulfonamide auristatin (mAb-ADCs) or to MMAE or DM4 controls (mAb-control ADCs) via cleavable linkers and were assessed for in vitro binding affinity and cytotoxicity. The therapeutic windows of mAb-ADCs and mAb-control ADCs were compared by assessing efficacy in mouse xenograft models and tolerability and pharmacokinetics in NHPs. mAb-ADCs had similar binding affinities to recombinant targets and/or to cancer cells expressing low to high levels of target antigen compared to the mAb-control ADCs. mAb-ADCs demonstrated similar in vitro cytotoxicity compared to mAb-control ADCs and this was recapitulated in vivo with similar tumor growth inhibition in mouse xenograft models. In a NHP tolerability/PK study, mAb-ADCs for all 3 targets were tolerated at doses up to 18 mg/kg (single dose IV infusion) compared to the mAb-control ADCs that showed severe to life-threatening neutropenia at lower doses. The increase in maximum tolerated dose for the mAb-ADCs over the mAb-control ADCs, together with comparable efficacy across 3 different targets, demonstrates the broad applicability of the novel N-acyl sulfonamide auristatin payload to expand the therapeutic window. This strategy, together with ongoing efforts to identify synergistic antibody paratopes that more efficiently deliver payload, could lead to next-generation biparatopic ADCs with improved activity.

#3913

Selective targeting of circulating tumor cells with agonistic EphA2 ligand.

Ahmed F. Salem,1 Si Wang,1 Sandrine Billet,2 Jie-Fu Chen,2 Parima Udompholkul,1 Luca Gambini,1 Carlo Baggio,1 Edwin M. Posadas,2 Neil A. Bhowmick,2 Maurizio Pellecchia1. 1 _University of California Riverside, Riverside, CA;_ 2 _Cedars-Sinai Medical Center, Los Angeles, CA_.

EphA2 is a tyrosine kinase receptor that is overexpressed in many cancer types like pancreatic, colon and breast cancers. Targeting cancer cells with an EphA2-targeting molecule conjugated with cytotoxic agents can spare normal cells from chemotherapy side effects. Here, we developed (123B9)2, a novel, potent ligand for the EphA2 receptor that binds to EphA2 ligand-binding domain and causes receptor activation at a sub micromolar concentration as evident in biochemical and cell-based assays. Furthermore, our molecule showed enhanced resistance to degradation, making it suitable for in vivo studies. Subsequently, we conjugated (123B9)2 with paclitaxel, a mitotic spindle inhibitor, and administered it in nude mice bearing breast cancer cells MDA-MD-231 in mammary glands. Remarkably, (123B9)2 paclitaxel-conjugate showed significant inhibition of breast cancer circulating tumor cells (CTC)s in orthotropic mice compared to paclitaxel alone. Finally, we are planning to use our molecule as a single agent to target cancer cells.

#3914

ZW49, a HER2-targeted biparatopic antibody-drug conjugate for the treatment of HER2-expressing cancers.

Kevin J. Hamblett,1 Phil W. Hammond,1 Stuart D. Barnscher,2 Vincent K. Fung,2 Rupert H. Davies,1 Grant R. Wickman,2 Andrea Hernandez,2 Tong Ding,2 Adam S. Galey,2 Geoffrey C. Winters,2 Jamie R. Rich,2 John S. Babcook2. 1 _Zymeworks, Seattle, WA;_ 2 _Zymeworks, Vancouver, British Columbia, Canada_.

Therapies targeting HER2 have transformed the treatment of patients with HER2-expressing breast and gastric cancers. Unfortunately, many patients recur following HER2-targeted treatments and new therapies are needed. Multiple antibody-drug conjugate (ADC) technologies are being explored in this setting, some of which utilize the anti-HER2 antibody trastuzumab. Here we present the preclinical characterization of a new anti-HER2 biparatopic ADC, ZW49, which is generated from the conjugation of a novel N-acyl sulfonamide auristatin payload to the inter-chain disulfide bond cysteines of the bispecific anti-HER2 IgG1 antibody ZW25, via a protease cleavable linker. A series of in vitro and in vivo experiments were performed to characterize ZW49 as a potential therapeutic candidate. In cellular binding assays, it was confirmed that the payload conjugation to ZW25 did not affect the antibody's binding to HER2-expressing cells. ZW49 displayed potent in vitro cytotoxicity in multiple cancer cell lines expressing HER2 and was efficacious in multiple patient-derived xenograft (PDX) models. In mice bearing the HBCx-13b HER2 3+ PDX, two doses of ZW49 administered two weeks apart generated tumor regressions. Furthermore, preliminary results from PDX models with lower levels of HER2 expression treated with ZW49 also generated regressions. In nonhuman primates ZW49 administered intravenously every two weeks for three doses was well tolerated. Based on these findings, we are proceeding with further development of ZW49 as a therapeutic candidate in HER2-expressing cancers.

#3915

Melatonin-tamoxifen hybrid ligands and their effects on breast cancer.

Mahmud Hasan,1 Mohamed Akmal Marzouk,2 Saugat Adhikari,1 Thomas Wright,1 Benton Miller,1 Brianna Peckich,1 Spencer Yingling,1 Robert Stratford,1 Darius Zlotos,2 Jane Cavanaugh,1 Paula Witt-Enderby1. 1 _Duquesne University, Bethel Park, PA;_ 2 _The German University at Cairo, Cairo, Egypt_.

Background: A dual melatonin and tamoxifen hybrid ligand (HL) was developed and shown to display uterine protective actions while co-administration of the same doses of melatonin and tamoxifen unlinked increased uterine weight (US Patent 8785501). The anti-cancer actions of this HL (C5) and 4 others (C2, C4, C9 and C15)—which differed only in their number of carbon linkers—were further tested in 5 phenotypically diverse breast cancer (BC) cell lines—MCF-7 (ER+/PR+), tamoxifen resistant MCF-7 (TamR), MMC (HER2+) and triple negatives (MDA-MB-231 with a raf-1 mutation and BT549 with PTEN mutation) for their effects on cell proliferation, migration, protein expression and binding affinity to melatonin receptors (MT1Rs) and estrogen receptors (ERs). Analysis of HL effects on pERK1/2, pERK5, NF-kB, Runx2, and β1-integrin expression in each BC line and use of the MEK1/2 inhibitor (PD98059) or the MEK5 inhibitor (Bix02189) in the presence of the HLs helped elucidate their mechanism of action. Also, we determined the pharmacokinetic parameters of C4 and C5 HL using both in vitro and in vivo models.

Results: All HLs demonstrated concentration-dependent inhibition of 2-[125I]-melatonin binding to MT1Rs, whereas C5 HL also showed equal binding affinity like melatonin. Only C4 and C5 HLs demonstrated equal affinity as tamoxifen or 4-OH-tamoxifen to ERs, and no concentration dependent inhibition of [125I]-estradiol/[3H]-estradiol binding occurred for C2, C9, and C15 HLs. All HLs were screened for their effects on BC cell proliferation and migration and it was determined that C4 and C5 demonstrated superior potency and efficacy compared to the other HLs (C2, C9, C15) and to co-exposure to melatonin and tamoxifen or 4-OH-tamoxifen (unlinked) for all BC cell lines including TamR MCF-7 cells. Acute (15 min) exposure to C4 and C5 HL increased pERK1/2 activity in MCF-7 cells and C5 HL increased NF-КB protein expression in MCF-7 and MDA-231 cells vs vehicle. Inhibition of MEK1/2 or MEK5 alone inhibited MMC, MDA-231 and BT549 cell migration. The addition of PD98059 enhanced C4-mediated inhibition of MCF-7 and BT549 cell proliferation and enhanced C5-mediated inhibition of MCF-7 cell proliferation and migration and BT549 cell migration. The addition of Bix02189 enhanced C4-mediated inhibition of MMC and BT549 cell proliferation and enhanced C5-mediated inhibition of MCF-7 and BT549 cell migration. Co-administration of Bix02189 and C4 HL blocked MDA-231 cell migration. C4 and C5 HLs displayed similar CYP-mediated loss profiles as tamoxifen in both mouse and human liver microsomes; including, the rate of metabolism being faster in mice vs human. Moreover, C4 HL showed better oral bioavailability than C5 HL in C57BL/6J female mice.

Conclusions: Novel melatonin-tamoxifen HLs linked by 4 or 5 carbons show promise as anti-cancer drugs in phenotypically diverse BCs including triple negative and tamoxifen resistant and may display less uterotropic effects.

#3916

Relationship of guanylyl cyclase C (GCC) expression and efficacy of TAK-164, a GCC-targeted antibody-drug conjugate in a panel of 68 subcutaneous HuPrime colorectal cancer PDX models.

Erik M. Koenig, Cong Li, Huyuan Yang, Andy Zhu, Pooja Shah, Kazuho Nishimura, Bret Bannerman, Mengkun zhang, Bradley Stringer, Brittany Bahamon, O. Petter Veiby, Adnan Abu-Yousif. _Takeda, Cambridge, MA_.

Guanylyl cyclase C (GCC) is a cell surface protein expressed in more than 95% of metastatic colorectal cancers and in the majority of gastric and pancreatic cancers, making GCC an attractive antibody-drug conjugate (ADC) target.

TAK-164 is an ADC comprised of a full-length, fully human IgG1 monoclonal antibody (mAb) directed toward the extracellular domain of GCC. The mAb is conjugated using ImmunoGen's peptide-linked indolinobenzodiazepine DNA alkylator DGN549. Previously presented preclinical studies using TAK-164 have demonstrated in vivo efficacy in a select number of GCC-expressing tumor models following a single administration of TAK-164. Here, we describe the selection of an appropriate dose for broad testing in a panel of patient-derived xenograft models of colorectal cancer. The efficacy data generated following a single administration of 15 µg/kg TAK-164 (payload concentration) were compared with molecular features that could aid in understanding drivers of TAK-164 activity, including GCC expression by immunohistochemistry (IHC).

Primary colorectal cancer models were derived from colorectal adenocarcinomas and implanted in immunocompromised mice. Once tumors reached a stratified mean volume of 150-250 mm3, animals were randomized into treatment groups (n = 1/group) and dosing was initiated on Day 0 of the study. There were three tumor groups (Vehicle treated, TAK-164 treated, and naïve), which were used to characterize genomic and molecular features of the model. The planned study endpoint was 60 days or until a humane endpoint was reached.

For all models included in the study, an IHC assay to detect GCC was used to explore the relationship between GCC expression and time to rise (TTR), a preclinical tumor progression measurement. TTR is the Delta duration in days on study for each PDX model (TAK-164 - Vehicle) to reach 1000 mm3 tumor volume. In the present study, a mixed effects Cox regression analysis, using a continuous or binary stratification of GCC IHC expression, resulted in a statistically significant (p<0.05) increase in TTR in TAK-164treated mice with higher expression levels of GCC.

Ongoing analysis aims to better understand additional molecular drivers of TAK-164 treatment that may be useful for patient enrichment strategies in the clinic. TAK-164 is scheduled to enter phase I evaluation in 2018 in GCC-positive colorectal cancer and other GI malignancies.

#3917

Potentiating effect of reovirus in anti-PD1 therapy in colorectal cancer.

Titto A. Augustine,1 Radhashree Maitra,2 Peter John,1 Sanjay Goel2. 1 _Albert Einstein College of Medicine, Bronx, New York, NY;_ 2 _Montefiore Medical Center, Bronx, New York, NY_.

Background: Microsatellite instability (MSI) high colorectal cancers (CRCs) have a deficiency in mismatch repair (MMR) and increased levels of PD-L1, LAG-3, and IDO, and respond positively to anti-programmed death (PD) therapy. MSI low or microsatellite stable (MSS) CRCs that make up majority of tumors in clinical practice have not seen any benefit with PD inhibition. MSS CRC have higher proportion of KRAS oncogenic mutations as compared to MSI CRC. Reovirus, a naturally occurring oncolytic double-stranded RNA virus, has intrinsic preference for replication in KRAS mutant cells causing apoptosis in CRC. Current study was designed to investigate if reovirus could potentiate a beneficial effect of anti-PD therapy in MSS CRC.

Methods: An array of CRC cell lines were screened for sensitivity to reovirus by MTT assay and expression of stem cell markers by RNA-seq and FACS. Based on MSI and KRAS status, four cell lines were explored further. Cells were treated with 5MOI reovirus for 48hr and expression of PD-L1 and PD-L2 with and without the potentiating effect of IFN-γ was assayed using FACS and qPCR. Combinatorial effect of reovirus with anti-PD-1 agent was studied in syngeneic models of BALB/c (CT26; KRASmut, MSS) and C57BL/6 (MC38; KRASwt, MSI) mice. The mice were grouped as control (PBS/IgG2A isotype control), reovirus, anti-mouse PD-1 antibody, and combination. Reovirus was used at a dose of 10 million/100 uL daily and anti PD-1 antibody was given i.p 200 ug/100 uL twice a week. Survival data and tumor volumes were recorded. HCT116 cells were xenografted in nude mice and treated with equivalent dose of reovirus. At the end point IHC was performed on excised paraffin-fixed tumor tissue with CD8 and granzyme antibodies.

Results: Administration of reovirus was found to be most effective in cell lines tested. HCT116 (MSI & KRASmut), SW620 (MSS, KRASmut), LIM2405 (MSI, KRASwt) and HT29 (MSS, KRASwt) were chosen based on expression of CD133, CD44 and CD24. HCT116, LIM2405 and SW837 revealed increased and HT29 reduced expression of PD-L1 upon treating with reovirus. Survival data and tumor volume measurements showed better potentiating effect of reovirus on anti-PD-1 therapy in CT26 syngeneic model when compared with MC38. While single-agent therapy did not increase survival, the combination did improve survival with significance vs. control in both BALB/c (median 42 vs. 16 days, p=0.003) and C57BL/6 (median 24 vs. 17 days, p=0.02). The reovirus-treated xenografted tumor tissue showed a higher infiltration of T lymphocytes as confirmed by CD8-positive and intensified granzyme staining.

Conclusion: Reovirus as single agent is more potent in KRASmut CRC. Syngeneic mice models proved synergistic anticancer effect of reovirus and anti-PD1 agent combination. Reovirus administration increased PD-L1 expression in CRC cells; possible mechanistic rationale for synergistic efficacy.

#3918

Targeting the CLCF1-CNTFR signaling axis using directed evolution for lung cancer therapy.

Cesar Marquez,1 Jun Kim,1 Amato Giaccia,1 Jennifer Cochran,1 Alejandro Sweet-Cordero2. 1 _Stanford University, Stanford, CA;_ 2 _UCSF, San Francisco, CA_.

Introduction:

While cancer-associated fibroblasts (CAFs) are known to promote and sustain the growth of tumors, the underlying mechanisms remain incompletely understood. Previous work in our lab identified a novel mechanism of communication in which CAFs secrete cardiotrophin-like cytokine factor 1 (CLCF1), a cytokine that binds ciliary neurotrophic factor receptor (CNTFR) on tumor cells and promotes neoplastic growth. CNTFR is a component of the tripartite receptor complex formed by CNTFR-gp130-LIFR and is capable of activating several oncogenic signaling cascades, including Jak-STAT. Here we demonstrate that targeting CNTFR represents a potentially novel strategy for treating non-small cell lung cancer (NSCLC).

Results:

Gene expression analyses reveal that increased CNTFR and CLCF1 levels correlate with poor patient outcomes and that CLCF1 expression is consistently higher in CAFs when compared to normal lung fibroblasts (NLFs) from the same patient. Elevated CNTFR expression was also seen in NSCLC cell lines by western blot, immunohistochemistry, and flow cytometry. Addition of recombinant CLCF1 increased the proliferation of NSCLC cell lines in culture (A549, H23, and H358), while CNTFR knockdown decreased cell proliferation and colony formation in vitro and tumor growth in A549, H23, and H2009 xenografts. CNTFR knockdown also decreased phosphorylation of STAT3 and ERK and decreased expression of several dedifferentiation markers. Therefore, CLCF1-CNTFR signaling may promote tumor growth by activating the Jak-STAT and Ras-Raf-MEK-ERK pathways, and by promoting the dedifferentiation of normal epithelial tissue. Finally, we tested a novel "ligand trap" to bind soluble CLCF1 thereby abrogating the CLCF1-CNTFR signaling axis. This ligand trap was designed using directed protein engineering to increase binding compared to the native CNTFR. In vivo treatment of cell lines and patient-derived xenografts (PDXs) led to decreased tumor growth. Currently we are testing this novel biologic in an autochthonous mouse tumor model. These results identify a pro-oncogenic role for CLCF1-CNTFR signaling in lung cancer and a potentially novel approach to therapy using a ligand decoy.

Perspectives:

We identify for the first time a critical role for CLCF1-CNTFR signaling in NSCLC progression. We validate this role in both a cell culture model and in PDXs, and we also demonstrate novel links between CLCF1-CNTFR and two well established oncogenic pathways. Our findings may shed light on oncogenic mechanisms in other cancer contexts and facilitate the development of novel therapeutic strategies.

#3919

Targeting urease to human VEGFR2 elicits antitumor activity in triple-negative breast cancer models.

Angelika Muchowicz,1 Anna Bujak,1 Beata Pyrzynska,1 Justyna Karolczak,1 Abdessamad Zerrouqi,1 Magdalena Ozga,1 Lukasz Szewczyk,1 Baomin Tian,2 Wah Wong,2 Marni Uger,2 Katarzyna Poplawska,1 Dorota Gierej,1 Pawel Wisniewski,1 Heman Chao,2 Magdalena Winiarska,1 Radoslaw Zagozdzon1. 1 _Helix Immuno-Oncology, Warsaw, Poland;_ 2 _Helix BioPharma Corp., Richmond Hill, Ontario, Canada_.

Introduction: Vascular endothelial growth factor receptor type 2 (VEGFR2) expression is one of the most prominent biomarkers of the tumor-associated neovasculature. Moreover, VEGFR2 can be aberrantly expressed on the surface of tumor cells. We have previously described the development of V21-DOS47, an immunoconjugate composed of the VHH-portion of a camelid single domain anti-VEGFR2 antibody (V21H4) and jack bean urease, which converts endogenous urea into ammonia and hydroxyl ions. V21-DOS47 is the second in a class of antibody-urease drugs. The first, L-DOS47, is currently in clinical testing for non-small cell lung cancer. In this study, we identified tumor cells with VEGFR2 expression, tested V21-DOS47 binding to tumor cells, and investigated in vivo activity in both immunocompetent and immunodeficient mice. Methods: Flow cytometry experiments were performed with biotinylated V21H4 antibody and anti-biotin fluorochrome-conjugated secondary antibody. Detection of urease by flow cytometry was performed with an anti-urease antibody followed by incubation with a fluorochrome-conjugated secondary antibody. Western blotting was used for the assessment of protein expression. For in vivo experiments, VEGFR2-overexpressing derivatives of human MDA-MB-231 and murine 4T1 breast cancer cell lines were generated. Balb/c or nude mice were inoculated with tumor cells: 2.5 x 105 4T1 or 1x106 MDA-MB-231, respectively, on Day 0 of each experiment. Intravenous injections of V21-DOS47 at a dose of 10 µg/kg were started on Day 3 and continued on Days: 5, 7, 9 and 11. Results: After screening human tumor cell lines for VEGFR2 expression by RT-PCR and Western blotting, we determined that two triple-negative breast cancer cell lines (MDA-MB-231 and MDA-MB-468) expressed the highest levels of VEGFR2. Binding of V21H4 to the cell surface of both cell lines was confirmed by flow cytometry. In vivo studies were conducted using both immunodeficient (with human MDA-MB-231-hVEGFR2 cells) and immunocompetent (with murine 4T1-hVEGFR2 cells) mice. In the syngeneic/immunocompetent model of Balb/c mice implanted with 4T1-hVEGFR2 cells, but not wild-type 4T1 cells, the antitumor effect of V21-DOS47 was significant and long-lasting. However, the results obtained from the initial experiment in the MDA-MB-231-hVEGFR2 model suggest that the antitumor effect of V21-DOS47 is transient in immunodeficient mice. Conclusions: Our data show successful targeting of the DOS47 platform to human VEGFR2 expressed on tumor cells. Our in vivo data indicate that the antitumor activity of V21-DOS47 is enhanced in immunocompetent mice, which suggests that the immune system is a significant component of the antitumor activity of the V21-DOS47 immunoconjugate.

#3920

**nfP2X** 7 **mediates survival of cancer cells in the tumor microenvironment.**

Simon Mark Gilbert,1 Christopher John Oliphant,1 Anne-Lise Peille,2 Peter Bronsert,3 Francesco Di Virgilio,4 Shaun McNulty,1 Romain Lara1. 1 _Biosceptre UK, Cambridge, United Kingdom;_ 2 _Charles River Discovery Research Services, Freiburg, Germany;_ 3 _University of Freiburg, Freiburg, Germany;_ 4 _University of Ferrara, Ferrara, Italy_.

The purpose of this study was to demonstrate the effect of the tumor microenvironment (TME) on expression of nfP2X7, a cancer specific form of P2X7 involved in cancer cell survival. P2X7 is an ATP-gated receptor, involved in inflammation, cell death, cell proliferation and cell migration. Alterations in these cellular processes are associated with the development of cancer. P2X7 is characterized by its capacity to open a large molecular weight pore in response to prolonged ATP activation. A mechanism that triggers membrane depolarisation and cell death. The TME contains high concentrations of ATP sufficient to cause opening of the P2X7 pore, while ATP is almost undetectable in healthy tissue. Secretion by cancer cells and release of intracellular ATP from dying cells are thought to be the main source of extracellular ATP in the TME. Non-functional P2X7 (nfP2X7) is a cancer-specific receptor in which formation of the large molecular weight pore, has been abrogated. Acquisition of nfP2X7 enables tumor cells to survive in the presence of high ATP concentrations. Using antibodies raised against nfP2X7, we assessed the expression of the receptor in a panel of cancer types and analysed the effect of the high ATP concentrations on nfP2X7 expression. We further analyzed the effect of siRNA mediated nfP2X7 depletion on cancer cell lines' viability. Our data shows that nfP2X7 is broadly expressed at the surface of many cancer types and supports cancer cell survival. Furthermore, we demonstrate that exposure to a high ATP concentration, equivalent to those in the TME, drives nfP2X7 expression. These data demonstrate that nfP2X7 is a cancer therapeutic target, whose expression is driven by the TME.

### Resistance and Biology

#3921

Inhibition of the ubiquitin proteasome system in cancer by b-AP15.

Arjan Mofers,1 Karthik Selvaraj,1 Ellin-Kristina Hillert,2 Paola Pellegrini,1 Pàdraig D'arcy,1 Stig Linder1. 1 _Linköping University, Linköping, Sweden;_ 2 _Karolinska Institute, Stockholm, Sweden_.

The ubiquitin proteasome system (UPS) is the primary method of the cell for regulation of protein stability and -recycling. Various types of cancer acquire defects in regulation of protein stability, and elevated protein synthesis rates in cancer cells causes accumulation of missfolded proteins - thus driving UPS addiction. These findings make the UPS an attractive target for the treatment of cancer. The 20S proteasome inhibitors bortezomib, carfilzomib and ixazomib have achieved FDA and EMA approval for clinical use for multiple myeloma and other cancers. Various other inhibitors of the UPS are in pre-clinical and clinical testing. We have previously reported that the small molecules b-AP15/VLX1570 inhibit proteasome deubiquitinase (DUB) activity, primarily inhibiting USP14 (D'Arcy et al., Nat Med 2011). Treatment with b-AP15 results in accumulation of ubiquitinated proteins, cell death and tumor growth delay in animal models. Of specific interest was the finding that bortezomib-resistant cells retained sensitivity to b-AP15. We here report that b-AP15 binds weakly and reversibly to a binding pocket in USP14, followed by covalent binding. Interestingly, selection for resistance to b-AP15 results in a modest degree of resistance, with a ~ 2-fold reduction in sensitivity. We found no correlation between b-AP15 resistance and expression of drug transporters while glutathione depletion potently attenuated resistance. We further report that b-AP15 is a cell cycle-active drug. Quiescent cancer cells were found to survive treatment, and when able to re-enter the cell cycle able to form drug-sensitive progeny. Overall these findings indicate that proteasome DUB activity is promising target for cancer therapeutics.

#3922

Ganetespib demonstrates strong anti-tumor effect in acquired EGFR-TKI resistance NSCLC cells.

Eisuke Kurihara, Kazuhiko Shien, Hidejiro Torigoe, Yuta Takahashi, Yusuke Ogoshi, Takahiro Yoshioka, Kei Namba, Hiroki Sato, Hiromasa Yamamoto, Junichi Soh, Shinichi Toyooka. _Okayama University Graduate School Of Medicine, Okayama, Japan_.

Purpose: Epidermal growth factor receptor (EGFR) -tyrosine kinase inhibitors (TKIs) are the standard first-line treatment of advanced non-small cell lung cancer (NCSLC). However, almost all patients eventually acquire resistance to EGFR-TKIs, and novel therapeutic strategies to overcome the acquired resistance have been required. Heat-shock protein 90 (Hsp90) is a chaperon protein involved in folding and stabilization of client proteins essential for cancer cell growth and survival. Ganetespib (STA-9090) is one of the second-generation Hsp90 inhibitors with strong anti-tumor effect on NSCLC cells. In this study, we evaluated the anti-tumor effect of ganetespib in EGFR-TKI sensitive and acquired resistance NSCLC cell lines.

Materials and Methods: We treated 4 EGFR-mutant NSCLC cell lines (HCC827, HCC4006, PC-9 and HCC4011), and 14 experimentally established EGFR-TKIs (gefitinib or afatinib) resistance cell lines with ganetespib. The EGFR-TKI resistance mechanism consisted of EGFR T790M second mutation, MET amplification, epithelial-to-mesenchymal transition (EMT) and cancer stem cell-like features. We determined cell proliferation by MTS assay and calculated the IC50 values. We also performed Western blotting to investigate downstream signaling pathways.

Results: The IC50 values in parental NSCLC cell lines ranged from 1.3nM to 15nM, and those in acquired EGFR-TKI resistant cell lines ranged from 0.87nM to 25nM, which suggests strong anti-tumor effect of ganetespib. In addition, this effect was observed regardless of the resistant mechanisms, including EMT. Ganetespib suppressed EGFR activation in EGFR-TKI resistance cells harboring EGFR T790M second mutation. Also, ganetespib suppressed MET activation in EGFR-TKI resistance cells harboring MET amplification.

Conclusion: Ganetespib showed strong anti-tumor effect in acquired EGFR-TKI resistance NSCLC cells regardless of the resistant mechanisms, suggesting that ganetespib could be a promising therapeutic option in the treatment of NSCLC with acquired EGFR-TKI resistance.

#3923

Insensitivity to c-Met inhibition at physiologic HGF concentrations in a prostate carcinoma model.

Veronica S. Hughes,1 Mumtaz Rojiani,2 Dietmar Siemann1. 1 _Univ. of Florida, Gainesville, FL;_ 2 _Medical College of Georgia-Augusta University, Augusta, GA_.

C-Met is a receptor tyrosine kinase critical for embryogenesis and liver repair. In tumors, including breast, prostate, glioblastoma, and thyroid cancer, Met protein levels are often elevated and associated with disease progression and metastasis. The c-Met pathway is activated by the endogenous ligand Hepatocyte Growth Factor (HGF). HGF is produced by mesenchymal cells and binds to the c-Met protein, leading to phosphorylation and upregulation of a variety of downstream signaling pathways that result in increased tumor cell migration, proliferation, survival, and induction of angiogenesis. C-Met is associated with disease progression and metastasis. Consequently, there has been significant interest in the development of c-Met inhibitors as anti-cancer therapeutics. The efficacy of such agents usually initially are determined in vitro utilizing HGF concentrations of 25-50 ng/mL. However, HGF serum levels in humans typically range from 0.4-0.8 ng/mL. In our studies, we examined the prostate carcinoma cell line DU145, and BMS-777607. Migration and phosphorylation of c-Met increased in a dose-dependent manner with HGF treatment. Additionally, c-Met inhibition with BMS-777607 reduced migration and invasion when in the presence of 25-50 ng/mL HGF. However, at physiologically relevant HGF concentrations the drug had no effect on migration or invasion of this cell line. In vivo models of c-Met inhibition have been limited mainly to mice genetically engineered to express HGF, on the basis that mouse HGF does not stimulate human c-Met. However, in our studies, mouse HGF did stimulate c-Met in the DU145 tumor cell line. Subsequent in vivo evaluation revealed treatment with BMS-777607 had no reduction on DU145 tumor cell-induced angiogenesis in cells pretreated with the drug, nor in mice receiving oral administration of the drug. An experimental metastasis model also resulted in no significant reduction in metastasis formation, as assessed by a tail vein injection model. To characterize HGF concentration in vivo, an ELISA was used to determine an average concentration of 7.7 ng/g of DU145 tumor tissue. Further evaluations of phospho-Met are underway utilizing immunofluorescence of FFPE samples. Preliminary results indicate low levels of phospho-Met in DU145 tumor sections that can be induced by treating tumor-bearing mice with exogenous HGF. Our results suggest that in vitro evaluation of c-Met inhibitors utilizing non-physiological concentrations of HGF may produce an abnormally high phospho-Met that can subsequently be reduced by treatment with c-Met inhibition. In vitro studies performed at high HGF concentrations may not accurately predict the in vivo drug efficacy. Further characterization of HGF concentrations within the tumor is necessary to properly conduct in vitro evaluation of c-Met inhibitors.

#3924

Aurora B, a potential new target in non-T790M lung cancer cells with acquired resistance to anti-EGFR therapy, is effectively blocked by the MET/AXL/FGFR inhibitor S49076.

Jordi Bertran-Alamillo,1 Valérie Cattan,2 Marie Schoumacher,2 Jordi Codony-Servat,1 Frederique Cantero,2 Ruth Roman,1 Sonia Rodriguez,1 Ana Gimenez-Capitan,1 Josep Castellvi,1 Cristina Teixido,1 Rafael Rosell,1 Miguel A. Molina-Vila1. 1 _Quiron Dexeus University Hospital, Barcelona, Spain;_ 2 _Institut de Recherches Internationales Servier, Suresnes, France_.

Background: Non-small cell lung cancer (NSCLC) tumors harboring mutations in the EGF receptor (EGFR) ultimately relapse to therapy with EGFR tyrosine kinase inhibitors (TKIs). S49076 is a potent ATP-competitive tyrosine kinase inhibitor of MET, AXL/MER and FGFR1/2/3 currently in a phase I clinical trial in combination with Gefinitib in NSCLC resistant to EGFR inhibitors.

Methods: We generated 6 resistant lines by treating EGFR-mutated, TKI sensitive PC9 cells with increasing concentrations of gefitinib (GR1-5) or erlotinib (ER). The six lines conserved the exon 19 deletion but the T790M resistance mutation emerged in two of them (GR1, GR4), which remained sensitive to osimertinib. Six additional cell lines resistant to osimertinib were derived from GR1 and GR4 by exposure to increasing concentrations of the inhibitor. The 12 resistant cell lines were genotyped and characterized for AXL, GAS6, MET, FGFR1 and FGFR2 expression by qRT-PCR, immunohistochemistry and Western blotting. The effects of S49076 and the aurora kinase B (AURKB) inhibitor barasertib were analyzed by MTT, flow cytometry and immunocytochemistry. Western blotting and ELISA of key signal transduction proteins and gene silencing was used to gain insight in the mechanism of action of the drug.

Results: Several mechanisms associated with resistance to EGFR TKIs were represented in our panel of resistant cell lines and they frequently co-occurred, including AXL/GAS6 and FGFR1 overexpression, MET activation or emergence and loss of the p.T790M. When using in vitro models, we discovered that the cell lines of our panel were relatively insensitive to single-agent treatment with inhibitors of AXL, MET or FGFR1. In contrast, resistant cells not harboring the p.T790M were sensitive in vitro and also in xenografts to S49076, a drug targeting AXL, MET and FGFR1/2/3. At higher doses (above 300 nM in cultured cells), S49076 blocks AURKB, which could therefore be considered as a further target of the compound. Since partial silencing of AURKB rendered cells resistant to S49076, we investigated the role of this protein. We found that the levels of phospho-histone H3 (pH3), but not Ki67, were increased in all the EGFR TKI resistant cells of our panel, revealing a widespread AURKB activation not related to increased cell proliferation. Treatment with S49076 or barasertib down-regulated pH3 and induced G1/S arrest and polyploidy. In cells harboring the p.T790M, polyploid cells underwent senescence. However, in absence of the resistance mutation, polyploidy was followed by cell death.

Conclusions: AURKB is a novel target in non-T790M NSCLC with acquired resistance to first and third line EGFR TKIs. Multitargeted agents such as S49076, which inhibit not only AXL, MET or FGFR1 but also AURKB, might be more effective in this setting than agents targeting the receptor tyrosine kinases alone.

#3925

Inhibition of oncogenic and drug-resistant PDGFRA and KIT alterations by DCC-2618.

Bryan D. Smith,1 Michael D. Kaufman,1 Anu Gupta,1 Cynthia B. Leary,1 Wei-ping Lu,1 Stacie L. Bulfer,1 Gada Al-Ani,1 Jarnail Singh,1 Subha Vogeti,1 Michael C. Heinrich,2 Daniel L. Flynn1. 1 _Deciphera Pharmaceuticals, Waltham, MA;_ 2 _Portland VA Medical Center and Oregon Health & Science University Knight Cancer Institute, Portland, OR_.

Introduction:

Activating mutations and other genetic alterations in KIT and PDGFRA receptor tyrosine kinases have been identified in certain cancers and proliferative diseases, including most cases of gastrointestinal stromal tumors (GIST) and systemic mastocytosis, and small percentages of gliomas, lung cancer, and leukemias. The treatment of metastatic GIST has been transformed with KIT inhibitors, but heterogeneous drug-resistant mutations arise during therapy, with individual patients often having multiple KIT mutations in different tumor sites. PDGFRA variants in GIST and other cancers also have a significant unmet medical need. DCC-2618 is a kinase switch control inhibitor that potently inhibits the spectrum of exon 9, 11, 13, 14, 17 and 18 mutations in KIT and exons 12, 14 and 18 mutations in PDGFRA. DCC-2618 has been designed to bind as a type II kinase inhibitor that forces the mutant kinases, including strongly activated mutants such as D816V KIT and D842V PDGFRA, into inactive conformations. DCC-2618 has been observed to be potent in enzyme and cell-based assays, and has demonstrated consistent efficacy in xenograft models driven by PDGFRA and KIT alterations.

Methods:

DCC-2618, and an active human metabolite, DP-5439, were tested for inhibition of PDGFRA and KIT mutants using standard enzyme and binding assays, and a variety of cell-based assays. Levels of phosphorylated PDGFRA and KIT were determined by Western blot or ELISA. Proliferation was measured using the fluorescent dye resazurin. An x-ray crystal structure of an analog of DCC-2618 was determined at Emerald Biostructures. The H1703 PDGFRA-amplified lung cancer and GIST T1 mutant KIT xenograft models were performed at MI Bioresearch. A GIST PDX exon 17 mutant KIT xenograft model was run at Molecular Response.

Results:

DCC-2618 and the metabolite DP-5439 inhibited KIT and PDGFRA variants with nanomolar potency. In CHO cells transfected with KIT or PDGFRA variants, DCC-2618 was shown to inhibit the full spectrum of the clinically relevant primary and refractory drug-resistant mutations tested. DCC-2618 also inhibited phosphorylation of KIT or PDGFRA in cell lines with various drug-resistant KIT mutations or PDGFRA alterations. DCC-2618 was compared to the FDA-approved KIT inhibitors imatinib, sunitinib, regorafenib, and midostaurin, as well as other KIT and PDGFRA inhibitors.

In vivo, treatment with DCC-2618 led to tumor regressions in KIT- and PDGFRA-driven xenograft models.

Conclusions:

DCC-2618 has been observed to be a potent inhibitor of KIT and PDGFRA alterations, including mutants, fusions, and amplifications. Based on this profile, DCC-2618 may have utility in the treatment of KIT and PDGFRA-driven cancers including GIST, systemic mastocytosis, and a subset of lung cancers, gliomas, and leukemias. DCC-2618 is currently in a Phase 1 clinical trial in KIT and PDGFRA driven cancers (ClinicalTrials.gov Identifier: NCT02571036).

#3926

Discovering a novel microtubule inhibitor overcoming tumor multidrug resistance.

Nannan Ning,1 Chengyong Wu,2 Min Wu,1 Yamei Yu,2 Changjun Zhu,3 Cyril Benes,4 Jianming Zhang,1 Xianming Deng,5 Qiang Chen,2 Ruibao Ren1. 1 _Ruijin Hospital affiliated to the Shanghai Jiaotong University School of Medicine, Shanghai, China;_ 2 _West China Hospital, Sichuan University, Chengdu, China;_ 3 _Tianjin Normal University, Tianjin, China;_ 4 _Massachusetts General Hospital, Charlestown, MA;_ 5 _Xiamen University, Xiamen, China_.

Microtubule inhibitors, as chemotherapeutic drugs, are widely used for cancer treatment in clinic. However, the development of multidrug resistance (MDR) in cancer is a major challenge for microtubule inhibitors in its clinical implementation. From a high throughput drug screening using cells transformed by oncogenic RAS, we identified a small molecule AR8-1006 that blocks cell proliferation. The analysis of structure-activity relationship indicated that this serial of scaffold represents a potent inhibitor of tumor cell growth. AR8-1006 shows activities against a larger panel of more than 1000 human cancer cell lines with a wide variety of tissue origins. Further analysis showed that this compound depolymerizes microtubules and affects formation of the spindle. Interestingly, it induces the spike-like conformation of microtubules in vitro and in vivo, which is different from typical microtubule modulators. Structural analysis revealed that this serial of compounds binds to the colchicine pocket at the intra-dimer interface, though mostly not overlapping with colchicine binding. More importantly, AR8-1006 displays favorable pharmacological properties on overcoming tumor MDR both in vitro and in vivo. Taken together, our data reveal a novel scaffold represented by AR8-1006 that could be developed as cancer therapeutics, especially for tumor multidrug resistance.

#3927

Clinicopathologic features and immune microenvironment of non-small cell lung cancer with primary resistance to epidermal growth factor receptor-tyrosine kinase inhibitors.

Yuta Takashima,1 Jun Sakakibara-Konishi,1 Yutaka Hatanaka,2 Kanako C. Hatanaka,2 Yoshihito Ohhara,3 Satoshi Oizumi,4 Yasuhiro Hida,5 Kichizo Kaga,5 Ichiro Kinoshita,3 Hirotoshi Dosaka-Akita,3 Yoshihiro Matsuno,2 Masaharu Nishimura1. 1 _First Department of Medicine, Hokkaido University School of Medicine, Sapporo, Japan;_ 2 _Department of Surgical Pathology, Hokkaido University Hospital, Sapporo, Japan;_ 3 _Department of Medical Oncology, Hokkaido University Graduate School of Medicine, Sapporo, Japan;_ 4 _Department of Respiratory Medicine, National Hospital Organization Hokkaido Cancer Center, Sapporo, Japan;_ 5 _Department of Cardiovascular and Thoracic Surgery, Hokkaido University Graduate School of Medicine, Sapporo, Japan_.

Background: Approximately 20-30% of non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) activating mutations are not responsive to EGFR-tyrosine kinase inhibitors (TKIs). Although, primary resistance to EGFR-TKI is attributed to various genetic alterations, little is known about the clinical and immunopathological features of patients with primary resistance. The tumor immune microenvironment including tumor infiltrating lymphocytes (TILs) and programmed death ligand 1 (PD-L1) has been reported to play an important role in tumor progression in NSCLC. However, few studies have directly focused on the relationship between the tumor immune microenvironment and primary resistance to EGFR-TKI.

Materials and Methods: Characteristics of 124 NSCLC patients with EGFR mutations who received EGFR-TKI were analyzed. Primary resistance was defined as disease progression within 3 months after EGFR-TKI treatment. Tumor specimens obtained before EGFR-TKI treatment were assessed for the density of TILs expressing CD4 or CD8, and for the expression rate of PD-L1 on tumor cells and tumor-infiltrating immune cells, immunohistochemically.

Results: Primary resistance was observed in 13.7% (17/124) of patients. Smoking Tobacco correlated significantly with primary resistance compared to non-primary resistance. Lower density of total TILs and negative PD-L1 expression as per immunohistochemical analysis correlated significantly with primary resistance. Moreover, immune ignorant phenotype of tumor microenvironment, negative PD-L1 expression with low TIL density, was significantly observed in primary resistance.

Conclusions: Smoking and immune ignorance in the tumor microenvironment might result in primary resistance to EGFR-TKIs.

#3928

Sertraline in melanoma treatment: TCTP as a therapeutic target.

Marianna Boia-Ferreira,1 Alana Basílio,1 Antonielle B. Baldissera,1 Fernando H. Matsubara,1 Marcia H. Appel,2 Cleber R. da Costa,1 Olga M. Chaim,1 Luiza H. Gremski,1 Silvio S. Veiga,1 Andrea Senff-Ribeiro1. 1 _Federal Univ. of Paraná, Curitiba, Paraná, Brazil;_ 2 _State University of Ponta Grossa, Ponta Grossa, Paraná, Brazil_.

Background: Sertraline (an SSRI antidepressant) binds directly to TCTP protein and decreases its intracellular levels. TCTP (Translationally Controlled Tumor Protein) is an antiapoptotic protein highly conserved through phylogeny. TCTP overexpression was detected in several tumor types. Silencing TCTP was shown to induce tumor reversion, a process overriding at the molecular level the malignant process. There is a reciprocal repression between TCTP and P53.

Methods: We evaluated the role of TCTP in melanoma using sertraline and siRNA. Cell viability, migration and clonogenicity were assessed in human and murine melanoma cell in vitro. Sertraline was evaluated in vivo in a murine melanoma model and compared to dacarbazine, a major a chemotherapeutic agent used in melanoma treatment. Long-term effect of sertraline was evaluated keeping cells on regular culture for 5 days after a 72h-treatment and then assessing proliferation, clonogenicity and in vivo growth.

Results: (i) In human melanoma cell lines, sertraline treatment and decrease of TCTP levels (by siRNA) are related to the inhibition of clonogenicity and migration; (ii) TCTP levels are related to melanoma malignancy and invasiveness (in B16F10/F1 murine model); (iii) knockdown of TCTP triggers decrease of proliferation and migration on murine melanoma cells; (iv) sertraline decreases TCTP mRNA levels in B16F10 cells and results in inhibition of clonogenicity; (v) sertraline presents remarkable in vivo antitumoral effects in B16F10/C57BL6 model, greater than dacarbazine, the main chemotherapeutic agent used for melanoma; (vi) sertraline treatment is capable of a long-term effect on melanoma cells, suggesting that the drug can trigger tumor reversion, a reprogram of tumor cells to a less malignant phenotype. Our data could pave the way for new approaches in the treatment of melanoma, a pathology whose prognostic is poor in late stages, and the efficiency of the current available chemotherapeutic treatment is very low.

Conclusion: Altogether, these results indicate that sertraline could be effective against melanoma and TCTP can be a target for melanoma treatment, opening up new therapies for melanoma treatment.

#3929

The FAD-directed LSD1 specific inhibitor, INCB059872, inhibits cell migration and metastasis by suppressing premetastatic niche formation in a spontaneous metastasis mouse model.

Sang Hyun Lee, Melody Diamond, Antony Chadderton, Huiqing Liu, Alla Volgina, Valerie Roman, Michael Weber, Chunhong He, Rebecca Stewart, Denise Hertel, Phillip Liu, Liangxing Wu, Julian Oliver, Swamy Yeleswaram, Alan Roberts, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Bruce Ruggeri. _Incyte Corporation, Wilmington, DE_.

The primary cause of cancer associated mortality is tumor metastasis. The concept of the tumor pre-metastatic niche is supported by evidence of changes at distal pre-metastatic sites that create a permissive environment to allow disseminated tumor cells to seed. Myeloid-derived suppressor cells (MDSCs) remodel the tumor microenvironment and function as immunosuppressive cells to promote tumor growth. Previously, we demonstrated that the clinical stage LSD1 specific inhibitor, INCB059872 significantly reshaped the myeloid compartment in the murine 4T1 syngeneic murine model of breast cancer. Treatment with INCB059872 significantly reduced the population of MDSCs in the tumor microenvironment. Since it has been reported that MDSCs promote establishment of a pre-metastatic niche, we hypothesized that INCB059872 could suppress or delay metastatic processes in the 4T1 model and thereby could impact spontaneous metastases to the lung. In vitro, INCB059872 significantly suppressed cancer cell migration of triple negative breast cancer cells, SUM145PT. In vivo, the effect of INCB059872 on forming the metastatic niche using the 4T1 mouse breast tumor model was explored. Vehicle treated animals exhibited a significant infiltration of MDSCs to the primary tumor and lungs prior to cancer cells metastasizing. In contrast, INCB059872 administration significantly suppressed the infiltration of MDSCs in primary tumor and lung tissues. Histological analyses further demonstrated the reduction of metastatic loci in lung with INCB059872 treatment. Plasma levels of CCL2, a cytokine which is required for the recruitment and functional specialization of MDSCs, were significantly reduced in animals treated with INCB059872. These data suggest a possible mechanism to reduce infiltration of MDSCs into lung tissues. Notably, analyses of molecular pathways using RNA-Seq identified that components of the EMT associated pathway are also downregulated in tumors treated with INCB059872, which further supports the role of INCB059872 in the inhibition of metastasis. Taken together, these preclinical data suggest that inhibition of LSD1 with INCB059872 can suppress metastasis through multiple molecular and cellular mechanisms, notably by inhibition of the formation of the pre-metastatic niche by modulating the population of MDSCs in the primary tumor and distal tissues.

#3930

Chemoprevention of metastatic colon cancer by a novel small molecule.

Deeksha Pal, Balaji Chandrasekaran, Ashish Tyagi, Becca Von Baby, Houda Alatassi, Murali Ankem, Chendil Damodaran. _Univ. of Louisville, Louisville, KY_.

Introduction: Colon cancer is a one of the leading cause of death in both men and women. Metastatic colon cancer is responsible for mortality due to resistance to conventional therapies. Our group has previously reported that activation of AKT and Notch1 plays an important role in metastasis of colon cancer cells. As Notch1 and AKT regulates cell proliferation and are upstream of epithelial to mesenchymal transition (EMT) cascade, it seems interesting to explore their inhibition for pharmacological intervention. In the present study we identified a sesquiterpene molecule, Verrucarin J (VJ), that inhibited Notch1 expression and downregulated AKT mediated EMT transition in colon cancer cells.

Methods: The anticancer effect of VJ was assessed on both colon cancer cells and stably AKT overexpressing colon cells by cell proliferation, apoptosis and Western blot analysis. For xenograft studies, pCMV/HCT-116 or AKT/HCT-116 cells (1.5 × 106) in a 50-μl final volume of phosphate-buffered saline (PBS) were injected subcutaneously into separate flanks of the mice. Also effects of VJ were determined in mouse model of colorectal cancer i.e. APC min+/_ mice. Statistical analysis was done by unpaired Student's t-test and one way ANOVA,*p≤0.05, **p≤0.01, ***p≤0.001.

Results: Molecular analysis of VJ treatment on colon cancer cells revealed that it inhibited colon cancer growth by down regulating AKT and Notch1 signaling. Western blot analysis revealed that VJ inhibits AKT/NF-κβ/ Bcl2- signaling axis in colon cancer cells. VJ also Inhibited migration and invasion of colon cancer cells that corresponds with downregulation of mesenchymal marker expression. In addition, VJ treatment induces apoptosis in colon cancer cells as well as AKT overexpressing cells. Intraperitoneal administration of VJ in pCMV/HCT116 and AKT/HCT-116 xenograft mice revealed decrease in tumor volume in comparison with control mice. We also examined the in vivo efficacy of VJ in ApcMin/+ mouse model. Treatment of ApcMin/+ with VJ (0.5 mg/kg/IP/twice a week) over 12 weeks significantly reduced the number of intestinal polyps (distal 84%; Middle 63% and Proximal 2%) as compared vehicle treated mice. In addition, we also observed 50% reduction in number of colon tumors in VJ treated mice.

Conclusion: In conclusion, our studies suggest that VJ inhibits both AKT and Notch1 signaling and induces apoptosis in colon cancer cells. In vivo studies revealed significant reduction in tumor formation in mice models of colon cancer. Hence VJ could be a viable therapeutic agent for treating patients with metastatic colon cancer.

#3931

Neratinib-induced gene expression profile in breast cancer cells: A comprehensive transcriptome investigation.

Yuxin Cui,1 Alwyn Dart,1 Richard E. Cutler,2 Alshad S. Lalani,3 Francesca Avogadri-Connors,3 Richard P. Bryce,3 Sioned Owen,1 Wen G. Jiang1. 1 _Cardiff University, Cardiff, United Kingdom;_ 2 _2Puma Biotechnology, Inc., Los Angeles, CA;_ 3 _Puma Biotechnology, Inc., Los Angeles, CA_.

Breast cancer can be classified into complicated malignant subtypes based on advances of clinical and research evidence. There are still unmet needs for the more effective treatment of breast cancer patients under specific pathological conditions (stages/subtypes). Neratinib is considered as a second-generation inhibitor of the EGFR family members (EGFR, HER2 and HER4) of receptor kinases. It can irreversibly inhibit the EGFR and HER2 tyrosine kinases by targeting a cysteine residue in the ATP-binding site of the receptor. Recently neratinib has been approved by FDA for the extended adjuvant treatment of early-stage HER2+ breast cancer. In this study, to further dissect the molecular mechanisms of neratinib, the global gene expression profiling analysis of breast cancer cells treated with neratinib was performed using RNA AmpliSeq transcriptome (including 20816 gene candidates) in the Ion PI sequencing system. We selected two breast cancer cell lines, the triple negative MDA-MB-231 cell line and the low HER2-expressing, but not-amplified, ER+ MCF-7 cell line. The RNA-Seq data show differential gene profiling heatmap from these two breast cancer cell lines. The RNA-Seq data confirmed the downregulation of HER2 in MCF-7 cells, but there was no change of gene expression of EGFR, HER3 and HER4. In both MDA—MB-231 and MCF-7 cell lines, there was a panel of upregulating genes including KIAA1024, ZNF550, MESDC1, TMC8, ZNF524, AGBL2, HIST2H2BC and PPARGC1B. In contrast, with response to neratinib, another cluster of genes was downregulated in both cell lines including TAS2R5, KRT14, VWCE, LY9, PAPLN and STK4-AS1. In MDA-MB-231 cells, neratinib intended to regulate LPS/IL-1 and eNOS mediated signaling pathways. However, in MCF-7 cells, neratinib regulated ephrin receptor (EPH) signalling and PPARα activation. We then investigated the possible HER2-independent effect of neratinib in breast cancer cells such as regulation of EPH. Our data using the electric cell-substrate impedance sensing (ECIS) indicated that MCF-7 cells responded to an EphB4 inhibitor, NVP-HPG-712, by a marked reduction of cellular migration, in contrast to MDA MB-231 which does not respond to the inhibitor. Similarly, MCF-7 cells also responded to the combinational treatment of neratinib and NVP-HPG-712 with a decrease in cellular migration. Again, MDA-MB-231 failed to yield a response. In summary, differential analysis of transcripts by high throughput RNA-seq could be valuable to provide deeper insight into the gene regulation and signaling pathway prediction of breast cancer cells with response to neratinib when the large scale of patient samples and multiple types of breast cancer cell lines are integrated.

#3932

Metabolic biomarkers predict the sensitivity to phosphatidylinositol 3-kinase pathway inhibitor in small-cell lung carcinoma.

Shigeki Umemura,1 Hideki Makinoshima,2 Genichiro Ishii,3 Takehiko Sasaki,4 Hiroyasu Esumi,5 Koichi Goto,1 Katsuya Tsuchihara3. 1 _National Cancer Center Hospital East, Kashiwa, Japan;_ 2 _National Cancer Center, Tsuruoka, Japan;_ 3 _National Cancer Center, Kashiwa, Japan;_ 4 _Akita University, Akita, Japan;_ 5 _Tokyo University of Science, Noda, Japan_.

Comprehensive genomic analysis has revealed that the PI3K/AKT/mTOR pathway is a feasible therapeutic target in small-cell lung carcinoma (SCLC). However, biomarkers to identify patients likely to benefit from inhibitors of this pathway have not been identified. Here we show that metabolic features determine sensitivity to the PI3K/mTOR dual inhibitor gedatolisib in SCLC cells. We analyzed the phosphatidyl lipid profile including the acyl groups of 11 SCLC cell lines using electrospray ionization mass spectrometry. We found that a specific phosphatidylinositol (3,4,5)-triphosphate (PIP3) subspecies lipid product, PIP3 (38:4), is predictive in assessing sensitivity to gedatolisib. We generated a large-scale metabolomic dataset for 28 SCLC cell lines. Notably, we found that higher amounts of purine-related aqueous metabolites such as hypoxanthine led to resistance to PI3K pathway inhibition. The levels of the mRNA encoding hypoxanthine phosphoribosyl transferase 1 (HPRT1), a key component of the purine salvage pathway, differed significantly between SCLC cells sensitive or resistant to gedatolisib. Loss of HPRT1 in SCLC cells decreased cellular proliferation in the presence of gedatolisib compared with a control. Further, complementation with purine metabolites could reverse the vulnerability to targeting of the PI3K pathway in SCLC cells normally sensitive to gedatolisib. These results indicate that the mechanism of resistance to PI3K pathway inhibitors is mediated by activation of the purine salvage pathway, which supplies purine resources for nucleotide biosynthesis. Metabolic profiles of clinical samples of SCLC revealed that purine-related metabolites were higher in SCLC tumor tissues, suggesting that higher amounts of purine-related aqueous metabolites might be characteristic of SCLC biology. Metabolomics is a powerful approach for identifying novel therapeutic biomarkers applicable to SCLC treatment.

#3933

Circadian timing regimen for alpelisib (NVP-BYL719), a selective inhibitor of the class Ia PI3K isoform alpha to maximize therapeutic index.

Christian R. Schnell, Thomas Ferrat, Daniel Wyss, Walter Tinetto, Sonja Tobler, Christine Fritsch, Michael Jensen. _Novartis Institutes for Biomedical Research, Basel, Switzerland_.

The gene encoding for the catalytic subunit of the phosphatidylinositol-3-kinase (PI3K), p110a (PIK3CA) is the most frequently mutated kinase in cancer. This discovery triggered the development of small molecule anti-PI3K inhibitors, such as NVP-BYL719. However, the PI3K / Akt signaling pathway plays not only an important role in promoting cell growth, proliferation and survival but also in regulating glucose homeostasis by directly mediating insulin-stimulated glucose uptake into insulin sensitive tissues (adipocytes and muscles). NVP-BYL719 (a selective inhibitor of the class Ia PI3K isoform alpha) given in the morning is facing on-target tolerability challenge (hyperglycemia) in clinical trials limiting the dose being administered to patient (1). As glucose metabolism is highly impacted by circadian rhythms in patients and rodents, we have decided to adopt an integrative circadian-timing approach in our pre-clinical models to interrogate the benefit of morning vs evening dosing for BYL719. By using a newly developed radio-telemetry technology (2), we were able to record in real time and 'around-the-clock' blood glucose levels in stress-free, freely moving rats dosed with NVP-BYL719 at different regimens. The dynamic profile of hyperglycemia observed after active or inactive period dosing of NVP-BYL719 (50 mg/kg qd p.o.) were similar. Dosing before the inactive phase (10 a.m.) allowed blood glucose to normalize in between 2 doses, which could not be achieved when dosing before the active phase (5 p.m.). After treatment discontinuation a significant hyperglycemia remained for a period up to 12h in the group dosed before the active phase (5 p.m.). Circadian "evening" NVP-BYL719 dosing is associated with a better control of glycaemia. Clinically this could potentially translate to better compliance and longer time on treatment hence better efficacy for patient. Based on these findings we could recommend optimized treatment schedules for future combination experiments in the clinic with NVP-BYL719. 1) Juric et al, "Phase I study of the PI3Kα Inhibitor BYL719, as a Single Agent in Patients with Advanced Solid Tumors (AST)", Annals of Oncology (2014), 25 (Supp. 4) 2) Brockway et al, "Fully Implantable Arterial Blood Glucose Device for Metabolic Research Applications in Rats for Two Months", J Diabetes Sci Technol (2015), 9(4):771-81

#3934

Determination of the PI3Kα selective inhibitor alpelisib mechanism of action and efficacy in ER+/ PIK3CA mutant breast cancer preclinical models.

Christine Fritsch, Estelle Pfister, Nicolas Ebel, Daniel Guthy, Christian Schnell, Francesco Hofmann. _Novartis AG, BASEL, Switzerland_.

Over the past years, a key focus has been on identifying inhibitors against components of pathways that drive tumor cell proliferation, survival, and metastasis such as the PI3K/mTOR pathway, known to be implicated in many human cancers through various mechanisms, such as, somatic PIK3CA missense mutations that occur at high frequency in a number of common solid tumors including ~40% of ER+ breast cancer patients. Based on these findings, cancer-specific mutants of PI3Kα appear to be ideal targets for PI3Kα selective inhibitors such as alpelisib which is unique in its relative selectivity for the α-isoform and previously reported to be efficacious in PIK3CA mutant cancer models. Recently, using the ER-negative/HER2-positive/PIK3CA mutant HCC1954 breast cancer cell line, the PI3K β-sparing inhibitor taselisib has been described to have a dual mechanism of action, both blocking PI3K signaling and inducing a decrease in p110α protein levels. Here, we investigated whether such dual mechanism of action could be associated with PI3Kα selective inhibition in ER+/PIK3CA mutant breast cancer pre-clinical models specifically. In vitro, using the T47D ER+/PIK3CA mutant breast cancer cell line, alpelisib displays dual MoA by inhibiting p-Akt and inducing a decrease of p110a protein levels in a dose-dependent manner. This effect is observed upon 24 hours of compound treatment and is maintained for at least 96 hours upon treatment. Interestingly, for both alpelisib and taselisib, p110α degradation becomes more pronounced at concentrations which produce strong PI3K pathway inhibition (>80% p-Akt inhibition). In contrast to T47D cells, we do not observe p110α degradation upon 24 hours treatment in 4 additional ER+/PIK3CA mutant breast cancer cell lines tested, despite a dose-dependent and robust inhibition of the PI3K pathway, suggesting the effect might be specific to certain cell lines or restricted to certain PIK3CA mutations or might require longer compound treatment periods. In the EFM19 cells, the PI3K inhibition-mediated p110α degradation occurs upon minimum 48 hours treatment for both alpelisib and taselisib. In vitro, when alpelisib and taselisib are then compared at concentrations that equally inhibit the PI3K pathway and equally degrade p110ɑ, both compounds display similar inhibitory effects on T47D and EFM19 cell proliferation and survival. In vivo, in an ER+/ PIK3CA mutant breast cancer patient derived xenograft (PTX) model, alpelisib and taselisib show comparable anti-tumoral efficacy when administered at doses that are equally well tolerated and induced comparable PI3K pathway modulation. Overall, the data demonstrate that in ER+/ PIK3CA mutant breast cancer pre-clinical models, selective PI3Kα inhibition can induce a dual mechanism of action and produces robust efficacy.

#3935

Differentiation of multi-kinase inhibitors by in vivo transcript profiling: Potential implications for HCC therapy.

Heidrun Ellinger-Ziegelbauer,1 Lukas Fiebig,1 Ludwig Schladt,1 Karl Ziegelbauer,2 Anette Sommer,2 Dieter Zopf2. 1 _Bayer AG, Wuppertal, Germany;_ 2 _Bayer AG, Berlin, Germany_.

Based on an improvement in overall survival (OS) the multikinase inhibitor (MKI) sorafenib is currently approved for front-line therapy of unresectable hepatocellular carcinoma (HCC). Brivanib another MKI failed to improve OS compared to sorafenib (Johnson et al, 2013). In order to identify factors which discriminate sorafenib from other MKIs beyond their different kinase profiles, we initiated an exploratory study to compare the effects of sorafenib with brivanib on the liver of healthy rats in vivo. Since the liver exhibits substantial metabolic and regenerative abilities, measureable molecular effects of MKIs were thought to become apparent using transcript profiling. Six male Wistar rats each were treated once daily with either sorafenib or brivanib at doses corresponding to respective clinically relevant doses, or corresponding vehicles. Liver samples were collected three hours after two and 15 treatments for transcript profiling on Rat ClariomD microarrays. Data were analyzed and statistically evaluated using Genedata ProfilerTM software. Genes with a minimum 1.5-fold change vs control were considered relevant. In addition, drug pharmacokinetics was analyzed from plasma after nine treatments. Clinical pathology data indicated minor effects on liver function for both compounds, consistent with previous observations for sorafenib. Mild histological changes in liver, pancreas and spleen were observed for brivanib. Sorafenib exposure was approximately 5-times lower than expected from former rat studies. Changes in gene expression were detectable in livers from treated vs. control animals supporting our hypothesis. The number of deregulated genes and the extent of deregulation were less pronounced for sorafenib probably attributed to its lower exposure. Transcript profile changes by both compounds were measurable early after two treatments and became stronger after 15 treatments. Both drugs induced changes in the expression of genes involved in vascular functions, consistent with their inhibition of VEGFR kinase, interferon / interleukin signaling, and of genes affecting metabolic pathways. Sorafenib, despite its lower exposure, selectively altered expression of genes involved in cell cycle progression and genes potentially promoting differentiation, such as vestigial-like family member 4. Vgll4 has been described as tumor suppressor by negatively regulating the formation of the YAP-TEAD transcriptional complex. In summary, the use of transcript profiling of liver tissue from healthy rats enabled us to identify genes whose expression is differentially regulated by sorafenib and brivanib and which have reasonable functional relevance. Further studies including more sophisticated models will be performed to confirm and expand these results.

#3936

Bromodomain testis-specific protein BRDT is expressed in a subset of esophageal squamous cell carcinomas and controls expression of differentiation-associated genes.

Xin Wang, Feda H. Hamdan, Madhobi Sen, Ana P. Kutschat, Steven A. Johnsen. _University Medical Center Göttingen, Göttingen, Germany_.

Esophageal cancer is one of the most malignant cancers, ranking as the sixth leading cause of cancer-related deaths worldwide. The poor survival rate and prognosis highlight the limitations in the biological understanding of esophageal cancer and the urgent need for identification of novel targeted molecular therapies. Recently, large scale genomic analyses have revealed the extensive alternations of epigenetic regulators which may be used as a basis for developing new "epigenetic drugs ". BRDT, bromodomain testis-specific protein, is a member of the bromodomain and extra-terminal (BET) family of epigenetic reader proteins. BET proteins can regulate gene expression by recognizing acetylated lysines, thus playing important roles in both normal development and disease progression. Inhibition of BET proteins has emerged as a potential therapy for many types of cancer. In normal human tissues, unlike the other members of BET family, BRDT is exclusively highly expressed in testes where it drives the meiotic and post-meiotic gene expression to promote spermatogenesis. We have identified BRDT to be expressed in over 20% of esophageal squamous cell carcinoma (ESCC), a predominant subtype of esophageal cancer. Knockdown of BRDT does not affect cell proliferation, but leads to alterations in the expression of differentiation markers. In addition, RNA-seq following BRDT knockdown also supports a role of BRDT in cell differentiation. Depletion of BRDT does not alter the cellular response to BET inhibition. Surprisingly, we also identified BRDT transcripts encoding two truncated isoforms lacking the first bromodomain, which could potentially alter its epigenetic reader function. The genome occupancy profile of BRDT showed a significant enrichment at transcription start site (TSS), which is distinct from BRD4. It also showed potential interactions between BRDT and E2F family and SMAD family. Together, we showed that BRDT is overexpressed in ESCC cell lines and may influence ESCC development or progression in a subset of tumors by regulating cell differentiation-associated genes.

#3937

Differential alteration of IL-8 in liver cancer stem cell enrichment in response to PI3K/Akt/mTOR inhibitors and sorafenib.

Deniz C. Kahraman,1 Tamer Kahraman,1 Rengul Cetin Atalay2. 1 _Bilkent University, Ankara, Turkey;_ 2 _Middle East Technical Univ., Ankara, Turkey_.

Liver cancer stem cells (LCSCs) are derived from damaged and transformed hepatic progenitor cells (HPCs) during precancerous cirrhosis stage. Ras/Raf/MAPK and PI3K/AKT/mTOR signaling pathways are significantly deregulated in liver cancer. The activation of PI3K/AKT/mTOR pathway in LCSC population is one of the reasons for acquired resistance to sorafenib in advanced hepatocellular carcinoma (HCC) patients. Therefore, identifying novel inhibitors targeting this pathway acting on LCSCs is highly essential. We, therefore, elucidated the bioactivities of small-molecule kinase inhibitors on LCSCs acting through PI3K/Akt/mTOR pathway in comparison with DAPT (CSC inhibitor), DNA intercalators and sorafenib. For this purpose, CD133+/EpCAM+ cells originated from HCC cells were analyzed by flow cytometry and effective inhibitors on LCSCs were further tested for their potential combinatorial effects. Treatment of cells with sorafenib and DNA intercalators resulted in enrichment of CD133+/EpCAM+ cells. Yet, rapamycin, LY294002 and DAPT significantly reduced CD133/EpCAM positivity. Combination studies revealed that sequential treatment strategy decreased the ratio of LCSCs as opposed to sorafenib treatment alone. The effects of the inhibitors were also demonstrated with LCSC sphere formation. Additionally, a large panel of genes involved in cancer pathways was analyzed using Nanostring® nCounter® Technology to identify the differentially expressed genes in rapamycin, sorafenib or DAPT treated cells. Systematic pathway analysis using Cytoscape ScoreFlow algorithm application allowed us to identify differential response genes involved in stemness. While the stemness genes were downregulated in cells treated with rapamycin or DAPT, JAG1 gene was found to be upregulated in sorafenib-treated cells. Interleukin 8 (IL-8) was downregulated upon treatment with DAPT, yet upregulated upon sorafenib treatment. Following IL-8 inhibition with repaxirin, CD133/EpCAM positivity of cells decreased significantly, indicating that IL-8 signaling is crucial for the conservation of stemness features of cancer cells. The combination of sorafenib and reparixin resulted in lower levels of SOX2 and Nanog proteins. Conclusion: PI3K/Akt/mTOR pathway inhibitors alter hepatic CSC composition and gene expression in favor or to the detriment of cancer stem cell survival. Blockade of IL-8 signaling provides a promising therapeutic approach for prevention of LCSC enrichment.

#3938

The alkylphosphocholine erufosine induces ER and mitochondrial stress in OSCC cells.

Shariq S. Ansari,1 Ashwini Sharma,1 Doaa M. Ali,1 Rainer Köng,2 Martin R. Berger1. 1 _German Cancer Research Ctr., Heidelberg, Germany;_ 2 _Jena University Hospital, Jena, Germany_.

Endoplasmic reticulum (ER) plays an essential role in cell function and survival. Accumulation of unfolded or misfolded proteins in the lumen of the ER activates the unfolded protein response (UPR), resulting in ER-stress and subsequent apoptosis. The alkylphosphocholine erufosine is a known Akt-mTOR inhibitor in oral squamous cell carcinoma (OSCC). In the present study we decipher a new facet of erufosine's mechanism of action and evaluate its role to induce ER and mitochondrial stress leading to autophagy, apoptosis and ROS induction. The cellular toxicity of erufosine was determined by MTT assay after 24h, 48h and 72h exposure in two OSCC cell lines, HN-5 and SCC-61. Based on these results, HN-5 cells were exposed to erufosine at concentrations corresponding to IC25, IC50 and IC75 concentrations, and then their mRNA was isolated and analyzed by Illumina Chip array. Gene expression and gene set enrichment analyses were performed to identify core KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways and GO (Gene Ontology) terms. A positive enrichment of ER stress upon erufosine exposure was observed, which was verified at protein level for the ER stress sensors and their downstream mediators. Upon erufosine exposure, calcium influx into the cytoplasm of the two OSCC cell lines was also observed. A differential regulation of genes and positive enrichment of processes pertaining to autophagy and apoptosis was also observed upon erufosine exposure. Autophagy was confirmed by increased acidic vacuoles and LC3-B levels. Apoptosis was confirmed by nuclear staining, annexin-V and immuno-blotting of caspases. The induction of mitochondrial stress upon erufosine exposure was predicted by gene enrichment analysis and shown by erufosine's effect on mitochondrial membrane potential, ATP and ROS production in OSCC cells. These data show that ER and mitochondrial targeting by erufosine represents a new facet of its mechanism of action as well as a promising new framework in the treatment of head and neck cancers. They furthermore support the future evaluation of erufosine as a therapeutic approach in cancer treatment alone, or in combination.

#3939

Activation of phosphatidylinositol-3′-kinase/Akt signaling in myxoid liposarcoma.

Marcel Trautmann,1 Magdalene Cyra,1 Christian Bertling,1 Ilka Isfort,1 Bianca Altvater,2 Claudia Rossig,2 Susanne Hafner,3 Thomas Simmet,3 Jessica Becker,4 Inga Grünewald,1 Pierre Åman,5 Reinhard Büttner,6 Eva Wardelmann,1 Sebastian Huss,1 Wolfgang Hartmann1. 1 _University Hospital Muenster, Muenster, Germany;_ 2 _University Children´s Hospital Muenster, Muenster, Germany;_ 3 _Ulm University, Ulm, Germany;_ 4 _University of Bonn, Bonn, Germany;_ 5 _University of Gothenburg, Gothenburg, Sweden;_ 6 _University Hospital Cologne, Cologne, Germany_.

Introduction: Myxoid liposarcoma (MLS) is the second most common type of liposarcoma and characterized by a high tendency to develop metastases. The molecular hallmark of MLS (≈90%) is a pathognomonic reciprocal t(12;16) (q13;p11) translocation, leading to the specific gene fusion of FUS and DDIT3. The resulting chimeric FUS-DDIT3 fusion protein is suggested to play a crucial role in MLS tumorigenesis, although its specific biological function and mechanism of action remain to be substantiated. While radiotherapy and chemotherapy with high-dose ifosfamide and doxorubicin represent established therapeutic options, prognosis in the metastasized situation is poor. Molecularly targeted therapeutic approaches are currently not available. Aiming at the preclinical identification of novel therapeutic options, we here investigate the functional relevance of phosphatidylinositol-3'-kinase (PI3-kinase)/Akt signaling in MLS. Experimental procedures: Immunohistochemical and FISH analyses of PI3-kinase/Akt signaling effectors were performed in a large cohort of clinical MLS tumor specimens. Mutational burden was studied by targeted next-generation sequencing (NGS; Illumina MiSeq). PI3-kinase/Akt-mediated signaling transduction was modulated by specific RNAi knockdown and a pharmacological approach applying the small molecule inhibitor BKM120 (Buparlisib; NVP-BKM120). Cell proliferation and FACS assays were performed in different MLS cell lines. An established MLS chorioallantoic membrane model (CAM) was employed for in vivo confirmation of the preclinical in vitro data. Results: In a significant subset of MLS tumor specimens, immunohistochemical staining revealed elevated phosphorylation levels of various signaling components, indicating that activation of PI3-kinase/Akt signaling is a frequent feature in MLS. Activating PIK3CA mutations and loss of PTEN as mechanism for PI3-kinase/Akt activation were detected in ≈15%. PI3-kinase inhibition significantly suppressed the signaling cascade, associated with reduction of MLS cell viability and induction of apoptosis in vitro and in vivo. Conclusions: Our preclinical study emphasizes the pivotal role of the PI3-kinase/Akt signaling cascade in MLS pathogenesis and indicates the occurrence of specific mutational aberrations apart from the pathognomonic FUS-DDIT3 gene fusion. Our in vitro and in vivo results suggest that targeting the PI3-kinase/Akt signaling pathway provides a rational, molecularly founded therapeutic strategy in the treatment of MLS.

#3940

Small molecule profiling uncovers the landscape uncovers the landscape of combinational PI3K inhibitor responses in HNSCC.

Nicole Lynn Michmerhuizen, Chloe Matovina, Elizabeth Leonard, Micah Harris, Caitlin Heenan, Gabrielle Herbst, Susan K. Foltin, Aditi Kulkarni, Thomas E. Carey, Carol R. Bradford, Hui Jiang, Chad Brenner. _Univ. of Michigan, Ann Arbor, MI_.

Recent sequencing studies of head and neck squamous cell carcinomas (HNSCCs) have identified the phosphatidylinositol 3-kinase (PI3K) pathway as the most frequently mutated, oncogenic pathway in this cancer type. Despite the frequency of activating mutations or amplification in PIK3CA (the gene encoding the catalytic subunit of PI3K), targeted inhibitors of PI3K have shown limited clinical efficacy as monotherapies. To identify factors that might predict sensitivity and resistance to PI3K inhibitors, we tested a panel of more than 20 patient-derived HNSCC cell lines and observed varying sensitivity to PI3K inhibitors. While all cell lines were much more sensitive than fibroblasts to these drugs, responses varied widely; for pan-PI3K inhibitor BKM120, IC50 values ranged from 0.4 to 7.4 uM across the OSCC cell line panel. Responses to BKM120 not correlate with genetic or phenotypic features, including: PIK3CA mutation status copy number, RNA expression, p110a protein levels, and AKT phosphorylation at baseline and after inhibitor treatment.

In order to characterize potential mechanisms of PI3K inhibitor resistance in HNSCC, we have also developed and optimized an unbiased, high-throughput screening approach. We have used this assay to test ~1400 inhibitors as monotherapies and in combination with PI3K inhibitors HS-173 and BKM120 in several HNSCC models. Our initial screening data suggested that the combination of PI3K inhibitor and EGFR/HER2 inhibitor afatinib is synergistic in a subset of cell lines. After testing this combination in additional models, we identified some cell lines that were responsive to the combination of HS-173 and afatinib, but not HS-173 and either EGFR inhibitor gefitinib or HER2 inhibitor CP-724714. In Detroit 562 cells, combination treatment with 0.25 uM HS-173 and 2.5 uM afatinib resulted in 35% more trypan positive cells than either drug alone. This also induced significantly more trypan positivity than combinations of 0.25 uM HS-173 with 5 uM gefitinib or 5 uM CP-724714. Likewise, the same HS-173 + afatinib treatment resulted in 35% or greater increases in Annexin V positivity compared to gefitinib and CP-724714 combinations. Western blot analysis demonstrated that these doses of gefitinib, CP-724714 and afatinib all inhibited target after 20 minute treatment in this cell line.

Future studies will also examine additional factors that may predict PI3K inhibitor responses and will seek to better understand promising combination treatments and advance them for effective use in HNSCC patients.

#3941

Heparin sulfate, a new target for platinum in metastatic TNBC.

Samantha J. Katner,1 James D. Hampton,1 Erica J. Peterson,1 Eriko Katsuta,2 Megan R. Sayyad,1 Kazuaki Takabe,2 Jennifer Koblinski,1 Nicholas P. Farrell1. 1 _Virginia Commonwealth Univ., Richmond, VA;_ 2 _Roswell Park, Buffalo, NY_.

Heparan sulfate (HS) are linear polysaccharides conjugated to proteins, heparan sulfate proteoglycans (HSPGs), located on the cell surface and extracellular matrix. The HS chains display varying degrees of sulfation. These sulfate clusters mediate the interaction of polynuclear platinum complexes (PPCs) with HSPG through a "sulfate clamp." Such PPC-HS interactions can be conceptualized as "polyarginine" mimics. Strong HS-PPC binding protects the oligosaccharide against sulfate loss through metalloshielding. The biological consequences of PPCs metalloshielding HS will in principle affect HS interactions with relevant enzymes and proteins such as heparanase and growth factors, similar in concept to inhibition of DNA-protein binding through modification of DNA structure and conformation. HSPGs, associated growth factors, and heparanase promote tumor progression by facilitating invasion, angiogenesis, and metastasis. High heparanase expression correlates with increased metastatic potential and poor clinical prognosis. PPC-HS interactions inhibit cleavage of a model pentasaccharide by heparanase and further modulated bFGF binding to HS, and bFGF-induced migration and signaling in colon cancer cells. The end-point of functional modulation of HS interactions is inhibition of angiogenesis and metastasis. We report proof-of-principle of strong in vivo anti-metastatic activity of PPCs in triple negative breast cancer (TNBC) models. Further, we examine the anti-metastatic and anti-angiogenic effects of PPC-HS metalloshielding in these in vivo models through multiple growth factor signaling pathways (bFGF, HB-EGF, VEGF), and heparanase activity in breast cancer and endothelial cells. PPCs represent a novel class of intrinsically dual-function agents combining platinum cytotoxicity through DNA targeting with anti-angiogenic effects through glycan targeting.

Chicago 14-17 April 2018

#3942

The fraction of garlic protein(s) affect HCC via regulation of angiogenesis.

Nabanita Chatterjee,1 Subhadip Das,1 Prajnamoy Pal,2 Krishna Das Saha3. 1 _Wexner Medical Center, The Ohio State University, OH;_ 2 _Fakir Chand College, Diamond Harbour, West Bengal, India;_ 3 _CSIR-Indian Institute of Chemical Biology, West Bengal, Kolkata, India_.

Cancers are leading cause of human mortality. Most of deaths are due cancer because of its high resistance to the chemotherapeutic drugs. Thus, there is an ongoing demand for generation of new potent and more effective therapies for cancer treatment. Hepatocellular carcinoma (HCC), is the most leading cause of cancer-related death in all over the world (seems to more than 80% of death cases in USA). It is frequently linked with continuous hepatocytes death, inflammatory cell infiltration, and compensatory liver regeneration with higher angiogenic rate. Considering the different signaling process during this tumorigenesis with the involvement of new 'lead' identification of various sources, desire to be less toxic and higher bioavailability. The use of natural or its active metabolic component(s) has begun to emerge as a potential innovative approach in medicinal research to deliver bioactive entities. Thus, garlic has already proven as the therapeutic implication on immune system and cancer due to presence of sulfur compound. But the role of active protein fraction present in fresh garlic is still not revealed yet. In our study, we are isolated an active garlic protein from fresh garlic aqueous extract with molecular weight about 40-45kD. This Garlic protein (GP) having non-cytotoxic to human/murine normal cells, could induce apoptosis in human HepG2 cells, as characterized by phosphatydylserine serine exposure, DNA fragmentation, alteration with loss of mitochondrial membrane potential and cytochrome C release, activation of series of caspases, other physiochemical changes lead to induce cancer cell death. This study also focused that the GP effectively suppressed DEN induced liver inflammation and hyperplasia in murine also decline in lipid peroxidation and changes in enzymic antioxidants and biochemical parameter with improvement of their survival rate. It reduced the elevated level of cytokines and growth factors in hepatocytes from liver fibrosis bearing mice. Thus, finding indicated that, GP may induce apoptosis and in hyper proliferating cancer cells via altering cytokines-chemokines level with tumor growth regulation in carcinogen exposed rodent system. Future direction, we are proposing to execute the experiments on p53-/- and cMyc-/- deficient mice to find out the role of GP in these circumstances.

#3943

Ceramide targets cell cycle and autophagy to suppress colon cancer growth.

Daniela Payne,1 Dafeng Yang,1 Chunwan Lu,1 Priscilla Redd,1 Thomas Albers,1 Iryna Lebedyeva2. 1 _Augusta University, Georgia Cancer Center, Augusta, GA;_ 2 _Augusta University, Augusta, GA_.

Ceramide, the central mediator of the sphingolipid metabolism pathway, plays a critical role in human cancer pathogenesis. Human colorectal carcinoma exhibits approximately 50% less ceramide than adjacent normal tissues. CD95 (Fas) is the death receptor for FasL of cytotoxic T lymphocytes (CTLs) and plays a key role in host cancer immune surveillance. Although CD95 has been shown to promote tumor growth under certain cellular contexts, the best known function of CD95 is mediating membrane-bound FasL-induced apoptosis under physiological conditions, and tumor suppression under pathological conditions. Loss of CD95 expression is associated with colon cancer progression. Ceramide is known to regulate CD95 clustering and activation, essential for CD95 function. We report here that a sub-lethal dose of a ceramide analog increases CD95 receptor clustering in human colon carcinoma cells to increase human colon carcinoma sensitivity to FasL-induced apoptosis in vitro. Ceramide analogs alone can induce apoptotic tumor cell death in a dose-dependent manner. However, inhibition of caspase activity does not decrease ceramide-induced apoptotic tumor cell death. Furthermore, inhibition of necroptosis did not decrease ceramide-induced apoptotic tumor cell death. Treatment of tumor cells with ceramide analogs induced ER stress and blocked autophagic flux. Furthermore, ceramide treatment induced chromatin condensation, DNA fragmentation and caused G0/G1 cell cycle arrest in colon cancer cells. These observations indicate that ceramide not only regulates CD95 receptor clustering to enhance FasL-induced tumor cell apoptosis, but also targets the cell cycle-autophagosome axis to induce tumor cell death through a caspase-independent mechanism.

#3944

Blockage of EGFR signaling repurposes tumor metabolism through suppression of glycolysis and Kreb cycle in head and neck cancer.

Ching-Chuan Kuo,1 Jang-Yang Chang,2 Hsing-Pang Hsieh,1 Hsing-Jien Kung,1 Hsih-Huei Chang,1 Chih-Hsiang Huang,1 Cheng-Chin Kuo,1 Yi-Yu Ke1. 1 _National Health Research Institutes, Zhunan, Miaoli County, Taiwan;_ 2 _National Cheng Kung University, Tainan, Taiwan_.

Head and neck squamous cell carcinoma (HNSCC), the most common malignant neoplasm arising in the mucosa of the upper aerodigestive tract, remains a significant cause of morbidity worldwide. With respect to the cancer treatment, HNSCC has a moderately good survival rate, however, the disease often recurs, leading to a poor prognostic disease course and tends to fail in treatment. The metabolic properties of cancer cells diverge significantly from those of normal cells. Emerging evidence suggests these metabolic alterations are also linked to therapeutic resistance in cancer treatment. EGFR targeted agents currently approved or under investigation for HNSCC. We recently identified a novel EGFR tyrosine kinase inhibitor (EGFR-TKI), BPR3K007S0, and found that this compound significantly inhibited EGFR phosphorylation in EGFR-overexpressing HNSCC cells in vitro and in vivo than that of gefitinib. BPR3K007S0 significantly suppressed the expression of a wide range of metabolic genes, including metabolic-related transcription factors, glycolysis, TCA cycle, and pentose phosphate pathway genes. Collectively, we also found that BPR3K007S0 significantly decreased glycolytic and mitochondria respiratory capacity. Pharmacological and genetic manipulation demonstrated that c-Myc/hexokinase 2 axis was one of downstream effector in response to EGFR inhibition. Furthermore, we found that EGFR-TKIs were able to suppress succinate dehydrogenase A leading to reduce fumarate, an oncometabolite generates from Kreb cycle, and contributed to EGFR-TKIs mediated antitumor effect. Taken together, these results revealed that blockage of EGFR signaling repurposes tumor metabolism through suppression of hexokinase 2 and succinate dehydrogenase A, in glycolysis and Kreb cycle, respectively, and demonstrated added benefits to treatment of HNSCCs.

#3945

CBT-102, an oral multi-kinase small molecule, demonstrates favorable activity in human hepatocellular carcinoma animal models and cardiac safety in rabbit Purkinje and beagle dogs compared to sorafenib.

Sarath Kanekal,1 Xiquan Zhang,2 Yanrui Song,2 Tan Wenmiao,2 Juan Zhang,2 Deyi Zhang,2 Henry Li,2 Mamatha Reddy,1 Gavin Choy,1 Sanjeev Redkar1. 1 _CBT Pharmaceuticals, Inc., Pleasanton, CA;_ 2 _Crown Biosciences, Inc., Taicang City, Jiangsu Province, China_.

Background: Cancer is a complex disease involving disruption of more than one receptor or signaling pathway. Inhibiting multiple oncogenic targets have been shown to be an important strategy in managing relapse and resistance. CBT-102 is an oral mTKI inhibiting several key oncogenic drivers including angiogenesis via VEGFR and PDGFR, MAPK pathway via B-RAF and C-RAF, in addition to inhibiting RET, CSF1R, c-KIT, and FLT3. Antitumor activity of CBT-102 was compared to sorafenib in two human primary HCC carcinoma xenografts in nude mice (LIMsh050 and PLCPRF5) and cardiac safety was evaluated in rabbit heart Purkinje fibers and in conscious telemeterized beagle dogs.

Methods: Female BALB/c nude mice were inoculated with LIMsh050 and PLCPRF5 tumors to establish the tumor models. In the LIMsh050 model, CBT-102 was administered orally QD x 21 days at 4, 10, and 25 mg/kg and sorafenib at 10 mg/kg (n=8/group). In the PLCPRF5 model, CBT-102 was administered orally QD x 14 days at 3, 8, and 20 mg/kg vs. 8 and 20 mg/kg sorafenib group (n=7/group). In both experiments, in addition to tumor volume, body weight and mortality were monitored. For the cardiac safety studies, rabbit Purkinje fibers were isolated from adult rabbit right heart perfused for 10 minutes with vehicle control, CBT-102 groups (1, 3, and 10 µM) or sorafenib and changes in action potentials were measured. In the second study, ECG parameters were measured after single oral administration of CBT-102 at 15, 45, and 67.5 mg/kg to beagle dogs (n=6/group) via telemetry for 24 h.

Results: In both the LIMsh050 and PLCPRF5 models, CBT-102 and sorafenib doses were well tolerated with no suspension of dosing due to weight loss or deaths. CBT-102 demonstrated inhibition of tumor growth in both models, to a significantly greater extent than sorafenib at comparable doses. In the PLCPRF5 model, CBT-102 demonstrated an ED50 of 2.5 mg/kg vs. 16.8 mg/kg of sorafenib. Importantly, unlike sorafenib, CBT-102 did not show a cardiac liability: there were no changes in action potential duration in rabbit Purkinje fibers nor any changes in cardiac conduction parameters (HR, QTc, RR, PR, and QRS intervals) in beagle dogs.

Conclusions: CBT-102 demonstrated improved efficacy in HCC xenografts models compared to sorafenib and does not appear to have the cardiac liability observed with sorafenib. CBT-102 will advance into GLP toxicology studies with a potential IND filing in 2H 2018.

#3946

Androgen receptor drives differential gene expression in KRAS-mediated non-small cell lung cancer.

Albert Roy Wang, Hope Beyer, Sean Brennan, Shannon Stiles, Dylan Wiese, Darya Buehler, Anwaar Saeed, Andrew M. Baschnagel, Gopal Iyer. _University of Wisconsin-Madison, Madison, WI_.

The Cancer Genome Atlas (TCGA) has identified alterations such as amplification, deletion, or mutation of androgen receptor (AR) in about 5% of human lung squamous cell carcinoma and adenocarcinoma. The expression of AR in human lung appears to play a crucial role in lung development and type II pneumocytes (PTII) maturation, but unlike in prostate cancer, the role of AR is not well known in non-small cell lung cancer (NSCLC). In our study, we demonstrated AR in NSCLC cell lines translocated into the nucleus over time when stimulated with synthetic androgen R1881, while addition of enzalutamide (MDV3100) or AR siRNA reduced AR nuclear localization. Using an NSCLC human tissue microarray also revealed that 10 out of 88 patients (11%) have positive AR immunohistochemical staining. Preliminary data from clonogenic assay indicated that enzalutamide might have radiosensitizing effect on certain NSCLC cells at 2 Gy, further suggesting the involvement of AR in lung cancer and its potential therapeutic value as a target. With the induction of R1881 for 30 minutes to 24 hours, certain NSCLC cells display decreased AR mRNA (0.5-0.8 fold), while others show modest increase (up to 1.5 fold), suggesting that AR regulation in these cells might be different due to their mutational landscapes. We then used predesigned 384 well panels from Bio-Rad to survey the effects of R1881, enzalutamide, and AR siRNA on mRNA expression levels of selected NSCLC cells. Distinct expression profiles were observed between cells that have wild-type and mutated KRAS. Taken together, these findings suggest that the AR signaling could be different based on KRAS mutational profiles of NSCLCs, and further work is required to reveal the underlying mechanism. Chromatin immunoprecipitation (ChIP) assay and RNA sequencing will be utilized to investigate AR interactions with androgen response elements and differential expressions in KRAS-driven NSCLC cells, respectively.

#3947

Glutaminolysis inhibitors benzo[d][1,2]selenazol -3(2H)-one dimers completely disrupt mitochondrial function and showed early promise as effective antitumor agents with little general toxicity.

Benfang Helen Ruan, Yan Yu, Zhao Chen, Yanhong Jin, Jinzhang Fang, Han Zhao, Jennifer Ruan. _Zhejiang University of Technology, Hangzhou, China_.

Targeting glutaminolysis is an emerging approach to solve the imminent problems of drug resistance and drug toxicity in cancer therapeutics. Ebselen was identified in the KGA coupled assay as a potent KGA inhibitor but showed very low potency in inhibiting the growth of glutamine - dependent cancer cells. For rigorous characterization, we developed a direct kinetic binding assay for KGA using Bio-Layer Interferometry (BLI) as the detection method; Ebselen was identified as a GDH inhibitor but not a KGA inhibitor. Proteomic analysis demonstrated that the ebselen derivative could crosslink with KGA protein, but the binding site is at the N-terminal appendix domain outside of the catalytic domain, which might explain why ebselen is not a potent KGA enzyme inhibitor in functional assays. Furthermore, we designed and synthesized several benzo[d][1,2]selenazol -3(2H)-one dimers which were subjected to SAR analysis by several glutaminolysis specific biochemical and cell based assays. Novel dual KGA/GDH inhibitors were discovered from the synthetic compounds; the dual inhibitors completely disrupt mitochondrial function, depleted cellular NAD(P)H levels, induced ROS mediated cell death, and inhibited the growth of various cancer cells but not the normal cells. Importantly, in an aggressive liver cancer mouse model, a KGA/GDH inhibitor reduced the tumor size by 50-60% and lowered plasma lactate dehydrogenase (a biomarker of tumor metastasis) by 2-3 times. Thus, the novel dual-specific KGA/GDH inhibitors showed early promise as effective antitumor agents with little general toxicity. 

### Targeting Oncogenes, Tumor Suppressors, or Gene Products

#3948

**Simultaneous** in vivo **PTEN rescue and KRAS silencing in cancer therapy.**

Gilles Divita,1 Veronique Josserand,2 Melanie Guidetti,2 Neil Desai1. 1 _Aadigen, LLC, Pacific Palisades, CA;_ 2 _Université Grenoble Alpes, Pacific Palisades, France_.

Background : The tumor suppressor PTEN and the KRAS oncogene play essential roles in tumorigenesis. PTEN mutations and resulting loss of function are common across variety of tumors and influence tumor cell proliferation. KRAS mutations occur in > 40% of cancer and have been associated with increased tumorigenicity. In numerous cancers, cooperative interactions between KRAS mutations and PTEN loss have been reported to promote metastatic development. However, to date there is no effective treatment for inhibiting KRAS activity or rescuing PTEN function in cancers. We investigated a new technique for simultaneous PTEN rescue and silencing of KRAS that may have therapeutic potential in PTEN/KRAS mutated pancreatic and other cancers. Methods : ADGN-technology is based on short amphipathic peptides that form stable neutral nanoparticles through non-covalent electrostatic and hydrophobic interactions with a large panel of nucleic acids. Self-assembled ADGN/nucleic acid nanoparticles remain stable over time in serum and plasma conditions. ADGN peptide complexes with wt PTEN mRNA or siRNA targeting KRAS were evaluated on pancreas (PANC1), ovarian (SKOV3), prostate (PC3) and glioblastoma (U25) cell lines. PTEN, KRAS and AKT phosphorylation were evaluated by western blot. Cell proliferation and apoptosis activation were determined by flow cytometry and Tunel assay. In-vivo Efficacy of IV-administered ADGN-peptides complexed with PTEN mRNA (0.25 mg/kg) and/or KRAS siRNA (0.5 mg/kg) was tested in PANC1-LUC mouse xenografts. Cytokine response to ADGN-nucleic acid complexes were measured in blood samples using Luminex cytokine 20 plex. Results : ADGN-mediated delivery of wt PTEN mRNA, rescued PTEN function in the cell-lines tested. Restoring PTEN resulted in ~90% reduction of cell proliferation, 3-8 fold activation of cell apoptosis, reduction of AKT phosphorylation and cell cycle arrest in G1. ADGN-nanoparticles containing wt PTEN mRNA resulted in tumor growth inhibition (TGI) of 80%. ADGN-mediated delivery of siRNA targeting KRAS, induced knock-down of KRAS expression by 60-70% in the cell-lines and significantly reduced viability by 50-70%. ADGN-nanoparticles containing siRNA KRAS resulted in a TGI of 50%. Combination mRNA PTEN and siRNAs KRAS ADGN nanoparticles, resulted in a TGI of 90% in PANC1 xenografts and also slowed development of distant metastates. No nonspecific cytokine response was observed following administration of ADGN-nucleic acid complexes.Conclusions: ADGN-peptides complexed with mRNA and/or siRNA were effective in targeting mutated PTEN and KRAS both invitro and invivo. This suggests a new strategy for targeting both tumor suppressors and oncogenes.

#3949

p53-restoring small molecule CB001 suppresses tumor growth and metastasis.

Shengliang Zhang, Lanlan Zhou, Jessica Wagner, David T. Dicker, Wafik S. El-Deiry. _Fox Chase Cancer Center, Philadelphia, PA_.

The uncontrolled growth and metastasis of tumor cells leads to morbidity and mortality in patients with cancer. The tumor suppressor p53, as a guardian of genome integrity, plays an important role in suppressing tumor growth and metastasis. However, loss of p53 pathway signaling frequently occurs in advanced tumors. Dysfunction of the p53 pathway is due to either p53 gene mutation or inactivation of p53. Mutations not only result in loss of wild-type p53 function, but also endow mutant p53 with a gain-of function, which further confers tumor aggressiveness and metastasis. Restoring p53 pathway signaling is an attractive strategy for suppressing tumor growth and metastasis in cancer therapy. Small-molecule CB001 restores p53 pathway signaling in p53-deficient colorectal cancer cells. We examined p53 pathway signaling based on the p53-responsive bioluminescence and the protein level of p53 targets. CB001 restores p53 pathway signaling in a p53-dependent and -independent manner in cancer cells. CB001 restores p53 pathway signaling via p73 in mutant p53-expressing cancer cells, and activates the p53 pathway in wild-type p53-expressing cancer cells. Correlated with restoration of p53 pathway signaling, CB001 induces cell death in cancer cells, but not in normal cells. Neither phosphorylation of H2AX in cancer cells nor DNA intercalation was observed upon CB001 treatment at the tested doses, suggesting little or no genotoxicity of CB001 in human colorectal cancer cells. Furthermore, both cell scratch assay and Boyden chamber assay reveal that CB-001 inhibits cancer cell migration. We further demonstrate that CB001 induces cell death and inhibits cell migration via p73 in p53-mutant colorectal cancer cells. These results taken together

suggest that CB001 is a promising lead for drug development with a strong capability to suppress tumor growth and metastasis through restoring p53 pathway in cancer cells.

#3950

Small molecule NSC59984 synergizes with ROS-generating agents to induce cell death via reduced GSH in cancer cells.

Shengliang Zhang, Lanlan Zhou, Wafik S. El-Deiry. _Fox Chase Cancer Center, Philadelphia, PA_.

NSC59984 is a small molecule with a duel capability to induce mutant p53 degradation and restore p53 pathway signaling in cancer cells. NSC59984 requires reactive oxygen species (ROS) to induce mutant p53 degradation via ERK2. ROS regulates cell growth and death with tumor cells having higher ROS levels than normal cells. Increasing ROS level is a promising therapeutic strategy applied in cancer clinical trials. However, the toxicity of high ROS levels is a limitation that needs to be solved urgently. We investigated the synergy between NSC59984 and ROS in cancer cells. To increase ROS, cells were treated with BSO, an ROS-generating agent. Our data reveal that ROS sensitizes cells to NSC59984 treatment including enhanced-p53 restoration and mutant p53 degradation induced by NSC59984. Combination of NSC59984 and BSO shows a synergy to induce cell death in cancer cells with an antagonism observed in normal cells at the tested doses. Consistent with the in vitro experiments, our in vivo experiments show that the combination of NSC59984 and BSO significantly suppresses tumor growth. Immunohistochemical staining shows a decrease in Ki67 and an increase in cleaved caspase 3 in xenografted tumors following combination treatment, suggesting that the tumor suppression is due to the induction of cell death and the inhibition of cell growth. Furthermore, the combination of NSC59984 and BSO shows a significant reduction in glutathione (GSH) levels in cancer cells, suggesting that the synergy between NSC59984 and BSO is due to the inhibition of the antioxidant system in mutant p53-expressing cancer cells. Our data indicate that a high level of ROS can be considered as a biomarker for NSC59984 administration in cancer therapy and supports further evaluation of the combination of NSC59984 and ROS-generating agents.

#3951

NVP-BEZ235 decreases mutant p53 and affects miR-23a-24a-27a to inhibit metastasis in breast cancer cells.

Jiajing Cai,1 Jingruo Xia,1 Dan Xiao,1 Chang Liu,1 Hebin Liao,1 Lei Xu,1 Dongsheng Wang,1 Xiaolan Guo2. 1 _North Sichuan Medical College, Nanchong, Sichuan, China;_ 2 _Affiliated Hospital of North Sichuan Medical College, Nanchong, Sichuan, China_.

Nearly half of human cancers harbor TP53 mutations, which are predominantly through missense mutations that result in accumulation of mutant p53 proteins in cancer cells. More and more findings indicate that the mutant p53 proteins acquire gain-of-function abilities to promote carcinogenesis, metastasis, tumor recurrence and chemoresistance. TP53 mutations are the most frequent genetic alterations in breast cancer, observed in 30% of breast carcinomas, especially the triple-negative breast cancer (TNBC), which most easily metastasizes and confers chemoresistance. Therefore, targeting TP53 mutation in breast cancer might be another promising approach of therapeutics. In this study, the AKT-mTOR dual inhibitor, NVP-BEZ235 (called BEZ235 hereafter) was employed to treat two TNBC cell lines, MDA-MB-231 and MDA-MB-468, which serve as TP53 mutation group at different dosages and time points. The results of CCK8, scratch, Transwell chamber and soft agar tests showed that BEZ235 could inhibit cell growth, migration, invasion and transformation abilities of the above TNBC cells. Most importantly, mutant p53 in these TNBC cells decreased significantly with BEZ235 treatment by Western blot. Therefore, we speculated that BEZ235 might inhibit the metastasis of TNBC cells through mutant p53 decline. Furthermore, we observed the autophagy marker LC3I/II conversion elevated obviously by WB, along with the upregulation of LC3 mRNA, which indicated that autophagy was induced after BEZ235 treatment in TNBC cells. To further understand the relationship of autophagy and p53 decrease upon exposure of BEZ235, autophagy inhibitor 3-MA and lentiviral shRNAs embedding ATG5/ATG7 were applied to deplete autophagy, and we found that mutant p53 was still decreased in the cells treated by NVP235 even though the autophagy was inhibited, which indicated that p53 degradation mediated by NVP235 may not relate with autophagy. In order to explore other potential regulatory mechanisms of the p53 downregulation upon BEZ235 exposure, microRNAs were considered to detect in the TNBC cells after treatment. Interestingly, miR-23a-24a-27a cluster was upregulated about 2-fold in TNBC cells after BEZ235 treatment in our study, while tumor growth and metastasis roles were discovered in some previous findings. More studies need to be done to illustrate miR-23a-24a-27a cluster in the TNBC cells treatment with NVP235, as well as the mechanisms with p53 regulation. In conclusion, our results suggest that BEZ235 might inhibit the metastasis abilities of triple-negative breast cancer cells targeting mutant p53 accumulation, which might provide an important direction to further study the breast cancer harboring mutant p53, and also have promising therapeutic benefits for cancer patients carrying p53 mutations.

#3952

Selective high affinity MYC-binding compound inhibits MYC-MAX interaction and MYC-dependent tumor cell growth.

Alina Castell, Qinzi Yan, Karin Fawkner, Per Hydbring, Fan Zhang, Vasiliki Verschut, Marcela Franco, Giovanna Zinzalla, Lars-Gunnar Larsson. _Karolinska Institutet, Stockholm, Sweden_.

The MYC family oncoproteins/transcription factors MYC, MYCN and MYCL (here referred to as MYC) are key players in tumor development and are particularly associated with aggressive disease and poor prognosis. Efficient and specific MYC-targeting drugs are therefore highly warranted, but no such drugs are available in the clinic at present. MYC is strictly dependent on heterodimerization with MAX for activation of transcription. In a cell-based Bimolecular Fluorescence Complementation protein-protein interaction screen for small molecule inhibitors we identified a molecule that exhibits strong selective inhibition of MYC-MAX interaction in cells as validated by Gaussia luciferase protein complementation assay, coimmunoprecipitation and in situ proximity ligation (isPLA) assay, reaching an IC50 at single-digit micromolar concentrations. The molecule was shown to inhibit MYC-MAX interactions in a biochemical FRET assay and binds selectively to the MYC bHLHZip domain with affinity in the single digit micromolar range as demonstrated by Microscale Thermophoresis and Surface Plasmon Resonance. Further, within the same concentration range, this molecule blocks MYC-driven transcription and efficiently inhibits tumor cell growth in a MYC-dependent manner, but spares normal cells. Moreover, the growth inhibitory responses to the molecule correlated significantly with MYC expression levels in a cohort of 60 human tumor cell lines. Importantly, utilizing a mouse tumor model of MYCN-amplified neuroblastoma, treatment with the molecule resulted in significant inhibition of the MYC-MAX interaction in tumor tissue, as shown by isPLA, and massive induction of apoptosis in the tumors. Since this molecule, unlike many experimental MYC inhibitors, is selective, has high affinity for MYC, has high efficacy in cells, reaches its target in vivo and does not affect MYC expression levels, it can be used as a chemical tool to specifically study the role of the MYC-MAX complex in MYC biology in normal and cancerous cells, and it has potential for drug development.

#3953

The role of USF1 in the regulation of lipogenesis and breast cancer tumor progression.

Jessica Lewis, Iqbal Mahmud, Daiqing Liao. _University of Florida, College of Medicine, Gainesville, FL_.

Breast cancer accounts for nearly one-quarter of all cancer diagnoses and is the principal cause of cancer-related mortality in women worldwide. Triple negative breast cancer (TNBC) is a clinically aggressive subtype of breast cancer commonly resistant to therapeutics that have been successful in increasing survival in patients with ER+, PR+ and HER2+ breast cancer subtypes. As such, identifying factors that contribute to poor patient outcomes and mediate the growth and survival of TNBC cells remain important areas of investigation. USF1 (upstream stimulatory factor 1), a gene linked to drive lipogenesis and cellular proliferation, is over-expressed in human malignancies, yet its contribution to cancer remains unclear. In analyzing large number of genomic datasets including The Cancer Genome Atlas (TCGA), we found that USF1 expression is significantly higher in TNBC tumor samples and cell lines. Also, USF1 gene expression positively correlates with key lipogenic enzymes. Significantly, we found that high expression of USF1 in breast cancer correlates with decreased patient survival. We found that USF1 shRNA expression resulted in cell death in breast cancer cell lines. We therefore hypothesize that USF1 drives de novo lipogenesis to promote breast tumorigenesis. Further studies are underway to determine the mechanisms by which USF1 promotes de novo lipogenesis, tumorigenesis and metastatic progression. Our studies will shed lights on the roles of USF1 in breast cancer tumor biology and undercover potential novel anticancer drug targets. (Supported by McKnight Foundation, Bankhead-Coley Cancer Research Program and James and Esther King Biomedical Research Program, Florida Department of Health.)

#3954

Anti-cancer agent P306 restores p53 pathway through PUMA.

Xiaobing Tian, Amriti R. Lulla, Shengling Zhang, Avital Lev, Philip Abbosh, Rahmat Sikder, David T. Dicker, Wafik S. El-Deiry. _Fox Chase Cancer Ctr., Philadelphia, PA_.

The tumor-suppressor p53 prevents cancer development via initiating cell-cycle arrest, cell death, repair, or anti-angiogenesis processes. Over 50% of human cancers harbor cancer-causing mutations in p53. p53 mutations not only abrogate its tumor-suppressor function, but also endow mutant p53 with a gain of function (GOF), creating a proto-oncogene that contributes to tumorigenesis, tumor progression, and chemo- or radiotherapy resistance. Thus, targeting mutant p53 and/or restoring a wild-type p53 signaling pathway provides an attractive strategy for cancer therapy. We previously showed that P01, a natural product, has potent anti-cancer activity against tumors with mutated p53. Based on the structure of the pharmacophore of the P01 family, we designed and synthesized new analogues. The novel synthetic analogue, P306, is potent in reducing both short-term and long-term proliferation in a broad panel of mutant p53 cell lines, including HT29, SW480, DLD-1, MDA-MB-231, MDA-MB-468, U251, FaDu, CAL-27, PANC-1, ASPC-1 and H1975, with IC50 values in the range of 33.9 to 242 nM. In this study, we have characterized a distinct mechanism of P306 that engages the apoptosis pathway by upregulating pro-apoptotic proteins including PUMA in a time-and dose-dependent manner in colorectal cancer cell lines. PUMA is necessary for the apoptotic effects of P306 in colorectal cancer cells with PUMA-mediated caspase 8 activation mediating P306-induced apoptosis. P306 not only restores p53 pathway but also depletes mutant p53 protein in various mutant p53-expressing cancer cells regardless of what kinds of mutation. P306 treatment potently downregulates c-Myc protein level, and apoptosis induced by P306 is dependent on the c-Myc-PUMA pathway. Our results provide novel mechanistic insights into the tumor cell death promoting activity of a candidate therapeutic agent for cancer.

#3955

PIK3R1 **loss activates AKT and STAT3 signaling in ovarian cancer.**

Xinran LI,1 Victor CY Mak,1 Yuan Zhou,1 Yiling Lu,2 Annie NY Cheung,1 Gordon B Mills,2 Lydia WT Cheung1. 1 _The University of Hong Kong, Hong Kong, China;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

The phosphoinositide 3-kinase (PI3K) regulatory subunit p85α is one of the critical gatekeepers of PI3K pathway activation through stabilizing and inhibiting the catalytic subunit p110. p85α is encoded by PIK3R1, which is frequently mutated or deleted in multiple cancer types including ovarian cancer, providing justification of exploring the approaches to target the aberrations. In this study, we observed a spectrum of oncogenic phenotypes in ovarian cancer cells upon knockdown of PIK3R1 in vitro and in vivo, indicating the functional significance of PIK3R1 loss in ovarian cancer. Strikingly, PIK3R1 loss not only led to AKT activation (canonical signaling of PI3K), but also unexpectedly JAK2/STAT3 signaling activation (non-canonical signaling of PI3K). PIK3R1 loss induced translocation of STAT3 into the nucleus, where gene transcription was promoted and thereby the malignant properties. Using in vitro 3D culture spheroid assay, we found that PIK3R1 loss sensitized ovarian cancer cells to AKT inhibitors and JAK2/STAT3 inhibitors. More importantly, the combination of AKT and STAT3 inhibitors resulted in synergistic loss of cell viability. The demonstrated signaling alterations and the associated therapeutic susceptibility downstream of PIK3R1 loss may open new avenue for the treatment of PIK3R1 loss-bearing cancer. [This study was supported by Research Grants Council of the Hong Kong SAR, China, Project No. 27103616]

#3956

Omomyc-based cell-penetrating peptides: From proof of concept to a clinically viable anti-Myc therapy.

Marie-Eve Beaulieu,1 Toni Jauset,1 Daniel Massó-Vallés,1 Sandra Martínez-Martín,1 Peter Rahl,2 Löika Maltais,3 Mariano F. Zacarias-Fluck,1 Sílvia Casacuberta-Serra,1 Erika Serrano del Pozo,1 Christopher Fiore,2 Laia Foradada,1 Virginia Castillo Cano,1 Matthew Guenther,2 Eduardo Romero Sanza,4 Marta Oteo,4 Cynthia Tremblay Tremblay,3 Génesis Martín,1 Danny Letourneau,3 Martin Montagne,3 Miguel Ángel Morcillo Alonso,4 Jonathan R. Whitfield,1 Pierre Lavigne,3 Laura Soucek1. 1 _VHIO, Barcelona, Spain;_ 2 _Syros, MA;_ 3 _University of Sherbrooke, Quebec, Canada;_ 4 _CIEMAT, Spain_.

Deregulation of the MYC oncoprotein drives tumorigenesis in most--if not all--cancers and generally correlates with poor prognosis, suggesting that its inhibition would be a useful therapeutic strategy. Indeed, we have shown that Myc inhibition displays extraordinary therapeutic benefit in various transgenic mouse models of cancer (i.e., skin, lung, pancreatic cancer and glioma), without eliciting resistance to therapy, and causes only mild, well-tolerated and reversible side effects in normal tissues. For these studies we employed a dominant negative inhibitor of Myc, called Omomyc, which is an effective inhibitor of Myc transactivation function both in vitro and in vivo. Omomyc has so far been utilized exclusively as a transgene, as a successful proof of principle whose application was believed to be solely limited to gene therapy. Here, though, we show that the purified Omomyc polypeptide itself spontaneously transduces into cancer cells and effectively interferes with MYC transcription, abrogating cell cycle and promoting apoptosis in different cancer cells, independently of their mutational profile. Efficacy of the Omomyc polypeptide in two complementary murine models of non-small cell lung cancer (NSCLC) establishes its therapeutic potential through both direct tissue delivery (intranasal) and systemic intravenous administration, providing for the first time evidence that the Omomyc polypeptide is an effective MYC inhibitor worthy of clinical development.

#3957

SRC inhibitors dephosphorylate and reactivate the DLC1 tumor suppressor: A new biomarker of clinical response.

Brajendra K. Tripathi,1 Meghan Anderman,1 Xiaolan Qian,1 Ming Zhou,2 Dunrui Wang,1 Alex G. Papageorge,1 Douglas R. Lowy1. 1 _National Institutes of Health, Bethesda, MD;_ 2 _Inova Schar Cancer Institute, Falls Church, VA_.

Although activation of oncogenes and inactivation of tumor suppressor genes frequently play cooperative roles in pathogenesis of cancer, most targeted cancer treatments are focused on oncogene inhibitors, with relatively little consideration given to their concomitant effects on tumor suppressors. In this study, we have identified a direct relationship between the SRC kinase and the tumor suppressor DLC1, determined that SRC phosphorylates and attenuates the tumor suppressor activity of DLC1, and explored whether reactivation of DLC1 may be used as a biomarker for the clinical response to SRC inhibition. Several human cancers have high SRC activity and constitutive up-regulation of RhoA-GTP, which is a pro-oncogenic driver that is implicated in several processes of tumor formation and its progression to metastasis. We determined that SRC directly phosphorylates two Tyrosines in DLC1, Y451 in the linker region and Y701 in the Rho-GAP domain, and inhibits the Rho-GAP activity and tumor suppressor functions of DLC1. SRC phosphorylation of DLC1-Y701 attenuates the hydrolysis of active RhoA-GTP to inactive RhoA-GDP, resulting in an increase in RhoA-GTP levels and its downstream signaling, while SRC phosphorylation of DLC1-Y451 abolishes the binding of tensin to DLC1, which further reduces DLC1 tumor suppressor activity independently of RhoA-GTP. The ability of SRC to phosphorylate its target Tyrosines in DLC1 is facilitated by the ERK-dependent phosphorylation of DLC1-S129, which enhances the binding of the SRC SH3 domain to DLC1 and increases the SRC-dependent phosphorylation of Y451 and Y701 in DLC1. The potentially reversible nature of the SRC phosphorylations of DLC1 may be useful as a biomarker of clinical response. In two models that have high SRC activity and express DLC1 (the mouse MMTV-PyMT breast cancer model and xenografts from a human lung cancer cell line), a 5-day treatment with the SRC inhibitors Saracatanib and Bosutinib have potent antitumor activity (~75% reduction in tumor weight) in conjunction with reducing phosphorylation of SRC target Tyrosines in DLC1 and reactivating DLC1 Rho-GAP activity, which reduces cellular RhoA-GTP levels and its downstream signaling. However, Saracatanib and Bosutinib have much less antitumor activity (~20% reduction in tumor weight) in isogenic versions of the lung cancer xenografts if the SRC inhibitors do not reactivate DLC1, as occurs when DLC1 is not expressed or the SRC phosphorylatable Tyrosines in DLC1 are mutated. Our results highlight the potential importance of DLC1 tumor suppressor reactivation as a biomarker for predicting and monitoring the response to SRC inhibitors.

#3958

Inhibiting the inhibitor: Exploring the p53-Rbm38 loop as a potential cancer therapeutic target.

Chris A. Lucchesi. _UC Davis, Davis, CA_.

Modulating Rbm38 negative regulation of p53 translation may be an effective therapeutic target in human cancers. As a focal regulatory component of RNA metabolism, RNA-binding proteins (RBPs) modulate all facets of RNA biogenesis. Given the fact that RBPs control RNA metabolism, defects in the function of RBPs can have far reaching implications including neurodegenerative disorders and cancer. Our group discovered that Rbm38 suppresses p53 mRNA translation by interacting with eukaryotic translation initiation factor 4E (eIF4E) on p53 mRNA. Conceptually, blocking the Rbm38 interaction with eIF4E employing synthetic peptides corresponding to their respective binding interface should relieve the translational repression caused by Rbm38. Indeed, treating cancer cells with synthetic peptides corresponding to the binding interface between Rbm38 and eIF4E resulted in robust p53 protein expression. Moreover, addition of peptide and low dose doxorubicin caused further accumulation of p53. Consequently, treatment of cancer cells with peptide alone or in combination with low dose doxorubicin leads to a decrease in colony formation as well as tumor sphere formation. Of significance, RKO xenografts with wild-type p53 were highly sensitive to treatment with a synthetic peptide comprising just 8 amino acids (Pep8) compared to the control group. Together, these data support that p53 expression can be modulated by using small peptides to inhibit Rbm38 from interacting with eIF4E, which may be used as a therapeutic approach in cancers carrying wild-type p53.

#3959

PRDM14 silencing by siRNA combined with an innovative nanoparticle reduced breast tumor formation and metastasis in vivo.

Hiroaki Taniguchi,1 Chiharu Moriya,1 Shiro Akinaga,2 Kozo Imai,1 Kazunori Kataoka3. 1 _The Institute of Medical Science, The University of Tokyo, Tokyo, Japan;_ 2 _AccuRna Inc., Tokyo, Japan;_ 3 _Kawasaki Institute of Industrial Promotion, Kawasaki, Japan_.

Triple negative breast cancer (hereafter TNBC) exhibits resistant to chemotherapy and radiotherapy, and develops distant metastases. Conventional treatments have had little impacts on TNBC. PRDI-BF1 and RIZ (PR) domain zinc finger protein 14 (PRDM14) is specifically expressed in embryonic stem cells (ESC) and primordial germ cells (PgC), and is required for the maintenance of ESC pluripotency and early differentiation in PgC. There is no expression of PRDM14 in the non-cancerous tissues, however PRDM14 is expressed in ~50% of TNBC, approximately. PRDM14 conferred the ability of resistance to chemotherapy, tumorigenicity and metastasis, in other words 'cancer stemness', on cancer cells (Taniguchi H, et al. Oncotarget 2017). PRDM14 was localized in nucleus, then we planned to develop an oligonucleotide therapeutics against PRDM14.

Our therapeutic methods are built on clinical application of basic science and technologies. #1. Methods of design the siRNA sequences with higher selectivity to its target and elimination off-target effects. #2. RNA-DNA chimera modification of small interfering RNA (chimera siRNA) exhibited stability in the bloodstream and low immunogenicity. #3. A polyion complex (PIC) nanocarrier exhibited high retention in blood, and accumulated siRNA in targeted cancer tissues, not in liver and spleen, due to the enhanced permeability and retention effect compared with typical nanocarriers, such as Lipid Nanoparticle.

Mice were orthotopically grafted with PRDM14+ HCC1937 (TNBC) cells. We also injected PRDM14+ MDA-MB-231 (TNBC) cells into mice via the tail vein for lung metastasis. PRDM14-specific chimera siRNA (1mg/kg) mixed with a PIC nanocarrier (N/P ratio = 5) was injected into mice tail vain 3 times a week for a month, after the HCC1937 tumor reached over 100mm3. This treatment caused 50.3% reduction of mean relative tumor volume, 98% reduction by synergistic effect with docetaxel (3.0mg/kg), and almost no lung metastatic lesions of MDA-MB-231 cells without any adverse effects.

GLP toxicology studies of PRDM14-specific chimera siRNA mixed with a PIC nanocarrier in female rats and monkeys showed no severe toxicity at 0.2, 1, or 2 mg/kg/day, twice a week for 4 weeks. Concomitant PK/TK studies revealed much higher exposure for PIC formulated siRNA than that for naked siRNA. Cmax was14900 ± 3200ng/ml, and AUC0-24h was 3730 ± 790 ng·h/mL for PIC formulated siRNA at 1mg/kg at day25.Naked siRNA was only detectable in plasma of animals for 2 h after administration of 1 mg/kg. We plan to submit an academic IND for First time in a human Phase I clinical trial for TNBC after finishing further extensive formulation works in 2019.

#3960

The allosteric inhibitor of CD44v6 AMC303 blocks c-MET, Ron and VEGFR-2 dependent signaling and cellular processes.

Thorsten Läufer, Martin Augsten, Vanessa Al-Rawi, Birgit Hotz, Yvonne Heneka, Oliver Coutelle, Alexandra Matzke-Ogi. _amcure GmbH, Eggenstein, Germany_.

Introduction: In cancer patients, the development of metastases is associated with a poor clinical prognosis. Metastatic dissemination requires cancer cells to detach from the primary tumor and invade adjacent or distant tissue. This process involves epithelial-to-mesenchymal transition (EMT), whereby tumor cells lose cell-cell adhesion mediated by E-cadherin repression.

The CD44 family of transmembrane glycoproteins comprises alternatively spliced variants that are involved in many cellular processes. The isoform CD44v6 has been shown to play a major role in tumor growth and metastasis. CD44v6 acts as a co-receptor for the receptor tyrosine kinases (RTKs) c-MET, RON and VEGFR-2, and is preferentially expressed in a variety of epithelial cancers. We have previously shown that the co-receptor function of CD44v6 for c-MET is the driving force for metastasis. c-Met and RON facilitate metastasis through MAPK signaling and VEGFR-2 plays a role in angiogenesis. AMC303, an allosteric and selective peptide inhibitor of CD44v6, prevents phosphorylation of the CD44v6 dependent RTKs and respective cellular processes.

Methods: The effect of AMC303 on cancer cell function in response to the growth factors HGF, MSP and VEGF was studied in various assays. Cell migration and invasion was analyzed in scratch assays and the Boyden Chamber. Morphological changes of EMT were monitored using microscopy in scattering assays in the presence of growth factors. EMT markers were analyzed by Western blot and immunohistochemistry. Tube formation was microscopically analyzed.

Results: AMC303 dose dependently and at low nanomolar concentrations (IC50 10 - 20nM) prevents HGF- or VEGF-induced activation of c-MET/VEGFR-2 in various cancer cells of pancreatic, lung and colonic origin or endothelial cells, which cannot be overcome by excess HGF and VEGF. HGF- and MSP-dependent cell migration, invasion and wound closure in scratch assays was prevented by AMC303 using different cancer cell lines. AMC303 had a strong inhibitory effect on HGF- and MSP- induced cell scattering, a morphological feature of EMT. Consistent with this, AMC303 inhibited the downregulation of HGF induced EMT markers (E-cadherin). In primary endothelial cells, AMC303 blocked activation of VEGFR-2 and VEGF-A induced formation of a tubular network.

Conclusions: Phosphorylation of CD44v6 dependent RTKs is inhibited in primary endothelial cells and different epithelial tumor cells by binding of AMC303 to the extracellular domaine of CD44v6. This novel mode of action results in strong anti-tumor and anti-metastatic effects of AMC303 involving inhibition of migration and invasion of tumor cells and prevention of EMT. AMC303 is currently tested in a First-in-Human Phase I/Ib study in cancer patients with solid epithelial tumors.

#3961

Role for neuropilin1 in mode of action of chlorotoxin.

Donna Kolber-Simonds,1 Jiayi Wu,1 Utpal Majumder,1 Daniel Custar,2 Danyang Li,1 Hong Du,1 Maarten H. Postema,1 Thomas Noland,1 Andrew Hart,1 George Lai,1 Sean Eckley,1 Vaishali Dixit,1 Karen Tendyke,1 Kenichi Nomoto,1 Mary Woodall-Jappe,1 Sharon McGonigle1. 1 _Eisai, Inc., Andover, MA;_ 2 _Mersana Therapeutics, MA_.

Chlorotoxin is an established tumor targeting peptide that naturally occurs in scorpion venom. 131I-labelled chlorotoxin assessed in early phase human glioma clinical trials achieved promising results. A peptide drug conjugate (PDC) composed of chlorotoxin peptide linked to a cytotoxic payload (cryptophycin analog) was used as a tool to probe the tumor targeting mechanism of chlorotoxin. The PDC proved efficacious, yet differential sensitivity was observed in multiple human tumor models. Previously described chlorotoxin targets did not align with observed PDC activity; therefore, studies to further elucidate its mechanism were undertaken. PDC treatment of distinct xenograft models led to a wide range of antitumor activity even though similar levels of active metabolite were present in tumor lysates; thus it was hypothesized that a role for chlorotoxin in uptake may be relevant.

We identified that the endocytic receptor Neuropilin1 (NRP1) binds to chlorotoxin peptide fragments following proteolytic digestion in vitro. NRP1 binding was selective for peptides with a free C-terminal arginine, while native chlorotoxin which has an amidated C-terminal arginine did not bind. Recovery of chlorotoxin from ex vivo tumor lysate revealed its metabolism to a carboxylated C-terminal arginine version of the peptide, capable of binding to NRP1. These data suggest that chlorotoxin acts as a cryptic peptide incapable of binding to NRP1 systemically and only when metabolized in the tumor microenvironment is NRP1 binding revealed.

The expression level of human NRP1 in tumors correlated to the PDC antitumor activity in multiple xenograft models; a wider therapeutic window was observed when NRP1 was highly expressed. Reduction of NRP1 levels in vivo through administration of NRP1 blocking antibodies or by NRP1 knockout in tumor cells blunted PDC antitumor activity while not affecting activity of the cytotoxic payload alone. Reduced PDC antitumor activity correlated with significantly lower levels of active metabolite detected in NRP1-deficient tumors. Together, our findings suggest that chlorotoxin metabolized in the tumor microenvironment binds NRP1 on tumor cells to increase uptake of active metabolite into cells, resulting in enhanced antitumor activity.

The identification of NRP1 as an uptake mechanism for chlorotoxin will enable selection of tumors for treatment with chlorotoxin-based therapeutics.

#3962

Antitumor effects of the antiparasitic agent ivermectin via inhibition of yes-associated protein 1 expression in gastric cancer.

Shotaro Kuramitsu,1 Sho Nanbara,1 Qingjiang Hu,1 Yousuke Kuroda,1 Takaai Masuda,1 Shuhei Ito,1 Hidetoshi Eguchi,1 Yoshihiko Mehara,2 Koshi Mimori1. 1 _Kyushu University Beppu Hospital, Beppu city, Oita, Japan;_ 2 _Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka city, Fukuoka, Japan_.

[Background] Yes-associated protein 1 (YAP1) acts as an oncogene through dephosphorylation and nuclear translocation, and nuclear accumulation of YAP1 is associated with poor prognosis in gastric cancer (GC). We previously identified ivermectin, an antiparasitic drug, as a YAP1 inhibitor. Here, we aimed to clarify whether ivermectin had antitumor effects on GC through inhibition of YAP1.

[Result] First, we evaluated the antiproliferative effects of ivermectin on human GC cells using in vitro proliferation assays and a xenograft mouse model. YAP1-knockdown assays were performed to assess whether the sensitivity to ivermectin depended on YAP1 expression. Next, we explored the mechanism through which ivermectin regulated YAP1 expression or localization by immunoblotting and reverse transcription-quantitative polymerase chain reaction for YAP1 and the downstream gene CTGF. Finally, the clinical significance of YAP1 expression was examined using three independent GC datasets. We found that MKN1 GC cells were most sensitive to ivermectin, whereas MKN7 cells were most resistant. In MKN1 xenografts, ivermectin suppressed tumor growth, and the sensitivity of MKN1 cells to ivermectin was decreased by YAP1 knockdown. Ivermectin inhibited YAP1 nuclear expression and CTGF expression in MKN1 cells but not MKN7 cells. Moreover, ivermectin decreased YAP1 mRNA expression, thereby inhibiting nuclear accumulation of YAP1 in MKN1 cells. In survival analysis, low YAP1 mRNA expression was associated with a better prognosis in three independent GC datasets.

[Conclusion] Ivermectin suppressed the growth of GC in vitro and in vivo by inhibiting YAP1 expression. Furthermore, GC with low YAP1 expression had favorable clinicopathological features and good prognosis. These findings provided insights into the antiproliferative effects of ivermectin as a YAP1 inhibitor and established a theoretical basis for preclinical evaluations of ivermectin for management of GC.

#3963

Cystine depleting human enzyme combined with auranofin synergistically inhibits pancreatic cancer cell growth in vitro and in vivo.

Sabin Kshattry, George Georgiou, Stefano Tiziani, Achinto Saha, Everett Stone, John DiGiovanni. _UT Austin, Austin, TX_.

Pancreatic ductal adenocarcinoma (PDAC) has one of the lowest 5-year survival rates of all cancers at 8%. Upregulated mechanisms to curb reactive oxygen species (ROS) accumulation are thought to help the growth of PDACs. Hence, perturbation of oxidative balance might be an effective therapeutic strategy for pancreatic cancer. To achieve this end, we used a human enzyme called Cyst(e)inase that degrades extracellular cysteine (Cys) and cystine (CSSC) - substrates that are necessary for synthesis of the antioxidant glutathione (GSH). Since tumor cells need to supplement intracellular Cys synthesis with extracellular import in order to fulfil their increased demand for GSH and protein synthesis, Cys/CSSC depletion can provide a therapeutic window. We validated this idea in our previous study where Cyst(e)inase treatment markedly inhibited tumor growth in several cancer models in vivo without any toxicity. In this study, we assessed the efficacy of Cyst(e)inase alone as well as in combination with auranofin in reducing pancreatic tumor growth. Cyst(e)inase treatment of cultured pancreatic cancer cells (Panc1, MIA-PaCa2, BxPC3) decreased intracellular Cys and GSH, and inhibited cell growth. Panc1 was very sensitive to Cyst(e)inase in vitro, exhibiting ROS accumulation and activation of stress-related signaling as well as G2-arrest and apoptotic cell death whereas BxPC3 and MIA-PaCa2 showed only a G1-arrest. Inhibition of the mTORC1-p70S6K-S6 ribosomal protein signaling pathway via the eIF2α-ATF4-Sestrin2 axis was observed in all 3 cell lines. In addition, MIA-PaCa2 but not Panc1 exhibited a rewiring of mitochondrial metabolism upon Cyst(e)inase treatment that bypassed succinate dehydrogenase. Combining auranofin and Cyst(e)inase caused synergistic increase in mitochondrial ROS and apoptotic cell death in the resistant cell lines MIA-PaCa2 and BxPC3 with associated activation of oxidative stress signaling. Surprisingly, the thioredoxin reductase system (the putative target of auranofin) was dispensable in producing synergy with Cyst(e)inase. Moreover, auranofin and Cyst(e)inase combination led to inhibition of mitophagy and accumulation of defective mitochondria. Supplementation with the antioxidant glutathione ethyl ester rescued the combination's cytotoxic effects. Biweekly intraperitoneal Cyst(e)inase treatment of nude mice harboring pancreatic cancer xenografts roughly recapitulated the in vitro sensitivity profile with Panc1 showing the most growth inhibition, albeit modest. BxPC3 xenografts were completely resistant to Cyst(e)inase alone but adding auranofin significantly inhibited their growth without causing any systemic toxicity. Collectively, the current data suggest that combining Cyst(e)inase and auranofin, an approved drug for rheumatoid arthritis, might constitute an effective therapy for pancreatic cancer.

#3964

Mesothelin targeting immunotoxin LMB-100 alters the profile of tumor-secreted proteins.

Salma El-Behaedi, Rebekah Landsman, Michael Rudloff, Emily Kolyvas, Rakan AlBalawy, Christine Alewine. _National Cancer Institute, National Institutes of Health, Bethesda, MD_.

Recombinant immunotoxins are antibody-based therapies that carry a bacterial toxin payload. LMB-100 is a next generation immunotoxin currently being tested in clinical trial. It targets the human cell surface antigen mesothelin (MSLN). MSLN is made by many solid tumors but has no expression in the parenchyma of vital organs. LMB-100 carries a Pseudomonas exotoxin payload that works by irreversibly modifying elongation factor-2 (EF-2), inactivating it and halting protein synthesis. To determine whether non-lethal doses of LMB-100 might inhibit tumor cell secretion of cytokines and growth factors that maintain the pancreatic cancer microenvironment, human pancreatic cancer cells were treated with LMB-100 and conditioned medium was assayed for tumor-secreted factors by ELISA and Luminex assays. Decreased levels of multiple secreted factors were detected including VEGF, PDGF, MMP-1, SPARC, and OPN. Conversely, levels of MIF, a stored cytokine released under stress conditions, increased dramatically upon LMB-100 treatment. To determine whether this also occurs in vivo, tumor-bearing athymic nude mice were treated with LMB-100 and levels of tumor-secreted factors in intratumoral fluid were measured. A statistically significant decrease in the same factors was found in treated mice compared to control mice. To determine whether these decreases in tumor-secreted factors could change the tumor microenvironment and alter the immune milieu, a mouse syngeneic model of pancreas cancer sensitive to LMB-100 was developed. LMB-100 treatment of this mouse cell model caused a statistically significant decrease in the angiogenesis factors VEGF and Angiopoietin-2 in conditioned medium. To determine if this also occurs in vivo, immune-competent mice bearing intraperitoneal or orthotopic tumors were treated with immunotoxin or vehicle then intratumoral fluid was assayed for secreted proteins contributed by both tumor and stromal cells. No detectable change in intratumoral fluid concentration was seen for VEGF, Antiopoietin-2 or a panel of 11 other secreted factors. In conclusion, LMB-100 does inhibit tumor cell production of secreted proteins, but this change is insufficient to alter the total concentration of secreted factors in the tumor microenvironment.

#3965

Sequential multiplex immunofluorescence technology (SMIFT): A new staining strategy for immunotherapy.

Joseph Vargas, David Tacha, Julio Masabanda, Cristin Douglas. _Biocare Medical, Pacheco, CA_.

Introduction: Tumor tissues exhibit a multitude of different cell types that have extravasated from other regions in the body. These cells are likely to represent heterogeneous populations of immune and effector cells. Assessing immunotherapy markers in tissues could be of significant value for patient care. We therefore have developed an innovative method that will enable the application of three or more sequential mouse monoclonal antibody (MMA) applications that can detect these immune cells on a single tissue. Previous methods using a sequential staining technique of MMA required a manual application and time-consuming heating and stripping between steps. The assessment of multiple targets can now be determined in approximately eight hours using full automation without heating or stripping procedures. This method may be valuable when the quantification of specific immunotherapy markers is required.

Materials/Methods: Two assays were developed using Sequential Multiplex Immunofluorescence Technology (SMIFT): FOXP3, CD163, PD-1, CD8 as well as p40, Desmoglein-3, TTF-1, CD8. Tissues were fixed in 10% NBF for 24 hours and processed into paraffin. Tissue sections were cut at 5 microns and adhered to glass slides. Sections were then heated in a citrate-based buffer at pH 6.0 within a pressure cooker at 95°C for 40 minutes. All antibodies were diluted in diluent and applied to tissues to determine optimal IF (and IHC) titers. Detection of each antibody was accomplished with commercially available anti-mouse/anti-rabbit HRP polymer followed by fluorophores 594, 488, 546 and 480 for IF (or by DAB for IHC comparison). A blocking reagent was incorporated into the protocol to prevent cross-reactivity of multiple detection fluorophores. Both assays were comprised of two sequential MMA followed by a third application of a mouse/rabbit monoclonal antibody cocktail to complete the procedure. Sections were then counterstained in DAPI (or hematoxylin).

Results: Multiplexed staining intensities were compared to individual antibody formats for both assays. Staining of each target was clearly identified even when multiplexed. Fluorescence intensities were scored on a subjective scale from 1-3+ with 3 representing the highest intensity. Both assays exhibited 3-3+ staining intensities for each target fluorophore as well as in multiplexed formats. Individual IHC staining results were also comparable to individual and multiplexed IF targets with identical staining patterns.

Conclusion: Multiplexing can be achieved using SMIFT, a sequential and multiplex approach for assays tested on a single tissue section. Three sequential applications of four antibodies can be performed in less than eight hours with full automation. This is in contrast to previous overnight methods. Most importantly, this approach could be used to assess immunotherapy markers in tissues and may provide prognostic or predictive value.

#3966

Mining the cancer immuno-responsome: The identification of functional antitumor antibodies from patients receiving checkpoint inhibitors.

Gilson Baia,1 Amy Manning-Bog,1 Alexander Scholz,1 Jeff DeFalco,1 Michael Harbell,1 Danhui Zhang,1 Felix Chu,1 Beatriz Millare,1 May Sumi,1 Patricia Zuno,1 Judevin Lugar Sapugay,1 Dongkyoon Kim,1 Yvonne Leung,1 Shuwei Jiang,1 Xiaobin Tang,1 Kevin Williamson,1 Xiaomu Chen,1 Sean Carroll,1 Christine Dowd,1 Ish Dhawan,1 Jonathan Benjamin,1 Gregg Espiritu Santo,1 Nicole Haaser,1 Ngan Nguyen,1 Eldar Giladi,1 David Minor,2 Yann Chong Tan,1 Jeremy B. Sokolove,3 Lawrence Steinman,3 Tito Serafini,1 Guy Cavet,1 Norman M. Greenberg,1 Jacob Glanville,4 Wayne Volkmuth,1 Daniel E. Emerling,1 William H. Robinson3. 1 _Atreca Inc, Redwood City, CA;_ 2 _California Pacific Medical Center Research Institute, San Francisco, CA;_ 3 _Stanford University School of Medicine, Stanford, CA;_ 4 _Stanford School of Medicine, Stanford, CA_.

Background: The role of B cells and antibodies in anticancer immune responses may correlate with improved prognosis in several types of cancer. Indeed, tumor-reactive antibodies are detected in the blood of cancer patients, tumor-infiltrating B cells have been shown to produce tumor-reactive antibodies, and tumor-reactive antibodies can cause tumor regression in several mouse models. Taken together, these observations support further identification, isolation and characterization of antitumor antibodies from patients demonstrating effective anticancer responses and defining the cognate targets and mechanisms whereby they contribute to tumor control.

Methods: We identified cohorts of patients with nonprogressing metastatic cancer who had received checkpoint immunotherapy and isolated their circulating plasmablasts. Antibody heavy and light chain paired sequences were obtained from individual cells using Atreca's Immune Repertoire Capture (IRCTM) technology. The expressed antibodies were then analyzed for their ability to bind to tumor cells as well as tumor tissue and their ability to mediate antitumor activity was explored in syngeneic mouse tumor models.

Results: Elevated plasmablast levels were observed in individuals with nonprogressing metastatic cancer, and analysis of plasmablast antibody sequences revealed clonal families of B cells that persisted over time with hallmarks of affinity maturation and class switching. We also identified antibody sequences with features common to more than one patient, consistent with convergent antibody selection. In particular, one antibody (AB-213) isolated from a NSCLC patient was found to demonstrate binding to unrelated human tumors as well as the mouse EMT6 tumor. When AB-213 was expressed with a mouse IgG2a constant region the chimeric antibody showed efficacy in vivo by reducing tumor volume and increasing survival in Balb/c mice harboring the syngeneic EMT6 model. Antitumor activity of the chimeric antibody was observed to be dose-dependent when administered as monotherapy or in combination with checkpoint inhibitors. We feel, based on these data, AB-213 could become a very important clinical therapeutic.

### Therapeutic Targeting

#3967

Potent treatment combinations with CIB1 depletion in triple-negative breast cancer.

Alexander H. Chung, Tina M. Leisner, Gabrielle Dardis, Marissa Bivins, Leslie V. Parise. _UNC Chapel Hill, Chapel Hill, NC_.

The current standard of care for triple-negative breast cancer (TNBC) patients is limited to chemotherapy, radiation, and/or surgery. Therefore, there is a pressing need for new targeted therapies to improve clinical efficacy, reduce toxicity, and overcome resistance. We previously found that CIB1 depletion induces cell death selectively in TNBC cells, while sparing normal cells. The purpose of this study was to ask whether depleting CIB1 further enhances tumor-specific killing when combined with either the commonly used chemotherapeutic, docetaxel, or the cell death-inducing ligand, TRAIL. To address this, we targeted CIB1 by RNA interference in MDA-MB-436, MDA-MB-231, MDA-MB-468 TNBC cells and ME16C normal breast epithelial cells alone or combination with docetaxel or TRAIL. Cell death was quantified via trypan blue exclusion using FACS and cell death mechanisms were analyzed by Western blotting. We found that the combination of CIB1 depletion with docetaxel significantly enhanced tumor-specific cell death relative to each treatment alone. The enhanced cell death strongly correlated with caspase-8 activation, a hallmark of death receptor-mediated apoptosis. Because CIB1 depletion also upregulated the death receptor, TRAIL-R2, we tested for sensitization to TRAIL. While TRAIL alone had no effect on cell viability, CIB1 depletion profoundly sensitized tumor cells to TRAIL, resulting in a synergistic increase in cell death. In addition to death receptor-mediated apoptosis, both combination treatments activated a non-apoptotic mechanism, called paraptosis. Interestingly, these combination treatments also induced nearly complete death of docetaxel-resistant MDA-MB-436 cells, again via apoptosis and paraptosis. In contrast, neither combination treatment induced cell death in normal ME16C cells. In conclusion, our results demonstrate that the novel combinations of CIB1 depletion with docetaxel or TRAIL significantly enhance naive and docetaxel-resistant TNBC cell death via increased death receptor-mediated apoptosis and paraptosis, suggesting that targeting CIB1 may also overcome resistance to chemotherapeutics. Taken together, combination therapies that target CIB1 could prove to be a safe and durable strategy for treatment of TNBC and potentially other cancers.

#3968

Bacterial heat labile enterotoxin LT-IIc effects autophagy and cell death in human TNBC.

Patricia A. Masso-Welch,1 Sofia Girald Berlingeri,2 Natalie D. King-Lyons,1 John C. Hu,1 Christopher Greene,3 Lorrie Mandell,1 Matthew Federowicz,4 Terry D. Connell,1 Yasser Heakal4. 1 _State University of New York, Buffalo, NY;_ 2 _NIH, Bethesda, MD;_ 3 _Roswell Park Cancer Institute, Buffalo, NY;_ 4 _D'Youville College, Buffalo, NY_.

Microbes and microbial products are being re-evaluated as potential therapeutic agents for breast cancer treatment. We previously observed the rapid induction of cell death in the mouse TNBC cell line 4T1, and mesenchymal TM12T, but not epithelioid TM12 cells, upon exposure to LT-IIc, a member of the bacterial type II subfamily of ADP-ribosylating toxins that bind to specific ganglioside subtypes. To test the hypothesis that LT-IIc may be effective against human breast cancer, we tested a panel of human breast cancer cells. LT-IIc exposure induced cytotoxicity in TNBC, particularly basal B (mesenchymal) TNBC (BT549 and MDA-MB-231), in a dose dependent fashion. The triple negative non-transformed breast epithelial line MCF10A was unaffected. Two ER+ cell lines (T47D and MCF7), and HER2 overexpressing SKBR3 cells showed limited cytotoxic response. We observed a distinctive rapid accumulation of large cytoplasmic vacuoles in TNBC, but not other normal or transformed cell lines. Combined cytotoxicity and vacuolization were not induced by cholera toxin, LT-IIa holotoxin, or forskolin, but still occurred upon treatment with the LT-IIc isolated B pentamer (which lacks the toxin's catalytic A subunit), suggesting that the effects of LT-IIc were not due to intoxification of the adenylate cyclase pathway. These vacuoles were not intracellular lipid droplets, as determined by lack of staining with Oil Red O. Instead, vacuoles were positive for punctate staining with LC3B, a marker of autophagosome formation. Western blotting of MDA-MB-231 and BT549 cells revealed an increase in LC3B and p62 (sequestosome), a marker of autophagic flux. Bafilomycin, an inhibitor of lysosomal fusion, augmented the cell death effect of LT-IIc, but blocked the accumulation of distended autophagic vacuoles, suggesting a negative autophagy feedback response to bafilomycin. Both necrosis and apoptosis were activated in response to LT-IIc exposure, as indicated by increased annexin/AAD staining and caspase 3/7 activity. Knockdown of ATG5 by siRNA, although blocking ATG5 expression, did not block LC3B lipidation, cell death or increased caspase 3/7 activity in response to LT-IIc, suggesting a noncanonical autophagic response. These results suggest that LT-IIc binding or signaling may represent a novel target of therapy for TNBC. Current studies are directed towards defining the ganglioside content in LT-IIc sensitive cell lines and identifying potential downstream proteomic targets.

#3969

Lipocalin-2-targeted small interference RNA for inflammatory breast cancer treatment.

Ginette S. Santiago-Sanchez,1 Fatma Valiyeva,2 Bisrat Debeb,3 Pablo E. Mejia1. 1 _University of Puerto Rico-Medical Science Campus, San Juan, Puerto Rico;_ 2 _UPR Comprehensive Cancer Center, San Juan, Puerto Rico;_ 3 _University of Texas MD Anderson Cancer Center, Houston, TX_.

Inflammatory Breast Cancer (IBC) is an aggressive form of invasive breast cancer with low survival rate. Available therapies to treat this type of cancer are almost limited to surgery and radiation therapy. Potential therapeutic targets for IBC treatment include the oncogene Her2 and the Epidermal Growth Factor Receptor (EGFR). However, these therapies have not been successful since not all IBC tumors are Her2-amplified and development of resistance to treatment has also been observed. Hence, there is a critical need for novel therapeutic targets for IBC treatment. One of such possibilities is Lipocalin-2 (LNC2). LCN-2 is a secreted glycoprotein and an adipokine superfamily member that posses roles in transport of small lipophilic molecules in circulation, innate immunity, and iron homeostasis. High levels of LCN2 have been observed in invasive breast cancer human tissue. Additionally, LCN2 has been linked with breast cancer tumorigenesis and metastasis. Therefore, the purpose of our study is to assess if LCN2 is a plausible therapeutic target to treat IBC. Western blot analysis was used to assess the LCN2 protein level in a panel of IBC (MDA-IBC-3, SUM-149) and non-IBC cell lines (MCF-7, MDA-MB-231, MDA-MB-438). IBC cell lines showed a significantly higher expression of the LCN2 protein in comparison to the non-IBC cell lines. We decreased the expression of the LCN2 protein with small interference RNA (siRNA) molecules. Silencing of IBC cells with LCN2-targeted siRNA molecules decreased the LCN2 protein expression levels in almost fifty percent. Colony formation assay demonstrated that siRNA-mediated LCN2 silencing in MDA-IBC-3 and SUM-149 cell lines resulted in a significant decrease in cell proliferation. Taken together these results suggest that LCN2 may become a right target for IBC treatment. Ongoing experiments include cell invasion assays to determine if LCN2-targeted siRNAs reduce the metastatic potential, increase apoptosis and block cell cycle progression. Acknowledgements: This work is supported by the University of Puerto Rico Comprehensive Cancer Center and the U54 partnership with MD Anderson Cancer Research Center.

#3970

Therapeutic targeting of estrogen receptor positive breast cancer with the BET bromodomain inhibitor ODM-207.

Julia Lindqvist,1 Mari Björkman,2 Reetta Riikonen,2 Daniel Nicorici,2 Elina Mattila,2 Chandrasekhar Abbineni,3 Mahaboobi Jaleel,3 John Eriksson,1 Pekka Kallio,2 Anu-Maarit Moilanen2. 1 _Åbo Akademi University, Turku, Finland;_ 2 _Orion Corporation Orion Pharma, Turku, Finland;_ 3 _Aurigene Discovery Technologies Limited, Hyderabad, India_.

Introduction: The bromodomain and extraterminal (BET) family of proteins are chromatin readers that recognize and bind to specific acetylated histones and promote the transcription of several important cell identity genes. BET bromodomain inhibitors have shown promising antitumor activity in a variety of pre-clinical cancer models, as BET inhibition abrogates the transcription of several key oncogenes in a cell type-specific manner. It is known that inhibition of BET proteins effectively inhibits the proliferation of estrogen receptor positive (ER+) breast cancer cells, at least in part through repression of ER and MYC signaling. However, many additional cancer-associated genes are likely to underlie the growth inhibitory effects of BET inhibitors in breast cancer. The purpose of this study was to determine the anticancer activity of the novel BET bromodomain inhibitor ODM-207 in pre-clinical ER+ breast cancer models, and further, to look for cancer-associated signaling pathways suppressed by BET inhibitors.

Methodology and results: ODM-207 is a novel, highly selective BET bromodomain inhibitor structurally distinct from JQ1 and its benzodiazepine-related derivatives. In this study, we show that ODM-207 effectively inhibits the proliferation of ER+ breast cancer cell lines when measured by cell viability assays as well as suppresses the growth of patient-derived xenograft tumors. Furthermore, we wanted to investigate the anticancer signaling pathways regulated by ODM-207 as well as the prototypical BET inhibitor JQ1 in breast cancer cells. For this purpose, we performed RNA sequencing on two ER+ breast cancer cell lines after 24h treatment with the aforementioned BET inhibitors. We found that both BET inhibitors targeted several genes and pathways important for breast cancer progression. For example, the targets included CDK4 and CDK6, two cell cycle kinases fundamental for the development and treatment of ER+ breast cancer. The RNA sequencing results were further validated in vitro, and were utilized as a basis for combination therapy assessment.

Conclusions: Our results indicate that the novel BET bromodomain inhibitor ODM-207, which is currently in Phase I clinical trials for treating solid tumors, causes significant growth inhibition and cell cycle arrest in pre-clinical models of ER+ breast cancer, and regulates multiple crucial signaling pathways involved in breast cancer cell cycle and survival.

#3971

Combination of neratinib and enzalutamide is an effective treatment for HER2-positive breast cancer metastasis to the brain.

Harshil D. Dhruv,1 Lauren Hartman,1 Chris Gooden,2 George Reid,1 Christophe Legendre,1 George Snipes,3 Cynthia Osborne,3 Joyce O'Shaughnessy,3 Bodour Salh4. 1 _TGen (The Translational Genomics Research Institute), Phoenix, AZ;_ 2 _University of Southern California, Los Angeles, CA;_ 3 _Baylor University Medical Center, Dallas, TX;_ 4 _Univeristy of Southern California, Los Angeles, CA_.

An estimated 10% to 30% of all breast cancer patients eventually develop brain metastases. Up to half of the patients with epidermal growth factor receptor 2 (HER2)-positive breast cancer will develop brain metastasis. Treatment options for patients with breast cancer brain metastases are limited to surgical resection and radiotherapy with dismal median survivals of 2 to 24 months. Efforts to identify clinically relevant targeted therapeutics have been limited by the lack of molecularly characterized model systems. We have developed a patient derived xenograft (PDX) model of a HER2-positive breast cancer brain metastasis and have genomically characterized the PDX and its -matched tumor. Whole exome and RNA sequencing data revealed high fidelity of the PDX tumor with its corresponding patient-matched tumor and confirmed retention of driver gene alterations such as the amplification and overexpression of HER2. Additionally, immunohistochemistry of the patient tumor revealed overexpression of the Androgen receptor (AR) (IHC Score 2+), which was also confirmed in the PDX. Given the importance of both HER2 and AR in aggressive tumor behavior, we developed a preclinical study to inhibit both targets. Neratinib and enzalutamide were selected because of their known blood brain barrier penetration properties. As proof of concept we first tested the combination strategy in the flank of NOG mice (n = 10 animals/group). Tumors were allowed to grow to ~250 mm3 in size and animals were subsequently treated with either vehicle control, neratinib (40 mg/Kg) alone, enzalutamide (30 mg/Kg) alone, or combination for 2 weeks. Neratinib alone and thecombination with enzalutamide demonstrated exceptional efficacy in suppressing tumor growth as compared to either the vehicle control or Enzalutamide alone (5 fold reduction in tumor size, p < 0.001). Interestingly, when the treatments were stopped, tumors treated with Neratinib alone grew significantly faster compared to tumor growth with the combination treatment (p < 0.05), suggesting more effective and sustained tumor suppression by the addition of Enzalutamide. Studies in an orthotopic setting and molecular profiling of treated and non-treated tumors are ongoing. In summary, our results demonstrated that Neratinib in combination with Enzalutamide could potentially be developed for patients with HER2 and AR-positive breast cancer metastasis to the brain and warrants further preclinical testing.

#3972

Combined inhibition of MCL1 and BCL-2 with AMG 176 and venetoclax induces anti-tumor effects in primary patient samples and models of acute myeloid leukemia.

Sean R. Caenepeel,1 Tao Osgood,1 Brian Belmontes,1 Jan Sun,1 Elaina Cajulis,1 Andrew Wei,2 Angela Coxon,1 Jude Canon,1 Paul Hughes1. 1 _Amgen Inc., Thousand Oaks, CA;_ 2 _Alfred Hospital and Monash University, Melbourne, Australia_.

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy involving the clonal expansion of immature myeloid cells. Dysregulated expression of the BCL-2 family of proteins has been implicated in AML pathogenesis. Specifically, the anti-apoptotic family members MCL1 and BCL-2 have been reported to play key roles in AML survival. The selective inhibition of these two proteins represents an emerging strategy in AML treatment. AMG 176 is a potent and selective MCL1 inhibitor currently being tested in an AML Phase I clinical trial. Here we describe the activity of AMG 176 and AM-8621, a structural analog, as single agents and in combination with the BCL-2 inhibitor venetoclax, in models of AML. AM-8621 and venetoclax were profiled as single agents against a panel of AML cell lines to characterize their dependency on MCL1 and BCL-2 for survival. A wide range of sensitivities to both compounds was observed, with several lines exhibiting dependency on both MCL1 and BCL-2 for survival, suggesting functional redundancy and a requirement for combined inhibition to maximize response. To test this hypothesis, we profiled a subset of cell lines with the combination of AM-8621 and venetoclax. A synergistic interaction was detected in each cell line, highlighting their codependence on MCL1 and BCL-2. We also evaluated the synergistic potential of this combination on primary AML patient samples. Here, freshly purified bone marrow aspirates were treated with equimolar concentrations of AM-8621 and venetoclax and compared against single agents in a flow cytometry based viability assay. Marked improvements in activity and potency were observed with the combination over either agent alone. We also tested the combination of AMG 176 and venetoclax in a MOLM-13 orthotopic xenograft model of AML. Mice were treated twice weekly with AMG 176 (60 mg/kg) and daily with venetoclax (100 mg/kg). While both single agents achieved significant reductions in MOLM-13 tumor burden (69% and 33% reduction in BLI respectively), the combination exhibited complete inhibition of tumor growth (100% reduction in BLI) and achieved tumor regression relative to the first day of dosing. We next characterized the effects of this combination on subsets of hematopoietic cells in vivo. The reduced affinity of AMG 176 for murine MCL1 (200-fold) required the use of a human MCL1 knock-in mouse for these studies. Terminal analysis of mice treated with the combination or AMG 176 alone showed significant decreases in peripheral blood B-cells and monocytes, whereas venetoclax alone exhibited significant reductions in B-cells only. Analysis of spleens revealed greater reductions in both cell types following treatment with the combination compared with either single agent. These data highlight the promise of combined MCL1 and BCL2 inhibition as a novel therapeutic strategy for the treatment of AML.

#3973

**Dabrafenib suppresses the growth of** BRAF-WT **cancers through inhibition of novel targets Nek9 and Cdk16.**

Manali S. Phadke, Lily Rix, Inna Smalley, Annamarie Bryant, Harshani Lawrence, Braydon Schaible, Ann Chen, Uwe Rix, Keiran Smalley. _Moffitt Cancer Center, Tampa, FL_.

Although the BRAF inhibitors dabrafenib and vemurafenib have both proven successful against BRAF-mutant melanoma, there seem to be differences in their mechanisms of action. Here, we show that dabrafenib is more effective at inhibiting the growth of NRAS-mutant and KRAS-mutant cancer cell lines than vemurafenib. Using mass spectrometry-based chemical proteomics we identified NEK9 and CDK16 as unique targets of dabrafenib. Both NEK9 and CDK16 were highly expressed in specimens of advanced melanoma, with high expression of both proteins correlating with a worse overall survival. A role for NEK9 in the growth of NRAS and KRAS-mutant cell lines was suggested by siRNA studies in which silencing was associated with decreased proliferation, cell cycle arrest associated with increased p21 expression, inhibition of phospho-CHK1, decreased CDK4 expression and the initiation of a senescence response. Inhibition of CDK4 but not CHK1 recapitulated the effects of NEK9 silencing, indicating this to be the likely mechanism of growth inhibition. We next turned our attention to CDK16 and found that its knockdown inhibited the phosphorylation of the Rb protein at S780 and increased expression of p27. Both of these effects were phenocopied in NRAS and KRAS-mutant cancer cells by dabrafenib but not vemurafenib. Combined silencing of NEK9 and CDK16 were associated with enhanced inhibition of melanoma cell proliferation and a greater induction of cell cycle arrest. In summary, we have identified dabrafenib as a potent inhibitor of NEK9 and CDK16, and suggest that inhibition of these kinases may have activity against cancers that do not harbor BRAF mutations.

#3974

Combination of birinapant and TRAILR2 agonist antibody enhances cell death in HPV-positive head and neck squamous cell carcinomas.

Yi An, Lillian Sun, Adeeb Derakhshan, Sophie Carlson, Zhong Chen, Carter Van Waes. _NIH, Bethesda, MD_.

Head and neck squamous cell carcinomas (HNSCC) induced by human papilloma virus (HPV) have increased recently in the US. Analysis of HNSCC TCGA datasets provide evidence for distinct alterations in expression of NF-κB components and death pathways in HPV(+) HNSCC. Previously, we have found that Birinapant, a novel SMAC mimetic that inhibits inhibitor of apoptosis proteins (IAPs), exhibits single agent activity and sensitizes a subset of HPV(-) HNSCC cell lines to death agonists like TNF-α and TRAIL. Birinapant exhibited anti-tumor effects in vivo for HNSCC with FADD amplification. However, Birinapant alone exhibited minimal activity against HPV(+) tumor in vivo. To explore how to enhance the sensitivity of HPV(+) cells to Birinapant, we treated a panel of cell lines with Birinapant alone, and in combination with TRAIL or TNF-α in vitro. Birinapant sensitized HPV(+) cell lines UPCI-SCC-90 and UM-SCC-47 to TRAIL or TNF-α. The IC50 for Birinapant was under 50nM when combined with TRAIL or TNF-α for UPCI-SCC-90 and UM-SCC-47 cell lines. To explore the therapeutic potential of enhancing TRAILR mediated death signaling in HPV(+) HNSCC cells, we investigated the effects of an agonistic polyclonal TRAILR2 antibody. Anti-TRAILR2, TRAIL, or TNF-α alone showed little or no inhibitory effect on proliferation of UPCI-SCC-90 and UM-SCC-47 cells in vitro. However, a combination of Birinapant and TRAILR2 antibody, and triple combination of Birinapant, TRAIL, and TRAILR2 antibody showed additive or synergistic effects to inhibit cell density in a dose dependent manner. Combination of Birinapant with TRAIL, TRAILR2 antibody, or triple combination led to cell death and subG0 DNA fragmentation, as demonstrated by propidium iodide staining and flow cytometry. To further analyze the mechanism of cell death, we treated cells with the inhibitor of pan-caspase (ZVAD), caspase-8 (ZIETD), or RIPK1 (necrostatin) inhibitors. In both UPCI-SCC 90 and UM-SCC 47 cells, caspase inhibition (ZVAD and ZIETD) completely abolished the effects of Birinapant treatment in combination with TRAIL, TRAILR2 antibody, and triple combination, whereas RIPK1 inhibition (necrostatin) did not reverse the effects significantly. Our data suggest that Birinapant and TRAIL receptor mediated cell death is predominantly caspase 8 dependent. Taken together, our study indicates that TRAIL or TRAILR2 agonist antibody enhances Birinipant anti-tumor activity, and triple combination exhibits the strongest synergistic effects. The triple combination of targeting death pathway by TRAIL and TRAILR2 antibody with Birinapant could be a potent strategy to treat aggressive subset of HPV(+) HNSCC, and warrants the testing in future clinical trials. (Supported by NIDCD intramural projects ZIA-DC-000016, 74).

#3975

Target engagement, thrombocytopenia, and efficacy induced by the dual Bcl2/xL inhibitor AZD4320 are quantitatively linked by a PK/PD model in leukemia xenografts.

Francis D. Gibbons, Ammar Adam, Marie-Eve Beaudoin, Eric Gangl, Paul Secrist. _AstraZeneca, Boston, MA_.

The proteins Bcl2 and Bcl-xL are often up-regulated in cancer, and hold in check the apoptosis that would normally be initiated by accumulation of the BH3-only proteins Bax and Bak in response to genomic dysregulation. AZD4320 potently disrupts that interaction, initiating the apoptotic cascade in Bcl-2 or Bcl-xL-dependent tumors. Because platelets are known to be dependent on Bcl-xL, thrombocytopenia is an expected on-target effect. AZD4320 was administered at dose levels 0.5-10 mg/kg, both intravenously and extravascularly, to immune-compromised mice inoculated subcutaneously with the RS4;11 model of acute lymphocytic leukemia (ALL). Drug concentrations were measured by liquid chromatography-mass spectrometry (LC-MS) in plasma and tumor. A cleaved-caspase-3 ELISA was used to assess apoptotic activity in the tumor. Parallel efficacy studies, were used to assess tumor growth compared to vehicle, following tumors from initial regression at tolerated doses to regrowth. Tumors were measured using calipers, and tumor volumes computed using an ellipsoid approximation. We present a mini-physiologically based pharmacokinetic/pharmacodynamic (mPBPK/PD) model that links drug concentrations in plasma and tumor to observed caspase activity and efficacy in an integrated manner, across multiple dose levels and schedules. The tumor is modeled as a pool of sensitive cells which can be triggered rapidly by AZD4320 to apoptose, from which point they transition gradually to death, reducing tumor volume. Cleaved caspase-3 is used as a marker of apoptosis, and modeled using a sigmoidal response function with steep slope parameter. In this way, we effectively capture the transient nature of the response, despite AZD4320's long residence in the tumor. Thrombocytopenia is described not as an effect on the megakaryocyte precursors, but as a linear concentration-dependent effect on circulating platelets. Feedback from the circulation to megakaryocytes drives increased production to fill the deficit. Parameters are well-estimated throughout. Together, these components provide a comprehensive means to investigate the effects of dose and schedule with a dual Bcl2/BCL-xL inhibitor.

#3976

Antihistamines as synergists with targeted therapies in chronic lymphocytic leukemia.

Aaron P. Chanas-LaRue, James B. Johnston, Spencer B. Gibson. _University of Manitoba, Winnipeg, Manitoba, Canada_.

Chronic Lymphocytic Leukemia (CLL) is a B-cell derived cancer and is the most commonly diagnosed leukemia in older adults. Several survival signals contribute to the accumulation of CLL cells including constitutive activation the B-cell receptor (BCR) signaling pathway. Approved treatments for CLL are nucleoside analogs such as Fludarabine, alkylating agents including Chlorambucil and Bendamustine, and targeted therapies inhibiting BCR-associated kinases such as Ibrutinib and Idelalisib. Unfortunately, CLL remains incurable, but other weaknesses of the disease have been identified. Recent studies have shown CLL cells to be sensitive to lysosomal membrane permeabilization (LMP) and release of lysosomal contents due to altered sphingosine metabolism. Drugs that induce LMP are referred to as lysosomotropic agents and include antidepressants and antimalarials. These drugs accumulate in lysosomes and inhibit enzymes in the sphingolipid metabolic pathway, causing lysosomal membrane damage. Effectors are released from lysosomes including cathepsin proteases and reactive oxygen species (ROS), which cause dysfunction in cellular machinery and cell death via apoptosis. Our data indicates that kinase inhibitors induce synergic death when combined with lysosomotropic agents in vitro in many cancer models. A non-small cell lung cancer study showed that antihistamines acted as lysosomotropic agents, and were correlated with better patient outcomes when combined with chemotherapy. Therefore, the objective of this study is to characterize the cytotoxicity of antihistamines in B-cell cancer models and identify synergistic interactions with clinically relevant drugs used in CLL. We have shown that three commonly prescribed over-the-counter antihistamines, Desloratadine, Loratadine and Clemastine, induce cell death at concentrations that are clinically achievable in the B-cell lines BJAB and I83, as well as CLL patient derived primary lymphocytes. Each antihistamine caused synergic cell death in combination treatments with Ibrutinib and Idelalisib, but not Fludarabine, Chlorambucil or Bendamustine, which may indicate that the synergy is specific to kinase inhibitors. In addition, the treatment with antihistamines was shown to induce cell death at significantly lower concentrations in primary CLL cells compared to normal lymphocytes from age-matched donors. Both the antihistamine-induced cell death and combination treatments depend on intracellular soluble ROS, but the main effectors of the apoptotic pathways remain to be determined. Taken together, this study intends to exploit the vulnerabilities of CLL by repurposing allergy drugs in combination with kinase inhibitors already available for treating CLL patients.

#3977

γ-Tocotrienol and metformin are cytotoxic to prostate cancer cell lines and exhibit synergy.

Christine A. Moore, Janet W. Lightner, Michelle Duffourc, Koyamangalath Krishnan. _East Tennessee State University, Johnson City, TN_.

Metformin and γ-tocotrienol (GT3) are promising non-cytotoxic drugs for prostate cancer chemoprevention and as adjunctive treatment options for prostate cancer. Multiple mechanistic pathways may be modulated. Studies suggest that GT3, a member of the vitamin E family, and the antidiabetic agent metformin have antineoplastic properties but their effects in prostate cancer are unclear. We compared the abilities of GT3 and metformin in preventing growth of two prostate cancer cells lines (LNCaP and PC-3) and a control prostate cell line (RWPE-1) by quantifying their effects on three signaling pathways known to be pivotal in prostate carcinogenesis (AKT, MAP Kinase [pERK], and p-c-JUN).Two prostate cancer cell lines, LNCaP (androgen dependent) and PC-3 (androgen independent), and prostate control cell line RWPE-1 were treated with increasing concentrations of GT3 and/or metformin in DMEM, and cytotoxicity determined by MTS cell culture experiments. Synergy was determined using the CompuSyn program. IC50 of LNCaP with GT3 and metformin were 76.6 µM and 100.6 mM, respectively. IC50 of PC-3 with GT3 and metformin were 53.8 µM and 73.5 mM, respectively. We found synergy in LNCaP at 5 µM/mM and 10 µM/mM GT3/metformin. We found synergy in PC-3 at 5 µM/mM GT3/metformin. RWPE-1 were unaffected. We determined the effect of GT3 and metformin on signaling pathways by analyzing β-actin, p-AKT, p-c-JUN, and p-ERK in combination via Western immunoblot. Initial experiments with LNCaP and PC-3 cells showed metformin inhibits the expression of p-c-JUN and p-ERK. GT3 inhibits the expression of p-ERK but not p-c-JUN in both prostate cancer cell lines. P-AKT was not activated by GT3 or metformin. GT3 and metformin inhibit cell growth in both prostate cancer cell lines, greater in combination compared to separately (more apparent in LNCaP compared to PC-3). GT3 and/or metformin caused less growth inhibition and no effects on expression of proteins p-AKT, p-c-JUN or p-ERK in the RWPE-1 prostate control cells. There is synergy with GT3 and metformin in inhibiting prostate cancer cell growth. GT3 inhibits expression of p-ERK, and metformin inhibits expression of both p-c-JUN and p-ERK. Although AKT is an important target in prostate cancer therapy, AKT is not modulated by either metformin and GT3 but modulates p-c-JUN and p-ERK. Cytotoxic effects of GT3 and metformin are independent of androgen sensitivity although androgen-independent PC-3 cell lines are more sensitive. GT3 and metformin are promising chemopreventive agents that need to be examined mechanistically. Recent literature suggests metformin influences methylation in breast cancer. We are in the process of analyzing GT3 and metformin and how they alter methylation pathways in prostate cancer.

#3978

Therapeutic evaluation of eprinomectin against advanced prostate cancer.

Angela Lincy Prem Antony Samy, Syed M. Ali, Subramanyam Dasari, Gnanasekar Munirathinam. _University of Illinois, Rockford, IL_.

Prostate Cancer (PCa) is the second most common cancer among men in United States after skin cancer. According to the American Cancer Society, in 2017 alone there will be 16,130 new cases of PCa and 26,730 deaths from PCa. Conventional chemotherapeutic drugs available for PCa treatment are limited due to toxicity and resistance issues. Therefore, there is an urgent need to develop more potent treatment for advanced PCa. Eprinomectin (EP) belongs to the class of avermectins which are lactone derivatives with potent anti-helminthic and anti-cancer properties. EP has been widely used to treat parasitic diseases in cattle and is found to be less toxic than the other avermectin class of drugs. The goal of our current study is to test the anti-cancer efficacy of EP on PCa cells. Initially cell viability assays were performed on PCa cell lines such as PC3, DU145, LNCaP, VCaP, and 22RV1 to assess the anti-cancer efficacy of EP. Among all the PCa cell lines tested, PC3 and DU145 showed more sensitivity to EP. Specifically, cell viability assays indicated that EP reduced the viability of PC3 and DU145 PCa cells by 50% at 25μM concentration. Next, in soft agar assay, EP effectively inhibited the anchorage independent growth of prostate cancer cells in vitro. Furthermore, wound healing assay results suggested that EP targeted the migratory property of PC3 and DU145 cell lines. In addition, apoptosis assay using Annexin-FITC and propidium iodide staining revealed that EP targets PC3 and DU145 cells by inducing apoptosis. Cell cycle analysis showed that EP arrested the PC3 and DU145 cells in G0 phase of the cell cycle. Interestingly, our results also showed that EP targets the PCa cells by inducing oxidative stress. Real time PCR analysis showed that EP effectively inhibited the expression of various cancer stem cell markers such as ALDH1, Sox-2, Nanog, Oct3/4 and CD44. In PC3 and DU145 cell lines, EP effectively inhibited the activity of Alkaline Phosphatase suggesting that EP could target pluripotent stem cells. In addition, treatment of PC3 and DU145 cells with EP resulted in the translocation of ß-catenin from the nucleus to the cytoplasm indicating that EP antagonizes Wnt/ß-catenin signaling pathway. Furthermore, EP also decreased the expression of the downstream target genes of ß-catenin such as cyclin D1 and c-Myc in PC3 and DU145 cells. EP also downregulated the expression of other key cell cycle markers such as cyclin D3, CDK4 and anti-apoptotic markers such as Bcl-2, Bcl-XL, XIAP, c-IAP2 and survivin in PC3 and DU145 cells. On the contrary, treating PC3 and DU145 cells with EP resulted in the activation of DNA damage marker, pH2AX and upregulation of pro apoptotic marker, Bad with concomitant activation of Caspase-9 and Caspase-3. Based on our results, EP appears to potently target advanced PCa cells by inhibiting tumorigenic and metastatic properties of advanced PCa cells in vitro. Further in vivo and preclinical studies are warranted to test the efficacy of EP on PCa.

#3979

**Active components of** Alpinia katsumadai- **induced cell apoptosis and autophagy in human pancreatic cancer cells.**

Weixiao An, Honglin Lai, Yangyyang Zhang, Xiukun Lin, Shousong Cao. _Southwest Medical University, Luzhou City,Sichuan Province, China_.

Alpinia katsumadai, as a traditional Chinese medicine, exhibited potent anti-oxidative and anticancer effects. Studies have shown that the effect of the active components of Alpinia katsumadai (ACAK) on anticancer is via apoptosis or autophagy in colorectal and glioblastoma cancer cells. However, the effects of ACAK on anticancer effect and inducation of apoptosis and autophagy in pancreatic cancer cells are still unknown. Therefore, we investigated the effects of ACAK on cytotocixity, apoptosis and autophagy as well as associated mechanisms in pancreatic cancers Panc-1 and Panc-28 cells. We found that ACAK showed greater inhibitory effects on the proliferation of Panc-1 and Panc-28 cells in a dose- and time-dependent manners (IC50 0.1692 and 0.1554mg/ml, respectively) compared to a variety of other cancer cells including breast cancer MDA-MB-468 cells, melamoma A875 cells, lung cancer A549 cells, colon cancer HCT-116 cells, and glioblastoma cancer U87 cells (IC50 0.2302, 0.2586, 0.2642, 0.3006, and 0.4298, respectively) by CCK-8 assay. Furthermore, ACAK induced cell apoptosis in pancreatic cancer Panc-1 and Panc-28 cells in a dose- dependent manner by Hochest 33342-PI and AnnexinV-PI dual staining analysis. Moreover, the expression levels of apoptosis related proteins casepase 3, casepase 9, and PARP were significantly up-regulated by Western blotting analysis. The study of cell autophagy was dectected by mRFP -GFP-LC3 adenovirus and western-blotting. and the results showed that the expression levels of autophagy-related protein beclin-1 and LC3-II were significanlt up-regulated by mRFP-GFP-LC3 adenovirus and western-blotting analysis. Our findings demonstrated that ACAK had greater cytotoxicity than a variety of other cancer cells by inducing apoptosis and autophagy in pancreatic cancer Panc-1 and Panc-28 cells. Therefore, ACAK may be developed as a novel anticancer agent for the treatment of pancreatic cancer clinically.

#3980

**Dual targeting mitochondrial and cytosolic one-carbon metabolism via the proton-coupled folate transporter with novel 5-substituted pyrrolo[3,2-** d **]pyrimidine antifolates.**

Aamod S. Dekhne,1 Gregory S. Ducker,2 Josephine Frühauf,1 Khushbu Shah,3 Md. Junayed Nayeen,3 Adrianne Wallace-Povirk,1 Carrie O'Connor,1 Zhanjun Hou,1 Lisa Polin,1 Aleem Gangjee,3 Joshua D. Rabinowitz,2 Larry H. Matherly1. 1 _Wayne State University, Detroit, MI;_ 2 _Princeton University, Princeton, NJ;_ 3 _Duquesne University, Pittsburgh, PA_.

Cellular one-carbon (1C) metabolism generates a host of metabolites especially critical to cancer cells. Classical 1C inhibitors often primarily target a single enzyme, commonly leading to drug resistance and necessitating the development of novel compounds targeting multiple enzymes. We synthesized several novel 5-substituted pyrrolo[3,2-d]pyrimidine compounds (AGF291, AGF320, and AGF347) with transport by the proton-coupled folate transporter (PCFT), a folate transporter with a narrow physiological niche, but commonly expressed in many solid tumors. These analogs showed substantial inhibition in proliferation assays with Chinese hamster ovary sublines engineered to individually express PCFT or the ubiquitously-expressed reduced folate carrier (the major tissue folate transporter), as well as in the PCFT-expressing human cancer cell lines H460 (large cell lung carcinoma), HCT-116 (colorectal carcinoma), and MIA PaCa-2 (pancreatic ductal adenocarcinoma). Full abrogation of inhibitory effects for all compounds required co-treatment with both adenosine and glycine, suggesting dual-targeting of cytosolic de novo purine biosynthesis and mitochondrial 1C/glycine metabolism. For cytosolic de novo purine biosynthesis, lack of protection by 5-aminoimidazole-4-carboxamide (AICA) suggested AICA ribonucleotide formyltransferase (AICARFTase), the second folate-dependent enzyme, as the likely enzyme target. AICARFTase inhibition was confirmed in intact H460 and HCT-116 cells by measuring accumulation of AICAR by targeted metabolomics. Targeting of mitochondrial enzymes in H460 and HCT-116 cells was suggested by selective inhibition of incorporation of [3-14C]serine over [14C]formate into the purine intermediate [14C]formyl glycinamide ribonucleotide. [2,3,3-2H]Serine isotope label scrambling analysis confirmed the mitochondrial target to be serine hydroxymethyltransferase2 (SHMT2). SHMT2 is a potentially promising anticancer drug target as its expression is highly correlated with the malignant phenotype across a broad spectrum of cancers beyond lung and colon, including breast, glioma, and liver. Indeed, our initial in-vivo studies with AGF291 suggest potent efficacy toward both MIA PaCa-2 and H460 xenograft mouse models. Our studies demonstrate the potential for tumor-selective dual targeting of both mitochondrial and cytosolic 1C metabolism to overcome resistance to classical 1C inhibitors.

#3981

Enhancement of the anticancer activity of methylenetetrahydrofolate dehydrogenase knockdown by folate depletion with carboxypeptidase G2.

Gulam M. Rather,1 John E. Kerrigan,2 Kathleen W. Scotto,1 Joseph R. Bertino1. 1 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ;_ 2 _Rutgers Biomedical Health Sciences, Rutgers, The State University of New Jersey, Piscataway, NJ_.

The mitochondrial folate enzyme, methylene tetrahydrofolate dehydrogenase (MTHFD2), is a promising anticancer target because 1) it is required for the generation of one-carbon units required for purine synthesis and generation of NADH/NADPH necessary for protection from ROS in the mitochondria; 2) it is markedly distinct from the cytoplasmic MTHFD1 enzyme with respect to location, catalytic activity and cofactors; 3) it is expressed at low levels in proliferating normal cells; 4) in contrast, it is highly expressed in a variety of rapidly proliferating malignant tumors; 5) knockdown (KD) of MTHFD2 has been shown to inhibit proliferation in a subset of tumor cells in vitro; and 6) MTHFD2 KD is predicted to have metabolic consequences (glycine auxotrophy, folate deficiency) that could be exploited for combination therapy. In this study we tested the hypotheses that downregulation of MTHFD2 will result in relative folate deficiency, which can be exploited by using a folate-depleting enzyme, carboxypeptidase G2 (CPG2), to enhance tumor cell kill. CPG2 is approved by the FDA for the treatment of methotrexate overdose in patients. It acts to inactivate MTX or naturally occurring folates by hydrolyzing the terminal glutamate from folates and MTX, generating a pteroate and glutamate. As pteroates cannot be used to resynthesize folates, or MTX, this treatment depletes cells from MTX or folates. The combination of CPG2 in cell lines, MCF7 and T47D with MTHFD2 KD, was done and the actual increase in growth inhibition showed enhanced antitumor effects, indicating that when effective inhibitors of MTHFD2 are available, this combination may have widespread clinical usefulness, especially in rapidly proliferating tumors that express high levels of MTHFD2.

#3982

Dual inhibition of HDAC and PI3 kinase signaling pathways with CUDC-907 in treatment of p53-driven medulloblastoma.

Jennifer S. Ronecker,1 Julia Pazniokas,1 Anubhav G. Amin,1 Raphael Salles Scortegagna de Medeiros,2 Sidnei Epelman,2 Chirag Ghandi,1 Nelci Zanon,2 Meic H. Schmidt,1 Meena Jhanwar-Uniyal1. 1 _New York Medical College, Valhalla, NY;_ 2 _Hospital Santa Marcelina, Sao Paulo, Brazil_.

Comprehensive studies of the medulloblastoma (MB) genome, epigenome and transcriptome have placed MB in four molecular subgroups: WNT, SHH, and two non-WNT/non-SHH: Group 3 and Group 4. Furthermore, studies suggest that p53-mutated SHH MBs frequently harbor genetic anomalies involving MYCN and GLI2 amplifications, which confer poor prognosis and resistance to treatments. The phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) intracellular signaling pathway that regulates cell growth, survival and invasion, is commonly deregulated in MB. Therefore, the PI3K/Akt/mTOR pathway may be a suitable target for therapeutic intervention. Moreover, Histone deacetylases (HDAC) regulate cancer initiation and progression, and studies have shown that small molecule inhibitors of HDAC can effectively limit MB proliferation. We aim to determine the status of tumor suppressor gene p53 in defining metastatic potential in SHH-MB and to establish the therapeutic efficacy of targeting these pathways in p53-driven MB cells with a dual agent or individual agents. Immunohistochemistry and FISH were used to determine the status of mutant p53 and MYC amplification or iso-p53, respectively, in MB tumors (n=41). p53-mutant MB cell line was used to investigate the signaling pathway that regulates proliferation, migration, and drug resistance using inhibitors of HDAC (LBH-589) and PI3K/mTOR (BKM-120/rapamycin) or CUDC-907 (dual inhibitor of HDACs and the PI3K/AKT). Results demonstrated that: 1) GAB-1 was highly expressed in the Shh group (82%) and KV1 expression was evenly distributed in all subtypes; 2) loss of p53 and overexpression of MYC varied in each subtype, but did not correlate with metastasis; 3) combined treatment with LBH-589 and BKM-120 or single treatment with CUDC-907 reduced cell proliferation, migration and S-phase entry; 4) MB cells were resistant to BKM -120, while LBH-589 or CUDC-907 caused apoptosis; 5) tumor formation was suppressed by BKM-120 given with mTOR inhibitors; 6) AKT and 4E-BP1 dephosphorylated following treatments with BKM-120 or CUDC-907. In conclusion, despite current clinical limitations, dual HDAC and PI3K inhibitors may benefit the understanding of aberrant signaling pathways in genetically driven MB, and may provide basis for future targeted therapies.

#3983

**Tumor-targeting with novel dual-targeted 6-substituted thieno[2,3-** d **]pyrimidine antifolates via cellular uptake by folate receptor α, and inhibition of de novo purine nucleotide biosynthesis.**

Adrianne Wallace-Povirk,1 Nian Tong,2 Carrie O'Connor,1 Zhanjun Hou,1 Aleem Gangjee,2 Larry Matherly,1 Xilin Zhou2. 1 _Wayne State University, Detroit, MI;_ 2 _Duquesne University, Pittsburgh, PA_.

Epithelial ovarian cancer (EOC) is the deadliest gynecologic cancer, with limited progress in targeted therapy or in combating drug resistance. Reflecting this, there is an urgent need to develop novel inhibitors with minimal toxicity. EOC is associated with increasing expression of folate receptor alpha (FRα) with increasing stage and grade of disease. Of particular interest are targeted therapeutics toward FRα which spare transport by the ubiquitously expressed reduced folate carrier (RFC). FRα is a viable target for tumor selectivity with limited expression in normal tissues, where it is only expressed on apical membranes. We previously described 6-substituted thieno[2,3-d]pyrimidine (6TP) benzoyl antifolates with bridge lengths of 2-8 carbons as potent and selective inhibitors of tumor cells including EOCs that express FRs. The most potent compound (AGF50) included a 4-carbon bridge and showed FRα selectivity and inhibition of de novo purine biosynthesis. We synthesized AGF50 analogs with various aryl side-chain modifications (e.g., thiophene, furan, pyridine) including 2'fluorine substitutions. Toward isogenic Chinese hamster ovary cell lines individually expressing FRα, RFC and the proton-coupled folate transporter (PCFT), the 6TP analogs were completely selective toward FRα over RFC and PCFT, manifested as inhibition of cell proliferation with nmolar IC50s. Activity of this series extended to KB human tumor cells and human EOC cell lines (e.g., IGROV1). Drug effects were abolished with excess folic acid, confirming FRα uptake. Adenosine completely protected cells from the inhibitory effects of 6TPs in vitro, suggesting inhibition of one-carbon transfers mediated by glycinamide ribonucleotide (GAR) formyltransferase (GARFTase) and/or 5-aminoimidazole-4-carboxamide (AICA) ribonucleotide formyltransferase (AICARFTase), leading to synthesis of purine nucleotides. Protection from 6TPs by AICA was incomplete (in contrast to a "pure" GARFTase inhibitor AGF94), suggesting inhibition of AICARFTase. Whereas AGF94 potently inhibited GARFTase (>98%) in KB cells, reflected in [14C]glycine incorporation into [14C]formyl GAR, inhibition by the 6TPs was incomplete (75-90%) up to 1 μM drug. AICARFTase inhibition was confirmed by measuring accumulation of AICA ribonucleotide (ZMP), although this varied for different 6TP analogs. Our studies establish an important structure-activity relationship for novel FRα-targeted 6TPs with aromatic side-chain modifications and imply the therapeutic potential of targeting AICARFTase for EOC.

#3984

Combination S6K and TAM tyrosine kinase targeting in PTEN-deficient glioma.

Kelli N. Ennis, Pranjal Sarma, Catherine A. Behrmann, Jie Wang, David R. Plas. _Univ. of Cincinnati, Cincinnati, OH_.

Loss of PTEN is one of the most frequent driver events in primary glioblastoma, conferring the ability to reconfigure metabolism and enhance apoptosis resistance to transformed glial cells. The ribosomal S6 protein kinases (S6Ks) are hyperactivated in PTEN-deficient settings, and genetic inactivation studies have shown that S6K1 deficiency can reduce the oncogenic growth of PTEN-deficient neoplasia. Investigating pharmacologic agents, we found that the S6K inhibitor LY-2584702 selectively elicits cytotoxic responses in PTEN-deficient cells when combined with the TAM kinase inactivating agent BMS-777607. Combined knockdown of TAM kinases sensitized cells to single agent treatment with LY-2584702. Conversely, expression of activated S6K1 or elevated AXL protected from combination treatment. These results indicate that both the TAMS and S6K1 are key regulators of survival in PTEN deficient cells.

#3985

Identifying and drugging glucuronidase targets in the human gut microbiome.

Jeffrey P. Hymes, Bret D. Wallace, Matthew R. Redinbo, Ward Peterson. _Symberix, Durham, NC_.

Background

The gut microbiome is a vast, untapped source of non-bactericidal drug targets with applications in therapeutic oncology. Enteric bacteria express β-glucuronidases (GUS) that contribute to tumorigenesis, neoplastic disease and cancer treatment outcomes. We previously reported in 2010 that a microbial-targeting, drug-like small molecule (SBX-1) protects mice from irinotecan (IRI)-induced diarrhea by blocking bacterial GUS-mediated reactivation of SN-38 from the inactive glucuronide (SN-38G). SBX-1 is >100-fold more potent against E. coli GUS than human GUS. We recently reported (2017) that E. coli GUS is one of nearly 300 distinct microbial GUS enzymes (GUSome) expressed in the human gut microbiome. The objectives of this work are two-fold: (1) Define for the first time the subset of GUS enzymes that can metabolize SN-38G; (2) Evaluate the selectivity of SBX-1 against GUS enzymes that metabolize SN-38G vs those that do not.

Methods

We exploited differences in the fluorescence properties of SN-38G and SN-38 to develop a high-throughput, cost-efficient assay that kinetically monitors GUS-mediated cleavage of SN-38G to SN-38. SN-38G concentrations were monitored at excitation/emission wavelengths of 230/420 nm, respectively. Cleavage of SN-38G to SN-38 results in a measurable decrease in 420 nm fluorescence. Thirteen distinct bacterial GUS enzymes representative of the structural diversity in the GUSome were expressed, purified, and screened to determine relative catalysis of SN-38G in the absence or presence of SBX-1. Enzymes were tested at a single concentration (20 nM) with a single concentration of SN-38G (15 µM). Initial velocities were calculated and normalized.

Results

The GUS enzymes tested exhibited a broad range of SN-38G catalysis. Rank-ordered from most efficient to least efficient: Escherichia coli, Eubacterium eligens, Bacteroides fragilis, Bacteroides uniformis (Type 2), Clostridium perfringens, H11G11, Bacteroides uniformis (Type 1), Faecalibacterium prausnitzii, Bacteroides uniformis (Type 3), Lactobacillus rhamnosus, Bacteroides ovatus, Ruminococcus gnavus, and Bacteroides dorei. SBX-1 is highly potent against E. coli, weakly potent against E. eligens, and not potent against other bacterial GUS orthologues. For each GUS orthologue, catalytic efficiencies for SN-38G and Ki values for SBX-1 will be presented.

Conclusions

In our study, we identified the subset of GUS enzymes that metabolize SN-38G most efficiently. We evaluated the selectivity of SBX-1, showing that it is highly potent against the most efficient enzyme (E. coli GUS), but not potent against other GUS enzymes. Identification of the specific bacterial targets responsible for SN-38G reactivation will guide the design of GUS inhibitors to improve IRI outcomes. Current efforts are underway to characterize a library of novel SBX-1 analogs that show differential selectivity across the GUSome.

#3986

HDAC4 degradation by combined TRAIL and valproic acid treatment induces apoptotic cell death of TRAIL-resistant head and neck cancer cells.

Bok-Soon Lee,1 Yeon Soo Kim,2 Jeon Yeob Jang,1 Hyo Jeong Kim,1 Myeong-Hoon Lee,1 Jae Won Chang,3 Bon Seok Koo,3 Ho-Ryun Won,1 Yong-Sung Kim,1 Chul-Ho Kim1. 1 _Ajou University, Suwon, Republic of Korea;_ 2 _Konyang University Hospital, Konyang University Myunggok Medical Research Institute,, Daejeon, Republic of Korea;_ 3 _Chungnam National University, Daejeon, Republic of Korea_.

Purpose: Although TRAIL can directly induce death in cancer cells via DR4 and DR5 receptors, it appears that TRAIL resistance exists in many cancers. This study focuses on anti-cancer drugs for TRAIL-resistant head and neck cancer (HNC) to provide a further step toward effective cancer therapy.

Materials and Methods: The combined effects of TRAIL and VPA on cell viability were assessed via MTT and flow cytometric assays. Western blotting was used to determine cell death signaling.

Results: The results indicate that combined TRAIL and VPA treatment greatly reduced the cell viability and thus induced cell death relative to treatment with TRAIL or VPA alone in TRAIL-resistant HNC cells. A caspase-dependent signaling pathway was shown, and combined treatment with TRAIL and VPA also significantly decreased the expression of HDAC4. When we pretreated cells with z-VAD followed by combined treatment with TRAIL and VPA, the cell death was blocked with no decreased expression of HDAC4. To confirm the cell death involved in HDAC4 in HNC cells, we pretreated siRNA of the HDAC 4, followed by treating the TRAIL and VPA. The results showed that the knock down of HDAC4 sensitized the TRAIL-resistant HNC cells to apoptotic cell death. Finally, we showed a high expression of HDAC4 in HNC tissues compared to normal tissues obtained from the same patients.

Conclusion: Combined VPA and TRAIL treatment may be a promising therapy for HNC via HDAC4 degradation.

#3987

CaMKK2 inhibition as a "dual-hit" strategy against ADT-induced osteoporosis and bone-metastatic prostate cancer.

Ushashi Dadwal, Justin Williams, Austin Pucylowski, Eric Chang, Khalid Mohammad, Theresa Guise, Uma Sankar. _INDIANA UNIVERSITY SCHOOL OF MEDICINE, Indianapolis, IN_.

Prostate cancer (PCa), the most frequently diagnosed cancer in men, will have an estimated 26,730 deaths in the US during 2017. Predominantly, these deaths will be caused due to metastatic disease. PCa preferentially metastasizes to the bone undermining its structural integrity. PCa cells rely on androgens for survival and proliferation. Consequently, androgen deprivation therapy (ADT) is the first line of defense against advanced-stage PCa. However, ADT contributes to significant decreases in bone mass, predisposing the patient to an increased risk of fractures. Hence, there is a critical need for therapies that protect against bone metastatic disease as well as reverse ADT-induced bone loss in PCa patients. Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a direct target of the androgen receptor (AR) and is highly over-expressed in androgen-dependent and independent forms of PCa. In vitro studies have shown that its inhibition suppresses the growth and migration of PCa cells. We observed that CaMKK2 inhibition suppresses 3-D spheroid formation by the androgen-independent PCa cell line C4-2 but not by the androgen-dependent PC3 cells. However, the exact downstream mechanism by which CaMKK2 regulates PCa growth remains unknown. More recently, CaMKK2 was shown to play a role in both the anabolic and catabolic pathways of bone remodeling. Genetic ablation and pharmacological inhibition of CaMKK2 via STO-609 stimulates osteoblasts and suppresses osteoclasts and confers protection from age- and ovariectomy-induced bone loss in mice. Based on these preliminary data, we hypothesize that inhibition of a single target, CaMKK2, will result in the therapeutic alleviation of two chief complications in advanced-stage PCa, i.e., bone metastatic tumor burden and ADT-induced bone loss. We performed sham or bilateral orchiectomy (ORX) on pretreated (saline/STO-609) 5-week-old male athymic mice (n=15 per cohort). Tri-weekly intraperitoneal (i.p.) injections were continued for 12 weeks. Additionally, two weeks after surgery, sham and ORX mice were intra-tibially injected with C4-2B cells. Micro-computed tomography analyses indicated a prevention of ORX-induced bone loss in STO-609 treated mice compared to saline treated controls (3-fold, p<0.05). Radiographic and histomorphomteric analyses reveal a decrease in C4-2B-initiated bone lesions in STO-609 treated mice compared to the saline treated cohorts. Taken together, our studies represent a highly novel and unique approach in the treatment of advanced-stage PCa.

#3988

Legumain regulates advanced prostate cancer via HSD17B4.

Sharon L. Eddie, Alice V. Ormrod, Rich Williams, Paul B. Mullan. _Queen's University Belfast, Belfast, United Kingdom_.

Prostate cancer is the most common male cancer diagnosis. However, current treatment is limited and patients often develop resistant, metastatic disease. Novel targeted therapies which prevent recurrence and metastasis are essential to improve patient prognosis. A possible novel therapeutic target, the enzyme Legumain (LGMN), is overexpressed in advanced prostate cancers. Conversely, cystatin E/M (CST6), the endogenous inhibitor of LGMN, is repressed, suggesting a role for uncontrolled LGMN activity in prostate carcinogenesis. Despite this implied role, little is known about LGMN functionality and how it may regulate to tumorigenesis.

To clarify how LGMN signalling impacts prostate cancer formation and metastasis, novel small molecule inhibitors were developed and tested across a panel of normal cell lines and prostate cancer lines. Our data demonstrate that prostate cancer cells are 'addicted' to LGMN signalling. Endogenous LGMN expression and enzymatic activity is heightened in prostate cancer cell lines, loss of which, via small molecule inhibitors or RNAi, is selectively toxic to cancer cells, with limited effect in normal cell lines. Further, LGMN appears to be pivotal to tumorigenic growth, with notable loss of colony formation in anchorage-independent growth assays when LGMN is depleted.

The biology surrounding LGMN is poorly characterised and to date few substrates have been confirmed. In order to identify novel LGMN interactors/substrates with potential roles in tumorigenic signalling, we performed BioID, a method which employs promiscuous biotin ligase labelling of proximal proteins. BioID identified a number of proteins which associate with LGMN including a known interactor, HSP90. A number of novel interactors were also identified including HSD17B4, a bifunctional steroidogenic enzyme previously been reported to associate with poor clinical outcomes in prostate cancer and to regulate resistance to anti-androgen therapies. Given the role of HSD17B4 in steroidogenesis we postulate that this LGMN-HSD17B4 interaction may represent one route through which LGMN promotes tumorigenesis and drug resistance (particularly to androgen deprivation therapies) in advanced disease. Reassuringly, knockdown of HSD17B4 reduced cell proliferation in a cancer-specific manner, similar to LGMN knockdown/inhibition. Ongoing work will delineate how LGMN and HSD17B4 interact to promote carcinogenesis and resistance to anti-androgen therapies.

There are limited options available to patients with advanced castrate-resistant prostate cancer. These data seek to clarify the role LGMN plays in prostate tumour growth and metastasis with the ultimate aim of improving patient outcomes and potentially providing novel therapeutic options, particularly for the clinical management of castrate-resistant prostate cancers.

#3989

Dual targeting of ErbB2/ErbB3 for treatment of SCL3A2-NRG1-mediated lung cancer.

Jeong Yeon Jo,1 Dong Hoon Shin,1 Ji-Youn Han2. 1 _Graduate School of Cancer Science and Policy, National Cancer Center, Goyang, Republic of Korea;_ 2 _National Cancer Center, Goyang, Republic of Korea_.

We previously identified an oncogenic fusion gene involving the 5′ region of SLC3A2 and the 3′ region of NRG1 in a patient with invasive mucinous adenocarcinoma (IMA) of the lung. Here, we characterized the SLC3A2-NRG1 fusion gene in non-small cell lung cancer (NSCLC) and established an effective therapy for patients with SLC3A2-NRG1 fusion-positive cancer. The SLC3A2-NRG1 fusion product was composed of the SLC3A2 transmembrane domain and the epidermal growth factor (EGF)-like domain of the neuregulin 1 (NRG1) protein. The NRG1 family is classified as a ligand of the ERBB family. We identified ERBB3 and ERBB4 in the ERBB family as binding partners of the SLC3A2-NRG1 fusion protein via ligand and receptor binding assays. We confirmed that SLC3A2-NRG1 increased formation of a heterocomplex of ERBB3, which lacks tyrosine kinase activity, with ERBB2 using immunofluorescence staining, FACS analysis, and immunoprecipitation. Activation of the ERBB2-ERBB3 heterocomplex by SLC3A2-NRG1 increased colony formation and tumor growth in vivo in a xenograft model through the downstream signaling pathways of PI3K-AKT and MAP kinase. siRNA specific for ERBB2 and ERBB3, pertuzumab (a monoclonal antibody for ERBB2), lumretuzumab (a monoclonal antibody for ERBB3), and afatinib (an ERBB2 kinase inhibitor) all decreased ERBB2-ERBB3 heterocomplex formation, phosphorylation of each protein, and their downstream signaling. In addition, single treatment with pertuzumab, lumretuzumab, or apatinib decreased tumor volume and weight, whereas combination treatment with these drugs and paclitaxel enhanced generation of cleaved-caspase 3, PARP, and TUNEL-positive cells compared with each single treatment. Thus, the SLC3A2-NRG1 fusion gene plays an important role in lung cancer cell proliferation and tumor growth by promoting generation of the ERBB2-ERBB3 heterocomplex, its phosphorylation, and activation of the PI3K/ERK/mTOR signaling pathway. Inhibition of either ERBB2 or ERBB3 alone did not completely shut down downstream signaling of ERBB2 and ERBB3; however, inhibition of both ERBB2 and ERBB3 blocked downstream signaling activated by SLC3A2-NRG1 fusion. ERBB2 and ERBB3 might be promising targets for treatment of SLC3A2-NRG1-positive tumors.

#3990

Clinicopathologic findings and in vitro efficacy of MYC-targeting agentsin NUT midline carcinoma.

Soyeon Kim,1 Minsun Jung,2 Heae Surng Park,3 Sun Och Yoon,4 Soon Won Hong,3 Weon-Seo Park,5 Bhumsuk Keam,6 Hyun Jik Kim,7 Dong-Wan Kim,6 Young Tae Kim,8 Dae Seong Heo,6 Tae Min Kim,6 Yoon Kyung Jeon2. 1 _Seoul National University, Cancer Research Institute, Seoul, Republic of Korea;_ 2 _Department of Pathology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea;_ 3 _Department of Pathology, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Republic of Korea;_ 4 _Department of Pathology, Yonsei University College of Medicine, Seoul, Republic of Korea;_ 5 _Department of Pathology, Center for Prostate Cancer, National Cancer Center, Goyang, Republic of Korea;_ 6 _Seoul National University, Cancer Research Institute, Department of Internal Medicine, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea;_ 7 _Department of Otorhinolaryngology, Seoul National University College of Medicine, Seoul, Republic of Korea;_ 8 _Department of Thoracic and Cardiovascular Surgery, Seoul National University College of Medicine, Seoul, Republic of Korea_.

Purpose: NUT midline carcinoma (NMC) is an aggressive and poorly differentiated carcinoma defined by chromosomal rearrangements of NUT gene, which is fused to the BET family genes (BRD3/4). Although NMC is extremely rare and mostly reported in a Western population, clinico-pathologic findings of Asian NMC patients are not well studied. Here, we performed the clinicopathologic analysis of Korean NMC cases and in vitro efficacy of MYC-targeting agents against patient-derived NMC cell lines.

Experimental Design: A total of 12 NMC patients (3 thoracic and 9 head and neck origins) were collected in Korean multi-centers by immunohistochemistry (IHC) and fluorescence in situ hybridization for NUT. IHC staining was performed for c-MYC, p53, EGFR, HER2, and PD-L1. NMC cell lines (SNU-2972-1, SNU-3178S, HCC2429, and Ty-82) were exposed to MYC-targeting agents including BET (I-BET, OTX-015, and AZD5153) and HDAC (vorinostat, romidepsin, panobinostat, and CUDC-907) inhibitors. Cell viability assays were performed and IC50 values were determined.

Results: Primary sites of tumor included sinonasal area (n=7), salivary gland (n=2), and thorax/lung (n=3). The median age was 47.5 years (range, 8-73 years) with equal male-to-female ratio. NUT immunostaining showed diffuse positivity in all except for one who had focal NUT positivity. C-MYC and p53 were expressed in all evaluated cases. PD-L1 expression was observed only in a patient (8.3%). Seven of 12 patients died at 3-24 months (median, 9 months) after diagnosis. Long-term survivor was observed in one patient with stage IV NMC who received mass excision plus metastasectomy followed by 6 cycles of docetaxel plus cisplatin and radiotherapy (survival > 27 months). Although BET and HDAC inhibitors showed limited in vitro efficacies against NMC cells, a dual HDAC/PI3K inhibitor, CDUC-907 exhibited the most potent efficacy with the IC50 of 6.2 to 9 pmol/L.

Conclusions: Korean NMC patients pursued grave prognosis like Western patients except one long-term survivor who received tri-modality treatments. Dual HDAC/PI3K inhibitor might be promising in NMC treatment.

#3991

Novel small molecule inhibitors of p300/CBP down-regulate androgen receptor (AR) and c-Myc for the treatment of prostate cancer and beyond.

Neil Pegg,1 Jenny Worthington,2 Barbara Young,3 Amy Prosser,3 Luke Gaughan,4 Gary Spencer,5 Tim Somervaille,5 Julie Burns,6 Margaret Knowles,6 Nigel Brooks1. 1 _CellCentric Ltd., Cambridge, United Kingdom;_ 2 _Axis Bioservices, Coleraine, United Kingdom;_ 3 _Sygnature Discovery, Nottingham, United Kingdom;_ 4 _Northern Institute for Cancer Research, Newcastle, United Kingdom;_ 5 _CRUK Manchester Institute, Manchester, United Kingdom;_ 6 _St. James's University Hopsital, Leeds, United Kingdom_.

Background: E1A binding protein (p300) and CREB binding protein (CBP), two paralogue histone acetyl transferase proteins, act as transcriptional co-activators of a variety of cancer related genes. We have developed CCS1477, a potent, selective and orally active small molecule inhibitor of the bromodomain of p300/CBP and investigated its role in regulating AR and c-Myc expression and function, for the treatment of prostate cancer and haematological malignancies. We have also examined the role of p300/CBP in driving synthetic lethality in tumours with loss of function mutations (eg. bladder cancer).

Methods: Potency and functional activity was evaluated in a panel of prostate cells lines representing hormone responsive, hormone independent and castration resistant disease. Effects of CCS1477 on AR, AR-V7 splice variant and c-Myc protein, as well as KLK3, c-Myc, UBE2C, CCNA2 and TMPRSS2 gene expression, were assessed. Inhibition of proliferation and function by CCS1477 was also examined in acute myeloid leukaemia cell lines and patient derived primary AML cells. In addition, potency was determined in bladder cell lines possessing a loss of function mutation in p300/CBP and compared to wild type.

Results: CCS1477 is a potent inhibitor of cell proliferation in castration resistant prostate cell lines (IC50 = 96nM 22Rv1; 49nM VCaP) with minimal effect in AR-ve lines (PC3 and DU145). Treatment of 22Rv1 and VCaP cells with CCS1477 significantly reduced expression of KLK3, UBE2C and CCNA2 in the presence and absence of enzalutamide indicating compromised signalling via AR and AR-SV. Furthermore, AR and AR-SV protein levels were inhibited in response to CCS1477 treatment. Utilising an enzalutamide-resistant cell line (LNCaP-ARF876L), CCS1477 treatment down-regulated both androgen and enzalutamide-stimulated KLK3 and TMPRSS2 gene expression. CCS1477 dosed at 20mg/kg qd caused complete tumour growth inhibition in a 22Rv1 xenograft model. CCS1477 is also a potent inhibitor of proliferation in AML cell lines (IC50 ~ 100nM; THP-1; MV4-11), with effects mediated by G1 cell cycle arrest and accompanied by myeloid differentiation. Comparable results are observed on patient derived primary AML cells. In bladder cancer cell lines, we observed differential sensitivity to CCS1477 with p300/CBP deficient lines (IC50 = 300nM VM-CUB-2 and 647V) compared with wild type (no activity at 30uM, RT112).

Conclusions: Taken together these data support the clinical testing of p300/CBP inhibition in patients in three settings; (i) castration resistant prostate cancer through down-regulating AR, AR-SV and c-Myc expression and function; (ii) haematological cancers by effects on cell cycle arrest and myeloid differentiation, and (iii) patients with loss of function mutations in p300 or CBP by driving synthetic lethality.

#3992

Varlitinib demonstrates tumor regression and vessel normalization in ErbB-dependent and mutated beta-catenin hepatocellular carcinoma patient-derived xenograft model.

Wai Ho Shuen,1 Richard Ong,1 Chloe Yeo,1 Rebecca Banu,1 Lip Seng Koh,1 Chit Lai Chee,1 Qihui Seet,2 Lisa Ooi,2 Mark McHale,2 Bertil Lindmark,2 Han Chong Toh,1 Huynh Hung1. 1 _National Cancer Centre Singapore, Singapore, Singapore;_ 2 _ASLAN Pharmaceuticals, Singapore, Singapore_.

Hepatocellular carcinoma (HCC) is the most common form of liver cancer and current therapies such as sorafenib, regorafenib and lenvatinib, prolong median overall survival by less than a year. Hence, there is an urgent need for additional HCC therapies. Varlitinib is a potent, reversible, small molecule pan – HER inhibitor that selectively binds to EGFR, HER2 and HER4. Varlitinib is currently being clinically tested across multiple indications including biliary tract cancer and gastric cancer. In HCC patient – derived xenograft (PDX) models, varlitinib has been shown to mediate potent anti – tumour activities (ASCO 2016). To understand the molecular profiles of HCC PDX sensitive to varlitinib, we studied the ErbB family signalling in 56 HCC PDX models and varlitinib was found to be efficacious in HCC PDX with activated ErbB2 / 3 as well as mutated beta – catenin. In HCC PDX models with activated ErbB2 / 3 , treatment with varlitinib inhibited multiple proliferation and anti – apoptosis pathways including AKT, PI3K and Survivin pathways. In addition, normalisation of blood vessels by varlitinib in the PDX models, as assessed by staining with biotinylated lectin and anti – CD31, also reduced hypoxia in the tumour microenvironment as well as enhanced the infiltration of immune cells. Activating mutations in the Wnt / beta – catenin pathway lead to tumour development in HCC and occur in a subset of HCC tumours. HCC PDX models with beta – catenin mutation were found to be sensitive to varlitinib. Varlitinib led to down – regulation of mutated beta – catenin signalling, and enhanced staining of beta – catenin at the membrane instead of in the nucleus. Since activating mutations in the Wnt / beta – catenin pathway are detected in 26 – 40 % of HCC samples, our data shows that varlitinib may represent an alternative treatment for a significant subset of HCC which are ErbB / beta – catenin& #8211; dependent.

#3993

Efficacy of SHP2 phosphatase inhibition in cancers with nucleotide-cycling oncogenic RAS, NF1 loss and RAS-GTP-dependent oncogenic BRAF.

Trever G. Bivona, Robert Nichols, Franziska Haderk, Carlos Stahlhut,Christopher Schulze, Golzar Hemmati, David Wildes. _UCSF, San Francisco, CA_.

Oncogenic alterations in the RAS-RAF-MEK-ERK pathway, including mutant forms of KRAS, BRAF, and loss of the tumor suppressor and RAS GTPase-activating protein (GAP) NF1, drive the growth of a wide spectrum of cancers. While BRAF and MEK inhibitors are efficacious against BRAFV600E-driven cancers, effective targeted therapies are lacking for most cancers driven by other pathway alterations, including oncogenic KRAS, NF1 loss, and non-V600E oncogenic BRAF. Here, we show that targeting the PTPN11/SHP2 phosphatase with a novel small molecule allosteric inhibitor is effective in human cancer models bearing nucleotide-cycling oncogenic RAS (e.g. KRASG12C), NF1 loss, or RAS-GTP dependent oncogenic BRAF (e.g. class 3 BRAF mutants). SHP2 inhibitor treatment decreases oncogenic RAS-RAF-MEK-ERK signaling and cancer growth by disrupting SOS1-mediated RAS-GTP loading. Our findings illuminate a critical function for SHP2 in promoting oncogenic RAS activation and downstream signaling in cancers with nucleotide-cycling oncogenic RAS, RAS-GTP dependent oncogenic BRAF, and NF1 loss. SHP2 inhibition is a promising molecular therapeutic strategy for patients with cancers bearing these oncogenic drivers.

#3994

Anti-metastatic drug MBQ-167 regulates cell polarity via Cdc42.

Michael John Rivera-Robles,1 Julia Medina-Velázquez,1 Gabriela M. Asencio-Torres,2 Sahily González-Crespo,1 Brian C. Rymond,3 José Rodríguez-Medina,1 Suranganie Dharmawardhane1. 1 _Univ. of Puerto Rico, School of Medicine, San Juan, PR;_ 2 _Univ. of Puerto Rico, College of Natural Sciences, San Juan, PR;_ 3 _University of Kentucky, Lexington, KY_.

Cdc42 (cell division control protein 42 homolog) is a Rho GTPase highly conserved among eukaryotes in structure and function. Mechanical or chemical cues in the microenvironment stimulate the localized activation of Cdc42, aiding cells to polarize and arrange their components accordingly. Its role in cell polarity was first described in budding yeast and subsequently in other eukaryotes, including animal cells determining the direction of motility. Accordingly, in cancer Cdc42 mediates migration, invasion, and spread of tumor cells. Therefore, we target Cdc42 as therapeutic strategy to treat metastatic breast cancer. Our group designed and characterized the small molecule MBQ-167 as a potent inhibitor against Cdc42 activation (IC50=0.1 µM), demonstrating its effectiveness in preventing tumor growth and cell migration in vitro and in vivo. Because in yeast S. cereviciae Cdc42 is well known for regulating the location of budding events, we used this well-characterized model of cell polarization to test our previous findings of MBQ-167 and its specificity for Cdc42. By using a tetracycline-controlled transcriptional inactivation (Tet-Off) system at the promoter region of the CDC42 gene of yeast, we knocked down Cdc42 supplementing the media with doxycycline (DOX; IC=10ug/uL). Next, we analyzed the growth, budding pattern, and Cdc42 activity of budding yeast exposed to DOX, MBQ-167 or in combination. The maximum growth, doubling time and budding polarity of recombinant yeast with the Tet-Off system were reduced when treated with DOX (EC50=10ug/mL), as expected. The same effect was seen in the group treated with MBQ-167 (ED50=100uM), both alone and in combination with DOX. Ultimately, pull-down assays of the active Cdc42-GTP complex confirmed our hypothesis that MBQ-167 disrupts cell polarity through impeding Cdc42 activation. Here we validated MBQ-167 as a Cdc42 inhibitor in another biologic context. and present a method for screening Cdc42 inhibitors and potential therapeutics against metastatic cancer.

#3995

Exploring non-small cell lung carcinoma cell lines' sensitivity to neratinib.

Sioned Owen,1 Dafydd A. Dart,1 Alshad S. Lalani,2 Francesca Avogadri-Connors,2 Richard P. Bryce,2 Wen G. Jiang1. 1 _Cardiff University, Cardiff, United Kingdom;_ 2 _Puma Biotechnology Inc, Los Angeles, CA_.

Introduction: Lung cancer is the second most common diagnosed malignancy and accounts for the biggest number of cancer related deaths worldwide. Despite increased health warnings associated with habits, such as smoking, ten year survival rates for lung cancer in the UK remain similar to those seen in the 1970's. In the USA, the 5 year survival rates dramatically reduce with late disease stage, from 49 percent at Stage 1A to 5 percent at Stage IIIB. Mutations in epidermal growth factor receptor (EGFR) appears to be quite common in lung cancer. ERBB2 mutations also appear, albeit at lower frequency. In addition, ERBB2 amplifications have also been reported, particularly in tumors that have become resistant to EGFR-TKIs. Neratinib is an orally available tyrosine kinase inhibitor that irreversibly binds and inhibits EGFR, ERBB2 and ERBB4 receptor tyrosine kinases. This study aimed to characterize in detail the expression profile of genes downstream of the EGFR family members in two NSCLC cell lines and their differential sensitivity to neratinib. Materials and Methods: A549 (adenocarcinoma), a cell line with intermediate sensitivity to gefitnib, and SKMES-1 (squamous cell carcinoma) NSCLC cell lines were purchased from American Tissue Culture Collection (ATCC). Cytotoxicity was assessed using an MTT assay. Changes in gene expression in response to neratinib were quantified by next generation sequencing using AmpliSeq technology and analysed using Ingenuity pathway analysis (IPA) software.

Results: SKMES-1 lung cancer cells were found to be more sensitive to neratinib than A549 cancer cells. Both cell lines exhibited similar low gene expression levels of ERBB2. SKMES-1 cancer cells showed higher gene expression profiles for the remaining HER family members, EGFR, ERBB3 and ERBB4, compared to the A549 cancer cells. IPA analysis showed that in SKMES-1 cells treated with neratinib differentially expressed genes were associated with inhibition of Akt and ERK pathways and their signal transduction whereas, this was not seen in the A549 cells.

Conclusions: This study has shown that although these two NSCLC cell lines have similar gene expression levels of ERBB2, there is a difference in profile amongst the other receptor family members. EGFR and ERBB4 mRNA transcript levels were twice as high in SK-MES-1 compared to A549. This variance in the ERBB family gene expression profile may account for the differing sensitivities these cell lines have to neratinib. Further analysis indicates that Akt/ERK signal transduction was inhibited in SKMES-1 cells treated with neratinib though the exact mechanism for this remains unknown.

#3996

Pyridoclax and its derivatives from oligopyridine family directly inhibit Mcl-1 and exert potent antitumor effects on ovarian cancer in vitro and in vivo.

Siham Hedir,1 Louis-Bastien Weiswald,1 Marcella De Giorgi,2 Jade Fogha,2 Martina De Pascale,2 Emilie Brotin,1 Bogdan Marekha,2 Peggy Suzanne,2 Fabien Gautier,3 Philippe Juin,3 Laetitia Ligat,4 Frédéric Lopez,4 Rémi Legay,2 Ronan Bureau,2 Sylvain Rault,2 Jana Sopkova-de Oliveira Santos,2 Anne-Sophie Voisin-Chiret,2 Laurent Poulain1. 1 _Normandie University, UNICAEN, INSERM U1086 ANTICIPE, Comprehensive Cancer Center F. Baclesse, BioTICLA group, Caen, France;_ 2 _Normandie University, UNICAEN, CERMN CNRS INC3M, Caen, France;_ 3 _Nantes-Angers Centre for Cancer Research, UMR 892 Inserm - 6299 CNRS, Nantes, France;_ 4 _Cancer Research Center of Toulouse, Plateau Interactions Moléculaires, INSERM-UMR1037, Toulouse, France_.

Ovarian cancer is the leading cause of death from gynecological malignancies worldwide. Although the patients are initially quite sensitive to the taxane and platinum-based first-line chemotherapy, most of them relapse and develop chemoresistance. Defects in apoptosis regulation in ovarian cancer allow the cancer cells to evade cell death and contribute to chemoresistance. Mcl-1 is an anti-apoptotic member of the Bcl-2 proteins family and its amplifıcation is one of the most frequent genetic aberrations found in human cancers. Its expression is at the origin of the acquired resistance to chemotherapy and to Bcl-2 and Bcl-xL inhibitors. In ovarian cancers, we previously demonstrated that Bcl-xL and Mcl-1 cooperate to prevent cancer cells from undergoing apoptotic cell death. Their concomitant inhibitions lead to massive apoptosis even in absence of chemotherapy. Moreover, in some cases, Mcl-1 inhibition is itself able to lead to apoptosis. If clinically relevant pharmacologic inhibition of Bcl-xL is available using ABT-263, selective direct inhibition of Mcl-1 remains problematic. In this context, our teams have designed and synthesized small compounds based on a pyridyl scaffold, named oligopyridines, which potentially target the Mcl-1 hydrophobic binding pocket. We demonstrated that the lead of the first generation of oligopyridines, named Pyridoclax, interacts directly with Mcl-1, releases its pro-apoptotic partners Bim and Bak and induces massive apoptosis at 25 µM concentration in combination with anti-Bcl-xL strategies in chemoresistant ovarian cancer cell lines (Gloaguen et al., J Med Chem 2015). In the present study, we investigated the antitumor activity of Pyridoclax hydrochloride in three subcutaneous xenograft models derived from the injection of chemoresistant ovarian cancer cell lines. Different routes of Pyridoclax hydrochloride administration were tested and its antitumor effect was analyzed at different doses as single agent or in combination with ABT-263. This study highlighted an effective antitumor activity of 20mg/kg of Pyridoclax administered intravenously as single agent in two of three xenograft models without side effects. In order to improve its biological activity, we evaluated the cytotoxic effects of a second generation of oligopyridines derived from the Pyridoclax. This allowed us to identify the MR31367, one of the most potent oligopyridines that shows a stronger pro-apoptotic activity in association with to Bcl-xL-targeting strategies in ovarian cancer cell lines. Further characterization showed that this derivative binds Mcl-1 and release Bim and Bak from it, leading to Bak-mediated apoptosis. Overall, these results open up interesting perspectives for the clinical use of Mcl-1 inhibitors as single agent or in combination with anticancer drugs to improve the clinical management of ovarian cancers.

## TUMOR BIOLOGY:

### Carcinogenesis 2

#3997

Somatic mutational processes in the cancerization field of the normal-appearing airway reveal early drivers in the development of non-small cell lung cancer.

Smruthy Sivakumar,1 Yasminka Jakubek,1 F Anthony San Lucas,1 Wenhua Lang,1 Christina McDowell,1 Zachary Weber,2 Carmen Behrens,1 Neda Kalhor,1 Cesar Moran,1 Randa El-Zein,3 Gareth Davies,2 Junya Fujimoto,1 Reza Mehran,1 Stephen G. Swisher,1 Jing Wang,1 Jerry Fowler,1 Steven Dubinett,4 Avrum E. Spira,5 Erik Ehli,2 Ignacio I. Wistuba,1 Paul Scheet,1 Humam Kadara6. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Avera Institute for Human Genetics, Sioux Falls, SD;_ 3 _Houston Methodist Research Institute, Houston, TX;_ 4 _David Geffen School of Medicine at University of California Los Angeles, Los Angeles, CA;_ 5 _Boston University, Boston, MA;_ 6 _American University of Beirut, Beirut, Lebanon_.

BACKGROUND: There are very few strategies to treat non-small cell lung cancer (NSCLC) at its primitive stages largely due to our poor understanding of molecular aberrations in development of the malignancy and that would be ideal targets for early treatment. Work from our group and others revealed that visually "normal" airway cells carry alterations (e.g. expression and copy number changes) that are characteristic of the nearby lung tumor, signifying a "field of cancerization" that is pertinent to the pathobiology of the tumor. Yet, the landscape of driver alterations in the cancerization field of the normal-appearing airway remains largely unexplored.

METHODS: 409 cancer-associated genes were surveyed by deep targeted sequencing in tumor adjacent (small airways) and distant (mainstem bronchi/large airways, nasal epithelia, normal lung parenchyma) normal-appearing tissues as well as in multi-region tissues from paired tumors contrasting events in blood, for a total of 500 samples from 48 patients with early-stage NSCLC (11 squamous cell carcinomas and 37 adenocarcinomas). Somatic point mutations were interrogated by aggregating multiple mutation callers. Mutation were then paired with our recent study of genome-wide copy number alterations (CNAs) inferred from high-density whole-genome SNP microarrays.

RESULTS: We identified somatic point mutations in 76 normal-appearing field samples (75% of patients) which were overall concordant with profiles in corresponding NSCLCs. Somatic mutation signatures associated with smoking, APOBEC activity and DNA mismatch repair were common to both airway field samples and NSCLCs from smoker cases. Airway field mutation burdens, while varied, increased significantly with proximity to the tumor. Most field mutations were observed in tumor-adjacent small airways (from 31 patients) some of which were in known drivers such as TP53, KEAP1, STK11 and KRAS. Somatic mutations were also identified in more distant normal samples including large airways (TP53, SETD2, CDKN2A), normal lung parenchyma (RB1, RET) and nasal epithelium (AKT1). We then integrated point mutation and CNA data and found "two-hit" gene alteration patterns in airway field samples that were consistent with their matched NSCLCs, including point mutated gene/CNA pairs such as KEAP1 and STK11/19p loss, TP53/17p loss and KRAS/12p gain. Some additional cases exhibited single hits (e.g., TP53) in their airway field progressing to "two-hits" (e.g., TP53/17p loss) in the matched NSCLC.

CONCLUSIONS: Our integrative high-throughput sequence and genome-wide SNP analyses implicate early mutational processes and putative drivers in the progression of the mutagenized "normal" airway field of cancerization to NSCLC thus offering insights into strategies for interception via early detection and personalized prevention.

#3998

Integrative molecular analysis uncovers key molecules and signaling pathways regulated by RKIP in gastrointestinal stromal tumors (GISTs).

Nathália C. Campanella,1 Matias Melendez,1 Leticia Ferro Leal,1 Adriane Feijo Evangelista,1 Vitor Marcel Faça,2 Rui Manuel Reis1. 1 _Barretos Cancer hospital, BARRETOS, Brazil;_ 2 _University of São Paulo, Ribeirão Preto, Brazil_.

Introduction: Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumor on the gastrointestinal tract, and are characterized by hotspot mutations in KIT and PDGFRA genes, which are predictive of imatinib-based therapy response. We showed previously that RKIP, a metastasis suppressor gene, is lost in about 17% of GIST, and it was associated with an adverse outcome. RKIP is plays a pivotal role in several signaling pathways, namely MAPK, NF-κβ, and GSK3β however a multidimensional integrative analysis to uncover its biological impact was never done in cancer and in particular in GIST.

Experimental procedures: We established a RKIP knockout GIST cell line (GIST-T1 KO) by CRISPR/Cas9 system. The differential gene and microRNA expression profile between GIST-T1 RKIP-KO and GIST-T1 negative control cells were assessed using the NanoString nCounter® technology and the nCounter® PanCancer Pathways and nCounter® miRNA Expression assays (NanoString Technologies). All procedures including sample preparation (100 ng of RNA), hybridization, detection and scanning were performed according to manufacture's instructions (NanoString Technologies). In a second step, we performed a global proteomic analysis using the Acquity UPLC M-Class chromatography system (Waters Co) linked to the mass spectrometer Synapt G2-Si (Waters Co). The protein

identification and quantification were performed in Progenesis QI for Proteomics (Waters Co) software.

Results: The nCounter gene expression analysis showed 56 genes differentially expressed: 18 downregulated and 38 upregulated genes in RKIP KO cells. The altered genes are especially involved in PI3k-AKT, MAPK and Ras signaling pathways. The global proteomic characterization showed 50 proteins differentially expressed: 5 were downregulated and 44 upregulated proteins in RKIP KO cells. In microRNA expression profile analysis we found 133 downregulated microRNAs in RKIP KO cells. Taking together, these findings uncover new molecules that are going to screen in a cohort of GIST patients.

Conclusion: For the first time, using a multidimensional integrative analysis we identified driver candidates and pathways, which will contribute the uncovered the biological role of RKIP in cancer.

#3999

Intracellular redox status determines self-renewal activity of breast cancer stem-like cells through activation of Nrf2-mediated FoxO3a-Bmi-1 signaling.

Do-Hee Kim, Jeong-Hoon Jang, Young-Joon Surh. _Seoul National Univ., Seoul, Republic of Korea_.

A subpopulation of cancer cells, termed cancer stem cells (CSCs), has a self-renewal property, which drives cancer recurrence and tumor resistance. It has been reported that CSCs possess enhanced capabilities to protect against oxidative stress caused by reactive oxygen species (ROS) compared with non-stem-like cancer cells. In the present study, we found that the redox-sensitive transcription factor NF-E2-related factor 2 (Nrf2) plays a role in self-renewal activity of human breast cancer cells, and that this was mediated by Bmi-1. The manifestation of stemness in breast CSC-like cells was associated with an elevated ratio of reduced to oxidized glutathione (GSH) and consequently, lowered ROS levels compared with parent breast cancer (MCF-7) cells. We observed that there was a concurrent increase in the expression of CD44, glutamate cysteine ligase (GCLC) and Bmi-1 in CD24low/CD44high breast cancer stem-like cells sorted from MCF-7 mammospheres. Treatment with buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis, led to a decrease in the number and the size of mammospheres, which was accompanied by the reduction of Bmi-1 expression. Conversely, treatment with a membrane permeable GSH ethyl ester (GSH-MEE) enhanced the size and the number of mammospheres. In addition, phosphorylation of AMPK (Thr 172) and FoxO3a (Ser 7) as well as expression of Bmi-1 was increased in mammosphere cells treated with GSH-MEE. When GSH-MEE-treated mammospheres were challenged with hydrogen peroxide, the phosphorylation of AMPK was abolished. Notably, FoxO3a directly bound to the Bmi-1 promoter as assessed by the chromatin immunoprecipitation assay. Moreover, elevated nuclear accumulation of Nrf2 protein was evident in cells growing under sphere-forming conditions. Under physiologic conditions, Nrf2 is sequestered in the cytoplasm as an inactive complex with Keap1. Sphere-forming breast cancer cells exhibited an enhanced interaction between Keap1 and CD44, which may account for an increased translocation of Nrf2 in nucleus. We found that expression of GCLC, FoxO3a and Bmi-1 was inhibited following introduction of Nrf2 siRNA into MCF-7 mammsophere cells. Knockdown of Nrf2 suppressed the xenograft growth of orthotopically injected breast cancer stem-like cells in athymic nude mice, and this was accompanied by decreased expression of GCLC, FoxO3a and Bmi-1. In conclusion, CD44 promotes Nrf2 accumulation in breast cancer stem-like cells, which, in turn, upregulates GCLC expression and consequently enhances GSH biosynthesis with concomitant reduction in intracellular ROS levels. The consequent upregulation of nuclear FoxO3a and its binding to the Bmi-1 promoter may stimulate self-renewal and growth of breast cancer stem-like cells.

#4000

Hepatitis B virus X gene accelerates the development of HBV-related hepatocellular carcinoma.

Yu ZHANG, Qian YAN, Dandan YU, Xinyuan GUAN. _HONG KONG University, HONG KONG, Hong Kong_.

Epidemic studies have shown hepatocellular carcinoma (HCC) is highly associated with chronic HBV infection. However, the underlying mechanism of how HBV integration leading to HCC remains elusive. We applied the ViralFusionSeq algorithm to aid our analysis of the raw whole-genome sequencing data from TCGA database and found HBX "re-integration" in tumor cells showed a relatively consistent pattern compared with the adjacent normal liver tissue. In order to further elucidate the oncogenic role of truncated HBX gene in HCC, full-length and two COOH-terminal truncated HBX (X120 and X134) genes were stably transfected into HCC cell lines to investigate its function. Results showed that both truncated forms (X120 and X134) overexpression increased HCC cell proliferation rate, the ability of foci formation and colony formation in soft agar, as well as migration and invasion, while full-length HBX showed the opposite effect, both in vitro and vivo. Furthermore, RNA-sequencing of HBX overexpression HCC cells revealed key cancer-related signaling pathways were altered compared with control cells. Here our findings give a novel insight into the HBV integration pattern in the host genome which contributes to HCC.

#4001

Gremlin-1 augments the estrogen-related receptor α signaling: Implications for progression of breast cancer in synergistic manner.

Sin-Aye Park, Bae-Jung Choi, Wonki Kim, Young-Joon Surh. _Seoul National University, Seoul, Republic of Korea_.

Gremlin-1 (GREM1), one of the bone morphogenic protein (BMP) antagonists, is known to be involved in organogenesis, tissue differentiation, and pathogenesis of several renal disorders including fibrosis, inflammation, and diabetes. Recently, it has also been reported that aberrant epithelial GREM1 expression initiates colonic tumorigenesis. However, the role of GREM1 in breast cancer remain largely elusive. We observed that GREM1 expression was highly increased in human breast cancer cells and tissues obtained from breast cancer patients. Furthermore, its overexpression is associated with poor prognosis of breast cancer patients, especially those with estrogen receptor (ER)-negative tumors. Suppression of GREM1 expression significantly inhibited the proliferation of SKBR3 human breast cancer cells in culture and xenograft mammary tumor growth while its overexpression enhanced their viability and invasiveness. Estrogen-related receptor α (ERRα) is an orphan nuclear hormone receptor and has recently attracted attention as a novel therapeutic target in the management of triple-negative breast cancers. We observed that ERRα binds directly to GREM1 promoter and modulates GREM1 expression. The mRNA level of GREM1 was reduced by an inverse agonist of ERRα, XCT790 and also by siRNA silencing of ERRα. Conversely, the overexpression of ERRα with its co-factor peroxisome proliferator-activated receptor gamma co-activator 1-α increased the mRNA level of GREM1. Surprisingly, the transcriptional activity of ERRα was found to be regulated by GREM1. The promoter activity of ERRα and the mRNA levels of ERRα target genes were highly increased in GREM1-overexpressing breast cancer cells. Treatment of MCF-7 cells with recombinant GREM1 protein also enhanced the ERRα promoter activity and transcription of its target genes. Taken together, our study identifies for the first time that the GREM1 can act as a downstream signaling molecule of the transcription factor ERRα and suggests a positive feedback loop between ERRα and GREM1, highlighting GREM1 as a potential diagnostic and therapeutic target for the treatment of ER-negative breast cancer.

#4002

EGR1 downregulation by miR-377-3p mediated lung tumorgenesis following benzo[a]pyrene exposure.

Xinxin Ke, Jing Shen, Jimin Shao, Hongyan Qi. _Zhejiang University, Hang Zhou, China_.

Polycyclic aromatic hydrocarbons (PAHs), especially Benzo[a]pyrene (B[a]P), contained in cigarette smoke and air pollution, belong to a category of important carcinogens for lung cancer. B[a]p exerts its carcinogenic function by causing mutations, cytotoxicity and inhibiting DNA synthesis. However, the early molecular events and related mechanisms of B[a]P-induced cell transformation and tumor initiation remains unclear. In this study, we showed that EGR1 was significantly downregulated during human bronchial epithelial BEAS-2B cell transformation and mice lung carcinogenesis after B[a]P and its active form B[a]PDE exposures, respectively. Using RNA-seq to screening and using qRT-PCR, western-blot and immunohistochemistry for validation. In contrast, overexpression of EGR1 inhibited the cell malignant transformation upon B[a]PDE exposure. Furthermore, miR377-3p was strongly increased by B[a]PDE/B[a]P in vitro and in vivo. And luciferase reporter assays showed that the exposure-induced miR377-3p was crucial for inhibition of EGR1 by targeting its 3'-UTR. Importantly, miR-377-3p antagomir reversed the effect of EGR1 downregulation in the cell malignant transformation and tumor initiation models. The target genes of EGR1, ANKRD1 and ATF3, which could be involved in B[a]P-induced lung tumorgenesis, were also analyzed in cell and mice model, respectively. Finally, the B[a]P exposure-induced molecular changes were examined by immunohistochemistry in clinical lung cancer tissues and evaluated with TCGA data. Taken together, the results demonstrated that Benzo[a]pyrene exposure may induce lung tumorgenesis through miR-377-3p mediated reduction of EGR1 expression, suggesting an important role of the tumor suppressor EGR1 in PAH-induced lung carcinogenesis.

Keywords: EGR1, miR-377-3p, malignant transformation, lung tumorgenesis, gene regulation

#4003

Comprehensive genomic profiling of Merkel cell carcinoma samples reveals bimodal distribution of tumor mutational burden and two mutually exclusive candidate mechanisms of malignant transformation.

Meagan Montesion,1 Ethan S. Sokol,1 Todd C. Knepper,2 Andrew S. Brohl,2 Garrett M. Frampton,1 Phil J. Stephens,1 James A. DeCaprio,3 Kenneth Y. Tsai,2 Lee A. Albacker1. 1 _Foundation Medicine, Inc., Cambridge, MA;_ 2 _H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL; _3 _Dana-Farber Cancer Institute, Boston, MA_.

Merkel cell carcinoma (MCC) is a rare but aggressive skin cancer. Etiologic risk factors include long-term UV exposure, Merkel cell polyomavirus (MCV) infection, age, and immunosuppression. Large-scale investigations of MCC are hindered by its rarity and comprehensive understanding of this disease is currently lacking. In a study of 275 patients, representing the largest sequenced cohort of MCC samples to date, we sought to characterize the genomic landscape of MCC as well as identify whether causality could be attributed to MCV infection, UV exposure, or a synergistic effect between the two.

Comprehensive genomic profiling of 395 cancer-related genes, including evaluation of tumor mutational burden (TMB) and mutational signatures, was performed on MCC tumors from 275 patients. The TMB of MCC exhibited a stark bimodal distribution between TMB high (≥20 mutations/mb) and TMB low (<6 mutations/mb) specimens with only 22 specimens having an intermediate TMB. Overall, 101/275 (37%) of MCC samples were TMB high (median 53.1 mutations/mb) with the majority carrying short variant mutations in TP53, RB1, or NOTCH1. Analysis of mutational signatures found that 94/101 (93%) of TMB high samples exhibited signs of UV damage. No signatures indicative of APOBEC activity or of intrinsic DNA repair defects such as microsatellite instability were observed in the TMB high group. In contrast, 152/275 (55%) of MCC specimens were TMB low (median 1.2 mutations/mb) with 39% of these samples having no known or likely genomic driver. We hypothesized that the primary driver in the TMB low group was MCV integration and examined MCV infection through de novo assembly of non-human sequencing reads in all specimens of the MCC cohort. We found 99/152 (65%) of TMB low specimens to be positive for MCV integration as opposed to 0/101 (0%) of TMB high specimens, which was a statistically significant difference (P = 2.20e-31, Fisher's exact test). Overall, 3 of the 22 TMB intermediate specimens were MCV-positive.

In a large cohort of 275 patient samples, we have described two genomically distinct subsets of MCC: a TMB high group defined by UV damage with mutations in well-known tumor suppressors and a TMB low group defined by MCV integration. We propose that MCV integration is the dominant driver in the TMB low cohort. Indeed, most MCV sequences that we observed appear to be derived from the large or small T antigens, two established viral oncogenes. These results are suggestive of two distinct etiologies of MCC that appear to be mutually exclusive, namely UV exposure vs. MCV integration. These etiologies may have significant impact on targeted and immuno-therapy options for MCC patients.

#4004

The H2 receptor antagonist nizatidine inhibits carcinogenesis in two rodent models of hepatocellular carcinoma.

Shen Li,1 Sarani Ghoshal,1 Gunisha Arora,1 Derek J. Erstad,1 Mozhdeh Sojoodi,1 Michael Lanuti,1 Yujin Hoshida,2 Thomas Baumert,3 Kenneth K. Tanabe,1 Bryan C. Fuchs1. 1 _Massachusetts General Hospital, Boston, MA;_ 2 _Mount Sinai Hospital, New York, NY;_ 3 _Strasbourg University Hospitals, Strasbourg, France_.

Introduction: The purpose of this study was to examine whether nizatidine could reduce liver fibrosis and subsequent tumor burden in two rodent models of hepatocellular carcinoma (HCC).

Methods: An in silico screen identified nizatidine as a compound that can reverse a previously identified gene signature associated with disease progression and HCC development in human cirrhosis patients. We tested the ability of nizatidine to inhibit HCC development in two rodent models. In the first study, male Wistar rats received weekly intraperitoneal injections of 50 mg/kg diethylnitrosamine (DEN). This model has previously been shown to reliably recapitulate the histologic and molecular features of human HCC development including fibrosis after 8 weeks, cirrhosis after 12 weeks, and HCC by 18 weeks. DEN-injured rats were randomized to receive oral gavage of nizatidine (n=7) or vehicle control (n=9) after 8 weeks. In the second study, male C57BL/6J mice received a single intraperitoneal injection of 25 mg/kg DEN at day 15 followed by initiation of a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) at 6 weeks of age. DEN+CDAHFD-injured mice were randomized to receive oral gavage of nizatidine (n=7) or vehicle control (n=9) at 12 weeks of age and were sacrificed at 30 weeks of age after development of HCC in the setting of nonalcoholic steatohepatitis (NASH).

Results: As expected, repeated injections of DEN in rats resulted in progressive fibrosis followed by HCC formation. Treatment with nizatidine resulted in a 35% reduction of tumor nodules relative to vehicle controls (p<0.18). Liver sections were stained by picrosirius red to assess fibrosis and nizatidine reduced collagen deposition in DEN-injured rats (collagen proportional area = 5±0.03 vs. 9.2±0.04; p<0.05). This histologic observation was further confirmed by gene expression analysis with reduction in several profibrotic markers, such as alpha-smooth muscle actin and collagen type I, after treatment with nizatidine. All mice receiving DEN+CDAHFD developed HCC. Treatment with nizatidine resulted in a 60% reduction in tumor nodules relative to vehicle controls (p<0.0001). Nizatidine treatment also resulted in a significant reduction in liver to body weight (p<0.01). Nizatidine treatment reduced collagen proportional area (11±0.05 vs. 15±.01; p<0.05) and expression of profibrotic markers. Nizatidine treatment also reduced the expression of several proinflammatory markers including CD68, interferon gamma, and interleukin-6.

Conclusion: Our data suggest that the H2 receptor antagonist nizatidine reduces fibrosis and subsequent HCC development. This could be beneficial in patient management given the low cost and ready availability of this agent.

#4006

Serine protease PRSS8 suppresses colorectal carcinogenesis and metastasis.

Yonghua Bao,1 Yongchen Guo,1 Yiqiong Yang,1 Xiaonan Wei,1 Xiangdong Zhu,2 Wei Zhang,3 Wancai Yang1. 1 _Jining Medical Univ., Jining, China;_ 2 _University of Illinois at Chicago, Chicago, IL;_ 3 _Northwestern Univ., Chicago, IL_.

PRSS8 is a serine protease and has important physiological and pathophysiological functions, but its roles in cancer initiation and progression are unclear. In this study, a conditional knockout mouse model of Prss8 fl/fl,p-Villin-Cre+ mice were developed and thoroughly characterized at the ages of 3, 6 and 9 months. Intestinal epithelial cell proliferation, migration, and differentiation, were determined. The approaches of gain- and loss of expression of PRSS8 in human colon cancer cells were performed to evaluate anti-cancer and anti-metastasis in vitro and in nude mice by mouse subcutaneous injection, tail vein injection and intra-spleen transplantation. Gene profile and gene set enrichment analysis and mechanistic studies were conducted in vitro and in human colorectal cancer tissues. Results: Genetic deficiency of the Prss8 gene caused spontaneous formation of colitis and inflamed rectum at the early age, but caused intestinal tumors and lymph node metastasis at late age, which was linked to increased intestinal cell proliferation and migration, but decreased cell differentiation. Increased PRSS8 expression showed inhibition of cancer cell growth and metastasis in nude mice, and inhibited migration, invasion, colony formation and tumor sphere formation in vitro, but reduced expression of PRSS8 facilitated malignancies. Mechanistic studies revealed that PRSS8 targets Wnt/β-catenin, epithelial-mesenchymal-transition, and stem cell signaling pathways, which was supported by the results from the Cancer Genome Atlas, and were validated in human colorectal cancers. Conclusion: PRSS8 is a novel tumor suppressor that plays critical roles in the suppression of carcinogenesis and metastasis via targeting Wnt/β-catenin, EMT and stem cell signaling pathway.

#4007

**Chemopreventive effect of** Dioclea reflexa **seeds dietary inclusion in experimental colon carcinogenesis.**

Uju D. Iliemene,1 Sunday Ene-Ojo Atawodi2. 1 _Bingham University Nigeria, Abuja, Nigeria;_ 2 _Ahmadu Bello University Zaria, Nigeria, Kaduna, Nigeria_.

The aim of the present study is to evaluate the colon cancer chemopreventive effect of dietary inclusion of Diocleareflexaseed, a popular Nigerian soup condiment in male albino rats following ten weeks of intra-rectal instillation with N-methyl-N-nitrosourea (MNU). Male albino wistar rats, randomly divided into 6 groups of 7 rats each, were fed D. reflexa diatary inclusion at 0%, 2.5%, 5%, and 10% for ten weeks with concomitant of intra-rectal instillation with MNU at MNU at a dose of 1.2mg/rat. At the end of feeding experiment, rats were sacrificed, blood and tissues were collected for biochemical, histological and immunohistochemical analysis. The flavonoids, total polyphenols and fiber content of the experimental diets were also assessed.The level of carcino-embryonic antigen (CEA) and malondialdehyde (MDA) were significantly (p<0.05) higher in the MNU control and the MNU group with 2.5% dietary inclusion when compared with animals on basal diet, but feeding with 5 and 10% Diocleareflexaseed inclusion, prevented this elevation. There wasaccompanied withstatistically significant (p<0.05) boost in the levels of glutathione s-transferase (GST), superoxide dismutase (SOD, and catalase activities in the colon of MNU groups fed the experimental diets when compared with the MNU control group. Histological examination revealed deep mucosal ulceration with moderate interglandular inflammation in the colon of the MNU control group, but feeding with the Diocleareflexaseedincluded diets significantly reduced or eliminated these effects. Immunohistochemicalexamination of the colon showed moderate no damage of mismatch repair protein Mutl homolog 1 (MLH1) in the MNU groupsfed 5 and 10% experimental diets as opposed to the MNU control and group. Total polyphenol content ranging from 83.2±0.08 to 287.8±0.01mg/ml gallic acid equivalent (GAE)/200mg dry matter and flavonoid from 21.8±0.04 to 54.6±0.002 µg/ml quercetin equivalent (QE)/200mg dry matter were observed increasing along with the percentage inclusion (2.5 to 100%) respectively. Also, some of our predictions were supported as there were strong correlation amonge the parameters assayed. In conclusion, the upregulation of endogenous antioxidant enzymes by D. reflexa inclusion was inversely correlated with down-regulation of markers of oxidative stress-related injuries, indicating D. reflexa contains constituents, presumably, fibers and polyphenols, especially, flavonoids that significantly blocked initiation of carcinogenesis and prevented the development of pathological lesions. Hence, D. reflexa seeds can be of great health benefits to the population that regularly consumes it.

Key words: Dioclea reflexa seeds; dietary inclusion; colon carcinogenesis; cancer chemoprevention; experimental carcinogenesis; dietary supplementation

#4008

**Activating** HER2 (ERBB2) **mutations lead to endocrine therapy resistance through S6K activation.**

Sarah Croessmann,1 Luigi Formisano,1 Teresa Dugger,1 Francesca Avogadri-Connors,2 Richard E. Cutler,2 Alshad S. Lalani,2 Carlos L. Arteaga1. 1 _Vanderbilt-Ingram Cancer Center, Nashville, TN;_ 2 _PUMA Biotechnology, Inc, Los Angeles, CA_.

Background: HER2 missense mutations are present in 2-4% of breast cancers, mainly in ER+ tumors (cBioPortal). Interrogation of METABRIC data revealed that ER+ cancers with HER2 missense mutations treated with endocrine therapy are associated with decreased survival compared to ER+ tumors with wild type (WT) HER2.

Methods: The HER2 activating mutations G309A, L755S and V777L, and HER2WT were incorporated into ER+ MCF7 cells using AAV-mediated gene targeting. We examined cell viability and ER transcriptional activity (ERE-luciferase reporter) in response to estrogen deprivation and treatment with fulvestrant. Signaling downstream mutant HER2 was examined by phosphokinase arrays and immunoblot analyses. Clonogenic growth assays and immunoblot analysis of cells treated with targeted inhibitors were used to examine mechanisms of estrogen independent growth. Inhibitors included neratinib (pan-HER tyrosine kinase), everolimus (TORC1) and selumentinib (MEK). In vivo anti-tumor efficacy of fulvestrant ± neratinib ± everolimus were assessed in ovariectomized athymic mice bearing MCF7/HER2V777L xenografts.

Results: MCF7 cells containing HER2 kinase missense mutations (L755S and V777L), but not cells with HER2WTor an extracellular domain mutation (G309A), were able to proliferate exponentially in estrogen-free medium. Cells with these mutations were also resistant to 1 μM fulvestrant, despite fulvestrant's ability to downregulate ER transcriptional activity and ER protein levels, suggesting the resistance to antiestrogens was independent of ER activity. MCF7 cells with HER2L755S and HER2V777L, but not cells transduced with HER2WT or HER2G309A, showed elevated levels of p70S6K and pS6K in estrogen-deprived medium or upon treatment with fulvestrant. These changes were not associated with increased pAKT, suggesting the upregulation was independent of PI3K activation. Treatment with neratinib or everolimus, each in combination with fulvestrant, resensitized HER2L755S and HER2V777L cells to fulvestrant while also downregulating pS6K levels. Addition of selumetinib to fulvestrant did not enhance the weak antitumor effect of fulvestrant in the HER2 mutant cells or downregulate S6K. Mice bearing MCF7/HER2V777L xenografts treated with fulvestrant and neratinib or with fulvestrant and everolimus but not with fulvestrant alone were equipotent at inhibiting tumor growth, further suggesting that S6K activation is causally associated with resistance to endocrine therapy in ER+/HER2 mutant tumors. Interestingly, the triple combination (everolimus, neratinib, and fulvestrant) induced complete regression of HER2V777L tumors.

Conclusions: These data suggest that, in ER+ breast cancers, HER2 kinase mutations abrogate dependence on ER function through S6K kinase activation and targeting HER2 and/or S6K in combination with an ER antagonist exhibits significant antitumor effect.

#4009

Biotinylated estrogens a novel tool for early detection of adduct in ovarian cancer and their carcinogenic effects in cultured human cells.

Kaushlendra Tripathi. _UAB Cancer Center, Birmingham, AL_.

In humans, endogenous estrogens such as Estrone (E1) and 17L-estradiol (E2) regulate many physiological processes including reproduction, primarily in females, and control the growth of certain responsive cells. However, unbalanced estrogen metabolism has been linked to development of several malignant diseases including breast, endometrial and ovarian cancer and has been identified as a risk factor for these cancers. Elevated estrogens exert their carcinogenic effects by at least three different mechanisms; they promote cell proliferation by transcriptional regulation of estrogen responsive genes, cause generation of reactive oxygen species and other free radicals, and directly react with DNA and form potentially mutagenic adducts. The metabolic breakdown of estogens is regulated by a series of reactions mediated by hepatic and peripheral enzymes that balances the induction and reduction of cellular stress. An imbalance in this enzymes and processes can lead to highly reactive catechol estrogen metabolites that can react with DNA, form carcinogenic DAN-adducts and lesions. Although several studies established a connection between estrogen-induced DNA damage and carcinogenesis, the underlying molecular mechanisms have been difficult to study because of the technical challenges in detecting and analyzing the variety of different DNA lesions that are formed by estrogen compounds. Moreover, detection and analysis of these adducts are important to directly monitor estrogen metabolites induced cellular responses in the cells. Towards this, we developed a novel method using biotinylated-estrogens that allows immunodetection of estrogen-induced DNA adducts by Slot-blot and single-cell molecular combing and proximity ligation assays. Using these modified estrogens we first time quantitatively detected these adducts on DNA by immune Slot-blot techniques and on DNA fibers. Furthermore, similar to other environmental carcinogens estrogens activates replication associated DNA damage responses and induces chromosomal instability. Thus, for the first time our studies demonstrate that biotin-labeled estrogens could be a powerful tool to detect estrogen adducts and to probe associated DNA damage responses and cellular responses.

#4010

Identification of signaling pathways involved in colorectal adenoma-to-carcinoma progression using phosphoproteomics.

Sanne Martens-de Kemp,1 Alex Henneman,1 Richard de Goeij-de Haas,2 Sander Piersma,2 Thang Pham,2 Gerrit Meijer,1 Beatriz Carvalho,1 Connie Jimenez2. 1 _Netherlands Cancer Institute, Amsterdam, Netherlands;_ 2 _VU University Medical Center, Amsterdam, Netherlands_.

Background Colorectal cancer (CRC) develops in a multi-step-process from normal epithelium, through a pre-malignant lesion (adenoma), into a malignant lesion (adenocarcinoma). A minority of about 5% of adenomas will ultimately progress into cancer. To improve clinical practice it is important to identify the subgroup of colorectal adenomas that is at risk of progressing to cancer. By extensive genomic analysis we have shown that specific copy number alterations are associated with risk of progression. An example of such an alteration is the gain of chromosome arm 20q and we identified AURKA and TPX2 as major drivers of this amplicon. Yet, the activation status of these and other genes during colorectal carcinogenesis and the downstream signaling pathways affected with gene (in)activation are not fully known. In order to better understand the biology of adenoma to carcinoma transition, we performed a comprehensive analysis of phosphoproteomes at different stages of colorectal carcinogenesis.

Methods Phosphotyrosine containing peptides were immunoprecipitated from 5 mg of colorectal adenoma (n=81) and colorectal carcinoma tissues (n=50) using agarose bead-coupled phosphotyrosine antibody P-Tyr-1000. A Q Exactive HF mass spectrometer was used to perform NanoLC-MS/MS. Spectral counts of phosphoproteins and ion intensities of phosphopeptides were defined by MaxQuant for relative quantitation of protein phosphorylation.

Results Phosphotyrosine-based phosphoproteomics of the 131 colorectal tissue samples yielded 6056 phosphopeptides, corresponding to 2745 unique phosphoproteins including 183 phosphokinases. Data analysis to identify regulated phosphorylation states and inference of kinase activity is ongoing.

Conclusions and future plans This is the first large phosphoproteomics dataset of colorectal adenomas and carcinomas. Preliminary data analysis shows promising differences in kinase activities between adenomas and carcinomas. After further analysis, we will manipulate the activity of the driver kinases in adenoma-derived organoids and monitor tumor outgrowth in mice.

#4011

**Deciphering the clonal evolution of recurrent tumor-initiating cells with a CRISPR/Cas9 genome-wide loss-of-function screen in an orthotopic HNSCC surgical model** in vivo **.**

Ali Nowrouzi,1 Mahdi Akbarpour,1 Vincent Roh,2 Anne Wursthorn,1 Agnés Hiou-Feige,2 Maximillian Knoll,1 Bouchra Tawk,1 Christian Schwager,1 Christian Simon,2 Jürgen Debus,1 Genrich Tolstonog,2 Amir Abdollahi1. 1 _German Cancer Research Center, Heidelberg, Germany;_ 2 _University Hospital of Lausanne, Lausanne, Switzerland_.

Local recurrence and distant metastasis constitutes a major obstacle in the curative treatment of cancer. The clonal evolution and molecular mechanisms governing these processes remain elusive. We utilized the CRISPR/Cas9 based genome-wide GeCKO library stably transduced in head and neck cancer (HNSCC) cells (FaDu) and orthotopically transplanted into submental area of the floor-of-mouth (FOM) of NMRI nude mice to identify candidate genes which drive molecular processes leading to the development of local recurrences. Primary tumor bulks were resected leaving a minimal residual disease (MRD) consisting of few loco-regionally disseminated tumor cells to form loco-regional recurrences and distant metastasis - the recurrent tumor initiating cells (R-TIC). Primary tumors (PT) developed within the first 14 days with well-defined borders. Following surgical resection of the primary tumors recurrences started to develop approximately 21 days post-surgery. The rate of local recurrences was 2-fold higher in mice which were transplanted with the GeCKO library compared to the parental FaDu line. From mice with local recurrence (LR), also the lymph nodes (LN) and the lung metastases (LM) were collected. Following multiplex low cycling quantitative PCR of the guide RNA sequences (gRNA-seq) by NGS, 270 million reads were extracted enabling a comprehensive evaluation of the GeCKO library representation and specific functional gene knockout (KO) in the PT and matching LR, LN and LM. In line with the proposed R-TIC hypothesis, we found a highly clonal evolution of few dominating "trunk" KO cells governing the locally recurrent, LN and distant metastases. In average, we could detect close to 13K gRNA-seq in PT indicating a polyclonal composition of the tumor bulk prior to surgery. In contrast, all recurrent tumors exhibited a highly clonal composition with >95% of the read counts originating from only 2-4 unique gRNA-seq. This indicates that the clonal expansion which has led to the growth of the recurrent tumor is initiated by few clones carrying loss-of-function mutations in 2-4 genes. Interestingly the abundancy of these clones was very low or below NGS detection limit in the corresponding PT and clones which initiated LR were also found in the corresponding LN and LM of the same mice. 14 loss-of function candidate genes were shared in all mice which developed LR post-surgery underlining their importance in mechanistically promoting the initiation of local recurrences. We provide here evidence for the existence and clonal evolution of an R-TIC population in HNSCC. Novel R-TIC specific molecular targets discovered in this study will facilitate rational design of treatment strategies aiming to prevent the development of loco-regional recurrences and distant metastasis.

#4012

**SAG/RBX2/ROC2 regulates pancreatic cancer initiation, cystic formation in mice driven by Kras** G12D **.**

Mingjia Tan, Qiang Zhang, Yi Sun. _Department of Radiation Oncology, University of Michigan, Ann Arbor, MI_.

BACKGROUND & AIMS: SAG, a small ring finger protein, a component of CRLs, might be involved in pancreatic tumorigenesis. SAG overexpression in human pancreatic ductal adenocarcinomas indicating that SAG could be promotes pancreatic tumorigenesis. We investigated how manipulated SAG (overexpression or disruption of SAG) cooperate KRAS to regulate pancreatic cancer progression in mice.

METHODS: We disrupted or overexpressed SAG specifically in pancreata of mice (Sagfl/fl;P48-Cre or SagTG;P48-Cre mice) and crossed them with LSL-KRASG12D mice, which express an activated form of KRAS and develop spontaneous pancreatic tumors. The resulting Sagfl/fl;LSL-KRASG12D;P48-Cre or SagTG;LSL-KRASG12D;P48-Cre mice were monitored; Pancreatic tissues were collected and analyzed by histology and immunohistochemical analyses. Protein was isolated from mouse tissues and primary acinar cells were prepared and cultured in 3D condition. Human PDA TMA was analyzed by immunohistochemistry.

RESULTS: Overexpression of Sag with KRASG12D in pancreata of mice increased the proliferation of pancreatic epithelial cells, lead to formation of acinar to ductal metaplasia and pancreatic intraepithelial neoplasias (mPanINs) and shorten mice survival. Disruption of Sag in LSL-KRASG12D;P48-Cre mice accelerated the growth of pancreatic cystic lesions compared to LSL-KRASG12D;P48-Cre mice, but did not change mouse survival. In mechanism, Sag promotes pancreatic tumorigenesis by regulating Deptor/mTOR/S6K axis and EGFR signaling pathway.

CONCLUSION: Sag is an oncogenic cooperator of activated KRAS for pancreatic tumorigenesis. Targeting SAG E3 ligase may, therefore, have therapeutic value for the treatment of prostate cancer associated with mutant KRAS.

#4014

APOBEC3B promotes hepatocarcinogenesis and metastasis through novel deaminase-independent activity.

Irene O L Ng, Wei Ma, Daniel WH Ho, Karen MF Sze, Yue M. Tsui, Lo K. Chan, Joyce MF Lee. _Univ. of Hong Kong, Pokfulam, Hong Kong_.

APOBEC3B (A3B), a cytidine deaminase, is known to play important roles in creating de novo genomic C-to-T mutations in cancers and contribute to induction of genomic instability. In this study, we evaluated the roles of A3B in the progression and metastasis of human hepatocellular carcinoma (HCC). Using whole-transcriptome and whole-exome sequencing and quantitative real-time PCR, we found that A3B was overexpressed in human HCCs and A3B expression was significantly correlated with the proportion of genomic C-to-A and G-to-T mutations. Upon clinicopathological correlation, higher A3B expression was associated with more aggressive tumor behavior. Wild-type A3B (wt-A3B) overexpression in HCC cells promoted cell proliferation, and cell migratory and invasive abilities in vitro, and tumorigenicity and metastasis in vivo. On the other hand, knockdown of A3B suppressed cell proliferation, migratory and invasive abilities of HCC cells with high endogenous A3B level. However, to our surprise, overexpression of A3B deaminase-dead double mutant (E68A/E255Q) led to similar results as wt-A3B in HCC both in vitro and in vivo. Furthermore, overexpression of wt-A3B and mutant A3B both enhanced cell cycle progression in HCC cells. Altogether, our data demonstrated a novel deaminase-independent role of A3B in contributing to HCC tumorigenesis and metastasis.

#4015

Histone-lysine methyltransferase up-regulation plays a causal role in hexavalent chromium-induced cancer stem cell-like property and cell transformation.

Zhishan Wang,1 Jianjun Wu,1 Brock Humphries,2 Kazuya Kondo,3 Yiguo Jiang,4 Xianglin Shi,1 Chengfeng Yang1. 1 _University Of Kentucky, Lexington, KY;_ 2 _University Of Michigan, Ann Arbor, MI;_ 3 _Tokushima University, Tokushima city, Japan;_ 4 _Guangzhou Medical Kentucky, Guangzhou, China_.

Chromium (Cr) and its compounds have been widely used in the manufacture of many consumer products. In addition to occupational exposure of millions of industrial workers, large numbers of general population are also commonly exposed to Cr through environmental pollution resulting from continuous industrial emissions and improper waste disposal, and the presence of Cr in urban particulate matters from automobile emissions. Cr exists in several valence and the most common forms of Cr found in occupational and general environment include Cr(0), Cr(III), and Cr(VI). Only hexavalent chromium [Cr(VI)], but not Cr(0) or Cr(III), has been recognized as a human carcinogen. Many epidemiological studies have established a link between Cr(VI) exposure and increased risk of cancer and other diseases, however, the mechanism of Cr(VI) carcinogenesis has not been clearly defined. It is generally accepted that Cr(VI) works as a genotoxic carcinogen due to the fact that Cr(VI) undergoes a series of metabolic reductions inside cells to generate various reactive Cr metabolites and reactive oxygen species (ROS), producing a variety of genotoxic effects. However, much less studies have been done to investigate its non-genotoxic mechanisms. Accumulating evidence indicates that Cr(VI) exposure also causes various epigenetic changes. The goal of this study was to determine the mechanism by which Cr(VI) exposure triggers epigenetic changes and whether Cr(VI)-caused epigenetic dysregulation plays a role in chronic Cr(VI) exposure-induced cancer stem cell (CSC)-like property and cell transformation. We exposed two immortalized human bronchial epithelial cell lines (BEAS-2B and 16HBE) to 0.25 µM of K2Cr2O7 for 20 and 40 weeks to induce cell transformation, respectively. It was found that chronic Cr(VI) exposure causes epigenetic dysregulation as evidenced by the increased levels of histone H3 repressive methylation marks and the related histone-lysing methyltransferases (HMTases) in Cr(VI)-transformed cells and Cr(VI) exposure-caused human lung cancer tissues. Using pharmacological inhibitors to inactivate HMTases or shRNA knockdown of HMTases significantly decreases histone H3 repressive methylation marks and malignant phenotypes of Cr(VI)-transformed cells. Moreover, knockdown of HMTases in parental BEAS-2B cells reduces chronic Cr(VI) exposure-induced CSC-like property and cell transformation. Together, our findings indicate that chronic Cr(VI) exposure increases histone H3 repressive methylation marks by increasing the related HMTases expression; and that increased HMTases expression contributes causally to Cr(VI)-induced CSC-like property and cell transformation.

#4016

The role of catechol estrogen in steroid hormone-induced prostate carcinogenesis in the Noble (NBL) rat.

Maarten C. Bosland, Michael J. Schlicht. _Univ. of Illinois College of Med. at Chicago, Chicago, IL_.

Results of studies with Noble (NBL) rats indicate that estrogens play a causative role in prostate carcinogenesis. In this strain, prostate adenocarcinomas develop after 6-12-mo treatment with testosterone (T), with and without estradiol-17β (E2) added. Dorsolateral prostate cancer incidence was 37% with T alone (~90 μg/d by subcutaneous [s.c.] Silastic implants), but 90-100% when E2 (~1.6 μg/d, by s.c. Silastic implants) was added to the treatment. T can be converted to E2 in the prostate by the enzyme aromatase and the catechol E2 metabolite 4-hydroxyestradiol (4OHE2) is thought to be carcinogenic. Previous short-term studies showed that 4OHE2 (5 μg/d, by s.c. slow-release implant) increased serum and prostate 4OHE2 levels 2-4-fold and increased prostatic levels of the N7-Gua and N3-Ade DNA adducts of 4OHE2. After 12-mo treatment with the same doses, 4OHE2 was slightly estrogenic, resulting in higher pituitary weights, and 4OHE2 and T+4OHE2 induced dysplastic lesions in the lateral prostate, but at a much lower incidence than with T+E2, and only cancer was found in the dorsolateral lobe, one in the anterior prostate, and one in the seminal vesicle, for a total tumor incidence of 23%. Thus, despite increasing DNA adducts and inducing dysplasia in the prostate, 4OHE2 did not induce prostate carcinomas, perhaps because it lacks strong estrogenic activity. To test whether both 4OHE2-mediated changes and estrogenic activity are required in addition to androgen for T+E2-induced prostate lesions, we used 2-fluoroestradiol (2FE2), which is estrogenic but cannot be converted to catecholestrogen. Animals were treated for 12 months with 2FE2 (~10 µg/d, by s.c. slow-release implant) or T alone or a combination of T+2FE2, 2FE2+4OHE2, or T+2FE2+4OHE2. Compared to historic controls, all 2FE2-treated rats had elevated pituitary weights and reduced prostate weights, indicative of estrogenic activity. Prostate weights were increased significantly in animals receiving T with or without 2FE2 or 4OHE2. Three of 18 animals treated with T alone developed large accessory sex gland adenocarcinomas, and a small cancer was found in the anterior prostate and the seminal vesicle in one rat each for a total cancer incidence of 28%, comparable to our previous findings with T treatment. Animals treated with T+2FE2 developed also only few prostate cancers and 2FE2+4OHE2 did not cause cancer. By contrast, 18 of 19 rats (95%) treated with T+2FE2+4OHE2 developed small adenocarcinomas in the periurethral ducts of the dorsolateral and anterior prostate; there were often multiple tumors with an average multiplicity of 2.7 tumors per rat. These findings strongly support the hypothesis that androgen and estrogen receptor-mediated action as well as catechol estrogen metabolite-induced DNA damage are all mechanistically required for a high tumor response to treatment with T+E2.

Supported by a Prevent Cancer Foundation grant.

#4017

**Inhibition of mutagenicity of PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine)by Chinese medicinal herbs** Scutellaria barbata **and** Oldenlandia diffusa **.**

Rayford Alva, Brian Yuen Yau Wong, John Tanner, Yanlingxue Wan, Padma Tadi Uppala, Ryan Hayes. _Andrews Univ., Berrien Springs, MI_.

Scutellaria barbata (SB) and Oldenlandia diffusa (OD) have been used in traditional Chinese medicine for treating liver, lung and rectal tumors. Recent research revealed that these two herbs were included in most of the herbal cancer treatment formulas in Taiwan hospitals. We previously showed that SB and OD inhibited mutagenesis, DNA binding and metabolism of aflatoxin B1 (AFB1) and benzo(a)pyrene (BaP) bioactivated by Aroclor 1254-induced rat S9. PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) is produced from high cooking temperature of creatine, sugars, and amino acids. It was demonstrated positive result in the Ames mutagenicity test. PhIP is one of the most abundant heterocyclic amines (HCAs) in cooked meat and has been announced as "reasonably anticipated to be a human carcinogen" by the U.S. Department of Health and Human Services National Toxicology Program. Cancer of the prostate, breast, lung, esophagus, stomach, and colorectum may be related to high intake and high exposure to PhIP. In this study, the effects of aqueous extracts of SB and OD on PhIP-induced mutagenesis using Salmonella typhimurium TA98 as the bacterial tester strain and rat liver 9000 x g supernatant (S9) as the metabolic activation system were assessed. Our results showed that SB and OD significantly inhibited the mutagenicity of PhIP (both 0.1 and 1µg/plate) bioactivated by S9 (800 mg/plate) in a dose-response fashion (p< 0.05). The percent inhibition of revertants formation of these two herbs against 0.1µg/plate of PhIP were 45%, 77%, and 87% for SB (1.5, 3, and 6 mg/plate) and 21%, 80%, and 95% for OD (1.5, 3, and 6 mg/plate), while the percent inhibition against 1 µg/plate of PhIP were similar to that of the 0.1 µg/plate (45%, 80%, 90% for SB and 44%, 81%, 93% for OD, respectively). There was a slight additive inhibition effect of the herbs in combination (SB + OD) at higher concentrations (60%, 85%, and 90%) against both concentrations of PhIP. These results suggested that OD and SB contain antimutagenic phytochemicals towards PhIP-induced mutagenicity. Further study of their modulation effects on metabolism and DNA binding of PhIP is warranted to reveal the potential anti-mutagenicity mechanism and their chemopreventive property against PhIP and potentially against other HCAs.

#4018

Mutations of HER2 at L755 residue results in HER2 nuclear accumulation and enhances breast cancer stem cell activity.

Wen-Ling Wang,1 Lei Nie,2 Kieu-Thanh Huynh,1 Jhen-Yu Chen,1 Jing-Han Yao,1 Mien-Chie Hung,2 Wei-Chien Huang1. 1 _China Medical University, Taichung, Taiwan;_ 2 _University of Texas MD Anderson Cancer Center, Houston, TX_.

Background: HER2 plays a critical role in tumor progression and cancer stemness in breast cancer cells. Somatic mutations in HER2 gene have been observed in low HER2-expressing cancer cells and are correlated with insensitization to tyrosine kinase inhibitors. One of the most frequently altered residue is L755, which is located at kinase domain and near a putative nuclear export signal sequence, increasing kinase activity and is associated with lapatinib resistance. Moreover, the correlation between HER2 nuclear localization and drug-resistance and poor clinical outcome has been demonstrated in cancer patients. However, the roles of HER2 mutations in its nuclear distribution and its ability to enhance cancer stemness remain unknown.

Methods: We hypothesized that activation of HER2 mutations on amino acid L755 might affect HER2 cellular localization. To investigate the cellular localization of HER2 and the role of L755 mutations in expansion of cancer stem cells, T47D cell line clones expressing wile-type (WT) and mutant HER2 (L755P and L755S) were established. The cellular fractionation, immunofluorescence staining, and western blot analysis were employed to verify the cellular distribution of HER2 L755 mutations. Biochemical strategies were further performed to examine the interaction of HER2 L755 mutant and cellular transporters nuclear importers or exporters.

Results: Our results showed that ectopically expressed HER2 L755P and L755S mutants exhibited a higher nuclear retention via interrupting nuclear exporting complex formation. HER2 L755P and HER2 L755S showed higher abilities to associate with CRM1 and RanBP3 but reduced the binding capacity with Ran. The increases in tyrosine phosphorylation of CRM1 and RanBP3 were found by HER2 mutants and cause the defect in the formation of nuclear exporting complex. Additionally, the HER2 L755P and HER2 L755S-expressing cell lines showed higher levels of cancer stem cell markers, CD44 and ALDH1 and increased mammosphere formation.

Conclusion: HER2 L755P and L755S mutations enhance the HER2 nuclear localization and breast cancer stemness. This study provided new insight into the role of HER2 mutation in cancer stemness, and was helpful to develop novel prognostic markers for the therapeutic efficacy in breast cancer patients.

#4019

The chronic intermittent hypoxia enhances cancer progression in lung cancer mouse model.

SangHaak Lee,1 In Kyoung Kim,1 Hyeon Hui Kang,1 Hee Young Kwon,1 Hye Sun Kang,2 Chang Dong Yeo3. 1 _St. Paul's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea;_ 2 _Incheon St.Mary's Hospital, College of Medicine, The Catholic University of Korea,, Incheon, Republic of Korea;_ 3 _Uijeongbu St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Uijeong bu, Republic of Korea_.

Background: Obstructive sleep apnoea (OSA) is a very prevalent disorder charaterized by chronic intermittent hypoxia (CIH) and some reports suggested that OSA is related to increased incidence of cancer as well as cancer progression. The purpose of this study was to evaluate whether intermittent hypoxia with a time course similar to that found in OSA influence lung cancer development and progression in metastatic lung cancer animal model.

Methods: C57/BL6 mice were divided into two groups; control condition (room air) or CIH. The mice of CIH group were exposed to IH for 2 weeks before and immediately for 2 weeks after tumour induction by tail vein injection of carcinoma cells. At day 14, the mice were sacrificed and measured cancer growth. Hypoxia inducible factor (HIF-1α), vascular endothelial growth factor (VEGF) and metastasis related matrix metalloproteinases (MMP) were measured by ELISA and gelatin zymography in lung tissue and serum, respectively. The mRNA expressions of epithelial mesenchymal transition markers were measured by real-time PCR in lung tissue.

Results: The marked increase in tumor number and volume was found in CIH group (p < 0.05). The accumulation of total cell and neutrophils in BAL fluids and the activation of MMP -2 and -9 in serum were also increased by CIH. The level of HIF-1α in the nuclear fraction of pneumocytes and the expression of VEGF were increased significantly in CIH group compared to the control (p < 0.05). The mRNA expression of E-cadherin was decreased in CIH group compared to the control group while vimentin was increased.

Conclusions: Our data shows that CIH is associated with enhanced proliferative and migratory properties of cancer. These results suggest evidence for the causal link between OSA and lung cancer progression.

Key words: chronic intermittent hypoxia, obstructive sleep apnea, lung cancer

#4020

Hyaluronan fragments as inflammation mediators in the early stages of melanomagenesis.

Piia Takabe, Leena Rauhala, Markku Tammi, Raija Tammi, Sanna Pasonen-Seppänen. _University of Eastern Finland, Kuopio, Finland_.

UV light exposure is the main risk factor for melanoma development. Skin is constantly exposed to UV light, which induces the formation of reactive oxygen species (ROS). ROS are able to degrade hyaluronan (HA), the main extracellular matrix component of the skin epidermis. HA participates in several biological and pathological processes like in inflammation and cancer. It is postulated that in skin an intact HA coat around the cells has a protective effect against UV-radiation, while HA fragments derived from HA degradation are biologically active and may have tumor promoting effects. The aim of the present work was to study the role of HA fragments in melanomagenesis. Does pericellular hyaluronan or its degradation products affect UVB tolerance, melanocyte viability, intracellular signaling and the expression of proinflammatory cytokines?

In our experimental model, primary human melanocytes were pretreated with Streptomyces hyaluronidase (Strept. Hyal) for 10 min to degrade the pericellular HA coat and thereafter exposed to UVB (30 mJ/cm2). After the UVB exposure Strept. Hyal was added again to accelerate HA degradation and HA fragment accumulation. The expression levels of proinflammatory cytokines were determined using qRT-PCR. Signaling pathways activated after UVB exposure were studied by western blotting. Cell viability was examined and the influences of UVB and HA coat degradation on cell cycle and apoptosis were analyzed with FACS.

In this study, we show that UVB induces the expression of the proinflammatory cytokines IL-1β and IL6 and the chemoattractant cytokines IL8 and CXCL-10. This induction was strongly elevated by the Strept. Hyal treatment suggesting that HA fragments produced by HA degradation are responsible for upregulation of these genes. The inhibition of p38 and NFkβ reversed the effect of UVB + Strept. Hyal treatment on IL6 and IL8 expression indicating that this signaling pathway is responsible for the upregulation of these genes. Moreover, silencing of hyaluronan synthase 2 (Has2) was also able to block the effect of UVB + Strept. Hyal on the studied genes suggesting that the fragmentation of endogenously produced HA generates biologically active, proinflammatory signals. Interestingly, blocking CD44, the main hyaluronan receptor, expression with siRNA was ineffective, suggesting that other upstream effectors than CD44 are involved. Regarding cell viability and apoptosis the Strept. Hyal –treatment showed only a slight difference compared to the UVB –treated melanocytes.

Taken together, our results demonstrate that HA fragments strengthen the inflammatory response induced by UVB exposure. UVB + Strept. Hyal activated the p38-NFkβ signaling pathway causing the upregulation of proinflammatory cytokines. The present data suggest that HA fragments mediate sustained inflammation, which may be a trigger for tumor formation.

#4021

The evaluation of carcinogenicity evidence of dibenzanthracenes.

Feng Tsai, Jennifer C. Hsieh, Gwendolyn Osborne, Martha Sandy. _Cal Environmental Protection Agency, Sacramento, CA_.

Dibenzanthracenes (DBAs) are identified as carcinogens and are on the California Proposition 65 list. DBAs are five-ring polycyclic aromatic hydrocarbons (PAHs). This chemical group consists of three isomers: DB[a,c]A, DB[a,h]A, and DB[a,j]A. DBAs are products of the incomplete combustion or pyrolysis of organic matter and can be formed during high temperature cooking. They are air pollutants present in engine exhaust, tobacco and marijuana smoke, and may also contaminate soil and water. DB[a,h]A is a well-studied International Agency for Research on Cancer (IARC) Group 2A carcinogen, whereas fewer studies relevant to carcinogenicity have been conducted with DB[a,c]A and DB[a,j]A. Carcinogenicity evidence from multiple data streams was integrated in the hazard identification of this chemical group. These data streams include animal cancer bioassays, mechanistic studies and other relevant data, including high-throughput studies (e.g., ToxCast), toxicogenomic studies, and information from quantitative structure-activity relationship (QSAR) models. Structure activity comparisons across the three DBAs and related carcinogenic PAHs served to bridge the data gaps for the two less studied isomers. The results of our analyses showed that each of the DBAs share similar biological activities in animals: all induce tumors in mice, are mouse skin tumor initiators, form DNA adducts, are genotoxic, and form metabolites that are also genotoxic and initiators of mouse skin tumors. All three DBAs activate the aryl hydrocarbon receptor (AhR) in reporter gene assays, and DB[a,h]A and DB[a,c]A have been shown to bind to the AhR. In addition to strong evidence of genotoxicity, other carcinogenic mechanisms are likely also operative, including receptor activation (e.g., AhR), immune suppression, altered cell proliferation, apoptosis, and cell cycle regulation. There are strong structure-activity similarities among the DBAs and several carcinogenic 4-, 5- and 6-ring PAHs, including benzo[a]pyrene and dibenzo[a,h]pyrene. In addition, DB[a,c]A and DB[a,j]A were predicted to be mutagenic and carcinogenic in several QSAR models, including VEGA, Lazar and OECD QSAR Toolbox. Integration of the evidence from these multiple data streams resulted in the identification of the DBAs as a chemical group as carcinogens.

### Cell Adhesion and Extracellular Matrix

#4022

Integrin β1 regulates doxorubicin resistance in breast cancer via FAK-mediated activation of ERK1 pathway.

Subhayan Das, Bikash Ch Jena, Swati Mundre, Aditya Parekh, Y Rajesh, Pralay Mitra, Mahitosh Mandal. _Indian Institute of Technology Kharagpur, Kharagpur, India_.

Introduction: Emergence of chemoresistance is one of the major concerns in cancer management. Development of chemoresistance is associated with several genotypical and phenotypical changes in cancer cells, making them less vulnerable to chemotherapeutic assaults. Integrins are the class of adhesion molecules which help to bind cells with extracellular matrix (ECM) and regulate key signaling pathways. Chemoresistant cells have altered interaction with ECM along with changes in their survival pathways which might have resulted from these altered interactions. Here we hypothesize that the state of chemoresistance is associated with the change in expression of integrin which triggers the intracellular signaling augmenting the chemoresistance in breast cancer.

Methodology: To model the chemoresistance, we developed chemoresistant clones from MDA MB 231 (231) breast cancer cell line by treating it with sub-lethal doses of the Doxorubicin over several cycles. To examine the immediate effect of Doxorubicin, the cell line was treated in a time-dependent manner. Xenograft model of breast cancer was developed and treated with Doxorubicin at 5mg/kg/week dose for two weeks. The residual tumor was considered as the in vivo model of chemoresistance. The initial screening of expression of all integrins was carried out by qPCR and confirmed by western blot and Immunohistochemistry (IHC). Downstream pathway analysis was carried out by western blot and IHC. Integrin β1 (ITGB1) was transiently overexpressed and down-regulated by appropriate plasmids containing either ITGB1 gene or shRNA.

Results: The initial screening showed that ITGB1 was significantly up-regulated in Doxorubicin resistant clones of 231 (DOX-R) which was confirmed by western blot. Similarly, the expression of ITGB1 was found to be increased in Doxorubicin treated 231 cells and xenograft model. The expression of ITGB1 was also increased in human breast cancer tissue subjected to neo-adjuvant therapy. Upon evaluating the mitogenic pathways in DOX-R, we found that elevated p-ERK1 expression correlated with ITGB1 expression. Further, we found that an interaction between ITGB1 and focal adhesion kinase (FAK) leading to Ras-Raf activation was crucial for regulating ERK1 phosphorylation. These findings were also confirmed in the xenograft model. Next, upon overexpressing ITGB1, we found a significant increase in phosphorylation of ERK1 as well as FAK-Ras-Raf pathway. These proteins seemed to be down-regulated on transient attenuation of ITGB1. The inhibition of ITGB1 also decreased the sensitivity of 231 and DOX-R cells to Doxorubicin.

Conclusion: Our study established that ITGB1 influence ERK1 pathway to augment Doxorubicin resistance in breast cancer and inhibition of ITGB1 causes a significant reversal of chemoresistance. These findings indicate that ITGB1 can be a valuable theranostic marker for breast cancer management.

#4023

Gap junctions contribute to anchorage-independent clustering of breast cancer cells.

Fabien Gava,1 Lise Rigal,1 Odile Mondesert,1 Elise Pesce,1 Bernard Ducommun,2 Valérie Lobjois1. 1 _Université de Toulouse, ITAV, CNRS, UPS, Toulouse, France;_ 2 _Université de Toulouse, ITAV, CNRS, UPS, CHU de Toulouse, Toulouse, France_.

Purpose: Cancer cell cluster formation is a key process involved during primary tumor growth, metastatic cells dissemination and secondary tumor evolution. We previously reported that cell-cell adhesion proteins such as E-Cadherin and desmosomal proteins are involved in cell aggregation independently of cell migration or matrix adhesion (Saias et al. 2015). Here we investigate the involvement of gap junction intercellular communication (GJIC) during anchorage-independent clustering of MCF7 breast adenocarcinoma cells.

Experimental Design: Live cell imaging and image processing was used to evaluate MCF7 clustering in the presence of pharmacological inhibitors and to perform a LOPAC® small bioactive compound library screening. Flow cytometry was performed to monitor calcein transfer assay and to evaluate GJIC.

Results: We first demonstrate that functional GJIC are established in the early phase of aggregation. We next show that pharmacological inhibition of GJIC using Tonabersat and Meclofenamate both reduces calcein transfer and delays clustering of MCF7 cells. We found that treatment with Latrunculin A, an actin cytoskeleton-disrupting agent, inhibits MCF7 clustering and impairs calcein transfer. In a screen for inhibitors of cell aggregation using a small-compound chemical library we identified Brefeldin A, an inhibitor of vesicular trafficking, and showed that it also impairs calcein transfer.

Conclusion: Our results demonstrate that GJIC are involved at the earliest stages of the anchorage-independent clustering process. They shed new light on the regulatory mechanisms that could modulate the formation of clusters of circulating tumor cells.

References:

Aceto N, Bardia A, Miyamoto DT, Donaldson MC, Wittner BS, Spencer JA, Yu M, Pely A, Engstrom A, Zhu H et al: Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis. Cell 2014, 158(5):1110-1122.

Saias L, Gomes A, Cazales M, Ducommun B, Lobjois V: Cell-Cell adhesion and cytoskeleton tension are key but opposing regulators of tumor cell aggregation. Cancer Research 2015, 75(12):1-8.

Gava, F., Rigal, L ., Mondesert, O., Pesce, E., Ducommun, B. and Lobjois, V. Gap junctions contribute to anchorage-independent clustering of breast cancer cells (Submitted).

#4024

TRAF2 Ser-11 phosphorylation confers resistance to AKT inhibitors in breast cancer cells.

Hasem Habelhah, Laiqun Zhang. _Univ. of Iowa, Iowa City, IA_.

The PI3K-AKT pathway is the most frequently aberrantly activated pathway in breast cancer (BC), with alterations in genes encoding the pathway components (e.g. RTKs, HER2, PTEN, PIK3C and AKT) occurring in ~80% of cases. However, inhibition of this pathway is either not effective or often results in development of resistance in BC. TRAF2 is a key effector protein downstream of the TNF receptor superfamily, and activates NF-κB to promote BC cell survival. We mapped two phosphorylation sites (Ser-11 and Ser-274) in TRAF2, and identified TBK1/IKKε and AKT as the kinases that directly phosphorylate TRAF2 at Ser-11 and Ser-274, respectively. TBK1 and IKKε are serine/threonine kinases activated by inflammatory stimuli and the oncogenic RTK-Ras-RALB pathway, and play a critical role in BC transformation and survival. IKKε and TRAF2 are overexpressed in a significant portion of BC, and their overexpression is associated with poor prognosis in patients with invasive breast carcinoma. We found that inhibition of AKT leads to increased TBK1 activation and TRAF2 Ser-11 phosphorylation in triple negative BC (TNBC) cells. Importantly, combination of AKT and TBK1/IKKε inhibitors synergistically induced apoptosis in all TNBC cell lines tested in vitro, suggesting that inhibition of PI3K-AKT derepresses the pro-survival TBK1-TRAF2 pathway, which in turn confers resistance to AKT inhibition in TNBC. We believe that any positive outcomes from our ongoing xenograft experiments will have an immediate translational impact in TNBC treatment.

#4025

An old foe with a new function: JAM-A regulates expression of EphB4 and inhibitor of apoptosis proteins in breast cancer.

Sri HariKrishna Vellanki,1 Rodrigo G. Cruz,1 Cathy E. Richards,1 Kieran Brennan,2 Ann M. Hopkins1. 1 _RCSI, Dublin 9, Ireland;_ 2 _University College Dublin, Dublin 4, Ireland_.

Over-expression of Junctional Adhesion Molecule-A (JAM-A) in breast tumors has been linked with increased risk of metastasis. Since JAM-A is co-expressed with the receptor tyrosine kinase (RTK) HER2 and regulates HER2 protein expression, we hypothesized that JAM-A regulates the expression and function of other RTKs. Using a joint RT-PCR array/western blot approach in three breast cell lines with modified JAM-A expression, we identified EphB4 as a downstream target of JAM-A. Interestingly, EphB4 gene silencing also cross-regulated the protein expression of JAM-A. Immunofluorescence analysis revealed colocalisation of JAM-A and EphB4 at the cell membrane. Since JAM-A contains a PDZ-binding motif and EphB4 contains a PDZ domain, we speculated that this might underlie the basis of an interaction. Accordingly, overexpression of a JAM-A mutant lacking the PDZ domain failed to upregulate EphB4 expression to the same extent as wild type JAM-A. Next we investigated the signalling events downstream of JAM-A and EphB4 and found downregulation of the tight junction protein ZO-1 in breast cells silenced for JAM-A or EphB4. Using a bioinformatics approach, we identified the transcription factor c-FOS as a potential regulator of JAM-A and EphB4 expression. In JAM-A- or EphB4-silenced cells, c-FOS expression was not localized in the nucleus (relative to cells transfected with negative control siRNAs). Furthermore, ERK5 phosphorylation was inhibited in JAM-A- or EphB4-silenced cells, suggesting a mechanism for reduced c-FOS phosphorylation on Serine-32 and its consequent exclusion from the nucleus. In the context of a proposed block on c-FOS transcriptional activity, we examined the genes affected and show for the first time downregulation of inhibitor of apoptosis proteins (IAPs) in JAM-A- or EphB4-silenced cells. In conclusion, our data are consistent with a model whereby JAM-A regulates EphB4 expression in a PDZ-dependent manner and influences apoptosis via a pathway involving ERK5 and c-FOS. Our data suggest that the pharmacological antagonism of JAM-A in JAM-overexpressing breast cancers merits consideration by virtue of its predicted ability to downregulate EphB4 expression and remove a blockade on apoptosis.

#4026

**Mechanism of cisplatin resistance and enhanced cell migration in cervical cancer cells expressing** PIK3CA **-E545K mutation.**

Arjumand Wani,1 Shiekh Tanveer Ahmad,2 Nicholas Jette,1 Siddhartha Goutam,1 Corinne M. Doll,1 Susan P. Miller1. 1 _Arnie Charbonneau Cancer Institute, Robson DNA Science Centre, University of Calgary, Calgary, Alberta, Canada;_ 2 _Clarke H. Smith Brain Tumour Centre, University of Calgary, Calgary, Alberta, Canada_.

Platinum-based drugs, in particular cisplatin, are among the most widely used chemotherapeutic agents, used in the treatment of a wide array of solid malignancies. Despite initial therapeutic success, cisplatin treatment often results in the development of resistance, which can lead to therapeutic failure. Despite significant advances in understanding mechanisms of cisplatin resistance in other tumor sites, there are substantial gaps in our molecular knowledge of resistance in cervical cancer. Our group has examined the role of the PI3K/AKT/mTOR pathway in cervical cancer patients treated with cisplatin and radiation therapy. The PI3K/AKT/mTOR pathway is a promising therapeutic target because it is activated in several human cancers, which is frequently mediated by "hotspot" mutations including E542K, E545K and H1047R in the PIK3CA gene. Moreover, multiple PI3K/AKT/mTOR pathway inhibitors have been developed and are currently being evaluated in clinical trials. We previously reported that PIK3CA mutation in patients with early stage (IB/II) cervical cancer was associated with poor survival after treatment with radiation and cisplatin (McIntyre et al. Gynecol Oncol. 2013, PMID: 23266353), and that PIK3CA-E545K mutation renders cervical cancer cells more resistant to cisplatin or cisplatin plus radiation and results in a more migratory phenotype than cell lines with wild type-PIK3CA. Moreover, these phenotypes were reversed by the PI3K inhibitor GDC-0941 (Wani et al. Oncotarget. 2016, PMID: 27489350). The aim of the present study is to explore the mechanism of cisplatin resistance and enhanced migration in cervical cancer cells engineered to express PIK3CA-E545K mutation. Gene expression analysis identified 161 genes that were up regulated and 189 that were down regulated in cervical cancer cells stably expressing PIK3CA-E545K, some of which are involved in pathways relevant to cisplatin resistance. On further validation, we found that Fibronectin1 (FN1) was upregulated at both the mRNA and protein level in cervical cancer cells expressing PIK3CA-E545K. FN1 is a glycoprotein that is widely expressed in multiple cell types and is involved in cellular adhesion and migration. Recent studies have reported that FN1 might have a role in regulating chemoresistance in tumors. However, the effect of FN1 on cisplatin resistance in cervical cancer cells expressing E545K mutation has not been investigated. The present study aims to determine the effect of FN1 expression on cisplatin resistance in cervical cancer cells expressing the PIK3CA-E545K mutation and to explore potential mechanisms of cisplatin resistance and enhanced cell migration in cervical cancer cells.

#4027

Fibroblast growth factor 2 (FGF2) is associated with the development of resistance to pemetrexed in lung cancer cells.

Kentaro Miura, Ken-ichi Ito, Takaaki Oba, Asumi Iesato. _Shinshu University School of Medicine, Matsumoto, Japan_.

Pemetrexed (MTA) is a folate antimetabolite used for treatment of non-small cell lung cancer and plural mesothelioma. Although several molecules such as thymidylate synthase (TS) or ATP-binding cassette transporter 11 (ABCC11) have been reported to be involved in the mechanisms of resistance to MTA, precise mechanisms underlying resistance to MTA remains unknown. To elucidate the mechanisms of resistance to MTA in lung cancer, we established MTA resistant clones in PC9 and H1993 lung adenocarcinoma cell lines (PC9-MTA, H1993-MTA). Then, gene expression profiles were compared between PC9 and PC9-MTA cells by using microarray analyses, and several genes including fibroblast growth factor 2 (FGF2) were identified. Elevated expression of FGF2 in PC9-MTA cells and H1993-MTA cells were confirmed by western blot analyses. Knockdown of FGF2 by siRNA partially restored the sensitivity to MTA both in PC9-MTA and H1993-MTA cells. In addition, anti-FGFR1 inhibitor (PD173074) restored MTA sensitivity particularly in PC9-MTA cells. On the other hand, the expression of TS was increased in PC9-MTA and H1993-MTA cells, and the inhibition of TS with the TS-targeting siRNA restored the sensitivity to MTA. However, with regard to the expression of FGF2 and TS, the inhibition of FGF2 did not modify the expression of TS, and vise versa. Furthermore, a simultaneous inhibition of FGF2 and TS by siRNAs in the MTA-resistant cells restored MTA sensitivity to approximately the level observed for the parental cell lines. With regard to the factors related to the signal transduction pathway, the phosphorylation of Erk and Akt in H1993-MTA cells was decreased compared with those in the parental cells. When H1993-MTA cells were treated with FGF2-targeting siRNA or anti-FGFR1 inhibitor, the increase of phosphorylation of ERK and AKT were induced. These data indicated the possibility that both FGF2 and TS could confer MTA resistance in the lung cancer cell lines in an independent manner, and that modification of signal transduction through FGF2-FGFR1 pathway might be involved in the development of MTA resistance in lung cancer cells.

#4028

Molecular mechanisms of magnolin resistance in ovarian cancer cells.

Sun-Mi Yoo, Cheol-Jung Lee, Seung-Min Kim, Seon-Yeon Cho, Juhee Park, Yong-Yeon Cho. _The catholic univeristy of Korea, college of pharmacy, Bucheon-si, Republic of Korea_.

Mitogen-activated protein kinase (MAPK) and PKB signaling pathways are highly activated in many human solid cancers and play a key role in carcinogenesis, tumor growth and cancer cell survival. Although regulation mechanisms of each signaling pathway are well-elucidated, interrelationship between both signaling pathways in cancer growth has not been understood. In this study, we found that magnolin, ERK1 and ERK2 inhibitor with 16 nM and 68 nM of IC50 value, suppressed cell proliferation and colony growth of ovarian cancer cells. In signaling pathway analysis, we found that TOV-112 and SKOV3 showed differential responsiveness by EGF stimulation. The differential responsiveness was caused by differential activation signaling through MAPK and PKB signaling pathway. Notably, TOV-112D sensitized by magnolin treatment in cell proliferation and colony growth in soft agar by alteration of cell cycle regulation, but not in SKOV3 cells. Notably, phosphorylation of ATF-1, a downstream target of RSK2, at Ser63 was inhibited in TOV-112 by magnolin treatment, resulted in cellular senescence. However, SKOV3 was vice versa. We further found that magnolin fraction suppressed TOV-112D cancer growth in xenograft animal model. Taken together, we concluded that signaling alteration might involve in magnolin resistance in SKOV3.

Keywords: MAP kinase, molecular targets, ovarian cancer, cancer growth

#4029

**The Wnt target gene** CLAUDIN-2 **regulates tumorigenesis and stemness in human liver cancer cells.**

Hironori Koga, Fumitaka Wada, Mitsuhiko Abe, Hideki Iwamoto, Toru Nakamura, Takahiko Sakaue, Atsutaka Masuda, Toshimitsu Tanaka, Hirohisa Yano, Takuji Torimura. _Kurume Univ. School of Medicine, Japan_.

Background: In our previous studies on T-cell factor (TCF)-4 isoforms, we identified a unique target gene CLAUDIN-2, which was upregulated by lack of SxxSS motif in exon 9 of TCF-4 (Exp Cell Res 2011, Liver Int 2013). This type of TCF-4 (TCF-4J) conferred high tumorigenic ability and hypoxia resistance to hepatocellular carcinoma (HCC) cells (PLoS One 2012). Of interest, the TCF-4J isoform-overexpressing HCC cells (J cells) strongly expressed the leaky tight junction protein CLAUDIN-2 under sphere-forming conditions, suggesting that J cells have cancer stem cell (CSC)-like features involving CLAUDIN-2 upregulation. Thus, the Aim of this study was to assess expression profile of CLAUDIN-2 and the CSC-relevant function of the molecule, such as tumorigenicity, in liver cancer cells.

Methods: The human liver cancer cell lines used in this study were as follows: HAK-1A, HAK-1B (Hepatology 1993), Huh-7, HepG2, Hep3B, HLF, KMCH-1 (Hepatology 1987), KMCH-2 (J Hepatol 1996), KYN-1, KYN-2, and HAK-5. KMCH-1 and -2 are unique combined hepatocellular-cholangiocarcinoma cell lines, and HAK-5 is a sarcomatous HCC cell line. Lentivirus-mediated delivery of siRNA for CLAUDIN-2 (Santa Cruz) and plasmid-mediated CLAUDIN-2 cDNA (Origene) were utilized to generate stable knock-down (KD) and overexpressing cells, respectively.

Results: CLAUDIN-2 was exclusively expressed in both KMCH-1 and KMCH-2 cells, showing 240~370-fold mRNA level of that in the well-differentiated HCC line HAK-1A. The protein expression was strongly observed at cytoplasmic membrane and cytoplasm in KMCH-1 and KMCH-2 cells, but not in the others examined. Gene silencing of CLAUDIN-2 resulted in slower cell proliferation. In xenograft experiment in nude mice, CLAUDIN-2-KD KMCH-2 cells formed much smaller tumors in size than the control cells. Of interest, overexpression of CLAUDIN-2 in constitutively low expressing cell lines, such as Hep3B and HLF cells, lead to high expression of Nanog, a stemness gene product.

Conclusion: As previously shown in transformed HCC cells by the Wnt transcription factor TCF-4J, CLAUDIN-2 was a key molecule regulating cell proliferation and tumorigenesis in non-gene-engineered human combined hepatocellular-cholangiocarcinoma cells. To elucidate precise mechanism of exclusively high expression of this molecule in such difficult-to-treat type of liver cancer may provide insights into generating novel therapeutic approaches.

#4030

Loss of E-cadherin expression is a critical step for peritoneal metastasis of gastric cancer with sub-serosal invasion.

Shingo Togano, Masakazu Yashiro, Kenji Kuroda, Tomohisa Okuno, Yuichiro Miki, Kosei Hirakawa, Masaichi Ohira. _Osaka City University Graduate School of Medicine, Osaka, Japan_.

Background: Peritoneal metastasis is the most frequent type of metastasis in gastric cancer. However, the actual mechanisms responsible for this metastasis are not understood clearly. The process of peritoneal metastasis requires some steps including the detachment of cancer cells from the primary tumor. E-cadherin, a calcium-dependent cell-cell adhesion molecule, is known to mediate intercellular adhesion. We examined the correlation between peritoneal metastasis of gastric cancer and reduced E-cadherin expression.

Methods: A total of 694 gastric cancer cases of which were resected at our department was enrolled in this study. The expression of E-cadherin of gastric cancer cells was evaluated by immunohistochemical staining. We analyzed the correlation between the E-cadherin expression and clinicopathological features. A case with less than 50% of E-cadherin expression was defined as E-cadherin negative.

Results: Among the 694 cases, 232 cases were E-cadherin negative. 171 cases developed peritoneal metastasis. Among the 171cases, 88cases were E-cadherin negative. E-cadherin-negative cancer was significantly (p<0.0001) associated with peritoneal metastasis. The 5-year survival rate of patients with E-cadherin-negative cancer (48.1%) was poor, in compared to that with E-cadherin-positive tumor (62.8%), while a multivariate analysis did not show significant difference. A multivariate analysis of sub-serosal invasion tumors (T3 stage tumor) indicated that only E-cadherin-negative was significantly associated with peritoneal metastasis (p=0.0005).

Conclusion: The loss of E-cadherin is a critical step in the promotion of cancer cells to a more malignant phenotype. E-cadherin is a useful prognostic factor for peritoneal metastasis in gastric carcinoma with sub-serosal tumor.

#4031

Novel tumor intrinsic vs. extrinsic mechanisms of resistance to chemotherapy in metastatic disease.

Madeleine J. Oudin,1 Lucie Barbier,2 Tatiana Kosciuk,2 Emanuel Kreidl,2 Frank Gertler2. 1 _Tufts University, Medford, MA;_ 2 _MIT, Cambridge, MA_.

For patients with metastatic triple-negative breast cancer (TNBC), where metastasis occurs in 30% of patients, treatment options are limited to chemotherapy, given the lack of any effective targeted therapies approved for this subtype. However, the response rate is low and the time to relapse is short. While most chemotherapeutic drugs target proliferating cells, we set out to investigate the effect of these drugs on metastatic cells and identify metastasis-specific mechanisms of resistance. In preliminary experiments, mice with triple-negative xenograft tumors and known metastatic disease were treated with taxol and doxorubicin, two drugs commonly used to treat metastatic TNBC patients. Both drugs significantly slowed tumor growth; however, taxol, but not doxorubicin, treatment reduced cell motility within the primary tumor as measured by intravital imaging. These data support the idea that chemotherapeutics can have different effects on growth and motility in vivo, and that effects on tumor size might not correlate with changes in cell motility. We first focused on identifying tumor-intrinsic mechanisms of resistance to chemotherapy. We found that cells expressing the proinvasive isoform of the actin regulatory protein Mena are resistant to the taxane paclitaxel, but not to the widely used DNA-damaging agents doxorubicin or cisplatin. Furthermore, paclitaxel treatment does not attenuate growth of MenaINV-driven metastatic lesions or reduce metastatic burden. Mechanistically, Mena isoform expression alters the ratio of dynamic and stable microtubule populations in paclitaxel-treated cells, while also increasing MAPK signaling in response to paclitaxel treatment. Our data indicate that highly metastatic cells may respond differently to chemotherapeutic drugs than nonmetastatic ones. Second, we investigated the role of tumor extrinsic factors on chemoresistance of metastatic cells, focusing on the extracellular matrix, which is known to drive local invasion and metastasis. We found that in vitro, plating TNBC cells on collagen renders cells more resistant to doxorubicin, but not taxol. In vivo, both taxol and doxorubicin treatments led to increases in collagen deposition. Our data suggest that chemotherapeutic treatments can therefore lead to changes in tumor ECM amount and can also change how cells respond to tumor ECM. Further studies are under way to identify the role of the matrix in chemoresistance. Together, these data indicate that chemotherapeutic drugs, commonly used to treat metastatic disease, have different effects on cell growth vs. cell migration, and that chemotherapy can lead to the generation of prometastatic environments.

#4032

Pharmacogenomic analysis of acquired resistance can reveal effective drug combination in glioblastoma.

Xiaolong Li, Juan Martinez-Ledesma, Chen Zhang, Feng Gao, W.K. Alfred Yung, Dimpy Koul. _MD Anderson Cancer Center, Houston, TX_.

Targeted cancer therapies have produced substantial clinical responses, but glioblastoma multiforme (GBM) has poor response due to developing resistance. The goal is thus to design strategies to overcome resistance to targeted drugs. To address this, we have embarked on a new type of screening for resistant targets, by developing acquired resistant glioma models and using pharmacogenomic platform to identify the resistant targets. We have established several patient-derived in vivo glioma stem cells (GSC) models using a PI3K specific inhibitor BKM-120 by a selective pressure after a chronic drug treatment. The resistant cells were subjected to RNA-seq analysis as well as pharmacologic analysis utilizing high-throughput kinase drug library screening. Analysis of integrated RNA-seq and kinase drug library screening of resistant cells identified multiple effective drug combination. Activation of Aurora kinase A (Aurora A)/Polo-like kinase 1 (Plk1)/cyclin-dependent kinase (CDK) signaling pathway was predominant in resistant GSCs. Concomitant targeting of PI3K together with Aurora A, Plk1 or CDK1 had a synergistic growth inhibitory effect. In addition, the combined inhibition of Plk1 and PI3K resulted in tumor growth suppression and significant delay of acquired resistance in vivo. These observations suggest that cell cycle pathway serves as a major survival signal upon PI3K inhibition and provides a rationale for the combined use of PI3K and cell cycle inhibition to prevent acquired resistance in human GBM.

#4034

Inhibition of AXL improves response to platinum and taxane in chemotherapy-resistant uterine and ovarian cancers.

Jeanne Quinn, Marguerite Palisoul, Lei Guo, Andrea Hagemann, Matthew Powell, David Mutch, Carolyn McCourt, Premal Thaker, Katherine Fuh. _Washington University School of Medicine, St. Louis, MO_.

AXL expression has been associated with decreased survival in uterine serous cancer (USC) and high-grade ovarian serous cancer (HGSOC). We determined whether therapeutic inhibition of AXL with BGB324, which is in Phase I/II clinical trials for solid tumors, would improve chemotherapy response in chemotherapy resistant uterine and ovarian cancers.

Taxane and platinum resistant ovarian and uterine cancer cell lines were used for in vitro cell viability (XTT) assays. Selective small molecule inhibition of AXL was achieved using BGB324. Western blotting was used to detect protein expression and activation. Tritium-labeled paclitaxel was utilized to measure chemotherapy accumulation in cancer cells. In vivo subcutaneous models were performed with the USC cell line ARK1 and patient-derived HGSOC xenografts (PDX). Statistical significance (p<0.05) and IC50 determination was assessed using Prism7.

Upon AXL inhibition by BGB324, the HGSOC cell lines OVCAR5, OVCAR3TP, and OVCAR3TPMes demonstrated a dose-dependent sensitization to paclitaxel and carboplatin chemotherapy. BGB324 treatment of OVCAR3TPMes cells improved response to carboplatin from untreated (IC50 of 73uM) to IC50 of 66uM, 51uM and 31uM at 0.25uM, 0.5uM, and 1uM, respectively (p<0.01). Similarly, paclitaxel response improved from untreated (IC50 of 15.4nM) to IC50 of 13.0nM, 11.3nM and 9.8nM at 0.25uM, 0.5uM, and 1uM, respectively (p<0.05).

To assess the dynamics of this chemoresponse, ARK1 USC cells were pre-treated with 0.5uM BGB324 and 1.0uM BGB324 and demonstrated a 31% and 43% increase in intracellular 3H-paclitaxel accumulation, respectively, when compared to cells without AXL inhibition (p<0.05). RT-PCR was utilized to detect changes in multidrug resistance protein transcripts; mRNA levels of P-Glycoprotein (PGP) had a 6.3-fold reduction in OVCAR3TPMES cells treated with 1uM BGB324 (p<0.05). This suggests that inhibition of AXL contributes to chemoresistance by increasing exposure of paclitaxel in tumor cells. The combination of paclitaxel and BGB324 therapy decreased USC tumor volume by 51-67% when compared to treatment with paclitaxel, BGB324, or vehicle control alone (p<0.05). In PDX models, BGB324 therapy improved tumor response with combined carboplatin and paclitaxel therapy when compared to chemotherapy alone, inhibitor alone, or vehicle control (77%, 87%, and 88% decrease in tumor volume at day 11, respectively, p<0.0001).

AXL expression contributes to platinum and taxane chemoresistance, and therapeutic inhibition of AXL with BGB324 restores chemosensitivity in ovarian and uterine cancer cell lines and patient derived xenograft models. Based on this data, chemoresistant tumors with AXL expression could be considered for treatment with BGB324 to restore chemosensitivity to carboplatin and paclitaxel.

#4035

Cetuximab combined with dasatinib reduces drug resistance in KRAS/BRAF mutated colorectal carcinoma cells.

Hsiang-Ling Chiu, Yi-Che Wu, Wan-Chen Wei, Ling-Yi Kao, Wei-Ting Chao. _Tunghai Univ., Taichung, Taiwan_.

Colorectal carcinoma is the most common cancer worldwide. Cetuximab is the first-line target drug for late-stage metastatic colon cancer treatment; however, resistance to it was found in 45% patients having KRAS mutation. Therefore, the alternative signal axis to target mutated cancer cells is urgent and investigated. The role and the importance of Src has emerged in colon cancer; this study is to investigate the drug combined efficacy of cetuximab and dasatinib, the Src inhibitor and the underling molecular mechanism in KRAS/BRAF mutated cells. In this study, KRAS and BRAF mutated colon cancer cell lines SW480 and HT-29 were treated with cetuximab, dasatinib and fluorouracil (5-FU) solitary or combined. Cell viability and migration ability were monitored, and the molecular mechanisms were analyzed by Western blot for signal protein expressions. The in vivo study was carried with subcutaneous xenograft mouse model to evaluate the drug efficacy. The result showed that cetuximab treatment induced Src activation dose dependently, and induced matrix-dependent cell migration in SW480 cells. Cetuximab, dasatinib and 5-FU combined treatment compared to solitary treatment can reduce cell viability in either wild-type or KRAS/BRAF mutated colon cells. In the signal protein expressions, Src expression was suppressed sufficiently with dasatinib treatment; the phosphorylation of MAPK in SW480 cells and the phosphorylation of PI3K in HT-29 cells were downregulated after treated with cetuximab together with dasatinib and 5-FU. The drug combination was also demonstrated to suppress subcutaneous tumor growth as well. These findings demonstrate the combination of cetuximab, dasatinib and 5-FU can be the potential treating strategy for colon cancer cells with KRAS/BRAF mutation.

#4036

Differential expression of claudins between benign and malignant salivary gland tumors.

Maria L. Arrojo,1 Claudia Arruda,1 Luiz P. Kowalski,1 Fernando A. Soares,1 Silvia V. Lourenço,2 Cláudia M. Coutinho-Camillo1. 1 _A.C. Camargo Cancer Center, Sao Paulo, Brazil;_ 2 _University of Sao Paulo, Sao Paulo, Brazil_.

Salivary gland tumors comprise a heterogeneous group of lesions presenting different histological features and diverse clinical behavior. Pleomorphic adenoma is the most common benign tumor of major salivary glands. It shows a marked histological diversity with epithelial, myoepithelial, and mesenchymal components in a variety of patterns. Although it presents a slow progression, without treatment can cause significant morbidity and even death. Mucoepidermoid carcinoma is the most common malignant tumor of the salivary glands. Local recurrence rates range from 7% to 26% and distant metastasis rates range from 6% to 15%. Tight junctions (TJ) are considered one of the major structures in the establishment and maintenance of cell polarity having important physiological function in the formation of tubular organs, including the salivary glands. Some studies have demonstrated an association between tumor behavior and protein expression of tight junctions, particularly claudins. Claudins are transmembrane proteins important for the maintenance of cell adhesion and polarity and act as selective barriers. Changes in claudin expression have been detected in several tumors and appear to be related to progression and invasion. However, few studies have evaluated the role of claudins in the development of salivary gland tumors. In this study, using Real Time RT-PCR we have analyzed the expression of claudins-1, -3, -4, -5, -7, -11 in a series of 40 salivary gland tumor samples (20 cases of pleomorphic adenoma and 20 cases of mucoepidermoid carcinoma) and 10 samples of normal salivary glands tissue samples. Increased expression of claudin-1 and claudin-7 was observed in mucoepidermoid carcinoma when compared to pleomorphic adenoma (p=0.010 and p=0.001, respectively). Decreased expression of claudin-3 and claudin-11 was observed in mucoepidermoid carcinoma when compared to pleomorphic adenoma (p=0.005 and p=0.030, respectively). Regarding mucoepidermoid carcinoma tissue samples, we have evaluated the presence/absence of CRTC1/MAML2 fusion and demonstrated that 57.9% of the cases were positive for the presence of the fusion. Claudin-7 expression was associated with the presence of the fusion (p=0.048) and the absence of the fusion was associated with high histological grade tumors (p=0.023). Our study provides evidence of alterations in the expression of claudins in salivary gland tumors demonstrating that regulation of tight junction components is an important feature in salivary gland tumors behavior. CNPq Research Grant 443243/2014-0

#4037

Interactions between STAT3 and TGFβ in the regulation of hyaluronic acid synthesis and signaling in immortalized human mammary epithelial cells.

Brenda G. Trevizo-Barresi, JoEllen Welsh. _University at Albany Cancer Research Center, Rensselaer, NY_.

Evidence suggests that growth of cancer stem cells (CSCs) drives intratumor heterogeneity, a major clinical problem that results in drug resistance, recurrence of tumors, and metastatic progression of breast cancer. Therapeutic strategies to eliminate CSCs require identification of signaling pathways that facilitate the emergence and/or expansion of CSCs. These signaling pathways are implicated in altering cell behavior, as well as promoting the expression of genes involved in epithelial-mesenchymal transition (EMT), cell migration and invasion. Previous work demonstrated that a cooperative STAT3/SMAD3 gene transcription program increased the expression of Hyaluronan Synthase 2 (HAS2), an enzyme that produces Hyaluronic Acid (HA) which serves as a CD44 ligand and facilitates EMT. We hypothesize that STAT3 is a critical regulator of HA synthesis and signaling which enhances EMT and stemness of human mammary epithelial cells, driving tumor progression. We tested this hypothesis in a TGFβ triggered model of EMT. Immortalized human mammary epithelial cells (HMLE) expressing either a control vector (pWZL-GFP) or a vector containing TGFβ1 (pWZL-TGFβ1-GFP) were characterized with respect to STAT3 signaling, HA synthesis, and mammosphere formation. Our data revealed that HMLE-pWZL-TGFβ1-GFP cells have significantly higher expression of active STAT3 (Y705) compared to control cells, but total STAT3 gene and protein expression were similar in the two cell lines. HMLE-pWZL-TGFβ1-GFP cells exhibited a significant increase in expression of HAS2 and HAS2-AS1, a long non-coding RNA that enhances the transcription of HAS2. Preliminary results show that overexpression of TGFβ1 increased HA secretion in media compared to control cells. HMLE-pWZL-TGFβ1-GFP cells demonstrated significantly higher mammosphere forming ability compared to control cells, suggesting that TGFβ increases the percentage of stem cells in the population. Initial results demonstrated changes in mammosphere size and morphology of HMLE-pWZL-TGFβ1-GFP cells treated with the HA synthesis inhibitor 4-Methylumbelliferone (4MU). Together, these results suggest STAT3 and TGFβ1 co-regulate HAS2 expression and HA production in a model of EMT supporting the use of the HMLE-pWZL cell series as a promising model to study STAT3 regulation of the HA-CD44 signaling pathway.

#4038

A functional genomics approach to the identification of genes involved in resistance to Oxaliplatin in colorectal cancer.

Federica Invrea,1 Francesca Cavicchioli,2 Consalvo Petti,3 Rosalia Russo,2 Claudio Isella,4 Enzo Medico4. 1 _Candiolo Cancer Institute, FPO-IRCCS Candiolo, University of Turin, Istituto Nazionale Biostrutture e Biosistemi, Rome, Italy;_ 2 _Istituto Nazionale Biostrutture e Biosistemi, Roma, Italy;_ 3 _Candiolo Cancer Institute, FPO-IRCCS IRCCS Candiolo, Torino, Italy;_ 4 _Candiolo Cancer Institute, FPO-IRCCS Candiolo, University of Torino, Italy_.

Currently, the first line therapy for metastatic colorectal cancer (CRC) is the FOLFOX regimen, which includes Oxaliplatin in combination with Fluorouracil and Leucovorin. Responses to FOLFOX range from overt regressions (35%) to full resistance (25%), and eventually all patients become resistant. Indeed, the molecular mechanisms of Oxaliplatin resistance remain mostly to be understood. To identify genes involved in sensitivity/resistance to Oxaliplatin, we designed an integrated approach including forward genetic screens and computational analyses. The pipeline includes: (i) stable transduction of CRC cells with pooled shRNA libraries; (ii) Oxaliplatin treatment for six weeks to select a drug-resistant subpopulation; (iii) identification of shRNA constructs enriched in the Oxaliplatin-resistant subpopulation by next-generation sequencing analysis. We focused the shRNA libraries targeting phosphatases and ubiquitin conjugation pathway genes. The first are involved in several biological processes, reversing the biochemical activity of kinases and typically acting as feedback loop regulators. The second direct proteins to degradation by the proteasome regulating homeostasis, cell cycle, and DNA repair pathways. HCT116 CRC cells, markedly sensitive to Oxaliplatin, were transduced in duplicate with both shRNA libraries. After Oxaliplatin treatment for six weeks, resistant populations emerged from the library-transduced cells, but not from control-transduced cells. To reveal shRNA constructs responsible of such phenotype, we extracted genomic DNA from transduced cells and, upon amplification of shRNA constructs by targeted PCR and next-generation sequencing, we compared the repertoire of shRNA sequences in HCT116 cells selected with Oxaliplatin with that of cells grown in standard medium. Screening hits were then prioritized for functional characterization, resulting in a phosphatase (CDC25C) and an ubiquitin ligase candidate (DCAF17). In conclusion, we successfully setup a functional genomic screening for acquired resistance to Oxaliplatin in CRC. Specific shRNA constructs from both the phosphatase and ubiquitin ligase libraries were enriched after Oxaliplatin treatment, highlighting candidate genes whose loss of function could potentially drive resistances to Oxaliplatin.

#4039

PAX8 increases migration and metastasis of high grade serous ovarian cancer through upregulation of Rho GTPases.

Laura Hardy, Melissa Pergande, Angel Hernandez, Stephanie Cologna, Joanna Burdette. _University of Illinois, Chicago, IL_.

High grade serous ovarian cancer, the most lethal subtype of ovarian cancer, can originate in either the fallopian tube epithelium (FTE) or ovarian surface epithelium (OSE). PAX8 is a lineage specific transcription factor that is ubiquitously expressed in HGSOC. We have shown that knockdown of PAX8 using shRNA in multiple ovarian tumor cells lines leads to apoptosis, suggesting that PAX8 plays an essential role in cancer survival. In this study, we used CRISPR technology to delete PAX8 from the OVCAR8 cell line. PAX8 deletion led to a decrease in migration and invasion in vitro and an increase in survival and a reduction in tumor volume in vivo. Previous work using RNA-sequencing and ChIP-sequencing identified cell adhesion as a top differentially expressed gene between malignant ovarian cancer and benign fallopian tube cell lines. We performed quantitative proteomic analysis of the OVCAR8-PAX8-/- cell line to define PAX8 altered proteins. We also performed quantitative proteomics and transcriptomic analyses on a previously generated murine OSE cell line with forced PAX8 expression (MOSE-PAX8). These analyses identified several genes that contribute to an increase in migration and EMT. Specifically, our data indicates PAX8 upregulates key drivers involved in altering cell morphology including the GTPases: RhoA, Cdc42, and Ras. Inhibition of RhoA led to a greater decrease in migration for both OVCAR8-PAX8-/- and MOSE-PAX8 when compared to control. Inhibition of Ras had a greater effect in OVCAR8-PAX8-/- while inhibition of Cdc42 had a greater effect in MOSE-PAX8. These data provide a mechanistic explanation for the role of PAX8 on increasing migration and metastasis in ovarian cancer.

#4040

**α** 2 **β** 1 **integrins are potential regulators of chemoresistance through modulation of biomechanical cues in pancreatic cancer.**

Ilaria Carnevale,1 Stefano Coppola,2 Niccola Funel,3 Erik H. Danen,4 Thomas Schmidt,2 Elisa Giovannetti1. 1 _Department of Medical Oncology, VU University Medical Center of Amsterdam, Amsterdam, Netherlands;_ 2 _Physics of Life Processes, Huygens-Kamerlingh Onnes Laboratory, Leiden University, Leiden, Netherlands;_ 3 _Cancer Pharmacology Lab, AIRC Start-Up Unit, University Hospital of Pisa, Pisa, Italy;_ 4 _Division of Toxicology, LACDR, Leiden University, Leiden, Netherlands_.

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant tumor characterized by intrinsic or rapidly acquired chemoresistance. The remarkable desmoplastic response has been reported as a major contributor to chemoresistance, suggesting that PDAC progression could be highly influenced by mechanical factors (1). A recent study showed a key role of epithelial tension and matricellular fibrosis in the aggressiveness of SMAD4 mutant PDAC (2). Here we aimed at evaluating new mechanopharmacology approaches to overcome chemoresistance. PDAC cells attached to collagen and fibronectin showed a decreased sensitivity to chemotherapy, while the malignant phenotype was highly correlated to α2β1 integrin(ITGA2)-mediated adhesion to type I collagen (3). We hence hypothesized that this integrin might modulate chemoresistance. In two cohorts of 50 radically resected patients treated with gemcitabine, high expression of ITGA2 (assessed by immunohistochemistry in tissue-microarrays with 4 cores for each tumor) correlated to significantly shorter progression and overall free survival. Our hypothesis was further investigated in a gemcitabine-resistant clone (PANC1-R). In this clone, ITGA2 was among the top 10 most differentially upregulated genes at next-generation sequencing analysis. Using migration assays, we found that PANC1-R were more invasive than PANC1 cells, and a specific siRNA for ITGA2 caused a significant reduction of invasiveness while enhancing both gemcitabine antiproliferative effects and apoptosis induction. Using elastic micropillar arrays of varying stiffness (20-150kPa) (4), we observed delayed spreading of PANC1-R compared to PANC1 while a 24-hour treatment with a non-cytotoxic dose of gemcitabine activated traction forces in both lines, albeit to a lesser extent in PANC1-R. Remarkably, these cells showed also a significant increase of CXCR4 and MMP2 expression when growing in spheroids embedded in stiff collagen, as well as a different trend in modulation of E2F1 after exposure to gemcitabine, when growing as 2D collagen monolayers on soft and stiff flat PDMS, suggesting that aggressive/invasive behavior and response to gemcitabine are altered by both collagen and stiffness. These results define ITGA2 as a new prognostic factor, exerting its function through the promotion of metastatic behavior and altered drug response of PDAC cells under differential biomechanical conditions, which holds a potential as a novel therapeutic target to overcome PDAC resistance.

References:

1. Coppola et al. Drug Resist Updates 2017;31:43.

2. Laklai et al. Nat Med 2016;22:497.

3. Grzesiak, Bouvet. Br J Cancer 2016;94:1311.

4. van Hoorn et al. Nano Lett 2014;14:4257.

#4041

K-ras transformation leads to increased HS-degradation.

Donghong Ju, Mary A. Kosir. _Wayne State University School of Medicine, Detroit, MI_.

INTRODUCTION: Degradation of glycosaminoglycans in the extracellular matrix impacts cell movement, angiogenesis and other cancer cell activities. The purpose of study was to detect and measure extracellular heparan sulfate (HS)-degrading enzyme in malignant ras-transformed cells where HS degradation occurred at attachment sites. METHODS: Serum free conditioned medium (CM) and lysate from subconfluent Balb/c3T3 and KiMSV (Ki-ras-transformed 3T3) cells were collected and protease inhibitors added. CM was concentrated by ultracentrifugal filters using 3K and 30K MWCO. Heparan degrading enzyme assay kit was use with lysate and CM for each cell line. Western blot analysis of lysate and CM used anti-HPSE, anti-NAP2, and anti-PPBP antibodies with actin control. mRNA for HPSE and PPBP was determined by RTPCR. RESULTS: The heparan degrading activity of CM from KiMSV cells was 12 fold higher than from Balb/c3T3 cells with 30K MWCO concentration. Further, HPSE was detected in the lysate, but not CM. Anti-NAP2 and anti-PPBP antibody detected protein greater than 30kD in CM and lysate for KiMSV cells. However, mRNA expression of PPBP/NAP2 was less in KiMSV than Balb/c3T3 cells. CONCLUSION: HS degradation in the CM of KiMSV cells is greater than for Balb/c3T3 cells from which they are derived after Ki-ras-transformation. The HPSE is not detected in the CM, but PPBP-like proteins at higher molecular weight are detected which may be responsible for the extracellular HS degradation.

#4042

Metformin-induced caveolin-1 expression promotes T-DM1 drug efficacy in breast cancer cells.

Wan-Chen Wei,1 Yuan-Chiang Chung,1 Ling-Yi Kao,2 Hsiang-Ling Chiu,2 Yi-Che Wu,2 Wei-Ting Chao2. 1 _Cheng Ching Hospital, Taichung, Taiwan;_ 2 _Tunghai University, Taichung, Taiwan_.

Trastuzumab emtansine (T-DM1, Kadcyla) is an antibody-drug conjugate (ADC) that was recently approved by the Federal Drug Administration (FDA) of the USA for the treatment of HER-2-positive metastatic breast cancer. The drug sensitivity of ADCs depends in the main on the internalization efficiency of the drug. Endocytic protein caveolin-1 was shown to promote T-DM1 internalization and enhance drug sensitivity. Whether caveolin-1 can be overexpressed to improve T-DM1 efficacy is interesting and has the potential for clinical application. In this study, the first-line diabetes drug metformin was investigated in terms of induction of caveolin-1 expression for increased efficacy of subsequent T-DM1 application. HER-2-positive BT-474 cells were pretreated with metformin, followed by combined therapy with metformin and T-DM1. The T-DM1 internalization and drug efficacy were determined, and the protein expressions for signal transduction were also monitored by Western blot to ascertain the molecular mechanism. Caveolin-1 shRNA was applied to suppress endogenous cellular caveolin-1 expression, and the ability of metformin to promote T-DM1 efficacy was investigated. The results showed that in BT-474 cells pretreated with metformin, cellular caveolin-1 overexpression was induced, which then promoted drug efficacy by enhancing T-DM1 internalization. As cellular caveolin-1 was suppressed by shRNA, the effect of metformin-enhanced T-DM1 cytotoxicity was decreased. This study demonstrated that metformin can be applied prior to T-DM1 treatment to improve the clinical efficacy of T-DM1 by enhancing caveolin-1-mediated endocytosis. 

### Determining How the Immune System Drives Tumor Progression

#4043

A novel role for Hedgehog signaling in macrophage-mediated immune evasion.

Ann Hanna, Dongquan Chen, Darshan S. Chandrashekar, Sooryanarayana Varambally, Rajeev S. Samant, Lalita A. Shevde. _University of Alabama at Birmingham, Birmingham, AL_.

In the tumor microenvironment, breast cancer cells participate in crosstalk with the surrounding stroma. This tumor-stromal interaction forms a balance that dictates tumor suppressing or promoting response mechanisms. Macrophages in the tumor microenvironment are plastic and can mediate several functions depending on their activation states. Tumor-associated macrophages co-exist as two major phenotypes: anti-tumorigenic and immune-eliciting classically activated M1 as well as tumor-promoting and immune-suppressive alternatively activated M2 macrophages. Alternatively activated macrophages are associated with more aggressive stages and poor clinical outcomes in breast cancer patients as they suppress the tumoricidal properties of the immune system, thus facilitating tumor cell proliferation and dissemination. Hedgehog (Hh) signaling is a critical developmental pathway; its activation modulates vital cellular processes such as cell proliferation, stem-cell differentiation, and angiogenesis. While the Hh signaling pathway is normally tightly controlled, it can often be dysregulated which fosters tumorigenesis and tumor progression. Aberrant Hh signaling activation is particularly implicated in breast cancer progression and metastasis. In this study, we investigate the role of Hh signaling in polarizing breast cancer-associated macrophages toward the detrimental M2 subtype. We report that targeting different steps of the Hh signaling cascade attenuates the cytokine profile associated with M2 macrophages. Additionally, we demonstrate that Hh signaling enables the molecular mechanism responsible for alternative macrophage polarization. We furthermore identify significant shifts in immune cell populations infiltrating the primary tumor when we administer an FDA-approved Hh inhibitor to an in vivo mammary tumor model. This altered immune profile is dynamically characterized by a reduction in immune-suppressive cells concomitant with enhanced antigen presentation properties and cytotoxic immune cell infiltration, overall culminating in reduced metastasis. Thus, we describe a novel role for Hh signaling in enabling pro-tumorigenic immunity by alternatively polarizing breast cancer-associated macrophages. This study identifies a novel strategy, which has potential clinical implications to treat breast cancer through targeting aberrant tumor cell Hh activation and eliciting a robust anti-tumorigenic immune response.

#4044

Draining lymph nodes in ovarian cancer patients have a naïve immune cell signature.

Kim Brunekreeft, Fenne Komdeur, Hagma Workel, Florine Eggink, Annechien Plat, Refika Yigit, Harry Hollema, Evelien Duiker, Hans Nijman, Marco Bruyn. _University Medical Center Groningen, Groningen, Netherlands_.

Purpose: Ovarian cancer (OC) is characterized by an immunosuppressive tumor micro-environment (TME). We speculated that the effects of this immunosuppressive TME extends to the draining lymph nodes (DLNs), hampering induction of a proper anti-tumor immune response. To address this, we compared the immune cell composition in OC tumors and DLNs using extensive flow cytometry.

Experimental design: OC tumor (n=16) and DLN samples (n=13) were collected during surgery after obtaining informed consent from the patients. All tissue samples were subjected to enzymatic digestion and single cells were isolated by density gradient centrifugation. Eight-color flow cytometry panels were used to cover various T cell and myeloid cell markers. Statistical analysis was performed as appropriate.

Results: CD8+ cytotoxic T cells (p<0.001) and T regulatory cells (Tregs, p<0.05) were significantly enriched in the tumor compared to the DLNs, while there were more CD4+ T helper cells in the DLNs (p<0.001). Interestingly, CD123+ plasmocytoid dendric cells (DCs) were largely absent from the tumor, whereas CD1a+ CD1c+ immature DCs (p<0.001) were enriched in the TME. No difference between the tumor and DLNs was seen for CD11c+ myeloid DCs (mDC). However, within the mDC population there were more CD1c+ myeloid type 1 DC (p<0.05) in the DLNs than tumor, but less CD1a+ myeloid type 2 DCs (p<0.001), no difference was seen for iDCs. Tumoral T cells had an exhausted effector memory T cell signature, upregulating HLA-DR, the memory T cell marker CD45RO and the T cell suppressing and exhaustion markers, PD-1, PD-L1, and CD27. Notably, tumoral CD4+ T cells highly expressed the co-stimulatory markers CD28 and OX40. In the DLNs, all T cells were predominantly CD45RA+ CCR7+ naïve T cells, concurrently expressing CD27 and CD28. In the tumor and DLNs, Tregs highly expressed CTLA-4 and ICOS, while CD8+ and CD4+ T cells heterogeneously expressed these markers. The proliferation marker Ki67+ was expressed by tumoral T cells and by all Tregs. The transcription factor EOMES was mainly expressed by Ki67+ CD8+ T cells, while the transcription factor Tbet was expressed by CD8+ T cells, independent of Ki67-expression or location in tumor or DLN. DLNs were largely devoid of Ki67+ T cells, with the exception of Tregs.

Conclusions: Cancer can elicit an antigen-mediated immune response via the DLN. However, our data shows a naïve immune cell composition in the DLNs of OC, possibly due to the immunosuppressive effects of the TME extending to the DLNs.

#4045

A novel transcriptomic-based immune classification of soft tissue sarcoma (STS) and its association with molecular characteristics, clinical outcome and response to therapy.

Florent Petitprez,1 Tom Wei-Wu Chen,2 Cheng-Ming Sun,3 Julien Calderaro,3 Li-Ping Hsiao,2 Laetitia Lacroix,3 Ivo Natario,3 Maud Toulmonde,4 Carlo Lucchesi,4 Yec'han Laizet,4 Antoine Italiano,4 Aurélien de Reyniès,5 Catherine Sautès-Fridman,3 Wolf H. Fridman3. 1 _Cordeliers Research Center / French League Against Cancer, Paris, France;_ 2 _National Taiwan University Hospital, Taipei, Taiwan;_ 3 _Cordeliers Research Center, Paris, France;_ 4 _Institut Bergonié, Bordeaux, France;_ 5 _French League Against Cancer, Paris, France_.

Soft tissue sarcomas (STS) form a group of rare cancers which accounts for around 1% of non-pediatric solid tumors. Recent clinical trials with check-point blockade immunotherapies reported a response in up to 15% of patients, but there are no biomarkers predicting response of STS to checkpoint blockade therapies yet. We analyzed transcriptomic data of publicly available cohorts, accounting for more than 600 complex genomic STS, including leiomyosarcoma (LMS, 35.4%), dedifferentiated liposarcoma (DDLPS, 33.9%) and undifferentiated pleomorphic sarcoma (UPS, 30.8%). Using a deconvolution method to estimate the tumor microenvironment composition with accurate scores depicting the immune profile of tumors, we established a robust immune classification that is consistent through different datasets. We divided the patients into 5 Sarcoma Immune Classes, labelled A1, A2, B, C1 and C2, which exhibit different extents and composition of immune infiltrate.

Two groups (A1: 23.3% and A2: 27.1% of all tumors) exhibit a very low to low immune infiltrate for all cell types. Another class (B: 14.5% of all tumors) displays moderate immune infiltrate and a strong presence of endothelial cells. Finally, the remaining two groups (C1: 19.4% and C2: 15.6%) are highly infiltrated by all immune cell types. All histologies were found in all five immune classes, although LMS were enriched in classes A1 and A2. The immune-high groups exhibit a significantly prolonged overall survival compared to the other groups.

Notably, the immune high group C2 is characterized by an extremely rich immune infiltrate containing both lymphocytes and myeloid cells, and is associated with a striking overexpression of several immune checkpoints genes: PDCD1 (PD-1), CD274 (PD-L1), PDCD1LG2 (PD-L2), LAG3, HAVCR2 (TIM-3), CTLA4. This group also contains tumors with a strong expression of the B cell attractant chemokine CXCL13, often associated with the presence of tertiary lymphoid structures (TLS).

We validated the relevance of TLS as a biomarker of this group by quantitative immunohistochemistry (IHC) using stainings for CD3, DC-Lamp, CD8, and CD20 on a 95-patient STS cohort (LMS, 34%; DDLPS, 33%; UPS, 34%). Tumors with visible TLS had a higher density of CD3+, CD8+ and CD20+ cells in the tumor core and the invasive margin, hence showing the possibility to identify the immune-high group by using IHC. The expression of immune checkpoint molecules was also detected by multiplex immunofluorescence for CD3, CD20, PD-1 and Lag3 in these tumors, confirming the presence of checkpoint molecules in the immune-high group.

To check whether the immune-high group C2 was associated with enhanced response to checkpoint blockade therapy, we intend to analyze tumors from a recently published phase 2 clinical trial in which patients underwent treatment with anti-PD-1 pembrolizumab.

#4046

The different immune evasion mechanisms in human prostate cancer.

Mayassa J. Bou-Dargham, Yuhang Liu, Qing-Xiang Amy Sang, Jinfeng Zhang. _Florida State University, Tallahassee, FL_.

Cancer development is associated with a number of genetic and epigenetic alternations. Normally, these alterations are detected by the immune system in a process called immune surveillance, where cancer cell killing is mediated by cytotoxic T lymphocytes (CTL) and natural killer cells (NK cells). Yet, tumor progression and development indicate that cancer cells have acquired mechanisms to avoid immune destruction. Various immune evasion mechanisms have been reported before such as impaired antigen presentation and activation of CTL and NK cells, immunosuppression, and induction of tolerance. Although current treatment methods are shifting towards more targeted strategies in prostate cancer, such as immunotherapy, a better understanding of the cancer-immune interactions and immune evasion mechanisms is yet to be established.

More immunotherapies are being developed and approved by the FDA for treating cancer. In prostate cancer, Sipuleucel-T, an autologous cellular immunotherapy, was approved by FDA for the treatment of asymptomatic or minimally symptomatic metastatic castrate resistant (hormone refractory) prostate cancer. Intensive investigation on other immunotherapies is ongoing in prostate cancer. These include blockade of negative checkpoint regulators (PD-1, CTLA4) and Ig superfamily member V-domain Ig-containing suppressor of T-cell activation (VISTA), chimeric antigen receptor-modified T (CAR-T) cells against prostate lineage-unique proteins (PLUPs), and monoclonal antibodies targeting immunoinhibitory molecules. However, the optimal choice of treatment for a prostate cancer patient needs to target their specific mechanisms of immune evasion, which are poorly studied. In this study, we will use The Cancer Genome Atlas RNA-seq data to check for immune evasion gene expression in prostate cancer patients to identify at the different mechanisms by which cancer avoids immune destruction. We will use a sequential biclustering algorithm developed by our group, to group prostate cancer patients based on their similarities in immune evasion gene expression. A similar unpublished work was done by our group on breast cancer where we identified 7 different groups of patients, each with different mechanisms or combination of evasion mechanism. We also showed that triple negative breast cancer can be immunogenic and non-immunogenic. This group-based identification of evasion mechanisms will not only provide a better understanding of the different immune evasion mechanisms in prostate cancer, but will also shed light on potential combinations of mechanism. This in turn, will help guide the choice of single and combined immunotherapeutic approches.

#4047

Loss of IFN-γ pathway gene expression in tumor cells as mechanism of immune escape of colorectal carcinoma.

Nathalie Britzen-Laurent,1 Julia Straube,1 Maximilian Waldner,1 Christoph Becker,1 Roland Croner,2 Christian Pilarsky,1 Susanne Merkel,1 Michael Stürzl1. 1 _University of Erlangen-Nuremberg, Erlangen, Germany;_ 2 _University of Magdeburg, Magdeburg, Germany_.

In colorectal carcinoma (CRC), the infiltration of tumor tissue by cytotoxic and T-helper 1 (Th1) T cells has been associated with an improved clinical outcome. This antitumor immune response is characterized by an elevated expression of interferon (IFN)-γ and IFN-stimulated genes such as IRF-1, CXCL-9, CXCL-10, caspase-1 or guanylate-binding protein 1 (GBP-1). Using GBP-1 as a marker of cellular response to IFN-γ in human CRC specimens, we observed that tumor cells, but not stroma cells, frequently lose GBP-1 expression in the context of a Th-1-dominated immune response. Similarly, 6 out of 11 colorectal carcinoma cell lines failed to express GBP-1 or other IFN-stimulated genes after treatment with IFN-γ, and were resistant to IFN-γ-induced apoptosis. This indicated that the loss of responsiveness to IFN-γ might represent a mechanism of immune escape in CRC tumor cells. We then investigated whether defects in RNA and protein expression of IFN-γ pathway genes (IFNGR1, IFNGR2, JAK1, JAK2, STAT1) might account for the loss of responsiveness to the cytokine. The most frequent deregulation observed was the downregulation of IFNGR1 expression at the RNA and protein level in 4 out of 6 resistant cell lines, where treatment with 5-Aza-deoxycytidine, a DNA methylation inhibitor, could restore IFNGR1 expression. In the two remaining resistant cell lines, IFNGR1 was expressed but the protein (IFNγRα) was mis-glycosylated and failed to locate at the plasma membrane. In addition, STAT1 protein expression was downregulated in two resistant cell lines. Using a Crispr/Cas9 silencing approach, we showed that the downregulation of IFNGR1 expression is sufficient to inhibit IFN-γ signaling in CRC cell lines. Moreover, long-term treatment of a sensitive cell line with IFN-γ resulted in the downregulation of IFNGR1, but not STAT1 expression. In human CRC samples, mRNA expression of IFNGR1 was decreased compared to normal tissue. Furthermore, low tumor expression of IFNGR1, but not STAT1, correlated with a reduced cancer-related survival in patients with CRC. Finally, mice harboring a specific knockout of the IFN-γ receptor in intestinal epithelial cells developed more tumors than control mice in a carcinogen-induced colon tumorigenesis model, indicating that the absence of IFN-γ receptor expression in intestinal epithelial cells fosters tumor growth. Altogether, our data suggest that the loss of IFN-γ pathway gene expression is a common event in CRC and represents an intrinsic mechanism of immune escape.

#4048

Comprehensive analysis of the suppressed tumor immune microenvironment of CIN gastro-esophageal adenocarcinomas.

Sarah Derks,1 Xinsen Xu,2 Leonie de Klerk,1 Tania Fleitas,3 Kevin Liu,2 Yang Liu,2 Claire A. Margolis,2 Felix Dietlein,2 Anna Maria Chiaravalli,4 Annacarolina da Silva,2 Gordon Freeman,2 Scott J. Rodig,2 Eliezer M. Van Allen,2 Bo Li,2 Jingxin Fu,2 Jin Wang,2 Shirley X. Liu,2 Adam J. Bass2. 1 _VU University Medical Center, Amsterdam, Netherlands;_ 2 _Dana Farber Cancer Institute, Boston, MA;_ 3 _Hospital Clínico Universitario de Valencia, Valencia, Spain;_ 4 _Ospedale di Circolo, Varese, Italy_.

Background and aims: Gastro-esophageal adenocarcinoma (GEA) is a common and lethal disease where immune checkpoint inhibitors have demonstrated efficacy within a minority of patients. Prior studies have identified distinct molecular classes of GEA, characterized by specific features that would be predicted to influence responses to checkpoint therapy. We here perform a comprehensive analysis of tumor immune associations in GEAs to further define the tumor immune microenvironment in distinct classes of GEA and to identify additional candidate mediators of immune evasion that may impact response to immunotherapy efficacy. Methods: Tumor immune interactions in GEAs were studied first using computational analyses to infer immune cell subsets from RNAseq data generated from The Cancer Genome Atlas (TCGA). These data were complemented by additional focused studies spanning immunohistochemistry, gene expression profiling and flow cytometry on archival tissue samples and from freshly resected GEA tumor samples. Results: Our results confirmed substantial heterogeneity in the composition of the tumor immune microenvironment between distinct GEA subtypes. As shown earlier, tumors with microsatellite instability (MSI) or Epstein-Barr virus (EBV) harbored larger T cell infiltrates in contrast to those with chromosomal instability (CIN), the most common class of GEA. We then focused greater attention on the CIN cohort, finding a spectrum of phenotypes including tumors with 'immune hot' and 'immune cold' states. The 'hot' cases were more often from Western patients and had greater degrees of DNA methylation. Notably, these tumors often showed co-expression of secondary immune checkpoint proteins, as confirmed by flow cytometry. There was no association between number of infiltrating T cells and mutational or neoantigen load within the CIN GEA subgroup. Immunological cold CIN GEAs were characterized by a higher level of chromosomal instability, enrichment of cell cycle pathways, specifically amplification of cyclin E1, CCNE1, and enrichment of an unfolded protein response.

#4049

Assessment of immune biomarkers by digital pathological analysis across a large colorectal cancer patient cohort predicts patient outcome and may provide a clinically relevant therapeutic index for immunotherapeutic treatment stratification.

Matthew P. Humphries,1 Stephanie Craig,1 Victoria Bingham,1 Maurice Loughrey,1 Helen Coleman,1 Liam Murray,1 Maughan Tim,2 Stephen McQuaid,1 Jacqueline James,1 Manuel Salto-Tellez1. 1 _Queens University Belfast, Belfast, United Kingdom;_ 2 _CRUK/MRC Oxford, Oxford, United Kingdom_.

Introduction: Colorectal cancer (CRC) is a heterogeneous malignancy, treatment for which has principally been cytotoxic chemotherapy. Herein we describe the assessment of immune-biomarkers in a large patient cohort, demonstrating the therapeutic potential of immunotherapy in CRC. Methods: By immunohistochemistry, employing REMARK criteria, using well-validated antibodies, 4 biomarkers (CD3, CD4, CD8, FOXP3) in over 1000 cases, represented across 33 TMAs, were assessed for their protein expression. Digital pathological assessment of immune biomarker densities were quantified via the application of QuPath, an open source digital pathology platform, developed at Queens University, Belfast (https://qupath.github.io.). Biomarker densities were dichotomised by R.O.C analysis. Kaplan-Meier curves (Log-rank p values) were used to assess the impact of immune cell densities on overall survival (OS) and progression-free survival (PFS). Hazard ratios were assessed using the Cox proportional hazards regression model for univariate analysis. Spearman's rank was used to assess correlations of the biomarkers. Results: Expression of two biomarkers were found to vary by stage and three significantly affected OS. Across the patient cohort CD3, CD4 and CD8 were individually shown to be positively prognostic for OS alone (log-rank p = 0.0007, p = 8.65e-06, p = 0.006, respectively). CD4 demonstrated a more significant impact on OS in later stage disease (Log-rank stage II p = 0.0017, stage III p = 0.0002), while conversely CD8 only showed significance in earlier stage disease (Log-rank stage II p = 0.009, stage III p = 0.546). Although significantly associated with OS and within all stages, CD3 showed no differential expression in a particular stage. Correlative analysis demonstrated a statistically significant association between biomarkers. Upon univariate analysis, all biomarkers remained significantly prognostic for OS. All analyses were performed using R. Conclusions: These data demonstrate the prognostic significance of adaptive immune markers in a large CRC cohort on OS. Moreover, stage specific expression maybe clinically relevant and assessment within stages II and III may provide important stratifications for specific immunotherapy treatment regimes. We additionally add weight to the beneficial utility of digital pathology and its potential for integration into routine tissue biomarker assessment. Additional analysis of immune checkpoint and adaptive immune biomarkers in CRC is warranted.

#4050

Immune microenvironment characteristics of primary tumor predict long-term disease-free survival in high-grade serous ovarian cancer (HGSOC).

Wendell Jones,1 Mahrukh Ganapathi,2 Chad Michener,3 Ram N. Ganapathi2. 1 _Q2 Solutions – EA Genomics, Morrisville, NC;_ 2 _Levine Cancer Institute, Carolinas HealthCare System, Charlotte, NC;_ 3 _Cleveland Clinic, Charlotte, NC_.

Epithelial ovarian cancer is a heterogeneous disease with HGSOC being the most common subtype and >70% of women are diagnosed at an advanced stage of the disease. Standard of care involving primary cytoreductive surgery followed by first-line adjuvant platinum/taxane based chemotherapy can lead to a favorable response in 80% of the patients, but the rate of recurrence is >70%. Patient outcome in serous ovarian cancer has been associated with presence of intra-tumoral T cells and an "immunoreactive" subtype. Further, chemotherapy can also affect the immune microenvironment in serous ovarian cancer. We hypothesized that in patients with HGSOC who have long-term disease-free survival the primary tumors harbor a more active adaptive immune cell response. To identify genes associated with immune activity we performed Pan-Cancer immunome analysis (Nature Methods 12, 453-457, 2015) of 4446 RNA-Seq tumor samples balanced for tissue and cancer subtypes, from The Cancer Genome Atlas (TCGA). This analysis yielded several highly correlated gene clusters. After mean-centering by gene and hierarchically clustering both genes and samples, gene-cancer type interactions as well as immunogenic tissue-independent characteristics in the clustered expression profile was observed. We identified a highly-correlated primary cluster of 57 genes associated with cytotoxic lymphocyte activity, referred to as the Cytotoxic Lymphocyte Immune Signature (CLIS). This signature was tested for association with disease-free survival (DFS) in a patient cohort (N=48) that received surgical cytoreduction of tumor followed by adjuvant platinum-taxane chemotherapy and was stratified in three risk-groups: a) Platinum-Resistant, median disease-free interval (MDFI) 0 months (range 0-12 months); b) Platinum-Sensitive, MDFI 15 months (range 13-30 months) and (c) Exceptional-Responders, MDFI >80 months (range 66 - >144 months). RNA isolated from frozen primary tumors was depleted of ribosomal RNA and sequenced using the Illumina TruSeq stranded Total Gold protocol yielding >50M 100 bp paired-end sequences per specimen. In this patient cohort CLIS was distinct in >66% of exceptional-responders compared to <25% in platinum-resistant patients and 44% of platinum-sensitive patients. Using Cox proportional hazards regression, CLIS (p=0.0031), as well as M1 macrophage (p=0.020) and B-cell (p=0.028) signatures were independently associated with DFS after accounting for age and stage. This novel association between CLIS, M1 macrophage and B-cell signature in DFS was replicated independently in HGSOC patient datasets from TCGA, N=164 (p < 0.05) and another (JNCI 106: dju249, 2014) HGSOC patient cohort, N=174 (p < 0.05). In summary, primary tumors in exceptional responders with HGSOC harbor a distinct gene profile/immune microenvironment for optimal treatment efficacy.

#4051

Different immune signatures in advanced lung tumors of Nrf2 wildtype vs. knockout mice.

Di Zhang, Jonathan Rennhack, Eran Andrechek, Cheryl Rockwell, Karen Liby. _Michigan State Univ., East Lansing, MI_.

Activation of Nrf2 and its downstream target genes serves as a master defense mechanism, protecting cells against oxidative and electrophilic stress. Numerous activators of Nrf2 inhibit carcinogenesis. However, genetic alterations resulting in constitutive Nrf2 activation in human lung tumors promotes tumor growth and chemoresistance. The dual role of Nrf2 in cancer has generated a high level of interest, but little is known regarding the effect of Nrf2 on immune cells during lung carcinogenesis. In this study, we used the potent carcinogen vinyl carbamate to induce lung tumors in mice. Tumor burden, the immunophenotype, and genetic signatures were compared in the lungs of Nrf2 knockout (-/-, KO) vs. wildtype (+/+, WT) mice. Nrf2 KO mice were more susceptible to vinyl carbamate; these mice developed tumors earlier than the WT mice and exhibited more and larger tumors over times ranging from 20-32 weeks after initiation. By 32 weeks, WT mice have an average of 6.5 ± 0.95 tumors per lung compared to 12.25 ± 2.6 tumors in the KO mice. Immune cell populations including T cells (CD4+ and CD8+), B cells, macrophages and myeloid derived suppressor cells were investigated in the lungs and spleens of Nrf2 WT and KO mice challenged with vinyl carbamate. T cell populations were significantly (P < 0.05) reduced in the lungs of Nrf2 KO mice, while tumor-promoting macrophages and myeloid derived suppressor cells were elevated in the lung and spleen, respectively, of Nrf2 KO mice compared to WT mice. Moreover, immune response genes, especially a series of cytokines (Cxcl1, Cxcl12, Csf1, and Ccl9) and MHC antigens that promote tumor growth, were significantly upregulated in tumors from Nrf2-/\- mice. Importantly, this expression profile is conserved in patients with lung cancer. Overall, our results confirmed a tumor protective role of Nrf2 in carcinogenesis and suggest an important regulatory role for Nrf2 in immune cells in both mouse and human lung tumors.

#4052

Analysis of types of tumor-infiltrating immune cells in oral squamous cell carcinoma (OSCC) and their association with survival in OSCC patients.

Manali Vora, Wei Sun, Noel Weiss, Chu Chen. _Fred Hutchinson Cancer Research Center, Seattle, WA_.

Background: The 5-year survival associated with oral cancer (OSCC) is relatively low. Recent investigations have implicated the presence of tumor infiltrating lymphocytes (TILs) as independent prognostic markers in several malignancies, including oral cancer. However, the studies of oral cancer evaluated only a limited number of immune cell types as prognostic markers. CIBERSORT, a computational software for in silico analysis of tumor microenvironment, provides an opportunity to measure 22 cell types of TILs in two previously-assembled cohorts of OSCC patients.

Methods: We used CIBERSORT to evaluate Gene Expression Profiles (GEPs) of OSCC tumor samples using data from The Cancer Genomic Atlas (TCGA) and Fred Hutchinson Cancer Research Center's Oralchip study to infer the types of immune cells that are present in the tumor tissue. Tumor samples were analyzed to evaluate a difference in immune cell composition of OSCC tumors by HPV status, location and by tumor stage. We also assessed the independent prognostic value of individual immune cell types by means of a multivariable Cox regression model which adjusted for patient's age at diagnosis, HPV status, tumor location, tumor stage, and smoking and alcohol consumption history. In addition, we did a subset analysis on 50 HPV-positive oropharyngeal (OPC) cases where we assessed the independent prognostic value of individual immune cell types through the same multivariable Cox regression model.

Results: The immune microenvironment of HPV-positive OSCC cases was significantly different compared to HPV-negative OSCC cases. The proportion of TILs such as naïve B cells, cytotoxic T cells, activated memory T cells, follicular helper and regulatory T cells was higher among HPV-positive OSCC cases and in HPV-positive OPC cases. Composition of some immune cell types differed by tumor stage. No significant association was observed between higher composition of individual immune cell types and overall survival in OSCC patients. Subset analysis showed that HPV-positive OPC cases with higher concentration of cytotoxic CD8+ T cells and activated memory CD4+ T cells were associated with better prognosis. On the other hand, higher concentrations of activated dendritic cells, mast cells and neutrophils were associated with relatively poor overall survival.

Conclusion: Our study suggests immune system is predictive of survival in HPV-positive OPC patients, and that the composition of immune cells in HPV-positive OPC is different from that of HPV-negative OPC. These findings may help guide clinical management of these patients.

#4053

Partial reversion of the tumor immunosuppressed environment by oncolytic adenoviruses armed with positive stimulators of the immune synapsis in breast cancer.

Francisco W. Puerta Martinez, Yisel A. Rivera, Teresa Nguyen, Xuejun Fan, Rehnuma Shifat, Mohammad Belayat Hossain, Hong Jiang, Chandra Bartholomeusz, Lang F. Frederick, Juan Fueyo, Candelaria Gomez-Manzano. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Immunotherapy is currently one of the primary focuses of cancer research and has the potential to become the standard treatment for oncologic patients over the next decades. Promising clinical responses have been observed in patients treated with a variety of immune-targeted therapies such as checkpoint inhibitors, modified dendritic cells, and T cell or NK cell-based immunotherapies. However, the efficacy of these treatments is still very limited due to multiple factors related to tumor nature and microenvironment. Hence, combination therapies aimed at modulating the strong tumor immunosuppressive microenvironment and stimulating T-cell infiltration into the tumor lesions are urgently needed to overcome cancer intrinsic resistance mechanisms. Preclinical and clinical studies have shown the efficacy of oncolytic virotherapy to elicit antitumor immune responses and to increase infiltration of both innate and adaptive immune cells. These capabilities of the oncolytic viruses can be enhanced and exploited to improve response of patients to different immunotherapies. Here we tested the ability of two modified oncolytic adenoviruses termed Delta-24-RGDOX and Delta-24-GREAT, expressing OX40L and GITRL respectively, to modify the tumor microenvironment in different murine triple-negative breast cancer models. We hypothesized that intratumoral injection of these armed oncolytic adenoviruses will decrease immunosuppression within the tumor microenvironment. To this end, mice harboring fat pad tumors were treated with intratumoral doses of these armed oncolytic adenoviruses. Leukocytes from spleen and tumor tissues were analyzed for the relative presence of lymphocyte and myeloid cell subsets. In all mice treated with Delta-24-RGDOX or Delta-24-GITRL, CD8+ T-cell populations were significantly higher compared with those observed in the control mice. Significantly lower percentages of cell populations with immunosuppressive phenotypes, such as PD-1\+ T cells (P<0.0001, Student t-test), CD4+CD25\+ Foxp3+ regulatory T cells (P<0.0001, Student t-test) and MDSC (P<0.0001, Student t-test) were found in the mice treated with the armed oncolytic adenoviruses. Taken together, our results confirmed the strong immunomodulatory effect of enhanced oncolytic viruses, which can be easily combined with immune-modulating checkpoints, for the treatment of deadliest human cancers.

#4054

Gene expression difference (GED) revealed immune function gene down-regulation as tumor-associated inflammatory cell (TAIC) infiltration in microenvironment in non-small cell lung cancer (NSCLC).

Ximing Tang,1 Wei Lu,1 Jiexin Zhang,1 Carmen Berhens,1 Edwin R. Parra,1 John Wineman,2 Jianjun Zhang,1 Don Lynn Gibbons,1 Mark Koeppel,2 BJ Kerns,2 Mark Stern,3 Boris Sepesi,4 Jack J. Lee,1 Ignacio I. Wistuba1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _HTG Molecular Diagnostics, Inc., Tucson, AZ;_ 3 _HTG Molecular Diagnostics, Inc, Tucson, AZ;_ 4 _UT MD Anderson Cancer Ctr., Katy, TX_.

Background Advances in molecular analyses of tumor tissues based on high-throughput technologies can contribute to a better prediction of disease prognosis and the efficacy of immunotherapies. We investigated the GED related to immune response in a large cohort of NSCLC. Methods Using low-input, high-throughput HTG EdgeSeq assay, we examined expression of a panel of 549 immune response-related genes (28 are categorized as immune function genes) in FFPE tumor tissues of 226 surgically resected, stage I to III NSCLCs (137 adenocarcinomas [ADC] and 89 squamous cell carcinomas [SqCC]) with annotated clinical, pathological characteristics. Molecular data were available on mutations of EGFR and KRAS (cases=126), STK11, PIK3CA and TP53 by whole exome sequencing (n=180), and TAICs density detected by IHC (CD3, CD4, CD68, CD8, FOXP3, GZB, PD-L1 and PD-1) (n=145). We associated GED to 12 clinical, pathological or molecular characteristics including age, gender, smoking history, histology, stage, size, lymph node status and gene mutations of tumors and to patients' outcome (recurrence free survival [RFS] and overall survival [OS]). Results Univariate analysis showed upregulations of 82 genes in ADC and 89 in SqCC by comparing each other (P≤0.001). In ADC, 23 GEDs [FDR (false discover rate) ≤5%] associated with ever smoking history (8 up- and 15 down-regulated). Some genes were identified to be associated with mutations of EGFR, KRAS, STK11, PIK3CA and P53 genes. GEDs were identified in the tumors with TAIC high density by IHC. In ADC, 192 genes were down- and 191 up-regulated; while among the 28 immune function genes 21 down- and, only the gene APP (in all 8 high density TAICs) up-regulated. In SqCC, 121 genes down- and 112 up-regulated; while in the 28 genes 17 were down- and only the gene CAP up-regulated slightly in the high FOXP3 tumors. Cox proportional hazard model using FDR 5% in multi-testing showed that in ADC treated with adjuvant chemotherapy high-expression in 10 genes such as CD52, CD74 and HLA-DRA having a beneficial and 5 genes such as HRT2 and SERPINB2 having worse effect on OS; in the ADC patients who did not receive adjuvant therapy, high-expression of PIK3CD showed beneficial and, TTK and CXCL8 showed detrimental effect on OS; no gene was found to be associated with RFS in ADC and with OS and RFS in SqCC. Conclusion Gene expression profiling analysis of a panel of immune-related genes using low-input mRNA from FFPE tumor tissues revealed a number of genes associating to tumor characteristics, gene mutation patterns and TAIC infiltration in NSCLC. Interestingly, almost of immune function genes were down-regulated in the tumor with TAIC high density in NSCLC. (Supported in part by grant CPRIT RP160668)

#4055

Genomic and immune characterization of triple-negative breast cancer brain metastases.

Benjamin G. Vincent, Maria Sambade, Shengjie Chai, Marni B. Siegel, Luz Cuaboy, Alan Hoyle, Joel Parker, Charles M. Perou, Carey K. Anders. _Univ. of North Carolina at Chapel Hill; Lineberger Comprehensive Cancer Center, Chapel Hill, NC_.

Introduction: Triple negative breast cancer (TNBC) is an aggressive subset of BC with high metastatic potential. Once metastatic, half of patients (pts) with TNBC will develop brain metastases (BM), commonly with progressive extracranial disease. While TNBC BM are routinely treated with radiotherapy, survival is generally less than one year. There are no approved systemic therapies to treat TNBC BM. Both the blood brain barrier and paucity of data on the biologic underpinnings and immune response of BCBM contribute to inadequate therapies for this disease. We sought to characterize the genomic and immune landscape of TNBC BM to foster the development of effective brain permeable anti-cancer agents.

Experimental Procedures: A clinically-annotated BCBM biobank of archival brain metastases and primary BC that eventually metastasize to the brain was created under IRB approval (LCCC1419). DNA (tumor/normal) and RNA (tumor only) was extracted. Following library preparation, whole exome (WES) and RNA sequencing (RNASeq) was performed. Common mutations were determined from WES as those co-identified by two variant callers (Strelka, Cadabra), while immune gene signature expression, molecular subtype identification, and B and T cell receptor repertoires were inferred from RNAseq data.

Results: Of the 26 enrolled pts, median age at BCBM diagnosis was 52 years (35-72); 23% were African American and 57% Caucasian. Additional non-brain metastatic sites included bone (23%), liver (20%), and lung (35%); 23% had a solitary BCBM. 35% of pts received chemotherapy after BCBM diagnosis, while 92% received radiation to the brain (21% WBRT, 5% SRS, 25% SRS plus WBRT and resection). The median survival from BCBM was 6 months. 40 (93%) of the tissues (21 primaries, 22 BCBM) were characterized as basal-like by PAM50; 1 HER2-enriched and 2 Luminal A. 34 genes were mutated in ≥20% of BCBMs, while only 8 were mutated in at least 25% of primaries. Commonly mutated BCBM genes included TP53, ATM and MYH9; in primaries only TP53. Many immune gene signatures were lower in BCBM compared to primary BC including B cell, dendritic cell, regulatory T cell, and IgG cluster (p<0.05). A signature of responsiveness to PD1 inhibition in melanoma was higher in BCBM compared with primary BC (p<0.05). Globally, the BCBM T cell receptor repertoires showed higher diversity (TCR evenness, p=0.006) and lower read count (TCR total read count, p=0.0267) compared to primary BC.

Conclusions: TNBC BM compared to primary BC that metastasize to the brain had more significantly mutated genes, lower immune gene signature expression, higher PD1 inhibition response signature expression, and T cell receptor repertoire features less characteristic of an active antigen-specific response. Given that checkpoint inhibitors are showing response in non-BCBM (i.e. lung and melanoma), these findings indicate that immunotherapy to treat patients with TNBC BM is worthy of exploration.

#4056

Immune regulation by miR-1227 LO.

Andrew R. Chin,1 Dolores Di Vizio,1 Valentina R. Minciacchi,2 Mariana Sobreiro,1 Cristiana Spinelli3. 1 _Cedars-Sinai Medical Center, West Hollywood, CA;_ 2 _Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt, Germany;_ 3 _McGill University, Montreal, Quebec, Canada_.

Immunotherapy offers great promise for the treatment of prostate cancer; however prostate cancer often acquires mechanisms to dampen the tumoricidal immune response. Understanding how prostate cancer dampens the immune system will facilitate the generation of more effective immunotherapies. Here we investigate how large oncosomes (LO)-mediated transfer of miR-1227 to immune cells modulates the immune response.

Extracellular vesicle (EV)-encapsulated miRNAs are being increasingly recognized for playing a key role in cancer progression and immune modulation. miR-1227 is a poorly characterized miRNA that is enriched in EV secreted by prostate cancer cells in comparison to non-malignant prostate epithelial cells. miR-1227 expression is increased in migratory glioblastoma cells in comparison to non-migratory glioblastoma cells, and in radiation resistant esophageal cells and colorectal cancer stromal cells indicating that miR-1227 is upregulated in different cancer types. However, the role of miR-1227 in cancer is poorly understood. We demonstrate here that EV obtained from prostate cancer cells overexpressing miR-1227, induce the migration of prostate cancer cells and fibroblasts. A comparative analysis between different EV subtypes indicates that miR-1227 is enriched in LO, a class of EV that are secreted by highly invasive amoeboid-migrating cells. We also show that LO carry more RNA than the more widely studied exosomes indicating that LO may be a more robust source of EV-encapsulated miRNA. RNA sequencing from miR-1227 stably expressing prostate cancer cells and 5 different in silico miRNA target prediction methods were used to identify putative miR-1227 targets. IPA pathway analysis from these predicted targets highlighted several functions related to the immune system, including T cell homeostasis, T cell development, and development of Th17 cells. A more in-depth analysis of the 51 genes involved in T cell homeostasis showed enrichment of genes associated with Th17 and Th1 T cell differentiation. Alterations in Th17 and Th1 differentiation modulates T cell cytokine secretion, which are important mediators of cancer growth and progression. Transient transfection of miR-1227 in immune cells altered the cytokine expression profile, resulting in the upregulation of several cytokines that promote cancer growth including CCL5 and IL6. We tested whether LO can be taken up by activated T cells to directly modulate cytokine secretion. To determine whether miR-1227 can be functionally transferred to recipient cells via LO we isolated LO from prostate cancer cells stably expressing miR-1227 using ultracentrifugation followed by density gradient purification. The isolated LO were used to treat immune cells to show that LO-encapsulated miR-1227 alters cytokine profiles. These results indicate that LO-encapsulated miR-1227 may be involved in cancer immune modulation through targeting specific genes involved in T-cell differentiation.

#4057

Identification of DNA repair genes that affect clonal heterogeneity and the immune microenvironment in breast cancers.

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

Background: While genetic aberrations of DNA repair genes are known to increase immunogenicity in breast cancer, the clinical significance of the tumor microenvironment including tumor-infiltrating immune cells in regard to DNA repair genes has not yet been elucidated in breast cancer patients. We sought to determine the significance of DNA repair genes and their impact on the immune microenvironment, which could provide insight for the development of immunotherapy in breast cancer. We aimed to correlate DNA repair gene deficiency with tumor-infiltrating immune cells and tumor immune microenvironment, and assess its clinical relevance.

Materials and Methods: Integrated and unbiased transcriptomics approach was conducted on genomic and clinical information of 3,614 breast cancer patients of The cancer Genome Atlas (TCGA) and the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) to assess the association between the aberration of DNA repair genes and the tumor immune microenvironment in breast cancer, as well as the clinical significance utilizing an integrated immunogenomic pipeline. Tumor-infiltrating immune cells (TICs) composition was calculated using the CIBERSORT system that estimates the fraction of 22 immune cell types in each tumor. Intratumor clonal heterogeneity and T-cell receptor diversity was evaluated utilizing mutant allele tumor heterogeneity (MATH) index and TCR repertoire analysis.

Results: We found that low expression of DNA repair genes, especially low BRCA1 expression, was significantly associated with improved survival and higher CD8+ cell composition. Tumors with low expression of BRCA1 enriched immune-response related gene sets, using gene set enrichment analysis, significantly associated to higher expression of the immune gene signature, such as HLA-A, PRF1, GZMA, and GZMB, as well as local immune cytolytic activity (CYT). Intratumor clonal heterogeneity was associated with low expression of BRCA1 with high composition of CD8+ T-cells. T-cell receptor (TCR) repertoire analysis elucidated that lower TCR diversity correlated to low BRCA1 expression in hormone-negative or triple-negative breast cancer patients. Utilizing the GEO database with clinical information of response to neoadjuvant chemotherapy (NAC) in breast cancer, patients with low BRCA1 expression showed significantly better response to NAC.

Conclusions: We conclude that the comprehensive and unbiased immunogenomic analysis clearly elucidated that low expression of BRCA1, was associated with higher intratumor immune response including immune gene signature, TICs, intratumor clonal heterogeneity, and TCR diversity, all of which have a significant impact on the clinical outcome in breast cancer.

#4058

Race-associated breast cancer disparities: The role of CRYBB2 and its pseudogene.

Maya A. Barrow,1 Megan E. Martin,2 Denise K. Reaves,1 Breanna Jeffcoat,1 Ginger R. Smith,1 Michael Tarpley,1 Kevin P. Williams,1 Jodie M. Fleming1. 1 _North Carolina Central University, Durham, NC;_ 2 _North Carolina State University, Raleigh, NC_.

Research demonstrates young African American (AA) women have higher mortality rates for triple-negative breast cancer (TNBC), despite lower incidence rates, when compared to Caucasian American (CA) women. These disparate poor patient outcomes may be attributed to access to quality care, socioeconomic status, education, or geographic location. However, numerous other studies have demonstrated that gene expression differences may also contribute to the higher mortality rates experienced by AA women, suggestive of biological differences among these two patient populations. Consequently, CRYBB2 is one gene that has consistently been associated with AA race in normal and malignant tissues. Although, its basic function in these tissues is currently unknown, higher expression of CRYBB2 is significantly correlated with poor outcome in breast, colorectal and prostate cancers in AAs. We analyzed all publically available gene expression datasets from studies focused on breast cancer health disparities and discovered that all probes detected CRYBB2 as well as its pseudogene, CRYBB2P1. It is becoming increasingly clear that pseudogenes have the ability to modify the mechanisms of their functional parental genes. However, it remains unclear whether the high expression of CRYBB2, CRYBB2P1, or both, affect cell behaviors to alter disease progression. Herein, we investigated the biological consequences of CRYBB2 and CRYBB2P1 in three basal-like TNBC cell lines (SUM159, MDA-MB-231, Hs578t) using CRISPR/Cas9 and overexpression technologies. The functional consequence of CRYBB2 and CRYBB2P1 expression were evaluated using in vitro and biochemical assays, as well as in vivo tumorigenesis studies. Our findings demonstrate overexpression of CRYBB2 and CRYBB2P1 alter cell proliferation, cytokine and growth factor production, and tumorigenesis. This data highlights the potential molecular mechanisms by which CRYBB2 and CRYBB2P1 contribute to poor patient outcome.

#4059

**Vaccination causes a clear subtype shifting of tumor infiltrate T** reg **in mouse breast cancer homografts.**

Xiaoyu AN, Davy Xuesong Ouyang, Jinping Liu, Li Chen, Henry Qixiang Li. _Crown Bioscience, San Diego, CA_.

Tumor-infiltrating lymphocytes (TIL), including CD8+ TIL-Teff (effector T-cells) and TIL-Treg (regulatory T-cells with immune suppression on self-restricted Teff), are believed to play a significant, but somewhat conflicting, role in the cancer prognosis and sensitivity to immune-oncology (I/O) treatments, including immune checkpoint inhibitors (ICI). Forkhead box P3 (FoxP3+) has long been considered a key marker of Treg while recent research has indicated significant heterogeneity of Treg with different levels of FoxP3 and the associated functional variations. The heterogeneity may explain some of the conflicting observations. The influence of the Treg heterogeneity on ICI treatment has yet to be investigated. We have previously created a mouse tumor homograft, mBR6004, by orthotopic-implanting breast adenocarcinoma spontaneously developed in MMTV-PyVT transgenic mice (GEMM) to the syngeneic FVB/N mice. The homograft has the similar growth, metastasis and histopathology as seen for the original GEMM tumors, and it responds to the CTLA-4 but not to the PD1/PDL1 inhibitors. Further, we also found that a prior vaccination treatment seemed to potentially render the tumor sensitive to PD1/PD-L1 inhibitors with increased CD8+ TIL in the treated tumors. To explore the underlying mechanism of vaccination, we are performing extensive immunophenotyping of TILs in the vaccinated tumors. Interestingly, our analysis clearly displayed a drastic shift of TIL-Treg from FoxP3lo to FoxP3hi population in the tumors with the prior vaccination as compared to the tumor without vaccination, which likely corresponds to the reported naïve FoxP3lo Treg and FoxP3hi effector Treg (eTreg). This FoxP3hi -shifted Treg is also largely absent in peripheral blood, even in the vaccinated mice, consistently to those seen in patients. Our data seem to suggest that the TIL-eTreg status, not naïve Treg, could play a critical role in tumor response to the checkpoint inhibitor treatment, and that the methods, such as vaccination to change TIL-eTreg, could be an important strategy to enhance checkpoint inhibitor treatment. In addition, the elucidation of TIL-Treg in animal model could also greatly help to investigate I/O mechanisms in the experimental settings.

#4060

In vitro **human tumor-associated macrophage model implicates macrophage proliferation as a mechanism for maintaining tumor-associated macrophage populations.**

Amber E. de Groot, Kenneth J. Pienta. _Johns Hopkins Univ. School of Medicine, Baltimore, MD_.

Prostate cancer tumor growth and disease progression is highly influenced by non-cancerous host cells within the tumor microenvironment. Tumor-associated macrophages (TAMs) are a critical component of this microenvironment and can comprise up to 50% of prostate cancer tumors. Additionally, high TAM infiltrate correlates with poor prostate cancer patient prognosis. Macrophages have different phenotypes and functions depending on the immune response in which they are involved. The majority of prostate cancer TAMs resemble alternatively-activated M2 macrophages which, in healthy individuals, participate in the Th2 immune response which is primarily involved in wound healing. Macrophages contribute to Th2 responses by promoting tissue remodeling, cell proliferation, angiogenesis, and immune suppression. These functions are taken advantage of by cancer cells to promote tumor growth, immune evasion, and disease progression. Techniques for studying TAMs in vitro are limited and are confounded by the lack of procedure consensus for in vitro macrophage differentiation and polarization. We have established an optimized protocol for mimicking TAMs in vitro using macrophages derived from human monocytes isolated from whole blood. (doi: 10.2144/000114435). These cell populations have been validated as an accurate TAM model by their marker expression and immunomodulatory functions. It has recently been widely accepted that tissue populations of macrophages are maintained by proliferation of tissue-resident macrophages seeded during embryogenesis rather than by recruitment of monocytes from the blood. Additionally, it has been reported that local macrophage proliferation is a prominent method for maintaining macrophage populations in Th2 diseases and TAM populations in breast cancer models. However, how and whether in vitro models recapitulate this macrophage behavior is has been largely ignored and remains unaddressed. To address this question, we investigated the proliferation status of our optimized in vitro TAM model by observing the expression of the proliferation marker Ki67 and EdU incorporation by flow cytometry and immunofluorescence. We found that subsets of these macrophages stain positively for Ki67 and incorporate EdU indicating that they proliferate in culture. These findings have wide implications for cancer as it suggests that the large M2 TAM populations found in prostate cancer tumors may be maintained by macrophage proliferation. This supports a mechanism for the origin and maintenance of TAM populations that is currently ignored by the majority of the cancer field. Further work will investigate the proliferation status of TAMs in vivo using MycCaP prostate cancer tumors in syngeneic mouse models.

#4061

The inflammasome of tumor cells modulates the biology of tumor-infiltrating T lymphocytes in colorectal cancer.

Linda Bilonda,1 Delphine Dansette,2 Cecile Deleine,1 Romain Oger,1 Nicolas Jouand,1 Juliette Podevin,3 Pierre Fourquier,4 Emilie Thibaudeau,5 Jerome Chetritt,6 Jean-François Mosnier,2 Celine Bossard,7 Nadine Gervois,1 Anne Jarry1. 1 _INSERM U1232, Nantes, France;_ 2 _CHU, Pathology Dept, Nantes, France;_ 3 _CHU, Digestive Surgery, Nantes, France;_ 4 _Nouvelles Cliniques Nantaises, Nantes, France;_ 5 _ICO, Saint-Herblain, France;_ 6 _Institut d'Histopathologie, Nantes, France;_ 7 _INSERM U1232 and Pathology Dept, Nantes, France_.

In colorectal cancer (CRC), little is known about mechanisms by which tumor cells can influence the biology of Tumor Infiltrating T lymphocytes (TILs) present in the tumor microenvironment. One of these mechanisms could be modulation of the inflammasome of tumor cells. The inflammasome is a molecular platform present in normal intestinal epithelial cells, whose effector protein, caspase-1, can rapidly mature IL-18 and generate a mucosal Th1 (IFNγ) response. However, the inflammasome status of tumor cells in CRC and its potential role in the biology of TILs are unknown yet. This prospective study aimed to determine in CRC patients (n = 50) : i) the status of the inflammasome (caspase-1 / IL-18 axis) in tumor cells according to their microsatellite status [unstable (MSI) or stable (MSS)], in relation with the density of Th1/Tc1 TILs (T-bet+) and with the levels of the prototype Th1 cytokine IFNγ, secreted in an ex vivo explant culture model of CRC we developed and ii) the impact of the inflammasome-dependent cytokines potentially secreted by tumor cells on the biology of isolated TILs. Our study delineates two major groups of patients. The first group (33% of cases, mostly MSS with a low immunoscore) featured no active caspase-1 in tumor cells, no or low levels of mature IL-18 and IFNγ and only few T-bet+ TILs in the tumor microenvironment. This profile could correspond to an immune escape mechanism facilitating tumor progression. The second group (66% of cases, both MSI and MSS with high immunoscore) featured active caspase-1 in tumor cells associated with mature IL-18 secretion, high density of T-bet+ TILs expressing IL-18Rα and IFNγ release in most cases. In addition, isolated IL-18Rα+ TILs cultured with recombinant IL-18 were able to secrete IFNγ, either left unstimulated or stimulated with anti-CD3. In these CRC, the inflammasome of tumor cells, maintained and active, can contribute to a Th1/Tc1 antitumor response elicited by TILs present in the microenvironment that can modulate tumor growth. Interestingly, in a few cases of this second group (MSI CRC), the numerous T-bet+ TILs were unable to generate a Th1 response. Noticeably, these TILs express numerous immune checkpoints including PD1 and TIGIT, potentially responsible for their exhaustion. This study is the first to delineate functional interactions between tumor cells and TILs in CRC, using ex vivo explant cultures. Altogether, our findings support the inflammasome of tumor cells as a potential new therapeutic target to strengthen the Th1/Tc1 immune response in CRC, in association with immune checkpoint blockers. This work is supported by the "Ligue contre le Cancer Grand Ouest"

#4062

Novel synthetic peptide targets CD206 receptor promoting the repolarization of prostate cancer (PCa) TAMs and increase the efficacy of conventional prostate cancer therapeutic.

Ahmad B. Salam,1 Benjamin Adu-Addai,1 Huixian Lin,1 Anghesom Ghebremedhin,1 Jason White,1 Ruksana Amin,1 Balasubramanyam Karanam,1 George Martin,2 Charles Garvin,2 Henry Lopez,3 Jesse M. Jaynes,1 Clayton Yates1. 1 _Tuskegee University, Tuskegee, AL;_ 2 _Riptide Bioscience, Inc., CA;_ 3 _Murigenics, CA_.

Background: Tumor associated M-φs (TAMs) have a complex etiology as a result of soluble factors secreted by prostate tumor cells, creating an immunosuppressive micro-environment (M-E) for the progression of late stage PC that has metastasized to the bone. The result is an M2-like polarization that expresses a number of M2-TAM specific markers, including CD206. Therapeutic targeting of TAMs has proven to be a challenge, due partly to a lack of understanding of the function of this biomarker and the bi-directional effects of TAMs on tumor cells. Our objective is to characterize the CD206 receptor in PC patients, and to test the potential of a novel engineered CD206 targeted peptide (T-P) to reverse the immune-suppressive M-Env in late stage PC. Methods: The TGCA Prostate Dataset was used for patient data and R software was used for informatics analysis. Small-angle X-ray scattering (SAXS) was used to generate the NMR structure of CD206. Peptide binding to the naïve CD206 receptor was determined in silico using PIPER, and was validated by Surface Plasmon Resonance (SPR). In vivo efficacy of CD206 T-P was determined in mice inoculated with PC-3 intratibially. Mice were treated with peptide alone, daily or twice a week at 10, 20, and 50mg/Kg, as a monotherapy or in combination with 20mg/Kg Docetaxel (DTX) over a 21day period. CD86 (M1) and CD206/CD163 (M2-TAM) markers and pro-inflammatory cytokines were determined by FACS. Bone osteolysis was determined by IHC of the entire bone, and bone turnover was determined by IHC analysis of RANKL. Results: TCGA data showed that over-expression of CD206 in Human PC is significantly associated with poor survival. In silico analyses showed the T-P exhibited strong affinity towards CD206 and was validated by SPR (Kd =3.57 µM). In bone-metastatic xenograft models of CRPC (PC-3), the flux differential of relative fluorescence in IVIS imaging showed peptide alone (at 20mg/kg qD sQ) decreased fluorescence by 64.36% compared with the untreated group. FACS analyses of M1/M2 markers CD86/CD206 in mice treated with peptide showed a 33.87 fold decrease in CD206 (M2-TAM) expression and a 7.6 fold increase in CD86 (M1-TAM) expression, compared with only a 4.4 fold decrease in CD206 expression and 0.44 fold decrease in CD86 expression in DTX treated mice. Stem cell marker CD133 showed a 4.7 fold decrease expression compared to the untreated group further suggesting that TAM repolarization reversed the immunosuppressive M-E. X-ray of tumors in bone suggests a reduction of osteolysis and was confirmed by IHC staining and analysis of whole bone showing fibrous and inflammatory remodeling patterns of tumor regression in the peptide only group. Conclusion: These results suggest that the novel engineered CD206 T-P repolarizes M2-TAM phenotype to M1-TAM which reverses the immunosuppressive cancer M-E in late stage PC.

#4063

Target immunosuppressive myeloid cells to enhance cancer immunotherapy.

Xin Lu. _University of Notre Dame, Notre Dame, IN_.

A significant fraction of advanced prostate cancer (PCa) patients treated with androgen deprivation therapy (ADT) experience relapse with relentless progression to lethal metastatic castration-resistant prostate cancer (mCRPC). Immune checkpoint blockade (ICB) using antibodies against cytotoxic-T-lymphocyte-associated protein 4 (CTLA4) or programmed cell death 1/ programmed cell death 1 ligand 1 (PD1/PD-L1) generates durable therapeutic responses in a significant subset of patients across a variety of cancer types. However, mCRPC showed overwhelming de novo resistance to ICB, motivating a search for targeted therapies that overcome this resistance. Myeloid-derived suppressor cells (MDSCs) are known to play important roles in tumor immune evasion. Circulating MDSC abundance correlates with PSA levels and metastasis in PCa patients. Mouse models of PCa show that MDSCs (CD11b+ Gr1+) promote tumor initiation and progression. These observations prompted us to hypothesize that robust immunotherapy responses in mCRPC may be elicited by the combined actions of ICB agents together with targeted agents that neutralize MDSCs yet preserve T cell function. Here we developed a novel chimeric mouse model of mCRPC to efficiently test combination therapies in an autochthonous setting. Combination of anti-CTLA4 and anti-PD1 engendered only modest efficacy. Targeted therapy against mCRPC-infiltrating MDSCs, using multikinase inhibitors such as cabozantinib and dactolisib, also showed minimal anti-tumor activities. Strikingly, primary and metastatic CRPC showed robust synergistic responses when ICB was combined with MDSC-targeted therapy. Mechanistically, combination therapy efficacy stemmed from the upregulation of IL-1ra and suppression of MDSC-promoting cytokines secreted by PCa cells. These observations illuminate a clinical path hypothesis for combining ICB with MDSC-targeted therapies in the treatment of mCRPC.

More recently, through mass spectrometry we uncovered a new mechanism by which MDSCs employ potent reactive nitrogen species (RNS) to inactivate key molecules in the T cell activation signaling pathway. Agents that neutralize RNS generate significant anti-tumor effect when combined with ICB in prostate cancer.

#4064

**Spatial re-distribution of natural killer cells targets drug resistance and anti-tumor response,** ex-vivo **.**

Munisha Smalley,1 Basava U. Shanthappa,1 Hans Gertje,1 Mark Lawson,1 Manjusha Biswas,2 Saravanan Thiyagarajan,2 Biswanath Majumder,2 D. C. Doval,3 Anurag Mehta,3 Neyaz Alam,4 Nabendu Murmu,4 S. P. Somashekhar,5 Aaron Goldman1. 1 _Mitra Biotech, Woburn, MA;_ 2 _Mitra RxDx, Bangalore, India;_ 3 _Rajiv Gandhi Cancer Institute & Research Centre, New Delhi, India; _4 _Chittaranjan National Cancer Institute, Kolkata, India;_ 5 _Manipals Hospitals, Bangalore, India_.

Background: It is now clear that response or resistance to therapy is contingent on the tumor microenvironment, which is comprised of malignant cells, normal stroma, and immune landscape; attributes that are unique to each individual patient. This is particularly true for emerging anticancer drugs, such as immune checkpoint inhibitors, which recalibrate the body's own immune defense largely by modulating exhaustion of cytotoxic lymphocytes including T cells and natural killer (NK) cells. However, clinical response to therapy varies enormously. There is a critical gap in our understanding for the mechanisms that drive response or resistance to conventional drugs and immunotherapies at the individual patient level. Methods: Here, we used in-vitro and in-vivo experiments to study the role of NK cells in models of drug tolerance. Next, we employed (CANscriptTM), a clinically-validated ex-vivo tumor model that recreates and preserves the native, patient tumor microenvironment, which integrates an algorithm-driven method to predict clinical response to therapy (M-Score). Utilizing tissue from patients diagnosed with luminal, HER2 positive, and triple-negative (ER- PR- HER2-) breast cancers (N=10), we studied spatial heterogeneity of the tumor-immune contexture, M-Score, and phenotypic alterations under pressure of conventional standard-of-care regimens and immunotherapies including immune-checkpoint inhibitors. To do this, we used a comprehensive panel of immunological assays to evaluate changes in cytotoxic lymphocytes by flow cytometry, immunohistochemistry and cytokine profiling (i.e. CD56, MHC class 1A/B, NKG2D/C, CD8, CD3, PD-1, CTLA-4, TIM-3, LAG-3, 4-1BB, granzyme A/B). Results: We identified that drug resistance is associated to downregulation of activating proteins on NK cells including NKG2D/C, reducing their cytotoxic capability. Furthermore, tumor response, predicted by M-Score, correlates to a unique pattern of spatially-distributed NK cells, which associated a pro-inflammatory cytokine signature from the tumor microenvironment. Interestingly, we determined that therapy-induced expression of exhaustion biomarkers on NK cell exhaustion inversely correlated to the expression of cytotoxic granzyme B in the tumor microenvironment. Conclusions: Taken together, these data demonstrate an integral role that NK cells contribute to resistance and the antitumor response to therapy including both conventional and immuno-modulatory drugs. We further demonstrate that CANscriptTM can be harnessed to study response and resistance of the innate and adaptive immune system. Such an advance in our preclinical methods to study anticancer drugs at the individual patient level can help guide treatment decisions for clinicians while simultaneously functioning as a platform to study clinical efficacy of novel and emerging agents.

#4065

Suppression of myeloid cell arginase activity leads to therapeutic response in Kras mutant lung cancer by activating anti-tumor immunity.

Juan J. Miret,1 Paul Kirschmeier,1 Min Wu,1 Wei Huang,1 William Walker,1 Sangeetha Palakurti,1 Nick Saccomano,2 Peter S. Hammerman,1 Kwok-kin Wong,3 Esra Akbay4. 1 _Dana Farber Cancer Institute, Belfer Institute, Boston, MA;_ 2 _Array Biopharma, CO;_ 3 _Division of Hematology and Medical Oncology, New York University, Boston, MA;_ 4 _UT Southwestern Medical Center, Dallas, TX_.

Metabolic changes in the tumor microenvironment impair the generation of an efficient anti-tumor immune response. Reduction of specific amino acids like arginine and tryptophan limit anti-tumor immune responses, contributing to a tumor generated immune suppressive environment. Arginine levels are critical for lymphocyte proliferation and function, and they are regulated by arginase 1 & 2 and nitric oxide synthase. Arginase 1 activity has been associated with most of the immunosuppressive effects resulting from arginine depletion and the role of arginase activity in lung tumor maintenance has not been investigated in clinically relevant established tumor models. RNA sequencing of sorted cell populations from immunocompetent genetically engineered KRAS G12D mutant mouse lung tumors, showed that arginase 1 expression is elevated in the myeloid cell population in the tumor microenvironment. Ex vivo analysis demonstrated that myeloid derived suppressor cell (MDSCs) from mouse tumors hindered T cell function by depleting arginine. These MDSCs expressed elevated levels of arginase 1, and inhibition of its activity by an Arg1/2 inhibitor, compound 9, restored effector T cell function. Treatment of a genetically engineered KRAS mutant mouse model with compound 9 increased arginine levels in blood and tumor and led to an increased number of tumor T cells and a significant reduction in tumor volume after 1 week of treatment. Compound 9 had no growth inhibitory activity across several murine and human KRAS mutant lung cancer cell lines in vitro. We also determined by immunohistochemistry that elevated levels of arginase 1 were expressed in granulocytic myeloid cells from KRAS mutant lung cancer patient samples. Given the data reported here and by others, an arginase inhibitor with excellent potency and in vivo target engagement could represent an important new immunotherapeutic agent. We have recently discovered and are fully characterizing molecules that demonstrate excellent potency and selectivity along with oral bioavailability in rodents that provide good arginase target coverage at modest doses.

#4066

AnnexinV: Multifunctional anticancer immunotherapeutic protein in tumor microenvironment.

Jung Hwa Park,1 Young Seob Kim,1 Hyun Jin Park,1 Gun Young Jang,1 T.c. Wu,2 Yeong-Min Park,1 Tae Heung Kang1. 1 _Konkuk University, Seoul, Republic of Korea;_ 2 _Johns Hopkins Medical Institutions, Baltimore, MD_.

Introduction: After cancer treatment using chemotherapy and radiotherapy, tumor microenvironment continually occurs inflammation, which depend on damage-associated molecular pattern (DAMPs) released from apoptotic tumor cells. In this case, tumor microenvironment is regarded as inflammation zone and induce various immune cells infiltration, pro- and anti-inflammatory cytokines production and interaction between immune cells and cancer cells. These, a dynamic and complex microenvironment induce a favorable state for tumor growth. Thus, recent studies assert that it is important to regulate excessively increased inflammatory response in the tumor microenvironment accordingly.

Annexin V is a Ca2+-dependent phospholipid binding pro[[Unsupported Character - Codename &shy;]]tein and known to bind to exposed phosphatidylserine on the apoptotic cells. Several groups and our study have recently demonstrated that annexin V has anti-inflammatory effects by inhibiting bone-marrow derived dendritic cell (BMDCs) activation and pro-inflammtory cytokine production. So, we suggest that Annexin V as a therapeutic approach for controlling inflammatory response in cancer patients having increased inflammation in tumor microenvironment.

Methods: The size and purity of recombinant proteins were assessed using SDS-PAGE. IVIS imaging system used to characterize the accumulation of Annexin V containing protein into tumor loci in tumor-bearing mice. Therapeutic efficacy was confirmed tumor-specific CD8+ T cells in PBMCs and splenocytes by Flow cytometry analysis. pro-, anti- inflammtory cytokines and various immune cells infiltration in tumor microenvironment were examined by ELISA and FACS. Cytotoxicity by tumor-specific CD8+ T cells of Luciferase-expressing tumor cells was observed by representative luminescence imaging system.

Results: The Targeting efficiency of Annexin V protein into tumor microenvironment was increased compared to control. Therapeutic efficacy was demostrated activation of tumor-specific CD8+ T cells (P < 0.001), inhibition of tumor growth (P < 0.05) and increase of survival (P < 0.05) in tumor bearing mouse compared to control. AnnexinV protein administration was decreased significantly TGF-beta 1 cytokine production (P < 0.001) and immunosuppressive cells infiltration (P < 0.001) in tumor microenviroment compared to non-treated group.

Conclusion: We identified multiple role of Annexin V protein, which has capacity of apoptotic tumor cell target, as regulator of excessive inflammation in tumor microenvironment.

This results could primarily pave the road for the development of anti-tumor immunotherapy.

#4067

Toll-like receptor 2 deficiency enhances KRAS-driven lung cancer.

Chia-Hsin Liu. _Univ. of Pittsburgh, Pittsburgh, PA_.

Chronic infection and inflammation have been suggested to have a strong association with lung cancer in clinical and epidemiologic studies. Toll-like receptor 2 (TLR2) mediates inflammatory response and is essential in regulating innate and adaptive immunity. TLR2 has also been reported to play either pro-tumorigenesis or tumor-suppressive role depending on the oncogenic driver mutations and cell types. However, the role of TLR2 in lung cancer, especially in the lung adenocarcinoma harboring KRAS mutations, remains unknown. In an analysis using human lung adenocarcinoma cohort (TCGA RNAseq dataset), our results revealed that KRAS-mutant lung tumors showed lower TLR2 mRNA expression than the paired adjacent normal lung tissue. Furthermore, lung adenocarcinoma patients with lower TLR2 expression displayed worse survival outcome. To functionally test the role of TLR2 during KRAS-driven lung tumorigenesis, we generated a mouse model of lung adenocarcinoma mutated for K-ras, with and without TLR2 inactivation (K-rasG12D/+ TLR2-/- and K-rasG12D/+TLR2+/+, respectively). Interestingly, the tumor number and tumor area significantly increased in K-rasG12D/+ TLR2-/- mice compared to K-rasG12D/+TLR2+/+ mice. In addition, the inflammatory cells in bronchoalveolar lavage (BAL), especially the alveolar monocytes, significantly increased in K-rasG12D/+ TLR2-/- mice. Microdissected lung tumors from K-rasG12D/+ TLR2-/- mice showed significantly higher inflammatory chemokines and cytokines gene expression (Ccl1, Ccl2, Cxcl1, Cxcl2, Cxcl5, Csf1, IL-6, Tnf) than those from K-rasG12D/+TLR2+/+mice. Additionally, Cxcl1 was significantly higher in K-rasG12D/+ TLR2-/- when BAL fluid was analyzed for cytokines/chemokines by Luminex assays. Our results suggest that TLR2 deficiency may promote KRAS-mutation driven lung tumorigenesis through increased cancer-related inflammation. Furthermore, the expression may serve as a prognostic biomarker for which the lower TLR2 expression is associated with worse survival outcome in lung adenocarcinoma patients with KRAS mutation.

#4068

Galectin-1 intensifies immunosuppression in head and neck cancer by boosting myeloid-derived suppressive cell (MDSC) expansion.

Dhanya K. Nambiar,1 Todd A. Aguilera,2 Joshua Daniel Bloomstein,1 Dadi Jiang,3 Hongbin Cao,1 Albert Koong,3 Quynh Thu Le1. 1 _Stanford Univ., Stanford, CA;_ 2 _U.T. Southwestern Medical Center, Dallas, TX;_ 3 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Head and neck squamous cell carcinoma (HNSCC), with more than 450,000 newly diagnosed cases annually, is the 6th most common cancer worldwide. Recent literature shows that tumor induced suppression of the host immune system is critical to HNSCC progression and metastases. Tumor secreted factors directly influence the expansion of myeloid-derived suppressor cells (MDSCs), which are considered important contributors for tumor to escape from immune surveillance. The presence of expanded MDSCs peripherally and within the tumor microenvironment is associated with metastasis and recurrence after definitive treatment in HNSCC patients. In this study, we evaluated at the role of tumor secreted Galectin-1 in recruitment of MDSCs in HNSCC. We utilized HPV-negative as well as HPV-positive HNSCC models to test the effect of Galectin-1 knockout (KO) in the tumor microenvironment. CRISPR/Cas9 knock out Galectin-1 in MOC2 and MEERL models resulted in a ~50% reduction in the tumor growth. We further analyzed the tumor microenvironment of these tumors for MDSC recruitment. Both MOC2 and MEERL tumors showed highly inflammatory phenotype, with significant presence of MDSCs (>70% of CD45+ cells were of myeloid origin). Knocking out Galectin-1 led to a significant decrease in CD11b+Gr1+ MDSCs in both MOC2 and MEERL tumors. We also observed high level of MDSCs in both the spleen and the peripheral blood of mice bearing Galectin-1 wildtype (WT) tumors in both models. In contrast, knocking out Galectin-1 from the tumor reduced systemic MDSCs levels. To investigate how Galectin-1 modulates MDSC levels, we compared the secreted cytokine profiles between MOC2 WT and Galectin-1 KO tumors. Galectin-1 null cells secreted significantly lower levels of monocyte and macrophage recruiting chemokines: CCL5, CXCL1, and G-CSF. Analysis of the gene expression signatures of MOC-2 WT and KO tumor cells revealed critical signaling pathways differences, including down-regulation of PI3K-AKT pathway in KO-tumors. In conclusion, the present study reveals that tumor derived Galectin-1 can stimulate the recruitment of MDSCs, thereby potentiating an inflammatory tumor microenvironment which supports HNSCC progression. (Acknowledgements: BMS for Anti-Gal1 Abs. Dr R. Uppaluri, for MOC-2 oral cancer cells, Dr. W. Spanos for MEERL cells)

#4069

Evaluation of inflammatory infiltration (tumor infiltrating lymphocytes - TIL) in malignant melanoma.

Kristyna Nemejcova, Ivana Ticha, Ondrej Kodet, Miroslav Dura, Michaela Bartu, Pavel Dundr. _First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague 2, Czech Republic_.

Assessment of tumor infiltrating lymphocytes (TIL) in malignant melanoma (MM) should be a standard part of biopsy examination. This parameter is of interest regarding its prognostic, but potentially also predictive significance. However, the usefulness of this marker in routine practice is limited due to unequal methodological approach and equivocal results of previous studies. The aim of our study is to compare 5 different methods of TIL assessment with respect to their prognostic significance.

TIL were double-blindly evaluated by two pathologists in 221 cases of primary cutaneous malignant melanoma. The sample set comprised of pT1 - 48 cases, pT2 - 61 cases, pT3 - 69 cases, and pT4 - 43 cases. Five different scoring methods were used, including: i) Clark scoring; ii) Melanoma Institute Australia (MIA) system; iii) scoring system used in the study of Saldanha et al.; iv) scoring system used in the TCGA study and modified by Park et al.; v) the system recently proposed by "International Immuno-Oncology Biomarker Working Group" for TIL scoring in all solid tumors. Cox's Proportional Hazard Method was used to test 10-year survival with three main outcomes - disease-specific survival (DFS), local recurrence-free survival (LFS), and distant metastasis-free survival (MFS).

The evaluation of TIL with respect to its prognostic value was statistically significant for all 3 outcomes when using Clark scoring (score 0, 1 vs. score 2; DFS p=0.027, LFS p=0.019, and MFS p=0.010); MIA system (groups 0, 1 vs. groups 2, 3; DFS p=0.060, LFS p=0.035, and MFS p=0.012) and the system proposed by "International Immuno-Oncology Biomarker Working Group". In this system, however, only the assessment of stromal TIL (not intratumoral or in the invasive margin) showed prognostic significance (DFS p=0.034, LFS p=0.033, and MFS p=0.033). Our results did not prove prognostic significance of the scoring systems used in the study published by Saldanha et al. and Park et al.

In conclusion, the results of our study showed that the evaluation of TIL in MM is a prognostic marker, but its significance is dependent on the chosen scoring method. From a practical point of view, application of a unified methodology is desirable. This would allow for a comparison across studies, and, even more interestingly, also for the assessment of the predictive value of TIL evaluation, especially with respect to immunotherapy. However, out of the five different methods used in our study, only 3 showed statistically significant results and none of the 3 systems seem to be superior to others.

This work was supported by Ministry of Health, Czech Republic (Conceptual development of research organization 64165, General University Hospital in Prague, and by project AZV 16-30954A).

#4070

The relationship between members of the canonical NF-κB pathway, components of the tumor microenvironment and cancer-specific survival in colorectal cancer patients.

Jean A. Quinn, Lindsay Bennett, Meera Patel, Lynette Loi, Mikaela Frixou, Antonia Roseweir, James H. Park, Paul G. Horgan, Donald C. McMillan, Joanne Edwards. _University of Glasgow, Glasgow, United Kingdom_.

The canonical NF-κB pathway regulates the transcription of many genes which may be involved in processes such as inflammation and proliferation, suggesting a potential link between the tumor, the tumor microenvironment and cancer-specific survival in colorectal cancer patients.

Immunohistochemistry was used to assess the expression of the upstream kinase TAK1/pTAK1 and four members of the canonical NF-κB pathway in tissue microarrays. Protein expression was determined using the weighted histoscore method. BRaf status, tumor stroma percentage, local inflammation, systemic inflammation and cancer-specific survival were examined in patients with colorectal cancer.

Cytoplasmic IKKβ was significantly associated with the inflammatory cell infiltrate (p=0.0015), systemic inflammation (p=0.03) and cancer-specific survival (p=0.046). In contrast, no significant association was found with the other three members. Cytoplasmic pTAK1 was associated with the tumor microenvironment (p=0.045) and cancer-specific survival (p=0.032). On multivariate analysis only cytoplasmic IKKβ associated with survival (HR1.75, 95%CI 1.05-291, p=0.033) and this was independent of TNM stage and markers of the tumor microenvironment. When cytoplasmic IKKβ was stratified with BRaf status (wild type (n=151) or mutant (n=48)), it was no longer associated with cancer-specific survival. However, when cytoplasmic pTAK1 was stratified with BRaf status its association with cancer-specific survival was strengthened. Cytoplasmic pTAK1 was significantly associated with cancer-specific survival in patients with wild type BRaf (p=0.014). In patients with BRaf mutations there was no significant association with cancer-specific survival (p=0.105), however the numbers were low and this is currently being investigated in a larger cohort.

The results of the present study show that high expression of cytoplasmic IKKβ was associated with decreased cancer-specific survival and with markers of the tumor microenvironment in patients with colorectal cancer. Expression of cytoplasmic pTAK1 was associated with cancer-specific survival and this was enhanced in patients with tumors expressing wild type BRaf.

#4071

Investigation of thyroid tumor microenvironment by immunohistochemical and bioinformatic analyses.

Emanuela Minna,1 Silvia Brich,1 Katia Todoerti,2 Silvana Pilotti,1 Federica Perrone,1 Nicholas Paielli,1 Luca Agnelli,3 Ilaria Bersani,1 Roberta Mortarini,1 Andrea Anichini,1 Paola Collini,1 Antonino Neri,3 Angela Greco,1 Maria Grazia Borrello1. 1 _Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy;_ 2 _IRCCS-CROB, Referral Cancer Center of Basilicata, Rionero in Vulture, Italy;_ 3 _Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico and Department of Oncology and Hemato-Oncology, University of Milan, Milan, Italy_.

Introduction

Tumor microenvironment plays an essential role in cancer development and progression. In the context of thyroid cancer (TC) it still represents a poorly investigated field. Recent data from in vitro and in vivo studies proposed a model of TC progression promoted by the converging action of thyroid tumor cells and cancer-associated fibroblasts (CAFs). According to this mouse model CAFs are recruited in tumor stroma where synthesize and deposit collagen (COL1A1) that is, in turn, cross-linked by the enzyme LOX, produced by the thyroid tumor cells. Collectively this coordinated action lead to matrix stiffness and TC progression. Interestingly, this process is reported to be specifically observed in murine thyroid tumors expressing BRAFV600E mutation, but not in those harboring RAS mutation. In this study we tested this cross-talk in human thyroid cancer tissues.

Experimental procedures

A series of thyroid specimens, comprising different tumor subtypes (PTC and PDTC) and histologic variants, was collected at our institution and investigated by immunohistochemical (IHC) analyses for the expression of α-SMA (marker for activated fibroblasts), collagen COL1A1 and LOX. Tumor samples were also screened for the most common genetic lesions reported in TC including BRAFV600E and N/H/KRAS mutations. Publicly available datasets of thyroid tumors and non-neoplastic thyroid controls, analyzed on Affymetrix HG-U133 Plus 2.0 array, were selected to investigate the expression profiles of α-SMA, COL1A1 and LOX genes. Robust Multi Array Average (RMA) normalization, Brain Array annotation and batch correction procedures were applied to obtain gene expression profiles across all samples.

Results

In IHC analyses α-SMA decoration was observed in the stroma of thyroid tumor tissues while was restricted to blood vessels in the adjacent non-neoplastic thyroid. Stromal α-SMA staining was not evenly distributed in the tumors but localized preferentially at the tumor invasive border, organized in peripheral structures as fibrous septa or capsule. In agreement with the proposed model, higher level of α-SMA staining were specifically observed in PTC tumors harboring BRAFV600E mutation compared with tumors harboring RAS mutation. IHC analyses on tissue serial sections confirmed the coordinated expression of α-SMA, COL1A1 and LOX; COL1A1 localized in tumor stroma and was closely correlated with α-SMA positive areas while LOX was expressed by thyroid tumor cells. The coordinated expression of the three genes was confirmed by meta-analysis of public datasets.

Conclusions

In this study we provide evidence of the coordinated presence of thyroid tumor cells, activated fibroblasts and proteins of extracellular matrix, such as COL1A1 and LOX, in the contest of human thyroid carcinoma.

#4072

Direct transdifferentiation of glioblastoma cells to antigen-presenting cells: A novel immunotherapeutic approach.

Mathew Sebastian, Son B. Le, Changwang Deng, Nagheme Thomas, David D. Tran. _University of Florida, Gainesville, FL_.

Cell-based immunotherapy has shown encouraging results in solid cancers. However, major limitations of this approach include difficulty with isolation and generation of effective antigen-presenting or immune effector cells and the high cost of cell-based vaccines. In glioblastoma (GBM), the most common and lethal brain cancer in adults, inadequate migration of immune cells across the blood-brain barrier represents another major obstacle to cell-based immunotherapy. One approach to overcome these barriers is to convert GBM cells directly into immune cells of interest. Transdifferentiation, the transformation of one cell type other than stem cells directly into another cell type without an intermediate pluripotent stage, has shown early promise in regenerative medicine, but has had a limited role as a cancer therapy, especially for solid tumors. This is in large part due to the laborious and time-consuming process of identifying master fate determinants of the cell types of interest in the transdifferentiation. To enable this novel application, we have developed and experimentally validated two tandem computational platforms, GeneRep and nSCORE. Applying this novel algorithm to large quantities of published and in-house gene expression datasets, we successfully predicted cell fate determinants of tens of different cell types with several having been validated in transdifferentiation experiments by others and us, such as the conversions of astrocytes to neuronal stem cells, of astrocytes to glioma stem-like cells, of fibroblasts to macrophages, etc. Here, we describe the process of combining a unique core set of hematopoietic stem cell, myeloid, and macrophage or dendritic cell (DC) fate determinants—some have not been described as critical in these cell fates—as predicted and highly ranked by GeneRep-nSCORE to efficiently transdifferentiate GBM cells into functional macrophages or dendritic cells, respectively, while neutralizing their malignant phenotype. This novel transdifferentiation has the potential for transformative impacts in many areas, including DC vaccine immunotherapy and cancer therapy in general, and pioneers the concept that solid tumor cells can be converted into another cell type, and not just any cell type, but one that potentially can elicit a therapeutic response against itself.

### Mechanisms and Models of Gastrointestinal Malignancies

#4073

Using patient-derived PDAC cells to understand c-MYC involvement in the drug resistance mechanisms of pancreatic cancer.

George S. Avetian,1 Erika M. Parasido,1 Jonathan Brody,2 Jordan Winter,2 Eric Londin,2 Michael Pishvaian,1 Eric Glasgow,1 Stephen Byers,1 Christopher Albanese1. 1 _Georgetown University, Washington, DC;_ 2 _Thomas Jefferson University, Philadelphia, PA_.

Background: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease that is typically incurable due to acquired resistance to systemic chemotherapy. A deeper understanding of the mechanisms involving drug resistance is needed to improve patient outcomes. Commercially available PDAC cell lines are the most used model in research but do not accurately recapitulate a given patient's tumor. Using the conditional reprogrammed cell (CRC) technique, we established patient-specific cell lines and created nab-paclitaxel (nab-PTX)-resistant clones, which allowed us to better study resistance mechanisms. In many cancers, c-MYC has a known function in tumor progression, and recent studies suggest that the c-MYC pathway is involved in PDAC. However, the role of c-MYC in the mechanisms leading to nab-PTX resistance has not been extensively explored.

Methods: Biopsies were collected from treatment-naive PDAC patients, and long-term cultures of PDAC CRCs were established. The PDAC origin of the cell lines was validated with genomic analysis. The IC50s for nab-PTX were calculated and used to generate drug-resistant clones. The Zebrafish model was used to verify the drug resistance in vivo, while subcutaneous injections of the parental and drug-resistant CRCs in nude mice were performed to test the cells' ability to form tumors. Exome sequencing, microarray, and Western blotting analyses were used to characterize and compare the parental lines with the drug-resistant clones and to identify key pathways involved in the evolution of drug resistance.

Results: We established KRAS-mutant primary cell lines from PDAC patients' biopsies. Using 2 parental lines, we generated 5 nab-PTX-resistant clones, with IC50s up to 800 times greater than those of the matched parents; resistant profiles were verified in Zebrafish. Subcutaneous injections in nude mice showed the CRCs' ability to form tumors that recapitulate human PDAC. A greater metastatic phenotype was observed in the tumors derived from the resistant clones. RNA microarrays, comparing the parental cells with the resistant clones, identified a possible resistance mechanism involving the induction of a pro-inflammatory pathway leading to c-MYC overexpression. Western blotting, as well as immunocytochemical and immunohistochemical staining, performed on the CRCs and CRC-PDX-derived samples confirmed the role of c-MYC overexpression in the evolution of nab-PTX resistance.

Conclusion: The conditional reprogrammed cell (CRC) methodology addresses the need for a reliable method for generating primary cell lines on a single patient basis. The ability to rapidly model in vitro, and verify in vivo, that the overexpression of c-MYC contributes to the development of nab-PTX resistance is a significant advancement in the field and provides a platform for discovery of more effective treatment of refractory PDAC.

#4074

Pten **haploinsufficiency promotes tumor invasion and carcinogenesis in mouse colon epithelium with** Apc **deficiency.**

Haruki Sada,1 Takao Hinoi,2 Hiroaki Niitsu,3 Masatoshi Kochi,1 Naoya Sakamoto,1 Kazuhiro Sentani,1 Naohide Oue,1 Wataru Yasui,1 Hideki Ohdan1. 1 _Hiroshima University, Hiroshima, Japan;_ 2 _National Hospital Organization Kure Medical Center and Chugoku Cancer Center, Kure, Japan;_ 3 _Vanderbilt University Medical Center, Nashville, TN_.

Background and Aim: The loss of PTEN (phosphatase and tensin homologue) expression in human colorectal cancer (CRC) is found in approximately 40% of cases, and its functional contribution is not fully understood. To address this question, the colon tumors from the Pten-deficient mice with a disruption of the Adenomatous polyposis coli (Apc) were compared with those from the mice with Apc deficiency alone.

Method: To recapitulate human CRC, mouse models carrying transgenes regulated by a 9.5-kb fragment containing sequences from the human CDX2 promoter (CDX2P9.5), which were previously shown to have tightly restricted transgene expression in the colon epithelium, were prepared. CDX2P9.5-NLSCre;Apcflox/+;Ptenflox/+ (referred to as CPC;Apc+Pten mouse) and CDX2P9.5-NLSCre;Apcflox/+(;Pten+/+) mice (referred to as CPC;Apc mouse) were generated, and the overall survival and tumor phenotypes (number, size and severity) at 4, 6, 9, 12 and 15 weeks of age were compared between the two models. In addition, the loss of heterozygosity status of the Apc and Pten wild-type alleles in tumor tissue and normal mucosa in the two mouse models were compared with multiplex polymerase chain reaction (PCR), and Pten transcripts and protein expression were evaluated by quantitative reverse transcription PCR and immunohistochemistry, respectively.

Results: CPC;Apc+Pten mice had a significantly shorter life span than CPC;Apc mice (median survival time of CPC;Apc+Pten mice and CPC;Apc mice: 14.4 weeks [n=20] vs. 21.6 weeks [n=20], respectively). The CPC;Apc+Pten mice had more tumors than CPC;Apc mice at all time points measured, but the tumor size in CPC;Apc+Pten mice was significantly larger than that in CPC;Apc mice only at 9 weeks of age. Invasion into the submucosa was more frequently observed in CPC;Apc+Pten mice than in CPC;Apc mice (CPC;Apc+Pten mice and CPC;Apc mice: 36.3%/58.3% and 4.9%/10% at 12/15 weeks of age, respectively). Regarding the findings on multiplex PCR, while the tumors from both models showed biallelic Apc inactivation, the tumors from CPC;Apc+Pten mice showed no loss of the wild-type allele of Pten. Regarding the Pten transcript levels, the levels in the tumors and mucosa from CPC;Apc+Pten mice were almost half of those in CPC;Apc mice. The Pten protein expression level in tumors from CPC;Apc+Pten mice was lower than that in tumors from CPC;Apc mice but not completely suppressed.

Conclusion: Pten promotes tumor invasion and carcinogenesis without two hits on the gene. Consistent with the previous finding that two hits on the PTEN gene was a rare event in human CRC with PTEN loss, our data strongly suggest the haploinsufficient tumor-suppressive activity of Pten. These findings show that this CPC;Apc+Pten mouse model recapitulates human CRC with PTEN loss.

#4075

Role of yes-associated protein 1 (YAP1) in human pancreatic ductal adenocarcinoma initiation and progression.

Xue Yin, Jun Liu, Naoki Akanuma, Michael Nipper, Pei Wang. _University of Texas Health Science Center at San Antonio, San Antonio, TX_.

Introduction: Understanding the molecular mechanisms underlying pancreatic ductal adenocarcinoma (PDAC) initiation and progression is an urgent need to achieve more effective diagnosis and treatment to this deadly cancer. We have developed a flow cytometry-based, high resolution lineage tracing method and 3D culture system to study PDAC initiation and progression using normal human pancreatic cells. Methods: Normal human acinar cells or cells undergoing acinar-to-ductal metaplasia (ADM, AD cells) are isolated by fluorescence-activated cell sorting (FACS), and cultured in 3D-culture system. Lentivirus carrying either YAP1-mCherry are transduced into acinar cells to study whether YAP1 is sufficient to induce ADM or is required during TGFβ-induced ADM. Lentivirus carrying either shYAP1-mCherry or shYAP1-KrasG12D-mCherry are transduced into AD cells to study whether YAP1 is required for AD cell proliferation and Kras-induced prolonged proliferation. ChIP-PCR and genome-wide ChIP-seq will be used to identify the mechanism through which YAP1 promotes PDAC development and progression. Lentivirus carrying inducible shYAP1 will be transduced into cancer producing, engineered AD cells and orthotopically transplanted into SCID mice to investigate whether inhibiting YAP1 could suppress PDAC progression. Results: We already found that TGFβ could induce ADM and oncogenic Kras could maintain the proliferation capacity of AD cells. Also, TGFβ induces YAP1 expression and TGFβ inhibitor blocks YAP1 induction during ADM process, and deleting YAP1 will suppress Kras-induced over-proliferation in AD cells, suggesting that YAP1 plays an important role in ADM, and is downstream of TGFβ as well as Kras signaling. Conclusion and significance: Successful completion of the project will give us a better understanding of how YAP1 functions in human PDAC initiation and progression. It will provide us opportunities to develop new methods for early detection and treatment of PDAC.

#4076

Gut microbiota and metabolite-driven phenotype modulation in a mouse model of colorectal cancer.

Jacob E. Moskowitz, Zhentian Lei, Susheel Busi, Marcia Hart, Craig Franklin, Lloyd Sumner, James Amos-Landgraf. _University of Missouri, Columbia, MO_.

Colorectal cancer (CRC) is a multifactorial disease that develops as a result of multiple genetic mutations, often including loss of the tumor suppressor Adenomatous Polyposis Coli (APC) gene. More recently, additional factors such as the host gut microbiota (GM) have also demonstrated an important role in tumor initiation and progression, yet our understanding of most commensal bacteria remains limited. We observed significant differences in intestinal tumor multiplicity between two ApcMin sub-strain mouse colonies; C57BL/6J-ApcMin (B6-Min/J) mice from The Jackson Laboratory had relatively lower adenoma burden while C57BL/6JD-ApcMin (B6-Min/D) mice from the University of Wisconsin develop twice the number of adenomas. To determine the underlying causes of this phenotypic variability, we used complex microbiota targeted rederivation (CMTR) to rederive embryos of the two Apc mutant sub-strains onto surrogate dams harboring distinct GM from two different vendors (GMJAX and GMHSD), generating four ApcMin groups: B6-Min/JGMJAX, B6-Min/JGMHSD, B6-Min/DGMJAX, and B6-Min/DGMHSD. We observed significant increases in intestinal tumor multiplicity in both B6-Min groups harboring the GMHSD compared to their GMJAX counterparts, suggesting that the GM accounts for a significant portion of variability in tumor phenotype between the ApcMin sub-strains. We characterized the GMJAX and GMHSD microbial populations using 16S rRNA gene sequencing to identify potentially causative and protective bacterial taxa. Relative abundances of several taxa including Bilophila sp., Desulfovibrio sp., and Akkermansia muciniphila, were significantly elevated in GMHSD suggesting pro-tumorigenic roles, while the family Peptococcaceae was more abundant in GMJAX, indicating a potential protective role. To establish the functional output of these distinct GM profiles contributing to tumor development, we used liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to evaluate differences in the intestinal metabolome of the four experimental groups. Notably, we found that the microbiome differences due to colonization with either GMJAX or GMHSD primarily accounted for distinct metabolomic profiles, whereas host genetic differences between the sub-strains had a minimal role in shaping the metabolomes. We detected 31 putative metabolites that were significantly modulated by the GM in both the B6-Min/J and B6-Min/D sub-strains, and were therefore predictive of the relative tumor burden. Moving forward, these metabolites have implications for both biomarker discovery and potential therapeutic and preventative targets. Overall, these results provide insight regarding environmental factors influencing tumorigenesis, and further demonstrate how host-environment interactions can modulate a given disease phenotype.

#4077

A new mouse model of gastric adenocarcinoma: A valuable tool for preclinical studies.

Jacob Till,1 Prince Addai,1 Neha Bhagwat,1 Stephanie Yee,1 Erica Carpenter,1 Sam Yoon,2 Sandra Ryeom1. 1 _University of Pennsylvania, Philadelphia, PA;_ 2 _Memorial Sloan Kettering Cancer Center, New York City, NY_.

We recently generated the first model of metastatic mixed-type gastric adenocarcinoma that closely recapitulates the human disease. Our model combines conditional loss of E-cadherin (Cdh1fl/fl) and p53 (Trp53fl/fl) with oncogenic Kras (KrasLSL-G12D/+) activation and yellow fluorescence protein (Rosa26LSL-YFP/LSL-YFP) expression in cells of the gastric parietal cell lineage (Atp4b-Cre). Median survival is 76.5 days (range 64-91 days, SD 7.2) with all mice developing local lymph node metastases and lung metastases, half developing paratracheal lymph node metastases, and 20% developing liver metastases. We previously provided proof of concept for the preclinical utility of our model by demonstrating extended survival upon treatment with a MEK inhibitor (PD0325901). Here we demonstrate the utility of this model to investigate the role of the microbiome in gastric carcinogenesis and to determine the use of circulating tumor cells (CTCs) as biomarkers of metastatic disease. The specific pathogen H. pylori is known to be causal in gastric adenocarcinoma. Further, many studies have implicated the microbiome in colon carcinogenesis. However, no studies have investigated the role of the normal microbiome in gastric carcinogenesis in the absence of H. pylori infection. We treated our model of mixed-type gastric adenocarcinoma with a commonly utilized antibiotic cocktail to deplete the gut microbiota and confirmed depletion of the microbiota by qPCR in stomach and fecal contents at necropsy. Microbiome depletion resulted in a 7.5-day increase in median survival (n=6) over vehicle treated mice (n=5, p=0.04). These data suggest a role for the normal microbiota in gastric cancer progression regardless of H. pylori infection. Given the broadly metastatic phenotype of our model, we examined whether the number of CTCs correlated with metastatic burden. Because our gastric cancer model incorporated a yellow fluorescence protein (YFP) as a marker of tumor cells, we used flow cytometry to measure CTCs in cardiac blood samples from terminal mice. We detected CTCs (defined as DAPI-;CD45-;YFP+) in all terminal mice and correlated CTC counts with lung metastatic burden. Mice with macrometastatic disease in the lung had >90 CTCs per 100 μl blood (n=3) as compared to mice with micrometastatic disease of the lung which showed <15 CTCs per 100 μl blood (n=4, p=0.008). These data suggest that our new mouse model of metastatic gastric adenocarcinoma offers a valuable tool for a variety of preclinical studies.

#4078

Sleeping Beauty transposon mutagenesis identifies genes involved in colorectal cancer progression.

Haruna Takeda. _Kanazawa University, Kanazawa, Japan_.

Cancer genome sequencing studies have identified numerous genes mutated in colorectal cancers (CRC). Majority of genes, however, were infrequently mutated between tumors. Sorting out infrequent driver genes from passenger genes is still challenging, but important since these infrequent driver genes sometimes seed for metastasis or confer drug resistance. In the previous study, we performed Sleeping Beauty (SB) transposon mutagenesis screens in mice and identified 1,333 candidate driver genes in CRC. These gene included known CRC genes such as Apc, Smad4 and Trp53 as well as novel CRC genes. Using publicly available datasets for genes mutated in human CRC, we compared the genes commonly mutated between human and mice, thus we could enrich a set of candidate driver genes. Among them, we especially focused on candidate tumor suppressor genes since tumor suppressor genes play a fundamental role in CRC development. To validate these candidate tumor suppressor genes, we set up the experimental system using the CRISPR-Cas9 system to knockout the genes in the intestinal organoid culture system. We obtained the mouse intestinal tumor organoid carrying Apc mutations and a KrasG12D activating point mutation (AK organoid), and also obtained organoids derived from human CRC. First, we introduced a Cas9 to obtain Cas9 expressing AK organoids and then introduced a set of 10 gRNAs targeting candidate tumor suppressor genes by lentivirus. These organoids were subsequently transplanted to NSG mice subcutaneously. AK organoid with gRNAs induced tumors, in contrast, AK organoids without gRNA did not induced tumors within 1 month after transplantation showing gene knockout by CRISPR-Cas9 induced tumor development. Next we purified genome from tumors and analyzed the ratio for each gRNA. The gRNA with an expanded ratio in tumors compared to organoids before transplantations are likely to be a novel tumor suppressor gene. So far we validated 30 candidate tumor suppressor genes by this approach and pick up Arid2, Acvr2a and Acvr1b. These 3 genes were mutated in ~5% of human CRC. Since single gene knockout for each of 3 genes in AK organoids also caused tumor development, we were able to confirm that these 3 genes were truly tumor suppressor genes in CRC, We are now validating using human CRC organoid. In summary, we have established the experimental system to validate candidate tumor suppressor genes in CRC using the CRISPR-Cas9 system and the organoid culture system. So far we have succeeded in identifying novel tumor suppresser genes such as Arid2, Acvr2a and Acvr1b. We will validate all candidate tumor suppressor genes and reveal the whole driver genes involved in CRC. Our results will give an important insight in finding new drug targets to cure cancers.

#4079

Perturbations in tat-interacting protein 30 (TIP30) levels contribute to pancreatic cancer aggressiveness.

Imade E. Imasuen-Williams, Francis Enane, Murray Korc. _Indiana University School of Medicine, Indianapolis, IN_.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer characterized by a high frequency of key driver mutations, (e.g. KRAS, TP53, CDKN2A, and SMAD4), as well many less frequent driver mutations. In addition, there is overexpression of growth factors such as TGF-βs, growth factor receptors such as the EGF receptor (EGFR), a markedly desmoplastic stroma, and a propensity to develop metastases and chemoresistance. Consequently, the 5-year survival rate for PDAC is 8-9% with current therapeutic modalities such as gemcitabine plus nab-paclitaxel or FOLFIRINOX. There is an urgent need, therefore, to devise novel therapies in PDAC. In this regard, we previously reported that high microRNA-10b (miR-10b) levels in the cancer cells from PDAC tissues are associated with decreased patient survival and earlier appearance of metastatic disease following neoadjuvant radiochemotherapy (Preis, et al, Clin Cancer Res, 2011), and that miR-10b targets the 3'UTR of tat-interacting protein 30 (TIP30) and downregulates TIP30 mRNA levels, and that these alterations enhance EGF- and TGF-β-mediated pancreatic cancer cell (PCC) invasion and migration (Ouyang et al, Oncogene, 2013). We therefore analyzed genetic alterations in TIP30 in PDAC data from The Cancer Genome Atlas (TCGA) and found that TIP30 exhibited both copy number gains (23%, n=109) and losses (26%, n=109), with parallel changes in TIP30 mRNA levels. Reduction of TIP30 mRNA levels correlated with increased PDAC survival when compared to PDAC with increased mRNA expression (p=0.004, n=182). However, in the absence of TIP30 expression levels in the normal pancreas in TCGA, the significance of this observation is not clear. We therefore evaluated the effects of TIP30 downregulation in human PCCs (hPCCs) using siRNA targeting TIP30 or a vector expressing miR-10b and empty vector controls. Both manipulations increased EGFR and AKT protein levels. To delineate the potential role of TIP30 in PDAC, we generated compound mutant Pdx1-Cre; LSL-KrasG12D; Tip30+/- (KTip30+/-C) mice carrying mutated KrasG12D and heterozygous Tip30 deletion in the pancreas. Heterozygous Tip30 deletion alone did not alter pancreatic cyto-architecture, but accelerated Kras-associated malignant transformation and progression to murine PDAC. Derived murine PCCs exhibited accelerated proliferation and increased EGFR expression. Our results indicate that TIP30 loss enhances PDAC aggression, in part through effects on EGFR. In conclusion, partial loss of TIP30 in miR-10b overexpressing hPCCs or heterozygous loss of Tip30 in a murine model of PDAC resulted in an enhanced PCC aggressive phenotype and increased EGFR levels. To advance our understanding of the role of TIP30 in PDAC we are now evaluating a cohort of mice with homozygous Tip30 deletion. Supported, in part by National Cancer Institute grant CA-075059 to M.K. and CA075059-17S2 Diversity Supplement to IIW.

#4080

An inducible zebrafish model to dissect the role of beta catenin signaling in hepatocellular carcinoma.

Srishti Kotiyal,1 Sharanya Maanasi Kalasekar,1 Kathryn Davis,1 Cindy Barba,1 Annika Young,2 Kimberley J. Evason1. 1 _University of Utah, Salt Lake City, UT;_ 2 _Academy of Math, Engineering and Science, Salt Lake City, UT_.

Hepatocellular carcinoma (HCC) is the third highest contributor to cancer-related mortality in the world. This disease is molecularly heterogeneous in nature, which obfuscates our understanding of the molecular etiology of the disease, giving rise to the essential and urgent need of delineating HCC mechanisms based on molecular subtypes.

Various signaling pathways have been implicated in HCC development. The Wnt/β-catenin pathway remains of particular significance with more than a third of all HCC cases characterized by aberrant activation of the Wnt/β-catenin signaling axis. About 20% of HCC cases are characterized by activating mutations in the β-catenin gene (ctnnb1). These activating mutations prevent β-catenin degradation and allow it to enter the nucleus and activate transcription of various genes involved in cell proliferation and apoptosis. The need to elucidate the role of aberrant β-catenin gene activation in the development of liver malignancy is thwarted by the lack of relevant animal models. In mice, β-catenin mutation alone is not sufficient to induce HCC. To address this bottleneck, we have designed a vertebrate HCC model system of activated β-catenin-driven HCC in an inducible background using zebrafish (Danio rerio).

In our previous work, we demonstrated that zebrafish expressing a mutated, phosphorylation-resistant, and constitutively-activated version of β-catenin develop HCC morphologically and transcriptionally similar to human HCC. Here, we describe a system in which spatially and temporally controlled expression of activated β-catenin is achieved using hepatocyte-specific, tamoxifen-induced expression of Cre recombinase.

Using hepatocyte-specific constitutive/induced Cre-recombinase zebrafish lines together with floxed β-catenin and/or floxed fluorescent reporter lines, we show here that Cre recombinase is successfully able to switch on the expression of floxed transgenes in fishes at larval, juvenile and adult stages. Through histology and longitudinal studies, we are determining the ability of our inducible system to increase cellular β-catenin levels and Wnt signaling in the liver and to stimulate HCC when activated β-catenin is turned on at different developmental stages including adulthood.

This inducible system will be useful for studying the impact of β-catenin activation on liver size and malignancy at different stages of liver development. The ability to induce the oncogene in adulthood will allow us to study HCC in a physiologically relevant setting. We will use this temporal and spatial control for further studies aimed at understanding the role of activated β-catenin in HCC initiation and developing treatment strategies.

#4081

Podoplanin expression in Kupffer cells and platelet deposition on the hepatic sinusoidal cells in the liver of transgenic mice with a hepatocyte-specific human BRAFV600E mutation.

Hiroki Tanaka,1 Kie Horioka,1 Masahiro Yamamoto,1 Asari Masaru,1 Katsuhiro Okuda,2 Seiji Ohtani,1 Kosuke Yamazaki,3 Keiko Shimizu,1 Katsuhiro Ogawa1. 1 _Asahikawa Medical Univ., Asahikawa, Japan;_ 2 _Asahikawa Medical Univ., Japan;_ 3 _Japanese Red Cross Hokkaido College of Nursing, Asahikawa, Japan_.

In hepatocarcinogenesis induced by diethylnitrosamine (DEN) treatment in neonatal B6C3F1 mice, the BrafV637E mutation, corresponding to the human BRAFV600E mutation, plays a pivotal role. We produced the transgenic mice expressing human BRAFV600E mutation specifically in hepatocytes by using the albumin-cre method. The Alb-Cre/BRAFV600E transgenic mice showed the enlarged liver whose weight was five times as large as that of normal mice, and the liver entirely consisted of small basophilic hepatocytes resembling the DEN-induced preneoplastic hepatocytes. Although these transgenic mice were healthy after birth, they spontaneously died showing renal and pulmonary diseases. In the transgenic mice, the liver showed thrombopoietin (TPO) overexpression, which is associated with eventual megakaryocytosis/thrombocytosis, and platelets were found activated in the peripheral blood and deposited in hepatic sinusoids. Simultaneous staining for CD61/CD31 or CD61/F4-80 revealed that platelets were sparsely adhered to liver sinusoidal endothelial cells (LSEC) and densely adhered to and incorporated into Kupffer cells. Because the interaction of podoplanin with C-type lectin receptor 2 (CLEC-2) on platelet membranes is an important mechanism to reciprocally activate platelets and the podoplanin-expressing cells, we investigated whether podoplanin is expressed in any of the hepatic cell components in the transgenic mice. Simultaneous staining for podoplanin and F4-80 revealed that podoplanin was expressed in some of the Kupffer cells, indicating that the platelet activation occurred via the interaction of podoplanin on Kupffer cell membrans with C-type lectin receptor 2 (CLEC-2) on the platelet membrane. Additionally, glomerulonephropathy and interstitial pneumonia associated with platelet deposition were observed. Because platelets contribute growth/survival of hepatocytes by activating hepatic sinusoidal cells and directly hepatocytes via releasing various factors, interaction of platelets with hepatic sinusoidal cells was considered to promote the growth/survival of BRAF mutated hepatocytes. Furthermore, podoplanin expression in part of Kupffer cells in the transgenic mice was an important mechanism for platelet activation in the transgenic mice. On the other hand, the aberrant platelet activation was thought to cause glomerulonephropathy and interstitial pneumonia and led to spontaneous death in some of the transgenic mice.

#4082

Contrasting role for claudin-2 in colitis-associated cancer (CAC) and spontaneous colon tumorigenesis (CRC) by regulating mucosal healing.

Rizwan Ahmad,1 Balawant Kumar,1 Narendra Kumar,1 Mary K. Washington,2 Punita Dhawan,1 Amar B. Singh1. 1 _University of Nebraska Medical Center, Omaha, NE;_ 2 _Vanderbilt University Medical Center, Nashville, NE_.

A link between chronic inflammation and colon cancer has long been appreciated. Accordingly, patients with ulcerative colitis are at higher risk for developing colitis-associated cancer (CAC). Mucosal barrier dysregulation allies with inflammation and colon cancer. Accordingly, claudin-2, a tight junction protein, is upregulated in ulcerative colitis, CAC and spontaneous colon cancer (CRC). Remarkably, claudin-2 expression is restricted to the colon crypt base, among undifferentiated and proliferative colonocytes, and its increased expression is responsible for the regulation of paracellular permeability, differentiation and proliferation. However, causal role of claudin-2 in CAC and CRC remains unclear. Considering the recent data from our laboratory and of others, that in vivo genetic manipulation of claudin-2 has inverse effects on colitis, we hypothesized an adaptive role for increased claudin-2 expression in colitis by promoting mucosal healing. We further postulated that mucosal healing can manifest in contrasting phenotypes for the CAC and CRC. To examine, Villin-claudin-2 transgenic (Cldn-2) mice were subjected to CAC by exposing them to AOM/DSS-induced cancer or interbred with the APCmin mice, the mouse model of spontaneous intestinal cancer. It was intriguing that the inflammation-driven colon cancer (AOM/DSS induced colon cancer) was significantly suppressed in Cldn-2 mice compared to WT littermates (p<0.001). These mice also showed immune suppressive milieu ideal for spontaneous colon tumorigenesis. Accordingly, we found contrasting disparity in results from APCmin/Cldn-2 mice, generated by the cross between Cldn-2 and APCmin mice. These mice showed significant increase in intestinal tumor burden (248+40%, p<0.001) and colonic tumor burden [Incidence (172.50±18%, p<0.001) and size (521.5±34%, p<0.001)] compared to APCmin mice. H&E analysis confirmed aggressive phenotype of APCmin-Cldn-2 mice tumors (vs APCmin mice tumors). APCmin/Cldn-2 mice also survived less than the APCmin mice. Further, inflammation induction by subjecting these mice to Dextran Sodium Sulfate (DSS) resulted in precocious colonic tumorigenesis only in APCmin mice, rejecting a role for the pro-inflammatory signaling in colon cancer progression in APCmin/Cldn-2 mice. High throughput transcriptome and oncogenic array analyses further suggested modifications of the key inflammatory and oncogenic signaling, in supporting these disparate phenotypes. Taken together, our data provide strong support for the key significance for the context and diverse immunogenic signaling in regulating CAC versus CRC. These studies also suggest that claudin-2 manipulated mice can provide the much needed mouse modelling to help identify key immunogenic signaling supportive of the colon tumorigenesis for therapeutic gains.

#4083

Intrahepatic cholangiocarcinoma and gallbladder carcinoma mouse model based on transplantation of syngeneic tumor-initiating cells.

Akiyoshi Kasuga, Takashi Semba, Hiromasa Takaishi, Takanori Kanai, Hideyuki Saya, Yoshimi Arima. _Keio University, School of Medicine, Tokyo, Japan_.

Biliary tract cancers (BTCs) are relatively rare in the Western world, but the rates of BTCs have risen worldwide in the past few decades. BTCs, which arise from epithelial cells in bile ducts, include intrahepatic and extrahepatic cholangiocarcinomas, gallbladder carcinomas, and ampullary carcinomas. The differences between these subtypes are not fully understood because of the limited number of genetically engineered mouse models for BTCs. Genetic mutations in the KRAS gene and the epigenetic changes in Ink4a/Arf genes have been frequently detected in BTCs, and it has been considered that those alterations are associated with the development and maintenance of human BTCs. To clarify the biological features of intrahepatic cholangiocarcinomas and gallbladder carcinomas, we developed syngeneic mouse models based on a combination of organoid 3D cultures and the transplantation of oncogene-expressed syngeneic bile duct epithelial cells. We isolated the epithelial cell adhesion molecule (EpCAM)-positive intrahepatic bile duct epithelial cells and the gallbladder epithelial cells from the Ink4a/Arf knockout C57BL/6 mice, and the cells were maintained as organoids under 3D culture conditions. Those cells were then infected with retroviruses expressing KRASG12V. Lethal metastatic adenocarcinomas having differentiated components formed in our mice when we transplanted the KRASG12V-expressing cells into the livers of wild-type C57BL/6 mice. The tumors resembled human desmoplastic intrahepatic cholangiocarcinomas and gallbladder carcinomas. Our mouse models can be useful for investigating pathogenesis and therapeutic strategies for human BTCs.

#4084

Identifying characteristics of orthotopic HCC mouse models to predict response to immunotherapy.

Carina Hage,1 Sabine Hoves,1 Mailin Ashoff,1 Leanne Strauss,1 Mario Perro,2 Frank Herting,1 Fabian Kiessling,3 Thomas Pöschinger1. 1 _Roche Innovation Center Munich, Penzberg, Germany;_ 2 _Roche Innovation Center Zurich, Zurich, Switzerland;_ 3 _RWTH Aachen University, Aachen, Germany_.

Introduction

Immunotherapy is a promising treatment strategy for hepatocellular carcinoma (HCC). A phase III trial for advanced HCC shows favorable results for nivolumab compared to sorafenib. In order to evaluate the outcome of different therapeutic strategies, we compared two HCC mouse models. The orthotopic transplanted HCC fragment Hep-55.1c model and the inducible HCC mouse model (iAST) were extensively characterized ex vivo and in vivo in response to immunotherapies, and were compared to the clinical situation of HCC patients.

Experimental Procedures

Fragments of mouse Hep-55.1c tumors were implanted in the left lateral liver lobe of C57/Bl6 mice and tumor growth kinetic was monitored by µ-CT. HCC tumor growth in iAST mice was induced via intravenous injection of the adenovirus expressing Cre recombinase. iAST mice express a loxP flanked stop cassette and the SV40 large T-antigen under control of a hepatocyte-specific albumin promoter. Both tumor models were characterized with respect to immune infiltration, cytokine release, somatic mutational load and histopathological characteristics. Mice were treated using different immunotherapeutic agents in monotherapy or in combination such as anti-PD-1, anti-CTLA-4 or the TLR7/8 agonist R848. Human HCC samples were analyzed for mutational load by sequencing, while tumor architecture and immune infiltrate were analyzed by histology.

Results

Hep-55.1c tumors showed high stroma content accompanied by a low and disorganized vasculature whereas multinodular iAST tumors were highly vascularized lacking stroma content. Furthermore, a high mutational load and a strong immune cell infiltrate including cytotoxic T cells, NK cells and myeloid cells were found in Hep-55.1c tumors. The iAST tumors were characterized by a relatively low immune infiltrate and a small number of mutations. Comparison of these baseline results with data obtained from immune-histopathological analysis and sequencing of human HCC samples confirmed this differentiated picture in the clinics. Following treatment, iAST mice showed no response to immune checkpoint monotherapies and only a marginal number of reactive T cells was found within the tumor. In contrast, tyrosine kinase inhibitor sorafenib led to tumor growth inhibition in iAST model. Treatment of mice bearing Hep-55.1c tumors with immunotherapy e.g. anti-PD-1 showed good response. Combination of anti-PD-1 with R848 increased therapeutic efficacy compared to monotherapies.

Conclusions

In this study, we have established two orthotopic HCC mouse models that reflect the diverse clinical situation of human HCC. Our findings demonstrate that the composition of the tumor microenvironment has a tremendous influence on the outcome of therapeutic strategies for HCC. Hence, thorough characterization of tumor patients is indispensable in order to predict response to immunotherapy.

#4085

Development of porcine hepatocellular carcinoma cell lines: Comprehensive in vitro and in vivo characterization.

Hanna H. Chen,1 Sulalita Chaki,2 Jordan L. Newson,1 Michele Obeid,1 Ramzy C. Khabbaz,1 Alvi Yasmin,1 Lauretta A. Rund,2 Mario F. Neto,1 Lawrence B. Schook,1 Kyle M. Schachtschneider,1 Ron C. Gaba1. 1 _University of Illinois at Chicago, Chicago, IL;_ 2 _University of Illinois Urbana-Champaign, Urbana, IL_.

Hepatocellular carcinoma (HCC) spans more than 780,000 new annual diagnoses, and causes 750,000 yearly mortalities. This deadly malignancy is expected to become the third leading cause of cancer death by 2030, highlighting the urgent need for new treatment strategies. While human clinical trials are the benchmark for advancing standard-of-care cancer therapeutics, preclinical animal models represent pivotal tools for translational investigations to develop and test novel therapeutics both in vitro and in vivo. The development of clinically relevant systems to serve as a bridge between preclinical murine studies and human clinical practice is thus of vital importance. The Oncopig Cancer Model (OCM) is a novel transgenic swine platform that recapitulates human cancer through development of site/cell specific tumors after Cre recombinase induced expression of heterozygous KRASG12D and TP53R167H transgenes. In this study, we tested the hypothesis that isolation and transformation of OCM hepatocytes from multiple individuals results in development of phenotypically consistent porcine HCC (pHCC) cell lines which faithfully recapitulate the in vitro and in vivo features of human HCC. Eight pHCC lines were established from primary hepatocytes isolated from resected liver specimens (median 9.4, range 4.9-15.0 g) of 4- to 8-week-old OCMs (n = 8), with a median yield of 2.5 x 106 (range 1.5-5.5 x 106) cells/g and 52% (range 30-95%) viability. At 24-hours post-isolation, porcine hepatocytes were transformed into pHCC with median 95% (range 82-98%) efficiency, and were maintained in culture for median 9 (range 6-15) passages. Morphological and behavioral phenotyping of pHCC cells performed using qualitative and quantitative assays were compared to the most widely used human HCC cell line for in vitro investigations (HepG2). Similar to human HCC, all pHCC cell lines exhibited Arginase-1 immunohistochemical positivity—indicating hepatocellular origin—and 100% purity. Eight of 8 (100%) pHCC cell lines showed RT-PCR proven transgene expression, confirming malignant transformation. Flow cytometry demonstrated a median pHCC cell cycle length of 13.0 (range 12.0-16.9) hours, similar to human HCC (15.1 hours). pHCC migration assay showed a median time to half gap closure of 6.0 (range 4.3-9.0) hours, comparable to HepG2 (3 hours). In vivo malignant capability was confirmed by subcutaneous xenograft growth in both SCID mice and Oncopigs, resulting in biopsy proven malignant masses within 7-14 days. The results of the current work indicate that pHCC cell lines may be consistently developed from OCMs, and validates OCM pHCC as a platform which accurately replicates human cancer for translational research.

#4086

Efficient primary culture model of patient-derived epithelial cells from colorectal cancer using a ROCK inhibitor and feeder cells.

Hyekyung Hong, Nakhyeon Yun, Taewon Kim, Yeosong Lee, Sujeong Song, Jaemoon Bae, Wooyong Lee, Yongbeom Cho. _Samsung Medical, Seoul, Republic of Korea_.

In vitro culture for patient-derived cells offers many advances in the development of novel therapies for colorectal cancer. Although various culture systems have been developed, experimental systems in which the long-term expansion of patient-derived epithelial cells still remained difficult. Here, we demonstrated that epithelial cells isolated from colorectal cancer patient-derived xenograft can be efficiently immortalized in conditioned medium from irradiated feeder cells containing a Rho kinase inhibitor Y-27632. Patient-derived tumor cells were rapidly proliferated to reach confluence in approximately 6 days. Short tandem repeat (STR) analysis of tumor tissue and cultured cells demonstrate that they have 13 identical STR loci and Amelogenin, Penta E, Penta D, D2S1338 and D19S433. The epithelial character was confirmed by staining for EpCAM, cytokeratin 20 and vimentin as a mesenchymal marker. Cells were continued to proliferate and formed well-defined tumor spheroids when transferred to 3D culture. To study the mechanism for promoting unrestricted cell proliferation, we investigated the expression of several cellular genes that fall into two designated pathways, induction of telomerase and cytoskeletal remodeling/p16 pathway. Moreover, patient-derived tumor cells were utilized for high-throughput screening of FDA approved drugs allowing detection of gene-drug associations. Collectively, we demonstrate that the long-term expansion of patient-derived tumor epithelial cells and these cells could be a useful tool in order to investigate a personalized treatment approach.

#4087

Investigating the novel role of Hippo signaling in mouse pancreas.

Ming Gao,1 Jun Liu,1 Francis E. Sharkey,1 Howard C. Crawford,2 Randy L. Johnson,3 Yidong Chen,1 Pei Wang1. 1 _UT Health San Antonio, San Antonio, TX;_ 2 _University of Michigan Health System, Ann Arbor, MI;_ 3 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Background: Large tumor suppressor kinase 1 and 2 are the core kinases of the Hippo signaling pathway, and play critical roles in regulating cell growth and organ size during animal development. Inactivation of the Hippo signaling pathway has been demonstrated to initiate tumor development in certain organs. However, the function of Lats1&2 in the pancreas is still elusive. Methods and results: To investigate the function of Lats1&2 in the pancreas, we generated mice with adult pancreatic acinar cell-specific deletion of Lats1&2 genes. Interestingly, instead of enlarging of the pancreas or pancreatic tumorigenesis, the deletion of Lats1&2 genes in pancreatic acinar cells resulted in severe inflammation of the pancreas. To further examine the mechanisms, we took advantage of a Rosa26 reporter to trace individual Lats1&2 null cells in vivo. We found that the loss of Lats1&2 did not affect cell growth directly but activated pancreatic stellate cells. This was followed by immune cell infiltration and acinar-to-ductal metaplasia in Lats1&2 null pancreas. Our RNA seq data revealed several cytokines and chemokine genes, such as ctgf, spp1, cxcl12, cxcl16, were gradually unregulated with time in Lats1&2 null pancreas before immune cell infiltration. Finally, we revealed the phenotype of Lats1&2 DKO mouse was in a YAP1/TAZ-dependent manner. Conclusion and significance: In our present study, our data suggested that deletion of the Lats1&2 genes in acinar cells caused pancreatitis through activation of pancreatic stellate cells directly. Our study discovered a new mechanism of the inflammatory response initiated by pancreatic epithelial cells and regulated by the Hippo signaling pathway, which is likely to be useful for the identification of new strategies for controlling pancreatic inflammation and prevention of pancreatic cancer.

#4088

Metabolic disease drivers of liver cancer, a new disease paradigm.

Michael R. Briggs, Dipti Deshpande, Nikole Siegmund. _Woodland Pharmaceuticals, LLC, Shrewsbury, MA_.

Much of the global burden of HCC until recently has been caused by alcohol abuse and hepatitis viruses (HepB & HepC). With a vaccine for HepB being widely used in Asian countries and multidrug cocktails to effectively cure much HepC, these drivers of HCC will be reduced in the future. Ironically, HCC continues to be one of the few cancers growing in incidence. It is postulated that the global obesity epidemic is fueling a new etiology of HCC, through fatty liver disease, nonalcoholic steatohepatitis (NASH) and cirrhosis. Liver and pancreatic cancers are projected to continue to increase in incidence for the foreseeable future, and distressingly, for the first time, children are being diagnosed with NASH. We have undertaken the development of a mouse model of NASH leading to HCC and performed a longitudinal analysis of liver samples through the phenotypes of obesity and hyperglycemia, inflammation and fibrosis leading to hyperproliferative disease and HCC to begin to map out the pathways and biomarkers of this metabolically driven liver cancer for comparison to human clinical data and biomarkers of disease progression to HCC.

#4089

Establishment and long-term in vitro culture of organoids derived from human biliary tract carcinomas.

Yoshimasa Saito,1 Toshihide Muramatsu,1 Yuko Sugiyama,1 Ryoei Uchida,1 Ryo Furukawa,1 Nao Yoshikawa,1 Tomoko Yamaguchi,1 Yae Kanai,2 Hidetsugu Saito1. 1 _Keio University Faculty of Pharmacy, Tokyo, Japan;_ 2 _Department of Pathology, Keio University School of Medicine, Tokyo, Japan_.

Background Biliary tract carcinomas (BTCs) are epithelial malignancies arising in the region between the intrahepatic bile ducts and the ampulla of Vater at the distal end of the common bile duct. Although patients with inoperable BTCs generally receive a chemotherapy regimen of gemcitabine and cisplatin, the effect of these drugs is limited and the 5-year survival rates of patients are very low. The newly developed 3D culture system known as "organoid culture" allows long-term expansion of stem cells into budding cyst-like structures (organoids) with properties resembling those of the original tissues. The aim of this study is to establish in vitro preclinical models for patients with BTCs using the organoid culture technology.

Methods We established organoids using cancer tissues obtained from patients with BTCs. Alterations of genome, gene expression profiles including microRNAs and drug sensitivity were analyzed in BTC organoids.

Results We were successful in establishment and long-term in vitro culture of five organoid lines derived from human BTCs including intrahepatic cholangiocarcinoma, gallbladder cancer and neuroendocrine carcinoma of the ampulla of Vater. These BTC organoids can be stably cultured over a year, whereas organoids derived from non-cancer bile duct and gallbladder tissues ceased proliferating after 15 passages or six months. H&E staining and immunohistochemistry of BTC organoids and the primary tissues demonstrated that BTC organoids closely recapitulate the histopathological features of the primary tumors. The tumor suppressor microRNA miR-34a was suppressed in organoids derived from one case of intrahepatic cholangiocarcinoma, and enforced expression of miR-34a markedly suppressed the growth of these organoids with down-regulation of its target oncogenes. We also examined the effect of the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) on BTC organoids. Treatment with 5-Aza-CdR suppressed the proliferation of all BTC organoids by inducing an anti-viral immune response including activation of interferon-responsive genes.

Conclusions Studies using organoids derived from various BTC cases could potentially pave the way for development of precision medicine for patients with BTCs. These preclinical models for refractory cancers may fill the gap between basic cancer research and patients trials and allow personalized cancer therapy.

#4090

Generation of a new mouse to model pancreatic cancer-induced cachexia.

Erin E. Talbert,1 Katherine J. Ladner,1 Maria Cecilia Cuitino,2 Gustavo W. Leone,2 Denis C. Guttridge1. 1 _The Ohio State University, Columbus, OH;_ 2 _Medical University of South Carolina, Charleston, SC_.

Of all cancers, pancreatic cancer causes the third most fatalities, and incidence rates of this devastating disease continue to rise. Further, pancreatic cancer presents with the highest incidence of cachexia, a syndrome of weight loss due to depletion of skeletal muscle and adipose tissue. Clinically, cachexia is associated with increased mortality, poor quality of life, poor treatment responses, and increased treatment complications. Although clearly the best way to prevent cachexia is to address the cancer, pancreatic cancer has proven challenging to treat, with a dismal 9% 5-year survival rate. We believe effective anti-cachexia therapy will improve the care of pancreatic cancer patients, but the development of these therapies will require animal models that accurately recapitulate the etiology of pancreatic cancer-induced cachexia. To improve on the current, non-pancreas tumor xenograft models of cachexia, we generated a genetically engineered mouse model (GEMM) of pancreatic cancer, where induction of Kras and loss of the tumor suppressor Pten occurs in the pancreas of postnatal mice, which we refer to as the KPP model. KPP mice progressively lose skeletal muscle and adipose mass as a result of their cancer. We find that KPP mice exhibit histologic features of pancreatic ductal adenocarcinoma and reach endpoint criteria at an average of 107 days of age. Beginning at approximately 75 days of age, KPP mice undergo progressive loss of existing skeletal muscle mass, resulting in decreased muscle function. We also find that muscle loss from KPP mice exhibits a gene expression signature that closely aligns with the gene expression signature in muscle from cachectic pancreatic cancer patients. In summary, we expect KPP mice to serve as a useful model to elucidate the underlying mechanisms of muscle loss in pancreatic cancer-induced cachexia and to evaluate potential anti-cachexia therapies.

#4091

Knockout of genes in the DNA mismatch repair pathway to model colorectal cancer hypermutation.

Abbey Y. Jin, Peter Westcott, Olivia Smith, Amanda Cruz, Mary Clare Beytagh, Tyler Jacks. _David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02142, Cambridge, MA_.

Human colorectal cancer (CRC) is characterized by a high burden of somatic mutations, particularly in tumors harboring defects in the DNA mismatch repair (MMR) pathway. However, genetically-engineered mouse models (GEMMs) of cancer to date harbor a very low burden of mutations. Importantly, recent clinical trials have shown that MMR deficient tumors respond favorably to anti-PD-1 immune checkpoint blockade, and it is thought that PD-1 suppression reinvigorates existing T cell clones against neo-antigens associated with hypermutation in these tumors. In order to develop a more clinically relevant GEMM of CRC in which to study the immune response, MMR genes will be knocked out via CRISPR-Cas9 to induce hypermutation. To identify optimal Cas9 single guide RNAs (sgRNAs) against the MMR genes Msh2, Msh3, Msh6, and Mlh1, sgRNAs were designed using the Broad Institute algorithm and knockout efficiency assessed in a reporter cell line by Western blot. In order to induce colon tumors in vivo, colonoscopy-guided submucosal injection was employed to deliver lentivirus encoding sgRNAs against a MMR gene and Apc into mice carrying a constitutive Cas9 allele. Apc is inactivated in over 80% of human CRC, and is an initiating event in CRC in humans and mice. Using a sgMsh2-sgApc dual targeting lentivirus, we were able to efficiently generate colon tumors harboring complete knockout of Apc and near complete knockout of Msh2. Deep sequencing of the targeted loci revealed predominantly loss-of-function frameshift mutations at Apc and Msh2, but also a high degree of polyclonality. Lower titer injections of virus will be explored to induce more clonal tumors. Mlh1, Msh3, and Msh6 will also be targeted in vivo, and the mutational effects of MMR gene loss will be assessed by whole-exome sequencing of tumors. This rapid and flexible GEMM of hypermutant CRC may facilitate a more clinically relevant assessment of the role of the immune system in CRC development, as well as provide improved platforms for testing immunotherapies.

#4092

Glycosyltransferase ST6Gal-I promotes pancreatic ductal adenocarcinoma progression and metastasis.

Asmi Chakraborty, Robert B. Jones, Christopher A. Klug, Lesley E. Smythies, Susan L. Bellis. _Univ. of Alabama at Birmingham, Birmingham, AL_.

Sialyltransferase ST6Gal-I adds α2-6 sialic acids to select N-glycosylated cell surface receptors, thereby modulating receptor function and intracellular signaling. ST6Gal-I is upregulated in multiple human cancers, including >70% of pancreatic ductal adenocarcinoma (PDAC) samples. ST6Gal-I confers cancer stem cell (CSC) properties evidenced by tumorspheroid growth, chemoresistance and tumor initiating potential. As an example, we previously reported that ST6Gal-I imparts gemcitabine resistance by dampening DNA damage. One possible mechanism by which ST6Gal-I promotes a CSC phenotype is by increasing the expression of the master stem cell transcription factor, Sox-9. Forced overexpression or knockdown of ST6Gal-I directly regulates Sox9 expression in a wide array of established cancer lines. Sox9 is known to be a potent driver of acinar to ductal metaplasia (ADM), a key PDAC initiating event. To interrogate the role of ST6Gal-I and Sox9 in ADM, ST6Gal-I was overexpressed in the nontransformed acinar cell line, 266-6. ST6Gal-I overexpression was found to induce expression of Sox9 and other ductal markers. Furthermore, ST6Gal-I overexpressing cells spontaneously formed foci in adherent culture, a hallmark of malignant transformation. Contrarily, ST6Gal-I knockdown in 266-6 cells decreased cell viability. Post ADM, PDAC progresses through the formation of PanINs. This process of progressive lesion formation leading to frank PDAC can be replicated in murine models of pancreatic cancer utilizing pancreas specific oncogenic K-Ras (KC). To investigate the role of ST6Gal-I pertaining to PDAC in vivo we generated ST6Gal-I knock-in (KI) mice with oncogenic K-Ras (KC-ST6Gal-I KI). The KC-ST6Gal-I KI mice presented with significantly decreased overall survival (median = 4.3 months) compared to the KC mice (median = 14 months). This was coupled with a marked increase in liver and lung metastasis in KC-ST6Gal-I KI mice compared to KC mice. To better understand the molecular events underlying ST6Gal-I's PDAC-promoting function, we generated organoids from KC, KC-ST6Gal-I KI, ST6Gal-I KI and WT mice. Preliminary data indicate that there is increased Sox-9 expression in ST6Gal-I KI organoids compared to WT controls. As well, the number of organoids formed in culture is increased in ST6Gal-I KI mice compared to the control. Since organoids are derived from the resident stem cells, this data further indicates increased stemness with enhanced ST6Gal-I expression. In ongoing studies we are defining the signaling mechanisms underpinning a ST6Gal-I/Sox9 axis in PDAC initiation events and other aspects of CSC behavior. These collective studies highlight a novel role for tumor glycans in PDAC pathogenesis, and implicate ST6Gal-I as a promising therapeutic target.

#4093

Preclinical evaluation of novel treatment strategies in patient-derived xenograft (PDX) models of pancreatic cancer.

Diana Behrens,1 Britta Büttner,1 Rita L. Lawlor,2 Christopher Heeschen,3 Malte Buchholz,4 Jens Siveke,5 Antje M. Wengner,6 Ashok Saluja,7 Jens Hoffmann1. 1 _EPO GMBH, Berlin, Germany;_ 2 _ARC-NET University of Verona, Verona, Italy;_ 3 _QMUL, Barts Cancer Institute, London, United Kingdom;_ 4 _Philipps University Marburg, Marburg, Germany;_ 5 _DKFZ, University Hospital Essen, Essen, Germany;_ 6 _Bayer AG, Berlin, Germany;_ 7 _University of Miami, Miami, FL_.

Pancreatic cancer (PC) remains a lethal disease with only 3 - 8% of patients surviving 5 years after diagnosis of the tumor (WHO, 2012). Within the EU project "CAM-PaC" a comprehensive panel of thirty patient-derived PC xenografts (PDX) was established and used for the efficacy screening of new therapeutic options. Within this study, responders to the MPS-1 inhibitor BAY1161909, the Super Enhancer disrupting agent Minnelide and the MEK inhibitor Trametinib were identified and analyzed for potential biomarkers.

Patient tumors were collected during surgery and circulating tumorigenic cancer stem cells were isolated from the peripheral blood using VAR2CSA-coated magnetic beads. Both were transplanted subcutaneously into NOD/SCID/IL2y- mice and propagated in NMRI:nu/nu mice after engraftment. These were morphologically and molecularly characterized by histopathological revision and with NGS panels, designed based on pathway aggregated genes identified with the International Cancer consortium (described by Bailey et al., Nature 531, 2016). Standard drugs were applied using clinically relevant dosages and schedule. MPS-1 inhibitor BAY1161909 was given in monotherapy and in combination with Abraxane. Minnelide (MTD) was applied second line after three cycles of chemotherapy (Cisplatin, Abraxane, Gemcitabine) and Trametinib was tested as monotherapy.

All PDX correlated with histopathological and molecular characteristics of patient tumours. BAY1161909 monotherapy showed moderate anti-tumor efficacy with an average tumor growth inhibition of 30% (p > 0.05). However, tumor relapse after the end of chemotherapy was delayed in mice treated with the combination of BAY1161909 and Abraxane compared to Abraxane alone. 13 out of 22 PDX models tested to date were identified as responders (tumor growth inhibition > 50%) to Minnelide and 5 out of 11 to Trametinib. While Minnelide induced tumor growth inhibition above 80% in 32% of the models, Trametinib achieved the same efficacy in only 9% of the tested PDX models.

The described PDX panel clearly reflects clinical situation of pancreatic cancer due to their histologic growth and detection of inherent and acquired treatment resistance as well as recurrent disease. In a few cases, the tested drugs induced complete remissions. We are currently analyzing the molecular data to determine response markers. Our approach may offer personalized treatment options for PC patients.

#4094

Oncopig and human hepatocellular carcinoma cell lines exhibit similar response to liver cancer chemotherapy agents.

Mario F. Neto, Hanna H. Chen, Jordan L. Newson, Lawrence B. Schook, Kyle M. Schachtschneider, Ron C. Gaba. _University of Illinois at Chicago, Chicago, IL_.

Preclinical animal models represent pivotal tools to develop novel therapeutics. While murine models are commonly used in preclinical drug research, many agents that show promise in mouse studies ultimately fail in human clinical trials. In contrast to mice, pigs share many genetic, physiological, and metabolic similarities with humans, which may facilitate relevant investigation of drug pharmacokinetic and therapeutic analyses. The Oncopig Cancer Model (OCM) is a novel transgenic swine platform that recapitulates human cancer through development of site/cell specific tumors after Cre recombinase induced expression of heterozygous KRASG12D and TP53R167H transgenes. Given similar drug metabolism between pigs and humans, we propose that the OCM can address unmet clinical needs by facilitating translation of results obtained in preclinical murine studies to human clinical practice. In order to investigate the feasibility of using the OCM for drug screening, we tested the hypothesis that OCM and human hepatocellular carcinoma (HCC) cell lines exhibit similar responses to chemotherapy agents commonly used in clinical settings. Responses to four cytostatic (sorafenib) and cytotoxic (doxorubicin, cisplatin, mitomycin C) HCC chemotherapies were compared between OCM derived porcine HCC cell lines and three representative human HCC cell lines (SNU-387, SNU-475, HepG2). Results of MTT time course assays (3x performance) revealed that OCM HCC and human HCC cell lines displayed similar sensitivities to all four chemotherapies when exposed to comparable, clinically relevant concentrations for 72 hours. Sorafenib had a cytostatic effect at 10-20 µM in all four cells lines tested, consistent with clinically relevant, 3-15 µM systemic therapeutic concentrations. Doxorubicin, usually administered at 1-5 µM for locoregional therapy in patients, had comparable half maximal inhibitory concentrations (IC50) for pHCC (0.19 µM), HepG2 (0.45 µM), and SNU-387 (0.94 µM), but was higher for SNU-475 (3.31 µM). Mitomycin C had similar IC50 for pHCC (1.77 µM), HepG2 (1.73 µM), and SNU-475 (2.93 µM), but was higher for SNU-387 (7.91 µM). Finally, IC50 for cisplatin was nearly identical for pHCC (7.54 µM) and HepG2 (8.34 µM), but higher for SNU-387 (25.89 µM) and SNU-475 (16.57 µM). These results show clinically relevant similarities in response to the tested chemotherapy agents between pHCC and HepG2 (the most widely used human HCC cell line for in vitro investigations). Notably, differential drug responses observed between human HCC cell lines are consistent with previous studies, and are likely reflective of differential pathway alterations driving oncogenesis in these lines. These results indicate the OCM represents a valuable tool for screening promising chemotherapy agents, providing a novel large animal bridge between preclinical investigation in small animal models and human clinical trials.

#4095

Active Kras induced metaplasia progression toward gastric neoplasia is suppressed by MEK inhibition during gastric carcinogenesis.

Eunyoung Choi, Amy C. Engevik, James R. Goldenring. _Vanderbilt Univ. Medical Ctr., Nashville, TN_.

Intestinal type gastric cancer develops within metaplastic mucosal fields and development of gastric neoplasia represents the most prominent example of gastric cancer developing within the morphologic changes of metaplasia. Recent investigations have noted activation of Ras activity in up to 40% of human gastric cancers and suggest. Our recent study has suggested that chief cells might be an origin of gastric cancer using a mouse model, Mist1-CreERT2;LSL-KRas(G12D) mouse (Mist1-Kras). The Mist1-Kras mice rapidly developed SPEM and IM within 3 months and invasive metaplasia within 4 months following induction of constitutively-active Kras(G12D) expression in the chief cells in the mouse stomach. We also observed regression of metaplasia by targeting downstream mediators of Kras signaling using a MEK inhibitor, Selumetinib. We therefore have now investigated how the progression and proliferation of metaplasia can be controlled. In this study, we have established metaplastic organoid lines (Meta3 and Meta4) from Mist1-Kras mouse stomach mucosa at 3 or 4 months after tamoxifen induction. We examined these organoids to understand the mechanisms of metaplasia progression through MEK inhibition. Both Meta3 and Meta4 organoids have formed distinguishable glandular structures in 3D cultures and maintained a stable phenotype observed in metaplastic glands in the Mist1-Kras mouse stomachs. The MEK inhibition with Selumetinib in the metaplastic organoids inhibited growth, but promoted differentiation of metaplastic cells toward intestinal absorptive cells with elaboration of an apical brush border. Single cell RNA sequencing data revealed that Meta-4 have distinct characteristics that differ from Meta3. In 3D culture, Meta4 organoids developed more aggressive phenotypes than Meta3 and expressed Cortactin, which has critical roles in cancer cell migration and metastasis. Cortactin localized at the lamellipodia in monolayer culture of Meta4. Meta4 organoids treated with Selumetinib produced significantly fewer colonies in soft agar and resulted in loss of lamellipodia localization of Cortactin. Our results indicate that gastric carcinogenesis can be controlled by a MEK inhibitor, Selumetinib, and support the concept that growth of pre-neoplastic metaplasia and early stage gastric cancer in humans might be controlled with administration of Selumetinib.

#4096

Stage-defined, transgenic immunocompetent mouse model (ASV-B) to investigate new drugs for hepatocellular carcinoma.

Annemilaï Tijeras-Raballand,1 Patricia Hainaud,2 Christian Hobeika,3 Clarisse Eveno,3 Marc Pocard,3 Philippe Bonnin,3 Valérie Paradis,4 Mohamed Bouattour,4 Eric Raymond,5 Armand de Gramont,1 Evelyne Dupuy,2 Sandrine Faivre4. 1 _AFR Oncology, Paris, France;_ 2 _Institut des Vaisseaux et du Sang, Paris, France;_ 3 _Inserm U965- Lariboisière University Hospital, Paris, France;_ 4 _Beaujon University Hospital, Clichy, France;_ 5 _Paris Saint-Joseph Hospital, Paris, France_.

Background: Hepatocellular carcinoma (HCC) is a complex multistep malignancy in need of new therapeutic options often arising on underlying chronic liver disease. HCC animal models relevant to clinical situations are crucial to investigate new anticancer drugs, alone or in combination. Mice bearing xenografts and transgenic mice are the two main models used for preclinical drug development, but most of them fail to mimic the different step of HCC observed in patients. In this study, we aim to describe a stage-defined, transgenic immunocompetent HCC mouse model.

Methods: ASV-B is a transgenic mouse model that spontaneously develops, upon SV40 T-Ag oncogene expression in hepatocytes, a reproducible stage-defined HCC. Hyperplasia at week(W)8 is followed by nodular stage at W12 (multiple well-delimited tumor nodules of about 1 mm, growing progressively up to 1 cm), then diffuse carcinoma stage at W16-20. HCC is restricted to male, backcrossed with C57BL/6J mice, the female littermates being used as controls.

Results: Liver volume assessed by ultrasound at each step of carcinogenesis showed a 2.7-, 2.7-, 3.9-, and 5.8-fold increase (p<0.001) in transgenic mice compared to controls (CTRL) at W8, 12, 16, and 20, respectively. ASV-B model displays marked arterialization with intense arterial flow in liver tumor similar to human HCC. Sinusoids in tumor nodules are tortuous and dilated, surrounded by activated hepatic stellate cells (HSCs) expressing smooth muscle actin (SMA) while HSCs surrounding normal sinusoids only express desmin. In addition, ASV-B livers develop along carcinogenesis a mesenchymal phenotype (increased vimentin and decreased e-cadherin staining), mimicking certain clinical situations of acquired resistance to VEGFR inhibitors. Angiogenesis, monitored using Doppler assessing blood flow velocity (BFV) in the hepatic artery, is enhanced by 29%, 51% and 156% at W8, W12 and W16, respectively, in transgenic liver animals as compared to CTRL. Increased angiogenesis was confirmed by CD31 staining in tumor specimen showing increased number of vessels per field. ASV-B model has been used to investigate the toxicity and efficacy of new drugs (galunisertib, MET/AXL inhibitor), including antiangiogenic agents (sorafenib and ramucirumab) alone or in combination, and may be relevant to explore the effects of immunotherapy agents. At the conference, we will display further characterization of liver tumors regarding vascularization, and immune cells localization using IHC.

Conclusion: ASV-B transgenic mouse model mimics several characteristics of human HCC developing on healthy liver including multinodular disease, vessel abnormalities, and dedifferentiation toward mesenchymal phenotype. Further steps will consist of characterizing the adjacent nontumor liver, and developing nonalcoolic steatohepatitis (NASH) on ASV-B model using specific diets.

#4097

Maintenance of primary human colorectal cancer microenvironment using a perfusion bioreactor-based 3D culture system.

Celeste Manfredonia,1 Manuele Giuseppe Muraro,1 Christian Hirt,1 Valentina Mele,1 Valeria Governa,1 Adam Papadimitropoulos,1 Silvio Daester,1 Savas D. Soysal,1 Raoul A. Droeser,1 Robert Mechera,1 Daniel Oertli,1 Raffaele Rosso,2 Martin Bolli,3 Luigi M. Terracciano,1 Giulio C. Spagnoli,1 Ivan Martin,1 Giandomenica Iezzi1. 1 _Univ. Hospital Basel, Basel, Switzerland;_ 2 _Canton Hospital Lugano, Lugano, Switzerland;_ 3 _Claraspital, Basel, Switzerland_.

Colorectal cancer (CRC) is a leading cause of cancer-related death, often diagnosed at advanced stages. Conventional chemotherapeutic regimens have limited success rates, and a major challenge is represented by the lack of adequate in vitro models predicting patient responsiveness to defined treatments, possibly guiding therapeutic decisions. Non-malignant cells, including mesenchymal and immune cells, critically affect development, progression and drug responsiveness of human CRC. However, tumor drug responses are still evaluated on culture systems, such as 2D tumor cell monocultures or on tumor xenografts, which do not preserve all cellular components of in vivo tumor microenvironment.In this work, we have investigated the suitability of a perfusion-based bioreactor for 3D culture of primary CRC samples. Freshly excised CRC specimens were cut into fragments, inserted between two collagen type I sponges in a "sandwich-like" format and cultured for three days in a perfused-based system or under static conditions.We show that cultures under perfusion result in significantly higher maintenance of tissue integrity as compared to static cultures, with preservation of whole tumor microenvironment components, including cancer cells, mesenchymal stromal cells and a fraction of immune cells. Tumor tissues cultured under perfusion displayed an almost intact architecture with viable and proliferating tumor cells. Stromal cells were also maintained in proportions similar to those of original tumors and were fully viable, as indicated by their responsiveness to microenvironmental stimuli, such as IL-17. In addition, immune cells were also partially preserved, and were capable to release effector cytokines upon activation. Importantly, perfusion-based cultures proved suitable for testing sensitivity of primary tumor cells to chemotherapies currently in use for CRC and revealed heterogeneous responsiveness across different samples.

[C.M. and M.G.M. contributed equally to this work.]

#4098

New strategy of drug response assessment using PDX organoid platform for non-resectable pancreatic cancer.

A-Ra Jeon,1 Sun Il Choi,1 Sang-Jae Park,2 Sung-Sik Han,3 Sun-Young Kong,4 Min Kyeong Kim,1 Yu-sun Lee,1 Jieun Im,1 Min Kyeong Lee,1 Sang Hyun Park,1 Joon-Ki Kim,1 Kyong-Ah Yoon,5 Young-Hwan Koh,3 Ju Hee Lee,3 Woo Jin Lee,2 Sang Myung Woo,2 Yun-Hee Kim1. 1 _Research Institute of National Cancer Center, Goyang-si, Republic of Korea;_ 2 _Liver and Pancreatobiliary Cancer Branch, Research Institute of National Cancer Center, Goyang-si, Republic of Korea;_ 3 _Center for Liver Cancer, National Cancer Center, Goyang-si, Republic of Korea;_ 4 _Department of Laboratory Medicine, Center for Diagnostic Oncology, Research Institute and Hospital, National Cancer Center, Goyang-si, Republic of Korea;_ 5 _College of Veterinary Medicine, Konkuk University, Goyang-si, Republic of Korea_.

To develop an efficient drug screening platform which overcomes the difference of drug response between initial screening and clinical trial stage is a pivotal issue for drug discovery. The patient-derived Xenograft (PDX) model has been reported as a screening system to reflect the microenvironment and heterogeneity of tumor. However, in pancreatic cancer that 80 % of patients is non-resectable, PDX is not be suitable for an initial screening model in terms of economic- and time cost of mouse-based amplification system as well as the lack of obtaining pancreatic tumor tissue from fine needle biopsy or percutaneous gun. To overcome this limitation, here we newly suggested organoids system, miniature organ culture on a dish, that are generated from tumor tissues of orthotopic PDX model, which has the advantages of reflection of each patient's characteristics as well as amplification of limited tumor tissue. Besides, it is possible to screen of drug responsibility with a little number of cells. 12 organoids derived from PDX using needle or gun biopsy tumor tissues showed EpCAM overexpression and each unique morphological phenotype. Moreover, from drug responsibility test, H #43 and H #44, an organoids derived from a gemcitabine-sensitive patients, were highly responsible to gemcitabine, whereas the organoids from gemcitabine-resistant patients, G #20 and H #19 showed a strong resistance to gemcitabine as measuring the IC50 value. In addition, combined treatment with gemcitabine and abraxane to the G #13 model which has no clinical information of drug response due to early death, it inhibited organoid formation significantly, showing a combination index below 1, which was proved through in vivo (PDX) validation. Taken together, the PDX-Organoid system might be able to reflect primary tumor characteristics as well as to overcome the quantitative limitations of the specimen and time cost, and thereby it is possible to predict drug response early in vitro, making it very efficient as an anti-cancer drug development platform for pancreatic cancer.

#4099

Novel patient-derived 3D (PD3D) cell models and matched patient-derived xenografts (PDX) from peritoneal metastasis of colorectal cancer for drug testing and biomarker analysis.

Christian RA Regenbrecht,1 Guido Gambara,1 Eva Pachmayr,2 Alessandra Silvestri,1 Mathias Dahlmann,3 Bernadette Brzezicha,4 Britta Buettner,4 Beate Rau,2 Ulrich Keilholz,5 Urike Stein,6 Wolfgang Walther6. 1 _cellular phenomics and oncology GmbH, Berlin, Germany;_ 2 _Charité University Medicine Berlin, Berlin, Germany;_ 3 _DKTK Deutsches Krebsforschungszentrum Heidelberg, Berlin, Germany;_ 4 _experimental pharmacology and oncology GmbH, Berlin, Germany;_ 5 _Charité Comprehensive Cancer Center, Berlin, Germany;_ 6 _ECRC, Charité and Max Delbrück Ctr. for Molecular Medicine, Berlin, Germany_.

For patients with colorectal cancer (CRC) peritoneal metastases (PM) represent a terminal tumor stage with limited therapeutic options. To increase therapeutic efficacy and overall survival, availability of appropriate patient-derived 3D cell culture (PD3D) and patient-derived xenografts (PDX) models of PM could help improving the predictability of drug response for a specific tumor, but also foster the identification of novel biomarkers and therapeutic targets for CRC-PM patients. In this context, we generated the first scaffold-based PD3D and matching PDX models of CRC-PM as platform to test for chemotherapy response and to identify novel biomarkers.

For model establishment, surgical specimens were processed either for PD3D cell culture or transplanted subcutaneously (s.c.) onto immunocompromized NOD scid gamma (NSG) mice. For 3D cell culture models, tissue was dissected, digested and filtered before embedding into a scaffold matrix. For PDX models engrafted tumors were transferred to NMRI nu/nu mice for further passaging. They were characterized by histopathology, immunohistochemistry and gene expression analyses using real-time RT-PCR. Chemosensitivity of both sibling models was evaluated on a panel of conventional chemotherapeutic and of targeted drugs.

The same panel of drugs was used in 15 matched PD3D/PDX models and revealed individual response patterns both in PD3D and PDX. Most interestingly, different drug response pattern was observed in models derived from tumor tissue of the omentum vs. tissue from the peritoneum of the same patient.

Our results demonstrate, that matched models maintain basic characteristics such as the morphology of the patient tumor in early passages, reflect heterogeneous response rates, and can be used as preclinical platform for translational studies of potential clinical use. 

### Molecular Imaging: Novel Probes and Preclinical Studies

#4100

Personalized medicine in pancreatic cancer using cellular-resolution optical metabolic imaging.

Joe T. Sharick,1 Tongcheng Qian,1 Jillian K. Johnson,2 Lingjun Li,2 Cheri A. Pasch,2 Dustin A. Deming,2 Alexander A. Parikh,3 Melissa C. Skala1. 1 _Morgridge Institute for Research, Madison, WI;_ 2 _University of Wisconsin-Madison, Madison, WI;_ 3 _University of South Carolina School of Medicine - Greenville, Greenville, SC_.

Pancreatic cancer has the worst prognosis of all cancers (5-year survival rate of 7%). There is a critical need for a tool to match individual patients with optimal drugs for their cancer. The goal of this work is to validate Optical Metabolic Imaging (OMI) of patient-derived pancreatic tumor organoids as a high-throughput predictive drug screen for patients.

Drug induced changes in cell metabolism precede changes in tumor volume and thus present an earlier marker of treatment response. OMI is sensitive to cellular metabolism because it measures the intrinsic fluorescent properties of NAD(P)H and FAD, coenzymes of metabolic reactions. OMI endpoints include the optical redox ratio (the fluorescence intensity of NAD(P)H divided by the fluorescence intensity of FAD), and the fluorescence lifetimes of NAD(P)H and FAD. The redox ratio reflects the cellular redox balance, and the fluorescence lifetimes report on the binding activity of these coenzymes. OMI, performed using high-resolution multiphoton microscopy, has the unique ability to non-invasively monitor metabolism in living, intact 3D samples on the single-cell level, and can thus quantify inter-cellular heterogeneity in drug response. OMI also allows for high-throughput screening of potential cancer drugs and drug combinations on patient biopsy samples cultured ex vivo. These patient-specific organoids are grown in a 3D matrix that mimics the natural tumor environment. Drug response can be quantified separately in tumor cells and tumor-associated fibroblasts. Organoids were successfully generated from 9 of 11 surgically resected pancreatic tumors and 2 of 2 pancreatic intraepithelial neoplasias. These organoids were treated with a panel of standard and experimental therapies, and early metabolic changes were measured using OMI. Changes at the single-cell level were quantified using the OMI Index, a linear combination of the optical redox ratio and the mean NAD(P)H and FAD fluorescence lifetimes. Patient survival data after surgery and drug treatment was compared to drug response in organoids to that same treatment. Early results have focused on the standard gemcitabine + 5-fluorouracil (G+F) adjuvant treatment. Organoids generated from a patient whose CA19-9 biomarker levels increased while on G+F adjuvant therapy contained a subpopulation of 68% of the cells which were resistant to G+F therapy. Moreover, no G+F resistant cell subpopulations were found in organoids generated from a patient with no signs of disease recurrence over 14 months of G+F adjuvant therapy. Follow-up with the remaining patients is on-going. The ability of OMI to capture heterogeneous cell-level drug responses shows promise for predicting treatment response of individual pancreatic cancer patients.

#4101

Molecular imaging of class-IIa histone deacetylases in pancreatic cancer as a biomarker of response.

Mei Gao, Miranda Lin, Nashaat Turkman, Joseph Kim. _Stony Brook University, Stony Brook, NY_.

Introduction: The histone deacetylases (HDACs) family of enzymes are overexpressed in human cancers including pancreatic cancer. Past issues with non-specific and off-target inhibition have limited the clinical utility of HDAC inhibitors, but class-specific inhibitors appear more promising. Targeting class-IIa HDACs 4, 5, 7 and 9 has been shown to reprogram the tumor-associated macrophages which triggers therapeutic immune responses. We previously targeted trifluoromethyloxadiazole (TFMO) for radiolabeling with F-18, since this chemical group has high selectivity to class-IIa HDACs. Then, we applied this radiolabeling strategy to TMP195, a potent class-IIa HDAC inhibitor which contains the TFMO moiety. Our objective was to test 18F-TMP195 for therapy and molecular imaging of pancreatic cancer.

Methods: A bromodifluoromethyloxadiazole precursor was developed and then treated with cesium 18F-fluoride (Cs18F) to generate 18F-TFMO. This methodology was applied to formulate 18F-TMP195. The expression of Class IIa HDACs in PDAC cell lines (AsPC-1, MIAPaCa-2, and PANC-1) and normal pancreatic duct cells (hTERT-HPNE) was measured by western blot assay. Cytotoxicity of TMP195 was measured in cultured PDAC cell lines and hTERT-HPNE cells. SAHA, a non-specific HDAC inhibitor, was used as control. PDAC uptake of radiolabeled drug (18F-TMP195) was measured to evaluate specific binding to endogenous Class IIa HDACs in PDAC cells. Finally, 18F-TMP195 uptake was evaluated in vivo by PET imaging in pancreatic cancers xenografted in NOD/SCID mice.

Results: All 3 PDAC cell lines exhibited high expression levels of Class IIa HDACs, whereas low expression was observed in hTERT-HPNE cells. TMP-195 exhibited cytotoxic effects in all 3 PDAC cell lines. 18F-TMP195 was successfully synthesized for the first time in our laboratory. Radio-ligand cellular uptake tests measured by radioactivity accumulated in cells demonstrated significantly higher uptake of 18F-TMP195 in PDAC cells than hTERT-HPNE cells. Treatment with cold TMP195, but not SAHA, blocked uptake of radiolabeled drugs by PDAC cells compared to 18F-TMP195 alone or 18F-TMP195 + SAHA, thus supporting the selective targeting of TMP195 to the active site of Class IIa HDACs. The in vivo 18F-TMP195-PET imaging showed significant accumulation of the radiotracer in tumor tissue compared to adjacent non-tumor tissue.

Conclusion: 18F-TMP195 was successfully formulated utilizing single radiochemical synthetic-step protocol. Our studies indicate that this class of drugs selectively targets Class-IIa HDACs in PDAC and suggest that selective class-IIa HDACs are promising targets with potential for molecular imaging of PDAC in vivo. Therefore, class-IIa HDAC expression patterns in PDAC present potential for development of diagnostic imaging agents to predict response to treatment.

#4102

Surgical imaging of pancreatic cancer using near infrared fluorescent hyaluronic acid nanomaterials.

Bowen Qi, Ayrianne Crawford, Samuel Cohen, Michael Hollingsworth, Aaron M. Mohs. _University of Nebraska Medical Center, Omaha, NE_.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) has an overall 5-year survival rate for all patients of approximately 7.1%. Surgery only increases the long-term survival rate of PDAC to 12.1%. Survival could potentially be increased with better detection of pancreatic cancer during surgical resection by improved margin detection, more effective debulking of disseminated disease, and earlier detection. In this work, we utilized nanoparticle formulations of the near infrared fluorophore indocyanine green (ICG) to provide optical detection of pancreatic tumors in an orthotopic, syngeneic mouse model of PDAC.

Methods: ICG was physico-chemically entrapped nanoparticles derived from amphiphilic hyaluronic acid, termed NanoICG and compared to ICG alone. Cytotoxicity and immunotoxicity of the imaging agents were determined by in vitro methods, while blood biochemistry, cell counts, and toxicological pathology, were examined in vivo. The ability to detect PDAC was determined in PDAC cells originating from KPC mice that were implanted orthotopically into syngeneic C57 mice. Contrast enhancement was determined using a combination of image-guided surgery systems and whole animal imaging systems.

Results: Neither Empty NP of NanoICG affected metabolic activity toward healthy pancreatic epithelial or pancreatic adenocarcinoma cells and NanoICG displayed did not have capacity to act as a chemoattractant. In vivo, NanoICG (normalized to maximum allowed ICG dose) did not alter blood biochemical markers including HGB, ALB, ALP, ALT, AMY, BUN, GLOB, and TP or cell counts including, WBC, RBC, PLT, or HCT. Histological examination showed no toxicity related to the administration of contrast agents in liver, lung, spleen, or kidneys. In the syngeneic, orthotopic PDAC model, NanoICG accumulated significantly within the pancreas and had a 2.1-fold stronger signal compared to ICG alone. Considering contrast within PDAC compared to uninvolved healthy pancreas, NanoICG was 2.34-fold stronger (P<0.05), while contrast between PDAC and healthy pancreas with ICG was minimally detectable. Using near infrared fluorescence microscopy, area strong signal from NanoICG localized to areas of PDAC.

Conclusions: NanoICG exhibited improved efficacy at detecting pancreatic tumors in an orthotopic pancreatic cancer model with negligible NP-derived toxicity, which shows NanoICG as a promising agent in pancreatic tumor diagnosis and surgical resection. Further research is being performed to minimize clearance organ uptake.<!--EndFragment-->

#4103

Utility of multispectral optoacoustic tomography in imaging pancreatic tumors using a uPA-probe.

Akiko Chiba, Abhilash Samykutty, Mary Smith, Lacey McNally. _Wake Forest, Winston-Salem, NC_.

Background: Pancreatic cancer has the lowest 5-year survival rate of all cancer types. Current methods of pancreatic cancer screening, diagnosis and treatment have failed to improve outcome in the last few decades. Potential explanation for this failure may be explained by the hypovascularized nature of pancreatic cancer, making conventional chemotherapy and imaging modality suboptimal. There is a potential for improved imaging using a new imaging modality, multispectral optoacoustic tomography (MSOT). We hypothesize that a using urokinase plasminogen activator (uPA) contrast agent while using MSOT imaging would improve identification of orthotopic pancreatic cancer in xenografts. Methods: Expression of uPA receptor (uPAR) were evaluated in pancreatic tumor cell lines, Panc1, S2CP9, MiaPaca-2 and S2VP10 using western blot. A uPA targeted contrast agent was created using standard bioconjugation methods with the Hilite 750 near infrared (NIR) dye. Five SCID mice were orthotopically implanted with S2CP9 (1.5 x 105) and 5 additional SCID mice were implanted with MiaPaCa2 (2.0 x 106) pancreatic tumor cells. When tumor size reached 3mm, 200µL of 100 nM uPA-750 probe or 750 dye alone was injected into mice. Biodistribution and accumulation of the uPA-750 probe was visualized using MSOT imaged at 2-hour intervals for 8 hours and at 24 hours. Accumulation of uPA-750 probe was evaluated in the tumor, liver, and kidney using NIR fluorescent imaging. Results: All pancreatic cancer cell line expressed uPAR with the highest in S2CP9 cells (3.0x), S2VP10 (2.5x), Panc1 (1.77x) and MiaPaca-2 (1.3x). In vivo, peak intensity of uPA-750 probe was successfully detected using MSOT at 4 hours, within the pancreas tumor in slices from 37mm-41mm. uPA-750 probe was undetectable within the tumor after 8 hours. Using ex vivo NIR fluorescence imaging, uPA-probe signal was detected only within the pancreatic tumor, however, no signal was detected in the liver or kidney. Conclusion: This study demonstrated MSOT imaging using uPA-probe as a contract agent may lead to improved pancreatic cancer detection which may lead to improvement in overall outcome.

#4104

**Evaluation of a novel LDH inhibitor efficacy in vivo** **in a glycolytic cancer model using hyperpolarized** 13 **C magnetic resonance imaging.**

Nobu Oshima,1 Shun Kishimoto,1 Kristin Beebe,1 Dan Crooks,1 Michael Moses,1 Kazutoshi Yamamoto,1 Jeffry R. Brender,1 Anastasia Sowers,1 Ganesha Rai,2 Daniel Urban,2 Goria Benavides,3 Giuseppe Squadrito,3 Victor Darley-Usmar,3 Matthew Hall,2 James B. Mitchell,1 Murali C. Krishna,1 Leonard M. Neckers1. 1 _NCI/NIH, Bethesda, MD;_ 2 _NCATS/NIH, Bethesda, MD;_ 3 _The University of Alabama at Birmingham School of Medicine, Birmingham, AL_.

Increased conversion of pyruvate to lactate by Lactate Dehydrogenase A (LDHA) is a feature of many neoplasms. Therefore, LDHA inhibition is considered a promising approach toward developing a new therapeutic strategy against cancers expressing the Warburg phenotype. To develop this strategy for clinical use, a feasible and sensitive noninvasive imaging approach that can dynamically evaluate LDHA activity in vivo would be highly beneficial since in vitro metabolic profiling dose not always predict in vivo cancer metabolism.

Hyperpolarized (HP) 13C Magnetic Resonance Imaging (MRI) can be used to perform dynamic 13C metabolic flux analysis in vivo. In particular, use of [1-13C]pyruvate (13C-pyr) with 13C MRI permits real-time monitoring of intratumoral LDHA activity through dynamic observation of conversion of 13C-pyr to [1-13C]lactate (13C-lac).

This study aimed to apply 13C MRI technology in support of a therapeutic strategy to explore efficacy of a newly developed and highly potent LDH inhibitor (LDHi) in a glycolytic tumor model using MiaPaCa2 xenografts in mice.

In vitro analysis showed that the LDHi dose-dependently inhibited human LDH activity and suppressed in vitro cell growth in MiaPaCa2 cells. By ex vivo assay, LDH activity in MiaPaCa2 xenografts was significantly suppressed (82.2 ± 5.6 % decrease compared to vehicle controls) following a single intravenous(IV) injection of 50 mg/kg LDHi.

Next, 13C MRI imaging of HP 13C-pyr metabolism was performed before and after a single LDHi IV injection to assess inhibitor impact on intratumoral metabolic flux in vivo. 13C MR spectroscopy confirmed that LDHi suppressed intratumor LDHA activity dose- and time-dependently. The maximum effective dose was 50 mg/kg and inhibitor impact in the tumor persisted for 10-12 hrs after a single injection. In addition, lactate production in the tumor was suppressed 30 minutes after IV administration of LDHi, as was the 13C-lac to 13C-pyr ratio decreased by 83.3 ± 4.4 % compared to vehicle controls. Importantly, the close correlation of these data with the results of the ex vivo LDH activity assay, suggests that 13C MRI can reliably monitor in vivo on target effects of LDHi without need for tissue sampling. Based on the data using 13C MRI, we developed a therapeutic strategy for using LDHi in an efficacy study of MiaPaCa2 xenografts. Intermittent IV administration of LDHi significantly suppressed tumor growth in this glycolytic model.

In conclusion, intratumoral inhibition of LDHA in vivo upon IV administration of a novel LDHi was readily visualized by HP 13C MRI, confirming the utility of this noninvasive method. This methodology can be of great value in developing new therapeutic strategies using LDHi and perhaps other metabolic inhibitors to treat cancers characterized by the Warburg phenotype. Further, HP 13C MRI should allow for selection of those patients likely to respond to such treatments.

#4105

Localized and triggered release of oxaliplatin for the treatment of colorectal liver metastasis.

Venkateswara R. Gogineni,1 Woo Ram Park,2 Jaidip Jagtap,1 Abdul Parchur,1 Gayatri Sharma,1 Amit Joshi,1 Dong-Hyun Kim,2 Andrew C. Larson,2 Sarah B. White1. 1 _Medical College of WIsconsin, Milwaukee, WI;_ 2 _Northwestern University, Chicago, IL_.

Though systemic chemotherapy is the standard of care for the treatment of unresectable metastatic colorectal cancer, new drug delivery platforms are needed to mitigate the associated toxicities. The aim of this study was to develop and evaluate a liposome formulation that delivers oxaliplatin under magnetic field stimulus in a rat model of colorectal liver metastases. The drug delivery platform was synthesized by encapsulating iron oxide nanocubes and oxaliplatin in sterically stabilized polyethylene glycol (PEG)-coated liposomes of ~140 nm size. Magnetic properties and efficacy of drug release were determined by exposing the liposomes to an alternating magnetic field (AMF). In vitro efficacy of the liposomes was tested by performing viability assay on rat colorectal liver metastasis cell line (CC-531 cell line). For in vivo studies, rats implanted with CC-531 cells were divided into various groups and the efficacy of systemically delivered oxaliplatin was compared to localized and triggered delivery. Localized delivery and triggered release of drug was imaged by quantitative magnetic resonance and optical imaging methods. The rats were euthanized after 3 weeks, tumors harvested and H&E and Prussian blue staining was performed. Kinetics of oxaliplatin bioavailability was performed by ICP-MS analysis for platinum at various time points and in different tissues. The analysis of feasibility and efficacy of multiple cycles of triggered release is currently in progress. Drug-loaded magnetic liposomes presented an oxaliplatin loading content of ~2 %. Magnetic liposomes separated from the solution within 30 min, when exposed to a permanent magnet. An increase in drug release (~18%) was observed by both HPLC and optical imaging methods when the samples were exposed to the AMF. Viability assay demonstrated that under high-dose oxaliplatin conditions (0.17-0.33 mg/mL), the cell viability after liposome treatment and exposure to AMF was significantly lower than those not exposed to AMF (p< 0.001). MR imaging revealed that all the rats developed tumors within 1 week. Optical imaging on rats showed that nanomaterial could be infused locally via mesenteric vein into liver and there is a consistent increase in the release of drug when exposed AMF. Although the tumors showed metastasis by the end of third week, triggered release of drug was well tolerated and shrinkage of primary tumor with necrotic regions was prominent in the MR imaging. Prussian blue staining and quantitative MR imaging showed delivery of liposomal cargo to the tumor site when exposed to AMF. H&E staining revealed marked necrotic regions in the tumor among the animals exposed to magnetic field. In conclusion, AMF triggered oxaliplatin release from liposome for increased cell death of CC-531 cell line. Triggering was well tolerated by rats and, further, can be employed to targeted killing of tumor by optimizing the dose of drug.

#4106

**Hyperpolarized [1-** 13 **C]-pyruvate/lactate magnetic resonance spectroscopic imaging of prostate cancer in vivo predicts response to lactate dehydrogenase inhibition.**

Bradley T. Scroggins,1 Masayuki Matsuo,1 Ayla O. White,1 Keita Saito,1 Jeeva P. Munasinghe,2 Carole Sourbier,1 Kazutoshi Yamamoto,1 Vivian Diaz,2 James B. Mitchell,1 Murali C. Krishna,1 Deborah E. Citrin1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _National Institute of Neurological Disorders and Stroke, Bethesda, MD_.

Purpose: Non-invasive magnetic resonance spectroscopic imaging (MRSI) of hyperpolarized (HP) 13C-labeled pyruvate and its metabolite lactate is being used to monitor the metabolic flux in solid tumors. In this study, we evaluate the potential of MRSI of HP [1-13C]-pyruvate and [1-13C]-lactate, in prostate cancer as a predictive biomarker for targeting lactate dehydrogenase.

Experimental Design: Two human prostate cancer cell lines (DU145 and PC3) were grown as xenografts. The conversion of pyruvate to lactate in xenografts was measured, after intravenous delivery of hyperpolarized [1-13C] pyruvic acid, by MRSI. Steady state metabolomic analysis of xenograft tumors was performed by mass spectrometry and steady state lactate levels were measured with proton (1H) MRS. Perfusion and oxygenation of xenografts were measured with electron paramagnetic resonance (EPR) imaging. Tumor growth was assessed after lactate dehydrogenase (LDH) inhibition with FX-11 (42 µg/mouse/day for 5 days x 2 weekly cycles). Lactate production, pyruvate uptake, extracellular acidification rates and oxygen consumption of the prostate cancer cell lines was analyzed in vitro. Protein levels of glycolysis regulators were assessed with immunoblotting.

Results: DU145 tumors demonstrated an enhanced conversion of pyruvate to lactate with HP [1-13C] MRSI relative to PC3 tumors (21% higher 13C-lactate/pyruvate p<0.05). In addition, DU145 xenografts have a 42% (p<0.05) reduction in 13C-lactate/pyruvate after FX-11 treatment while PC3 xenografts demonstrate no sensitivity to FX-11 treatment. In correlation FX-11 significantly delayed DU145 tumor volume doubling time by 3.4 days (p<0.05). By comparison no difference was observed between DU145 and PC3 xenografts in steady state measures of lactate, oxygenation, or perfusion. The two cell lines also exhibited similar pyruvate uptake, lactate production, extracellular acidification and oxygen consumption rates and sensitivity to FX-11 in vitro. Difference in the expression of glycolysis regulators such as Hif-1α, LDHA, MCT1, MCT4, HK2 and PFKP were observed in vitro but did not correlate with HP [13C]-lactate/pyruvate MRSI results or FX-11 sensitivity.

Conclusions: Hyperpolarized [1-13C]-pyruvate MRSI of prostate cancer xenografts predicted the efficacy of targeting LDH when steady state markers of glycolysis, in vivo and in vitro, did not.

#4107

Tumor multifunctional targeting polymeric nanomicelles with polypharmacy payload for effective therapy and imaging of resistant renal cell carcinoma.

Hashem O. Alsaab,1 Samaresh Sau,1 Vino T. Cheriyan,2 Ulka Vaishampayan,3 Arun K. Rishi,2 Arun K. Iyer1. 1 _Use-inspired Biomaterials & Integrated Nano Delivery (U-BiND) Systems Laboratory Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI; _2 _John D. Dingell VA Medical Center, Wayne State University, Detroit, MI;_ 3 _Karmanos Cancer Institute, Wayne State University, Detroit, MI_.

Renal cell carcinoma (RCC) is very difficult to treat as the cells are mostly resistant to many current therapies. Therefore, newer treatments including better ways of drug delivery are urgently needed to fight this malignant disease efficiently. The ongoing work was done by generating and characterizing RCC cells that are resistant to everolimus, a front-line mTOR-targeted therapy, and tested whether a class of CARP-1 functional mimetic (CFM) compounds inhibits parental and everolimus-resistant RCCs in combination with sorafenib. CFMs inhibited RCC cell viability in a dose-dependent manner that was comparable to everolimus treatments as well as the combination with sorafenib. CFM-4.16 inhibited viabilities of everolimus-resistant RCC cells, albeit CFM-4.16 combined with sorafenib was more effective than CFM-4.16 alone. CFM-dependent loss of RCC cell viabilities was due in part to reduced cyclin B1 levels, activation of pro-apoptotic, stress-activated protein kinases (SAPKs), and apoptosis. However, CFMs and sorafenib are hydrophobic, and their dose escalation for in vivo studies remains challenging. We addressed the solubility and delivery concerns of CFM-4.16 and sorafenib by utilizing a nanotechnology-based approach. Our data thus far revealed that we have been successful in overcoming the solubility concerns of CFM-4.16 and sorafenib by encapsulating it in water-soluble vitamin E TPGS-based micellar nanoformulation that enables high drug loading and affords its intravenous administration for animal testing. We also use our polymeric NPs formulation for in vitro efficacy in parental, wild-type and everolimus-resistant RCC. After confirming the anticancer activity of CFM-4.16-loaded nanoformulations in vitro, we finally examined the intravenous (i.v.) administration of the CFM-4.16-loaded polymeric NPs in a clinically relevant RCC mouse model. The CFM-4.16 formulation inhibited the viability of parental and everolimus-resistant RCC cells in vitro and suppressed the growth of parental A498 RCC-cell-derived xenografts (RCC animal model), in part by stimulating apoptosis. After optimizing its potency and biologic stability, we performed imaging of animal inoculated with RCC tumor and PDX model by conjugation with NIR dye. The results showed high binding affinity and specific tumor uptake, faster normal tissue clearance and less non-target organ uptake. These findings make CFM-4.16 an important potential therapeutic in combination with sorafenib using nanoformulation in the treatment of RCCs.

#4108

Longitudinal diffusion-weighted MRI assessment of NRAS mutant melanoma response to dual RAF-MEK inhibition reveals differences associated with collagen deposition.

Teresa Delgado-Goni, Slawomir Wantuch, Teresa Casals-Galobart, Udai Banerji, Nina Tunariu, Simon P. Robinson, Martin O. Leach, Mounia Beloueche-Babari. _Institute of Cancer Research, London, United Kingdom_.

Background and Purpose: Oncogenic NRAS, which drives the constitutive activation of the RAS-RAF-MEK-ERK pathway, is expressed in 15–20% of melanomas and is the second most common driver mutation in this disease. Inhibition of oncogenic RAS has proven extremely challenging with no agents clinically approved to date. RO5126766 is a dual RAF/MEK inhibitor with activity in mutant RAS driven cancers currently in early phase clinical trials.

Here the potential of non-invasive diffusion-weighted MRI (DW-MRI) to detect response to acute and long-term treatment with RO5126766 was investigated in NRAS mutant human melanoma xenografts.

Experimental procedures: Female NCr/nu mice with established D04 (NRAS(Q61L) melanoma) subcutaneous tumors (~300mm3) were treated with either vehicle or RO5126766 (5mg/kg once daily) for 3 days (acute protocol, n=4 /group) or 10 days (long-term protocol, n=5/group). DW-MRI was performed prior to and at the end of each treatment protocol to determine tumor apparent diffusion coefficient (ADC), a parameter proportional to tissue water diffusivity. MRI data were correlated with immunohistochemical analysis of tumor tissue stained with H&E (cellularity and necrosis), Masson's trichrome (MTC, collagen) and P-ERK (target inhibition). In addition, D04 cells were treated in vitro with RO5126766 (1µM, 10xGI50) for 24h and mRNA expression of genes involved in collagen synthesis (COL1A1 and COL1A2) assessed with qRT-PCR.

Results: RO5126766 induced a significant reduction in D04 tumor volume, relative to pre-treatment values, after both acute (76 ± 14%, p=0.04) and long-term treatments (63 ± 20%, p=0.01). Baseline tumor ADC equaled ca. 700x10-6mm2/s and showed no changes in the 3-day treatment cohort relative to pre-treatment values (95 ± 10%). In contrast, a significant decrease in ADC was observed in the long-term RO5126766 treatment cohort (to 81 ± 16% of day 0 value, p=0.04). Whilst H&E staining revealed no change in tumor necrosis following either treatment protocol, a significant increase in collagen deposition, shown by MTC staining, was detected in long-term RO5126766-treated tumors compared to vehicle controls (p=0.01). qRT-PCR analysis in D04 in vitro cell cultures following exposure to RO5126766 revealed a significant increase in COL1A1 expression (up to 2.6-fold, p=0.006), indicating that the increase in tumor collagen observed in vivo was, at least in part, due to increased synthesis by melanoma cells.

Conclusions: Our data show that response to the dual RAF-MEK inhibitor RO5126766 in mutant NRAS melanoma xenografts is associated with extracellular matrix remodeling via collagen deposition following long-term treatment. This process can be monitored non-invasively using DW-MRI indicating potential for clinical translation.

#4109

Multi-modality imaging to interrogate lipidome changes during melanoma progression in zebrafish.

Fiona Henderson,1 Hannah Johnston,1 Emrys Jones,2 Duncan Foster,1 Raghavendar T. Nagaraju,1 Michael Green,1 Michael Fairclough,1 Irene Barinaga-Rementeria Ramirez,1 Shuning He,3 B.Ewa Snaar-Jagalska,3 Katherine A. Hollywood,4 Warwick Dunn,4 Herman P. Spaink,3 Paul Lorigan,1 Emmanuelle Claude,2 Kaye Williams,1 Adam Hurlstone,1 Adam McMahon1. 1 _University of Manchester, Manchester, United Kingdom;_ 2 _Waters Corporation, Wilmslow, United Kingdom;_ 3 _Leiden University, Leiden, Netherlands;_ 4 _Central Manchester University Hospitals NHS Foundation Trust, Manchester, United Kingdom_.

Lipids play diverse roles in the body, including cell structure, cell signalling, and energy production; all of which are altered in cancer. Lipid metabolism is therefore a potential therapeutic target, and here has been investigated using a transgenic zebrafish model. This study combines transcriptome and lipidomic analyses for the first time, with in-vivo positron emission tomography (PET) and ex-vivo desorption electrospray ionisation-mass spectrometry (DESI-MS), to investigate lipid metabolism changes in melanoma. Most lipids are made of fatty acids, and the methods by which tumours acquire fatty acids can be altered compared to normal tissue. Here, the PET tracer [18F]-FTHA (a fatty acid analogue) has been used to demonstrate free fatty acid uptake in melanoma tumours in-vivo. Additionally, the excellent spatial resolution of mass spectrometry imaging has been utilised by imaging the FTHA ex-vivo by DESI-MS, allowing more precise spatial correlation with the histology, and revealing the heterogeneous nature of this fatty acid uptake. These imaging data complement the findings of the transcriptome analyses which show upregulation of genes associated with fatty acid uptake. Integrating transcriptome and lipidome data analyses revealed dysregulation of glycerophospholipid pathways, and was supported by DESI-MS data, which revealed heterogeneous changes in lipid metabolism.

#4110

Metabolic imaging of glioblastoma using hyperpolarized 13C-MRI: Glycolytic metabolism in cancer stem cell-like cell-derived tumor model.

Tatsuya Kawai, Murali C. Krishna, Jeffery R. Brender, Jennifer Lee, Tamalee Kramp, Kazu Yamamoto, Shun Kishimoto, Tomohiro Seki, Kevin A. Camphausen. _National Cancer Institute/NIH, North Bethesda, MD_.

OBJECTIVES:

Since the cancer stem-like cells (CSCs) play an important role in the resistance against radiation and/or chemotherapy due to its different metabolic profile, to investigate the specific metabolic characteristics of CSCs is valuable. Dynamic nuclear polarization (DNP) technique using 13C-labeled substrates enables magnetic resonance imaging (MRI) to monitor specific enzymatic reactions in tumors. In this study, DNP-MRI along with hyperpolarized [1-13C] pyruvate was conducted to evaluate the difference in glycolytic profile between a glioblastoma cell line and a CSC-derived glioma using orthotopic xenograft mouse model. The imaging findings were compared to those in immunohistology and cell cultures.

METHODS:

A 3T MRI scanner along with a custom-made head coil was used for imaging. To obtain hyperpolarized [1-13C] pyruvate, 30 μL of [1-13C] pyruvic acid containing 15 mM of OX063 (trityl radical compound) and 2.5 mM of the gadolinium chelate were polarized at 3.35 T and 1.4 K in the Hypersense DNP polarizer. The hyperpolarized sample dissolved in a buffered medium was injected via the tail vein followed by a 13C-two-dimensional spectroscopic imaging. During the acquisition, the body temperature was kept in the range of 35.0 to 36.9 Celsius degrees.

Orthotopic brain tumor model was developed in nude mice by intracranial implantation of glioblastoma cell lines, U251 and glioma stem-like cells, NSC11. DNP-MRI was performed when the tumor size ranged 50 + 10 mm3 on T2-weighted MRI. Independently, immunofluorescent analyses of monocarboxylate transporter 1(MCT1) and MCT4 was performed using paraffin-embedded tumor tissue.

Expressed protein level of lactate dehydrogenase A (LDHA), LDHB, MCT1 and MCT4 under the normal cell culture conditions were evaluated by Western blotting.

RESULTS:

13C-MRI showed an increase in lactate-to-pyruvate ratio (Lac/Pyr) both in U251 and NSC11

compared to the contralateral normal brain tissue, suggested elevated anaerobic glycolysis (P<0.05). The extent of the increase in Lac/Pyr compared to the normal brain tissue was higher in U251 than NSC11 except for one U251 case. Western blotting showed both LDHA and LHDB expression in U251 was lower than NSC11. The expression of MCT4 was higher in U251 than NSC11, whereas they were almost the same for MCT1. Immunohistochemistry showed relative weak staining for MCT4 in NSC11, consistent with the result of Western blotting.

CONCLUSION:

The quality of 13C-DNP-MRI using [1-13C] pyruvate was validated and considered to be feasible to apply to the brain tumors. The result was reasonable in that malignant tumors enhance anaerobic glycolytic pathway even in the aerobic environment. 13C-DNP-MRI also demonstrated a difference in the extent of Lac/Pyr in relation to the normal brain between the two tumor models probably related to expression levels of glycolysis-related proteins.

#4111

Water content based Electrical Properties Tomography (wEPT) for modelling delivery of Tumor Treating Fields to the brain.

Catherine Tempel-Brami, Cornelia Wenger, Hadas S. Hershkovich, Moshe Giladi, Ze'ev Bomzon. _Novocure, Haifa, Israel_.

Objective: The purpose of this study was to investigate the application of Water content based Electrical Properties Tomography for mapping electrical properties (EPs) of brain tissues in the frequency range of 100-1000 kHz.

Background: TTFields are electric fields with frequencies of 100-500 kHz that disrupt mitosis. TTFields are approved for the treatment of glioblastoma multiforme. Determining the EPs of brain tissues is important for understanding how TTFields distribute within the head. The EPs of tissues are heterogeneous, especially in the region of the tumor. Therefore methods that map EPs within the brain with high spatial resolution are desired. Water content based EP tomography (wEPT) is a method that utilizes the ratio of two T1w MRI images with different relaxation times (TRs) to map EPs based on empirically derived relationships between T1, water content (WC) and EPs. wEPT has been applied to map EPs of healthy brain at 128 MHz using typical WC and EP values of healthy tissues reported in the literature to derive the empirical models. Here we adapted wEPT to map EPs in the 100-1000 kHz range utilizing in-house measurements of WC and EPs from healthy bovine and tumor-bearing rat brain tissue.

Methods: The empirical model connecting MRI images, WC and EPs in the 100-1000 kHz range were created using 32 tissue samples derived from three 3 calf brains and 1 CSF sample of a pig. For each sample, T1w MRIs with TRs {700, 4000} ms were acquired and the image ratio (Ir) between the images was calculated. EPs of samples were measured using parallel plates, and WC was measured by the wet-dry weight method. Curve fitting yielded empirical models connecting Ir, WC and EP. Next, T1w MRIs of in-vivo tumor-bearing rat brains and 6 ex-vivo pieces of calf brain were acquired, and the empirical curves described above were used to map WC and EP within the rat brains and the pieces of calf brain. EPs and WC were measured on 6 small samples excised from each imaged brain. For each sample, measured values were compared to the median WC and EPs extracted from the corresponding Regions of Interest (ROIs) in the wEPT map.

Results: Anatomical structures and the tumor were clearly visible in wEPT maps. WC estimated using wEPT agreed well with measurements on excised samples. There was a clear connection between EPs estimated with wEPT and the measured values. However, in some samples large differences between wEPT-derived EP values and measurements were found. In particular, the differences between tumor and healthy tissues conductivity estimated using wEPT was significantly higher than the measured difference in conductivities within the corresponding excised samples.

Conclusion: wEPT maps WC in healthy and tumor brain tissues and provides information on local electrical properties at frequencies of 100-1000 kHz. Further investigation is needed to clarify the relationship between WC and EP within this frequency range.

#4112

Synthetic luciferin, CycLuc1, improves bioluminescence imaging for intracranial glioblastoma xenografts.

Shiv K. Gupta,1 Minjee Kim,2 Bianca-Maria Marin,1 Lihong He,1 Katelyn Swanson,1 Tuma Tuma,1 Brett L. Carlson,1 Mark A. Schroeder,1 William F. Elmquist,2 Jann N. Sarkaria1. 1 _Mayo Clinic, Rochester, MN;_ 2 _University of Minnesota, Minneapolis, MN_.

Background: Bioluminescence imaging (BLI) of firefly luciferase expressing cells is widely used for analysis of growth, metastasis and drug response in tumor xenografts. D-luciferin, a commonly used luciferase substrate, has limited brain distribution that may undermine BLI for intracranial xenografts. The synthetic luciferin cyclic alkylamino-luciferin (CycLuc1) is a purported brain penetrant luciferase substrate not yet tested in intracranial xenografts. We compared BLI with CycLuc1 and D-luciferin in a glioblastoma (GBM) xenograft model.

Methods: GBM6 cells were lentivirally transduced to express LUC2=tdTomato fusion protein. In vivo BLI were performed in orthotopic and heterotopic xenografts established in athymic nude mice. Concentrations of D-luciferin and CycLuc1 were measured by LC-MS/MS.

Results: In a dose-response analysis, CycLuc1 at 5 mg/kg provided robust and reproducible imaging; however, an increased photon flux was observed with 10 mg/kg, while escalation to 15 or 20 mg/kg provided marginal additional gains in photon flux. In comparison of CycLuc1 (5 or 25 mg/kg) and D-luciferin (30 or 150 mg/kg, respectively) with serial, crossover BLI at both dose levels, CycLuc1 resulted in less variability and significantly higher photon flux as compared to D-luciferin in GBM6 intracranial xenografts (15 days after implantation). On average BLI with 25 mg/kg CycLuc1 resulted in ~8 fold greater photon flux from these early-stage xenografts as compared to 150 mg/kg D-luciferin (2.9±0.6x106 vs. 3.3±2.8x105 p/sec/cm2, p<0.001). Interestingly, bioluminescence emission with CycLuc1 and D-luciferin was comparable in xenografts imaged 28 days after implantation. In samples harvested after last imaging session, there was no significant difference in substrate distribution in intracranial tumors (tumor to plasma ratios for CycLuc1 and D-luciferin were 0.012 ± 0.020 and 0.012 ± 0.015, respectively; p=0.89). These results suggest that intracranial tumors at early stage may comprise relatively intact blood-brain barrier, which is disrupted at later stage. Consistent with notion that suboptimal delivery may interfere with D-luciferin mediated BLI in intracranial xenografts, photon flux in heterotopic tumors with D-luciferin or CycLuc1 was comparable (4.9±4.6x108 vs. 1.3±0.8x108 p/sec/cm2, p=0.10).

Conclusions: These findings demonstrate that CycLuc1 may be a superior BLI substrate as compared to D-luciferin for monitoring tumor growth of intracranial GBM xenografts.

#4113

A multimodal (PET/NIRF) smart probe for selectively identifying, grading, and visualizing astrocytic gliomas.

Kenneth S. Hettie, Eben L. Rosenthal, Frederick T. Chin. _Stanford University, Stanford, CA_.

Astrocytic gliomas are the most common glioma subtype and constitute the majority of all primary brain tumor types. The malignancy of an astrocytic glioma is classified into one of four grades (I-IV) based on its infiltrative and proliferative characteristics, wherein the extent of this diffuse tissue and depth of embedment into normal brain tissue correlates to the degree of difficulty in accurately identifying and directly visualizing the requisite tumor tissue for achieving its complete resection. To date, no single- or multimodal imaging agent can noninvasively identify intracranial gliomas or accurately distinguish its grade as well as facilitate the direct visualization of its diffuse and deeply-embedded tissue in real-time.

The standard imaging modalities for lesion detection (MRI and CT) provide anatomical information which have limited application in the pre- and post-surgical setting for identifying and grading tumor types as well as in monitoring for early-stage and recurrent growth, thereby creating a critical gap in evaluating invasive brain tumors that these techniques cannot successfully fill. Positron emission tomography (PET) is the ideal imaging modality that could fill this gap due to its ability to report on biological processes with superior sensitivity. However, conventional PET probes for gliomas operate on ubiquitously-expressed targets which provide insufficient or unreliable contrast levels between tumor and normal brain tissue or amongst low-grade gliomas.

The standard of care for treating brain tumors entails unguided surgical resection followed by a tailored radio- and/or chemotherapy regimen that depends on tumor type and grade. As such, the extent of surgical resection largely governs patient survival time. Fluorescence-guided surgery (FGS) improves maximal resection of tumor tissue while preserving eloquent brain tissue. Primarily 5-aminolevulinic acid (5-ALA) is used in the FGS of brain tumors. However, its fluorescent product emits at short wavelengths and operates via nonselective signal accumulation, which affords limited tissue penetration and false-positive demarcations. The advent of near-infrared fluorescence (NIRF)-guided surgery leverages conventional (nontargeted and/or unactivatable) NIR fluorophores for tumor tissue visualization at 10-fold greater penetration depths, but poorly delineate tumor margins due to generating minimal signal accumulation and high background.

To these ends, we have worked to develop a PET/NIRF smart probe for noninvasively identifying and grading intracranial astrocytic gliomas as well as affording direct visualization of its diffuse and deeply-embedded tumor tissue upon its application to NIRF-guided surgery. The multimodal smart probe uses a molecular logic gate design strategy to target a select active enzyme that serves as a validated prognostic factor for astrocytic gliomas.

#4114

**Development and application of** 18 **F-labeled Tenascin-C aptamer for cancer imaging.**

Jun Young Park,1 Ye Lim Cho,1 Ju Ri Chae,1 Ga Eul Chu,2 Won Gil Cho,2 Sang Wun Kim,1 Won Jun Kang1. 1 _Yonsei University College of Medicine, Seoul, Republic of Korea;_ 2 _Yonsei University Wonju College of Medicine, Wonju, Republic of Korea_.

Aptamers are synthetic single-stranded DNA or RNA that can be held together with complementary sequences by base-pairing. Tenascin-C is an extracellular matrix protein that is overexpressed in cancer tissues. Herein, we investigate the biological characteristics of Tenascin-C aptamer as potential tumor imaging agents. Cy5 or F-18 labeled Tenascin-C aptamers were prepared through hybridization-based labeling method using complementary oligonucleotide platform. Immunoblot analysis was performed on U-251 MG, U-87 MG, C6 and NCI-H460 cell lines to confirm the expression of tenascin-C. In vitro binding specificity of Tenascin-C aptamers was evaluated using confocal microscopy. In vivo biodistribution properties and PET Imaging of the 18F-labeled Tenascin-C were determined in C6 tumor-bearing nude mice. High levels of tenascin-C protein were detected in C6, U-251 MG. and U-87 MG cell lines. However, tenascin-C protein levels were undetectable in NCI-H460 cells. Confocal fluorescence microscopy images showed that Cy5-labeled Tenascin-C aptamer bound specifically to tenascin-C protein expressing cell lines including C6, U-251 MG and U-87 MG, however, not bound to NCI-H460 cells. Tumor uptake of 18F-labeled Tenascin-C aptamer was 1.64 ± 0.36 at 30 min after injection in C6 tumor xenografts. C6 tumors were clearly visualized by microPET imaging in a tumor-bearing mouse at 60 after injection. Tenascin-C aptamer was successfully labeled with F-18 labeled complementary oligonucleotide by base-pair hybridization. In vitro data showed that the cODN platform-hybridized Tenascin-C aptamer was selectively bound to target tumor cells. In addition, C6 tumor xenografts in mice were clearly visualized on microPET imaging. Our study demonstrated that Tenascin-C may be a useful target molecule for the imaging of malignant gliomas.

#4115

Comparison of IL-4 receptor targeting elastin-like polypeptide based on size and structure.

Boyeon Seo, Vijaya Sarangthem, Aena Yi, Rang-Woon Park. _Kyungpook National Univ., Daegu, Republic of Korea_.

For novel drug delivery systems, in order to improve clinical outcomes clear optimization of size, shape, multifuntionality, and site-specificity are highly considered. As molecular size significantly influenced the biodistribution, precise control of molecular sizes must be considered for optimum targeting effect in cancer therapy. Elastin like polypeptide (ELP), has been utilized as an attractive vehicle for cancer therapy as it provide unparalleled control over sequence and structure in genetic level thereby allowing generation of various polymers with defined architectures and functional properties. In this study, we designed various multivalent elastin-like polypeptide based tumor targeting polymer in which multiple copies of IL-4 receptor (IL-4R) targeting ligands, AP1 peptide were subjected into ELP polymer backbone periodically to enhance affinity and avidity towards IL-4R- highly expressed tumor cells. Several ELP with different molecular sizes and structure from unimer to micelle forming polymer have been designed to investigate the pattern of accumulation in tumor, as well as their bio-distribution in vivo. Different percentage of cell binding and uptake depend on size, number of targeting peptides, or based on unimer compared with micelle were determined. The high molecular weight AP1-ELP showed superior binding activity with faster entry and efficiently processed in the IL-4R dependent endocytic pathway compared to low molecular weight polypeptides. In addition the high molecular weight, micelle forming AP1-ELP displayed better tumor penetration and extensive retention in tumor tissue with decrease non-specific accumulation in vital organs compared to low molecular weight AP1-ELP in xenograft model. It was suggested that superior binding activities shown by A86 and A100 may depends on multiple presentation of ligands upon transition to micelle like structure rather than increased number of targeting ligands and molecular weight. Thus this study have significance in demonstrating different pattern underlying unimer and micelle forming ELP mediated tumor targeting as well as biodistribution in vivo.

#4116

**Rapid imaging of tumor cell death in vivo** **using C2Am.**

Andre A. Neves, Kevin M. Brindle. _University of Cambridge, Cambridge, United Kingdom_.

Cell death is an important target for imaging the early response of tumors to treatment. We describe here the validation of C2Am, a phosphatidylserine-binding agent based on the C2A domain of synaptotagmin-I, for detecting tumor cell death in vivo.

Methods: The capability of (99m)Technetium-, (111)Indium-labelled or near- infrared fluorophore-labelled derivatives of C2Am for imaging tumor cell death was evaluated in implanted and genetically engineered [Fig.1] mouse models of lymphoma and in a human colorectal xenograft, using single photon CT (SPECT) and planar near-infrared fluorescence imaging, respectively [1,2].

Results: (99m)Tc-C2Am and (111)In-C2Am showed favorable biodistribution profiles, with predominantly renal clearance and low nonspecific retention in the spleen and liver at 24 h after probe administration. (99m)Tc-C2Am and (111In)-C2Am generated tumor-to-muscle ratios in drug-treated tumors of 4.3× and 2.2×, respectively, at 2 h and 7.3× and 4.1×, respectively, at 24 h after administration. A fluorophore-labelled C2Am derivative also showed predominantly renal clearance and high specificity and sensitivity for detecting low levels of tumor cell death (<5%). There was a significant correlation (R=0.9) between fluorescently labelled C2Am binding and histologic markers of cell death (cleaved caspase-3); no such correlation was observed with a site-directed mutant of C2Am that does not bind phosphatidylserine (iC2Am).

Conclusions: Given the favorable biodistribution profile of radiolabelled C2Am derivatives, and their ability to produce rapid and cell death-specific image contrast, these agents have potential for clinical translation, particularly as PET tracers. We have initiated GMP manufacture and toxicology studies required for a Phase 1 trial.

References:

[1] Neves AA, Xie B, Fawcett S, Alam IS, Witney TH, de Backer MM, Summers J, Hughes W, McGuire S, Soloviev D, Miller J, Howat WJ, Hu DE, Rodrigues TB, Lewis DY, Brindle KM. Rapid Imaging of Tumor Cell Death In Vivo Using the C2A Domain of Synaptotagmin-I. (2017) J Nucl Med. 58:881-887.

[2] Xie B, Tomaszewski MR, Neves AA, Ros S, Hu DE, McGuire S, Mullins SR, Tice D, Sainson RCA, Bohndiek SE, Wilkinson RW, Brindle KM. Optoacoustic Detection of Early Therapy-Induced Tumor Cell Death Using a Targeted Imaging Agent. (2017) Clin Cancer Res. 23:6893-6903.

#4117

Imaging hypoxia: Development of a PET-optical smart probe.

Jessica L. Klockow, Kenneth S. Hettie, Frederick T. Chin. _Stanford University, Stanford, CA_.

Hypoxia is a characteristic trait of many aggressive tumors. Identification of hypoxic tissues is important for establishing patient treatment plans and prognoses. We show progress towards the development of a hypoxia-activated molecular probe that combines near-infrared (NIR) fluorescence and positron emission tomography (PET) imaging. The fluorescence emission enables facile scrutiny of probe function in vitro and positron emission facilitates translation for clinical use. The probe is a small molecule that possesses a nitro-aromatic group which is sensitive to hypoxic environments. The caged smart probe is non-fluorescent due to photo-induced electron transfer from the nitro-aromatic group. Nitroreductase, an enzyme which is upregulated in hypoxic environments, activates the fluorescence by reducing the nitro group, thereby disabling the quenching effect. Wet chemistry techniques were used to develop the probe which was analyzed by UV/Vis and fluorescence spectroscopies. In vitro and in vivo imaging experiments were also performed. The probe demonstrated membrane permeability and lysosomal accumulation in glioblastoma cells. Additional favorable characteristics include NIR emission and low toxicity to cells. The fluorescence was detected in subcutaneous murine cancer models providing evidence of potential clinical translation toward applications like image-guided surgery. In short, the probe was synthesized in good yield from commercially-available materials and showed promising optical characteristics in cells and animal models. Future work involves F-18 radiolabeling of the probe followed by microPET imaging.

#4118

Rapid, non-subjective characterization of disease in preclinical cancer research using desorption electrospray ionization mass spectrometry.

Michael Woolman,1 Alessandra Tata,1 Isabelle Ferry,2 Claudia Kuzan-Fischer,2 Megan Wu,2 Sunit Das,3 Michael D. Taylor,2 James T. Rutka,2 Howard J. Ginsberg,4 Arash Zarrine-Afsar1. 1 _University Health Network, Toronto, Ontario, Canada;_ 2 _Hospital for Sick Children, Toronto, Ontario, Canada;_ 3 _University of Toronto, Toronto, Ontario, Canada;_ 4 _St. Michael's Hospital, Toronto, Ontario, Canada_.

Cancer tissue smears are routinely used in rapid intraoperative pathology workflows using quick staining methods to characterize cancer in surgical margin assessments or tumor pathology. Mass spectrometry (MS) is a sensitive analytic platform that can detect the presence of cancer from the pattern of cancer-specific molecules present in the mass spectrum of the tissue under examination. In particular, mass spectrometry analysis with desorption electrospray ionization (DESI-MS) is shown to have utility in research models for cancer characterization or even for grading different subclasses of disease based on tumor-specific small molecule lipid or metabolites. DESI does not require extensive tissue preparation, and the data collection and analysis can be done within a few seconds. In this work, we evaluate the combination of rapid DESI-MS detection with rapid tissue smear preparation for research use in preclinical xenograft models of breast cancer and pediatric medulloblastoma requiring only seconds of sampling, and an overall preparation and analysis time of less than one minute. Principal component analysis (PCA) was performed to evaluate the concordance between DESI-MS profiles of breast cancer from tissue slices and smears prepared on various surfaces. PCA suggested no statistical discrimination between DESI-MS profiles of tissue sections and tissue smears prepared on glass, polytetrafluoroethylene (PTFE), and porous PTFE. However, the abundances of cancer biomarker ions varied between sections and smears, with DESI-MS analysis of tissue sections yielding higher ion abundances of cancer biomarkers compared with smears. The coefficient of variance (CV) analysis suggests DESI-MS profiles from tissue smears are as reproducible as the ones from tissue sections. The limit of detection with smear samples from single pixel analysis is comparable to tissue sections that average the signal from a tissue area of 0.01 mm2. The smears prepared on the PTFE surface possessed a higher degree of homogeneity compared with the smears prepared on the glass surface. This allowed single MS scans (~1 s) from random positions across the surface of the smear to be used in rapid cancer typing with good reproducibility, providing useful pathologic information at speeds suitable for research use. Likewise, DESI-MS enabled the rapid classification of subgroups of medulloblastoma in these preclinical models.

#4119

Spatial mapping and molecular phenotyping of heterogeneous breast cancer lesions with multi-spectral short wave infrared emitting rare-earth nanoprobes.

Harini Kantamneni,1 Michael Donzanti,1 Xinyu Zhao,2 Shuqing He,2 Mei Chee Tan,2 Mark Pierce,1 Charles M. Roth,1 Shridar Ganesan,3 Vidya Ganapathy,1 Prabhas V. Moghe1. 1 _Rutgers Univ., New Brunswick, NJ;_ 2 _Singapore University of Technology and Design, Singapore;_ 3 _Cancer Institute of New Jersey, New Brunswick, NJ_.

Breast cancer is made up of distinct heterogeneous subpopulations that influence treatment responses. Currently, molecular phenotyping of a tumor involves post-biopsy histology, which makes quantification and assessment of spatial heterogeneity difficult. While there has been moderate success in radiographic imaging of heterogeneity, by PET and MRI, there is an urgent need for non-invasive imaging modalities to quantitatively index tumor heterogeneity in real-time. Our study utilizes rare earth(Re) nanoprobes which absorb near infrared radiation (980nm) and emit in the short wave infrared (SWIR) region (1000- 3000nm), allowing for improved tissue penetration and detection depth. We designed Re nanoprobes encapsulated in albumin to form Rare-Earth Albumin NanoComposites (ReANCs), which can be administered in vivo to target and detect deep tissue microlesions (~18mm3) at a depth of ~1cm. Additionally, ReANCs have been shown to detect multi-organ micro-lesions early and have excellent safety and tolerability profile. Albumin encapsulation not only creates a biocompatible nanoparticle, but also allows for increased biodistribution, pharmacokinetics, and the possibility of functionalization. Most notably, the availability of different Re dopants, Erbium and Thulium, with distinct emission spectral bands allows for accurate indexing of cellular subsets. In this study, we first demonstrate the ability of multi-spectral, ReANCs to distinguish and map a heterogeneous tumor lesion. 3D spheroids made of varying ratios of prelabeled populations of MDA-MB-231 cells (erbium-doped) and HCC1954 (thulium doped) were engineered and imaged to obtain a training set for spatial mapping of the different cell populations. Ratiometric analysis of the spheroids was performed to develop an algorithm for indexing. Subsequently, cancer-targeted ReANCs were engineered and target validation was performed in a 3D spheroid model followed by spatial mapping of targeted populations leading to ratiometric indexing of the different populations. Briefly, erbium doped ReANCs (MDA-MB-231 cells) and thulium doped ReANCs (HCC1954 cells), were deployed to detect distinct subpopulations in a 3D heterogeneous spheroid model of Her2+/- breast cancer. Images were obtained using in vitro microscopy platforms, using 980 nm excitation sources. Separate band-pass filters isolated emissions from the distinct REANC dopants, allowing for quantification of each pre-labeled cell in the training set. This was followed by development of an algorithm to find the percentage of subsets with unknown proportions allowing indexing of unknown subsets in a single tumor spheroid. This novel in vivo imaging modality forms the early basis for real-time on molecular aspects of the tumor and real-time tumor response tracking.

#4120

A novel HDAC-targeted probe for triple-negative breast tumor imaging.

Chu Tang,1 Yang Du,2 Jie Tian2. 1 _Xidian University, Xi'an, China;_ 2 _Chinese Academy of Sciences, Beijing, China_.

Purpose: Due to lack of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2), there was no specific imaging probes can be applied to detect triple-negative breast cancers (TNBCs). Currently, several lines of evidences support that overexpression of histone deacetylases (HDACs) has been associated with TNBC. Here, we sought to develop HDAC-targeted probe for TNBC imaging.

Experimental Design: The pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) was chosen as the targeting ligand, and was labeled with a near-infrared dye IRDye800CW for the development of HDAC-targeted probe IRDye800CW-SAHA. Subsequently, HDAC binding affinity and specificity of probe IRDye800CW-SelSA to TNBC MDA-MB-231 cells was evaluated by HDAC inhibition assay, flow cytometry and confocal laser microscope experiments. Moreover, the in vivo fluorescence molecular imaging (FMI) was performed to monitor the biodistribution and tumor targeting effects of the probe in MDA-MB-231 tumor xenografts.

Results: The fluorescence probe IRDye800CW-SAHA was successfully developed, and conjugating IRDye800CW to SAHA did not alter the HDAC binding affinity. The cellular uptake and the fluorescence intensity of probe on MDA-MB-231 cells were significantly higher compared to normal MCF-10A cells (P < 0.05). Meanwhile, the cellular uptake of probe can be effectively blocked by co-incubation with SAHA. The in vivo imaging results showed that IRDye800CW-SelSA was able to target tumor rapidly, and the tumor to normal tissue (T/N) fluorescence intensity ratio reached the peak around 12 h (T/N = 3.4).

Conclusions: Our study demonstrated that IRDye800CW-SAHA is an efficient probe for the targeted imaging of TNBC and may possess clinical translation in the future.

CT and YD contributed equally to this work

#4121

Differences in optoacoustic signal reflect different characteristics of the vasculature and response to antiangiogenic therapy in breast cancer models.

Isabel Quiros-Gonzalez, Michal Tomaszewski, Laura Ansel-Bollepalli, Sarah J. Aikten, Michael Gill, Sarah E. Bohndiek. _University Of Cambridge, Cambridge, United Kingdom_.

Optoacoustic imaging has the potential to monitor tumour neo-vascularization, essential for disease progression and also a target for therapy. While promising in some settings, anti-angiogenic therapy has yet to fulfil its promise and mechanisms of resistance are poorly understood. In breast cancer, for example, resistance has been linked to hormonal status, oxygen consumption and alternative mechanisms of neo-vasculogenesis, including vascular mimicry (VM). Here, we use two mouse xenograft models: estrogen-dependent MCF-7 and estrogen independent MDA-MB-231. We analyse their characteristics in vivo using Multispectral optoacoustic tomography (MSOT) to detect oxy- and deoxy- haemoglobin (Hb) during tumour growth. We also analysed their histological and biochemical characteristics for blood vessel density (CD31) and maturity (aSMA, PAS staining), tumour hypoxia (CAIX), angiogenesis, inflammation and VM. Anti-angiogenic therapy response was also tested using Bevacizumab (BV, 10 mg/Kg) in two cohorts. While MSOT reveals similar blood content in both tumours, estrogen-dependent MCF-7 tumours show higher blood oxygenation than estrogen-independent MDA-MB-231 (55.8±7.5 % vs. 45.6±5.1%, p=0.0008) with values similar to healthy tissue (55.8±7.5 % vs. 57.8±5.3%, p=0.917). Ex vivo histological analysis shows that while MDA-MB-231 tumours are more hypoxic compared to MCF-7, they have a higher blood vessel density, however, the vasculature of MCF-7 tumours is more mature (vessel wall thickness and aSMA coverage). Based on molecular biomarkers, MCF-7 presents a more pro-inflammatory endothelial-like response, marked by an increase of vascular endothelial growth factor (p<0.0001) of host origin (mVEGF), macrophage Nitric Oxide Sinthase (iNOS) and circulating NO, a mediator in inflammation and endothelial homeostasis (p=0.0031). VE-Cadherin of human origin is detectable in MDA-MB-231 tumours, which present CD31-absent blood vessels; together, these findings indicate presence of VM in MDA-MB-231 tumours. Interestingly, these 2 biological profiles respond differently to the anti-angiogenic drug BV: we observed a partial response in MDA-MB-231 tumours (CR=2/22, PR=3/22), but no effect in tumour size or survival was observed in MCF-7 tumours. While no change was observed in mVEGF upon BV treatment in any cohort, changes in optoacoustic signal of total Hb were observed in the MDA-MB-231 tumours after treatment. In conclusion, optoacoustic imaging delineates the vascular function of the blood vessel networks generated by angiogenesis and vascular mimicry respectively in our estrogen-dependent and estrogen-independent breast cancer xenografts. This study highlights the potential application of this technique in monitoring tumour vasculature development and in the future could be used to assess response to therapy.

#4122

Acidic tumor microenvironment targeted wormhole-shaped mesoporous silica nanoparticles to detect ovarian cancer by multispectral optoacoustic tomography.

Abhilash Samykutty,1 Molly W. McNally,1 William E. Grizzle,2 Akiko Chiba,1 Alexandra Thomas,1 Lacey R. McNally1. 1 _Wake Forest Univ. Comp. Cancer Ctr., Winston-Salem, NC;_ 2 _University of Alabama Birmingham, Birmingham, AL_.

Purpose: The implementation of the innovative technologies remains the top priority for the development of potential modalities for the diagnosis and treatment of various cancers. Despite all the recent advances, ovarian cancer is considered as a lethal gynecologic malignancy in which vast majority of the cases are diagnosed at the late metastatic stage at which the prognosis is poor. Because of the few apparent early symptoms, significant effort was made for developing efficient methods to identify early progression of the disease. We demonstrated that the bioconjugation of the V7 peptide on 27nm wormhole mesoporous silica nanoparticles (V7-CWMSN) particles specifically release the IR780 imaging probe at the orthotopically implanted early-stage ovarian tumor to detect by multispectral optoacoustic imaging technology (MSOT).

Methods: Wormhole mesoporous silica nanoparticles (WMSN) were synthesized by sol-gel chemistry. The WMSN were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The surface of the WMSN particle was functionalized with pH sensitizer chitosan to obtain CWMSN. The CWMSN particle was loaded with propidium Iodide (PI) or IR780 infrared imaging dye. Further, the conjugation of the CWMSN with V7 pHLIP peptide (V7-CWMSN) developed pH sensitive cargo release from the nanoparticles. Female athymic mice were orthotopically implanted with ES-2 ovarian cancer cells. Following 10 days of orthotopic implantation of the tumor cells, mice were intravenously injected with V7-CWMSN particles containing IR780 dye and were imaged with MSOT.

Results: In the current study, we have synthesized the wormhole-shaped mesoporous silica particles (WMSN) with the 27 nm diameter carrying IR780 imaging probes for the detection of early-stage ovarian cancer. The V7 peptide undergoes a conformational change upon contact with the acidic tumor microenvironment. Also, the chitosan crosslinking on the surface of the particles (V7-CWMSN) acts as a gatekeeper that is degradable upon contact with acidic pH tumor to prevent off-target release. As a model for ovarian cancer, we have used athymic mice orthotopically implanted with ovarian cancer cells. This model closely resembles the human ovarian cancer pathophysiology. Our results have demonstrated that the intravenous delivery of V7-CWMSN particles could detect the orthotopically implanted early-stage ovarian tumors (p<0.0001,n=5).

Conclusion: The development of the distinct nanoformulations with potent imaging technology enables the visualization of early-stage ovarian tumors. Translating these modalities may allow clinicians to identify early-stage malignancies that are currently undetectable through conventional imaging techniques.

#4123

Pancreatic cancer imaging and interventional PTT with a human bispecific scfv antibody via click chemistry.

qian wang,1 dan li,1 kun wang,2 chunyan liu,3 mingyuan gao,3 xinming zhao,1 jie tian2. 1 _National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical, beijing, China;_ 2 _Institute of Automation Chinese Academy of Sciences, beijing, China;_ 3 _Chinese Academy of Sciences, beijing, China_.

Early diagnosis and therapy intervention remains a meaningful task for prolonging pancreatic cancer lifetime and improving prognosis. Successful development of so many highly specific targets emerge, for accurate diagnosis and precise treatment. Herein, we generated bispecific antibody fragment by connecting anti-EGFR scfv and anti-VEGF scfv, via click ligation of TCO and tetrazine. Firstly, Cellvizo imaging was acquired by AF488/647 labeled anti-EGFR scfv and anti-VEGF scfv, respectively, displayed overexpression and metabolic process in real time. IVIS and 3D MSOT imaging of mice bearing anthropogenic cell line of bxpc3(EGFR+/VEGF+) subcutaneous and orthotopic and tumors with Irdye800 labeled Bs-(scfv)2 revealed a more significantly enhancement and long circulation of tumor uptake and tumor-to-background ratio compared with each special scfv group(T/B ratio of 12.1±3.4 at 4h post-injection, n=5 in IVIS image). Thus, we validated that dual targeting of VEGF and EGFR provides a high affinity and specificity by Irdye800-labeled-Bs-(scfv)2. Finally, we successfully replaced irdye800 with PLA-PEG-NHS@ICG (combined or uncombined GEM) and conjugated Bs-(scfv)2 for interventional PTT, compared with GEM treatment, a 18% higher median survival rate of PTT combined GEM has been achieved. Our finding offer a preclinical proof concept for Bs-(scfv)2 PTT in clinical trial in future, due to effective bio-distribution and high affinity.

### Pediatrics 3: Signaling, Transcription, and Metastasis

#4124

YAP1 fusion proteins mediate oncogenic activity in ependymoma via interaction with TEAD transcription factors.

Kristian W. Pajtler,1 Konstantin Okonechnikov,1 Mikaella Vouri,1 Sebastian Brabetz,1 David T. Jones,1 Andrey Korshunov,2 David Capper,3 Lukas Chavez,1 Stefan M. Pfister,1 Marcel Kool,1 Daisuke Kawauchi1. 1 _German Cancer Research Center, Heidelberg, Germany;_ 2 _Heidelberg University Hospital, Heidelberg, Germany;_ 3 _Charité – Universitätsmedizin Berlin, Berlin, Germany_.

Ependymomas are neuroepithelial tumors of the central nervous system (CNS), presenting in both adults and children but accounting for almost 10% of all pediatric CNS tumors and up to 30% of those in children under 3 years. In a previous study we identified two molecular groups of supratentorial (ST) ependymoma in pediatric patients driven by distinct gene fusions involving either the NF-κB effector RELA or the HIPPO signaling regulator YAP1, designated ST-EPN-RELA and ST-EPN-YAP1, respectively. The lack of adequate models for ST-EPN-YAP1, which predominantly occurs in very young children, has so far hindered the development of effective targeted therapies for these tumors. In an attempt to model this group, the most frequent fusion type, YAP1-MAMLD1, was cloned into a Luciferase-carrying transposable vector. Oncogenicity was subsequently tested using an electroporation-based in vivo gene transfer following injection of the vector into the lateral ventricle of E13.5 wildtype mouse embryos. After birth, YAP1-MAMLD1-expressing tumors, monitored using luciferase-based in vivo bioluminescence imaging, developed rapidly with 100% penetrance, indicating that the fusion alone is sufficient to initiate tumors. To further investigate the role of this fusion in human EPNs, we performed YAP1 ChIP-seq analyses on human ST-EPN-YAP1 and ST-EPN-RELA primary tumors. Despite similar gene expression levels of YAP1 in both molecular groups, our comparative analyses found that putative binding sites of TEADs, transcriptional factors interacting with YAP1, were significantly enriched in identified YAP1 peaks in ST-EPN-YAP1. In vivo validation further confirmed that interaction between YAP1-MAMLD1 and TEADs is crucial for oncogenicity of the fusion, since prevention of YAP1-TEAD binding did not result in tumor formation. Thus, targeting the YAP1-TEAD interaction might represent a promising therapeutic approach for this devastating infant disease.

#4125

YB1-phosphorylation mediates YB1-PARP interaction to regulate DNA-repair post-radiation in Shh medulloblastoma.

Abhinav Dey, Anshu Malhotra, Anna Kenney. _Emory University, Atlanta, GA_.

The Sonic hedgehog (Shh)-subgroup of the pediatric brain tumor medulloblastoma (MB) has the highest frequency of post-radiotherapy local recurrence, which is fatal. Recently, there have been major advances in characterizing the genomic divergence during MB tumor recurrence (1) and the role of spatial heterogeneity in MB (2). However, there is no evidence that demonstrates actionable targets in the Shh-pathway for prevention of tumor recurrence post-radiation. Previously we have shown that Shh-target Y-box binding protein 1 (YB1) drives MB cell proliferation (3) and controls the expression of IGF2, which contributes to radiation resistance in MB (4). Stem-like tumor cells occupying the peri-vascular niche (PVN) are proposed to drive MB recurrence through their radiation-resistant properties (4, 5). In the SmoA1 mouse model for Shh MB, we observe that these cells feature elevated levels of the oncogene YB1. Ectopic expression of YB1 in this model significantly reduced survival, while phosphomutant YB1 inhibited tumor formation. Using mouse organotypic MB slice cultures we found that YB1 is essential for expansion of the cancer stem-like cell population from the PVN post-radiation. We also demonstrate localization of endogenous phosphorylated YB1 to sites of DNA damage post-radiation, and we show that ectopic expression of YB1 in primary MB cell (MBC) cultures drives rapid DNA repair in a PARP-dependent manner. Mechanistically, we show that inhibiting YB-1 phosphorylation using the flavonoid Fisetin disrupts the physical interaction of YB1 and PARP, thus, derailing the DNA repair process post-radiation in MBCs. Subsequently, using a novel ex vivo assay of tumor Neurosphere-implantation on a cerebellar Brain Slice (NoBS) culture, we demonstrate the effectiveness of Fisetin in preventing the survival of cancer stem-like cells in the MB tumor microenvironment. Our findings demonstrate a novel role for YB1 in promoting Shh MB tumor growth, and they reveal a critical requirement for YB1-phosphorylation in driving radiation resistance through PARP-mediated DNA repair, enabling escape from DNA damage-induced apoptosis. Moreover, our results imply that YB1 phosphorylation inhibitors can be used to enhance radiation responsiveness, thereby reducing incidence of medulloblastoma recurrence. Reference: 1. Morrissy AS, Garzia L, Shih DJ, Zuyderduyn S, Huang X, et al. 2016. Nature 529: 351-72. 2. Morrissy AS, Cavalli FMG, Remke M, Ramaswamy V, Shih DJH, et al. 2017. Nat Genet 49: 780-83. 3. Dey A, Robitaille M, Remke M, Maier C, Malhotra A, et al. 2016. Oncogene 35: 4256-684. 4. Fernandez LA, Squatrito M, Northcott P, Awan A, Holland EC, et al. 2012. Oncogene 31: 1923-375. 5. Hambardzumyan D, Becher OJ, Rosenblum MK, Pandolfi PP, Manova-Todorova K, Holland EC. 2008. Genes Dev 22: 436-48

#4126

DNA methyltransferase inhibitors target a YES1-YAP1 signaling axis in childhood rhabdomyosarcoma.

Katherine Slemmons,1 Choh Yeung,2 Josh Baumgart,2 Amy McCalla-Martin,2 Lee Helman1. 1 _Children's Hospital Los Angeles, Los Angeles, CA;_ 2 _NIH, Rockville, MD_.

We have previously shown that the SRC family kinase (SFK) member, YES1, is highly expressed and rapidly activated upon IGF-1R blockade in rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children and adolescents. YAP1 (YES-associated protein) is also highly expressed in human RMS tumors where it functions as an oncoprotein when the upstream Hippo tumor suppressor pathway is silenced, driving cell growth and survival. Thus far, efforts to pharmacologically inhibit YAP1 have been unsuccessful. One activator of the Hippo pathway, RASSF1a, is epigenetically silenced in many cancer types including sarcomas. Here we show a loss of RASSF1a expression in RMS cell lines via promoter methylation. Treatment with DNA methyltransferase inhibitors (DNMTis) can re-activate RASSF1a expression, resulting in YAP phosphorylation and inactivation. DNMTi treatment also significantly inhibits RMS cell growth, which can be rescued by overexpression of a constitutively active YAPS127A. In the alveolar variant (aRMS) in particular, DNMTis increase apoptosis and decrease both YES1 expression and activation. This suggests DNMTi treatment can inhibit both YAP1 and YES1 signaling. To further investigate the connection between YES1 and YAP1 we performed proximity ligation assays and discovered that YES1 and YAP1 interact in the nucleus of RMS cell lines. Furthermore, dasatinib treatment, a known SFK inhibitor, prevents YAP1 nuclear accumulation suggesting a role for YES1 in regulating YAP1 cellular localization. YAP1 is also tyrosine phosphorylated, which may be one way YES1 regulates YAP1. Lastly, cells expressing a constitutively active YAPS127A have increased activation of SFK suggesting a positive feedback loop whereby YAP1 activity can further activate YES1 through an unknown mechanism. These data uncover a signaling axis between the YES1 and YAP1 oncoproteins in RMS and suggest an alternative way to silence YES1 and YAP1 through use of DNMTis. We will further analyze this signaling through shRNA knockdown of YES1 and evaluate combination treatment of a DNMTi and dasatinib in vitro and in vivo.

#4127

A tumor evolution screen in zebrafish identifies PlexinA1 as a driver of metastasis and growth in human rhabdomyosarcoma.

Alessandra M. Welker,1 Madeline N. Hayes,1 Qin Tang,2 David M. Langenau1. 1 _Massachusetts General Hospital, Harvard Stem Cell Institute, Charlestown, MA;_ 2 _Dana Farber Cancer Center, Boston, MA_.

Relapse and metastasis are major clinical problems facing patients with rhabdomyosarcoma (RMS), a devastating pediatric malignancy of the muscle. Alveolar Rhabdomyosarcoma (ARMS) is characterized by genetic translocations while Embryonal Rhabdomyosarcoma (ERMS) is characterized by Ras pathway activation. Treatment for either RMS subtype requires surgical resection, chemotherapy, and radiation with overall poor prognosis for patients with high-risk features including metastasis and relapse. Thus, there is great interest in elucidating key molecular pathways and genetic factors that are involved in continued RMS growth and tumor maintenance. In the Langenau lab, we utilize a powerful transgenic zebrafish model of kRASG12D- induced ERMS and have recently developed a p53-/- (null) zebrafish model. p53 is one of the most common tumor suppressor genes, and upwards of 1/3 of RMS patients have mutations or Loss-of-Heterozygosity (LOH) of this locus. This model accurately mimics the molecular underpinnings and histopathology of human ERMS and has been used to discover molecular pathways that drive relapse. Using our tp53-/- (null) zebrafish along with large-scale cell transplantation assays, we have assessed metastatic potential of our ERMS tumors following serial passaging and acquisition of new driver mutations. Specifically, we performed a tumor evolution screen using serial engraftment of ERMS into recipient fish followed by RNA sequencing to discover novel pathways that are associated with elevated tumor growth and metastasis. Plexin A1 was one of our top hits in this screen, being expressed at >8 fold in metastatic lesions of the zebrafish and expressed in >80% of human RMS. Plexins are a large family of receptors for transmembrane, secreted and GPI-anchored semaphorins. Knockdown of PLXNA1 decreased human ERMS cell proliferation, increased differentiation and impaired anchorage-independent growth and migration. Collectively, our data supports the hypothesis that PLXNA1 is an important moderator of tumor growth in a large fraction of human RMS and likely regulates local invasion and metastasis. This work will provide much needed data for discovery of novel therapeutically-actionable pathways for relapsed ERMS and likely inform future treatment for high-risk patients.

#4128

Loss of p53 and Rb enhances ligand-dependent hedgehog signaling in development and cancer.

Catherine R. Cochrane,1 Anette Szczepny,1 Samantha W. Jayasekara,1 Vijesh Vaghjiani,1 D. Watkins,2 Jason E. Cain1. 1 _Hudson Institute of Medical Research, Melbourne, Victoria, Australia;_ 2 _Garvan Institute of Medial Research, Sydney, New South Wales, Australia_.

The evolutionary conserved Hedgehog (Hh) signaling pathway plays fundamental morphogenic and mitogenic roles in tissue development and homeostasis, as well as in the initiation and progression of various cancers. Signaling is driven by the binding of Sonic hedgehog (Shh) ligand to the receptor Patched (Ptch1), to initiate primary cilia dependent Smoothened (Smo)-Gli activation. Although mutations in Hh pathway components are described in some cancers, the majority of Hh tumors exhibit ligand-dependent Hh signaling, where Hh ligand is produced by the stroma and/or tumor cells to maintain pathway activation and self-renewal. Here, we have utilized various Hh-dependent developmental models to better understand the tumorigenic mechanisms of Hh ligand-dependent signaling in cancer. During cerebellar development, Shh is secreted from cerebellar purkinje cells for rapid expansion of the cerebellar granule cell precursor (GCP) population in the external granule layer (EGL). Inactivation of Shh in purkinje cells (Pcp2Cre;Shhfl/fl) leads to reduced GCP proliferation, premature eradication of the EGL and reduced cerebellar size. In contrast, Shh overexpression by purkinje cells (Pcp2Cre;ShhTg) results in increased GCP expansion, a sustained EGL and increased cerebellar size. Indeed, signs of remnant EGL persist into adulthood, similar to the pre-neoplastic lesions observed in Ptch1+/- mice, believed to be the origin of SHH medulloblastoma. During skeletal development, Indian hedgehog secreted from pre-hypertrophic chondroblasts is required for osteoblast progenitor specification from multipotent mesenchymal stem cells. Sustained Hh signaling in osteoblast precursors (OsxCre;SmoM2 or OsxCre;ShhTg) results in reduced ossification of the calvaria, maxilla and mandible, reduced long bone width, and reduced expression of mature osteoblast markers. Together, these models implicate sustained Hh expression in the maintenance of an immature cell state at the expense of differentiation. Moreover, in a mouse model of osteosarcoma, inactivation of Smo (OsxCre;p53fl/fl;Rbfl/fl;Smofl/fl) abolished the malignant phenotype of OsxCre;p53fl/fl;Rbfl/fl mice, leading to benign osteoid osteoma consistent with osteoblast differentiation. Increased Hh signaling in cancers with prevalent p53 and Rb mutations led us to investigate a role for p53 and Rb in Hh ligand-dependent signaling. Genetic inactivation of p53 and/or Rb in the developing mouse neural tube resulted in expansion of the Hh-dependent Nkx2.2 ventral domain, consistent with a Hh gain of function phenotype. Similarly, siRNA knockdown of p53 and/or Rb in mouse mesenchymal stem cell line C3H10T1/2 markedly increased Hh-dependent osteoblast specification in response to Hh ligand. Taken together, we propose that p53 and Rb mutations enhance ligand-dependent Hh pathway activation that maintains cells in a primitive state and promotes tumorigenesis.

#4129

RUNX2 **as a regulator of high-risk T-ALL.**

Nitesh Devinarayan Sharma,1 Christian K. Nickl,1 Stuart S. Winter,2 Huining Kang,1 Ksenia Matlawska Wasowska1. 1 _The Univ. of New Mexico, Albuquerque, NM;_ 2 _Childrens Minnesota Research Institute, Minneapolis,, MN_.

For patients with T-cell lineage acute lymphoblastic leukemia (T-ALL) who fail induction or relapse, event free survival is <10%. Mixed lineage leukemia gene rearrangements (KMT2A-R) are recurrent translocations found in ~3-5% of T-ALL and are determinants of high-risk disease. However, the roles of downstream targets of KMT2A chimeras such as Runt-related transcription factor 2 (RUNX2) remain uclear. We assessed RUNX2 mRNA levels in 100 pediatric T-ALL samples derived from the COG AALL0434 study. RUNX2 expression was upregulated in patients harboring KMT2A-R (p <0.0005) and those who failed induction therapy (p <0.005). Therefore, we hypothesized that RUNX2 is a critical determinant of high-risk T-ALL. To investigate the relationship between RUNX2 and KMT2A chimeras we utilized the chromatin immunoprecipitation assay to assess primary T-ALL samples with KMT2A-R (n = 3). We found that RUNX2 targets KMT2A-R (p < 0.0005) and conversely, that KMT2A chimeras target the promoter region of RUNX2 (p < 0.0005). We next demonstrated that KMT2A-MLLT4 induces RUNX2 expression. To further test the effects of RUNX2 on KMT2A-R, we performed shRNA mediated silencing of RUNX2 expression in primary T-ALL cells (n =3). RUNX2 silencing decreased KMT2A levels, suggesting the functional cross talk between KMT2A-R and RUNX2. To test the regulatory role of RUNX2 on T-ALL signal transduction we knocked down RUNX2 in T-ALL primary samples and cell lines (Loucy and Jurkat). RUNX2 silencing led to a decrease in cellular proliferation (p < 0.0005) indicating that RUNX2 positively regulates T-ALL cell growth in vitro. We next utilized flow-based assays to examine the effects of RUNX2 on cell death and cell cycle. RUNX2 inactivation induced apoptotic cell death and delayed cell cycle progression as demonstrated by increased G1 and decreased S/G2/M phases of the cell cycle. Apoptotic cell death was associated with increased expression of cleaved caspase 3, 7 and 9. We next examined the effects of RUNX2 on T-ALL cell survival and found that RUNX2 depletion reduced activation of Akt, active β-catenin and survivin levels but had no effect on p53 and Bcl-2. Similar experiments showed that RUNX2 inactivation leads to decreased expression of c-Myc and Ras. In addition, treatment with the specific allosteric Akt inhibitor, MK2206 (10 μM, 24 hrs) reduced RUNX2, active β-catenin, and survivin protein levels suggesting reciprocal activation between RUNX2 and Akt. To study the role of RUNX2 in leukemia burden, Jurkat cells transduced with RUNX2 shRNA and control plasmid were transplanted intravenously into the NSG mice (5 mice/group). RUNX2 silencing significantly decreased leukemia burden in spleen (p <0.001) and bone marrow (p <0.01) of the tested animals compared to the negative control group. We propose upregulation of RUNX2 expression as a novel mechanism governing high-risk T-ALL. We postulate that the Akt/β-catenin/survivin axis serves as a functional driver of RUNX2-mediated T-ALL survival.

#4130

TRIM37 expression levels dictate susceptibility to centrosome removal, supporting Plk4 inhibition as a potential new strategy for targeting neuroblastoma.

Franz Meitinger,1 Robert L. Davis,2 Ruth Kabeche,1 John V. Anzola,2 Yao Liang Wong,1 Andrew K. Shiau,2 Arshad Desai,1 Karen Oegema1. 1 _Ludwig Institute for Cancer Research/UCSD, La Jolla, CA;_ 2 _Ludwig Institute for Cancer Research, La Jolla, CA_.

Centrosomes are the major microtubule-organizing centers in animal cells. Centrosomes catalyze microtubule assembly to accelerate formation of the bipolar mitotic spindle that segregates chromosomes during cell division. To analyze the effect of centrosome removal in normal and cancer cells, we developed centrinone, a potent, specific and cellularly-active inhibitor of the protein kinase polo-like kinase 4 (Plk4), which is essential for centrosome duplication (Wong et al., 2015, Science 348:1155-60). Centrosome-less cells take longer to execute mitosis, and trigger a mitotic stress signaling pathway that results in p53 stabilization and apoptosis and/or senescence of the resulting daughter cells. Failure to form a spindle can also lead to apoptosis during mitosis or exit from mitosis without chromosome segregation (mitotic slippage). In a genome-wide CRISPR/Cas9 loss-of-function screen for resistance to centrinone, we had previously shown that loss of the ubiquitin ligase TRIM37 makes spindle assembly more robust to centrosome removal (Meitinger et al., 2016, J Cell Biol. 214:155-66), raising the possibility that high levels of TRIM37 might increase sensitivity to centrosome removal. Here, we describe the use of single-cell live imaging assays to identify determinants of vulnerability of cancer cells to centrosome removal. Among a panel of 37 tested cell lines, neuroblastoma-derived cell lines were the most sensitive to centrosome removal, exhibiting high levels of apoptosis and rapid loss of viability following treatment with centrinone. We found that sensitivity to centrosome removal correlated with TRIM37 expression levels. Upon centrosome depletion, cells with high TRIM37 levels, particularly neuroblastoma cells, exhibited greatly increased mitotic duration and frequency of mitotic failure. p53 positive neuroblastoma cell lines exhibited apoptosis resulting from mitotic stress-mediated p53 activation and from mitotic failure. Surprisingly, p53 negative neuroblastoma cell lines, which are unable to sense mitotic stress, were also highly sensitive to centrosome removal, with cell death in this case resulting from high rates of mitotic failure. TRIM37 deletion restored the ability of cells to proliferate in centrinone. Thus, centrosome removal via Plk4 inhibition appears to be a promising strategy for therapeutic treatment of neuroblastomas and potentially other cancers with high levels of TRIM37 expression.

#4131

Plasticity of transcriptional and epigenetic cellular states in neuroblastoma is driven by core lineage transcription factors.

Johan V. Nes, Tim V. Groningen, Jan Koster, Linda Valentijn, Danny Zwijnenburg, Ellen M. Westerhout, Mohamed Hamdi, Rogier Versteeg. _Academic Medical Center, Amsterdam, Netherlands_.

Background: Core Regulatory Circuitries (CRCs) of lineage transcription factors associate with super-enhancers and are drivers of cellular identity. We recently showed that most neuroblastomas include two types of tumor cells with divergent gene expression profiles. Undifferentiated mesenchymal (MES) cells and committed adrenergic (ADRN) tumor cells resemble cells from developmental stages of the adrenergic lineage.

Results: We identified the unique super-enhancer (SE) landscapes of MES and ADRN-type cells. SEs associated with lineage transcription factors that identified the Core Regulatory Circuitries (CRC) for each cell type. MES cells were resistant to chemotherapy. Accordingly, MES cells were strongly enriched in post-treatment samples, suggesting that MES cells could play a role in resistance and relapse development. MES and ADRN cells of isogenic origin spontaneously interconverted, showing the transcriptional plasticity of CRCs. We studied the mechanism of MES and ADRN transdifferentiation. The MES TFs PRRX1 and NOTCH were expressed as single genes in ADRN cells. Each of the genes induced a step-wise reprogramming of the ADRN transcriptome towards a MES state. The transcriptional switch was accompanied by genome-wide remodeling of the epigenome to a MES enhancer state. MES TFs repressed super-enhancers of ADRN core TFs, leading to transcriptional downregulation of the ADRN CRC. Deregulated enhancers associated with Polycomb repression. The transdifferentiation was initially reversible, but became gradually stabilized. The transgenes activated an endogenous feed-forward loop including ligands, receptors and co-factors from the NOTCH signaling route. Ultimately, this endogenous NOTCH-cascade maintained a transgene-independent MES state. Super-enhancers in stable MES cell lines associated with NOTCH receptors and co-factors, consistent with NOTCH driving MES lineage identity. Both NOTCH-induced MES cells and ADRN cells were tumorigenic in vivo.

Conclusions: Our results demonstrate that single TFs from the MES CRC impose transdifferentiation via remodeling of the epigenetic and transcriptional landscape of ADRN cells, mimicking spontaneous interconversion. Plasticity of CRCs and lineage identity may have profound implications for treatment strategies in neuroblastoma.

#4132

ASO screen uncovers splicing as a therapeutic vulnerability in theinsulin-like growth factor (IGF) signaling pathway.

Safiya Khurshid,1 Matias Montes,1 Brianne Sanford,1 Chelsea Brown,1 Daniel Comiskey,2 Frank Rigo,3 Peter Houghton,4 Dawn Chandler1. 1 _The Research Institute Nationwide CHildren's Hospital, Columbus, OH;_ 2 _Ohio State University, Columbus, OH;_ 3 _Ionis Pharmaceuticals, San Diego, CA;_ 4 _The Greehay CCRI, San Antonio, TX_.

Alternative splicing is a process contributing to structural transcript variation and proteome diversity but is often disrupted in cancer. Like most other transcripts, the insulin receptor (IN-R) undergoes alternative splicing to produce two isoforms: the full-length IN-RB and exon 11 skipped IN-RA isoform. The expression of these isoforms is tightly regulated during development, however there is an aberrant increase in IN-RA expression in childhood cancers like Rhabdomyo- & Osteo-sarcoma. This increased IN-RA expression is deleterious to the cells because it encodes for a receptor which has high affinity for both insulin & IGF2 growth hormones and it exploits the IGF pathway to accelerate the onset of tumor-cell hallmarks like proliferation & angiogenesis. Rhabdosarcoma is the 3rd most frequent solid tumor and the most common soft-tissue sarcoma in children. Despite improvement in the treatment for this disease, there is substantial inadequacy of less toxic alternative therapies.

We found that there is a significantly increased expression of IN-RA levels in a cohort of 40 Rhabdosarcoma (RMS) patients (embryonal & alveolar) as compared to the control samples. We confirmed this increased expression in multiple RMS cell lines. Furthermore, we went on to show that cellular stress such as hypoxia increases alternative splicing to produce more IN-RA isoform. In order to get a mechanistic insight into this phenomenon, we utilized this hypoxia-inducible splicing system and performed an antisense-oligonucleotide (ASO) screen to characterize sequence elements and splicing factors involved in the regulation of IN-R splicing. We found that sequence elements flanking exon 11 are critical to the increased alternative splicing we see under hypoxic conditions. We then performed a refined ASO walk to target the regions important for exon inclusion or exclusion and identified a region known to be a binding site for the splicing-factor CUGBP1. RMS derived cell lines almost exclusively express IN-RA but when treated with our lead ASO compound that targets this region, they show a dramatic decrease in the IN-RA expression. More importantly, the ASO treated cells exhibit a significant reduction in cell proliferation and migration. Application of ASO compound attenuates the IGF signaling pathway measured by decreased pAKT staining. Additionally, when tested in RMS xenograft models, we see decreased extravasation of blood vessels in the grafts cells transfected with the ASO that blocks IN-RA expression compared to controls.

Our current data shows promising insight into how we can impede the IGF2 pathway by reducing IN-RA expression and consequently mitigate tumor hallmarks like cell-proliferation, migration and angiogenesis. The goal is to use these ASO compounds as therapeutic interventions in conjunction with already established anti-IGF1 receptor therapies to treat pediatric Rhabdomyosarcoma.

#4133

Increased I2PP2A compromises TP53 function by stabilizing MDM2 in Shh medulloblastoma.

Yun Wei, Victor Maximov, Anna Kenney. _Emory Univ., Decatur, GA_.

Medulloblastoma is the most common malignant pediatric brain tumor. Currently, the standard therapy comprising radiation, surgery and chemotherapy "cures" about 70% of medulloblastoma patients. However, these aggressive and non-differential treatment modalities cause lifelong side effects in patients. Medulloblastoma patients are classified into four different subgroups based on transcriptional and molecular profiles, namely WNT, SHH, Group 3, and Group 4. The SHH subgroup accounts for approximately 30% of all medulloblastoma cases and is further subcategorized into TP53 wild type and TP53 deficient subtypes. The importance of TP53 status in SHH medulloblastoma patients has been recently recognized by the association between TP53 mutation and poor prognosis for these patients. However, research findings illustrating TP53 signaling in preclinical models of SHH medulloblastoma are quite limited. Apart from the fact that TP53 mutation is associated with poor survival, 80% of medulloblastoma cases are TP53 wild type. TP53 protein is a tumor suppressor which acts as a checkpoint protein for all types of cells. Cancer cells either mutate TP53 gene or employ other mechanisms such as overexpressing MDM2 to compromise TP53 protein function. Therefore we hypothesize that TP53 function is compromised by highly active MDM2 in TP53 wild type Shh medulloblastoma.

Using the SmoA1 mouse model, which closely recapitulates human SHH medulloblastoma, we aim to investigate whether TP53 signaling has been disrupted in Shh medulloblastoma. Surprisingly, we observed relatively higher TP53 protein levels in tumor tissue lysates as compared to that in neighboring normal cerebellum using SmoA1 mice. However, MDM2, the major suppressor of TP53, showed higher phosphorylation at Ser166, which would stabilize MDM2, resulting in degradation of newly-produced TP53 protein. We also found significantly increased protein levels of I2PP2A, an endogenous inhibitor of phosphatase 2A, which can dephosphorylate MDM2 at Ser166. Using COG112, a small molecule inhibitor of I2PP2A, we showed the reactivation of PP2A, concurrent with a decrease of p-MDM2 and an increase of TP53 in a time-dependent manner. From these experiments, we conclude that 1) TP53 pathway is functional in our SmoA1 mouse model; 2) I2PP2A upregulation could be the reason for rapid degradation of TP53 protein in SmoA1 mice. These findings have translational implications as they suggest that targeting I2PP2A in TP53 wild type SHH medulloblastoma patients could be a method of driving tumor cell death, thus resulting in better clinical outcomes for these patients.

#4134

HELLS is upregulated in Sonic hedgehog-associated medulloblastoma and proliferating cerebellar progenitors in a YAP-dependent manner.

M. Hope Robinson, Anna M. Kenney. _Emory University, Atlanta, GA_.

Aberrant hedgehog signaling has been implicated in many human cancers, including medulloblastoma (MB), the most common malignant pediatric brain tumor. In fact, 30% of MB tumors are driven by aberrant activity of the Sonic hedgehog (SHH) signaling pathway. Our efforts focus on unraveling the downstream components of this pathway to better understand medulloblastoma and better inform therapeutic treatment development and decisions. While some of the signaling alterations in SHH MB are due to gene mutations or amplifications, others may have their roots in epigenetics. Therefore, we examined expression levels of several candidate epigenetic regulators in our systems and identified lymphoid-specific helicase (HELLS) as a gene whose expression is markedly induced by SHH. HELLS is a unique member of the SNF2 family of chromatin remodelers with multiple epigenetic functions in DNA methylation, histone acetylation and methylation, and chromatin remodeling. Additional roles in transcription activation and DNA repair have also been reported. Of interest, HELLS was shown to delay senescence by inhibiting the expression of CDKN2A, the genetic locus of P16INK4A, a key tumor suppressor whose inactivation has recently been reported as critical to MB progression. Confirming our initial finding, we observed considerably higher levels of HELLS in primary cultures of cerebellar granule neuron precursors (CGNPs) treated with SHH and in SmoA1 mouse MB tumor tissue when compared to adjacent normal cerebellum. Our preliminary analysis of a large cohort of MB patients also indicates overexpression of HELLS in human SHH MB. Bioinformatic promoter analysis and experiments with SHH pathway inhibitors suggest regulation of HELLS expression through members of the SHH proliferation program. Downstream effectors of SHH include GLI1/2 and YAP1. Inhibition of GLI1 and GLI2 with Gant61 in both CGNPs and cultured mouse MB cells resulted in a reduction of HELLS, but an increase of apoptosis markers may indicate this reduction is due to cell death rather than specific downregulation of HELLS. In contrast, verteporfin, an inhibitor of the interaction between the transcriptional co-activator YAP1 and the transcription factor TEAD1, resulted in a reduction of HELLS at the mRNA and protein level without a concomitant increase of apoptosis markers. These results suggest that transcriptional upregulation of HELLS in mouse cerebellar progenitors and MB tumors is mediated by the SHH effector YAP1. Experiments to confirm direct involvement of YAP1 in HELLS gene transcription are underway. Future studies to understand the role of HELLS in SHH MB have the potential to provide a better understanding of the disease and new avenues for development of targeted treatments to improve the lives of these patients.

#4135

c-MYC **drives a subset of high-risk pediatric neuroblastoma and is activated through mechanisms including enhancer hijacking and focal enhancer amplification.**

Mark W. Zimmerman,1 Yu Liu,2 Shuning He,1 Adam D. Durbin,1 Brian J. Abraham,3 John Easton,2 Ying Shao,2 Xu Beisi,2 Shizhen Zhu,4 Xiaoling Zhang,4 Zhaodong Li,1 Nina Weichert-Leahey,1 Richard A. Young,3 Jinghui Zhang,2 A. Thomas Look1. 1 _Dana-Farber Cancer Inst., Boston, MA;_ 2 _St. Jude Children's Research Hospital, Memphis, TN;_ 3 _Whitehead Institute, Cambridge, MA;_ 4 _Mayo Clinic, Rochester, MN_.

The amplified MYCN gene serves as an oncogenic driver in approximately 20% of high-risk pediatric neuroblastomas. Here we show that c-MYC itself is a potent transforming gene in a separate subset of high-risk neuroblastoma cases (~10%), based on (i) its upregulation by focal enhancer amplification or genomic rearrangements leading to enhancer hijacking, and (ii) its ability to transform neuroblastoma precursor cells in a transgenic animal model. The aberrant regulatory elements associated with oncogenic c-MYC activation include focally amplified distal enhancers or translocation of highly active enhancers from other genes to within topologically associating domains containing the c-MYC gene locus. The clinical outcome for patients with high levels of c-MYC expression is virtually identical to that of patients with amplification of the MYCN gene, a known high-risk feature of this disease. Together, these findings establish c-MYC as a bona fide oncogene in a clinically significant group of high risk childhood neuroblastomas.

#4136

Marked decrease of BIRC5/Survivin by haploinsufficiency does not inhibit neuroblastoma in transgenic mice: Implications for survivin as a therapeutic target in neuroblastoma.

Carmen Dorneburg,1 Volker Rasche,2 Andre Lechel,3 Sarah-Fee Katz,3 Klaus-Michael Debatin,1 Christian Beltinger1. 1 _University Medical Center Ulm, Dept. of Pediatrics and Adolescent Medicine, Ulm, Germany;_ 2 _University Medical Center Ulm, Dept. of Internal Medicine II, Ulm, Germany;_ 3 _University Medical Center Ulm, Dept. of Internal Medicine I, Ulm, Germany_.

Overexpression of survivin, a crucial regulator of the mitotic spindle checkpoint with additional antiapoptotic effects, is associated with poor prognosis in neuroblastoma (NB) and other cancers. Thus, survivin is a promising therapeutic target in NB. Transcriptional inhibitors of survivin show preclinical efficacy and have been tested in clinical trials. The results of these trials, however, have been disappointing, due to modest efficacy and unexpected off-target effects. We thus investigated the consequences of marked, yet incomplete decrease of survivin on NB in a novel transgenic mouse model.

To this end, we crossed mice haploinsufficient for survivin with TH-MYCN mice to generate BIRC5+/- MYCN tg/+ mice. These mice developed NB with markedly decreased mRNA and protein expression of survivin. Surprisingly, incidence and latency of the NB that developed in the BIRC5+/- MYCN tg/+ mice were not different from those in the TH-MYCN mice. Growth of tumors was not decreased in BIRC5+/- MYCN tg/+ mice, as assessed by serial MRI imaging, and survival of the mice was not increased. Immunohistochemistry of the NB did not show decreased proliferation (Ki67), increased apoptosis (activated caspase 3) or a change in vascularity (CD31). No difference in copy number variations between the two NB mouse models was discernible using CGH arrays. To determine whether dysfunctional T cells described in mice haploinsufficient for survivin could have masked any loss of aggressiveness of NB cells haploinsufficient for survivin, NB of BIRC5+/- MYCN tg/+ mice were retransplanted into survivin sufficient mice. No difference in tumor incidence, latency and growth was detected.

Taken together, despite a marked decrease in the expression of BIRC5/survivin by haploinsufficiency development and growth of NB were not inhibited. While a further decrease of survivin beyond that seen in haploinsufficiency will inhibit NB, it most likely will also elicit marked side effects, given that survivin is essential for normal cells. Our results cast doubt on the efficacy of inhibitors of survivin expression in NB and call for targeting survivin by alternative approaches, such as inhibiting protein-protein interactions of survivin.

#4137

Inhibition of protein synthesis with polyamine depletion in high-risk neuroblastoma.

Andrea T. Flynn, Kangning Liu, Annette Vu, Michael D. Hogarty. _Children's Hospital of Philadelphia, Philadelphia, PA_.

The MYC family of oncogenes coordinately deregulate cellular programs that link cell cycle progression with the requisite biomass and energy creation. Neuroblastoma is a MYC-driven, commonly lethal pediatric tumor, with a high proportion of tumors containing MYCN amplification. Polyamine synthesis is a pathway downstream of MYC, with polyamines functioning, in part, to support protein synthesis. ODC1 encodes ornithine decarboxylase, the rate-limiting enzyme in polyamine synthesis, and we found that ODC1 is co-amplified with MYCN in a subset of high-risk neuroblastomas (~8%). Difluoromethylornithine (DFMO) is an irreversible inhibitor or ornithine decarboxylase and is an approved agent for the treatment of Trypanosomiasis. We have previously shown that DFMO inhibits tumor progression and synergizes with chemotherapy in murine mouse models of neuroblastoma, and we postulate that inhibition of protein synthesis is a predominant mechanism of anti-tumor activity. The effects of polyamine depletion on specific components of protein translational machinery have yet to be defined. Here, we show inhibition of global protein synthesis of up to 90% after treatment with various concentrations of DFMO in vitro, with a larger decrease in protein synthesis generally correlating with the presence of MYCN amplification. We found that colony formation is inhibited at DFMO exposures as low as 150 to 500 μM DFMO, which are achievable concentrations in adults based on previous clinical trials. We also show via isoelectric focusing that treatment of neuroblastoma cells in vitro with DFMO leads to a marked decrease in the proportion of hypusinated, or active, eIF5A (an elongation factor important in translation of polyproline stretches and possibly broader influence on translation initiation). To interrogate influences of polyamine depletion on the cap-dependent translation eIF4F complex, we evaluated for changes in the phosphorylation of 4E-BP1 in DFMO treatment conditions, though no changes in phosphorylation were seen compared to controls. Studies to further elucidate the protein synthetic components affected by polyamine depletion are ongoing, as are studies to assess for selective effects of DFMO on polyproline-containing proteins via eIF5A. Further elucidation of the mechanisms of DFMO activity will identify opportunities for drug synergy and provide a responder hypothesis to test in upcoming pivotal Phase 2 and 3 clinical trial of DFMO in the treatment of neuroblastoma.

#4138

CK1δ-BRD4 pathway as novel therapeutic target for SHH subtype of medulloblastoma.

Sze Kiat Tan,1 Clara Penas,1 Cheng-Ming Chiang,2 David Robbins,3 Nagi Ayad1. 1 _Sylvester Comprehensive Cancer Center, Center for Therapeutic Innovation, University of Miami, Miami, FL;_ 2 _Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX;_ 3 _Sylvester Comprehensive Cancer Center, University of Miami, Miami, FL_.

Background: Medulloblastoma is the most common malignant pediatric brain tumor with variable prognosis due to its clinical and genomic heterogeneity. Despite decades of treatment advances, nearly 40% of children experience tumour recurrence, and 30% will die from this disease. Thus, new drugs and drug combinations need to be developed that effectively treat medulloblastoma. Casein kinase 1δ (CK1δ) is a serine/threonine kinase that controls cell cycle progression, signal transduction and neurogenesis, and we previously reported high levels of CK1δ in mouse models of medulloblastoma. BRD4 is an epigenetic reader protein that controls expression of several oncogenes. We wanted to determine the mechanism through which CK1δ regulates BRD4 activity in medulloblastoma and assess whether a combination of CK1δ and BRD4 inhibitors could be more effective in decreasing medulloblastoma progression in vivo.

Methods: Granule cell progenitors (GCPs), medulloblastoma Ptch-/-P53-/- mouse cells and SUFU-/- mouse embryonic fibroblasts were incubated with the CK1δ inhibitor SR-1277 and the phosphorylation levels of BRD4 were determined by WB and its binding to chromatin by ChIP. In vitro phosphorylation studies with human BRD4 purified from E. coli were performed and whether genetic disruption of CK1δ reduces BRD4 phosphorylation was tested. We combined BRD4 and CK1δ inhibitors (JQ1 and SR-1277, respectively) and analysed Gli1 mRNA expression and EdU incorporation. We deleted CK1δ in GCPs in Ptch-/-;P53-/- mouse models of medulloblastoma by breeding these mice with a Tg(Atoh1-Cre);CK1δfl/fl strain. Furthermore, we intracranially transplanted human SHH medulloblastoma PDX cells expressing luciferase in mice, to validate the combination therapy.

Results: CK1δ inhibitor treatment and CK1δ knockout reduced BRD4 phosphorylation, suggesting that CK1δ phosphorylates BRD4 and is required for BRD4 recruitment to chromatin. In vitro phosphorylation studies with purified human BRD4 and CK1δ confirmed CK1δ-mediated BRD4 phosphorylation and CK1δ is required for BRD4 phosphorylation at serine 492 and 494 in vivo. Using loss of function studies in GCPs, both CK1δ and BET inhibition reduced BRD4 association with Gli1 promoter, thereby reducing Gli1 mRNA levels. In addition, SR-1277 suppressed SHH signaling downstream of SUFU of medulloblastoma cells, in vitro and in vivo. We also found synergy by combining these compounds in vitro and in reducing tumor burden in the in vivo model with intracranial allografts.

Conclusion: Together, our studies validate the CK1δ-BRD4 pathway as a novel target in medulloblastoma. The significance of our work is underscored by the possibility that simultaneous CK1δ-BRD4 inhibition could overcome the resistance observed with BRD4 inhibitors and enhance therapeutic benefit to patients. We are also the first group to demonstrate that CK1δ acts downstream of SHH signalling in SHH-driven medulloblastoma.

#4139

SNAI2 inhibition promotes myogenic differentiation and prevents tumorigenic features of embryonal rhabdomyosarcoma.

Silvia Pomella, Elena Carcarino, Cristina Cossetti, Franco Locatelli, Rossella Rota. _Ospedale Pediatrico Bambino Gesu', Roma, Italy_.

Pediatric Rhabdomyosarcoma (RMS) is a soft tissue sarcoma of childhood that in its high risk form has a dismal prognosis. RMS includes two histological subtypes, embryonal and alveolar, which differ clinically and molecularly. Both are characterized by the expression of Myogenic Regulatory Factors (MRFs) such as MYOD and MYOG but tumor cells are unable to differentiate in skeletal muscle cells and proliferate indefinitely. For this reason, differentiation therapies have anticancer potential in this tumor. Interestingly, one of the cause of the inability of MRFs in inducing myogenic-like differentiation is related to the loss of transcriptional activity of these factors partly due to the lack of availability of DNA binding sites and/or of capability to form transcriptional complexes. We demonstrate here that the repressor SNAI2 is upregulated in embryonal RMS (ERMS) patients and cells lines compared to their normal counterparts. Silencing of SNAI2 in 2 ERMS patient-derived cell lines using a puromycin pLKO lentiviral vector expressing a SNAI2 shRNA results in a slowdown of cell proliferation associated to p21Cip1 and Myogenin upregulation starting 3 days post-selection, as compared to control vector. These molecular changes are followed by the induction of Myosin-Heavy Chain and the formation of myogenic-like multinucleated structures reminiscent of myogenic fibers. Moreover, colony formation and anchorage independent growth are both strongly impaired. Finally, SNAI2 depleted cells form smaller spheres when cultured in cancer stem cells conditions. Therefore, SNAI2 down-regulation could have therapeutic value in ERMS. Studies are ongoing to verify the restoring of MYOD transcriptional activity and to identify genes regulated by SNAI2 in our system.

The work is supported by Associazione Italiana Ricerca sul Cancro project IG15312 to RR.

#4140

The dual role of MDM2-ALT1 as both a suppressor and driver of oncogenesis is highlighted in a new RMS mouse model.

Daniel F. Comiskey, Aishwarya G. Jacob, Matias Montes, Krista La Perle, Prosper N. Boyaka, Dawn S. Chandler. _The Ohio State University, Columbus, OH_.

MDM2 is an important negative regulator of protein stability and transcriptional activity of tumor suppressor p53. We have previously shown that the alternatively spliced variant of MDM2, MDM2-ALT1, is induced specifically after exposure to ultraviolet light (UVC) and cisplatin treatment and is associated with various soft tissue sarcomas, including rhabdomyosarcoma (RMS). We have also shown that MDM2-ALT1 interferes with the normal heterodimer formation between MDM2 and MDM4 that is required for efficient p53 degradation by MDM2-mediated ubiquitination. Consistently, we have shown upregulation of p53 activity as a result of transient transfection of this isoform in wild-type p53 background cells. Furthermore, we observed increased levels of p53 and its downstream targets, p21 and Bax, when MDM2-ALT1 is expressed in cells, as well as the expected cell-cycle arrest. In contrast, we have also shown the MDM2-ALT1 can promote cell proliferation and metastatic behavior when transfected into tumor cells lacking an intact p53 pathway. These seemingly opposing roles of MDM2-ALT1 are poorly characterized in vivo, and models to better understand them are not available. To investigate the effects of MDM2-ALT1 on cell cycle and the progression of tumorigenesis in the context of the whole animal, we generated an inducible MDM2-ALT1 mouse model. When MDM2-ALT1 is ubiquitously expressed in mice lacking p53 expression, we see an increased incidence of spindle cell sarcomas, including RMS. Hence, constitutive MDM2-ALT1 expression is oncogenic and exacerbates the tumorigenesis induced by p53 loss. We next assessed the role of MDM2-ALT1 in the presence of wild-type p53. Surprisingly, when MDM2-ALT1 expression was limited to B cells in the presence of wild-type p53 expression, it also gave rise to increased tumorigenesis, albeit with an extended latency (after 18 months). Interestingly, flow cytometric analyses for B-cell markers prior to the onset of tumorigenesis revealed an MDM2-ALT1-associated decrease in the B-cell population of the spleens of these animals. Our data suggest that the B-cell loss is a p53-dependent response to persistent MDM2-ALT1 expression. In conclusion, the MDM2 ALT1 splice variant is a modifier of both p53-dependent and p53-independent tumorigenesis in vivo. Our findings underscore the importance of MDM2 post-transcriptional regulation in controlling the p53 pathway and in tumorigenesis. Furthermore, the MDM2-ALT1-expressing p53 null mice represent a novel mouse model of fusion-negative RMS, a cancer type with few animal models available for therapy design and testing.

#4141

EWS-FLI regulates mitotic kinesins in Ewing sarcoma family of tumors.

Rebecca J. Wates,1 Yan Ma,1 Jennifer Crow,1 Glenson Samuel,2 Andrew K. Godwin3. 1 _University of Kansas Medical Center, Kansas City, KS;_ 2 _Children's Mercy-Kansas City, Kansas City, MO;_ 3 _University of Kansas Cancer Center, Kansas City, KS_.

The current situation faced by children and young adults diagnosed with Ewing sarcoma family of tumors (ESFT), the second most common pediatric bone malignancy in the U.S., is bleak. Although the oncogenic phenotype and master regulator of ESFT is driven by an underlying pathognomonic chromosomal translocation (EWS-Ets), therapeutic targeting of this molecular Achilles' heel has been notoriously challenging. For pediatric patients with progressive (refractory) or recurrent ESFT, the overall survival continues to remain poor. To date, no established second-line (salvage) treatment regimen exists for these pediatric sarcoma patients. The failure to improve the overall survival for these patients highlights the need to better understand the molecular points of vulnerability of these diseases, which can be translated into novel treatment strategies. To address this challenge, we have identified KIF11, KIF15 and TPX2, part of the cellular mitotic machinery, as druggable downstream targets of EWS-Ets in ESFT. Our recent studies sought to identify drugs that reverse a gene expression profile similar to the pattern obtained by the silencing of EWS/FLI1 via RNAi approaches (siEWS/FLI1) (Pessetto et al., 2017). This work identified KIF15 (gene rank #1, p=0.00005) and its potential binding partner TPX2 (gene rank #14, p=0.01830) as significantly downregulated genes within this signature. These genes encode KIF15 and TPX2, which are postulated to play a compensatory role in resistance to KIF11 inhibitors in the clinic. We found that KIF11, KIF15 and TPX2 proteins are abundantly expressed in ESFT cell lines as compared with benign controls (7.7-, 5.5-, and 8.5-fold, respectively). We also discovered that RNAi-mediated downregulation of KIF15 increases sensitivity of ESFT cell lines to treatment with KIF11 inhibitors (KIF11i). We have for the first time, shown that silencing EWS-FLI1 (the most common EWS-Ets translocation), in ESFT cells decreases KIF11, KIF15 and TPX2 promoter activity by 99%, 97%, and 87%, respectively. We propose that this activity is a function of EWS-FLI1-mediated E2Fs; EWS-FLI1 disrupts the E2F-regulated proliferation balance such that the suppressive E2F4 transcription factor is displaced by the activating E2F3. Our data show that E2F3 is enriched at KIF15 (8.9-fold) and TPX2 (2.9-fold) promoters. Further, when EWS-FLI1 is siRNA depleted, enrichment at the KIF15 and TPX2 promoters is lost (3.9-fold or 2.6-fold decrease, respectively). These data strongly suggest that aberrant expression of the genes encoding these proteins is controlled by oncogenic EWS-FLI1. Taken together, we have demonstrated that KIF11, KIF15 and TPX2 are aberrantly expressed in ESFT and are putative therapeutic targets. Our ongoing work includes development of KIF11/KIF11/TPX2 axis-targeting compounds toward the goal of implementing pharmacologic approaches to provide new therapies for children diagnosed with these deadly diseases.

#4142

Conformational dynamics of the chimeric kinase DNAJB1-PRKACA, the driver for fibrolamellar hepatocellular carcinoma.

Michael D. Tomasini,1 Yingiie Wang,2 Adak Karamafrooz,2 James Hall,3 Susan S. Taylor,3 Thijs Beuming,4 Gianluigi Veglia,2 Sanford M. Simon1. 1 _Rockefeller University, New York, NY;_ 2 _University of Minnesota, Minneapolis, MN;_ 3 _University of California, San Diego, San Diego, CA;_ 4 _Schrödinger Inc, New York, NY_.

In fibrolamellar hepatocellular carcinoma (FLC) there is a single common genetic alteration in all tumors: A deletion in one copy of chromosome 19 resulting in the fusion of the first exon of DNAJB1, encoding the J-domain of heat shock protein 40, with exons 2-10 of the catalytic subunit of protein kinase A, PRKACA [1]. This produces an enzymatically active chimeric protein. Expression of the chimera in mouse liver either by recreating the deletion by CRISPR/Cas9, or expression, in trans, by a transposon is sufficient to produce FLC [2]. We used molecular dynamics simulations, NMR, and SAXS to analyze the conformational dynamics of the native and chimeric kinase. The most significant differences were in the amino domain with the chimera in an ensemble of conformations. Some structures had the J-domain tucked under the large lobe of the kinase, similar to that reported in the crystal structure, and in others the J-domain was dislodged from the core of the kinase, swinging free in solution. These simulated dislodged states were captured experimentally both by NMR and small angle X-ray scattering. Modeling of the different conformations onto the RIIβ holoenzyme revealed no obvious steric interactions suggesting that the J-domain does not preclude formation of the holoenzyme. The flexible conformations appear to be independent of the nucleotide/substrate binding mode as they were observed in ATP, ADP, Apo, and ATP-PKI bound states.

[1] Honeyman, J. N. et al. Detection of a recurrent DNAJB1-PRKACA chimeric transcript in fibrolamellar hepatocellular carcinoma. Science 343, 1010-1014 (2014).

[2] Kastenhuber, E. R. et al. DNAJB1-PRKACA fusion kinase interacts with β-catenin and the liver regenerative response to drive fibrolamellar hepatocellular carcinoma. Proc. Natl. Acad. Sci. 201716483 (2017). doi:10.1073/pnas.1716483114

#4143

The role of neuropeptide Y and its Y5 receptor in RhoA-mediated regulation of cytokinesis and cell motility.

Nouran Abualsaud,1 Congyi Lu,1 Akanksha Mahajan,2 Abrar Bakr,1 Shiya Zhu,1 Lindsay Caprio,1 Sung Hyeok Hong,1 Joanna Kitlinska1. 1 _Georgetown University, Washington, DC;_ 2 _Northwestern University, Chicago, IL_.

Treating metastatic disease remains a major obstacle in oncology. Neuropeptide Y (NPY) and its Y5 receptor (Y5R) are expressed in a number of malignancies, including pediatric tumors Ewing sarcoma (ES) and neuroblastoma (NB). Our previous clinical and experimental data implicated NPY/Y5R axis in ES and NB metastasis. We have shown that Y5R is highly expressed in angioinvasive NB cells, while elevated release of NPY from NB tumors associates with metastatic disease and poor clinical outcome. Additionally, hypoxia-induced over-activation of NPY/Y5R pathway in ES tumors, which express particularly high levels of Y5R, led to formation of polyploid cells that give rise to chromosomally unstable, metastatic progeny. This phenomenon was reproduced in CHO-K1 cells over-expressing Y5R, which became polyploid due to a cytokinesis defect. Altogether, our data suggested a role of the NPY/Y5R pathway in the regulation of cell migration and cytokinesis, processes essential in tumor growth and dissemination. Thus, the goal of our study was to determine mechanisms of NPY/Y5R actions. We have found that in both CHO-K1/Y5R transfectants and ES cells, Y5R stimulation activated an essential cytoskeleton regulator, RhoA, activity of which is tightly controlled in a spatial and temporal manner during both cell migration and cytokinesis. This data was confirmed by strong co-localization of Y5R and active RhoA in all cell types. In non-dividing cells, expression of Y5R and active RhoA was limited, while their levels were higher in mitotic cells. Notably, in CHO-K1 cells over-expressing Y5R, both Y5R and RhoA accumulated in the narrow zone of the cleavage furrow at the abscission phase of cytokinesis. Such RhoA activation at this stage was previously shown to trigger cytokinesis defects. The concurrent changes in subcellular localization of Y5R and active RhoA were also observed in migratory cells. In single cell migration, both Y5R and active RhoA accumulated on the trailing and leading edges of the cells, while in the cells migrating collectively their expression was elevated in those present at the front of the moving colony. Strong Y5R/active Rho-A co-localization was also observed in membrane blebs of cells in amoebal migration mode. The role of Y5R in stimulation of cell migration was confirmed by transwell assay, in which Y5R antagonist significantly inhibited NPY-induced migration of NB and ES cells. Altogether, our data support the role of NPY/Y5R/RhoA pathway in regulation of cytoskeleton functions. The stimulation of this pathway promotes cell migration, while its over-activation may lead to cytokinesis defects and chromosomal instability, both of which are essential processes in tumor progression. Further studies are required to determine if these functions of the NPY/Y5R/RhoA pathway contribute to metastasis and validate Y5R as a potential therapeutic target.

#4144

The role of GABA metabolism in medulloblastoma spread.

Vahan Martirosian, Michelle Lin, Thomas C. Chen, Josh Neman. _University of Southern California, Los Angeles, CA_.

Central nervous system (CNS) tumors remain the leading cause of cancer-related childhood mortality. Amongst these, medulloblastoma (MB), a highly malignant cancer originating in the cerebellum, remains the most common pediatric CNS tumor. Patients are clinically stratified by age at diagnosis, extent of resection, and metastatic status. Patients with incomplete tumor resection or leptomeningeal spread at the time of diagnosis are considered high-risk. Standard of care for all patients consists of surgical resection followed by radiation and chemotherapy. MB tumors have recently been molecularly stratified into 4 groups, with each group associated with a particular effected pathway. Group 1 and 2 have deregulated WNT and SHH pathways, respectively. Group 3 and 4, however, are less characterized, but have been shown to be associated with a GABAergic and glutamanergic phenotype, respectively. While Group 1 and 2 MBs have an overall 5-year survival rate of 75%, Group 3 and 4 have a worse prognosis, with survival rates closer to 30-40%. This is correlated with the latter 2 groups having an increased rate of metastasis at diagnosis, as metastatic lesions are more advanced and therapeutically difficult to treat. Therefore, efforts must be made to elucidate mechanisms and treatments of metastasis, as well as finding subgroup specific therapeutic targets.

Although canonical cancer therapy counters the tumors' effects on the surrounding environment, manipulating the effects of the tumor microenvironment on the tumor has also shown promise as a treatment regimen. Given that the cerebellum, the initiation site for MB, has high concentrations of GABA, and Group 3 tumors have upregulation of GABAergic properties, we explored GABA's role in this context. For these studies, we interrogated the GABA shunt pathway - the principal pathway for GABA metabolism - in two Group 3 MB cell lines. We utilized lentivirus mediated gene knockdown of GABA Transaminase (ABAT), the initial protein in the GABA shunt, to deduce phenotypic and genotypic changes in these cells. Cells supplemented with GABA had an increased ability to detach and form spheres. These spheres thrived in GABA supplemented floating conditions, while no similar phenomenon was seen in cells cultured in control medium or GABA supplemented ABAT knockdown cells. Genotypic analysis of these cells using RT-qPCR revealed an increase in the epithelial-to-mesenchymal transition profile of GABA supplemented spheres as compared to control conditions. To determine if knocking down ABAT changes tumor seeding capabilities in vivo, we transplanted MB cells into the lateral ventricle of athymic nude mice. Control MBL cells regardless of prior exposure to GABA or floating conditions, were able to seed and form tumors; While ABAT KD cells were unable to do so. These findings provide evidence that GABA metabolism in MB metastatic cells may be imperative for survivability in the CSF and leptomeningeal spread.

#4145

Characterization of the ODZ receptor family in neuroblastoma.

Teodora Andonova, Per Kogner, John Inge Johnsen, Malin Wickström. _Karolinska Institutet, Stockholm, Sweden_.

Neuroblastoma is the most common extracranial solid tumor of childhood, causing 6% of all childhood cancers but 9% of deaths from malignant tumors in children. It is a tumor with highly heterogeneous clinical behavior, believed to originate from undifferentiated neural crest cells that form the sympathetic nervous system. Neuritogenesis is the process during embryonic development when neural crest cells migrate and differentiate into multiple cell types. The Rho/Rac signaling cascade is fundamental for this process. We performed whole genome sequencing of 40 neuroblastoma tumor samples, 32.5% of the samples contained a mutation in a gene associated with Rho/Rac signaling. The most frequently mutated receptor family among the Rho/Rac associated genes was the tenuerins (ODZ1-4). The ODZ genes were mutated in three out of 40 patients, i.e. 7.5%. The ODZ proteins are not fully characterized, however it is known they are highly expressed during the development of the central nervous system and play role in differentiation of the drosophila fly.

Evidence suggests a function for dysregulated expression of the ODZs in human tumors, but investigations of a deeper cellular and molecular understanding in neuroblastoma are missing. The aim of this study is to investigate the ODZ receptors to determine the role and function of these proteins in neuroblastoma tumor development, progression and metastasis. When investigated in publically available and validated neuroblastoma cohorts, low expression levels of ODZ1, ODZ2 and ODZ3 were associated with poor overall survival, while low ODZ4 expression correlated with high overall survival. Our data demonstrated that the ODZ receptors were differently expressed in neuroblastoma cell lines. Preliminary results also showed that siRNA knockdown of ODZ4 in a neuroblastoma cell line led to a 50% decrease in cell viability compared to control, together with morphological changes such as more neurite extensions, suggesting that an inhibition of ODZ4 may result in differentiation. Knockout of the four different ODZ transcripts also showed that the ODZ receptors might have compensatory mechanisms for each other. Our data suggest that activity of the ODZ receptors may be important for neuroblastoma development and that manipulation could offer a possible therapy for neuroblastoma.

#4146

**N** 6 **Methyladenosine RNA demethylase ALKBH5 as a novel therapeutic target for osteosarcoma.**

Pooja Yadav, Panneerdoss Subbarayalu, Nourhan Abdelfattah, Vijay K. Eedunuri, Yidong Chen, Manjeet K. Rao. _UT Health San Antonio, San Antonio, TX_.

Osteosarcoma (OS) is the most prevalent primary bone malignancy that affects children and young adults. Despite several years of research, survival outcome of OS patients has not improved in last three decades. OS is treated with multi-modal chemotherapy, which is highly toxic and does not work well for metastatic and chemo-resistant tumors. Currently there is no FDA approved drug that can serve as an alternative to chemotherapy, warranting an urgent need to find more efficacious and targeted therapeutics for OS. Here, we report that RNA demethylase AlkB Homolog 5 (ALKBH5) may serve as a novel therapeutic adjuvant for treating OS. N6 Methyladenosine (m6 A) is the most common internal mRNA modification, which is modulated by the multi-component RNA methyltransferase complex, RNA demethylase (ALKBH5) and m6A readers . We show that ALKBH5 is amplified in sarcomas and its expression is highly elevated in osteosarcoma patients. We demonstrate that silencing of ALKBH5 inhibits the OS growth and migration without affecting the viability of normal human fetal osteoblast cells. Our results reveal that ALKBH5 depletion impairs the cell cycle progression and induces apoptosis in OS cells. Interestingly, we demonstrate that reduction in ALKBH5 levels suppresses the DNA damage repair capacity of osteosarcoma cells rendering them sensitive to DNA damaging agent like Doxorubicin. Using DR-GFP reporter-based homologous recombination (HR) assay, we show that ALKBH5 depletion leads to reduced HR-mediated DNA repair capacity of osteosarcoma cells. Supporting this, we observed significantly reduced expression of several genes that are known to play critical roles in cell cycle progression and DNA damage repair. In summary, this study shows that ALKBH5 is a critical regulator of OS growth and chemosensitivity. Approaches aimed at silencing ALKBH5 can be potentially used to inhibit osteosarcoma growth and progression as well as sensitize osteosarcoma cells to DNA damaging agents.

#4147

Neuropeptide Y promotes osteolytic activity during bone invasion and metastasis in Ewing sarcoma.

Richard Garner, Emily Hong, Shiya Zhu, Mina Adnani, Sung-Hyeok Hong, Susana Galli, Joanna Kitlinska. _Georgetown Univ., Washington, DC_.

Ewing sarcoma (ES) is a pediatric malignancy affecting bones and soft tissues. The presence of metastases is associated with poor prognosis, particularly in patients with dissemination to the bone. ES cells produce and secrete high levels of neuropeptide Y (NPY), a 36-amino acid neurotransmitter normally released from peripheral sympathetic neurons, and express its receptors. Our previous studies in an ES xenograft model demonstrated that the severity of bone destruction in primary tumors and incidence of osseous metastases positively correlate with levels of NPY release and are significantly decreased with NPY shRNA. Thus, the goal of our study was to determine the mechanism of this NPY-induced osteolytic effect. Since tumor bone invasion and metastasis is associated with changes in osteolytic and osteogenic activity within bone, we assessed osteoblast and osteoclast content in bones adjacent to the ES xenografts. No significant difference in osteoblast number was observed between bones within ES xenografts with high and low NPY levels. However, TRAP assay showed that ES tumors secreting high NPY levels had significantly increased osteoclast density on the border of the bone-tumor interface, as compared to tumor tissues with minimal NPY secretion or treated with NPY shRNA. To identify the mechanisms underlying these osteolytic actions of NPY, we tested its effect on osteoclast recruitment and differentiation. Transwell migration assay showed that osteoclast precursors, RAW 264.7 murine macrophages, were recruited at significantly higher levels to the conditioned media from ES cells with high NPY release, as compared to those cells exposed to conditioned media from NPY-low ES cells. Similarly, ES-conditioned media with high NPY content had a higher ability to stimulate differentiation of RAW 264.7 cells into osteoclasts than those obtained from NPY-low ES cells. Both of these effects were exacerbated by hypoxic conditions, known to upregulate NPY and its Y5 receptors (Y5R) in ES cells. Blocking the expression of NPY and Y5R in ES cells with shRNA inhibited macrophage recruitment and osteoclastogenesis, and decreased RANKL content in conditioned media from NPY-high ES cells. Altogether, these data suggest that the NPY/Y5R autocrine loop stimulates the release of RANKL from ES cells, promoting macrophage recruitment and consequent osteoclast differentiation. Therefore, increased NPY secretion could have a significant impact on shifting bone homeostasis to promote osteolysis, bone invasion, and osseous metastasis. Further studies are needed to identify other factors released by tumor cells secreting high NPY levels and to test Y5R antagonists as a potential therapeutic option to inhibit these processes. Importantly, aside from ES, the above findings may be relevant to other NPY-rich tumors metastasizing to the bone, such as neuroblastoma.

#4148

Role of the photoreceptor gene, CRX (cone-rod homeobox) in pathogenesis of non-WNT, non-SHH medulloblastoma.

Shalaka Masurkar,1 Aliasagar Moiyadi,2 Epari Sridhar,3 Rakesh Jalali,3 Neelam Shirsat1. 1 _Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre (TMC), Homi Bhabha National Institute, Navi Mumbai, India;_ 2 _Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre (TMC), India;_ 3 _Tata Memorial Hospital (TMH), Tata Memorial Centre (TMC), India_.

Central nervous system (CNS) tumours are the second most common cancers diagnosed in children after leukemia and continue to be the leading cause of cancer-related mortality in children. Medulloblastoma (MB) is a highly malignant primary brain tumour that occurs in posterior fossa region of the brain. The current standard treatment regimen includes surgical resection, craniospinal irradiation and adjuvant chemotherapy. Although this has resulted in significant increase in survival, approximately one-third of medulloblastoma patients still die because of the disease. Genome-wide gene expression profiling studies have identified four molecularly distinct subgroups of medulloblastoma namely WNT, SHH, Group 3, and Group 4. Among the four subgroups, Group 3 patients exhibit highest incidence of metastasis and worst overall survival rates. Molecular mechanism underlying the pathogenesis of Group 3 medulloblastomas is not understood. Group 3 tumors often express retina photoreceptor-specific genes like CRX (Cone Rod Homeobox) which is a homeodomain transcription factor and it is essential for the normal development and maintenance of cones and rods cells of the retina. We have noted that this expression is also retained by the medulloblastoma cell lines like D425, D283 and D341 which have characteristics of Group 3 tumours. Downregulation of CRX in these cell lines brought about inhibition of cell proliferation and anchorage-independent growth as studied by soft agar colony formation assay. CRX gene, therefore, seems to act as an oncogene similar to OTX2, another homeobox gene overexpressed in medulloblastomas. Transcriptome profiling and CHIP-seq analysis is being carried out to identify genes regulated by CRX transcription factor to delineate the molecular mechanism underlying the oncogenic role of CRX in medulloblastoma pathogenesis.

#4149

Inhibition of ribonucleotide reductase (RNR) in Ewing sarcoma cells results in suppression of protein synthesis.

Stacia Koppenhafer, Kelli Goss, David Gordon. _University of Iowa, Iowa City, IA_.

Ewing sarcoma is a highly aggressive bone and soft tissue cancer that is caused by the EWS-FLI1 fusion protein. EWS-FLI1 is required for tumor growth and survival. However, directly targeting EWS-FLI1 with drugs has been challenging and an alternative therapeutic approach is to identify unique vulnerabilities incurred by the oncoprotein. In previous work, we identified that Ewing sarcoma cells are uniquely vulnerable to drugs that inhibit ribonucleotide reductase (RNR), which is the rate limiting enzyme in the synthesis of deoxyribonucleotides. Furthermore, we also showed that blocking the function of checkpoint kinase 1 (CHK1), a major regulator of the response to impaired DNA replication, using either a small molecule inhibitor or siRNA-mediated knockdown causes synergistic toxicity with gemcitabine, an irreversible inhibitor of the RRM1 subunit of RNR, in Ewing sarcoma cells. In the current work, we identifiy that inhibition of RNR in Ewing sarcoma cells in vitro and in vivo results in the suppression of protein synthesis. Moreover, we have found that the treatment of Ewing sarcoma cell lines with gemcitabine results in an increase in the active (unphosphorylated) form of 4E-BP1, which is a well-described inhibitor of protein translation. However, in contrast to the Ewing sarcoma cells, treatment of other cell types with gemcitabine did not alter 4E-BP1 phosphorylation or protein synthesis. Currently, ongoing work is focused on the in vivo testing of gemcitabine, alone and in combination with CHK1, ataxia telangiectasia and rad3-related protein (ATR), and protein synthesis inhibitors, as a novel therapeutic approach for the treatment of Ewing sarcoma.

#4150

MDM4 expression is increased in fibrolamellar hepatocellular carcinoma with PRKACA-DNAJB1 fusion protein.

Anju Karki, Juan Putra, Michael LaQuaglia, Antonio Perez-Atayde, Khashayar Vakili. _Boston Childrens Hospital, Harvard Medical School, Boston, MA_.

Introduction: Fibrolamellar hepatocellular carcinoma (FL-HCC) is a rare variant of hepatocellular carcinoma (HCC) that preferentially affects young adults with no underlying liver disease. Overall patient outcome is poor due to the lack of effective treatment options. Recent studies have shown a 400 kb pair deletion resulting in DNJAB1-PRKACA fusion transcript and protein in majority of FL-HCC tumors. The exact oncogenic mechanism of this fusion protein is yet to be elucidated. In our previous studies we had noted upregulation of p53 and phosphorylated p53 in tumor samples compared to normal liver tissue without expected p53-induced apoptosis. Since MDM4 (a.k.a. MDMX or HDMX), a known negative regulator of p53, is overexpressed in various human cancers, we sought to examine its role in dysregulation of p53 signaling pathway in FL-HCC containing PRKACA-DNAJB1 fusion.

Methods: We analyzed 7 FL-HCC tumors containing DNAJB1-PRKACA fusion protein and 5 non-neoplastic samples. Immunohistochemistry (IHC) on formalin-fixed and paraffin-embedded sections was performed using primary anti-MDM4 antibody. Diaminobenzidine (DAB) substrate kit was utilized to detect the signal and the slides were counterstained with hematoxylin. The nuclear MDM4 expression was assessed using immune reactive score (IRS) in a blinded manner. The IRS gives a range of 0-12 as a product of multiplication between positive cells proportion score (0-4) and staining intensity score (0-3). We also analyzed transcript levels of MDM4 from tumor and non-neoplastic liver samples on 4/7 patients. Reactions were performed in duplicates and MDM4 was normalized to GAPDH.

Results: We found that the percentage of positive MDM4 cells ranged from 0 to 80 in normal liver samples (5/7 patients) and 10 to 100 in tumor samples (7/7 patients). The IRS score of MDM4 ranged from 0 to 6 in non-neoplastic liver tissue and 2 to 12 in tumor tissue. Statistically, the IRS score of MDM4 protein expression was significantly upregulated in tumor samples in comparison to non-neoplastic samples (P=0.0098). Additionally, the MDM4 transcript levels in 2/4 tumors examined were increased by 2- and 180-fold, respectively, compared to non-neoplastic liver tissue.

Conclusion: The overexpression of MDM4 protein in FL-HCC patients with PRKACA-DNAJB1 suggests that the p53 effector function of DNA repair or induction of apoptosis may be dysregulated by increased MDM4 expression. Further studies targeted at blocking MDM4 inhibition of p53 activity in FL-HCC will be important in determining its contribution to tumor cell survival and proliferation.

### Radiation Studies Using in Vivo and Clinical Models

#4151

Fucoidan reduced radiation-induced fibrosis and secondary primary cancers.

Szu-yuan Wu. _Taipei Medical University, Taipei, Taiwan_.

Radiotherapy (RT) causes tissue fibrosis and secondary cancers. Fucoidan is a sulfated polysaccharide that is mainly found in brown algae and brown seaweed. In this study, we assessed the radioprotective effect of oligo-fucoidan on zebrafish. Embryos were exposed to RT (5, 10, or 20 Gy) at 4 hours post-fertilization and compared with an oligo-fucoidan pretreatment group. We found that oligo-fucoidan protects embryos from death and embryonic defects. Using oligo-fucoidan oral feeding adult zebrafish prior radiation, we found that oligo-fucoidan treatment of wild-type fish yields much lower expression of apoptosis and fibrosis marker genes in liver compared to RT (40 Gy) only. Liver fibrosis was diagnosed from Sirius stain, proved oligo-fucoidan reduce fibrosis caused by high dose of radiation. Using a diet-induced obesity HBx,src(p53-) triple transgenic fish liver cancer model, we found that oligo-fucoidan can lower the expression of cell cycle/proliferation markers, as well as fibrosis markers and lipogenic enzymes, compared with that in fish with radiation exposure only. Furthermore, oligo-fucoidan can effectively decrease liver cancer formation, as observed by hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) staining of PCNA proliferation marker. Genomic transcriptome profiling by GeneTitan reveals the main genes/pathways activated by oligo-fucoidan pre-treatment which have protection effect for the radiation-induced fibrosis and secondary primary cancers. Our results provide evidence for the ability of oligo-fucoidan to prevent steatosis, hepatocellular carcinoma and secondary cancers induced by radiation in zebrafish model.

#4152

Synergistic gold nanostar-mediated photothermal and immunotherapy for cancer metastasis treatment.

Yang Liu,1 Paolo Maccarini,1 Gregory M. Palmer,2 Brant A. Inman,2 Tuan Vo-Dinh1. 1 _Duke University, Durham, NC;_ 2 _Duke University Medical Center, Durham, NC_.

Cancer has become a severe thread to human health resulting in more than eight million deaths each year. Cancer metastasis is the main mechanism in more than 90 percent of cancer deaths. However, current therapeutic options including chemotherapy and radiation therapy have limited therapeutic effects to treat cancer metastasis. Therefore, there is an urgent need to develop novel methods for cancer treatment. Our group has developed a unique method to synthesize star-shaped gold nanoparticles, gold nanostars (GNS), without using toxic surfactant, which is very suitable for in vivo applications. The synthesized GNS has tip-enhanced plasmonics and tunable strong in the near-infrared "tissue optical window", enabling it a superior photon-to-heat nanotransducer. The GNS nanoparticles accumulate preferably into tumor with enhanced permeability and retention (EPR) effect after intravenous administration. We have applied GNS for photothermal therapy with 808 nm laser to specifically ablate tumor. Here we report a new cancer treatment method by combing nanophotothermal therapy and anti-PD-L1 immunotherapy. Immunogenic cell death is often insufficient to trigger effective anti-cancer immunity due to the presence of immunosuppressive factors occurring in the tumor microenvironment such as hypoxia, impaired leukocyte trafficking and inhibitory immune checkpoint signaling. The GNS-mediated photothermal ablation can reduce hypoxia and improve leukocyte trafficking to the tumor. In addition, the administration of anti-PD-L1 antibody reverses another critical immunosuppression factor. Experiment results using a mouse bladder cancer model demonstrated that the combined photothermal and immunotherapy have synergistic effect and can not only eradicate primary tumors with laser treatment but also distant metastasis without laser treatment. Furthermore, delayed rechallenge in the cured mice didn't result in new tumor generation, indicating that our combined therapy induced memorized immune response against cancer. The combined GNS-mediated photothermal therapy and anti-PD-L1 immunotherapy has promise to improve therapeutic effect on not only primary tumor but also cancer metastasis.

#4153

Commissioning and radiobiological verification of the small animal radiotherapy research platform (SARRP).

Jonathan L. Kane. _Xstrahl Inc., Suwanee, GA_.

Preclinical radiation biology has become increasingly sophisticated due to the implementation of advanced small animal image guided radiation platforms into laboratory investigation. Small animal radiotherapy devices enable state-of-the-art image guided therapy (IGRT) research to be performed in small animal cancer models by combining high-resolution cone beam computed tomography (CBCT) imaging with an isocentric irradiation system. The technological evolution from simple, broad field irradiation configurations, to more sophisticated dose deliveries for preclinical radiobiology experiments has introduced new dosimetry challenges for preclinical small animal cancer research. Because of this, robust QA and dosimetry techniques are a key part of using novel treatment platforms using very small irradiation fields. In this study, we present a dosimetric study of the small animal radiotherapy research platform (SARRP, Xstrahl Inc.) focusing on the small field dosimetry. Physical dosimetry was assessed using ion chambers and radiochromic film, investigating the impact the beam focus size has on the dose rate output as well as beam characteristics (beam shape and penumbra variations). Two film reading modalities have been used to assess the dose output using the 0.5 mm diameter aperture. This study establishes that FilmQA Pro is a suitable tool for small field dosimetry, with a sufficiently small sampling area (0.1 mm) to ensure an accurate measurement. Furthermore, all small field dosimetry data generated in this study are equally comparable with our Monte Carlo simulations for both focal spot sizes. Overall, it was observed that the small focal spot, while having a lower dose-rate output was shown to produce a more stable and homogenous beam with minimum penumbra over time when compared to the larger focal spot. These findings suggest that when preclinical stereotactic irradiation fields are used, a practical compromise (time to deliver a desired dose and field homogeneity) needs to be considered when deciding the optimum treatment plan and beam configuration used.

#4154

1-Palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) exhibits the therapeutic effect in chemoradiation-induced oral mucositis mouse model.

Ha-Reum Lee,1 Solji Choi,2 Kwang Hoon Yang,2 Do Young Lee,1 Byoung-Gon Moon,1 Ki-Young Sohn,1 Sun Young Yoon,1 Jae Wha Kim2. 1 _Enzychem Lifesciences Corp., Daejeon, Republic of Korea;_ 2 _Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea_.

Oral mucositis is a common complication of chemoradiation therapy and is often accompanied by erythema, ulceration, pain, weight loss. It could delay remission and limit the effectiveness of cancer therapy and increase the risk of infections. However, no specific therapy for protection against mucositis is currently available. In previous study, PLAG (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol, acetylated diglyceride) was shown to exert a therapeutic effect with pegfilgrastim to treat chemotherapy-induced neutropenia by modulating neutrophil transmigration and chemotherapy-induced megakaryocyte/erythrocyte progenitor decrese was significantly alleviated following PLAG administration. In this study, we investigated the therapeutic effect of PLAG in 5-fluorouracil (5-FU) and radiation-induced oral mucositis mouse model. Following 5-FU (100 mg/kg) injection on Day 0, mice were exposed to whole-body irradiation with 1 Gy gamma-ray radiation on Day 2. In order to facilitate the risk of infection, tongue was scratched 0.2 cm wound at using the tip of an 18-gauge needle at an equal force and depth on Day 4. PLAG was orally administered at 250 mg/kg/day. 5-FU, Radiation and scratching-induced oral mucositis mice exhibited ulceration, fibrosis, and festering wounds. PLAG treatment group decreased ulcer formation and diminished the degree of wound festering form on Day 5. Although CCRT-induce mice declined 16% of body-weight, PLAG only suppressed 2% of body-weight compare to control mice on Day 8. PLAG supported to mouse survival and inhibited mucositis-induced inflammatory responses in the tongue and serum. For head only radiation, custom-made lead shield was used for mice to limit the radiation to the head. After 5-FU (100 mg/kg) injection, mouse's head were received 20 Gy x-ray radiation and PLAG was administrated with 250 mg/kg daily. In toluidine blue (TB) staining, PLAG suppressed concurrent chemoradiotherapy (CCRT)-induced oral mucositis and inflammatory responses. At Day 10, CCRT-induced oral mucositis mice were shown the weight loss and tongue wound festered, and had still not fully recovered and resulted in death. PLAG administration significantly reduced CCRT-induced mucositis and recovered scar better quicker. PLAG also decreased body-weight loss and increased mice survival rate. In histological analysis, CCRT-induced inflammation was recovered in PLAG-treated mice. These data suggest that PLAG enhances recovery from 5-FU/radiation-induced oral mucositis and cachexia. From these data, PLAG could be therapeutically useful in reducing the complications associated with chemotherapy and radiation, and thus may be an excellent supplementary agent for anti-cancer therapy.

#4155

USP11 regulates UV-induced DNA damage repair and cell survival associated with skin cancer.

Palak Shah, Lei Qiang, Seungwon Yang, Keyoumars Soltani, Yu-Ying He. _University of Chicago, Chicago, IL_.

UV radiation is a major risk factor for skin cancer, the most common cancer in the United States. UV exposure leads to DNA damage by formation of dimers between adjacent pyrimidine bases. The nucleotide excision repair (NER) process is responsible for removal of UV-induced DNA damage. When the damage is left unrepaired due to inefficient NER in disorders like xeroderma pigmentosum, it leads to skin tumorigenesis. Although the main players of the NER pathway have been characterized, the molecular regulation of the pathway is less understood. Understanding the regulation, particularly post-translational regulation, of the NER pathway has the potential to yield modulators of NER efficiency to prevent skin cancer. We found that chronic UV irradiation led to down-regulation of ubiquitin specific peptidase 11 (USP11) in mouse skin tissue. We also found that USP11 is decreased in skin tumors in mice and humans. Moreover, high-throughput proteomics analysis had predicted that USP11 may interact with NER factor XPC. Hence, we undertook this study to determine the role of USP11 in NER and cellular response to UV. We found that USP11 promoted the NER pathway by regulating XPC activity. USP11 promoted XPC deubiquitination and retention at the DNA damage sites. We also found that USP11 interaction with ubiquitinated XPC was enhanced post-UV irradiation, and that USP11 was recruited to the chromatin post-UV damage. Further, squamous cell carcinoma cells showed reduced survival with inhibition of USP11 after UV exposure. Our findings indicate that USP11 plays an important role in maintaining NER capacity, and suggest that USP11 acts as a tumor suppressor via its role in DNA repair.

#4156

Inhibition of NF-kB improves sensitivity to radiation and EGFR inhibitor and decreases radiation-induced lung toxicity in lung cancer.

Wei Wang, Rong Wang, Shunli Peng, Qi Li, Xiaojuan Zhang, Yueyun Ma. _Nanfang Hospital, Southern Medical University., GuangZhou, China_.

Combination with EGFR-TKIs and radiation seems to be a good strategy against lung cancer, since radiation could enhance EGFR expression. However, the results of clinical trials were controversial. MET activation was involved in resistance to both radiation and EGFR inhibitor, while NF-kB was reported to closely relate to HGF/MET signaling axis. Furthermore, NF-κB has been proved to play a role in bleomycin-induced pulmonary fibrosis, which could be ameliorated by IKKB inhibitor (IMD-0354). Therefore, we evaluated the influence of NF-kB/MET inhibition to the sensitivity of radiation and EGFR-TKIs in lung cancer cells in vitro and in vivo. we also examined the role of NF-κB in radiation-induced lung toxicity. Our data showed that radiation enhanced activation and expression of MET and EGFR, whereas only activation of MET could be reversed by IMD-0354 and p65 depletion, suggesting that radiation-induced MET activation was mediated by NF-kB signaling. Furthermore, NF-kB inhibition increased radiation-induced DNA damage and apoptosis in lung cancer cells. On the other hand, radiation decreased sensitivity to EGFR inhibitor in EGFR mutant lung cancer cells, while inhibition of NF-kB or MET overcame the resistance. Importantly, IMD-0354 increased sensitivity to radiation, EGFR-TKIs, or their combination in mouse xenograft tumors by arresting cell proliferation and inducing cell apoptosis. Interestingly, IMD-0354 significantly reduced the lung toxicity in a mouse model of radiation-induced pneumonia and lung fibrosis. Our preclinical findings indicated that inhibition of NF-kB/MET could improve sensitivity to radiation and EGFR inhibitor and decrease radiation-induced lung toxicity in lung cancer.

#4157

Second tumor type showing hypoxia targeting with radiation improves tumor control in a mammal tumor model.

Howard J. Halpern,1 Boris Epel,1 Matthew C. Maggio,1 Martyna Krzykawska-Serda,1 Gage H. Redler,1 Richard C. Miller,1 Eugene D. Barth,1 Ralph R. Weichselbaum,1 Victor M. Tormyshev2. 1 _Univ. of Chicago, Chicago, IL;_ 2 _Novosibirsk State University, Novosibirsk, Russian Federation_.

Living tissue hypoxic resistance to radiation, has been known for over a century. Our research has validated the hypothesis that radiation dose boosts focused specifically on hypoxic regions of tumors would improve tumor curability in a fibrosarcoma mouse model. We used Electron Paramagnetic Resonance (EPR) imaging of absolute pO2 in volume elements of murine tumors with 1 torr pO2 resolution and 0.7 mm spatial resolution in FSa fibrosarcomas in the legs of C3H mice. Hypoxia was defined as subvolumes or voxels with pO2 less than 10 torr. This showed EPR pO2 images as a reliable identifier and locator of relevant radiobiologically hypoxia. For the first time in mammalian tumors pO2 based dose painting was shown to improves tumor cure. This required gantry based x-ray treatment with an XRAD225Cx system to deliver dose to mouse tumors accurately registered with EPR pO2 images. Tumors were pretreated with a dose of radiation sufficient to cure 15% of tumors, a TCD15. For hypoxic boosts or hypoxia avoidance of the same volume, 3D printing Tungsten loaded, highly conformal plastic blocks were used. This allowed comparison of treating 100% of hypoxic tumor voxels with hypoxia avoidance boosts. This showed significant (p=0.02) tumor control differences between hypoxic boosts and hypoxia avoiding boosts. This is the first validation of the curative effectiveness of focusing hypoxia based dose painting in mammalian tumors. This was motivated by a conclusive failure to demonstrate a difference between treating ~ 85% of hypoxic voxels with a hypoxic boost compared with radiation avoiding hypoxia which proved tenets of radiation biology: a few surviving hypoxic clonogens cause radiation treatment failure.

To further validate this approach, we have applied the same techniques to a second mouse mammary carcinoma, the MCa4. We have determined the dose response of 325 μl IM tumors in C3H mouse legs with a 50% control at 180 days with 52 Gy radiation (the TCD50). We have begun the study of tumor control using a whole tumor TCD15 of 49 Gy and boosts of 11 Gy to hypoxic and well oxygenated tumor of the same volume. With a median follow up of35 days of 10 animals, none of the hypoxic boost cohort have failed. All failure has been in the well oxygenated tumor boost cohort. Log-Rank significance is ~0.2. Our past experience has been that these early results portend future significance.

Support: R01 CA098575, P41 EB002035, R50 CA211408

#4158

**Differential intestinal tumorigenesis and DNA repair response in APC** Min/+ **mice after acute and fractionated proton radiation.**

Kamal Datta, Shubhankar Suman, Santosh Kumar, Albert J. Fornace. _Georgetown Univ., Washington, DC_.

Ionizing radiation (IR) exposure is a risk factor for colorectal cancer (CRC) development. With increasing interest in exploring outer space, astronauts on prolonged space missions such as a mission to Mars are expected to receive IR doses that could heighten the risk of CRC. It is estimated that about 90% of the IR in outer space is energetic protons, and the radiation dose during solar particle events (SPEs) could reach up to 2 Gy behind shielding. However, the risk of CRC after energetic proton radiation is not yet fully defined due to lack of human or animal model data. The purpose of the current study was to quantitatively and qualitatively compare and characterize differences in intestinal tumorigenesis between acute and fractionated proton irradiation in APCMin/+ mice, a well-established mouse model for human CRC. Mice were exposed to 1.88 Gy of proton radiation delivered in a single fraction or in 4 equal daily fractions (0.47 Gy x 4). Since proton dose to astronauts are variable based on space environment, in this initial study we chose a high single dose and a lower daily dose pattern to assess intestinal tumorigenesis. Intestinal tumor frequency and grade were noted, and tumor samples were collected from irradiated and control mice euthanized 100 to 110 days after radiation exposure. Molecular analysis was focused on DNA damage and repair, and cellular proliferative signaling pathways relevant to CRC using immunoblots, immunohistochemistry, and qPCR. Relative to control and fractionated-protons, there was significantly higher intestinal tumor frequency and grade after acute (single dose) proton irradiation. Decreased cellular differentiation and increased oxidative damage to DNA was observed after acute proton radiation. At the molecular level, our data showed increased DNA double strand breaks associated with decreased levels of DNA repair proteins, and upregulation of proliferative β-catenin and Akt signaling after acute proton exposure. Since increased DNA damage was not associated with concomitant increased apoptosis, our data suggest continued proliferation of cells bearing sub-lethal damage to promote tumorigenesis. In the fractionated proton group, tumor frequency and grade as well as molecular changes were comparable to sham-irradiated control group. When considered along with decreased tumor frequency and upregulation of DNA repair pathways after fractionation, our data are suggestive of marked differences in carcinogenic effects of acute vs. fractionated proton radiation. In summary, our data suggest that acute exposure to proton radiation is associated with higher risk of intestinal tumorigenesis in APCMin/+ mice with implications for gastrointestinal cancer risk in astronauts undertaking long duration space missions and in patients undergoing proton radiotherapy.

#4159

Circadian clock protects against radiation-induced dermatitis and cardiomyopathy in mice.

Panshak P. Dakup, Kenneth I. Porter, Zhaokang Cheng, Shobhan Gaddameedhi. _Washington State Univ., Spokane, WA_.

Radiation therapy (RT) is commonly used in cancer treatment. Despite technological advances to improve precision external-beam RT in treating breast, lung, and esophageal tumors, a substantial number of patients receive radiation to their hearts and the external skin region. This radiation insult results in acute and chronic toxicities including dermatitis and cardiomyopathy, affecting the patient's quality of life and survival. Hence, reducing toxicities associated with RT are desirable outcomes. With the knowledge that the natural circadian clock, present in almost every cells of the body including the skin and heart, regulates DNA repair, cell death and proliferation, and cardiac function processes, we hypothesize that molecular defensive mechanisms of the circadian system play regulatory roles to minimizing RT-induced skin and heart toxicity in cancer patients. To investigate acute toxicity of RT, studies were performed using SKH-1 hairless mice with healthy clock (wild-type day shift (DS)) and environmentally disrupted clock (wild-type rotating shift (RS) and genetically disrupted clock (Per1/2 knockout (Per1/2-/-)) conditions. We treated all groups (n=9 mice per group) with a single dose RT (6 grays of ionizing radiation) to the whole body. We used body weight as an indicator of overall toxicity, red spots as a measure of dermatitis, and left ventricular ejection fraction (LVEF) by ultrasound echocardiography as a readout for cardiomyopathy, relative to the measurements pre-RT as controls. After sacrifice, genotoxicity was detected using alkaline comet assay in PBMCs. Worse toxicities were observed until 2 weeks post-RT in mice with circadian clock disruption compared to mice with healthy clocks. Body weights of the disrupted clock groups were significantly reduced by up to 10% on day 2, unlike the DS group which reduced by only 2%. However, there was body weight loss recovery from day 6 in clock disrupted mice but this recovery was not sustained in Per1/2-/- mice. Dermatitis was counted where visible and there was 2.4- and 1.7- fold increases in RS and Per1/2-/- mice compared to DS mice. In the heart, LVEF was significantly decreased in RS and Per1/2-/- mice by days 1 and 4, and began to recover by day 8 in RS. Comparatively, the LVEF in DS mice was not significantly reduced. On a cellular level, genotoxicity in blood cells were significantly increased in RS and Per1/2-/- mice relative to DS mice. Taken together, these findings strongly suggest that the healthy circadian clock protects the whole body, skin, and heart from RT-induced toxicity. Future work includes mechanistic understanding of how the natural circadian clock system can be harnessed to minimize toxicity against RT. The successful completion of this project will provide a novel mechanistic understanding into the protective role of the circadian clock against acute and chronic skin and heart toxicities for cancer patients undergoing RT.

#4160

A new orotopically applied vasoconstrictor drug significantly confers normal oral mucosa resistance to radio-damage.

Amanda Graul-Conroy,1 Margo Hoover-Regan,1 Paul M. Sondel,1 Natalie S. Callander,1 Walter L. Longo,1 Ningfeng F. Li,2 William E. Fahl1. 1 _Wisconsin Institutes of Medical Research, University of Wisconsin-Madison, WI;_ 2 _Barts & The London School of Med. & Dent., London, United Kingdom_.

Introduction: Oral mucositis (OM) represents one of the most debilitating and troublesome adverse effects of chemoradiotherapy frequently encountered in patients. Most ionizing radiation-induced oral mucosa cell death results from radiation-induced reactive oxygen species (ROS) attack of DNA bases and molecular oxygen (O2) "fixation" of the ROS damage, which results in DNA breakage. Our earlier work showed complete suppression of radiation-induced oral mucositis in rodents by transient reduction of ROS related DNA damage when the animals were pre-treated with a topically-applied vasoconstrictor before irradiation. In this study, we have initiated a Phase IIa clinical investigation to determine whether orotopical vasoconstrictor application conferred the same resistance to radio-damage in normal oral mucosa of patients receiving whole-body radiation prior to bone marrow transplantation. Patients and Methods: In two years, 58 patients received the cyclophosphamide +TBI conditioning regimen. A clinical trial (NCT02434146) was approved by the University of Wisconsin Review Board to test the ability of five increasing doses of the NG-11-1 topical vasoconstrictor formulation to suppress the incidence and severity of OM. For a control cohort, records of five patients who received the cyclophosphamide+TBI regimen at UW were analyzed, records including weights, daily OM scores, use of total parenteral nutrition (TPN) were collected for comparison to the NG-11-1 treatment group. For the "topical NG-11-1 treated" group, each patient received the lowest of the five projected concentrations of NG-11-1 vasoconstrictor. The study physician directed 30 spray pulses to the buccal, lingual and sub-lingual surfaces. The formulation contains a blue coloring that enables directing and judging drug coverage. Orotopical NG-11-1 was applied: i) 15-20 min before IV cyclophosphamide was initiated, ii) 25-30 min after IV cyclophosphamide was initiated, and iii) 15-20 min before every 1.5 Gy irradiation. Pain score and OM score were collected before and after treatments and during follow-up clinic re-visits for 2 weeks. Results: The mean of "Grade 3 oral mucositis duration (days)" in NG-11-1 vasoconstrictor drug treated patients was significantly lower (P=0.047) than that of control patients. And, the mean of "total mucositis duration (days)" in NG-11-1 treated patients was significantly lower (P=0.043) than the level of control patients. Thirdly, either nasogastric feeding or total parenteral nutrition was required in 60% of control patients, however, none of the NG-11-1-treated patients required this intervention. Conclusions: These clinical results showing significant suppression of OM severity are consistent with the same degree of NG-11-1 dose-dependent suppression of radiation-induced oral mucositis that we observed in our previous mouse OM model studies.

#4161

Protecting Sphingomyelin Phosphodiesterase Acid Like 3B (SMPDL3b) enhances kidney function and reduces concurrent chemoradiotherapy-induced nephrotoxicity.

Anis Ahmad, Alla Mitrofanova, Saba Ansari, Junwei Shi, Yidong Yang, Sandra Merscher, Alessia Fornoni, Youssef Zeidan, Brian Marples. _UM, MIAMI, FL_.

Background: Cisplatin is a widely used radiosensitizer. Dose-related and cumulative renal insufficiency, including acute renal failure, are the primary dose-limiting toxicities of cisplatin. The lipid-modulating enzyme sphingomyelin phosphodiesterase acid-like 3B (SMPDL3b) and Sphingosine 1-Phosphate Receptor-1 (S1P1) are critical determinants of renal podocyte injury. In the current study, we investigated the functions of SMPDL3b and S1P in cisplatin and radiation-induced renal injury, and the use of rituximab (RTX) to attenuate nephrotoxicity.

Material/Methods: Podocytes were incubated for 2 h with S1P (100 nM) or vehicle (2% dodecane/98% ethanol) and irradiated with 0-8 Gy, or podocytes were irradiated (8 Gy) in the presence of RTX (100 mg/ml) or IgG (100 mg/ml). In other experiments, 10-14-week old C57BL/6 mice were given bilateral kidney X-irradiation (4 Gy) using an image-guided small animal arc radiation treatment system (iSMAART), 6mg/kg cisplatin or combined cisplatin plus radiation. Some animals received a single intravenous (IV) injection of rituximab mAb or IgG (50 mg/kg) prior to treatment. Functional kidney parameters and immuno-histopathological assessments of renal damage (e.g. oil red staining, H & E, Periodic Acid-Schiff (PAS) and SMPDL3B expression) were measured post-RT. iSMAART dynamic contrast-enhanced (DCE) imaging was used for estimating glomerular filtration rate 20 weeks post radiation.

Results: Exogenous S1P rescued podocytes from concurrent chemoradiotherapy-induced cellular cytoskeletal remodeling, and RTX pretreatment mitigated podocytopathy in vitro. iSMAART dynamic contrast-enhanced (DCE) imaging analysis showed that rituximab pretreatment improved the GFR and kidney function parameters post concurrent chemoradiotherapy. SMPDL3b expression at the protein level and podocyte counts dropped significantly in irradiated kidney post-RT in cisplatin-treated mice. Rituximab pretreatment in cisplatin-treated mice, improved GFR, kidney functional parameters, vascular structure, normalization of pericyte coverage, suppress the development of fibrosis and tubular damage post-irradiation.

Conclusion: This study shows that rituximab pretreatment protects mice against concurrent chemoradiation induced kidney injury. SMPDL3b and Sphingosine 1-Phosphate Receptor-1 may be important therapeutic targets for radiation-induced kidney injuries in cancer patients.

#4162

Lentivirally delivered shRNA knockdown of CXCL12 is effective at preventing radiation fibrosis in normal tissues.

James T. Paget,1 Martin McLaughlin,1 Joan N. Kyula,1 David Mansfield,1 Henry G. Smith,1 Victoria Roulstone,1 Paul A. Harris,2 Alan A. Melcher,1 Kevin J. Harrington,1 Aadil A. Khan1. 1 _Institute of Cancer Research, London, United Kingdom;_ 2 _The Royal Marsden NHS Trust, London, United Kingdom_.

Late Adverse Effects (LAEs) following adjuvant radiotherapy (RT) are common sequelae in free flaps used for breast reconstruction and result in ongoing healthcare burdens during the phase of cancer survivorship.

Intra-arterial delivery of gene therapy to an isolated vascular region permits localized, targeted genetic modulation of reconstructed tissue, avoiding potential off-target effects in microscopic residual disease.

We studied the immunological component of LAE development in a validated accelerated rodent model recapitulating autologous flap reconstruction, across 90 days after RT (50 Gy/3). CXCL12 was significantly up-regulated from 2-21 days after RT, peaking at 7 days (p<0.001), and corresponded to an increase in CXCR4 expression (p=0.047). Multiplexed confocal imaging co-localised CXCL12 and γH2AX in fibroblast stromal cells after RT, confirming that CXCL12 was a flap-originating signal suitable for our therapy modality. This was associated with increased macrophage ingress and fibroblast activation as characterized by immunohistochemical (p=0.002) and flow cytometric end-points (p=0.035). In vitro work corroborated the relevance of the CXCL12 axis in the development of LAEs, demonstrating significant increase in CXCL12's transcriptome expression (p=0.013) and protein secretion (p=0.03) in primary rat fibroblasts stimulated with RT, high expression of CXCL12's receptors, CXCR4 and CXCR7 on M1 and M2 rat macrophages, and increased fibroblast activation, assayed with αSMA expression, with co-stimulation of CXCL12 and TGFβ.

We hypothesized that LAEs in animals with flap-targeted knockdown of CXCL12 would be reduced. Flaps were infected with vascularly-delivered, lentiviral particles encoding shRNA against CXCL12 (LVshCXCL12), scrambled controls (LVSCR) alongside sham (phosphate-buffered saline (PBS)) and un-irradiated controls.

Flaps infected with LVshCXCL12 exhibited reversal of LAE-mediated flap contracture and were not significantly different from un-irradiated flaps at 90 days post-RT, but were significantly greater in area compared to sham flaps or those infected with LVSCR (p<0.0001). Radiation Therapy Oncology Group (RTOG) LAE scores were also significantly improved compared with LvSCR (p<0.0001) and PBS groups (p=0.0011). Endpoint assays demonstrated significant vascular preservation (p=0.049) and reduced tissue collagen deposition (p=0.019).

In conclusion, targeted knockdown of CXCL12 is effective at preventing the development of LAEs in flap tissue exposed to RT and is a promising strategy for clinical translation.

#4163

Space radiation-induced persistent DNA damage and alterations in cytoskeletal remodeling pathways affect intestinal epithelial cell migration in mice.

Santosh Kumar, Shubhankar Suman, Albert Fornace, Kamal Datta. _Georgetown Lombardi Comp. Cancer Ctr., Washington, DC_.

Lombardi Comprehensive Cancer Center, and Department of Biochemistry and Molecular & Cellular Biology Georgetown University, Washington, DC 20057, USA.

Heavy ion space radiation is considered as a risk factor of gastrointestinal (GI) cancer and perturbed intestinal epithelial cell (IEC) migration has been implicated in GI carcinogenesis. The purpose of the current study was to assess rate of IEC migration as well as dissect key IEC migration signaling events after exposure to heavy ion 56Fe irradiation and compare the results to sham- and γ-irradiation. Male C57BL/6J mice were exposed to 0.5 Gy of either γ or 56Fe radiation and control mice were sham irradiated. IEC migration distance was measured 7 and 60 d after exposure using BrdU pulse labeling. We also analyzed cell polarity, microtubule dynamics, and adhesion dynamics using immunoblots and immunofluorescence. Measure of IEC migration rate showed a greater decrease after 56Fe relative to γ radiation at both the time points. At the molecular level, compared to sham and γ radiation, 56Fe radiation exhibited significantly greater impairment of cell adhesion dynamics evidenced by altered integrins and Rock1 as well as cytoskeletal dynamics evidenced by altered MLCK expression. Similarly, perturbed cell polarity, tight junction, and microtubule dynamics was revealed by the deregulation of Par3, Claudin1, Scribble, Occludin, ZO-1, phospho-Tau and MAP1B. Also, the Wnt/β-catenin and EphB/EphrinB pathways critical for IEC migration consistently showed greater deregulation at both the time points tested after 56Fe radiation relative to γ -rays. Taken together, our molecular findings correlate with the BrdU pulse labeling data, and demonstrate greater effects of heavy ion 56Fe on IEC migration relative to γ -rays. In summary, our results have demonstrated for the first time that radiation can disrupt cell migration dynamics through alteration of signaling molecules involved in crucial cellular processes and these results have implications for heavy ion space radiation-induced gastrointestinal homeostatic deregulation and carcinogenesis.

#4164

Fasting protects mice from lethal radiation by promoting small intestinal stem cell survival.

Marimar de la Cruz Bonilla, Kristina M. Stemler, Tara N. Fujimoto, Sabrina Jeter-Jones, Jessica M. Molkentine, Gabriela M. Asencio Torres, Cullen M. Taniguchi, Helen Piwnica-Worms. _MD Anderson Cancer Center, Houston, TX_.

Pancreatic cancer is the fourth leading cause of cancer-related deaths in the US. Surgical resection is the only potentially curative treatment; however, only 15%-20% of patients present with tumors that can be resected. There is no consensus regarding standard of care in unresectable cases, however many academic centers use stereotactic body radiotherapy (SBRT) to give tumor-directed radiotherapy (RT). Unfortunately, even this conformal technique can still cause severe gastrointestinal (GI) toxic effects caused by the proximity of the pancreatic head to the duodenum. Protecting the intestine from the toxic effects of radiation may enable dose escalation that could achieve more effective local control of disease. We and others have previously shown that a prolonged fast of 24 hours protects mice from lethal doses of etoposide. In this study, we extend and build on our previous finding to demonstrate that a similar 24 hour fast also protects from lethal doses of total abdominal radiation. Histologic analyses, using the Withers-Elkind microcolony assay, show that fasting protected small intestinal (SI) stem cells from radiation damage and promoted early regeneration. To show a proof-of-principle for the use of this radioporotective maneuver in cancer therapy, we developed an orthotopic model of pancreatic cancer using KPC tumor cells syngeneic to C57BL/6. Here, we show that fasting-mediated intestinal protection enabled dose escalated SBRT for treatment of these orthotopic tumors. RT with fasting radioprotection delayed tumor growth and improved survival compared to controls. Given this robust phenotype, we developed a 3D culture ex vivo assay using intestinal stem cell enriched epithelial spheroid cultures. We modified these intestinal spheroids with a bioluminescent reporter and used these cells to develop a modified clonogenic assay for 3D culture that can be used to identify novel radioprotectors, such as a fasting mimetic. Taken together, these results suggest that fasting protects small intestinal stem cells sufficiently to allow animals to receive potentially curative doses of abdominal radiation that would other wise be lethal. Future work will aim to identifying the mechanism by which fasting confers intestinal protection and drug candidates that can be used to mimic this fasting-mediated protection.

#4165

Novel genetic rat models to identify factors that modulate cardiac and tumor radiation sensitivity.

Rachel A. Schlaak,1 Anne Frei,1 Aronne M. Schottstaedt,2 Brian L. Fish,1 Leanne Harmann,1 Tracy Gasperetti,1 Michael J. Flister,1 Meetha Medhora,1 Jennifer L. Strande,1 Carmen R. Bergom1. 1 _Medical College of Wisconsin, Milwaukee, WI;_ 2 _Case Western Reserve University, Cleveland, OH_.

Purpose/Objectives: Over 50% of breast cancer patients receive radiation therapy, but radiation doses can be limited by normal tissue toxicity. Radiation therapy can improve breast cancer-specific survival, but cardiac morbidity can be increased in patients with left-sided tumors. We used rat genetic models to identify targets to improve the therapeutic ratio of radiation. We assessed the radiation responsiveness of mammary tumors and the heart in genetically similar consomic rats conducive to genetic mapping.

Materials/Methods: Female SS rats and SS.BN3 consomic rats, which are genetically identical to SS rats except that chromosome 3 is inherited from the BN strain, have previously been shown to exhibit different vascular dynamics and breast tumor growth. Human MDA-MD-231 cells or syngeneic mammary tumor cells developed from DMBA-induced mammary tumors were implanted orthotopically into immunodeficient or immunocompetent SS and SS.BN3 rats, respectively, and tumors were treated locally with mock or 5x4 Gy. To examine cardiac toxicity, adult female SS and SS.BN3 rats received image-guided localized whole-heart radiation to a dose of 24 Gy or 9 Gy x 5 (AP and 2 lateral fields, weighted 1:1:1). Echocardiograms with strain analysis were performed at baseline, 3 months and 5 months. The Student's t-test was used to compare values.

Results: The BN strain-derived genetic variant(s) on rat chromosome 3 is important for tumor radiation sensitivity. Tumors in SS.BN3 rats were significantly more radiosensitive than tumors in the parental SS strain. A supra-additive effect was seen with both tumor cell lines, with recurrence-free survival of 30% vs. 67% at 137 days in SS vs SS.BN3 rats (p=0.02) in xenografts, and recurrence-free survival of 9% vs. 100% at 141 days in SS vs. SS.BN3 rats (p<0.0001) in syngeneic tumors. The SS female rats that received 24 Gy exhibited enhanced cardiac toxicity compared to SS.BN3 rats, with larger pericardial effusions in SS vs SS.BN3 rats (p<0.05), significantly elevated end diastolic volume (EDV) and end systolic volume (ESV) at 5 months (EDV: 0.62 vs. 0.49 mL, p<0.01; ESV: 0.12 vs. 0.03 mL, p<0.01), and increased cardiac mortality (5/11 SS vs. 0/7 SS.BN3 rats). Fractionated heart radiation yielded similar results. Taken together, the SS.BN3 tumors are more sensitive to radiation, while the hearts of SS.BN3 rats are protected against radiation toxicity, when compared to the SS strain.

Conclusions: These results demonstrate that genetic variants on rat chromosome 3 alter the sensitivity to radiation therapy, enhancing tumor responses to radiation and protecting the heart, thus improving the therapeutic ratio. Gene expression analysis and genetic mapping will be performed to identify the causative target(s). This project has the potential to enhance the effectiveness and toxicity profile of radiation therapy in breast cancer.

#4166

Hair follicles as a minimally invasive resource of epithelial cells in mouse studies of ionizing radiation.

Hanus Slavik,1 Martin Mistrik,1 Pavlina Duskova,1 Tomas Furst,2 Josef Srovnal,1 Marian Hajduch1. 1 _Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital in Olomouc, Olomouc, Czech Republic;_ 2 _Faculty of Science, Palacky University and University Hospital in Olomouc, Olomouc, Czech Republic_.

Introduction

Hair follicles are relatively accessible as a biological material allowing repeatable and minimally invasive sampling of predominantly epithelial cells for biomarker studies. The utility of such a material is promising for example in murine experiments, where repeated sampling would require multiple biopsies. As a proof of concept, the ionizing irradiation response in murine follicular cells was chosen as a suitable and common external factor for studying cellular senescence, apoptotic pathways, neoplastic transformation, tumor development and response to cancer therapy.

Methods and material

Hair follicles were collected from 30 healthy C57Bl/6 mice into the RNA later for RT-qPCR and into the 4% formaldehyde for immunofluorescence with specially developed vacuum collector. Follicles were collected before X-ray irradiation and 30 min, 3 h, 6 h and 24 h after irradiation. Each group of 10 mice obtained one dose (2, 6 or 10 Gy). RNA was extracted from the samples using miRNeasy mini kit (Qiagen) and two-tube RT-qPCR was performed for SESN1, p21, MDM2 and HPRT gene expression analysis. Immunofluorescence was done for γ-H2AX and quantitatively evaluated by in-house developed software routine.

Results

Total RNA extracted from one pluck was usually less than 35 ng but it had good quality (RIN ≥ 7.5) even after the storage at room temperature. We determined p21 as the best marker for the uniformity and specific dynamic respond for each of the doses. The strongest responds were found after 30 min with 2 Gy (average fold change FCav = 3,24). In case of higher doses, longer remaining p21 overexpression was detected. For instance, FCav = 2,29 even after 24h at dose 10 Gy. Our data were corroborated by immunofluorescence for direct marker of ionizing radiation induced DNA damage γ-H2AX, where 1-3 hair follicles proved to be efficient for microscopic analyses. Strong γ-H2AX signal was detectable within 3h after irradiation followed by rapid decrease within the next 24h.

Conclusion

Collected murine hair follicles proved to be a feasible alternative material for molecular analyses which in logistic aspects outreaches phlebotomies or skin biopsies because of repeatable, minimally invasive and accurate sampling and non-laborious processing.

Dedication

This work was financially supported by grants IGA_LF_2017_013, EATRIS-CZ, TACR TE02000058 and VG20102014001.

#4167

MUC3A promotes lung cancer progress via activating NFκB pathway and attenuates radiosensitivity.

Yingming Sun, Chengcheng You, Shijing Ma, Yuan Luo, Yan Gong, Yu Xiao, Junhong Zhang, Conghua Xie. _Zhongnan Hospital of Wuhan University, Wuhan, China_.

MUC3A is highly expressed in 40% of lung adenocarcinoma, but its functions and effects on clinical outcomes are not well understood. Tissue microarray of 92 patients' tumor samples from Chinese National Human Genetic Resources Sharing Service Platform indicated that highly expression of MUC3A was associated with poor prognosis, advanced staging and low differentiation. IP-MS and co-IP results revealed that MUC3A interacted with RELA and activated the NFκB pathway via promoting RELA phosphorylation and interfering the binding of RELA to IκB. MUC3A knockdown significantly suppressed cell proliferation and induced G1 arrest via down-regulating cell cycle checkpoints such as CDK2/4/6 and Cyclin D1. MUC3A deficiency increased γ-H2AX phosphorylation and increased radiation-induced DNA double strain breaks. MUC3A knockdown inhibited the BRCA-1/RAD51 pathway and nuclear translocation of P53 and XCRR6, suggesting that MUC3A promote DNA damage repair and attenuate radiation sensitivity. Moreover, MUC3A deficiency induced autophagy in lung cancer cells and increased the protein levels of LC3 II and Beclin-1. MUC3A knockdown significantly suppressed tumor growth in H1975 xenograft model and had a synergistic effect with radiation. Less nuclear translocation of RELA and P53 was also observed in tumor tissue in vivo. Our studies indicated that MUC3A was a potential oncogene and its highly expression was associated with unfavorable clinical outcomes in patients with lung adenocarcinoma, who should be more frequently follow-up, and might benefit less from radiotherapy.

#4168

Radioresistant anaplastic thyroid cancers display a unique gene signature and respond to hypofractionated radiotherapy.

Ayman J. Oweida, Andy Phan, Benjamin Vancourt, Tyler Robin, David Raben, Bryan Haugen, Nikita Pozdeyev, Rebecca Schweppe, Sana D. Karam. _University of Colorado Denver - Anschutz Medical Campus, Aurora, CO_.

Purpose: Patients diagnosed with anaplastic thyroid (ATC) cancer display heterogeneous tumor responses to radiation therapy. It remains unclear how the molecular profiles of ATC subtypes mediate a radiosensitive or radioresistent phenotype. Furthermore, the optimal fractionation for ATC remains controversial. In this study, we sought to identify genetic determinants of tumor radioresistance in ATC subtypes. We further compared the role of hypofractionated radiotherapy to conventional fractionation in a novel orthotopic model of ATC.

Methods: Eight human ATC and PDTC cell lines (C643, SW1736, BCPAP, T238, CAL62, KHM-5M, 8505C, and KTC-2) were profiled using microarray analysis. Clonogenic survival assays were performed for each cell line. Irradiation (IR) was performed at 0, 2, 4, 6, and 8 Gy and cells were incubated for 8-10 days post-IR. Data were fitted to the linear quadratic model. Cell lines were classified as radioresistant (RR) or radiosensitive (RS) based on SF2 and SF4 values. Hierarchical clustering was performed to determine the genomic profile of RR and RS cell lines. For in vivo studies, mice were surgically implanted with the luciferase-tagged 8505C ATC cell line. Mice were randomized on the day of treatment initiation (day 14-15) based on BLI photon radiance signal to receive hypofractionated (HF), conventional fractionation (CF) or no irradiation. A total dose of 20Gy was delivered in 2 weekly doses for the HF group and daily 10 doses for the CF group.

Results: We identified five radioresistant cell lines and three radiosensitive cell lines. Microarray gene-expression analysis revealed an inflammatory gene signature associated with radioresistance. In particular, CXCR4 was increased by more than 2-fold in RR cell lines. CXCR4 has been shown to play a role in mediating tumor growth, metastasis and resistance to therapy and its targeting has shown efficacy in various tumor animal models. Longitudinal analysis of weekly BLI revealed significant differences in tumor growth in the HF group compared to the control and CF groups. On the last day in which all mice were alive (day 36), average photon radiance in the HF group was significantly lower compared to mice in the control group (125-fold decrease) and mice in the CF group (16-fold decrease). In addition to its effect on primary tumor growth, HF resulted in significantly improved survival compared to mice in the control and CF groups.

Conclusions: Our study provides evidence that hypofractionated radiotherapy is superior to conventional radiotherapy in a radioresistant model of ATC. Hypofractionated RT significantly retarded tumor growth and prolonged survival. Gene-expression analysis revealed targetable pathways that may be associated with radioresistance. These findings have direct clinical implications for enhancing treatment outcomes and survival in ATC patients.

#4169

Inhibition of EphB4-ephrin-B2 signaling results in enhanced sensitivity to cetuximab-radiation therapy in head and neck cancers.

Shilpa Bhatia, Jaspreet Sharma, Sanjana Bukkapatnam, Ayman Oweida, Shelby Lennon, Parkash Gill, Antonio Jimeno, David Raben, Lynn Heasley, Sana D. Karam. _University of Colorado Denver, Aurora, CO_.

Introduction: The management of locally-advanced head and neck cancer patients relies on the combination treatment involving 7 weeks of radiation therapy (RT) with Cetuximab, an EGFR-inhibitor. Only a fraction of HNSCC patients, however, show response to cetuximab-radiation with an estimated 5-year overall survival of 46% compared to 36% with radiotherapy alone. This suggests potential involvement of redundant or compensatory tumor survival pathway that comes into action and blunts the therapeutic response. Based on our previous findings and published reports, elevated expression of EphB4, a member of Eph family of receptor tyrosine kinases and its membrane-bound ligand, ephrin-B2 might play a role in mediating this effect. In the current study, we hypothesized that EphB4-ephrin-B2 favors pro-tumorigenic survival signaling by altering the sensitivity to EGFR inhibitor and radiation. Therefore, combined inhibition of EphB4-ephrin-B2 signaling is critical to enhance sensitization of HNSCC towards anti-EGFR and radiation therapy.

Materials and methods: To test the underlying hypothesis, we used a blocking protein targeting EphB4-ephrin-B2 axis in the absence and presence of EGFR inhibitor with radiation in HNSCC cell lines and patient-derived xenograft (PDX) model. Alteration in the protein levels in control and treatment groups was detected by Western blotting and immunohistochemistry. Circulating levels of cytokines was analyzed in vivo by Mesoscale U-plex assay. Tumor tissues were subjected to human PhosphoRTK array to assess changes in the activation status of downstream targets of EphB4-ephrin-B2 signaling mediating response in the triple combination groups.

Results: Our data from locally advanced HNSCC patients treated with standard of care definitive Cetuximab-RT show high levels of both EphB4 and ephrin-B2 after failure of Cetuximab-RT. We observed significant tumor growth delay and increased response towards Cetuximab and radiation therapy following EphB4-ephrin-B2 inhibition resulting in better overall survival in PDX tumors and HNSCC cells. The tumor growth inhibition effects observed in vivo was accompanied by decrease in the levels of proliferation marker, angiogenic regulators, and increased apoptosis. Changes in levels of phopsho-receptor kinases and in circulating immune profile was also evident in tumors subjected to triple combination strategy.

Conclusions: Overall, our data suggest that EphB4-ephrin-B2 and EGFR pathway cross-talk with each other to circumvent the therapeutic response resulting in uncontrolled tumor growth, apoptotic evasion, and immune escape. Therefore, development of combinatorial approaches targeting Eph-ephrin family of proteins might show promising outcome to enhance sensitivity towards anti-EGFR therapeutics and radiation in head and neck cancers.

#4170

The RNA helicase, DDX3, modulates DNA damage repair in Ewing sarcoma.

Marwa Afifi,1 Breelyn A. Wilky,2 Catherine Kim,1 Venu Raman,1 David Loeb3. 1 _Johns Hopkins school of medicine, Baltimore, MD;_ 2 _Sylvester Comprehensive Cancer Center, FL;_ 3 _Albert Einstein College of Medicine, NY_.

Introduction: Ewing sarcoma (ES) is a primary bone sarcoma occurring in adolescents and young adults, accounting for 15% of childhood/adolescent cancer. We previously reported that an RNA helicase, DDX3, regulates the translation of a variety of proteins in ES cells, including those modulating DNA damage repair. Therefore, we hypothesized that inhibiting DDX3, using a small molecule called RK-33, would radio-sensitize ES cells by preventing DNA damage repair.

Methods: ES cell lines (MHH-ES and TC71) were transfected with DDX3- targeted shRNA or a control, scramble shRNA. RT-PCR and western blotting confirmed knockdown of DDX3. RK-33 cytotoxicity was evaluated using cell viability assay. DNA double strand breaks (DSB) were quantified by immunofluorescent staining for γH2AX, and subcellular localization of DDX3 was determined by immunofluorescence. ES patient derived xenografts (PDX) were subcutaneously implanted in 4 cohorts of NSG mice.

Results: Expression of DDX3 mRNA and protein was decreased in DDX3-knockdown cells compared with controls. To validate that DDX3 is inhibited by RK-33, we compared the cytotoxicity of this agent in control cells and in the DDX3 knockdown cell lines. Parental ES and control cells (MHH-ES, TC71, MSD-10) were more sensitive to RK-33, with IC50 values that were significantly lower than their corresponding DDX3-knockdown lines (M2C7, M1F6; 4 μM, 2.7 μM and 2.6 μM vs 42 μM and 11.6 μM, respectively, p<0.05). To determine the effect on DNA damage repair, DSB were quantified in DDX3- knockdown cells and controls, treated with or without RK-33, at various time points after irradiation. At early time points, treatment with RK-33 had no impact on the number of ES and control cells (MHH-ES, TC71, MSD-10) with >10 γH2AX foci, but after 24 hours, the average number of irradiated cells with >10 foci was significantly higher in RK-33-treated cells than in cells treated with vehicle alone (p<0.01). The time needed for full recovery of radiation-induced DSB in untreated cells was less than 6 hr, while RK-33 pre-treated cells failed to recover even after 24 hr. Irradiated DDX3-knockdown cells demonstrated impaired DNA damage repair as well. To expand our investigation into the mechanism by which DDX3 regulates DNA damage repair, we studied the subcellular localization of the enzyme in response to radiation. We found that DDX3 translocates into the nucleus and colocalizes with γH2AX in irradiated cells, suggesting a dual role, direct and indirect, in DNA damage repair. In vivo, we found that animals treated with subtherapeutic external beam radiation concurrently with RK-33 had a substantial increase in tumor response. Our results suggest that DDX3 plays a crucial role in regulating DNA damage repair dynamics in ES, both indirectly by affecting the translation of involved proteins, and directly by localizing to sites of DSB. Therefore, inhibiting DDX3 using RK-33 may represent a novel approach to radiosensitization of cancer cells.

#4171

Preferential radioresistance of glioblastoma stem-like tumor cells that migrate to the olfactory bulb.

Cindy R. Timme, Barbara H. Rath, Kevin Camphausen, Philip J. Tofilon. _NCI-NIH, Bethesda, MD_.

Brain tumor xenografts initiated from human glioblastoma (GBM) stem-like cells (GSCs) simulate many of the characteristics of GBMs in situ including extensive invasiveness and migration. Using a GSC orthotopic brain tumor model, we tested the hypothesis that location within the brain microenvironment influences the radiosensitivity of GBM cells. At 35 days after implantation of CD133+ NSC11 GSCs into the right striatum of nude mice, tumor cells are detectable throughout the striatum, the corpus callosum and the olfactory bulb. To evaluate radioresponse of tumor cells in each brain region, mice were injected with the halogenated thymidine analog CldU (5-chloro-2'-deoxyuridine), which is incorporated into DNA during S-phase and allows for the identification of proliferating cells. Specifically, NSC11 brain tumor xenografts received a single dose of 10Gy, which results in an approximate 13-day survival advantage, followed by 3 daily intraperitoneal (IP) injections of CldU initiated every 4 days following irradiation. Subsequent immunohistochemical analyses of CldU incorporation along with the GSC marker SOX2 was then used to determine the percentage of proliferating tumor cells in the corpus callosum, striatum, and olfactory bulb. When CldU injections were initiated 4 days post-10Gy, 4% of tumor cells in the corpus callosum, 0.75% in the striatum, and 35% in the olfactory bulb were CldU positive. These values can be compared to 55%, 53%, and 56% CldU positive tumor cells in the corpus callosum, striatum, and olfactory bulb, respectively, of the sham irradiated tumors. These data indicate that by 4 days after irradiation significantly more tumor cells in the olfactory bulb were proliferating than in the corpus callosum or striatum. CldU injections initiated 12 days post-10Gy resulted in 7% of tumor cells in the corpus callosum, 4% in the striatum, and 60% in the olfactory bulb being CldU positive. Finally, when CldU delivery was begun 20 days post-10Gy approximately 17% of tumor cells in the corpus callosum, 15% in the striatum, and 85% in the olfactory bulb were CldU positive. These data suggest that after irradiation, GBM tumor cells located in the olfactory bulb recover and begin to proliferate sooner than those in the striatum or corpus callosum. As a measure of radiation-induced DNA double strand breaks, γH2AX foci were quantified in the tumor cells located in the corpus callosum, striatum, and olfactory bulb on day 35 post-implantation of NSC11 cells. At 0.5h after irradiation, no difference was detected in the mean number of γH2AX foci per tumor cell in the corpus callosum, striatum, or olfactory bulb; no consistent differences were detected out to 6h post-irradiation. While the mechanism remains to be defined, analysis of tumor cell proliferation indicates a preferential radioresistance of GBM cells located in the olfactory bulb.

#4172

Radiation-induced blood-tumor barrier permeability in a preclinical model of lung cancer brain metastasis.

Samuel A. Sprowls, Neal Shah, Afroz S. Mohammad, Marc Pinti, Rachel M. Tallman, Devin U. John, Paul R. Lockman. _West Virginia University, Morgantown, WV_.

BACKGROUND: Lung cancer has the highest mortality rate among all cancers and accounts for over half of newly diagnosed brain metastases each year. Standard therapeutic advancements are prolonging lives of lung cancer patients with each new approved formulation, however patients with brain metastases have limited options and no curative measures currently exist. Current treatment modalities for brain metastases from primary lung cancer include differing methodologies of radiotherapy (whole brain radiotherapy and/or stereotactic radiosurgery), systemic chemotherapies, and neurosurgery. Radiation in many cases is not ablative, but palliative. Herein we demonstrate the ability of radiotherapy to influence blood-tumor barrier permeability in a novel preclinical model of lung cancer brain metastasis.

METHODS: Athymic female nude mice were intracardially injected with our novel PC-9-BR brain specific lung cancer cell line. Tumors were allowed to develop and monitored using bioluminescent imaging. Using the Xstrahl XenX small animal irradiator, we were able to deliver 26 Gy to the right hemisphere of the immobilized mouse. 4 hours post radiotherapy, mice were perfused with the passive permeability marker Oregon Green, followed by an indocyanine green (bound to albumin) washout to infer vascular space. Brains were then extracted, snap frozen in isopentane (-50°C), and sliced to 20μm. Florescence was quantified using a MVX microscope and cellsense software.

RESULTS: Analysis of irradiated brain regions demonstrated statistically significant more accumulation of the passive permeability marker, Oregon Green, compared to the contralateral hemisphere.

CONCLUSIONS: Radiation induced permeability was witness in mice after a single dose of 26 Gy when compared to non-irradiated tumor tissue 4 hours post treatment.

FURTURE DIRECTIVES: Experiments utilizing ionizing radiation to simulate treatment of CNS metastases could reveal better adjunctive chemotherapeutic regimens and or provide insight to the mechanism underlying this enhanced permeability.

#4173

Inhibition of EphB4-ephrinB2 signaling increases efficacy of radiation in pancreatic ductal adenocarcinoma.

Shelby Lennon,1 Shilpa Bhatia,1 Ayman Oweida,1 Jaspreet Sharma,1 Andy Phan,1 Sanjana Bukkapatnam,1 Nomin Uyanga,1 Todd Pitts,1 Stephen Leong,1 Karyn Goodman,1 David Raben,1 Elena Pasquale,2 Jorge Martinez-Torrecuadrada,3 Sana Karam1. 1 _University of Colorado Anschutz Medical Campus, Aurora, CO;_ 2 _Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA;_ 3 _Centro Nacional de Investigaciones Oncologicas, Madrid, Spain_.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer related death in the United States. Survival rates are dismal, at only 8% five year survival. Radiation is one treatment option for PDAC, although historically its efficacy has been controversial. Our research goal is to improve the efficacy of radiation for PDAC patients. An ancillary study of the recent LAP07 trial showed that patients with neutrophilia were more resistant to chemoradiation. Additionally, when patients with systemic inflammation were excluded, one year local control was 80% in the chemoradiation arm compared to 54% in the chemotherapy arm. The EphB4 receptor and ephrinB2 ligand have been shown to play a role in neutrophil mobilization from the bone marrow and infiltration into melanoma tumors. We hypothesize that inhibition of EphB4-ephrinB2 signaling will enhance the efficacy of radiation in PDAC by reducing inflammation by neutrophils.

Materials/Methods: To study tumor growth delay, Athymic nude mice were implanted in the flank with a patient derived xenograft (PDX) model of PDAC. To inhibit EphB4-ephrinB2 signaling, mice were administered with 20mg/kg of B11, a human single chain Fv antibody fragment against ephrinB2. Tumors were irradiated with 12Gy and volume was monitored by caliper. For mechanistic studies, C57/BL6 mice were injected in the flank with 10^6 FC1242 cells, which are derived from the pancreata of KPC mice. To inhibit EphB4-ephrinB2 signaling, mice received a hydrodynamic tail vein injection of 20ug/2mL TNYL-RAW-Fc plasmid. TNYL-RAW is a peptide antagonist of EphB4, and it is fused to human Fc. Tumors were irradiated with 12Gy and harvested four days later for flow cytometry analysis. Blood was also collected at various time points and analyzed by blood film white blood cell differential and flow for neutrophils. Plasmid transduction efficiency was confirmed by analyzing plasma samples collected at different time-points on an Fc protein-based ELISA assay.

Results: We observed a significant tumor growth delay in PDX implanted mice who received B11 and radiation. An ELISA for human Fc confirmed transduction of the TNYL-RAW-Fc plasmid in FC1242 tumor bearing mice. Flow analysis on FC1242 tumors showed a significant decrease in percentage of infiltrating neutrophils when treated with TNYL-RAW and radiation, and blood film white blood cell differential and flow analysis on blood showed a significant decrease in circulating neutrophils in the same mice.

Conclusions: Our findings support the hypothesis that EphB4-ephrinB2 signaling contributes to inflammation by neutrophils in PDAC, and inhibiting this signaling will increase the efficacy of radiation. This data can contribute toward a clinical trial design for combination therapy of EphB4-ephrinB2 inhibition and radiation in patients with pancreatic ductal adenocarcinoma.

#4174

Differential response of non-small cell lung cancer harboring different epidermal growth factor receptor mutations to ablative radiation therapy.

Areej Al Rabea,1 Brian Meehan,1 Paul Daniel,1 Siham Sabri,1 Chaitanya Nirodi,2 Janusz Rak,1 Bassam Abdulkarim1. 1 _McGill University, Montreal, Quebec, Canada;_ 2 _USA Mitchell Cancer Institute, AL_.

Background: ablative radiation therapy (ABR) serves as the treatment of choice for early stage non-small cell lung cancer (NSCLC) patients who are not surgical candidates. NSCLC patients with mutations in the tyrosine kinase domain (TKD) of the epidermal growth factor receptor (EGFR) had a significant response to tyrosine kinase inhibitors (TYIs). The most common mutations of EGFR in NSCLC are present in the TKD domain and include: deletion (DEL) in the exon 19 and a missense mutation (L858R) in the exon 21. The role of extracellular vesicles (EVs) has been under extensive investigation due to its contribution in preparing the distant site through a process named pre-metastatic niche formation. Release of irradiation-induced EVs in EGFR mutated NSCLC and their responses to ABR have not been well investigated. We aim to assess EVs release and tumor growth of NSCLC harboring different EGFR mutations post- ABR. Materials and methods: We used A549 that were transduced with different EGFR status: EGFR-WT (WT), EGFR-DEL (DEL) or EGFR-L858R (L858R) and irradiated them at 0, 12 or 34Gy. The condition media were then collected at 24hrs post-irradiation and used to measure release of extracellular vesicles (EVs) using nanosight. We transduced the cells with lentivirus expressing luciferase. Cells were irradiated at 0Gy (Ctrl group) or 34Gy (IR group) and injected subcutaneously in yellow fluorescent protein -severe combined immunodeficiency (YFP-SCID) mice. Tumor volume and animal weight were measured regularly and bioluminescence imaging (BLI) was used to evaluate tumor growth and metastasis. Results: L858R-expressing cells had an increase in EVs release post-ABR (12 and 34Gy), compared to WT-expressing cells which did not have difference in EVs release following ABR. DEL-expressing cells had an increase in EVs release only at 34Gy. Furthermore, in vivo data revels that ABR caused a decrease in tumor growth of IR-WT and IR-DEL groups when compared to Ctrl-WT and Ctrl-DEL, respectively. Interestingly, both Ctrl-L858R and IR-L858R groups presented similar tumor growth. Further investigations are undergoing assessing the EVs release and their function in the occurrence of distant metastasis post-ABR. Conclusion: in our study, we report a differential response of non-small cell lung cancer to ABR that could be caused by the differences in EGFR status. As a result, the standard use of ABR should not only be based on the patients' comorbidity status, but should also be based on his/her genetic background in order to determine the optimal treatment. 

### Therapeutic Approaches to Metastasis

#4175

Endoscopic stent insertion for obstructive colorectal cancer may induce tumor cell dissemination into the peripheral circulation, resulting in viable circulating tumor cell detection.

Shinya Yamashita,1 Masahiro Tanemura,2 Yosuke Shimizu,3 Toshio Kuwai,3 Yasuo Urata,4 Kiyomi Taniyama3. 1 _Nissay hospital, Osaka, Japan;_ 2 _Osaka Police Hospital, Osaka, Japan;_ 3 _National Hospital Organization, Kure Medical Center and Chugoku Cancer Center, Kure, Japan;_ 4 _Oncolys BioPharma Inc., Tokyo, Japan_.

The placement of a self-expanding metallic stent (SEMS) in obstructive colorectal cancer (OCRC) is acknowledged to be a safe and effective procedure for the relief of obstruction. However, there is concern that shear forces acting on the tumor during stent expansion may release cancer cells into the circulation, resulting in distant metastasis. The aim of the present study was to determine whether colonic stent insertion increases viable circulating tumor cells (v-CTCs) in the peripheral blood. For detecting v-CTCs of colorectal cancer (CRC), we employed a novel method, TelomeScan F35 detection system. The system was constructed with a GFP-expressing attenuated adenovirus, in which the telomerase promoter regulates viral replication to detect only viable cancer cells. 7.5 ml of peripheral blood samples were obtained before/after stent insertion and after operation. GFP-positive and CD45-negative cells were counted as v- CTCs. This study was a single institution trial, approved by the Kure Medical Center IRB (No. 25-40). Between October 2013 and June 2015, 8 patients, aged 62-85 years (5 males and 3 females) were enrolled. Median follow-up was 28.1 months (range 15.5-34.6 months). All patients underwent successful SEMS insertion. Five patients had an Eastern Cooperative Oncology Group performance status of 0, and the remaining 3 patients had performance status of 1 or 2 (PS1 1, PS2 2). OCRCs were located in the sigmoid colon in 5 patients, descending colon in 2 patients, and transverse colon in 1 patient. Four patients were diagnosed as TNM stage II, and 4 patients were diagnosed as stage III. Stent insertion was performed as a bride to surgery in 7 patients and for palliation in one patient. Surgical resection (R0) was performed in 7 patients. No patients required a permanent stoma and have any complications. Four patients had no v-CTCs before SEMS placement, two of four measurable patients had an increased number of v-CTCs after SEMS placement (1-3 v-CTCs), and one of two patients with increased v-CTCs developed distant lymphatic metastasis 24 months after surgery despite curative resection and receiving systemic chemotherapy with UFT+LV. Four patients had v-CTCs (1-19 cells) before SEMS placement, and two of these four patients had an increase in the number of v-CTCs (20-21 cells) after SEMS placement, while one of the four patients died early with distant metastasis. In conclusion, the present study demonstrated that endoscopic stent insertion may result in tumor cell dissemination into the peripheral circulation and may induce early distant metastasis. Although stenting has some advantages as a BTS in OCRC, the oncological risk and long-term prognosis of this approach have not been clarified. The conclusions of this study may open a window of opportunity for raising an alarm about SEMS placement in OCRC.

#4176

Abrogation of desmoglein 2-mediated c-met activation as a novel therapeutic strategy for reducing anaplastic thyroid cancer metastasis.

Dong G. Lee, Jeong-Ki Min. _Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea_.

Anaplastic thyroid cancer (ATC) is a fast growing and highly metastatic cancer with limited therapeutic options. Thus, targeted therapies need to be developed. This study aims to determine desmoglein 2 (Dsg2) expression in ATC and to elucidate its biological role and potential as a therapeutic target in ATC. Dsg2 expression was low or abnormal in ATC tissues. The ATC patients with reduced Dsg2 expression also had distant metastasis. Dsg2 depletion significantly increased cell migration and invasion, with relatively little effect on ATC cell proliferation in vitro, and increased distant metastasis in vivo. Dsg2 knockdown induced cell motility through the hepatocyte growth factor receptor (HGFR, c-Met)/Src/Rac1 signaling axis, with no alterations in the expression of EMT-related molecules. Further, specific targeting of c-Met significantly inhibited the motility of shDsg2-depleted ATC cells. Decreased membrane expression of Dsg2 increases the metastatic potential of ATC cells. Dsg2 plays an important role in migration and invasiveness in cell lines derived from ATC. Therapies targeting c-Met might be effective for ATC patients with decreased membrane expression of Dsg2. Thus, analysis of Dsg2 expression may provide a novel therapeutic strategy for ATC treatment.

Keywords Anaplastic Thyroid Cancer, Desmoglein 2, Metastasis, c-Met, Tivantinib

#4177

An IL13Rα2 peptide inhibits colorectal cancer metastatic progression.

Ignacio Casal, Marta Jaén, Rubén A. Bartolomé. _Centro de Investigaciones Biológicas (CIB), Madrid, Spain_.

Interleukin 13 receptor α2 (IL13Rα2) is an excellent tumoral marker for metastatic colorectal cancer and glioblastoma. Our goal was to block IL-13 binding to IL13Rα2 by using IL13Rα2 synthetic peptides in order to delay or suppress metastatic spread and progression. Peptides were selected from the D1 binding region.

Synthetic peptides were synthesized using solid phase chemistry. Two metastatic colorectal cancer cell lines (KM12SM and SW620) plus two glioblastoma cell lines were used for testing proliferation, adhesion, migration and invasion. We used Biacore assays to demonstrate direct interaction between the peptides and IL-13. In vivo experiments with nude mice were performed to study mice survival to metastasis after peptide treatment.

Peptides containing the highly preserved WKTIITKN motif of the D1 binding domain of IL13Rα2 demonstrated a strong capacity to inhibit migration, invasion and proliferation in metastatic colorectal cancer and glioblastoma cells. Alanine scanning experiments indicated that, at least, residues 82K, 83T, 85I and 86T were essential for IL-13 binding to IL13Rα2. IL13Rα2 D1 peptide inhibited IL-13-mediated cell signaling through FAK, Src, AKT and ERK1/2 in colorectal cancer and glioblastoma cell lines. Moreover, the D1 peptide abolished ligand-mediated receptor internalization and degradation. To improve bioavailability and resistance to proteolytic digestion for in vivo administration, we prepared the D enantiomer version of the peptide. Mice inoculated with metastatic colorectal cancer cells and treated with the enantiomer version of the D1 peptide showed a remarkable increase in survival.

In conclusion, the D1 enantiomer peptide blocked the IL-13/IL13Rα2 binding, paving the way to develop novel and promising therapeutic strategies to inhibit metastatic progression in colorectal cancer and other solid tumors, including glioblastoma.

#4178

AKT-ERK axis in circulating tumor cells is a target for metastatic suppression.

Hung-Chi Cheng, Ying-Jan Wang, Li-Hsin Cheng. _National Cheng Kung Univ., Tainan, Taiwan_.

Cytotoxic cancer therapies often result in unwanted side effects, drug resistance, and cancer metastasis. To avoid this, an anti-metastatic therapeutic strategy independent of apoptosis is alternatively desired. Malignant circulating tumor cells (CTCs) may escape from anoikis by sustaining AKT survival signals. Moreover, cancerous fibronectin polymers (polyFN) are known to mediate the adhesion of CTCs to endothelia and lung metastasis. Depletion of polyFN from CTCs without lowering AKT signaling may thus be an ideal anti-metastatic approach. Screening several phytochemical compounds, we found that Pterostilbene (PS), a resveratrol derivative, potently reduced polyFN on suspended tumor cells, whereas it significantly elevated AKT phosphorylation (pAKT), apparently in contrast to its well-known anti-apoptotic effect on adherent tumor cells. Inactivation of ERK signaling in suspended tumor cells was responsible for the enhanced pAKT by PS, leading to suppressions of polyFN assembly and tumor metastasis. Pretreatment with a PI3K inhibitor, LY294002, effectively reduced PS-activated AKT, rescued PS-inactivated ERK, and consequently reversed the PS-suppressed polyFN assembly which was then overturned by the ERK inhibitor U0126. Indeed, PS-suppressed lung metastasis was counteracted by LY294002, which was reversed with U0126. Dietary PS may serve as a novel complementary medicine, for it could be orally administered and efficiently blockaded tumor metastasis by targeting AKT-ERK signaling axis in CTCs.

#4179

A novel approach for antimetastatic therapies against TNBC utilizing a physiologic suppressor.

Ali Ekrem Yesilkanal,1 Daniel C. Rabe,1 Payal Tiwari,1 Casey Frankenberger,1 Gary L. Johnson,2 Marsha Rosner1. 1 _Univ. of Chicago, Chicago, IL;_ 2 _University of North Carolina, Chapel Hill, NC_.

Metastatic progression of tumors is the major cause of death in patients with triple-negative breast cancer (TNBC). However, since metastasis is a multistep process, unraveling its complexity is a major challenge. One effective way of tackling this question is to study natural blockers of the metastatic process, metastasis suppressors, and identify the mechanisms by which they regulate metastasis. Raf kinase inhibitory protein (RKIP), a protein that regulates kinase activity, is a suppressor of TNBC metastasis. Although RKIP inhibits the activity of key kinases such as Raf-1, GRK2, NIK/IKK in cultured cells, the kinase targets of RKIP in tumors are not known. To address this question, we used a mass spectrometry approach involving inhibitor-conjugated beads to identify kinases that are downregulated by RKIP in human TNBC xenograft tumors. Our results identified a network of stress kinases targeted by RKIP, including kinases that have not been previously reported as RKIP targets. In order to unravel the effect of this stress network on metastatic gene expression, we investigated genes that correlate with RKIP expression in TCGA breast cancer patient data set. We identified prometastatic genes such as APC and DOCK4 as novel RKIP targets in in vitro and in vivo models of TNBC. We also demonstrated these genes are downstream of the RKIP-stress network. Finally, by using a high-throughput invasion assay, we developed a low-dose multidrug cocktail of small-molecule kinase inhibitors that mimic RKIP's antimetastatic role in TNBCs. Elucidating RKIP function at a systems level reveals the interplay between key metastatic signaling cascades, particularly in relation to cell motility and invasion. Our findings suggest that the low-dose multidrug combination that targets a network of stress kinases is a viable antimetastatic therapy for TNBC patients.

#4180

Game-changing restraint of Ros-damaged phenylalanine, upon tumor metastasis.

Nicolás Anselmino,1 Paula Chiarella,2 Emiliano G. Ortiz,1 Daiana B. Leonardi,1 Felipe M. Jaworski,1 Sofia Lage Vikers,1 Jimena Giudice,3 Anna Woloszynska-Read,4 Roberto P. Meiss,5 Raul Rugiero,2 Geraldine Gueron,1 Elba S. Vazquez1. 1 _Univ. of Buenos Aires, CABA, Argentina;_ 2 _IMEX-CONICET, CABA, Argentina;_ 3 _School of Medicine University of North Carolina, Chapel Hill, NC;_ 4 _Medical Oncology The University of Texas, M.D. Anderson Cancer Center, Houston, TX;_ 5 _Institute of Oncological studies (IEO)-National Academy of Medicine, CABA, Argentina_.

An abrupt increase in metastatic growth as a consequence of the removal of primary tumors suggests that the concomitant resistance (CR) phenomenon might occur in human cancer. CR occurs in murine tumors and ROS-damaged phenylalanine, meta-tyrosine (m-Tyr), was proposed as the serum anti-tumor factor primarily responsible for CR. Herein, we demonstrate for the first time that CR happens in different experimental human solid tumors (prostate, lung anaplastic, and nasopharyngeal carcinoma). Moreover, m-Tyr was detected in the serum of mice bearing prostate cancer (PCa) xenografts. Primary tumor growth was inhibited in animals injected with m-Tyr. Further, the CR phenomenon was reversed when secondary implants were injected into mice with phenylalanine (Phe), a protective amino acid highly present in primary tumors. PCa cells exposed to m-Tyr in vitro showed reduced cell viability, downregulated NFκB/STAT3/Notch axis, and induced autophagy; effects reversed by Phe. Strikingly, m-Tyr administration also impaired both, spontaneous metastasis derived from murine mammary carcinomas (4T1, C7HI, and LMM3) and PCa experimental metastases. Altogether, our findings propose m-Tyr delivery as a novel approach to boost the therapeutic efficacy of the current treatment for metastasis preventing the escape from tumor dormancy.

#4181

Simultaneous inhibition of the Wnt/Β-catenin and Ras pathways by KY1022 effectively suppresses tumor initiation and progression of colorectal cancer.

Eun Ji Ro, Jeong-Ha Hwang, Yong-Hee Cho, Kang-Yell Choi. _Yonsei University, Seoul, Republic of Korea_.

APC (80-90%) and K-Ras (40-50%) mutations frequently occur in human colorectal cancer (CRC) and these mutations cooperatively accelerate tumorigenesis including metastasis. In addition, both Β-catenin and Ras levels are highly increased in CRC, especially in metastatic CRC (mCRC). Targeting these signaling pathways may therefore be an ideal strategy for treating mCRC; however, therapeutics targeting both of these pathways have not been developed because the mechanisms regulating these pathways are not well understood. Based on our recent identification of the Β-catenin destruction complex-mediated Ras protein stability mechanism, we screened a small-molecule library to identify compounds that destabilize both Β-catenin and Ras proteins via inhibition of the Wnt/Β-catenin pathway. Among the effective small molecules destabilizing both Β-catenin and Ras proteins via inhibition of the Wnt/β-catenin pathway, we characterized KY1022, a small molecule that most effectively inhibits EMT, an initial process of metastasis. As shown by in vitro and in vivo studies using APCMin/+/K-RasG12DLA2 mice, KY1022 effectively suppressed the development of mCRC at an early stage of tumorigenesis. Our results suggest that a small molecular approach degrading both Β-catenin and Ras via inhibition of the Wnt/Β-catenin signaling would be an ideal strategy for treatment of CRC including mCRC.

#4182

Impacting early events in metastatic spread: Targeted therapeutics to disrupt formation of the pre-metastatic niche and development of lung metastases.

Khalid A. Mohamedali, Lawrence H. Cheung, Michael G. Rosenblum. _UT MD Anderson Cancer Ctr., Houston, TX_.

There is now growing interest in understanding the early events which lead to the successful formation of solid tumor metastatic spread because this information may lead to early interventional approaches. Bone marrow derived cells (BMDCs) of various lineages are gradually being identified as central to the ongoing growth of the primary tumor as well as the successful development of metastasis. Upon distant stimulation from the primary tumor, BMDCs migrate from the bone marrow to organ sites and self-assemble into a pre-metastatic niche that hosts migrating tumor cells from the primary site. Thus, disruption of this niche or selective targeting the migratory tumor cells may inhibit metastatic spread. Using GFP+ BMDC's from genetically engineered mice, we characterized the role that recruitment of BMDCs may play in breast cancer metastasis. We assessed the BMDC recruitment profile in lethally irradiated female nu/nu mice transplanted with GFP+ BMDCs from donor mice, followed by orthotopic placement of MDA-MB-231/luc cells or injected with MDA-MB-231/luc conditioned media for 30 days. Flow cytometry results show a gradual increase in the recruitment of CD11b+VEGFR-1+ cells in all the tissues examined from tumor-bearing mice, particularly lungs and lymph nodes. Recruitment of these cells to the liver in mice treated only with MDA-MB-231/luc conditioned media was also apparent and at higher levels than to livers of mice with the orthotopically-placed tumors. Significant recruitment of CD11b+VEGFR-2+ BMDCs was observed to liver, lung and lymph nodes. Interestingly, MDA-MB-231/luc conditioned media appeared to recruit this subset of cells more strongly to liver and lung tissues than did signaling from orthotopic tumors, while spleen and lymph node showed less recruitment of CD11b+VEGFR-2+ BMDCs. Treatment with GrB/VEGF fusion construct - a novel granzyme B-based pro-apoptotic fusion protein which specifically targets cells harboring VEGFRs - indicated that GrB/VEGF normalized recruitment of VEGFR-1+ or VEFGR-2+ cells to lungs when assessed two weeks after the final treatment. GrB/VEGF treatment also increased recruitment of F4/80+ macrophages to the lung, including those with the CD11b+Gr-1- signature, which were otherwise significantly reduced. GrB/VEGF also impacted the BMDC profile of several cell lineages in the primary tumor. Studies are ongoing to understand the impact of GrB/VEGF systemic administration on lung metastasis. Identifying key players in modulating the formation of the pre-metastatic niche and the early development of metastasis will be critical in designing targeted therapeutic approaches to inhibit the metastatic process. Research conducted, in part, by the Clayton Foundation for Research.

#4183

Gold nanorods-assisted photothermal therapy inhibits metastasis by decreasing the mammary tumors bleeding in canine and felines.

Moustafa Ragab Khalil Ali. _Georgia Inst. of Technology, Atlanta, GA_.

Metastasis enables cancer cells to migration, which is responsible for most cancer-related deaths. It usually begins with local invasion to the surrounding tissues, followed by intravasation into the lymph and blood microvasculature before the cancer cells finally colonize within the microenvironment of other locations in the patient's body. Recently, we discovered that gold nanorods assisted photothermal therapy (AuNRs-PPTT) inhibits cancer cell migration and prevent metastasis. AuNRs-PPTT is a therapy in which AuNRs are injected to a tumor before near-infrared light is transiently applied to the tumor producing localized heat. However, AuNRs-PPTT has great potential in replacing the current ineffective anti-metastasis treatments but most of the studies has been performed in vitro. The objectives of this study to understand the AuNRs-PPTT impacting blood and blood vessels in naturally occurring mammary glands tumor in cats and dogs. Our histopathology results showed AuNRs-PPTT caused infiltration of neoplastic cells into lymphatic vessels. Furthermore, we observed that AuNRs-PPTT significantly cause blood coagulation around the tumor, which could be incorporated in the surgery to decrease the bleeding and potentially avoiding the risk of metastasis caused by the surgery. This observation might explain why AuNRs-PPTT inhibited metastasis for mammary gland metastasis upon AuNRs-PPTT. After 24 months, the optimized doses of 2.5 nm of AuNRs irradiated by near-Infrared laser with 0.5 w/cm2 intensity for 2 minutes, have shown no any toxicity signs in liver and kidney functions.

#4184

Novel parathyroid hormone antagonist prevents breast cancer bone metastasis and cortical bone destruction.

Murali Anbalagan,1 Tulasi Ponnapakkam,2 Yibin Kang,3 Robert C. Gensure,4 Brian G. Rowan1. 1 _Tulane Univ. School of Medicine, New Orleans, LA;_ 2 _Xavier University, New Orleans, LA;_ 3 _Princeton University, Princeton, NJ;_ 4 _Tufts Medical Center, Boston, MA_.

The majority of patients (~70%) with advanced breast cancer will develop bone metastases and suffer from severe pain, bone fracture and eventually death. Current palliative treatments have only limited efficacy highlighting an urgent need for development of targeted agents. Bone metastatic breast cancer cells secrete parathyroid hormone related peptide (PTHrP) that promotes autocrine tumor growth, induces bone turnover that releases additional tumor stimulatory factors, and creates cavities in the bone that permit tumor outgrowth. Previous PTH/PTHrP antagonists failed clinically due to short half-life and inadequate targeting to bone. We created a novel bone-targeted PTH antagonist drug, PTH(7-33)-CBD, by fusing an N-terminally truncated analog of PTH (aa 7-33) with the collagen binding domain (CBD) from ColH collagenase (Clostridium histolyticum). Our previous preliminary study demonstrated in vivo efficacy of PTH(7-33)-CBD against tumor burden and cortical bone destruction using an established model of breast cancer bone metastasis by injecting a bone-trophic variant of estrogen receptor-negative MDA-MB-231 breast cancer cells expressing luciferase (MDA-MB-231-BM/luc+) into the tibia marrow of nude mice. PTH(7-33)-CBD treatment did not result in hypocalcemia or reduce animal body weight. We extended this previous study by using increased animal numbers, administration of a control drug, and assessment of apoptosis of breast cancer cell in vitro by PTH(7-33)-CBD. PTH(7-33)-CBD reduced PTH agonist-stimulated cAMP accumulation in SaOS-2 osteosarcoma cells by 71% confirming the PTH antagonist activity of the compound. PTH(7-33)-CBD exhibited a direct cytotoxic effect towards MDA-MB-231 breast cancer cells in vitro by increasing apoptosis as assessed by the Caspase Glow Assay. To assess in vivo efficacy, on Day 0 nude mice were treated IP with either vehicle, 1000 µg/kg control drug PTH-7-34 (without CBD domain), or 1000 µg/kg PTH(7-33)-CBD (N=8/treatment group). On Day 1 mice were injected with 2x106 MDA-MB-231-BM/luc+ cells into the tibia marrow and tumor burden and bone density were assessed weekly by bioluminescence imaging and X-ray imaging, respectively. PTH(7-33)-CBD significantly reduced tumor burden in bone from weeks 4-8 post-tumor cell inoculation compared to vehicle (P<0.05), and from weeks 6-7 compared to PTH-7-34 control drug (P<0.05). PTH(7-33)-CBD reduced cortical bone destruction from weeks 3-7 weeks compared to both vehicle and PTH-7-34 control drug (P<0.05). Taken together, these data demonstrate that PTH(7-33)-CBD reduces both bone metastatic breast tumor burden and osteolytic lesions in nude mice, and induces apoptosis of MDA-MB-231 breast cancer cells in vitro.

Support: RCG and BGR are co-principal investigators for Breast Cancer Research Program (BCRP) Breakthrough Award, BC150685.

#4185

The novel Rac inhibitor HV-107 as a potential therapeutic for metastatic breast cancer.

Grace E. Velez Crespo,1 Eliud Hernández,2 Cornelis Vlaar,2 Suranganie Dharmawardhane,3 Linette Castillo-Pichardo1. 1 _UCC, Bayamon, PR;_ 2 _UPR School of Pharmacy, San Juan, PR;_ 3 _UPR Medical Sciences Campus, San Juan, PR_.

Breast cancer is the first cause of death in women globally. Metastatic breast cancer, which occurs when cancer cells migrate from the primary tumor and establish in distant organs, is estimated to affect more than a quarter of a million of women in the US. Regrettably, metastatic breast cancer still lacks effective treatment and remains the principal cause of breast cancer mortality. Therefore, it is imperative to develop new and more effective strategies to inhibit metastasis. A promising target for anti-metastatic cancer therapy is the Rho GTPase Rac because it plays a key role in the regulation of cellular adhesion, migration, proliferation and survival; processes which promote tumorigenesis and metastasis. Recently, our group developed Ehop-016, a small molecule that inhibits Rac in metastatic breast cancer cells with an IC50 of 1µM and reduces the activity of its downstream effector p21-activated kinase (PAK). Ehop-016 also inhibits tumor growth, angiogenesis, and metastasis in a mouse model of metastatic breast cancer. However, its relative bioavailability is moderate and should be further improved. Therefore, in an effort to find a compound with greater inhibitory capacity and bioavailability, we tested several Ehop-016 derivatives for their activity against Rac and their toxicity towards metastatic breast cancer cells. Using Rac pulldown assays we show that HV-107 at 250nM inhibits Rac activation by 55% in MDA-MB-231 and MDA-MB-435 metastatic breast cancer cells. The effects of HV-107 on cell viability were tested by MTT assays and our results indicate HV-107 at ≥500nM significantly inhibits metastatic breast cancer cell viability, while showing minimal toxicity towards non-cancerous mammary epithelial cells. We also measured caspase 3 activity, in order to evaluate apoptosis as a potential mechanism of HV-107-induced cell death. Our results show HV-107 at 1000nM causes a significant 1.5 fold increase in caspase 3 activity in MDA-MB-231 cells, indicating HV-107 affects cell viability by inducing apoptosis. Finally, we tested HV-107 in a mouse model of metastatic breast cancer and found a 25% reduction in tumor growth in mice treated with 5mg/kg BW HV-107. Taken together, our results suggest HV-107 is approximately four times more efficient than the parent compound Ehop-016 and has potential as anti-breast cancer metastasis therapeutic.

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

#4186

Monensin is selectively cytotoxic to cancer cells in an EMT-like state.

Marion Vanneste, Huang Qin, Devon Moose, Mengshi Li, Michael Schultz, Meng Wu, Michael Henry. _University of Iowa, Iowa City, IA_.

Metastatic, drug-resistant cancer is lethal. Epithelial-to-mesenchymal transition (EMT) is a type of phenotypic plasticity that has been implicated in cancer metastasis and drug resistance. One potential therapeutic approach is to specifically target cancer cells in an EMT-like state. In this study, we have developed a cell-based high-throughput screening (HTS) protocol using the high content Operetta imaging platform to identify EMT cytotoxic compounds. This screen was performed on epithelial (PC-3E) and mesenchymal-like (TEM 4-18) cell lines, both isolated from the parental PC-3 prostate cancer cell line based on their differential ability to invade an endothelial monolayer. A library of 2,640 compounds was screened on co-cultured PC-3E GFP cells and TEM 4-18 mCherry cells. After 72h exposure to compounds, relative numbers of GFP- and mCherry-positive cells were quantitated. Dose-response curves were established for each compound exhibiting a greater toxicity against EMT-like cells. The mechanisms of action of one of the most potent and selective compound against EMT-like cells, monensin, were further evaluated. Among the 2,640 compounds tested, salinomycin and monensin, both monovalent cation ionophores, displayed a potent and selective cytotoxic effect against EMT-like cells. A closely related compound absent from the compound library, nigericin, was also evaluated and appeared to be the most potent of the three compounds tested. Monensin (10nM) induced apoptosis, cell cycle arrest, and an increase in ROS production in TEM 4-18 cells. In addition, monensin rapidly induced swelling of Golgi apparatus, a previously known effect of monensin, in multiple cell lines most likely resulting in a blockage of intracellular protein trafficking and cell death. We then evaluated the toxicity of monensin in 24 human cancer cell lines and classified them as resistant or sensitive based on IC50 cutoff of 100nM. Supporting these findings, Gene Set Enrichment Analysis identified EMT as the top of the gene sets enriched in the sensitive group. Increased monensin sensitivity in EMT-like cells is associated with elevated uptake of H3-monensin compared to resistant cells. In conclusion, using a high-throughput screening approach, we identified monensin as a potent and specific inhibitor of cancer cells in an EMT-like state where it rapidly disrupts Golgi function. At least part of selectivity of monensin for EMT-like cells is due to increased uptake of this compound.

#4187

Targeting the cytoskeleton protein ezrin sensitizes metastatic breast cancer cells to anthracycline based chemotherapy.

Victoria Hoskin, Abdi Ghaffari, Xiaolong Yang, Yolanda Madarnas, Sandip SenGupta, Sonal Varma, Peter A. Greer, Bruce E. Elliott. _Queen's Univ., Kingston, Ontario, Canada_.

The main cause of cancer-associated deaths is the spread of cancer cells to distant organ sites. Despite recent advances in treating primary tumors, modern chemotherapeutic strategies are relatively ineffective at treating metastasis, with clinical trials showing minimal improvements in overall survival for patients with metastatic disease. This is in large part due to chemotherapy resistance which remains a major clinical challenge limiting therapeutic responses for metastatic cancer patients. The cytoskeleton crosslinker protein ezrin has been shown to promote cancer metastasis in multiple preclinical models and is associated with poor prognosis in several cancer types, including breast cancer (BC). Ezrin also promotes pro-survival signaling, particularly in disseminated cancer cells, to facilitate metastatic outgrowth. However, whether ezrin plays a role in chemoresistance in BC is not yet known. In this study, we sought to determine whether ezrin can predict response to chemotherapy in BC patients and whether pharmacologic inhibition of ezrin alters the sensitivity of metastatic BC cells to anthracycline-based chemotherapy in preclinical models of metastasis. Ezrin protein expression was assessed in a BC patient cohort by tissue microarray immunohistochemistry (IHC) using the automated quantitative platform HaloTM. Among patients treated with systemic chemotherapy across all prognostic groups, high ezrin levels were associated with reduced disease-free, distant metastasis-free, as well as overall survival, compared to patients with lower ezrin levels. Next, we sought to determine whether targeting ezrin using a small molecule inhibitor (NSC668394) could enhance the efficacy of systemic doxorubicin treatment in vivo. Using an experimental lung metastasis model, we showed that the addition of NSC668394 sensitized metastatic BC cells to doxorubicin treatment, compared to either agent alone. We also tested the efficacy of these agents in targeting microscopic metastasis using neoadjuvant and adjuvant treatment models. Our results show that in both treatment modalities, NSC668394 or doxorubicin treatment alone was not able to reduce metastasis, however the addition of the ezrin inhibitor markedly sensitized metastases to doxorubicin and reduced overall lung metastatic burden. Taken together, our data suggest that ezrin may be a novel predictive marker of treatment response in BC patients and provide rationale for potential targeting of ezrin in patients with metastatic disease as an adjunct to chemotherapy. (Supported by OMPRN, CRS and BCAK).

#4188

Preoperative supplementation of branched-chain amino acids suppresses Osteopontin expression in patients with hepatocellular carcinoma.

Yukihiko Hiroshima, Yasuhiro Yabushita, Yutaro Kikuchi, Daisuke Kawagushi, Takashi Murakami, Yasushi Ichikawa, Itaru Endo, Kuniya Tanaka. _Yokohama City University, Yokohama City, Japan_.

Background: Recently, the new effect of branched-chain amino acid (BCAA) on Osteopontin (OPN), which correlates with cancer progression, has been reported. The aim of this study was that preoperative administration of BCAA could improve prognosis via degradation of OPN expression in hepatocellular carcinoma (HCC).

Methods: Human hepatocellular carcinoma cell lines were treated with BCAA (2nM), and investigated OPN expression, cell proliferation and invasion in vitro. Next, patients undergoing elective liver resections for HCC (n=72) were randomized to receive or not receive preoperative oral supplementation with BCAA. OPN protein levels in HCC patients were examined using immunohistochemistry and compared with the clinicopathologic features of the tumors.

Results: Experimentally, the expression levels of OPN were significantly decreased in HCC cells treated with BCAA. BCAA treatment significantly decreased cell invasion. In the clinical setting, OPN expression correlated with HBV or HCV infection (p = 0.03), venous invasion (p = 0.02) and liver fibrosis (p = 0.07). There was no difference in both DFS and OS between OPN-positive and OPN-negative group, and preoperative BCAA administration did not improve prognosis, either. For the patients without late recurrence, OPN-positive group showed significantly poor outcome in DFS (p = 0.047), and preoperative BCAA administration improved DFS (p = 0.062).

Conclusions: OPN expression was associated with cancer cell invasion and intrahepatic metastasis. BCAA treatment suppressed HCC cell invasion and preoperative BCAA administration prevented early intrahepatic recurrence by decreasing the expression level of OPN in HCC.

#4189

Cationic polymer inhibits pancreatic cancer invasion and metastasis.

Ibtehaj A. Naqvi,1 Ruwan Gunaratne,1 Jessica McDade,2 Douglas Rouse,1 Jen Jen Yeh,3 David Pisetsky,1 Jaewoo Lee,1 Rebekah White,4 Bruce Sullenger1. 1 _Duke University Medical School, Durham, NC;_ 2 _University of Washington, Seattle, WA;_ 3 _University of North Carolina Chapel Hill, Chapel Hill, NC;_ 4 _University of California San Diego, San Diego, CA_.

Background and Hypothesis: Pancreatic cancer (PC) has the poorest prognosis of all major cancers, with a 5-year survival of less than 5%. Cell-free DNA (cfDNA) has been well validated as a biomarker that correlates with tumor burden. More recently, a role for cfDNA in tumor progression has also been identified. The latter occurs through activation of Toll-like receptors (TLRs) in both tumor cells and the host environment, which can upregulate prometastatic signaling pathways. In addition, tumor-derived extracellular vesicles (EVs) such as microparticles and exosomes have also been implicated in promoting metastasis by activating proinvasive pathways in tumor cells and preconditioning secondary sites for metastatic establishment. Our laboratory has previously shown that cationic polymers can neutralize cfDNA and abrogate inflammation in disease models of autoimmunity and infection. We investigated the ability of the cationic polymer, PAMAM-G3, to bind and inhibit cfDNA and EVs and thereby inhibit tumor invasion in vitro and metastasis in vivo.

Methods and Results: Transwell-Matrigel invasion assays were performed using multiple PC cell lines. Cells were plated in the upper chamber with no serum +/- cfDNA, EVs, or PAMAM-G3. The bottom chamber was plated with complete media as a chemo-attractant. Cells treated with cfDNA, EVs, or PAMAM-G3 were also analyzed for activation of pro-inflammatory pathways (NFkB, MAPK, etc.). The in vivo model was performed in C57B6 mice that were injected with bioluminescent murine PC cells into their spleens and followed for liver metastases with bioluminescent imaging. Experimental mice were treated biweekly with intraperitoneal PAMAM-G3 (20 mg/kg) or saline starting 48 hours after pancreatic tumor cell implantation. PAMAM-G3 significantly inhibited in vitro invasion of pancreatic cancer cell lines in response to cancer patient serum-derived cfDNA and extracellular vesicles. Moreover, PAMAM-G3 (20 mg/kg) treatment led to a significant reduction in liver metastasis without affecting primary tumor growth in vivo. Serum derived from saline-treated mice induced higher levels of invasion than serum from polymer-treated mice and exogenous polymer blocked this effect (**** = p<0.0001, *** = p<0.001, ** = p<0.01, NS = not significant by t-test).

Discussion: Cationic polymers such as PAMAM-G3 may represent a novel class of therapeutics to combat pancreatic cancer metastasis. These polymers can bind and neutralize cfDNA and tumor-derived EVs, thereby blocking activation of pro-inflammatory pathways within tumor cells, and reducing tumor invasion and metastasis. Cationic polymers will be tested in other preclinical models of cancer and in dosing studies for potential progression to clinical translation.

#4190

Synergistic anticancer effect of sulforaphane and cisplatin on sirtuins-mediated epithelial-to-mesenchymal signaling in triple negative breast cancer.

SONAM SINHA, Neeta Shrivastava. _B.V. Patel Pharmaceutical Education & Research Development (PERD) Centre, Ahmedabad, India_.

Purpose:

Triple negative breast cancer (TNBC) overexpresses sirtuins (SIRTs), a class III HDACs, which induces epithelial-to-mesenchymal (EMT) transition and facilitates Cisplatin (CIS) resistance. We hypothesized that Sulforaphane (SFN), an HDAC inhibitor, when combined with CIS, could potentiate CIS efficacy against TNBC metastasis and overcome its resistance.

Material and Methods:

The effect of SFN and CIS combination on cellular proliferation and apoptosis of two TNBC cell lines, MDA-MB-231 and MDA-MB-468 were assessed by MTT assay and Annexin and PI method followed by FACS analysis respectively. The anti-metastatic capabilities of the compounds were evaluated using wound healing assay, invasion assay, chemotaxis assay, colony formation and tumorsphere assay. The regulation of key proteins involved in sirtuin-mediated EMT signaling and metastasis was determined by zymography and western blot analysis.

Results:

The results revealed that SFN in combination with CIS synergistically inhibited TNBC cells through inhibition of cellular proliferation, induction of apoptosis, in-silico and in-vitro modulation of SIRTs (1-7) expression. Anti-metastatic capabilities of the combinatorial treatment regimen were associated with inhibition of migratory, invasive, mammosphere forming potential and downregulation of matrix metallopeptidase (MMP-9 and MMP-2) activity of TNBC cells. When TNBC cells were treated with SFN, the expression of epithelial marker, E-cadherin was up-regulated, while the expression of mesenchymal markers, ZEB1, SNAIL, TWIST and associated SIRTs were down-regulated. In contrast, when the cells were treated with CIS, the expressions of mesenchymal markers were up-regulated. Interestingly, the combinatorial treatment of SFN and CIS synergistically inhibited EMT transition in TNBC cells. In-vivo studies are warranted to confirm our results.

Conclusion:

Collectively our findings demonstrated a SIRTs-mediated EMT-suppression leading to synergistic antitumor effect by combination of SFN and CIS in TNBC cells. This combination therapy may serve as a novel therapeutic strategy for TNBC management.

#4191

Effect of a novel orally bioavailable CXCR4 inhibitor, AMD070, on the metastasis of oral cancer cells.

Nobuyuki Kuribayashi, Daisuke Uchida, Makoto Kinouchi, Kyoko Kuribayashi, Tomonori Hasegawa, Shuma Yagisawa, Hitoshi Kawamata. _Dokkyo University School of Medicine, Tochigi, Japan_.

We have previously demonstrated that the stromal cell-derived factor (SDF)-1/CXCR4 system is involved in the metastasis of head and neck cancer. Additionally, it has been shown that the blockade of CXCR4 by subcutaneous daily injection with AMD3100, a CXCR4 antagonist, may be effective in preventing metastasis in CXCR4-related head and neck cancer. Recent investigations have suggested that AMD070, a novel orally bioavailable inhibitor of CXCR4, may be minimally invasive compared with AMD3100. In the present study, we examined the effect of AMD070 on metastasis induced by the SDF-1/CXCR4 axis in B88-SDF-1 oral cancer cells, which express high levels of SDF-1 and CXCR4. Although treatment with AMD070 did not affect the anchorage-dependent growth of B88-SDF-1 cells, it significantly suppressed the anchorage-independent growth. Moreover, the SDF-1/CXCR4-dependent migration and invasion of B88-SDF-1 cells was significantly inhibited following treatment with AMD070. Subsequently, we performed an experimental therapy using AMD070 to prevent the distant metastasis of B88-SDF-1 cells in vivo. Daily oral administration of AMD070 significantly inhibited the lung metastasis of B88-SDF-1 cells in nude mice. Furthermore, AMD070 treatment did not induce body weight loss despite the tumor bearing-condition of the mice. In addition, we could not detect the apparent macroscopic organ abnormalities associated with the daily oral administration of AMD070. These results indicated that AMD070 could be useful as a novel orally bioavailable inhibitor of oral cancer metastasis.

#4192

C-terminal HSP90 inhibitor, L80, targets cancer stem cell (CSC)-like property and suppresses metastasis via JAK/STAT3 inactivation in triple-negative breast cancer.

Tae-Min Cho, Eunhye Oh, Daeil Sung, Yoon-Jae Kim, Seojin Jang, Jeewoo Lee, Ji Young Kim, Jae Hong Seo. _Korea University, Seoul, Republic of Korea_.

Purpose: Triple-negative breast cancer (TNBC) harbors high level of cancer stem cell (CSC)-like populations, which is associated with aggressive phenotype leading to tumor cell dissemination and poor clinical outcomes. Therefore, new drugs that effectively target both CSCs and tumor bulk cells are needed to improve clinical outcomes. The 90-kDa molecular chaperone heat-shock protein, HSP90 has been implicated in cancer progression and metastasis by modulating the stabilization and maturation of many oncogenic proteins. Our objective was to investigate the effects of L80 on cancer stem-like characteristics, cell proliferation and metastasis in TNBC in vitro and in vivo.

Experimental Design: The effect of L80 on TNBC cell lines in vitro was evaluated in terms of cell viability, apoptosis and mammosphere-forming ability and expression of JAK/STAT3 signaling-related factors. An orthotopic injection model with 4T1 mammospheres with self-renewability was used to examine the effect of L80 on tumor growth, angiogenesis and metastasis.

Results: L80 treatment caused a significant induction of apoptosis, accompanied by caspase-3/-7 activation and the dysregulation of JAK/STAT3 activation in TNBC cells. The latter phenomenon was associated with impairment of cancer stem-like trait, concomitant with a significant suppression of mammosphere formation. To confirm the physiological relevance of our in vitro findings, we examined the impact of L80 on allografted tumor growth, angiogenesis and metastasis in vivo. L80 administration impedes tumor growth and subsequently suppresses angiogenesis, as evidenced by a marked reduction in number of CD31-positive microvessels in the L80-treated groups compared to their counterparts. L80 administration leads to a significant reduction in lung and liver metastases, coinciding with decreased MMP-2 and MMP-9 levels in circulating blood. These responses were intimately associated with dysregulation of JAK/STAT3 activation.

Conclusion: C-terminal HSP90 inhibitor, L80 suppresses tumor growth and metastasis via suppression of JAK/STAT3 activity and CSC-like property. These findings suggest that HSP90 could be a promising target for treatment of metastatic TNBC.

#4193

Biochanin A in pancreatic cancer.

Vilas Desai,1 Satya Murthy Tadinada,2 James C K Lai,3 Alok Bhushan1. 1 _Philadelphia University + Thomas Jefferson Univ., Philadelphia, PA;_ 2 _University of Iowa, Iowa City, IA;_ 3 _Idaho State University, Pocatello, ID_.

Pancreatic cancer, with its dismally low survival rate (<5%), is one of the most aggressive forms of cancer and is the fourth leading cause of cancer-related deaths in the U.S. Gemcitabine, the first-line drug approved by the FDA, has been marginally effective as it improves survival by only six months. Epidemiologic studies have implicated a role for dietary isoflavones in lowering the incidence of different cancers in the Asian population. Isoflavones such as genistein, biochanin A and daidzein have been tested for their anticancer potential in different cancers; however, the clastogenic effect associated with genistein limits its therapeutic potential. Primarily used in management of hyperlipidemia and cardiovascular diseases, statins have been documented to exert antiproliferative effects. We hypothesized that a combination treatment of biochanin A and atorvastatin exerts enhanced anticancer effects on pancreatic cancer cells viz. AsPC1, PANC-1 and MIAPaCa-2. Our studies showed a decrease in cell viability on combination drug treatment with differential effects on mitogenic and cell signaling pathways in the cell lines investigated. In PANC-1 cells, biochanin A and atorvastatin combination treatment decreased invasiveness, dampened the expression of activated STAT3, and critical mediators of cell cycle progression. We also observed the involvement of enhanced proapoptotic effect of the combination treatment in AsPC-1 cell line and potential cell cycle arrest phenomenon in PANC-1 cells. Cell metabolism studies using seahorse XFp analyzer showed an increase in mitochondrial respiration and a decrease in glycolytic activity on combination treatment. Further, the XF-Plasma Membrane Permeabilizer (XF-PMP) assay used to evaluate the mitochondrial ETC complex activity showed a complete impairment of complex I and II (prominently), with MIAPaCa-2 being more susceptible to the combination treatment than AsPC1 and PANC-1. Together, the combination treatment-induced effects on pancreatic cancer cell lines may have potential implications in developing novel and improved therapies for the treatment of pancreatic cancer.

#4194

Tumor Treating Fields (TTFields) affect invasion properties and cell morphology of various cancer cells in vitro.

Rosa S. Schneiderman, Moshe Giladi, Einav Zeevi, Anna Shteingauz, Tali Voloshin, Yaara Porat, Mijal Munster, Eilon D. Kirson, Yoram Palti. _Novocure, Haifa, Israel_.

Tumor Treating Fields (TTFields) therapy is an approved modality for the treatment of glioblastoma. TTFields are delivered via continuous, noninvasive application of low intensity, intermediate frequency alternating electric fields. The antimitotic effects of TTFields have been extensively investigated. We explored other processes that may be affected by TTFields such as cellular invasion. Human glioma, human and mouse melanoma, human breast cancer and human colon cancer cell lines were treated with TTFields using the inovitro system. Invasion assays were performed using a modified Matrigel coated Boyden chamber. TTFields effect on cells size, focal adhesions and stress fibers morphology was studied using immuno-fluorescent confocal microscopy.

Invasion was significantly reduced compared to untreated cells in all tested cell lines.

Furthermore, the optimal frequency for inhibition of invasion in tested cells was 300 kHz while the optimal anti mitotic frequency leading to glioma cell death is 200 kHz. The inhibitory effect on migration was observed at electric fields intensities of 0.6 V/cm RMS which is below the 1 V/cm RMS threshold previously reported for induction of cell death. Combining TTFields with Bumetanide, an agent that inhibits cellular motility, resulted in further inhibition of glioma cell invasion.

TTFields application resulted in an increase in cell size, as well as in focal adhesion size, number and peripheral distribution of the adhesion sites. Also, treated cells adopted a more flattened and spread shape in comparison with control cells.

TTFields treated cells exhibit reduction in appearance of stress fibers and a dense meshwork of actin filaments around the entire cell periphery.

Our results suggest that cancer cell motility is impaired by exposure to TTFields. The TTFields parameters (intensity and frequency) which led to the maximal inhibition of cellular motility are different from those required for exerting anti-mitotic effects. The alterations in cells morphology during exposure to TTFields may contribute to reduction in cell motility. Further studies are needed to elucidate the mechanism by which TTFields disrupts cellular motility in glioma cancer cells.

#4195

β-elemene inhibits the metastasis of multidrug-resistant gastric cancer cells through the Cbl-b-EGFR-ERK/Akt signaling pathway.

Ruoxi Yu,1 Mingming Deng,2 Huicong Song,1 Dan Zou,1 Ling Xu,1 Xiujuan Qu,1 Yunpeng Liu,1 Yuee Teng,1 Xuejun Hu,2 Ye Zhang1. 1 _Department of Medical Oncology, the First Hospital of China Medical University, Shenyang, China;_ 2 _Department of Respiratory and Infectious Disease of Geriatrics, the First Hospital of China Medical University, Shenyang, China_.

Background/Aims: β-elemene, a Curcuma wenyujin plant extract, has been widely used as an adjuvant therapeutic agent in cancer treatment. The emergence of drug-resistant tumor cells is defined as multidrug resistance (MDR), and is one of the major ways cancer therapy can fail. Our previous study indicated that, compared to chemo-sensitive human gastric cancer cells, drug-resistant cells have a stronger ability for migration. The aim of this study was to explore the anti-metastasis effects and mechanism of β-elemene in multi-drug resistance human gastric cancer cells.

Methods: A cell viability assay was used to assess the resistance of adriamycin-resistant human gastric cancer SGC7901 cells (SGC7901/ADR) and the cytotoxic effects of β-elemene on SGC7901/ADR cells. A wound healing assay and transwell assay were used to explore the anti-metastasis effects of β-elemene. Human matrix metalloproteinase (MMP) antibody array and Western blot analysis were used to explore the potential mechanism. A murine tumor metastasis assay was used to confirm the anti-metastasis effects in vivo. Immunohistochemistry analysis was performed to examine the expressions of related proteins in xenograft tumors.

Results: At sub-lethal concentrations (1 μg/ml and 5 μg/ml), β-elemene significantly inhibited cell migration and invasion of SGC7901/ADR cells. The human MMP antibody array indicated that β-elemene could regulate almost all proteins of the MMP family, and changes of MMP-2 and MMP-9 were the most obvious. Western blotting showed an increase of E-cadherin and decrease of vimentin in β-elemene-treated cells. Downregulation of transcription factor ZEB1 and ZEB2 was also detected. Treatment of SGC7901/ADR cells with β-elemene led to downregulation of Akt, ERK, and EGFR phosphorylation, and significant upregulation of the E3 ubiquitin ligase, Cbl-b. Changes of these proteins were confirmed in xenograft tumors. Hematoxylin and eosin staining showed significantly decreased numbers of metastatic tumor colonies in the lungs of nude mice injected with SGC7901/ADR cells and β-elemene compared with those only injected with SGC7901/ADR cells.

Conclusions: The results strongly suggest that β-elemene inhibits the metastasis of multidrug-resistant gastric cancer cells in vitro and in vivo by regulating the Cbl-b-EGFR-ERK/Akt signaling pathway. β-elemene may be a promising treatment for multidrug resistant human gastric cancer.

#4196

**Aspirin inhibits metastasis in the intravascular phase through the blockade of COX-1-TXA** 2 **pathway in platelets.**

Serena Lucotti,1 Camilla Cerutti,2 Magali Soyer,2 Ana M. Gil-Bernabé,1 Ana L. Gomes,1 Philip D. Allen,1 Sean Smart,1 Bostjan Markelc,1 Karla Watson,1 Paul C. Armstrong,3 Jane A. Mitchell,4 Timothy D. Warner,3 Anne J. Ridley,2 Ruth J. Muschel1. 1 _CRUK & MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, United Kingdom; _2 _Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom;_ 3 _Centre for Immunobiology, Blizard Institute, Barts & the London School of Medicine & Dentistry, Queen Mary University of London, London, United Kingdom; _4 _Cardiothoracic Pharmacology, Vascular Biology, National Heart and Lung Institute, Imperial College London, London, United Kingdom_.

Evidence from experimental studies and clinical trials has shown that aspirin reduces the incidence of distant metastases. Aspirin inhibits cyclooxygenase (COX)-1 and COX-2, triggering anti-thrombotic and anti-inflammatory effects, respectively. However, the mechanisms underlying the anti-metastatic effect of aspirin are still largely unknown.

By employing mouse models of experimental and spontaneous metastasis, we have shown that aspirin and a selective COX-1 inhibitor dramatically reduced metastatic lung nodules, an effect that was not replicated by selective inhibition of COX-2. Metastasis was impaired in COX-1 deficient mice as well, suggesting a pivotal role of microenvironmental COX-1 in the metastatic process. In particular, COX-1 activity was essential during the intravascular phase of pulmonary metastasis. Using a model of platelet depletion and re-infusion we identified platelet-derived thromboxane A2 (TXA2) as the main product of COX-1 responsible for its permissive effect on metastasis. The inhibition of the COX-1-TXA2 pathway in platelets decreased the aggregation of platelets on tumor cells and was associated with a reduction in endothelial activation, in tumor cell adhesion to the endothelium, in recruitment of metastasis-promoting monocytes/macrophages and in transendothelial migration. Thus, platelet-derived TXA2 orchestrates the generation of a favorable intravascular metastatic niche that promotes tumor cell seeding and metastasis.

Taken together, our data suggest that COX-1 inhibition in platelets by aspirin is sufficient to exert an anti-metastatic effect and shed a new light on COX-1-TXA2 signalling in the context of tumor cell dissemination. From this perspective, TXA2 might present a more selective therapeutic target for the prevention of metastasis.

#4197

Sulforaphane suppresses metastasis via autophagic degradation of cathepsin S in oral cancer cells.

Chiao-Wen Lin,1 Chang-Tai Chen,1 Shun-Fa Yang2. 1 _Institute of Oral Sciences, Chung Shan Medical University, Taichung, Taiwan;_ 2 _Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan_.

Sulforaphane has been demonstrated to exert numerous biological effects, such as neuroprotective, anti-inflammatory, and anticancer effects. However, the detailed effects of sulforaphane on human oral cancer cell metastasis and the underlying mechanisms remain unclear. In this study, we observed that sulforaphane attenuated SCC-9 and SCC-14 cell motility and invasiveness by reducing cathepsin S expression. Moreover, sulforaphane increased microtubule-associated protein 1 light chain 3 (LC3) conversion, and the knockdown of LC3 by siRNA increased cell migration ability. Mechanistically, sulforaphane inhibited the cell motility of cervical cells through the extracellular signal-regulated kinase (ERK1/2) pathway, and blocking of the ERK1/2 pathway reversed autophagy-mediated cell motility and CTSS inhibition. In conclusion, sulforaphane exhibits antimetastatic activity against oral cancer cells by repressing cathepsin S through the ERK1/2 signaling pathway. Thus, cathepsin S and LC3 may be new targets for oral cancer treatment.

#4198

2-Hydroxymelatonin shows antitumor activity against human colorectal cancer cells.

Yi Yang,1 Rui Zhou,1 So-Yeon Park,1 Kyung Keun Kim,2 Hangun Kim1. 1 _Sunchon National Univ., Sunchon, Republic of Korea;_ 2 _Chonnam National University Medical School, Gwangju, Republic of Korea_.

2-Hydroxymelatonin is a predominant hydroxylated melatonin metabolite in plants. To investigate whether it has potent cytotoxic effects on colorectal cancer cells, four colorectal cancer cell lines, Caco2, HCT116, DLD1, and CT26, were treated with 2-hydroxymelatonin and melatonin. 2-Hydroxymelatonin had a much lower IC50 value than melatonin in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cytotoxic effect of 2-hydroxymelatonin was much stronger than that of melatonin at high concentrations (1000 or 2000 μM) in HCT116, DLD1, and CT26 cells, but only at intermediate concentrations (250 or 500 μM) in Caco2 cells. The cytotoxicity of 2-hydroxymelatonin was induced through activation of the apoptotic signaling pathway, as confirmed by Hoechst staining and Annexin V-FITC/propidium iodide double labeling of cells treated with a lethal dose (1 mM). However, sub-lethal doses of 2-hydroxymelatonin inhibited the invasive ability of Caco2 cells. Epithelial-mesenchymal transition (EMT) markers were significantly regulated by 2-hydroxymelatonin. Overall, the anti-cancer activity of 2-hydroxymelatonin is more potent than that of melatonin. Taken together, 2-hydroxymelatonin exhibits potent anti-cancer activity against human colorectal cancer cells via induction of apoptosis and inhibition of EMT.

#4199

**Resveratrol induced** REG III **and inhibited head and neck cancer progression.**

Ichiro Ota, Shinji Mikami, Takashi Masui, Takahiro Kimura, Hirokazu Uemura, Hideyuki Okamoto, Shin Takasawa, Takashi Kitahara, Ryogo Shobatake. _Nara Medical Univ., Kashihara, Japan_.

Head and neck squamous cell carcinoma (HNSCC) is one of the 10 most common malignancies in the world and known for clinical progression and poor prognosis. Despite the recent progress of the treatment for improving locoregional control in HNSCC patients, that for recurrence and metastatic control remains insufficient, indicating that there is a continued need for improved treatment strategies. Therefore, the key molecules that enhance chemo- and radio-sensitivity in HNSCC and identification of reliable biomarkers for predicting the recurrence and metastasis would be desirable to improve the prognosis of HNSCC. The human regenerating gene (REG) expression has been reported to be associated with progression of various cancers. Recently, we have reported that REG III expression is associated with an improved survival rate for HNSCC patients as a reliable biomarker. Herein, we investigated the inducer for REG III expression in HNSCC cells and found that resveratrol significantly increased the REG III promoter activity and REG III mRNA. In addition, we demonstrated that the effects of resveratrol on cancer cell progression in HNSCC in vitro and in vivo. To reveal the effects of resveratrol on HNSCC cell proliferation, cell proliferation was measured by using WST-8 assay. Resveratrol significantly inhibited cell growth in FaDu and HSC-4 HNSCC cells. Secondly, to check the effects of resveratrol on chemo- and radio-sensitivity in HNSCC cells, HNSCC cells were expose to cisplatin and radiation, respectively. Resveratrol significantly increased the sensitivity of cisplatin and radiation in HNSCC cells. We also examined the effects of resveratrol on cancer invasion using 24-well Matrigel-coated Transwells. Resveratrol significantly attenuated the cancer invasion of HNSCC cells. Moreover, to assess resveratrol-induced chemo-sensitivity in vivo, we established a xenograft model of HNSCC in BALB/c nude mice. The tumor volumes in the mice receiving combined administration of resveratrol and cisplatin were significantly lower than those of resveratrol or cisplatin alone. These results strongly suggest that resveratrol can inhibit head and neck cancer progression through the REG III expression pathway and can be a potential sensitizer for anticancer therapy for patients with HNSCC.

#4200

New potential therapeutic applications of WX-UK1, as a specific and potent inhibitor of human trypsin-like proteases.

Emil Oldenburg,1 Christine R. Schar,1 Eva Lange,2 Terry F. Plasse,3 Danielle T. Abramson,3 Reza Fathi,3 Eric M. Towler,3 Mark Levitt,3 Jan K. Jensen1. 1 _Aarhus University, Aarhus, Denmark;_ 2 _Aarhus University, Bala Cynwyd, PA;_ 3 _RedHill Biopharma, Tel Aviv, Israel_.

WX-UK1 (the active metabolite of upamostat) was originally developed as an inhibitor of the serine protease urokinase (uPA) with a Ki ~1 uM. To identify more sensitive targets, we performed a bioinformatic analysis of the ~200 human trypsin-like serine proteases, many of which play crucial roles in homeostasis and disease. Among these we selected a subset for biochemical analysis based on an inspection of modelled 3D structures of WX-UK1:protease complexes and sequence alignment of binding site residues. Samples of the selected proteases were prepared and characterized for their binding to WX-UK1; enzymatically with respect to inhibition constant (Ki) and by surface plasmon resonance with respect to dissociation constant (kd). We now report that WX-UK1 is a potent and specific inhibitor of five human serine proteases (trypsin-3, trypsin-2, trypsin-1 and matriptase-1 and trypsin-6), with Ki's down to the low nanomolar range, 19 nM for trypsin-3. Several of these serine proteases are known to be associated with cancer progression and metastasis. As a compound with an established clinical safety profile, targeted use of upamostat in oncology and extension to non-oncology indications may be assessed.

#4201

Dp44mT, an iron chelator, suppresses cell growth and invasion by inducing N-myc downstream-regulated gene 1 and inhibiting Wnt/β-catenin signaling in human glioma cells.

Peng Luo,1 Ye Zhang,2 Yang Jiang,1 Ying Xu,3 Zhitao Jing1. 1 _Department of Neurosurgery, The First Affiliated Hospital of China Medical University, Liaoning 110001, Shenyang, China;_ 2 _The First Laboratory of Cancer Institute, The First Affiliated Hospital of China Medical University, Liaoning 110001, Shenyang, China;_ 3 _Biochemistry and Molecular Biology, University of Georgia, GA_.

The iron-chelating agent di-2-pyridylketone 4, 4-dimethyl-3-thiosemicarbazone (Dp44mT) can inhibit cell growth and invasion in some human cancers. However, the precise molecular mechanisms involved remain obscure in the context of glioma. Recent evidence suggests that N-myc downstream-regulated gene (NDRG)1 has an oncogenic role function. In the present study, we investigated whether Dp44mT regulates the NDRG1 expression and thereby inhibits glioma cell growth and invasion. We found that Dp44mT inhibited glioma proliferation and invasion in vitro, and reduced average tumor size and wet weight in a xenograft model. Dp44mT overexpression increased NDRG1 expression; NDRG1 silencing stimulated cell growth and invasion and triggered epithelial-to-mesenchymal transition (EMT). We also found that Dp44mT exerts its anti-tumor activity via inhibition of Wnt/β-catenin signaling. Taken together, these findings suggest that the iron chelator Dp44mT inhibits glioma development and progression both in vitro and in vivo by inducing the expression of NDRG1 and suppressing Wnt/β-catenin signaling. Thus, targeting NDRG1 with Dp44mT can be an effective treatment for glioma.

#4202

Resection of primary osteosarcoma tumors terminates self-seeding, increasing lung metastasis.

Ryan D. Roberts,1 Amanda J. Saraf,1 Helene Le Pommellet,2 Randall Evans,1 Amy C. Gross1. 1 _Nationwide Children's Hospital, Columbus, OH;_ 2 _The Ohio State University College of Veterinary Medicine, Columbus, OH_.

Like many solid tumors, osteosarcoma (OS) kills patients through metastatic spread. In the case of OS, metastasis occurs almost exclusively to lung. Metastases most often arise after both resection of a primary tumor and completion of chemotherapy. In seeking to understand this long-appreciated pattern of spread, we sought to explore whether the presence of a primary tumor protects mice from developing OS lung metastasis through the process of self-seeding, and whether resection then faciliates metastatic lung colonization. Our data show that resection of a primary orthotopic tumor triggers the spread of OS tumor cells into the lung. Mice bearing a primary tumor have fewer labeled tumor cells recovered in the lung after IV inoculation. Many labeled cells can be recovered in the primary tumor. Supernatants from OS cell cultures drive OS cell chemotaxis with greater potency than serum. Several specific soluble factors produced by OS cells also drive chemotaxis of those same cells. Disruption of these signals may prevent self-seeding. Manipulation of this system may be used to remove OS cells from circulation.

#4203

Alpha 9 nicotinic acetylcholine receptor promotes tumor progression in triple negative breast cancer.

Yung-Che Kuo, Jan-Show Chu, Kha-Liang Lee, Hsiao-Feng Wang, Victor James Drew, Wei-Zhan Zhuang, Chi-Long Chen, Yuan-Soon Ho, Yen-Hua Huang. _Taipei Medical University, Taipei, Taiwan_.

Purpose: To assess the potential therapeutic target on the alpha 9 nicotinic acetylcholine receptor (α9-nAChR) and the insulin-like growth factor (IGF)-I receptor (IGF-IR) for preventing distant metastasis in TNBC patients. Experimental Design: Expression levels of stemness-related genes, α9-nAChR and IGF-IR in clinical tissues (n = 67) were analyzed by RT-qPCR and the Pearson correlation analysis. The α9-nAChR, OCT4, IGF-1R, and p-IGF-1R protein in tissues were detected by immunohistochemical staining (n = 200). The effects of either α9-nAChR activation or IGF-1R transactivation on stemness expression in TNBC were examined using Western blot, ALDH activity assay with flow cytometry, shRNA silencing, wound closure/migration/invasion assay, as well as microCT scanning and IVIS assay for animal models. Results: A high positive correlation between the gene expression levels of OCT4/NANOG/CD24/CD44 and α9-nAChR/IGF-IR in human breast cancer tissues was observed. IGF-1R and OCT4 highly expressed in tumor part compared than that of in normal part. Moreover, IGF-1R positively correlated with OCT4 in tumor tissues. We also show that nicotine induced α9-nAChR activation trans-activates the expression of IGF-1R and pluripotent protein OCT4, which mediated stemness-related properties in TNBC cell lines. Our results reveal that knockdown α9nAChR and IGF-1R diminish the pernicious effects of nicotine, inhibit stemness activities, and suppress metastasis both in vitro and in vivo. Conclusions: A crosstalk between α9-nAChR and IGF-IR signal mediates the stemness-related properties and metastasis therefore convene poor prognosis in TNBCs.

#4204

Structure-activity relationship of diphenylamine derivatives to target epithelial to mesenchymal transition in triple negative breast cancer.

Akshita B. Bhatt,1 Thomas D. Wright,1 Katie Anna,1 Mohit Gupta,1 Patrick Flaherty,1 Van Hoang,2 Matthew Burow,3 Jane E. Cavanaugh1. 1 _Duquesne University, Pittsburgh, PA;_ 2 _Tulane University, LA;_ 3 _Tulane University, New Orleans, LA_.

Triple negative breast cancer (TNBC) is characterized by the loss of hormone receptors and high invasive potential. TNBC cells express high levels of the mesenchymal markers vimentin, fibronectin, N-cadherin, and cadherin11, which are involved in cell invasion and metastasis, and low levels of the epithelial markers E-cadherin, occludins and cytokeratins, which are involved in cellular contact and stability of the cell membrane. Mitogen activated protein kinases, including ERK5 and ERK1/2 are important pro-survival proteins and are known to be upregulated in most cancers. Of these pathways, the MEK5-ERK5 pathway is understudied in triple negative breast cancer, and there are few research tools available to selectively inhibit this pathway. The diphenylamine moiety was derived from the parent molecule Trametinib, a FDA approved MEK1/2 inhibitor for melanoma. Structural modifications were made on the lead molecule to gain selectivity towards MEK. SC-1-151, one of the compounds from this diphenylamine series was identified as a dual MEK1/2 (98.6% inhibition) and MEK5 (59% inhibition) inhibitor. Additionally, we have shown that SC-1-151 attenuates tumor growth in severe combined immunodeficient (SCID) xenograft mice and causes a mesenchymal to epithelial transition (MET) in MDA-MB-231 TNBC cells. The goal of this research is to determine the structural features of diphenylamines that are responsible for MET.

E-cadherin and cadherin-11 protein expression and cell morphology were examined to study MET after the treatment of MDA-MB-231 cells with different structural analogs of SC-1-151 for 5 days. Alkyl or N-Methyl piperazine substituents on the amide of ring 1 produced similar result as SC-1-151, and substituting the amide group with acid or ester also induced MET. In contrast, ortho-fluoro, para-iodo functional groups of the arene ring 2, when replaced with a meta-bromo substituent did not induce MET. Therefore, our data suggest the necessary functional groups for inducing MET include the hydrogen atom on the amine linkage, and the iodine atom on the arene ring 2. Future studies will be performed to determine the specific protein interactions of the promising compounds.

## EPIDEMIOLOGY:

### Biomarkers of Prognosis and Pharmacoepidemiology

#4205

Colorectal cancer molecular markers and subtypes in relation to disease survival.

Amanda I. Phipps,1 Peter T. Campbell,2 Barbara Banbury,3 Daniel Buchanan,4 Andrew T. Chan,5 Dallas English,6 Alton B. Farris,7 Graham G. Giles,8 Tabitha Harrison,3 Roger Milne,4 Polly A. Newcomb,3 Martha L. Slattery,9 Melissa Southey,4 Shuji Ogino,10 Ulrike Peters3. 1 _University of Washington, Seattle, WA;_ 2 _American Cancer Society, Atlanta, GA;_ 3 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 4 _University of Melbourne, Melbourne, Australia;_ 5 _Massachusetts General Hospital, Boston, MA;_ 6 _University of Melbourne, Australia;_ 7 _Emory University Hospital, Atlanta, GA;_ 8 _Cancer Council Victoria, Victoria, Australia;_ 9 _University of Utah, Salt Lake City, UT;_ 10 _Dana Farber Cancer Institute, Boston, MA_.

Colorectal cancer (CRC) is a biologically heterogeneous disease. Such biological heterogeneity reflects diversity in tumor etiology and has implications for survival. Previous studies have demonstrated a strong association between the presence of microsatellite instability (MSI) in CRC and longer disease-specific survival. The influence of other tumor markers, including the CpG island methylator phenotype (CIMP) and BRAF and KRAS mutation status, on survival is less clear. However, these four markers are correlated. Thus, consideration for these markers in combination, with recognition for the distinct etiologies those combinations may reflect, is important to further our understanding of CRC survival. We pooled data from 7 observational studies of CRC: the multisite Colon Cancer Family Registry (CCFR), the Cancer Prevention Study-II (CPS-II), the Diet Activity and Lifestyle Study (DALS), the Health Professionals Follow-Up Study (HPFS), the Nurses' Health Study (NHS), the Melbourne Collaborative Cohort Study (MCCS), and The Cancer Genome Atlas (TCGA). All studies collected data on MSI, CIMP, and somatic mutation status for BRAF and KRAS. Participants with complete tumor marker data were classified into five subtypes based on tumor marker combinations previously proposed to reflect different etiologic pathways: Type 1 (MSI-high, CIMP+, BRAF-mutated, KRAS-wildtype); Type 2 (microsatellite-stable/low-instability (MSS/MSI-low), CIMP+, BRAF-mutated, KRAS-wildtype); Type 3 (MSS/MSI-low, non-CIMP, BRAF-wildtype, KRAS-mutated); Type 4 (MSS/MSI-low, non-CIMP, BRAF-wildtype, KRAS-wildtype); and Type 5 (MSI-high, non-CIMP, BRAF-wildtype, KRAS-wildtype). We used Cox regression to estimate hazard ratios (HR) and 95% confidence intervals (CI) for associations with disease-specific (DSS) and overall survival (OS) for each tumor marker individually (N=4727 to 5105) and for the above tumor marker combinations (N=118 to 2109); analyses were adjusted for age, sex, and study population. When considered individually, MSI-high status was associated with significantly longer (HROS=0.74, HRDSS=0.39, p<0.001) and KRAS-mutated status with significantly shorter survival (HROS=1.11, HRDSS=1.31, p=0.04 and <0.001, respectively); BRAF-mutation and CIMP status were not associated with survival. When markers were combined into the subtypes listed above, relative to those with the predominant Type 4 tumor subtype, participants with Type 2 and 3 CRC had significantly shorter DSS (HRType2=1.73, p=0.002; HRType3=1.28, p=0.002) and those with Type 1 and 5 CRC had longer DSS (HRType1=0.30, p<0.001; HRType5=0.46, p=0.003). Associations were similar for OS. In this large, collaborative study, CRC subtype classified by proposed etiologic pathways were associated with marked differences in survival. These findings highlight the clinical importance of molecular heterogeneity in CRC.

#4206

Pretreatment markers of cellular-mediated inflammation and association with survival vary significantly among cancers.

Rachel Howard, Kathleen Egan, Peter Kanetsky. _H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL_.

Background.

The neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) are established markers of systemic inflammation, and they are believed to have prognostic value in various diseases including many common cancers. These indicators of the cellular-mediated inflammatory response are easily obtained from patient blood samples and hold promise as simple and inexpensive tools for risk stratification.

Methods.

In the present study, 4509 patients spanning multiple cancer types (breast N=979; colorectal N=1024; esophageal N=594; hepatocellular N=287; melanoma N=349 and pancreatic N=1276) treated at Moffitt Cancer Center and with valid hematology lab values, demographic information, clinical disease characteristics, and outcome data were identified. We compared marker counts and ratios across all cancer types to identify disease-specific patterns in pre-treatment host immune response and evaluated the similarities with other established measures of disease-specific immunogenicity. An initial evaluation of the association between pre-treatment NLR, PLR (and corresponding absolute counts of lymphocytes (L), neutrophils (N) and platelets (P)) and disease-specific survival was conducted, controlling for age, sex, race, disease stage, treatment modality, and additionally for primary site and histological and molecular subtypes as appropriate.

Results.

Mean levels of N, L, P, NLR, and PLR all demonstrated significant differences across cancer types. Pre-treatment NLR was significantly associated with disease-specific death in four of our six cancer sites (colorectal: hazard ratio (HR) = 1.47, 95% confidence interval (CI) = 1.21-1.79; esophageal: HR = 1.38, 95% CI = 1.05-1.82; melanoma: HR = 1.87, 95% CI = 1.18-2.95; pancreatic: HR = 1.69, 95% CI = 1.43-1.99). Pre-treatment PLR was significantly associated with disease-specific death in two sites (colorectal: HR = 1.34, 95% CI = 1.10-1.64; pancreatic: HR = 1.53, 95% CI = 1.30-1.79). This suggests that the utilization of markers of the cellular-mediated inflammatory response for clinical prognosis and decision-making may require disease-specific risk classification criteria.

#4207

Androgen receptor expression and ER+ breast cancer prognosis in the BIG 1-98 trial.

Kevin H. Kensler,1 Meredith M. Regan,2 Yujing J. Heng,3 Michael E. Pyle,3 Stuart J. Schnitt,4 Beat Thürlimann,5 Giuseppe Viale,6 Marco Colleoni,6 Myles Brown,2 Rulla M. Tamimi7. 1 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 2 _Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA;_ 3 _Beth Israel Deaconess Medical Center, Boston, MA;_ 4 _Brigham and Women's Hospital, Boston, MA;_ 5 _Cantonal Hospital, St. Gallen, Switzerland;_ 6 _European Institute of Oncology, University of Milan, Milan, Italy;_ 7 _Brigham and Women's Hospital and Harvard Medical School, Boston, MA_.

Background The androgen receptor (AR) is emerging as a potential biomarker of breast cancer prognosis and therapeutic target. AR is expressed in 60-80% of breast cancers, with higher prevalence among estrogen receptor (ER) positive tumors. Treatment with dihydrotestosterone has been found to inhibit ER signaling in ER+/AR+ breast cancer cell lines, and epidemiologic studies have shown AR expression to be associated with improved survival among women with ER+ breast cancer. However, it is unknown if AR expression modifies the efficacy of selective ER modulators or aromatase inhibitors in patients with ER+ cancers.

Methods We evaluated AR expression among 3,103 postmenopausal patients with ER+ breast cancer in the Breast International Group (BIG) 1-98 trial (NCT00004205). The BIG 1-98 study was a four-armed randomized double-blind phase III clinical trial that compared 5 years of tamoxifen or letrozole monotherapy, or sequences of 2 years and 3 years treatment with one drug and then the other. Tissue microarrays were constructed from tumor tissue from trial participants. AR expression was measured by immunohistochemistry and the percentage of AR positive nuclei was digitally quantified. The association between AR expression and prognosis was evaluated through use of Cox proportional hazards models, adjusting for patient, tumor, and treatment factors. Continuous AR-by-treatment interactions were assessed by use of Subpopulation Treatment Effect Pattern Plots (STEPP).

Results In the univariate Kaplan-Meier analysis, AR expression (≥1% vs. <1%) was associated with longer breast cancer-free interval (247 events) over a median 8.2 years (IQR: 7.3-9.1) of follow-up (p=0.029 from log-rank test). Patients with AR+ cancers were more likely to have smaller, lower-grade tumors, as well as tumors with greater ER and PR expression and lower Ki67 expression. However, upon adjustment for patient, tumor, and treatment factors, AR expression (≥1% vs. <1%) was not associated with breast cancer-free interval (HR=1.06, 95% CI 0.82-1.36). A 10% increase in AR expression similarly did not predict breast cancer-free interval (HR=1.02, 95% CI 0.96-1.07). The letrozole vs. tamoxifen treatment effect was non-significantly stronger among AR- tumors (HR=0.42, 95% CI 0.24-0.74) than among AR+ tumors (HR=0.65, 95% CI 0.48-0.87). STEPP analysis also suggested no heterogeneity of treatment effect across the continuum of AR expression (p=0.280). Findings were likewise null when evaluating the secondary endpoints of distant recurrence free interval, disease free survival, and overall survival.

Conclusions After adjustment for tumor characteristics, AR expression did not predict prognosis among postmenopausal patients with ER+ breast cancer, nor did it modify the effect of tamoxifen or letrozole treatment. These findings suggest that AR expression may not be an informative biomarker for the treatment of postmenopausal ER+ breast cancers.

#4208

HOXA9 methylation detection by droplet digital PCR and blood vessel invasion using FFPE tissues for prognostic stratification of stage I lung adenocarcinoma patients.

Delphine Lissa,1 Teruhide Ishigame,2 Rintaro Noro,3 Marguerite J. Tucker,1 Valery Bliskovsky,1 Steven Shema,1 Elise D. Bowman,1 Curtis C. Harris,1 Ana I. Robles1. 1 _NIH/NCI, Bethesda, MD;_ 2 _Fukushima Medical University, Fukushima, Japan;_ 3 _Nippon Medical School, Tokyo, Japan_.

Surgical resection is the only recommended curative treatment for stage I non-small cell lung cancer (NSCLC) patients, as the benefit of adjuvant chemotherapy remains controversial. After complete resection patients with disease at the same stage experience different outcomes, and within 5 years a third of patients relapse. With the implementation of low-dose computed tomography scan for lung cancer screening, the number of lung cancer patients diagnosed at stage I is projected to rise. Therefore, prognostic biomarkers are urgently needed to more accurately predict recurrence following surgery and potentially guide the decision to administer adjuvant chemotherapy for high-risk patients. In the present study, we evaluated the prognostic significance of two biomarkers, namely HOXA9 promoter methylation and blood vessel invasion (BVI), for risk stratification of stage I lung adenocarcinoma patients. The type of biospecimens and the choice of assay platform are key issues to foster translation of biomarkers to the clinic. Here, we demonstrated the application of a droplet digital PCR (ddPCR)-based assay to analyze HOXA9 promoter methylation in formalin-fixed, paraffin-embedded (FFPE) tumor specimens, generated during routine pathologic assessment of resected patients. In recent years, ddPCR has become increasingly used clinically due to its ability to reliably detect and quantitate rare alleles, as well as its technical simplicity, rapidity and cost effectiveness. We replicated previous observations that HOXA9 promoter is methylated de novo in stage I tumors (P <0.0001). Using 177 FFPE tumor samples, we showed that high methylation was associated with worse cancer-specific survival (Hazard Ratio [HR], 3.37; P=0.0002) and identified high-risk stage IA and IB patients. Importantly, addition of this molecular marker improved a risk model comprising clinical and pathologic parameters (Nested likelihood ratio test; P=0.003). HOXA9 promoter methylation was associated with a transcriptome signature enriched in genes marked by Polycomb in Embryonic Stem Cells, a signature previously associated with poor differentiation and worse overall patient survival. Moreover, BVI was independently associated with poor outcome (HR, 2.62; P=0.054). A score that combined BVI with HOXA9 promoter methylation further stratified high-risk patients (Trend P=0.0001 comparing 0, 1 or 2 positive markers). Collectively, our results support the use of ddPCR to quantify HOXA9 promoter methylation and BVI determination from routine pathology FFPE specimens, to identify patients at high risk of recurrence. If validated in a larger independent study, our findings could help inform patient management in prospective clinical trials that evaluate the benefit of adjuvant chemotherapy in early-stage invasive lung adenocarcinoma to prevent recurrence.

#4209

First-line doublet chemotherapy for mTNBC: circulating tumor cell analysis of the tnAcity trial.

Minetta C. Liu,1 Wolfgang Janni,2 Vassilis Georgoulias,3 Denise A. Yardley,4 Nadia Harbeck,5 Xin Wei,6 Desmond McGovern,6 Robert Beck6. 1 _Mayo Clinic, Rochester, MN;_ 2 _Universitätsklinikum Ulm, Germany;_ 3 _School of Medicine, University of Crete, Greece;_ 4 _Tennessee Oncology, Nashville, TN;_ 5 _Universität München, Germany;_ 6 _Celgene Corporation, Summit, NJ_.

Introduction Circulating tumor cells (CTCs) have been shown to be prognostic in patients (pts) with metastatic breast cancer (MBC) (Pukazhendhi, Gluck, J Carcinog. 2014). The tnAcity trial evaluated the efficacy/safety of 3 chemotherapy combination regimens (nab-P/carboplatin [C], nab-P/gemcitabine [G], or G/C) as first-line treatment for pts with mTNBC. nab-P/C resulted in a significantly longer PFS & numerically higher ORR vs nab-P/G or G/C (Yardley. SABCS 2016; manuscript in process). CTC levels were assessed at baseline (BL) & during treatment to determine correlation with clinical benefit.

Methods Pts with pathologically confirmed mTNBC & no prior cytotoxic chemotherapy for MBC received (1:1:1) nab-P 125 mg/m2 with C area under the curve (AUC) 2, nab-P 125 mg/m2 with G 1000 mg/m2, or G 1000 mg/m2 with C AUC 2, all given d1 & 8 q3w. Primary endpoint: investigator-assessed PFS. Secondary endpoints included ORR & OS; CTC enumeration was a prespecified exploratory objective. CTC levels, assessed (CELLSEARCH®) at BL & start of cycles 3 & 5 (post-BL), were grouped as follows: 0 CTCs/7.5 mL blood at BL (−−), ≥ 1 CTCs/7.5 mL blood at BL & post-BL (++), & ≥ 1 CTCs/7.5 mL blood at BL & clearance of CTCs in ≥ 1 post-BL measurement (+−).

Results In total, 126 pts were included: 48, 24, & 54 in the −−, ++, & +− groups, respectively. Mean BL CTC levels for the ++ & +− groups were 55 & 66 CTCs/7.5 mL blood. Pts with BL CTCs of ≥ 1 vs 0 had numerically similar median PFS (7.0 vs 5.9 mo; HR 1.45 [95% CI 0.93 - 2.25]) & OS (15.0 vs 16.0 mo; HR 0.90 [95% CI 0.57 - 1.42]) & higher ORR (64% vs 44%). Mean cycle 3 & 5 CTC levels in the ++ group were 17 & 111 CTCs/7.5 mL blood. Pts in the −− & +− groups had longer PFS vs the ++ group (median 5.9, 8.5, & 4.7 months). Similar improvements in OS & ORR were noted (Table).

Conclusions Absence of CTCs at BL & during treatment & clearance of CTCs from BL at least once during treatment were generally associated with improved efficacy outcomes in pts with mTNBC treated with combination chemotherapy.

Table. Outcomes by Change in CTC Level from Baseline

---

|

Group

Outcome | −−

n = 48 | +−

n = 54 | ++

n = 24

PFS, median, mo

HR (95% CI) | 5.9

0.60 (0.34-1.06)a | 8.5

0.30 (0.17-0.54)a | 4.7

OS, median, mo

HR (95% CI) | 16.0

0.40 (0.22-0.73)a | 17.8

0.35 (0.20-0.62)a | 9.8

ORR, % | 43.8 | 79.6 | 29.2

CR | 6.3 | 16.7 | 4.2

PR | 37.5 | 63.0 | 25.0

SD | 35.4 | 20.4 | 54.2

PD | 16.7 | — | 16.7

NE | 4.2 | — | —

CR, complete response; HR, hazard ratio; NE, not evaluable; ORR, overall response rate; OS, overall survival; PD, progressive disease; PFS, progression-free survival; PR, partial response; SD, stable disease.

a vs ++ group.

#4210

HP and EBVaGC in Peruvian patients.

Carolina Belmar López, Miluska Castillo Garcia, Carlos A. Castañeda Altamirano, Luis F. Barreda Bolaños, Daniel Valdivia Leonardo, Eduardo Payet Meza, Valeria Villegas Bernaola, Jais Nieves Prado, Joselyn R. Sanchez Sifuentes, Luis A. Bernabé Monsalve, Manuel A. Chumpitaz La Rosa Sanchez, Omar Mejía Dionisio, Ivan Chavez Passiuri. _Instituto Nacional De Enfermedades Neoplasicas, Lima, Peru_.

BACKGROUND: Gastric cancer (GC) is the second most common cancer and first leading cause of cancer-related deaths in Peru. Infection by Helicobacter pylori (HP) and Epstein-Barr virus (EBV) are accepted carcinogenic and infection is widely spread in the Peruvian population. The aims of this study were to evaluate HP and EBV-associated GC (EBVaCG), to assess the prevalence rate and to define the characteristics of Peruvian patients. METHODS: GC samples were prospectively collected from patients who underwent gastroscopy or surgical resection with no preoperative treatment at INEN between 2015 and 2017. Tumor tissue (T), proximal healthy tissue (P) and distal healthy tissue (D) samples were assessed for HP and EBV by quantitative PCR (qPCR) using specific primers. HP and EBV status were analyzed along with clinicopathologic parameters of the tumor. Kaplan-Meier estimation curves overall survival (OS) was applied. Comparison between qPCR detection and current standard methods of detection was also performed. All tests were two sided, and a p≤0.05 was considered statistically significant. RESULTS: A total of 150 patients were studied with a mean age 65 years and predominance of males (51.3%). Intestinal-mixed histological subtype rate was 72.5% in cases. Most frequent clinical stage was III-IV (74.6%). HP+ patients were determinate with at least one ureA/hspA gene result and EBV+ patients were divided in high viral load (>100copy/μl) and low viral load (<100copy/μl) groups. Results found HP in 87.3% (n=131/150) and EBV in 10% (n=15/150) of the population. Cases and HP concentration were higher in D (ureA: 62.8%, [703.58±245.47pg]; hspA: 73.8%, [42.77±10.17pg]) than P (ureA: 59.0%, [539.69±121.32pg]; hspA: 71.0%, [31.90±8.64pg]) and T (ureA: 49.7%, [296.32±164.98pg]; hspA: 70.5%, [4.6±1.33pg]) (p<.001). High viral load EBV was found in 40% (n=6/15) of cases. Co-infection detection rate was 7.3% (n=11/150). Correlation between qPCR and H&E evaluation of HP was found in only 72.9% (n=19/57) of cases without evidence of HP (p<.001) and no detectable expression of EBV was found by EBER in qPCR positive cases (n= 15). Cases were defined in 4 groups: HP+EBV+ (n=9), HP+EBV- (n=98), HP-EBV+ (n=6), and HP-EBV- (n=37). Results showed median age (67, 66, 67 and 66), most frequent III-IV clinical stage (75%, 69.5%, 72.7% and 62.1%), histologic grade found was GII (50%, 28.8%, 50% and 43.3%) and intestinal-mixed histological subtype (66.6%, 56.6%, 100% and 62.1%) in four groups respectively. EBVaGC was associated to OS (38.3% vs 72.3%) at 1 year, p=0.033). Based on a mean follow-up of 24 months, the 1-year survival was lower in EBV+ [45.5% (HP+ EBV+) and 25% (HP- EBV+)] than in EBV- [73.1% (HP+ EBV-) and 61.5% (HP- EBV-)] groups. CONCLUSION: HP infection is frequent in Peruvian CG patients. Presence of HP and EBV infection evaluated by qPCR define 4 groups with different features and survival. Study supported by CIENCIACTIVA-CONCYTEC, under the contract #196-2015-FONDECYT.

#4211

The role of GDF15 (growth/differentiation factor 15) during prostate carcinogenesis.

Sudha M. Sadasivan,1 Yalei Chen,1 Nilesh S. Gupta,1 Kanika Taneja,1 Scott A. Maresh,2 Alysa Gonzalez,3 Xiaoxia Han,1 Kuan-Han Hank Wu,1 Dhananjay Chitale,1 Andrew Rundle,4 Deliang Tang,4 Benjamin A. Rybicki1. 1 _Henry Ford Health System, Detroit, MI;_ 2 _Wayne State University, Detroit, MI;_ 3 _Michigan State University, Lansing, MI;_ 4 _Columbia University, New York, NY_.

GDF15 (growth/differentiation factor 15), also known as MIC-1 (Macrophage inhibitory cytokine 1), is a divergent member of the TGFβ superfamily of cytokines and is highly expressed in prostate tumors, but its role in prostate carcinogenesis and utility as a prognostic biomarker is unclear. We studied 91 prostate cancer cases that underwent surgery as their primary treatment and were then subsequently followed for biochemical recurrence (BCR). These cases also had a benign prostate biopsy at least one year before their prostate cancer diagnosis. In both the benign biopsy and tumor specimens, we quantified the intensity of GDF15 expression and characterized the presence of tumor associated macrophages by measuring the density of CD68-positive stained cells and the M2 macrophage marker CD204 by immunohistochemical analysis. Marker expression was measured in a) benign biopsy, b) tumor-adjacent benign and c) tumor tissue using an automated multi-image processing macro developed in the ImageJ software. Expression measurements were log2 transformed and high-low cut-off points were selected that optimized the association of biomarker expression with BCR-free survival. A Cox proportional hazards model was used to test the association of time to BCR with low vs high biomarker expression. During follow-up, 23 cases (25.2%) experienced BCR (96% of men without BCR had at least one year of follow-up). An increased hazard ratio (HR) for BCR was found in men with a higher ratio of GDF15 expression in their tumor vs. tumor-adjacent benign tissue (HR: 3.74; 95% confidence interval (CI) = 1.27-10.99) Adjusting for tumor grade, pathological tumor stage and PSA at diagnosis did not alter risk estimates significantly. In these same prostate tumor specimens, increased hazard ratios for BCR were found among men who had elevated CD204 expression in tumor (HR = 5.24; 95% CI = 2.02-13.62) and in tumor-adjacent benign tissue (HR = 3.29; 95% CI = 1.32, 8.23). We found no association of BCR-free survival with either GDF15 or CD204 expression in pre-diagnostic benign biopsies. Our results suggest that men who have a larger difference in GDF15 expression levels between prostate tumor and tumor-adjacent benign tissue, and with increased levels of M2 macrophages in both tumor and tumor-adjacent benign tissue, are at greater risk of disease recurrence. Further evaluation of the differences in the prostate immune cellular profile in the pre-malignant and malignant state may offer additional insight into inflammatory-mediated prostate carcinogenesis.

#4212

**PD-L1 expression,** EGFR **and** KRAS **mutations in unresectable stage III non-small cell lung cancer (NSCLC) patients.**

Deirdre Cronin-Fenton,1 Tapashi Dalvi,2 Elizabeth Hedgeman,3 Mette Norgaard,1 Lars Pedersen,1 Hanh Hansen,1 Jon Fryzek,3 Jill Walker,4 Anders Mellemgaard,5 Torben Rasmussen,6 Norah Shire,2 James Riggas,2 Danielle Potter,2 Stephen Hamilton-Dutoit,1 Henrik Sørensen1. 1 _Aarhus University Hospital, Aarhus, Denmark;_ 2 _AstraZeneca, Gaithersburg, MD;_ 3 _EpidStat Institute, Rockville, MD;_ 4 _AstraZeneca, Cambridge, United Kingdom;_ 5 _Herlev Hospital, Herlev, Denmark;_ 6 _Danish Lung Cancer Group, Odense, Denmark_.

Background: Anti-PD-L1 therapy may improve prognosis in advanced NSCLC. We examined the relation of PD-L1 expression, KRAS and EGFR mutations, with survival in unresectable stage III NSCLC patients.

Methods: We obtained data on unresectable stage III NSCLC patients (defined via TNM AJCC staging and without surgery up to 120 days after diagnosis) diagnosed 2001-2012. We retrieved medical data from Danish population-based registries and paraffin-embedded tumor tissue from pathology archives. We assessed PD-L1 expression using the Ventana IHC (SP263) validated assay. We genotyped KRAS and EGFR using PCR-based kits. Follow-up began at NSCLC diagnosis and continued to death, emigration, or 31/12/2014. We used Cox regression to compute hazard ratios (HRs) and associated 95% confidence intervals (95%CI) for PD-L1, EGFR, and KRAS.

Results: Among 305 patients, 183 (60%) were men, 167 (55%) were aged >65 years at diagnosis and none used immunotherapy; 148 (49%) had adenocarcinoma, 117 (38%) squamous histology, 96 (31.5%) had PD-L1 positive tumors (>=25%), 6 (2%) had EGFR mutations, and 69 (23%) had KRAS mutations. Among PD-L1 positive tumors, 55% had stage IIIA, 45% IIIB disease; 1% had EGFR and 34% had KRAS mutations. Among PD-L1 negative tumors, 54% had stage IIIA and 46% IIIB; 3% had EGFR and 16% had KRAS mutations. Median survival was similar in patients with KRAS wild-type and KRAS mutations (Hazards ratio (HR)=1.07; 95% CI=0.76-1.51). Patients with an EGFR mutation had a lower non-statistically significant risk of death (HR=0.75; 95% CI=0.28-2.06). Tumor cell positivity for PD-L1 (>=25% versus <25%) yielded an HR=0.80 (95%CI=0.60-1.07). Immune cell positivity for PD-L1 >=1% and >=25% yielded HRs of 0.50 (95%CI=0.39-0.66) and 0.45 (95%CI=0.30-0.67), respectively. Tumor infiltration by immune cells with PD-L1 measured as a continuous variable yielded an HR=0.96 (95%CI=0.93-0.99).

Conclusion: Our findings suggest that PD-L1 expression and EGFR mutations, but not KRAS mutations, are associated with survival in this real-world Danish study of unresectable Stage III NSCLC patients.

#4213

Molecular biomarker study of programmed death receptor ligand 1 (PD-L1) in Korean patients with lung adenocarcinoma.

Xiaoqiao Liu,1 Jinseon Lee,2 Soo Jung Lee,2 Jong Ho Cho,2 Jong-Mu Sun,2 Lu Zhang,1 Yunfei Pei,1 Lan Chen,1 Marisa Dolled-Filhart,1 Kenneth Emancipator,1 Jared Lunceford,1 Ronghua Chen,1 Wei Zhou,1 Jhingook Kim2. 1 _Merck & Co., Inc., Kenilworth, NJ; _2 _Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea_.

Purpose We explored the association between different molecular biomarkers and PD-L1 mRNA and protein expression in Asian patients with lung adenocarcinoma.

Experimental design PD-L1 protein expression level was evaluated using a prototype immunohistochemistry (IHC) assay with the 22C3 antibody in tumor samples from 157 patients with lung adenocarcinoma from Samsung Medical Center. Other biomarkers, including PD-L1, PD-L2 and IFN-γ mRNA expression detected using the Affymetrix array, EGFR and KRAS mutation detected using the Sequenom platform, ALK fusion detected using the NanoString system and tumor mutation burden (TMB) calculated as total number of non-synonymous single nucleotide substitution from Illumina HiSeq 2000 sequencing platform were provided as part of the Asian Cancer Research Group collaboration. Spearman correlation test, t test, Fisher exact test, and multivariate regression models were utilized to test the association between different biomarkers and PD-L1 mRNA/protein expression. PD-L1 IHC tumor proportion score was analyzed as a continuous or categorical variable, where PD-L1 strong and weak positivity were defined to be traceable to the 1% and 50% cutoffs used in the clinical trial version of the assay.

Results The median age was 61 years (range, 20-84), 50% were female, 40% were smokers, and 77% had stage I/II disease. There was strong correlation between PD-L1 mRNA and protein expression (N = 79, Spearman R = 0.762, P < 0.0001). Absence of EGFR mutation was associated with higher PD-L1 expression (for mRNA, N = 166, t test P = 0.023; for protein, N = 83, Fisher test P = 0.051), and presence of ALK fusion was associated with higher PD-L1 expression (for mRNA, N = 229, t test P = 0.001; for protein, N = 144, Fisher test P = 0.002). Similar associations between EGFR mutation, ALK fusion, and PD-L1 mRNA and protein expression were observed after adjusting for age, sex, smoking status, and disease stage. No association was found between PD-L1 expression and KRAS mutation (for mRNA, N = 140, t test P = 0.305; for protein, N = 78, Fisher test P = 0.243). mRNA expression of PD-L2, the other PD-1 ligand, was highly correlated with PD-L1 expression (for mRNA, N = 253, Spearman R = 0.650, P < 0.0001; for protein, N = 79, Spearman R = 0.444, P < 0.0001). As evidence for IFN-γ-inducible biology, IFN-γ mRNA expression was significantly correlated with PD-L1 expression (for mRNA, N = 253, Spearman R = 0.57, P < 0.0001; for protein, N = 79, Spearman R = 0.44, P < 0.0001). TMB was not significantly correlated with PD-L1 expression (for mRNA, N = 31, Spearman R = 0.092, P = 0.62; for protein, N = 8, Spearman R = 0.19, P = 0.65), which suggested independent predictive ability for anti-PD-1 therapy.

Conclusion Our study provides evidence that DNA and RNA features, including PD-L1 mRNA, lack of EGFR mutation, and ALK fusion are associated with PD-L1 protein expression for Asian lung adenocarcinoma patients.

#4214

Characteristics of carcinoembryonic antigen-producing colorectal cancers: A population based study.

Sarah Groover,1 Pragatheeshwar Thirunavukarasu,2 Mark E. Payton,3 Asha Karippot,2 Rashmi Kaul1. 1 _Oklahoma State University Center for Health Sciences, Tulsa, OK;_ 2 _Cancer Treatment Centers of America, Tulsa, OK;_ 3 _Oklahoma State University, Stillwater, OK_.

Serum Carcinoembryonic Antigen (CEA) is a tumor marker often found to be elevated in colorectal cancer patients. Elevated CEA has been strongly associated with poor prognosis. However, not all colon cancer tumors produce CEA, and little is known about the patient and tumor characteristics between CEA-producing and non-CEA-producing tumors. We performed a retrospective analysis of all patients (n=120,571) diagnosed with colorectal adenocarcinoma from 2010 to 2014 in the National Cancer Institute's Surveillance, Epidemiology and End Results (SEER) database. All patients were designated as either positive/elevated (C1) or negative/normal (C0) based on the pretreatment serum CEA level. We performed univariate and multivariate analyses to identify variables associated with CEA-producing tumors. Of the 68,856 (57.1%) with available CEA information, 33,426 (48.5%) were C1. Median age was 65 years and 36,479 (53.0%) were male. Compared to C0 cancers, C1 cancers were significantly more likely (P<0.001) to be female (49.3%% vs. 47.9%), black (58.4%), separated or never married (55.0% and 54.8%, respectively), higher grade (39.0%, 46.2%, 51.1% and 52.3% of well, moderately, poorly, and undifferentiated cancers, respectively), and of signet ring cell histology (61.1%). Multivariate analysis showed age, sex, race, marital status, and TNM stage to be independent prognostic factors associated with the diagnosis of CEA-producing tumors. This is the first report to describe differences in patient and tumor characteristics between CEA-producing and non-CEA-producing tumors in a large American population. About half of all colorectal adenocarcinomas are associated with elevated pre-treatment serum CEA levels. CEA-producing tumors are associated with female gender, non-Caucasian race, separated or never married marital status at diagnosis, and more extensive local, regional and metastatic disease.

#4215

Comprehensive investigation of programmed death receptor ligand 1 (PD-L1) expression and associated molecular features in gastric cancer patients.

Xiaoqiao Liu,1 Jeeyun Lee,2 Kyoung-Mee Kim,2 Seung Tae Kim,2 Se Hoon Park,2 Won Ki Kang,2 Razvan Critescu,1 Senaka Peter1. 1 _Merck & Co., Inc., Kenilworth, NJ; _2 _Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea_.

Purpose

We explored PD-L1 expression and analyzed its association with molecular subtypes, clinical features and other molecular markers in Korean gastric cancer (GC) patients.

Experimental design

As with the Asian Cancer Research Group (ACRG) GC cohort, 300 primary tumor specimens were procured from Samsung Medical Center. Tumor PD-L1 expression was measured by immunohistochemistry (IHC) using the 22C3 PD-L1 antibody pharmDx (Dako) kit. PD-L1 positivity was defined as CPS ≥1%, where CPS is PD-L1+ cells (tumor cells, macrophages, lymphocytes) over the total number of tumor cells, expressed as a percentage. ACRG and The Cancer Genome Atlas (TCGA) molecular subtypes, and other molecular and clinical features including PD-L1 mRNA expression by microarray, tumor mutation burden (TMB) by targeted sequencing, microsatellite instability (MSI) status by IHC, Epstein-Barr virus (EBV) infection by in situ hybridization, Helicobacter pylori (H. pylori) infection by microscope, ERBB2 (HER2) and EGFR overexpression by IHC, signet ring cell and Lauren classification were provided as part of the ACRG collaboration. Spearman correlation test, Wilcoxon rank-sum test, Kruskal-Wallis test were utilized to test the association with PD-L1 expression, and survival was analyzed by Kaplan-Meier method and log-rank test.

Results

The median age was 64 years (range, 24-86), 101 (34%) were female, and 127 (42%) had stage I/II disease. Prevalence of PD-L1 positivity was 59.3%, with significantly higher expression in stage I samples (P = 0.002). PD-L1 positivity was associated with good prognosis (P = 0.008) especially for early stage (I/II) subjects (N = 127, P = 0.007). PD-L1 mRNA was moderately correlated with protein expression (R = 0.430, P < 0.001). For molecular subtype, PD-L1 expression was associated with ACRG subtype (P < 0.001) with significantly higher expression in MSI subtype (P < 0.001). And for TCGA subtype, the association with PD-L1 expression also existed (N = 238, P < 0.001) with significantly higher expression in MSI and EBV subtype (P < 0.001). H. pylori infection was also associated with higher PD-L1 expression (N = 127, P = 0.001). Signet ring cell carcinoma had significantly lower PD-L1 expression (P < 0.001). PD-L1 expression was also significantly higher in ARID1A or PIK3CA mutant samples (N = 233, FDR adjusted P = 0.038). TMB was found to be weakly correlated with PD-L1

expression (N = 233, R = 0.266, P < 0.001). No significant association was observed between PD-L1 expression and overexpression of ERBB2 or EGFR.

Conclusion

Our study provides evidence that several molecular features, especially MSI and EBV infection, are significantly associated with higher PD-L1 protein expression, which suggests that PD-L1 IHC may be an appropriate biomarker to screen GC patients for immune checkpoint inhibitors.

#4216

Adiponectin, leptin and breast cancer in high risk postmenopausal women: Results from a nested case-control study.

Debora Macis,1 Ivana Sestak,2 Valentina Aristarco,1 Harriet Johansson,1 Aliana Guerrieri-Gonzaga,1 Andrea Decensi,3 Bernardo Bonanni,1 Jack Cuzick,2 Sara Gandini1. 1 _European Institute of Oncology, Milan, Italy;_ 2 _Centre for Cancer Prevention Wolfson Institute of Preventive Medicine Queen Mary University of London, London, United Kingdom;_ 3 _Galliera Hospital, Genoa, Italy_.

Introduction: Obesity is a risk factor for postmenopausal breast cancer (BC) and evidence supports the pivotal role of adipokines in the association between obesity and BC. In the present study, we aim to investigate the role of adiponectin and leptin as independent risk markers for postmenopausal BC in a prospective nested case-control study within the cohort of women from the International Breast cancer Intervention prevention Study II (IBIS-II). Methods and Results: In the IBIS-II Prevention trial, 3864 healthy postmenopausal women at increased risk for BC were randomized to receive 1 mg/day oral anastrozole or matching placebo for 5 years. We measured serum adiponectin and leptin at baseline and 12-months in 119 cases and 336 controls in the placebo arm, and 57 cases and 165 controls in the anastrozole arm. We performed the adipokines measurements on the automated immunoassay platform ELLA (ProteinSimple, Biotechne). Baseline characteristics (age, body mass index (BMI), smoking status, and oophorectomy) were not different between cases and controls in both trial arms, except for hormone replacement therapy (HRT) use (38.7% of cases vs 51.5% of controls) and hysterectomy (25.2% of cases vs 37.5% of controls) in the placebo arm. We did not observe any statistically significant difference in adiponectin and leptin at baseline between cases and controls in both trial arms. When we considered the change in adiponectin levels between baseline and 12-months of follow-up, we observed a borderline significant higher proportion of BC cases in subjects with a decrease in adiponectin (36.4%) compared to subjects with an increase in adiponectin (25.7%). The multivariate Cox model indicated that women on placebo with a decrease in adiponectin between baseline and 12-months had a 36% significant increased risk in BC (HR=0.64; CI 0.42-0.98; adjusted for age, BMI, previous HRT use, smoking, oophorectomy, hysterectomy). Changes in leptin were not significantly associated with BC incidence overall. However, the multivariate Cox model for the anastrozole arm, indicated a significant higher BC risk for women with the highest increase (>10 ng/ml) vs the lowest decrease (<-10 ng/ml) in leptin: HR=4.45; CI 1.08-18.30; adjusted for age, BMI, previous HRT use, smoking, oophorectomy, hysterectomy. Conclusion: Our results did not support a prognostic role of baseline adiponectin and leptin levels in postmenopausal BC development. However, we observed an increased BC risk in women with a 12-month adiponectin decrease and leptin increase. These data suggest further investigations of changes of adiponectin and leptin levels as attractive targets for BC prevention in postmenopausal women at high risk for BC.

#4217

Prognostic gene expression signatures of immune responses in the colon cancer microenvironment.

Youngchul Kim, Hannah J. Hoehn, Yunyun Chen, Mollie E. Barnard, Amanda Bloomer, Sean Yoder, Domenico Coppola, Stephanie L. Schmit. _Moffitt Cancer Center, Tampa, FL_.

Colorectal cancer remains the 2nd leading cause of cancer deaths in the United States. This suggests that traditional prognostic factors are not optimally refined for predicting survival outcomes and guiding therapeutic decisions for some patients. Mounting evidence supports that quantifying the strength and diversity of host immune responses in the tumor microenvironment may improve prognostication and clinical decision-making; however, standard pathological assessment of T cell infiltration is time-consuming and difficult to standardize for clinical utility. The goal of this study was to develop a molecular classifier associated with CRC prognosis based on the expression of 770 immune-related genes measured on the Nanostring (NS) nCounter PanCancer Immune Profiling Panel. This panel includes markers of immune cell types, common cancer antigens, and diverse categories of immune responses (e.g. T cell function, cytokines). We also aimed to assess the validity of combining gene expression data derived from different tissue types (FFPE, fresh frozen) and mRNA profiling platforms (NS, Rosetta/Merck human RSTA Custom Affymetrix 2.0 microarray). FFPE (N=24) and fresh frozen tumor tissues (N=28) from 50 primary stage II colon cancers from the Moffitt Cancer Center Total Cancer Care cohort were profiled using the NS platform, and microarray data were generated on frozen tissues from all patients. Geometric mean-normalized NS data of FFPE and frozen tumor tissues were merged by the ComBat algorithm that adjusted for different RNA source types. 634 (87%) genes in the NS dataset had expression values that positively correlated with those of the microarray data. A 2-way hierarchical cluster analysis of these genes in NS data revealed two clusters of patients with non-overlapping overall survival (OS) curves, but no statistically significant difference due mainly to a lack of events (Log-rank P=0.12; 5-year OS probability=91.3% vs 74.1% for cluster 1 (N=23) and cluster 2 (N=27)). To examine cross-platform predictability of the 2 clusters, a 5-gene classifier was trained on NS data using penalized logistic regression. Applying this classifier to microarray data on the same patient set (N=49) significantly discriminated the clusters (AUC=0.8, P<0.01). Functional annotation of the 5 genes (CD27, CD37, ITGAL, KLRG1 and LAG3) revealed enrichment for T cell receptor signaling, hematopoietic cell lineage, and natural killer cell mediated cytotoxicity (FDR P<0.001). This pilot study provides early evidence that an immune gene expression panel may capture the prognostic value of intratumoral host immune responses. It also supports the feasibility of combining different RNA sources and expression platforms. Expanded future studies that pool across sample types and publicly-available expression datasets are needed to validate the 9-gene classifier from this study and examine its broader prognostic impact.

#4218

Breast cancer survival by age and subtype (ER/PR/HER2) in a nationwide population-based cohort.

Anna L. Johansson,1 Cassia B. Trewin,2 Kirsti V. Hjerkind,2 Merete Ellingjord-Dale,3 Tom B. Johannesen,2 Therese Sorlie,4 Giske Ursin2. 1 _Karolinska Institutet, Stockholm, Sweden;_ 2 _Cancer Registry of Norway, Oslo, Norway;_ 3 _Imperial College, London, United Kingdom;_ 4 _Institute for Cancer Research, Oslo University Hospital, Oslo, Norway_.

Purpose: We assessed the effect of breast cancer subtype, as defined by estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) status, on breast cancer survival among young (20-39y), middle-aged (40-49y), screen-aged (50-69y) and elderly (70-89y) women, to elucidate the role of tumor biology for differences in survival across age. Material and methods: A population-based cohort of 21,385 women with a breast cancer diagnosis at ages 20 to 89 between 2005 and 2015 was identified in the Cancer Register of Norway. Subtype was defined as luminal A-like (ER+ PR+ HER2-), luminal B-like HER2-negative (ER+ PR- HER2-), luminal B-like HER2-positive (ER+ PR+/PR- HER2+), HER2-positive (ER- PR- HER2+) and triple-negative (TRN) (ER- PR- HER2-). Cox regression models estimated hazard ratios (HR) for the association between age at diagnosis, subtype and breast cancer specific mortality, while adjusting for year, grade and TNM stage. Results: Young women had higher rates of HER2-positive and TRN tumors compared to middle-aged, screen-aged and elderly women who more often had luminal A-like tumors. Compared to women aged 50-59y, young women had doubled mortality rate (20-39y: HR=2.3 [95% CI 1.8-2.8]), while elderly had two to five times higher mortality rate (70-79y: HR=2.3 [1.9-2.7]; 80-89y: HR=5.2 [4.2-6.4]). After adjustment for subtype, grade and stage the association was reduced among young women (20-39y: HR=1.3 [1.1-1.6]) but less so among the elderly (70-79y: HR=2.1 [1.7-2.5]; 80-89y: HR=3.9 [3.2-4.9]). Among young women, luminal A-like and luminal B-like HER2-negative subtypes were still associated with increased mortality after adjustment for grade and stage. Among the elderly, all subtypes were associated with increased mortality after adjustment for grade and stage, with stronger effects for TRN, HER2-positive and luminal B-like HER2-positive subtypes. Conclusion: Tumor-associated factors, such as subtype, grade and stage, may explain a large part of the poorer survival among young patients, whereas comorbidities and less intensive treatment may explain the persisting poorer survival across subtypes among the elderly.

#4219

Breast cancer distribution and survival among Buddhist and Muslim women in southern Thailand.

Kali Defever,1 Katie M. Rentschler,1 Ana Khazan,1 Elizabeth Pinkerton,1 Shama Virani,2 Hutcha Sriplung,2 Judy Pang,1 Celina G. Kleer,1 Carlos F. Mendes de Leon,1 Justin A. Colacino,1 Laura S. Rozek1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _Prince of Songkla University, Hat Yai, Thailand_.

Objective: Thailand is undergoing a period of rapid economic growth, which has led to an epidemiologic transition from infectious to chronic diseases such as cancer. Preliminary research shows a difference in rates of breast cancer incidence and mortality between Buddhist and Muslim women in southern Thailand. This study examines differences in breast cancer and potential explanations for this disparity. Methods: Demographic, diagnostic, and outcome data from breast cancer patients diagnosed from 2001-2015 were collected from a population-based cancer registry in southern Thailand and matched with formalin-fixed paraffin-embedded blocks from Songklanagarind Hospital. This region was selected due to the relatively high proportion of Muslim women. Of 424 cases selected, 369 were Buddhist and 55 were Muslim. Formalin-fixed paraffin-embedded blocks were sent to Michigan Medicine and histological and subtype analyses were performed by two pathologists. Statistical analysis was performed in RStudio using chi-square tests of independence, logistic regression, and Cox proportional hazards models. Results: Muslim women were generally diagnosed at a younger age than Buddhist women (Muslim mean: 46.6 years, Buddhist mean: 52.7 years) and with later stage cancers (Muslim: 36% stage 3 and 14% stage 4, Buddhist: 20% stage 3 and 8% stage 4). Buddhist and Muslim women had similar distributions of Luminal A and B subtypes, but Muslim women were more likely to be diagnosed with Triple Negative breast cancer (Muslim: 30%, Buddhist: 18%), the histological subtype with the worst prognosis. Invasive ductal carcinoma was most common in both groups, but Buddhists had more heterogeneity in subtypes. There were no significant differences in tumor histology or subtype by religion. Conclusions: These analyses show differences in the distribution of breast cancer between Buddhist and Muslim women. Muslim women are diagnosed younger with later stage breast cancers, and with histological differences, which generally lead to lower survival rates. Lack of statistical significance may be due to small sample size, necessitating further studies.

#4220

Heterogeneous distribution of prognostic protein markers in glioblastoma.

Lindsay C. Stetson, Quinn T. Ostrom, Peter Liao, Andrew E. Sloan, Mark R. Chance, Jill S. Barnholtz-Sloan. _Case Western Reserve Univ., Cleveland, OH_.

Background: Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor in adults. The heterogeneity of the disease leads to significant variability in response to standard therapy (surgery plus concurrent radiation and temozolomide). An accurate and reliable predictor of patient prognosis represents an unmet need to improve the care of GBM patients. While protein markers are an effective readout of cellular function, proteomics has rarely been utilized in GBM prognostic marker discovery. Experimental Procedures: GBM patients were prospectively recruited (Ohio Brain Tumor Study) and proteomics discovery using liquid chromatography mass spectrometry analysis (LC MS/MS) was performed in a discovery set of 27 patients including 13 short-term survivors (< 9 months, STS) and 14 long-term survivors (>= 18 months, LTS). Statistically significant proteins were evaluated in two independent datasets, including in18 samples micro-dissected from multiple tumor areas of 6 GBM patients. Results: Proteomics discovery identified 11,941 peptides in 2,495 unique proteins, with 172 proteins exhibiting significant dysregulation when comparing STS and LTS. Proteins involved in glycolysis/TCA cycle were up-regulated in STS compared to LTS by examination of individual targets as well as upon application of a novel protein and peptide pathway enrichment analysis. Validation of these dysregulated proteins and other protein markers from the literature in our first validation set (18 samples micro-dissected from n=6 patients) demonstrated that they were very unevenly distributed throughout individual patients' tumors. Several proteins overcame the heterogeneous nature of the tumors and were both prognostic markers differentially expressed between LTS and STS, as well as potential drug targets owing to their even distribution throughout the tumor. Protein abundance of significant marker proteins were verified in a second independent validation set using Western immunoblots. Conclusion: The current study verified the importance of metabolism in GBM pathology and demonstrated the heterogeneous nature of GBMs at the protein level.

#4221

Association between SOX9 expression in Mexican patients with early colon cancer stage.

Erika Ruiz-Garcia,1 Tatiana Galicia,1 Edith Fernandez Figueroa,1 Saul Lino-Silva,1 Cesar Lopez-Camarillo,2 Laurence Marchat,3 German Calderillo,1 Juan Zinser,1 Abelardo Meneses-Garcia,1 Horacio Astudillo-de la Vega4. 1 _National Cancer Institute, Mexico City, Mexico;_ 2 _Posgrado en Ciencias Genómicas. Universidad Autónoma de la Ciudad de México, Mexico City, Mexico;_ 3 _Programa en Biomedicina Molecular y Red de Biotecnología, Escuela Nacional de Medicina y Homeopatía, Instituto Politécnico Nacional, Mexico City, Mexico;_ 4 _Laboratorio de Investigación Traslacional en Cáncer y Terapia Celular, Hospital de Oncología, Centro Médico Siglo XXI, Mexico City, Mexico_.

Introduction. Colon cancer is the fourth leading cause of mortality worldwide. Between 30-40% of patients are diagnosed at early stage (I or II), sadly 20% will relapse. It is necessary to have a biomarker that identifies patients at high risk. SOX 9 is a transcription factor required for differentiation of different cells and is important for cell proliferation, senescence and lineage commitment. The exact role of SOX9 in carcinogenesis and cancer progression is controversial because could have oncogenic and tumor-suppressing functions. Reports show that strong levels of SOX 9 in colon cancer stage II are linked to low risk of relapse. Objective. The aim of this study was to investigate if the protein expression of SOX9 was associated with the stage, clinical characteristics and relapse of Mexican colon cancer patients. Methods. We obtained 97 samples embedded in paraffin. A pathologist blinded to the clinical information determined the immunoreactivity of SOX9 into 2 groups: low and high expression (by evaluating the percentage of positive nucleus and the tissue staining intensity). All statistical analyses were conducted with SPSS v.22 Statistics. Results. We include stage I (N= 34) and stage II (N=63) patients. The median follow-up period was 42 months. We found statistical difference between SOX9 expression and age (p=0.003), tumor localization (p=0.048) and stage (p&lt0.001) for whole patients group. Twelve patients (12.3%) relapsed, 7 (at locally level) and 5 with distant metastasis: liver (N=3) and retroperitoneal nodes (N=2), all had low SOX9 expression; one patient with local relapse had died (because of surgical complications). In the relapse group, we found a significant correlation between SOX9 expression and stage (p=0.038) but not with relapse free survival (RFS). Conclusion. We found interesting data even though this is a retrospective study with limitations. This study could not associated SOX9 and RFS, because there are few events. Nevertheless, we observe that 41% patients had a systemic relapse. In the other hand, the youngest patients (&lt40 y) and the right colon had the highest SOX9 expression. With this result, we have decided to increase the sample size before any assumption, and perform NGS to the five systemic relapses.

#4222

The prognostic impact of human cytomegalovirus infection in breast cancer.

Joel Touma,1 Afsar Rahbar,2 Belghis Davoudi,2 Jonas Christoffer Lindstrom,1 Torill Sauer,1 Ida Rashida Bukholm,1 Katja Vetvik,1 Cecilia Soderberg-Naucler,2 Jurgen Geisler1. 1 _Akershus University Hospital, Oslo, Norway;_ 2 _Karolinska Institue, Stockholm, Sweden_.

Emerging evidence demonstrates a frequent presence of Human Cytomegalovirus (HCMV) proteins and nucleic acids in several primary malignant tumours including breast cancer (BC). HCMV maintains latency in monocytes and reactivates in an inflammatory or immune-suppressive state. The virus infection expresses more than 750 proteins, many has revealed both oncogenic and onco-modulatory properties which fulfil all the criteria for the hallmarks of cancer. HCMV is not believed to transform the host cell itself, but HCMV-encoded proteins are involved in cell cycle progression, interact with key proteins in the extrinsic and intrinsic apoptotic signalling cascade, and can induce drug resistance to chemotherapeutic agents. Earlier studies from our lab detected HCMV-IE (immediate early) and HCMV-LA (late antigen) proteins in >90% of analysed primary breast cancer tumours, sentinel lymph node and brain metastasis, and that their prevalence is higher in advanced breast cancer. We have reported a significant correlation between HCMV and hormone receptor expression in a certain subgroup of breast cancer patients. The purpose of this study is to retrospectively investigate the prognosis and overall survival of 120 patients diagnosed and treated for breast cancer at the Akershus University Hospital, Norway, between 1996 and 2010. Material/methods: Formalin-fixed, paraffin-embedded, consecutively collected breast tumour samples and metastases, from clinically well-characterized patients were examined for HCMV-IE and HCMV-LA, using immuno-histochemical techniques. HCMV values was then correlated with BC subtypes: HER-2 positive, luminal A (LUM-A), luminal B (LUM-B) and triple negative breast cancer (TNBC). As well as time to: distant metastasis, loco-regional relapse and overall relapse. All data were defined in Kaplan-Meier curves for survival and a Cox regression model was fitted to each subgroup. Results: Both HCMV-IE and HCMV-LA was present in >99% of the tissue samples analysed. We found a significant association between HCMV-LA and overall survival in TNBC patients (Hazard ratio 2.502; P-value 0.025). In addition, HCMV-LA was significantly correlated to the risk of local relapses in TNBC patients (Hazard ratio 4.149; P-value: 0.026). Finally, we also found the risk for local relapses and distant metastasis to be significantly tied to HCMV-LA levels in TNBC patients (Hazard ratio 4.126; P-value: 0.032). HCMV-LA and HCMV-IE levels were not found to be associated with either survival or risk for relapses in any other BC subtype. Conclusion: Our findings reveal a significant correlation between HCMV-LA expression and TNBC subtype concerning both risk of recurrence and overall survival. While no other BC subtype was found to be significantly influenced by the presence of HCMV-related proteins, the presence of HCMV-LA that imply an active HCMV infection seems to indicate a more aggressive disease when detected in TNBC patients.

#4223

Personal history of non-melanoma skin cancer and the risk of death from invasive cutaneous melanoma in men.

Fengju Song,1 Jiali Han2. 1 _Tianjin Medical University Cancer Institute & Hospital, Tianjin, China; _2 _Indiana University, Indianapolis, IN_.

BACKGROUND: Previous studies have found an increased risk of invasive cutaneous melanoma (CM) among those with a history of non-melanoma skin cancer (NMSC). OBJECTIVE: The aim of this study is to evaluate the risk of CM death after NMSC. METHODS: The study was based on the Health Professionals Follow-up Study (HPFS). Cox proportional hazards model was used to examine the hazard ratio (HR) of CM death associated with personal history of NMSC, among the entire study population (primary analysis) and invasive CM cases (secondary analysis) respectively. RESULTS: We documented a total of 908 invasive CM cases over a total of 0.7 million person-years of follow-up. Among all participants, the risk of developing either lethal or non-lethal invasive CM increased for those with NMSC history. The risk of melanoma death based on NMSC history was non-significantly increased, with HR (95% CI) of 1.53 (0.95 - 2.46). In the case-only analysis, those with NMSC history had a significantly lower risk of melanoma death compared with those with no such history, HR=0.60 (0.35-0.94). LIMITATIONS: Because HPFS consists exclusively of male health professionals, the results of this study may not be extended to the entire population. CONCLUSION: Personal history of NMSC is associated with a decreased risk of melanoma-specific death among male patients with invasive CM. KEYWORDS: invasive cutaneous melanoma; non-melanoma skin cancer; mortality; cohort study

#4224

Psychotropic polypharmacy during the initial phase of care among cancer patients in the United States.

Ami M. Vyas, Stephen J. Kogut, Hilary Aroke, Ashley Buchanan. _University of Rhode Island College of Pharmacy, Kingston, RI_.

Objectives: Limited evidence exists regarding the rates of psychotropic polypharmacy (PP) during the initial phase of care among cancer patients. We describe the characteristics, rates of PP and its predictors during the initial phase of care among cancer patients using administrative claims data.

Methods: A retrospective cohort study was conducted in patients age 18 and above diagnosed with the most commonly occurring cancers (breast, prostate, lung, colorectal) during 2011-2012 using the Optum ClinformaticsTM Data Mart (OptumInsight, Eden Prairie, MN) commercial claims database. PP was defined as concurrent use of two or more psychotropic medications for at least 90 days during the initial phase of care (defined as the first year after cancer diagnosis). Psychotropic medications included antidepressants, antipsychotics, anticonvulsants, anxiolytics-sedatives-hypnotics, and central nervous system stimulants/psychostimulants. The average number of psychotropic medications per patient with PP during the initial phase of care and the proportion of patients on PP were determined. An adjusted logistic regression identified significant predictors of PP in this vulnerable population.

Results: Among 5,604 cancer patients included in the study, 52.6% had breast cancer, 30.6% had prostate cancer, 11.4% had colorectal cancer and 5.5% had lung cancer. During the initial phase of cancer care, PP was found in 7.4% of cancer patients, with highest among those with lung cancer (14.4%), and least among those with prostate cancer (2.9%). Cancer patients on PP had an average of 10.7 (standard deviation=9.6) psychotropic medications prescriptions during the initial phase of cancer care, with highest among lung cancer patients (mean=12.4) and least among prostate cancer patients (mean=9.4). The significant predictors associated with an increased risk of PP were gender (adjusted odds ratio (AOR) for females=2.1, 95% confidence interval (CI):1.29, 3.41), age (AOR for age 55-64 years=1.7, 95% CI: 1.09, 2.65), colorectal cancer (AOR=2.18; 95% CI:1.29, 3.69), lung cancer (AOR=2.46; 95% CI:1.39, 4.35), and presence of psychiatric (AOR for two or more psychiatric conditions=11.7, 95% CI: 6.75, 20.3) and physical chronic conditions (AOR for two or more physical conditions=2.2, 95% CI: 1.63, 3.05).

Conclusion: Psychotropic polypharmacy differed by type of cancer, and it was highly prevalent among those with lung, breast and colorectal cancer patients during their initial phase of care. These findings underscore the importance of evidence-based psychotropic prescribing and close surveillance of adverse drug interactions between psychotropic medications and between psychotropic medications and anti-cancer treatment regimen, in order to improve quality of care and clinical outcomes in this vulnerable population.

#4225

Myelodysplastic syndrome and acute myeloid leukemia following use of chemo-immunotherapy and granulocyte colony-stimulating factors among elderly non-Hodgkin lymphoma patients.

Kellyn M. Moran,1 Zhaoju Wu,1 Sruthi Adimadhyam,1 Todd A. Lee,1 Brian C. Chiu,2 Gregory S. Calip1. 1 _University of Illinois at Chicago, Chicago, IL;_ 2 _The University of Chicago, Chicago, IL_.

Purpose The purpose of this study was to examine use of granulocyte colony-stimulating factors (G-CSF) in relation to risk of myelodysplastic syndrome and acute myeloid leukemia (MDS/AML) in a population-based cohort of elderly patients with non-Hodgkin lymphoma (NHL).

Methods We conducted a retrospective cohort study of adults ages 66 years and older diagnosed with first primary NHL between 2001 and 2011 using the Surveillance Epidemiology and End Results (SEER)-Medicare linked database. Patients were included if they were alive and without documented secondary primary cancer at three months post-diagnosis.

Using a validated algorithm with Medicare claims and SEER records, we identified MDS/AML cases, primary NHL treatment received (i.e., chemo-immunotherapy and radiation) and type and number of doses of G-CSF received (i.e., filgrastim and/or pegfilgrastim). We determined the risk of developing MDS/AML at 12 months or longer after NHL diagnosis. We estimated adjusted hazard ratios (HR) and 95% confidence intervals (CI) for MDS/AML risk associated with G-CSF use in cause-specific Cox proportional hazards models, stratified by treatment to account for confounding by indication.

Results A total of 18,245 patients were included with a mean age of 76 years and median follow up of 3.5 years. Most patients were diagnosed with diffuse large B-cell lymphoma (34%) and follicular lymphoma (22%). Fifty-six percent of all patients received chemo-immunotherapy, among whom most received rituximab-based chemotherapy (74%) or monotherapy (18%). G-CSF was most commonly used among those who received rituximab plus chemotherapy (77%). Subsequent MDS/AML was observed in 666 (3.7%) patients. Overall, we found a modest increased risk of MDS/AML with any use of G-CSF (HR=1.28, 95% CI 1.01-1.62); although, a linear trend was observed with increasing number of G-CSF doses (P<0.001). Among patients receiving rituximab plus chemotherapy, risk of MDS/AML was highest in those receiving 10 or more G-CSF doses (HR=1.64, 95% CI 1.14-2.37). When analyzing specific G-CSF agents, this increased MDS/AML risk was consistently observed with filgrastim (10+ doses: HR=1.67, 95% CI 1.25-2.23), but not with pegfilgrastim (10+ doses: HR=1.11, 95% CI 0.84-1.45).

Conclusions In this study of elderly NHL patients, we observed a higher MDS/AML risk among those receiving chemo-immunotherapy and G-CSF that was specific to higher number of filgrastim doses but not pegfilgrastim. The benefits of preventing complications from therapy-related neutropenia in patients receiving highly myeloablative treatments likely outweigh the risk of this rare but serious second primary cancer. Understanding differential risk by type of agent warrants further study with the increasing use of G-CSF and FDA-approved biosimilar products (filgrastim-sndz) newly available.

#4226

Association between NSAID, statins, and bisphosphonates and prostate cancer survival during androgen deprivation therapy.

Paavo V. Raittinen,1 Kirsi Talala,2 Kimmo Taari,3 Teuvo L. Tammela,4 Pauliina Ilmonen,1 Anssi Auvinen,4 Teemu J. Murtola4. 1 _Aalto University, Helsinki, Finland;_ 2 _Finnish Cancer Registry, Helsinki, Finland;_ 3 _University of Helsinki, Helsinki, Finland;_ 4 _University of Tampere, Tampere, Finland_.

We study the association between non-steroidal anti-inflammatory drugs (NSAIDs), Statins, and Bisphosphonates (BPs), and prostate cancer (PCa) survival during androgen deprivation therapy (ADT). STAMPEDE trial has demonstrated better PCa-specific survival in men using combination of celecoxib (CEL) and zoledronic acid (ZA) during ADT compared to ADT alone. The mechanism is unclear. ZA inhibits mevalonate pathway (MevP) previously linked with cancer growth. We evaluated PCa survival among men on ADT and simultaneously using BPs including ZA or statins, another drug group inhibiting MevP, and NSAIDs including CEL. We hypothesized that combined use of a MevP inhibitor and NSAID would be associated with improved PCa survival.

Our study cohort includes 4,428 men from the Finnish Randomized Study of PCa Screening (FinRSPC) initiating ADT in 1995-2015. Cox proportional hazards model with adjustment for age, FinRSPC study arm, tumor clinical characteristics and co-morbidities (obtained from national registries) was used to calculate HRs and 95% CI for PCa death. Medication use was analyzed as time-dependent variable.

Compared to non-users, the risk of PCa death was increased in users of NSAIDs or acetaminophen, and lowered in statin users. Use of BPs or coxibs alone were not associated with the risk. Coxibs and statins together were associated with lowered risk to a similar degree as statins alone. No statistically significant risk differences were observed for other combinations.

Statin users with high-risk prostate cancer undergoing ADT have lowered risk of PCa death. NSAID users have increased risk of PCa death, which becomes statistically insignificant when used with statins. Statin and BP use together shows no statistically significant evidence of negating the effects of statin. No clear additive benefit was observed for statins and coxibs together over statins alone. Our findings do not support additive benefits of MevP inhibitor and NSAIDs.

Statistical significance codes: *** : p = 0.001, ** : p = 0.01, * : p = 0.1

---

Drug | HR | 95 % CI | Significance

Statin | 0.78 | 0.68 | 0.90 | ***

Acetylsalisylic acid | 0.90 | 0.76 | 1.07

|

Coxib | 1.07 | 0.94 | 1.22

|

NSAID | 1.17 | 1.04 | 1.31 | **

Acetaminophen | 1.66 | 1.51 | 1.82 | ***

Bisphosphonate | 0.79 | 0.38 | 1.64

|

Bisphosphonate and NSAID | 0.89 | 0.55 | 1.42

|

Statin and NSAID | 1.07 | 0.90 | 1.27

|

Statin and Bisphosphonate | 1.14 | 0.86 | 1.50

|

Coxib and Bisphosphonate | 0.85 | 0.42 | 1.74

|

Coxib and Statin | 0.80 | 0.62 | 1.02 | *

EAU tumor Risk Group | 2.66 | 2.35 | 3.00 | ***

#4227

Treatment regimens, treatment duration, and between-treatment duration in patients with small cell lung cancer across lines of therapy: An analysis of electronic health records data.

Rui Jiang,1 Scott H. Gulbranson,1 Philip Komarnitsky,1 Martina M. Koch,2 Fabio A. Lievano,1 Ravi Potluri,3 Charity Scripture,2 Jerzy E. Tyczynski1. 1 _Abbvie, North Chicago, IL;_ 2 _AbbVie Stemcentrx, South San Francisco, CA;_ 3 _SmartAnalyst Inc., New York, NY_.

While platinum-based combination chemotherapy is recommended for first-line treatment of small cell lung cancer (SCLC), the treatment for relapsed and refractory SCLC is heterogeneous with limited clinical benefit. Topotecan is the only approved second-line agent, and no drug or combination regimen is approved in the third- or later line settings. The purpose of this study was to describe treatment regimens, treatment duration, and between treatment duration in SCLC patients across lines of therapy (LOT) in recent clinical practice. We analyzed treatment patterns in 8,291 SCLC patients who had a diagnosis between 01/01/2008 and 09/30/2016 and met our inclusion criteria using Optum's electronic health records (EHR) database. The database consists of de-identified, patient-level EHR data from a network of healthcare provider organizations in the United States. Of these 8,291 newly diagnosed patients, 3,633, 1,211, 428, 138, 45, 17, 5, 1 patients received the first-, second-, third-, fourth-, fifth-, sixth-, seventh- and eighth- line of therapy, respectively. Platinum-based chemotherapy (carboplatin or cisplatin) plus etoposide was the most frequently administered first-line treatment (51%+29%=80%) in SCLC (Table 1). Cisplatin has been largely replaced by the less toxic carboplatin after first line therapy. The most frequent second-line treatments were topotecan and carboplatin+etoposide. Topotecan, paclitaxel, gemcitabine, docetaxel, and carboplatin+etoposide were the most frequently administered chemotherapy drugs in third or later lines. As the disease progressed/became more severe, time to subsequent LOT decreased with increasing number of therapy lines (Table 1). In conclusion, this analysis provides valuable insight into real-world SCLC treatment characteristics, particularly for later lines, and indicates a need for more effective novel therapies for SCLC.

Table 1. Regimens, treatment duration, and between treatment duration across lines of therapy (LOT)

---

|

LOT1 (N=3,633) | LOT2 (N=1,211) | LOT3 (N=428) | LOT4+ (N=206)

Carboplatin+Etoposide | 51% | 21% | 7% | 10%

Cisplatin+Etoposide | 29% | 5% | 1% | 0%

Topotecan | 2% | 31% | 29% | 13%

Carboplatin+Paclitaxel | 5% | 4% | 5% | 2%

Etoposide | 4% | 4% | 3% | 4%

Paclitaxel | 1% | 8% | 18% | 13%

Gemcitabine | 0% | 2% | 7% | 15%

Irinotecan | 0% | 4% | 4% | 5%

Carboplatin | 2% | 2% | 0% | 0%

Docetaxel | 0% | 2% | 7% | 10%

Carboplatin+Pemetrexed | 2% | 0% | 0% | 0%

Nivolumab | 0% | 3% | 4% | 6%

Carboplatin+Irinotecan | 0% | 3% | 3% | 4%

Pemetrexed | 0% | 3% | 2% | 1%

Cisplatin+Irinotecan | 0% | 2% | 2% | 1%

Cisplatin | 1% | 1% | 0% | 0%

Cyclophosphamide+Doxorubicin+Vincristine | 0% | 1% | 3% | 5%

Carboplatin+Gemcitabine | 1% | 1% | 1% | 1%

Other regimens | 2% | 3% | 4% | 7%

|

Treatment duration | |

|

|

Mean (SD), Days | |

|

LOT1 | 60.8 (47.7) | |

|

LOT2 | 56.2 (67.8) | |

|

LOT3 | 54.7 (69.1) | |

|

LOT4 | 52.5 (54.1) | |

|

LOT5 | 64.5 (111) | |

|

LOT6 | 28.5 (30.1) | |

|

LOT7 | 68.4 (55.9) | |

|

LOT8 | 36 (0) | |

|

|

Duration between LOTs | |

|

|  | |

|

LOT1 to LOT2 | 199 (165) | |

|

LOT2 to LOT3 | 149 (131) | |

|

LOT3 to LOT4 | 134 (131) | |

|

LOT4 to LOT5 | 139 (97.8) | |

|

LOT5 to LOT6 | 125 (88.7) | |

|

LOT6 to LOT7 | 91.4 (34.9) | |

|

LOT7 to LOT8 | 201 (0) | |

|

#4228

Incidence of pericardial and pleural effusions in patients with small cell lung cancer treated with third-line or later-line therapy: An analysis of electronic health records data.

Rui Jiang,1 Scott H. Gulbranson,1 Mondira Bhattacharya,1 Fabio A. Lievano,1 Philip Komarnitsky,1 Martina M. Koch,2 Ravi Potluri,3 Jerzy E. Tyczynski1. 1 _AbbVie, North Chicago, IL;_ 2 _AbbVie Stemcentrx, South San Francisco, CA;_ 3 _SmartAnalyst Inc., New York, NY_.

Small cell lung cancer (SCLC) is an aggressive, rapidly fatal disease. Pericardial and pleural effusions are, to a large extent, part of the natural history of lung cancer and the occurrences in SCLC may have differed due to novel therapies. We estimated the incidence of treatment-emergent pericardial and pleural effusions in SCLC patients treated with third-line or later-line therapy using Optum's electronic health records (EHR) data. The database consists of de-identified EHR data from a network of healthcare provider organizations in the United States. Natural language processing (NLP) was used to identify SCLC. Of 8,291 patients with newly diagnosed SCLC between 01/01/2008 and 09/30/2016 who met our inclusion criteria, 428 patients received 3rd line of therapy (LOT). 206 LOTs were 4th or later lines. Pericardial and pleural effusions were identified by a combination of ICD-9/ICD-10, procedures, or terms from NLP structured data. At start of 3rd LOT, median age was 65, 46% were male, 97% had extensive disease (excluding n=52 with missing staging). The incidence rate was 1.58 (95% CI, 1.17-2.07) per 1,000 person-days for pericardial effusion and 4.40 (95% CI, 3.67-5.25) for pleural effusion in patients with 3rd LOT while on-treatment (Table 1). The incidence rate was 1.15 (95% CI, 0.88-1.49) for pericardial effusion and 3.68 (95% CI, 3.12-4.32) for pleural effusion in patients with 3rd LOT while on-treatment and off-treatment. Patients treated with 4th or later lines had higher incidence of pericardial and pleural effusions compared to patients treated with 3rd LOT. The incidence proportions of the effusions (Table 1) should be interpreted with care, as the patients had varying length of follow-up. In conclusion, this analysis is the first, to our knowledge, to provide population-based estimates of incidence for pericardial and pleural effusions in SCLC patients treated with third- or later-line therapy.

Table 1. Incidence of pericardial and pleural effusions

---

|

Incidence Proportion % (95% CI) | Incidence Rate per 1,000 Person-Days (95% CI)

On-treatment*  | |

3rd LOT (n=428) | |

Pericardial (n=51) | 11.9% (9.00-15.4%) | 1.58 (1.17-2.07)

Pleural (n=125) | 29.2% (24.9-33.8%) | 4.40 (3.67-5.25)

4th\+ LOT(n=206) | |

Pericardial (n=32) | 15.5% (10.9-21.2%) | 2.14 (1.46-3.01)

Pleural (n=66) | 32.0% (25.7-38.9%) | 5.49 (4.25-6.99)

On-treatment and off-treatment** | |

3rd LOT(n=428) | |

Pericardial (n=59) | 13.8% (10.7-17.4%) | 1.15 (0.88-1.49)

Pleural (n=152) | 35.5% (31.0-40.3%) | 3.68 (3.12-4.32)

4th\+ LOT(n=206) | |

Pericardial (n=37) | 18.0% (13.0-23.9%) | 1.49 (1.05-2.05)

Pleural (n=80) | 38.8% (32.1-45.9%) | 4.14 (3.28-5.15)

*from start of LOT to earlier of (30 days after end of LOT or start of next LOT); **from start of LOT to the day prior to start of next LOT or earlier of (365 days from end of LOT or loss to follow-up date), if there is no subsequent LOT; for those with the event, time to first occurrence was used

#4229

Preliminary results from the Pharmacogenetics Ovarian Cancer Knowledge to Individualize Treatment (POCKIT) study.

Ayush Giri,1 Rebecca T. Levinson,1 Spencer Keene,1 Gwendolyn Holman,1 Stacy D. Smith,1 Leshaun Clayton,1 Whitney Lovett,1 Samantha P. Stansel,1 Malcolm-Robert Bringhurst Snyder,1 Jason T. Fromal,1 Gabriella D. Cozzi,1 Dineo Khabele,2 Alicia Beeghly-Fadiel1. 1 _Vanderbilt University Medical Center, Nashville, TN;_ 2 _Kansas University Medical Center, Kansas City, KS_.

Introduction: With an overall five-year survival rate of only 46%, ovarian cancer is the most lethal gynecologic malignancy. Treatment includes surgical cytoreduction (debulking) followed by chemotherapy with platinum and taxane agents, and prognosis generally depends upon clinical characteristics, such as stage of disease at diagnosis, success of surgical debulking, and histologic subtype. However, even among women with comparable clinical characteristics, there can be variation in survival. Part of this variability may be due to inherited genetic variation in genes related to the absorption, distribution, metabolism, and excretion (ADME) of pharmacologic agents commonly used to treat ovarian cancer.

Approach: To test the hypothesis that pharmacogenetic variants influence ovarian cancer prognosis, we assembled a clinical cohort of confirmed epithelial ovarian cancer cases from electronic medical records (EMR) at the Vanderbilt University Medical Center (VUMC) with banked DNA samples available. Clinical characteristics were abstracted from EMR using natural language processing (NLP)-assisted EMR review and a REDCap data collection instrument. Genotyping was conducted on the Sequenom iPLEX ADME PGx panel at the Vanderbilt Technologies for Advanced Genomics (VANTAGE) core facility. Associations with overall survival were evaluated using multivariable proportional hazards regression.

Results: We identified a total of 391 epithelial ovarian cancer cases with banked DNA from VUMC EHR. Clinical characteristic abstracted by NLP followed expected distributions; the majority of cases were Caucasian (87%), with serous histology (63%), late-stage (62%), high-grade (60%) disease. Most common treatments included surgical cytoreduction (93%) and chemotherapy with a platinum (83%) and/or taxane agent (82%) agent. DNA was successfully pulled, plated, and genotyped for 327 cases (81.3%) for 73 common ADME variants in 30 genes. To prevent population stratification, genetic analyses were restricted to 287 Caucasians, where five nominally significant overall survival associations were identified: ABCB1 rs1045642, ABCC2 rs2273697, CYP2A6 rs1801272, CYP2E1 rs2070673, and SLCO2B1 rs2306168. Additional analyses, including for gene and drug scores, are currently under way.

Conclusions: Individual variation in ADME genes may contribute to variation in ovarian cancer survival. Future steps include testing associations for replication, and evaluation of response to treatment. In addition, this research demonstrates that EMR-based study populations, in concert with linked biorepositories, can facilitate research on ovarian cancer.

### Cancer in Minority Populations, Health Disparities, and Survivorship Research

#4230

Influence of sociodemographic factors on treatment decisions in lung cancer.

Narjust Duma,1 Urshila Durani,1 Ryan Frank,1 Jonas Paludo,1 Gustavo Westin,1 Yanyan Lou,2 Leventakos Konstantinos,1 Julian Molina,1 Ronald Go,1 Aaron Mansfield,1 Sikander Ailawadhi,2 Alex Adjei1. 1 _Mayo Clinic, Rochester, MN;_ 2 _Mayo Clinic, Jancksonville, FL_.

Background: In stage IV non-small cell lung cancer (NSCLC), survival has significantly improved with the use of novel therapies and better supportive care. Despite such trends, it has been noted that patients (pts) frequently refuse cancer treatment. Therefore, we explored the factors associated with treatment refusal in pts with stage IV NSCLC.

Methods: Utilizing the National Cancer Database (NCDB), we identified all incident cases with stage IV NSCLC from 2004 to 2014. Patients who received cancer treatment outside of the reporting facility or died before receiving treatment were excluded. Multivariable logistic regression models were used to determine associations with treatment refusal.

Results: A total of 341,993 pts were identified. On average, 5.4% of pts refused radiation therapy and 10.3% refused chemotherapy despite provider recommendations. The proportion of pts refusing radiation and chemotherapy increased over the study period from 4.2% to 7.3% and 7.9% to 15%, respectively (p<0.001 for both). In multivariable analysis, men were less likely to refuse radiation or chemotherapy compared to women (OR: 0.82, 95%CI: 0.76-0.83, p<0.001; OR: 0.84, 95%CI: 0.81-0.86, p<0.001, respectively). Factors associated with radiation therapy refusal included: Medicaid or Medicare as primary insurance (OR: 1.95, 95%CI: 1.76-2.15, p<0.001; OR: 1.25, 95%CI: 1.17-1.34, p<0.001, respectively) or a >2 Charlson Comorbidity Index (OR: 1.97, 95%CI: 1.85-2.10, p<0.001). Moreover, receiving care at an academic center (OR: 0.57, 95%CI: 0.53-0.62, p<0.001) was associated with less likelihood of refusing radiation. Regarding chemotherapy, uninsured pts (OR: 2.45, 95%CI: 2.26-2.66, p<0.001), pts with Medicaid as primary insurance (OR: 2.17, 95%CI: 2.03-2.32, p<0.001) or with a high comorbidity index (OR: 1.76, 95%CI: 1.68-1.84, p<0.001) were more likely to refuse chemotherapy. Contrary to findings from previous studies, Non-Hispanic Blacks and Hispanics were less likely to refuse chemotherapy compared to non-Hispanic whites (OR: 0.86, 95%CI: 0.82-0.91, p<0.001; OR: 0.78, 95%CI: 0.72-0.89, p<0.001). Patients receiving care at an academic center and residing in high income neighborhoods were less likely to refuse chemotherapy (OR: 0.76, 95%CI: 0.72-0.80, p<0.001; OR: 0.76, 95%CI: 0.71-0.81, p<0.001, respectively). Distance from residence to treating facility and living in a rural county did not affect treatment refusal.

Conclusions: Socioeconomic factors rather than race/ethnicity appear to influence refusal of cancer treatment in pts with stage IV NSCLC in the NCDB. Assessing socioeconomic challenges should be an essential part of patients' evaluation when discussing treatment options.

#4231

Breast and prostate cancers harbor common somatic copy number alterations that consistently differ by race-ethnicity.

Yalei Chen,1 Jia Li,1 Sudha Sadasivan,1 Ruicong She,1 Indrani Datta,1 Dhananjay Chitale,1 Nilesh Gupta,1 Melissa B. Davis,1 Craig G. Rogers,1 Lisa A. Newman,1 Pamela L. Paris,2 Benjamin A. Rybicki,1 Albert M. Levin1. 1 _Henry Ford Health System, Detroit, MI;_ 2 _University of California at San Francisco, San Francisco, CA_.

Pan-cancer studies of somatic copy number alterations (SCNAs) have demonstrated shared SCNAs across cancer types, but whether these shared SCNAs vary by race-ethnicity has not been explored. Utilizing data from The Cancer Genome Atlas (TCGA), we identified SCNAs in breast and prostate tumors, two cancers with racially-disparate outcomes, and then tested for differences in SCNA magnitude by self-reported African American and European American race-ethnicity, as well as by regional chromosomal African ancestry within African Americans. GISTIC2 was applied to high density SNP array data to map SCNA regions in 712 European and 174 African American female breast tumors and 267 European and 42 African American prostate tumors derived from the TCGA dataset. For each tumor, SCNA magnitude was quantified by the area under the logarithm-base 2 copy number curve, and the germline ancestral origin of SCNAs was inferred using RFMix. A linear model was used to assess the association between SCNA magnitude and race-ethnicity (or regional African ancestry) while adjusting age-at-diagnosis and tumor severity. Race-differentiated SCNAs common to breast and prostate were found at chromosomes 5q11-21, 6q12-14, 6q16-22, 8q21-24, 11q22, 13q12-21, and 16q21-24, with 8q21-24 being the only amplification. African American breast and prostate tumors had higher magnitude alterations in the regions on 5q11-21, 8q21-24, 11q22, and 13q12-21, and among African Americans, this higher magnitude at 8q21-24 and 13q12-21 was consistent with increasing regional African ancestry. Within these regions with higher magnitude SCNAs in African Americans, expression analysis revealed 18 cancer genes, including RB1 and PVT1, differentially expressed by race-ethnicity in both tumors types that were consistent with the observed SCNA differences. While differences in SCNAs by race-ethnicity have been studied in single cancers, this is the first study to identify race-differentiated SCNAs shared by two hormonally-driven cancers and to explore the potential of germline genetic ancestry as a mechanism leading to this differentiation. The differentially expressed genes within SCNAs common to both tumor types could provide further insight into the racially disparate outcomes in breast and prostate cancers.

#4232

Multivariate analysis of breast cancer patients showing superior outcome in Hispanics compared to non-Hispanics.

Aurash Khoobehi,1 Runhua Shi,1 Amanda Burton,2 Gary V. Burton1. 1 _LSU Shreveport, Shreveport, LA;_ 2 _George Mason University, Fairfax, VA_.

Background

Breast cancer incidence and mortality varies relative to ethnicity and socioeconomic status. Prior research has shown that Hispanics appear to have a lower mortality rate from breast cancer when compared to Non-Hispanic Blacks (NHB) and Non-Hispanic Whites (NHW). To further delineate the influence of Hispanic ethnicity on breast cancer outcomes, we compared ethnicity and outcome while adjusting for multiple variables using data from the National Cancer Data Base (NCDB) collected between 2004 and 2013.

Methods

Data were analyzed from 984,512 women registered in the NCDB who were diagnosed with stage I to stage IV breast cancer. Overall survival was the primary outcome variable; the primary predictor variable was ethnicity (NHW, NHB, and Hispanic). Variables addressed and adjusted for included: stage, age, Charlson Comorbidity index, estrogen/progesterone receptor (ERPR) status and HER2 receptor status. Multivariate Cox regression was used to investigate the effect of ethnicity on overall survival while adjusting for secondary predictive factors.

Results

The majority of patients were NHW (85.6%), NHB (12.5%), and Hispanic (1.85%). The median overall survival for all patients was greater than 12 years.

Hispanics, compared to NHW, had a higher percentage of patients with triple negative (TN) (7.6 vs 5.6%), HER2 positive (19.3 vs 14.2%), and stage III/IV (23.1 vs 15.7%). In univariate analysis, the hazard ratio (HR) of death in Hispanics, compared to NHW, was lower for stage III (0.744), stage IV (0.842), HER2 positive (0.748) and TN (0.862) patients (all p-value <0.05). Hispanics, compared to NHW, had a lower percentage of patients with ERPR positive (77.3 vs 82.9%) and stage I (39.3 vs 52.3%). However, the HR of death in Hispanics, compared to NHW, was lower for stage I (0.607) and ERPR positive (0.779) patients (all p-value <0.05).

All patients with a Charlson score of 1 or more, regardless of ethnicity, are 65% more likely to die compared to patients with no co-morbidities. Triple negative patients are 2.5 times more likely to die compared to patients of non-TN status. Patients that are stage II, III, and IV are 1.9, 4.6, and 19.2 times more likely to die than patients with stage I disease, respectively.

In multivariate Cox regression, effects of ethnicity on survival were significant. Hispanic patients were 17% less likely and NHB patients were 34% more likely to die when compared to NHW breast cancer patients.

Conclusion

The Hispanic, breast cancer population has a statistically significant better survival than NHW patients regardless of stage, age, co-morbidities, or receptor (ERPR, HER2 or TN) status. These results indicate that Hispanic ethnicity is a predictor of superior patient outcomes. Further investigation will be required to delineate the factors which contribute to this outcome difference.

#4233

Racial differences in patterns of cytogenetic abnormalities in multiple myeloma.

Meytal B. Chernoff,1 Madina Sukhanova,1 Liz Stepniak,1 Wei Zhang,2 Brian C. Chiu1. 1 _University of Chicago, Chicago, IL;_ 2 _Northwestern University, Chicago, IL_.

Introduction: African American (AA) patients with Multiple Myeloma (MM) are diagnosed at younger ages and show an age-adjusted incidence twice as high as that seen in European Americans (EA); however, a study by Waxman et al found higher disease specific survival in AA. A recent study showed differences in chromosomal aberrations between AA and EA, but further work is needed to understand racial differences in cytogenetics through Fluorescent In Situ Hybridization (FISH) using an extensive array of probes.

Methods: We identified 470 consecutive patients with MM from the University of Chicago Cancer Cytogenetics Laboratory from October 2003-January 2017. Of those, 127 (22%) patients had abnormal karyotypes and 343 were evaluated by FISH using a panel of 18 probes. Twenty-two patients who self-identified as Hispanic, Multiracial, Asian, or Indian were dropped from the analysis due to low numbers. For the remaining 311 patients, we obtained data on IGH translocations (partner genes included IGH/FGFR3 fusion [t(4;14)(p16.3;q32)]; IGH/CCND1 fusion [t(11;14)(q13;q32)]; IGH/MAF fusion [t(14;16)(q32;q23)]); TP53 loss [17p13.1]; CDKN2C loss [1p32.3]; CKS1B gain [1q21]; ATM losss [(11)(q22.3)11(q22.3)]; trisomy of chromosomes 3, 7, 9, 12, and 15; and chromosome 13 loss or interstitial deletion of 13q. We grouped the FISH results as high (loss of TP53, loss of CDKN2C; CKS1B gain, IGH/MAF fusion), intermediate (IGH/FGFR3 fusion), or standard (Trisomies 3, 7, 9, 15; Gain of TP53, Gain of ATM, chromosome 13 loss) prognostic risk markers.

Results: Of 311 patients, 172 (55.3%) are self-reported EA, 130 (41.8%) are AA. Compared with EA, AA patients were more likely to be females (44.8% vs. 59.2%, respectively) and older than 65 years at diagnosis (47.7% vs. 56.2% respectively). Overall, 6.3% of patients were younger than 45 years at diagnosis, but there was no racial difference in the proportion of young patients. Compared with EA, AA patients assessed with standard risk probes were less likely to have standard risk cytogenetic features (48% vs. 66%; p-value=0.02). AA patients showed a lower percentage of hyperdiploidy, 36.5% of AA patients assessed were positive for hyperdiploidy compared with 57.1% of assessed EA (p=0.02). Despite the lower proportion of AA with cytogenetic markers associated with favorable prognosis, there was no significant difference between EA and AA patients in the prevalence of adverse prognostic markers, such as TP53 deletion, IGH/MAF fusions, or CKS1B gain. However, we did observe that AA patients were more likely to have loss of CDKN2C (16.7% vs 3.6%, p=0.02).

Conclusions: We found that compared with EA, AA patients are less likely to have cytogenetic features associated with favorable prognosis (i.e. hyperdiploidy), yet AA do not have a higher prevalence of high risk cytogenetics. These findings suggest that cytogenetics alone may not fully explain racial/ethnic differences in MM mortality.

[Meytal Chernoff and Madina Sukhanova are co-first authors of this abstract.]

#4234

Association between educational level and mortality: A pooled analysis of over 694,000 individuals in the Asia Cohort Consortium.

Keming Yang,1 Ying Zhang,2 Eiko Saito,3 Yu Chen,4 Prakash C. Gupta,5 Yu-Tang Gao,6 Akiko Tamakoshi,7 Yong-Bing Xiang,6 Woon-Puay Koh,8 Norie Sawada,3 Kemmyo Sugiyama,9 Yasutake Tomata,9 Atsuko Sadakane,10 Chisato Nagata,11 San-Lin You,12 Renwei Wang,13 Myung-Hee Shin,14 Habibul Ahsan,15 Wen-Harn Pan,16 Mangesh S. Pednekar,5 Jian-Min Yuan,17 Xiao-Ou Shu,18 Hui Cai,18 Shoichiro Tsugane,3 Ichiro Tsuji,9 Seiki Kanemura,9 Keiko Wada,11 Yoon-Ok Ahn,19 Kotaro Ozasa,10 John D. Potter,20 Wei Zheng,18 Hongmei Nan1. 1 _Department of Epidemiology, Indiana University Richard M. Fairbanks School of Public Health, Indianapolis, IN;_ 2 _Department of Biostatistics, Indiana University Richard M. Fairbanks School of Public Health, Indianapolis, IN;_ 3 _Epidemiology and Prevention Group, Center for Public Health Sciences, National Cancer Center, Tokyo, Japan;_ 4 _Department of Population Health, Department of Environmental Medicine, New York University School of Medicine, New York, NY;_ 5 _Healis-Sekhsaria Institute for Public Health, Navi Mumbai, India;_ 6 _Department of Epidemiology, Shanghai Cancer Institute, Shanghai, China;_ 7 _Graduate School of Medicine, Hokkaido University, Sapporo, Japan;_ 8 _Duke-NUS Medical School, Singapore, Singapore;_ 9 _Tohoku University Graduate School of Medicine, Miyagi Prefecture, Japan;_ 10 _Radiation Effects Research Foundation, Hiroshima, Japan;_ 11 _Graduate School of Medicine, Gifu University, Gifu, Japan;_ 12 _School of Medicine, Big Data Research Centre, Fu-Jen Catholic University, Taipei, Taiwan;_ 13 _University of Pittsburgh Cancer Institute, Pittsburgh, PA;_ 14 _Department of Social and Preventive Medicine, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea;_ 15 _Department of Public Health Sciences, University of Chicago, Chicago, IL;_ 16 _Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan;_ 17 _Department of Epidemiology, University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA;_ 18 _Division of Epidemiology, Department of Medicine, Vanderbilt Epidemiology Center, Vanderbilt–Ingram Cancer Center, Vanderbilt University, Nashville, TN;_ 19 _Department of Preventive Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea;_ 20 _Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA_.

Background: Higher educational levels have been linked to reduced risk of death in previous studies. Most of these studies, however, were conducted in the United States and Europe.

Methods: We performed a pooled analysis to evaluate the association between educational levels and risk of death among 694,434 Asian individuals from 15 prospective cohort studies that participated in the Asia Cohort Consortium. The analysis included 103,023 deaths during a mean follow-up period of 12.5 years, among which 33,945 were due to cancer and 34,645 were due to cardiovascular diseases (CVD). We employed Cox proportional hazards regression models to estimate hazard ratios (HRs) and 95% confidence intervals (95% CIs) for the association between educational level and the risk of death after adjusting for potential confounders.

Results: Higher educational levels were significantly associated with lower risk of death from all causes; compared to a low educational level (≤ primary school), HRs and 95% CIs for secondary education, trade/technical education, and ≥ university degree were 0.86 (0.83-0.90), 0.80 (0.71-0.89), and 0.68 (0.61-0.75), respectively (Ptrend <0.0001). Similar associations were observed for cancer or CVD-specific mortality, with HRs of 0.93 (0.89-0.97), 0.87 (0.78-0.97), and 0.79 (0.71-0.87) for risk of cancer deaths with increasing levels of education (Ptrend = 0.003) and HRs of 0.86 (0.81-0.91), 0.75 (0.64-0.87), and 0.64 (0.57-0.73) for risk of CVD deaths with increasing levels of education (Ptrend <0.0001). The pattern of the association among East Asians and South Asians was similar; compared to ≤ primary school, HR (95% CI) for all-cause mortality associated with ≥ university degree was 0.68 (0.61-0.76) among 539,724 East Asians (Chinese, Japanese, and Korean) and 0.61 (0.54-0.69) among 154,710 South Asians (Indians and Bangladeshis) included in this analysis.

Conclusions: Higher educational level was associated with substantially lower risk of death in Asian populations including both East and South Asians.

#4235

Race differences in treatment and survival of ovarian cancer.

Janet S. Hildebrand, Kristin Wallace, Linda E. Kelemen. _Medical University of South Carolina, Hollings Cancer Center, Charleston, SC_.

Black women have lower incidence rates of ovarian cancer than white women in the U.S., but their cancers are more often fatal. Factors associated with lower survival among blacks include advanced stage at diagnosis and fewer women receiving a surgery-chemotherapy sequence consistent with national guidelines for treatment of primary ovarian cancer. We examined treatment and survival among 365 white and 95 black ovarian cancer patients captured by the Hollings Cancer Center Cancer Registry in Charleston, S.C. from 2000 to 2015 with the aim of identifying factors to explain observed racial disparities. Outcomes of interest were receipt of a cytoreductive surgery-chemotherapy sequence, with or without neoadjuvant therapy, and overall survival (OS) calculated as months from diagnosis to death. We used unconditional logistic regression to estimate odds ratios (ORs) of black vs. white race associated with receipt of surgery-chemotherapy, controlling for clinical stage, histology, insurance status, smoking status, and severity of comorbidities (none, mild, moderate, moderate-severe, severe), assessed using the age-adjusted Charlson index. For survival comparisons, we plotted Kaplan-Meier curves by race and estimated the death hazard ratio (HR) using Cox proportional hazards regression. We assessed statistical interaction between race and comorbidities using −2 log likelihood tests. Age, stage and histology distributions were similar between blacks and whites; however, white women were more likely than blacks to be current or former smokers (44% vs. 32%) and to have private health insurance (38% vs. 26%), while blacks were more likely to have moderate-severe or severe comorbidities (37% vs. 30%), although differences were not statistically significant. There were significant racial differences in receipt of surgery-chemotherapy (blacks 44% vs. whites 69%, p<0.0001). Women who did not receive a surgery-chemotherapy sequence were 3 times more likely to be black (adjusted OR 3.13, 95% confidence interval (CI) 1.90−5.16), but the disparity was observed only in women with comorbidity as opposed to none (interaction p=0.01). Median OS was 29.3 months among black women and 49.2 months among whites (log rank p<0.001). The survival disadvantage among blacks persisted after controlling for treatment, comorbidities, stage, smoking, and insurance (HR 1.87, 95% CI 1.41−2.48), and no statistical interaction between race and comorbidities was evident (p=0.17). In this study, black women, particularly those with comorbidities, were significantly less likely than whites to receive a surgery-chemotherapy sequence for treatment of ovarian cancer. Black race was associated with an 87% higher fatality rate independent of treatment, stage, comorbidities, and insurance status. More research is needed to understand race-specific reasons for nonadherence to guideline based care and racial disparities in survival after ovarian cancer diagnosis.

#4236

Charlson comorbidity index scores before and after breast cancer diagnosis among a racially diverse cohort of women.

Alpana Kaushiva,1 Susan Hong2. 1 _University of Illinois at Chicago School of Public Health, Chicago, IL;_ 2 _University of Illinois Cancer Center, Chicago, IL_.

Background: We examined racial/ethnic differences in Charlson comorbidity index scores (CCI) pre and post breast cancer (BC) diagnosis among Non-Hispanic White (NHW), Hispanic, and African-American (AA) women.

Methods: We conducted a retrospective chart review to calculate CCI scores (excluding age and BC) for 387 women diagnosed with BC between December 2013 and June 2017 at the University of Illinois Hospital and Health Sciences System. Using all available electronic records, we used International Chronic Disease 9 and 10 codes to determine presence or absence of a chronic condition pre and post BC diagnosis. Data was collected on race, ethnicity, stage, age at diagnosis, and vital status. Logistic regression was used to calculate odds ratios (OR) and 95% confidence intervals (CI) for associations between race/ethnicity and CCI among BC patients.

Results: Of the 387 BC patients, 63% were AA, 15% were Hispanic, and 16% were NHW. Mean ages were 56, 56, and 57 years, respectively. 217 women had a CCI of 0 (no comorbidity), 101 women had a CCI ≥1 at their BC diagnosis (mean CCI 4.8), and 69 women developed at least one chronic condition after their BC diagnosis (mean CCI 5.2; with a mean duration of follow-up of 1.9 years). AA women had a higher average CCI score compared to Hispanic and NHW women (CCI= 4.3 versus 3.9 & 3.3, p=0.006). Although not statistically significant, mean CCI scores were higher for Hispanic versus NHW women (CCI= 3.9 vs 3.3, p=0.08). At the time of their BC diagnosis, AA and Hispanic women were 2.2 times more likely to have a CCI ≥ 1 compared to NHW women [(AA vs NHW 95% CI (1.3-3.9), p=0.004); (Hispanic vs NHW 95% CI (1.1-4.3), p=0.02)]. Overall, compared to NHW women, AA women were 3.1 times more likely to have a cancer diagnosis other than breast [95% CI (1.1-8.9), p=0.04] and 5.4 times more likely to suffer from heart failure [95% CI (1.3-23.3), p=0.02]. Compared to NHW women, rates of diabetes were 2.3 times higher among AA women [95% CI (0.95-5.7), p=0.06] and 3.9 times higher among Hispanic women [95% CI (1.4-10.9), p=0.008].

Of the women with a CCI=0 at the time of their BC diagnosis, AA women were 3.3 times more likely [95% CI (1.3-8.1), p=0.01] and Hispanic women were 2.9 times more likely [95% CI (1.0-8.6), p=0.05] to go on to develop at least one chronic condition compared to NHW women. Additionally, of the women who died (n=16), AA and Hispanic women were diagnosed with BC at significantly younger mean ages compared to NHW women [(AA mean age=55 years; Hispanic mean age=64 years; NHW mean age=88 years; p=0.05)].

Conclusion: AA and Hispanic women enter their BC diagnosis with higher mean CCI scores and are more likely to develop ≥1 chronic health condition after their BC diagnosis. Our findings suggest that the burden of chronic disease both before and after BC diagnosis is greater for minority vs. NHW BC survivors. Future studies examining how this impacts survivorship for minority women are warranted.

#4237

**Differences in the frequencies of tumor** VHL **mutation and HIF-2α expression between black and white patients with clear cell renal carcinoma.**

Catherine L. Callahan,1 Lee E. Moore,1 Petra Lenz,2 Kendra Schwartz,3 Julie Ruterbusch,3 Faith Davis,4 Wong-Ho Chow,5 W. Marston Linehan,6 Maria J. Merino,6 Stephen M. Hewitt,6 Nathaniel Rothman,1 Jonathan N. Hofmann,1 Michael L. Nickerson,1 Mark P. Purdue1. 1 _National Cancer Institute, Rockville, MD;_ 2 _SAIC-Frederick, Inc., Frederick, MD;_ 3 _Wayne State University, Detroit, MI;_ 4 _University of Alberta, Edmonton, Alberta, Canada;_ 5 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 6 _National Cancer Institute, Bethesda, MD_.

Background: Black Americans have a poorer prognosis for clear cell renal cell carcinoma (ccRCC) than white Americans, potentially due in part to differences in tumor biology. In a recent analysis of The Cancer Genome Atlas (TCGA), tumors from black ccRCC patients had a lower rate of mutation in the VHL tumor suppressor gene and lower expression of hypoxia inducible factors (HIF) than tumors from white ccRCC patients. However, as this study was based on a small number of black patients (N=19) and had limited information on patients' medical histories and risk factors profiles, further investigation is needed.

Objective: We evaluated differences in the frequencies of somatic VHL gene mutations and HIF-1α and -2α protein expression between tumors from black and white ccRCC patients.

Methods: The investigation utilized formalin-fixed tissue and data collected from patients participating in a case-control study conducted in Chicago and Detroit. We sequenced VHL using the Ion Torrent platform for tumors from 69 black and 98 white patients, and measured tumor HIF-1α and -2α protein expression for 88 black and 240 white patients using immunohistochemistry.

Results: Black patients' tumors had a lower frequency of VHL mutation than those of white patients (32% vs. 49%; P = 0.03) as well as a lower frequency of above-median HIF-2α expression (33% vs. 56%; P=0.002). HIF-1α expression did not differ by race (P=0.14). These racial differences persisted after multivariable model adjustment for age, sex, hypertension, chronic kidney disease, body mass index, smoking status, stage, grade, and tumor size [VHL mutation: odds ratio (OR) = 0.44, 95% confidence interval (CI) = 0.19, 0.98; HIF-2α expression: OR = 0.33, 95% CI = 0.18, 0.61].

Conclusions: Our observation that VHL mutation and high HIF-2α expression are less frequent in ccRCC tumors of black vs. white patients confirms the earlier TCGA finding. These findings suggest that ccRCC in black patients is a fundamentally different disease than ccRCC in white patients.

#4238

Impact of diabetes mellitus on adenoma detection rates in three disparate institutions.

Yakira David,1 Lorenzo Ottaviano,2 Sadat Iqbal,1 Brandon Lung,2 Michelle Likhtshtegyn,1 Samir Kumar,1 Ellen Li,2 Laura Martello-Rooney,1 Shivakumar Vignesh,1 Joshua Miller,2 Evan Grossman1. 1 _Downstate Medical Center, Brooklyn, NY;_ 2 _Stony Brook University, Stony Brook, NY_.

Background: Racial disparities persist in the incidence and mortality of colorectal cancer despite the availability of screening tools. Adenoma detection rates have been associated with diabetes in some studies. This study seeks to evaluate the effect of diabetes mellitus on adenoma detection rates (ADR) in initial screening colonoscopies performed at three distinct institutions.

Methods: A retrospective chart review was performed on all initial average risk screening colonoscopies performed on patients between 45-75 years at an Urban Safety Net Hospital (USNH), an Urban University Hospital (UUH) and a Suburban University Hospital (SUH) from January 1st to December 30th 2012 to coordinate existing databases at the three institutions. Patients were excluded if they had a history of colon cancer, polyps, alarm symptoms, or if the current colonoscopy was incomplete or had poor bowel prep. Data points collected included sex, age, race, insurance, BMI, smoking status, diabetic status and attending provider. Univariate analysis was performed comparing ADRs between the three institutions using Graph Pad Prism. Further comparisons were made between current smoking and diabetes status with ADR.

Results: There were a total of 2225 initial screening colonoscopies (SUH, n =647; UUH, n = 444; USNH, n = 1134) excluding 135 incomplete and/or poor prep colonoscopies (SUH, n = 34; UUH, n = 50; USNH, n = 51). USNH and UUH patients were more likely to be African-American (93% and 88% vs 7%, p<0.0001) and diabetic (29.7 and 29.8% vs 12%, p <0.0001) than the SUH patients. UUH and SUH patients were more likely to be male (39% and 40% vs. 32%, p<0.0001), more likely to be current smokers than the USNH patients (11.9% and 12.8% vs 5.8%, p<0.0001), and were less likely to be uninsured (7% and 3% vs 43%, p<0.0001). In 2012 the ADR was significantly lower in patients at the USNH compared to both the UUH and SUH( 17% vs 30% and 26%, p<0.0001), but with the institution of ADR monitoring at the USNH, the ADR has since increased to 29% in 2017. Across all three institutions diabetics were noted to have higher ADRs (26% vs 18%, OR 1.6 95% CI 1.3-2.0, p<0.0001) and current smokers were noted to have higher ADRs (30% vs. 20%, OR 1.8 95% CI 1.3-2.4, p = 0.001)

Discussion: Diabetes and current smoking are associated with increased ADR across all three institutions despite differences in race and baseline ADR. Patients should be counseled on the increased risk associated with diabetes and smoking. Initiatives should be implemented to insure improved screening rates for adenomas among diabetics and also to screen for diabetes so that interventions can be made early to reduce the impact of diabetes on colon cancer risk. Further prospective studies are needed to validate these findings.

#4239

Infection, inflammation and cancer: Understanding Alaska Native cancer health disparities.

Holly Martinson,1 Steven Alberts,2 Matthew Olnes3. 1 _Univ. of Alaska Anchorage, Anchorage, AK;_ 2 _Mayo Clinic Cancer Center, MN;_ 3 _Alaska Native Medical Center, Anchorage, AK_.

Gastric cancer is a leading cancer health disparity among the Alaska Native (AN) people, occurring at a 3-fold higher incidence and 4-fold higher mortality rate in comparison to U.S. Non-Hispanic whites (NHW). AN gastric cancer patients have significant differences in tumor location and appearance compared to NHW patients. In addition, AN patients are younger at the time of diagnosis, 60 versus 69 years and have a lower 5-year survival rate of 10% compared to 22% of NHW gastric cancer patients. The purpose of this research was to understand the role of gene mutations, inflammation, and infection as possible factors in the promotion of gastric cancer in the AN population. In this study, we examined previously obtained tissue samples from AN gastric cancer patients diagnosed between 2006-2014 at the Alaska Native Medical Center. Using next-generation sequencing, immunohistochemistry and in situ hybridization, we evaluated for 50 cancer related gene mutations, the presence of the virus, Epstein-Barr virus (EBV), the bacterium, Helicobacter pylori (H. pylori), and inflammatory factors. Cancer related gene mutations were observed in 69% of gastric tumors from AN patients. The most commonly mutated genes were TP53 (30%), PIK3CA (13%), STK11 (6%), KRAS (5%), and PTEN (5%). Of particular interest, gene mutations were associated with patients who were younger at time of diagnosis (less than 55 years old) and had a history of chronic gastritis. Additionally, EBV is associated with 23% of AN gastric cancers, which is significantly higher than the 10% of EBV associated gastric cancer cases observed in other populations. EBV associated AN gastric cancer patients were more likely to have a history of chronic gastritis, prior or current H. pylori infection, and tobacco use. Notably, aberrant overexpression of the inflammatory factor cyclooxygenase 2 (COX-2) and the glycoprotein Mucin 1 (MUC1) in gastric cancer cells was significantly associated with a poorer prognosis among AN gastric cancer patients. Collectively, these findings indicate infection and inflammation are potential driving factors in the pathogenesis of gastric cancer among the AN people, which may have important clinical implications.

#4240

Breast cancer characteristics and survival among different Indigenous American communities in Peru.

Lizeth I. Tamayo,1 Tatiana Vidaurre,2 Jeannie Navarro Vásquez,2 Sandro Casavilca,2 Jessica I. Aramburu,2 Monica Calderon,2 Daniel Cherry,3 Sikai Song,4 Garth H. Rauscher,1 Laura Fejerman4. 1 _University of Illinois at Chicago, Poplar Grove, IL;_ 2 _Instituto Nacional de Enfermedades Neoplasicas, Lima, Peru;_ 3 _University of California San Diego, San Diego, CA;_ 4 _University of California San Francisco, San Francisco, CA_.

Background: Breast cancer prognosis depends on stage at diagnosis and varies by intrinsic tumor subtype. In the US, the distribution of tumor subtypes has been shown to differ between racial/ethnic groups with African American and Latina women more likely to be diagnosed with the more aggressive "triple negative" breast cancer (TNBC) compared to Non-Latino White women. Latinos in the US originate from different countries with different cultures and ancestral genetic backgrounds, demonstrating the heterogeneity that exists. Information about the distribution of tumor subtypes in Latin American regions is limited. Methods: Data for these analyses come from The Peruvian National Cancer Institute . Includes clinical information for 303 patients diagnosed with breast cancer between 2010 and 2015 and who are members of Indigenous American communities. We analyzed women from different groups: the Quechuas (Group 1; N=223), Aimara (Group 2; N=9), Ashankinka/Nomatsigenga/Yenesha (Group 3; N=17), Awajun/Kichwa/Shawi/Shipibo-Konibo (Group 4; N=29) and other communities (Group 5; N=20). Some communities were combined based on previous literature describing their genealogical proximity. We compared tumor characteristics and survival between these groups using Fisher exact tests, T-tests, and a Cox Proportional Hazards model with predictors age at diagnosis, stage, tumor subtype, and treatment. Breast cancer subtype was defined as luminal A (ER/PR+/HER2-), luminal B (ER+/HER2+), HER2 overexpressing (ER/PR- HER2+) and triple negative (ER/PR- HER2-) based on immunohistochemistry. Results: Overall, tumors from the 303 Indigenous American women included in the present study were 37% luminal A, 20% luminal B, 23% HER2 overexpressing and 19% triple negative. Our analyses showed that women from group 1 were diagnosed at an older age (55 vs. 48-50, p<0.0001) and less frequently with TNBC compared to women from other groups (45% vs. 55-78%, p=0.06). Compared to group 1, women from the other groups a non-significant trend towards higher mortality (HR 1.5-1.9, p=0.272). In the full model, including age, stage, tumor subtype and treatment, the trend was no longer apparent. Whether the women had surgery had the strongest effect on survival (p=0.001) followed by stage (p=0.0012) and TNBC (p=0.0023). Conclusion: Differences in survival between the women of different indigenous communities with breast cancer in Peru are most likely due to differences in access to care. There could be environmental factors or subtle sub continental genetic differences influencing the risk of TNBC. However, the lower frequency of TNBC among the Quechuas could also be due to a reduced set of logistic barriers. The limited power of this study demonstrates the need for larger data sets for subgroup analysis in Latinas. The more we learn by analyzing diverse populations, the better equipped we will be to provide adequate care for all women.

#4241

Associations between perceived medical device-related radiation harm and adherence to mammography screening guidelines.

Kekoa Taparra,1 Kay M. Pepin,2 Alexandra J. Greenberg-Worisek2. 1 _Mayo Clinic School of Medicine, Rochester, MN;_ 2 _Mayo Clinic, Rochester, MN_.

Introduction: Many forms of cancer screening involve imaging technologies utilizing radiation. Broad media coverage of radiation-related health risks may shape the public's perceptions of imaging-based cancer screening, such as mammography. In turn, this may impact patient outcomes by affecting cancer screening decisions. Our primary aim was to evaluate population differences in beliefs about medical radiation exposure in the United States and associations with mammography screening rates in women.

Methods: We analyzed data from the National Cancer Institute's Health Information National Trends Survey (HINTS) 2012 administration. Bivariate and multivariate regression analyses were used to evaluate associations of sex, ethnicity, age, annual income, and education with perceptions of harm from medical test radiation exposure. Additional analyses evaluated the role of radiation perception on compliance with current mammography screening recommendations from the United States Preventive Services Task Force.

Results: Approximately one-third of respondents indicated that they worried about medical imaging-related radiation exposure. Respondents who perceived medical imaging radiation as harmful to health were more likely to be female, be diagnosed with cancer, have less formal education, report lower socioeconomic status, and identify as an ethnic minority. Most female participants were not in compliance with current breast cancer screening recommendations (weighted percentage = 83.8%). Annual income (p = 0.042) and ethnicity (p = 0.0478) were two variables that were significantly associated with adherence to the recommended mammography screening paradigm. Women without health insurance were more likely to worry about medical imaging-related radiation (p=0.073). Neither health insurance (95%CI = 0.29, 4.99) nor excess worry about radiation exposure from medical imaging devices (95%CI=0.42, 1.89) predicted compliance to mammography screening, after adjusting for relevant sociodemographic variables.

Conclusions: These results indicate that some populations may be more concerned about harm from medical device-related radiation than others. Identifying these populations may help improve future cancer patient outcomes by promoting targeted health interventions and communication strategies for these individuals. Women may be more likely to perceive medical imaging radiation exposure as harmful to health, however this alone could not account for why the majority of female respondents were not adhering to recommended mammograms screening guidelines. Future studies are needed to identify the major contributor to low breast cancer screening rates in the United States.

#4242

Ethnic disparities in the risk of cardiac death following cancer diagnosis: A population-based study.

Mohamed M. Gad,1 Anas M. Saad,1 Muneer J. Al-Husseini,1 Abdelrahman I. Abushouk,1 Haitham M. Ahmed2. 1 _Ain Shams University Faculty of Medicine, Cairo, Egypt;_ 2 _Cleveland Clinic Foundation, Cleveland, OH_.

Background: Cancer-related cardiac complications and mortality have been well studied. However, the disparities among racial and ethnic groups have not been addressed on a large scale. In this study, we aimed to investigate the racial and ethnic disparities associated with cardiac death in cancer patients.

Methods: Data were collected from The Surveillance, Epidemiology and End-Results program of the National Cancer Institute. We defined an "event" as death due to a cardiac cause within ten years after cancer diagnosis, and observed patients who experienced the event during that period. Then, we calculated the Observed/Expected (O/E) ratio and the excess risk per 10,000 person-years to estimate the change of risk following the diagnosis in different ethnicities, when compared to the general population.

Results: We reviewed 4,671,989 cancer patients, of whom 163,255 died due to a cardiac cause within the ten years following cancer diagnosis. The overall risk of cardiac death increased significantly in this period with an O/E ratio of 1.16 and an excess risk of 11.66 per 10,000. The sample was divided into 2 groups: Whites with a total O/E ratio of 1.11, and non-Whites with a total O/E ratio of 1.38. When examined for the risk in the first 5 years after diagnosis, Whites and non-Whites demonstrated significant increase in the risk with O/E of 1.12 and 1.39, respectively. However, when examined for the long-term risk more than 5 years after diagnosis, only non-Whites had a significant increase with an O/E of 1.21, while Whites with a cancer diagnosis had the same risk of cardiac death as Whites without cancer diagnosis with an O/E of 1.00. The sample was then further subdivided according to ethnicity into non-Hispanic Whites, Blacks, American Indians (or Alaska natives), Asians (or Pacific islanders), and Hispanics. All groups showed a significantly increased risk of cardiac death following a cancer diagnosis (O/E=1.13, 1.36, 2.33, 1.44, and 1.05, respectively).

Conclusion: Non-White patients with a prior cancer diagnosis had a significantly higher long-term risk of cardiac death compared to noncancer patients. Accordingly, careful attention should be paid to the cardiovascular status assessment of those patients to mitigate the added risk of cardiac mortality due to cancer-related morbidities. Further studies are warranted to determine why cardiac event risk increases at a discrepantly higher rate in minority patients with a cancer diagnosis.

#4243

Association of oral health with lung cancer risk: Results from the Southern Community Cohort Study.

Hyung-Suk Yoon, Wanqing Wen, Jirong Long, Wei Zheng, William J. Blot, Qiuyin Cai. _Vanderbilt University Medical Center, Nashville, TN_.

Increasing evidence suggests that oral health may be associated with cancer and other chronic diseases. Oral bacterial infections, which induce inflammation of the gums and tooth loss, can progress to systemic inflammation. In addition, poor oral health is often the result of smoking and/or limited access to regular dental care. Previous studies have suggested that poor oral health may increase the risk of lung cancer among individuals of European and Asian descent, but the evidence remains elusive. In this nested case-control study, we investigated whether oral health status was associated with lung cancer risk among African Americans and European Americans and whether the association varied by race and smoking habits. A total of 403 incident lung cancer cases and 1,612 matched controls from the Southern Community Cohort Study were included in the current study. The controls were matched to cases on age, sex, race, and recruitment site. Multivariate conditional logistic regression models were fitted to evaluate the risk of lung cancer linked to tooth loss, tooth decay, and history of periodontal disease (gingivitis or periodontitis). Stratified analyses were conducted by race, smoking status, and pack-years, and the interactions of these variables with oral health were further evaluated. Tooth loss was significantly associated with an increased risk of lung cancer. Compared to those without any loss of teeth, the odds ratio (OR) of loss of >10 teeth was 1.64 (95% confidence interval [CI]: 1.00-2.69) after adjusting for confounders. Tooth decay was also significantly associated with increased lung cancer risk. Those with ≥ 6 decaying teeth had an OR of 1.65 (95% CI: 1.18-2.31), compared to those with no tooth decay. An increased lung cancer risk was significantly associated with a history of periodontal disease (OR: 1.44, 95% CI: 1.09-1.19). While there was no evidence of interaction by race, the increase in risk associated with tooth loss, tooth decay, and periodontal disease was observed only among smokers who had smoked more than the race-specific median pack-year. Results from our study indicate that poor oral health, as measured by tooth loss, tooth decay, and periodontal disease is associated with an increased risk of lung cancer and that this association is more evident among heavy smokers.

#4244

Hookah pipe awareness and perceived harmfulness: Prevalence, correlates, and association with smoking cessation outcomes.

Kahee A. Mohammed, Martin W. Schoen, Nosayaba Osazuwa-Peters, Divya S. Subramaniam, Lauren D. Arnold, Leslie Hinyard, Thomas E. Burroughs. _Saint Louis University, St. Louis, MO_.

Introduction: Hookah smoking is becoming an increasingly popular choice of tobacco use and is remarkably common among young adults in the United States. This study aimed to evaluate the prevalence and correlates of hookah awareness and perceived harmfulness among US adults and to examine its association with cigarette smoking quit intention and attempt. Methods: Data from the Health Information National Trends Survey 2013 to 2017 were analyzed. The sample included 6,711 adults aged ≥ 18 years. Weighted multivariable logistic regression analyses were performed and adjusted for age, gender, race/ethnicity, education, household income, region, and cigarette smoking status. Results: Overall, 78% of US adults were aware of hookah. Of these, 72% believed hookah pipes were equally harmful as cigarettes, whereas 17.6% believed otherwise. Those who were aware of hookah were less likely to be older (aOR=0.12, 95% CI=0.09 - 0.16), Black (aOR=0.56, 95% CI=0.43 - 0.72) and Hispanic (aOR=0.61, 95% CI=0.48 - 0.79), and lesser-educated (aOR=0.40, 95% CI=0.29 - 0.54) adults. Compared to never smokers, former smokers were 64% (95% CI=1.37 - 1.96) more likely to be aware of hookah. Among those aware of hookah, older (aOR=0.49, 95% CI=0.37 - 0.65), Black (aOR=0.73; 95% CI=0.58 - 0.97), and lesser-educated (aOR=0.69; 95% CI=0.48 - 0.95) were less likely to perceive hookah as less harmful than cigarettes. Those who perceived hookah to be just as harmful as cigarettes were less likely to try to quit cigarette smoking in the past year (aOR=0.49, 95% CI=0.25 - 0.93). Those who perceived hookah to be less harmful than cigarettes were more likely to consider quitting cigarette smoking in the next 6 months (aOR=2.86, 95% CI=1.29 - 6.33). Conclusions: Young, White, and college graduate adults are more likely to believe that hookah smoking is less harmful that cigarettes. These disparities can be used to target behavioral interventions aimed at increasing individuals' perceived risk, knowledge, and perceived harmfulness of hookah smoking.

#4245

Racial disparities in comorbidities, income levels, and surgical procedures observed in hispanic patients with pancreatic cancer compared to non-hispanic patients results from the national inpatient sample database.

Miren Peña, Jswanth Raj Kintada, Hegde Sheetal, Sammira Rouhani, Edgar Munoz, Joel Michalek, Ali Seifi, Amelie Ramirez, Pratap A. Kumar, Dimpy Shah. _University of Texas at San Antonio, San Antonio, TX_.

Background: Higher incidence and lower survival rates have been linked to racial/ethnic and socioeconomic (SES) disparities in patients with pancreatic cancer. However, systematic evaluation of the interaction between race/ethnicity, SES, comorbidities and type of surgical procedures to predict outcomes associated with pancreatic cancer are missing. Hence, we conducted this study to investigate these risk factors and outcomes of pancreatic cancer discharges, with a focus on Hispanic population.

Methods: We surveyed Healthcare Cost and Utilization Project (HCUP) National Inpatient Sample (NIS) database for year 2014 to identify patients with pancreatic cancer (ICD9: 230.9, 157.1-157.4, 157.8, 157.9). To identify racial disparities, we examined age, median household income at zipcode level, various comorbidities (e.g. alcohol abuse, diabetes, obesity, liver disease, hypertension, chronic pulmonary disease, etc.), types of surgical treatments (surgical vs non-surgical options; Whipple vs. others) as a treatment choice, and death during hospitalization.

Results: From approximately 7 million records, we identified 18,069 pancreatic cancer-related discharges. The median age of patients was 68 years (range: 0 to 90 years). Compared to NHW, Hispanics had a significantly higher prevalence of diabetes (40% vs. 30%, p<0.001) and liver disease (8% vs. 5%, p<0.001), whereas no significant differences were observed for obesity, hypertension, congestive heart failure, or alcohol abuse. Majority of Hispanic patients (39%) with pancreatic cancer belonged to lowest income quartile ($1 - $39,999 per year) compared to NHW (20%), P value <0.001. The only two factors associated with the difference in receiving Whipple procedure were having underlying liver disease and the income level, stratified by race/ethnicity. We did not identify any factors associated with differences in mortality rates.

Conclusions: We identified significant racial disparities such as higher prevalence of comorbidities, lower income levels, and different types of surgical procedures in Hispanic versus NHW patients hospitalized for pancreatic cancer. Future studies quantifying the impact of stage of illness at presentation, multiple comorbidities including smoking, rural/urban care setting, SES, insurance coverage on treatment choices and outcomes associated with pancreatic cancer in different racial/ethnic groups are needed.

#4246

Characterizing breast cancer health disparities through molecular and genomic profiling of a diverse breast cancer cohort.

Jung S. Byun,1 Tingfen Yan,1 Adam Sowalsky,2 Jack Zhu,2 Paul Meltzer,2 Nasreen Vohra,3 Peter Kragel,3 Anna Napoles,1 Eliseo Perez-Stable,1 Kevin Gardner4. 1 _NIMHD, National Institutes of Health, Bethesda, MD;_ 2 _NCI, National Institutes of Health, Bethesda, MD;_ 3 _East Carolina University, Greenville, NC;_ 4 _Columbia University Medical Center, New York, NY_.

The major indicators of breast cancer health disparities may include higher incidence, earlier onset, faster progression, and poorer outcomes. The ultimate goal of health disparities research is to elucidate actionable mechanisms of these differences to define the means of intervention that will help reduce the disparity. Breast cancer is the most common cancer and second leading cause of cancer death in American women and the new cases of invasive cancer have been increased lately in the United State. It is a heterogeneous disease with few modifiable risk factors, characterized by multiple subtypes that influence different populations with varying incidence, morbidity and mortality. Though recent genomic advances have enabled the stratification of breast cancer into numerous subtypes with differing therapeutic and prognostic values, it remains unclear why the incidence and outcome of certain breast cancers show an uneven distribution across different race/ethnic populations. For example, the higher frequency of more aggressive forms of breast cancer in young women of African descent is well known, yet why this occurs remains to be determined. To understand the mechanisms of breast cancer health disparity, we performed a molecular and genomic profiling on a unique cohort of racially diverse breast cancer patients from a catchment area in the northern part of east North Carolina and found earlier onset of breast cancer in women of African Ancestry (AA) with 32.1 % occurring before age 50 compared to 22.5 % for women of European Ancestry (EA). Furthermore, women of AA have higher levels of obesity with 35.5 % having a Body mass index (BMI) above 35 compared to 17.9 % for women of EA. Notably, women of AA also showed presence of higher stage of breast cancer with more than 26 % having stage 3 or 4 compared to 16.5 % for women of EA. We also found that women of AA showed poorer overall survival outcome from breast cancer compare to EA with Hazard Ratio of 1.67. The panel of exome sequencing of 147 tumors from this cohort revealed differential mutational frequencies in African Ancestry versus European Ancestry. The insertion/deletion mutated genes found only in African Ancestry included PAX5, CYP2A7, UGT2B17, CYP4A11, ACAA1, KIT and NAT2 with high percentage of cases with mutation account, whereas those genes unique to European Ancestry included GSTP1, PRSS53, AURKA, ATF1, BIRC2, PGAP3 and GSTA4. In addition to this, there are differentially mutated genes across specific breast cancer subtypes where ABL2, UBA1, CYP4F8, PIK3CG, CTNNA1, CYP39A1 genes are only associated with Triple Negative Breast Cancer patients. By characterizing the risk of breast cancer among ethnically diverse groups, we provide needed insights that will not only improve the development of personalized prognostic and predictive biomarkers but may also enhance breast cancer screening, treatment, survival and prevention.

#4247

Racial/ethnic differences in all-cause and cause-specific mortality patterns among a cohort of invasive U.S. breast cancer patients.

Alyssa N. Troeschel, Yuan Liu, Lindsay Collin, Kevin Ward, Keerthi Gogineni, Lauren McCullough. _Emory University, Atlanta, GA_.

It is important to explore racial differences in mortality patterns due to breast cancer (BC), cardiovascular disease (CVD) and all-causes (AC) among women diagnosed with breast cancer in the U.S. Data from 891,635 non-Hispanic black (NHB) and white (NHW) women diagnosed with malignant breast cancer from 1990-2014 were obtained from the National Cancer Institute Surveillance, Epidemiology, and End Results (SEER) database. Cumulative incidences for mortality due to BC and CVD were calculated at 2, 5, 10 and 20 years post-diagnosis by race and age (dichotomized at the study mean, 62.5 years). A Cox hazard model was used to obtain hazard ratios (HR) for all-cause mortality and the subdistribution hazards model was used for mortality due to BC and CVD, accounting for the presence of competing risks, along with their corresponding 95% confidence intervals (CI). A total of 300,445 deaths occurred over 6,319,054 person-years. Among women aged <62.5 years at diagnosis, the cumulative incidence of BC death and CVD death at 2, 5, 10, and 20 years was 5.2%, 10.6%, 15.3%, 19.5% and 0.2%, 0.6% 1.2%, 2.5%, respectively, for NHWs and 10.0%, 19.9%, 27.9%, 34.8% and 0.5%, 1.3%, 2.7%, 5.3% for NHBs. Results for BC were similar among women diagnosed >62.5 years, while there appeared to be no racial differences in CVD-death. Among women diagnosed at age <62.5 years, NHBs had an increased hazard of mortality due to BC (HR=2.03, 95% CI: 1.97, 2.08), CVD (HR=2.64, 95% CI: 2.38, 2.94), and AC (HR=2.84, 95% CI: 2.77, 2.91) during the first 2 years post-diagnosis. This association decreased over time for mortality due to BC (5-10 years: HR=1.76; 10-15 years: HR=1.58) and AC (5-10 years: HR=1.54; 10-15 years: HR=1.07, 15+ years: HR=0.93) but remained relatively stable for CVD (15+ years: HR=2.47). Among women diagnosed at age >62.5 years, NHBs had an increased hazard of mortality due to BC (HR=2.13, 95% CI: 2.06, 2.19), CVD (HR=1.33, 95% CI: 1.25, 1.40) and AC (HR=1.51, 1.48, 1.55) during the first 2 years post-diagnosis. The association decreased for BC (2-5 years: HR=1.55; 5-10 years: HR=1.15; 10-15 years: HR=0.88; 15+ years: HR=0.64) and CVD (2-5 years: HR=1.1.06; 5-10 years: HR=1.00; 10-15 years: HR=0.85; 15+ years: HR=0.65) while the association with AC mortality initially decreased, then remained relatively stable (5-10 years: HR=1.20). Results suggest NHB women diagnosed at younger ages have the highest cumulative incidence of mortality due to BC, CVD and AC over the 25-year follow-up period. In addition, among women diagnosed at younger ages, NHBs appeared to have a two-fold increase in hazards of AC, BC and CVD mortality during the years immediately following BC diagnosis compared to NHWs. The increased hazard for CVD mortality among younger NHBs remained relatively stable over time, whereas the hazards for AC and BC mortality attenuated over time. Results were less pronounced among women diagnosed at older ages.

#4248

Small round blue cell tumour of childhood. A case of health disparity in Nigeria in the survival of patients.

Kasimu U. Adoke, Faruk Mohammed. _AHMADU BELLO UNIVERSITY ZARIA NIGERIA, ZARIA, Nigeria_.

Introduction

Nigeria is Africa's most populous country and the 9th most populous country in the world. The country is the 6th largest producer of petroleum in the world. Health care and general living conditions in Nigeria are poor, especially for children and women. At least 70 to 75 percent of health expenditure comes from out of pocket expenses.

Childhood cancers as a growing public health challenge and is increasingly being recognized worldwide, including the developing nations. Tremendous progress has been made in the treatment and cure of childhood cancers mostly in the developed world, most cases die of childhood cancers in our society. We look at fifty two cases of small round blue cell tumour of childhood (SRBCT) a diverse group of childhood cancers that have considerable overlap in epidemiology, morphology and immunophenotype over a three year period.

Methods

We reviewed the histology report and paraffin blocks of patients diagnosed as SRBCT of childhood in the department of pathology, from 2014-2017. We identified fifty two patients diagnosed as SRBCT during the study period. The information extracted includes age, sex, cancer type, treatment modality and outcome of all patients. We subjected the blocks to nine

immunohistochemical stains to rule out differentials using Genemed biotechnology protocol. The stains include Cytokeratin, EMA, S-100, CD99, Desmin, CD10, CD20, BCL2 and Synaptophysin..

Results

The age range in this study was 2-15 years with a mean of 7.2 years. The most common site for SRBCT was the head and neck region accounting for more than 46%. This is followed by the abdomen accounting for 19%. The commonest SRBCT after immunohistochemical stains was Burkitt's lymphoma 16(30.7%), retinoblastoma 8(15.4%), alveolar rhabdomyosarcoma 7(13.5%), Follicular lymphoma 5(9.6%), ES/PNET 5(9.6%), nephroblastoma 4(7.7%), neuroblastoma 4(7.7%) and synovial sarcoma 3(5.8%). All patients 52(100%) undergo chemotheraphy, 36(69%) undergo both surgery and chemotheraphy while 8(15.4%) sign against medical advice. All patients pay through out of pocket expences with no access to insurance. Thirty three patients (63%) died during chemotheraphy. Most patients are lost to follow up due to financial burden and unavailability of standard oncology services.

Conclusion

While tremendous progress has been made in the treatment of childhood cancers in the developed world, it still remains an important health problem in our societies largely because of multifactorial reasons creating a huge health disparity and contributing to under five mortality in our environment. Reducing poverty and creating better health facilities through knowledge sharing and collaboration with reduce this disparity.

#4249

Disparities in cancer screening between Latino and non-Latino firefighters: Evidence from the Sylvester Firefighter Cancer Initiative.

Alberto J. Caban-Martinez,1 Natasha Schaefer Solle,1 Tulay Koru-Sengul,1 Katerina M. Santiago,1 Kevin J. Moore,1 Feng Miao,2 David J. Lee,1 Erin N. Kobetz2. 1 _University of Miami, Miller School of Medicine, Miami, FL;_ 2 _Sylvester Comprehensive Cancer Center, University of Miami, Miller School of Medicine, Miami, FL_.

Firefighters face an increased risk for work-related exposures to hazardous environmental carcinogens during fire incidence response. A growing body of epidemiologic studies suggest that firefighter site-specific cancer incidence and mortality rates are higher when compared to the general US population. As the fastest-growing ethnic subgroup in the US, Latinos have been increasingly represented in the first responder workforce. Latinos relative to non-Latinos vary in cancer-related risk factors including rates of smoking, alcohol consumption, physical activity patterns, and rates of cancer morbidity and mortality. Despite these differences in risk factors, there is limited literature on the cancer screening behavior of Latino firefighters who may be at an increased risk for cancer. In the present study, we characterize sociodemographic and work characteristics in addition to cancer screening behaviors between Latino and non-Latino active firefighters. The Annual Cancer Survey (ACS) research project of the Firefighter Cancer Initiative launched with a 127-item comprehensive cancer questionnaire administered online to a non-probabilistic sample of firefighters employed in South Florida. We present ACS data collected during the first 12-months of this cross-sectional, convenience sample. Firefighters were invited and consented by the study team to complete the ACS using a secure iPad device during their regular work shift. Univariable and multivariable logistic regression models were fitted to predict history of cancer screening behaviors. Among the 3,152 study participants, the mean age was 40.4 years (± 9.1 years standard deviation), 34.3% were Latino, 10% female, and had been employed on average 14.0 years (± 8.6) as a firefighter. The number of Latino firefighters who had not seen a doctor in the past 12 months (5.1%) or visited a primary care physician for routine care (25.5%) was significantly more relative to non-Latino Firefighters (4.2% and 19.3%, respectively; both p<0.01). Across cancer screening behaviors, the number of Latino firefighters who ever had a PSA test to screen for prostate cancer (24.7% vs 38.1%, p<0.01) and a colonoscopy (20.0% vs. 29.0%, p<0.01) was significantly less than non-Latino firefighters. Latino firefighters are significantly less likely than their non-Latino colleagues to report a full body skin exam by their doctor (adjusted odds ratio 0.35; 95% CI 0.29-0.42) while controlling for age, gender, race, educational attainment, household income, health insurance type, routine access to primary care and tenure as a firefighter. Florida firefighters of Latino ethnicity tend to participate less in routine cancer screening practices. Strategies at the fire station or at the individual worker-level are needed to improve cancer screening disparities within this high-risk minority workforce.

#4250

Mediation of black/white race disparity in triple-negative breast cancer by socioeconomic position, parity and p53 status.

Keith A. Dookeran. _UWM, Milwaukee, WI_.

Rauscher et al. (2016) recently suggested that the black/white race disparity in aggressive ER/PR-negative breast cancer (BC) might be transmitted through social and reproductive influences. Hence, we examined to what extent the black/white race disparity in aggressive triple-negative (TN) BC might be transmitted through social, reproductive and molecular influences. Our study sample is a previously reported historical cohort of 419 consecutively enrolled (331 non-Hispanic (nH) black and 88 nH white) women with primary BC aged 24-94 years, treated between 2000-2005 at an NCI-MBCCOP facility in Chicago, IL (Dookeran et al. 2012). US 2000 census ZIP Code Tabulation Area (ZCTA) data matched to patient address zip codes at diagnosis was used to develop a single binary measure of socioeconomic position (SEP) (> US poverty mean vs. referent of US poverty mean </= 9.2%; based on national census data for the number of families living below the poverty level threshold in 1999). Binary parity (parous vs. nulliparous) was used as a single measure of reproductive status. Binary p53 status (positive vs. negative) was used as a single indicator of molecular influence. We estimated average controlled direct associations (ACDA) using logistic regression with model-based standardization (predictive margins) in order to estimate the age/BMI-adjusted prevalence difference with bias-corrected bootstrapped 95% confidence intervals (CIs) for TN vs. luminal A type BC by race, and performed a series of ACDA (with comparison of rescaled coefficients) controlling for SEP, parity, and p53 status, both individually and together. In age/BMI adjusted models, nH black women were 15%-points more likely than nH white to have TNBC (p=0.039). This disparity was reduced to 13%-points after adjustment for p53 status, 12%-points after adjustment for parity, 11%-points after adjustment for SEP, 9%-points after adjustment for both parity and SEP (95% CI: -0.09, 0.24), and 7%-points after adjustment for p53, parity and SEP together (95% CI: -0.1, 0.79). Based on the method of rescaled coefficients, parity and SEP accounted for approximately 19% and 24% respectively of the black/white disparity in TNBC when modeled together, accounting in combination for 44% of the disparity (p=0.08). Further, p53, parity and SEP accounted for approximately 10%, 12% and 28% respectively of the race disparity in TNBC when modeled together, accounting in combination for 51% of the disparity (p=0.13). Our findings support the hypothesis that at least part of the observed black/white disparity in aggressive TNBC might be transmitted through social influences on tumor biology, in particular via SEP.

#4251

Neighborhood data system to enable health disparities research in Chicago.

David J. Press,1 Scarlett Lin Gomez,2 Briseis Aschebrook-Kilfoy,1 Habibul Ahsan1. 1 _The University of Chicago, Chicago, IL;_ 2 _University of California, San Francisco, San Francisco, CA_.

Background: Chicago has become the focus of national concern following findings of striking disparities in cancer incidence and mortality, with worse outcomes for Non-Hispanic Black persons. Chicago also has communities with very high crime rates, racial/ethnic segregation, and resource-poor communities. Social stressors, including crime, social isolation, low socioeconomic status (SES), interpersonal and institutional discrimination, and residence in resource-poor settings intersect at individual- and neighborhood- levels across the lifespan and are associated with poor health outcomes. However, the extent to which these social determinants separately and together contribute to cancer health disparities is unclear.

Purpose: To elucidate health disparities attributable to neighborhood social stressors in the Chicago setting, a pilot Chicago Neighborhood Data System (ChNDS) is currently in production, using the Chicago Police Department crimes database, data from the 2010 Census short form, 2008-2012 American Community Survey (ACS), University of Chicago MAPSCorps project, and Environmental Systems Research Institute (ESRI) geodatabases.

Approach: We use principal components analysis (PCA) to generate composite indices of neighborhood-level factors at the small area level (census tract or block group): racial/ethnic composition; SES; immigration/ acculturation; other sociodemographic characteristics; crime; and community assets including density/proximity to types of businesses, retail food, and recreational facilities.

Future directions: Data record linkages at the small area-level will be performed between the pilot ChNDS and observational and population-based datasets to examine the impact of neighborhood-level characteristics on research study participation, disease occurrence, healthcare utilization, and disease outcomes in Chicago.

#4252

Antihypertensive medication use and ovarian cancer survival.

Tianyi Huang,1 Anil K. Sood,2 Shelley S. Tworoger3. 1 _Brigham and Women's Hospital and Harvard Medical School, Boston, MA;_ 2 _MD Anderson Cancer Center, Houston, TX;_ 3 _Moffitt Cancer Center, Tampa, FL_.

Background: Both experimental and clinical studies suggest that beta-blockers, a common antihypertensive agent, may reduce ovarian tumor aggressiveness, inhibit angiogenesis and metastasis, and improve survival. However, no study has considered the potential role of other antihypertensive medications in conjunction with beta-blockers in ovarian cancer survival. Methods: We evaluated the associations of pre-diagnostic (n=899) and post-diagnostic (n=683) use of individual antihypertensive medications with ovarian cancer survival in the Nurses' Health Study (NHS; diagnosed between 1994-2012) and NHSII (diagnosed between 2001-2013), with at least 4 years of follow-up for death. Cox proportional hazards models were used to estimate hazard ratios (HR) for ovarian cancer mortality according to antihypertensive medication use. Results: In analyses of individual drugs adjusted for age, BMI and tumor characteristics, pre-diagnostic use of calcium-channel blockers (CCB) was associated with higher ovarian cancer mortality (HR: 1.52; 95% CI: 1.15, 2.00), whereas lower mortality was observed for post-diagnostic use of beta-blockers (HR: 0.76; 95% CI: 0.58, 1.01) and angiotensin-converting enzyme (ACE) inhibitors (HR: 0.64; 95% CI: 0.44, 0.93). While hypertension itself was not associated with ovarian cancer mortality (HR: 0.99; 95% CI: 0.83, 1.17), the associations between antihypertensive medications and ovarian cancer mortality were suggestively stronger among women with history of hypertension. Conclusion: Our findings suggest that post-diagnosis use of beta-blockers and ACE inhibitors is associated with lower ovarian cancer mortality and pre-diagnosis use of CCBs may be associated with higher mortality. These results warrant further investigation, and ongoing work is exploring whether the observed associations may be explained by changes in antihypertensive medication prescription after ovarian cancer diagnosis or changes in overall prescription patterns over time.

#4253

Anal cancer survivorship: Clinical and nonclinical factors.

Eric Adjei Boakye,1 Matthew Simpson,2 Rebecca Rohde,2 Lauren Anderson,3 Betelihem B. Tobo,3 Thomas E. Burroughs,3 Nosayaba Osazuwa-Peters4. 1 _St. Louis Univ. Center for Health Outcomes Research, Saint Louis, MO;_ 2 _St. Louis Univ. School of Medicine, Saint Louis, MO;_ 3 _St. Louis Univ. College for Public Health and Social Justice, Saint Louis, MO;_ 4 _St. Louis Univ. Cancer Center, Saint Louis, MO_.

Purpose: An estimated 8,200 cases of anal cancer will be diagnosed in 2017, accounting for nearly 1,100 deaths. Though anal cancer is a relatively rare malignancy, its increasing incidence has coincided with the increase in prevalence of risky sexual practices associated with anal human papillomavirus (HPV) infection, especially among men. At least 9 in 10 anal cancers are associated with HPV. Due to the rarity of anal cancer, little is known about factors affecting survivorship of anal cancer patients. The aim of this study was to evaluate clinical and nonclinical factors associated with anal squamous cell carcinoma (ASCC) survival in the United States.

Methods: A retrospective cohort study of patients aged ≥18 years with ASCC in the National Cancer Institute Surveillance, Epidemiology, and End Results registry (2004-2014) was analyzed. ASCC (site codes C21.0-C21.9 and C20.9), were coded for invasive squamous cell carcinomas per the International Classification of Diseases for Oncology, third edition. The outcome was survival (alive or dead) and the exposures were clinical (stage of presentation and treatment modality), and nonclinical (marital status, age, sex, race/ethnicity). Fine and Gray proportional hazards regression model were used to assess differences in survival among sociodemographic and clinical factors.

Results: There were 13,142 patients diagnosed with ASCC between 2004 and 2014. Mortality rate during the study period was 34.9% and median survival time was 113 months. In the adjusted model, a one-year increase in age was associated with 2% increased risk of death (adjusted hazard ratio (aHR) =1.02; 95% confidence interval (CI): 1.02-1.03). Compared with female patients, male patients (aHR=1.54; 95% CI: 1.40-1.70) had higher hazard of death, as did patients who were non-Hispanic black (aHR=1.40; 95% CI: 1.23-1.60) or non-Hispanic other (aHR=1.34; 95% CI: 1.09-1.65) versus non-Hispanic white. Patients who were never married (aHR=1.29; 95% CI: 1.15-1.44) or widowed (aHR=1.37; 95% CI: 1.18-1.59) had higher hazard of death compared to married patients. Patients had higher hazard of death if they were had stage II (aHR=2.01; 95% CI: 1.62-2.49), stage III (aHR=4.58; 95% CI: 3.70-5.67) or stage IV cancer (aHR=13.43; 95% CI: 10.74-16.78); and had not received surgery (aHR=1.41; 95% CI: 1.26-1.57).

Conclusion: Besides the clinical factors known to impact survival, including stage and treatment modality, several nonclinical factors are associated with the survival of anal cancer in the United States: age, sex, race/ethnicity, and marital status. It is important that interventions considering these factors are developed to improve survivorship, especially among the unmarried, males, and minority patients.

#4254

Associations of sleep, quality of life and energy level with breast cancer mortality and recurrence: The Shanghai Breast Cancer Survival Study.

Jingjing Zhu,1 Pingping Bao,2 Hui Cai,1 Liang Shi,2 Ying Zheng,2 Wei Zheng,1 Xiao-ou Shu1. 1 _Vanderbilt University, Nashville, TN;_ 2 _Shanghai Municipal Center for Disease Control and Prevention, Shanghai, China_.

Background: Sleep disorder is highly prevalent among breast cancer patients. Whether sleep disorder is associated with breast cancer prognosis has rarely been investigated. The objective of this study is to prospectively assess the association of self-reported sleep difficulty and sleep quality with total mortality and breast-cancer specific mortality/recurrence in the Shanghai Breast Cancer Survival Study.

Methods: This study included 4,219 women with stage I-III breast cancer diagnosed at an age between 20 to 75 years. Information on sleep attributes was collected at approximately 18 months and 36 months after breast cancer diagnosis. Information on disease recurrence and survival status was obtained via in-person follow-up and via record linkage with vital statistics registry data. Cox proportional hazard model was used to derive hazard ratios (HR) and confidence intervals (CIs) for all-cause mortality and breast cancer-specific mortality/recurrence. Stratified analyses according to self-reported overall quality of life, depression and energy level assessed at 18 months were also conducted.

Results: During a median follow-up of 9.8 years (range: 0.1-12.2 years), 757 deaths due to any causes and 672 breast cancer recurrences or related deaths were recorded after the 18-month post-diagnosis survey. Sleep difficulty and low sleep quality were not significantly associated with total or breast-cancer specific mortality/recurrence. The respective HRs were 0.98 (95% CI: 0.79, 1.22) and 1.01 (95% CI: 0.77, 1.31) for total mortality and 1.04 (95% CI: 0.82, 1.31) and 0.93 (95% CI: 0.69, 1.26) for breast cancer-specific mortality/recurrence. On the other hand, low quality of life, depression and low energy level were significantly associated with increased total mortality, with HRs of 3.19 (2.45, 4.15), 1.61 (1.21, 2.14) and 1.53 (1.23, 1.91), respectively. These three factors did not modify the association of sleep measurements with breast cancer outcomes (all P-interactions > 0.10).

Conclusions: In a large population-based cohort study, we did not observe any significant association of sleep difficulty and poor sleep quality with increased risk of death and recurrence in breast cancer patients. Instead, low quality of life and energy level were found to be associated with disease prognosis and thus they could be important indicators for targeting population with poor breast cancer prognosis.

#4255

Differences in the sociodemographic correlates of HPV-associated cancer survival in the United States.

Nosayaba Osazuwa-Peters,1 Matthew C. Simpson,1 Eric Adjei Boakye,2 Kahee A. Mohammed,1 Longwen Zhao,3 Sai D. Challapalli,1 Rebecca L. Rohde,3 Vy T. Pham,3 Sean T. Massa,1 Mark A. Varvares4. 1 _St. Louis University School of Medicine, Saint Louis, MO;_ 2 _St. Louis University Center for Health Outcomes Research (SLUCOR), Saint Louis, MO;_ 3 _St. Louis University College for Public Health and Social Justice, Saint Louis, MO;_ 4 _Harvard Medical School, Boston, MA_.

Purpose: Human papillomavirus (HPV) is the most common sexually transmitted infection in the United States. One in every four individuals-nearly 80 million-is infected. More than 38,000 new cases of HPV-associated cancers are diagnosed annually. However, factors related to HPV-associated cancer survivorship, based on primary anatomic site, remain understudied. The aim of this study was to assess sociodemographic factors related to survival following diagnosis of HPV-associated cancers in the United States.

Methods: Patients ≥18 years diagnosed with first-primary HPV-associated cancer between 2007 and 2014 were identified from the Surveillance, Epidemiology, and End Results 18. HPV-associated cancers sites were defined as anal, cervical, oropharyngeal, penile, vaginal, and vulvar per the International Classification of Diseases for Oncology, third edition codes. Kaplan-Meier curves showing cancer-specific survival (CSS) from each HPV-associated cancer site stratified by sex with differences assessed by log-rank tests. Fine and Gray proportional hazards regression models for each HPV-associated site controlled for clinical covariates and estimated sociodemographic predictors of hazard of death from cancer.

Results: A total of 63,329 patients with HPV-associated cancers were included in the analyses. The most common sites were cervix for females (58%) and oropharynx for males (78%). Overall 8-year survival at the end of follow-up was 56%. For males, anal cancer had the lowest CSS (62%) compared to oropharyngeal (69%) and penile (72%) cancer (p<0.01). For females, vaginal cancer had the lowest CSS (46%) compared to anal (71%), cervical (67%), oropharyngeal (57%), and vulvar (72%) cancer. Final adjusted model showed significant CSS differences based on sociodemographic factors, including sex, age, marital status, race/ethnicity, and insurance status. Males were more likely to die from anal cancer compared to females (aHR=1.53, 95% CI 1.39, 1.68), while less likely than females to die from oropharyngeal cancer (aHR=0.91, 95% CI 0.84, 0.98). Blacks were more likely to die from anal (aHR=1.33, 95% CI 1.16, 1.52), cervical (aHR=1.13, 95% CI 1.05, 1.22), and oropharyngeal cancer (aHR=1.54, 95% CI 1.41, 1.68) compared with Whites. Each increasing year of diagnosis was associated with a 1-3% increase in hazard of cancer-specific death for all cancers.

Conclusions: There is marked variability in sociodemographic correlates among HPV-associated cancer survivors in the United States, based on sex, age, insurance and marital status, race/ethnicity, and cancer type. This has important implications for clinical decision making and identification of populations at greater risk of death from HPV-associated cancers.

#4256

Handgrip strength and cognitive function in elderly cancer survivors.

Lin Yang,1 Lee Smith,2 Liang Hu,3 Graham A. Colditz,4 Adetunji T. Toriola,4 Davy Vancampfort,5 Mark Hamer,6 Brendon Stubbs,7 Thomas Waldhör1. 1 _Medical University of Vienna, Wien, Austria;_ 2 _Anglia Ruskin University, Cambridge, United Kingdom;_ 3 _Zhejiang University, Hangzhou, China;_ 4 _Washington University School of Medicine, St. Louis, MO;_ 5 _University of Leuven, Kortenberg, Belgium;_ 6 _Loughborough University, Loughborough, United Kingdom;_ 7 _King's College London, London, United Kingdom_.

Aim: Cancer and cancer treatment-related cognitive impairments are frequently reported in cancer survivors, presenting as lack of attention, impaired executive function and short-term money loss. The aetiology of these impairments is largely unknown. Recently, serum level of brain-derived neurotrophic factor (BDNF) has been associated with cancer related cognitive impairments. BDNF is a neurotrophin, secreted in responses to muscle contraction. Meanwhile, muscle dysfunction, characterized by muscle strength and muscle composition, is common in cancer survivors. Yet its impact on cancer survivors' cognitive function is unknown. To address this gap we evaluated for the first time the associations of muscle strength determined by handgrip with two cognitive function outcomes in cancer survivors using data from the National Health and Nutrition Examination Survey (NHANES). Method: Data in two waves of NHANES (2011-2014) were aggregated. Handgrip strength in kilogram (kg) was measured with the Takei Digital Grip Strength Dynamometer, defined as the maximum value achieved using either hand. Data on two cognitive function tests were extracted, both were conducted among adults 60 years and older: the Animal Fluency Test (AFT) which examines categorical verbal fluency (a component of executive function), and the Digital Symbol Substitution test (DSST) which assesses processing speed, sustained attention and working memory. Survey analysis procedures were used to account for the complex sampling design of the NHANES. Gender-specific multivariable linear regression models were used to estimate associations of handgrip strength with cognitive test scores, adjusting for confounders (age, gender, race/ethnicity, education, marital status, smoking status, depression and leisure time physical activity). For explorative purpose, we further adjusted for handgrip strength squared. Results: There were 383 cancer survivors (mean age=70.9 years, mean BMI=29.3 kg/m2) with detailed data for analyses. Prevalent cancer types were breast (22.9%), prostate (16.4%), colon (6.9%) and cervix (6.2%). The prevalence of handgrip strength defined sarcopenia was 10.9% in men (<30kg) and 20.4 % in women (<20kg). Scores on AFT and DSST were 18.0 (s.e. 0.4) and 51.7 (s.e. 1.0), respectively. We observed significant linear associations of handgrip strength with both test scores in women. Each increase kg of handgrip strength was associated with 0.20 (95% CI: 0.08 to 0.33) higher score on AFT and 0.83 (95% CI: 0.30 to 1.35) higher score on DSST. In men, we observed inverted U-shape association between handgrip strength and DSST score, which peaked at handgrip strength of 40-42 kg. Conclusion: Handgrip determined muscle strength appears to be associated with aspects of cognitive functions in cancer survivors. Prospective studies are needed to identify the role of muscle on cancer related-cognitive impairments in men and women cancer survivors.

#4257

Predicting fatigue levels of head and neck cancer patients with gene expression using machine learning.

Ronald C. Eldridge, Andrew H. Miller, Deborah W. Bruner, Jonathan J. Beitler, Kristin A. Higgins, Evanthia C. Wommack, Linh Kha Huynh, Nabil F. Saba, Dong M. Shin, Canhua Xiao. _Emory University, Atlanta, GA_.

Introduction: Cancer-related fatigue is physical, emotional, or cognitive exhaustion which can affect treatment adherence and quality of life, and predict survival. Head and neck cancer (HNC) patients experience high levels of fatigue due to the degree of radiotherapy (RT), but that alone does not fully explain it. Recent studies suggest that fatigue may be related to a patient's personalized metabolic and inflammatory response, suggesting a patient's gene expression (GE) may be used to predict and monitor fatigue - a precursor to managing it. In this analysis, we examined whether GE is predictive of pre-RT patient reported fatigue using cross-validated penalized Lasso regression - a machine learning approach.

Study population: From Emory University Clinics, 44 HNC patients donated blood samples before undergoing RT. GE was assessed using an Affymetrix Clariom S Human microarray which measures gene transcripts for roughly 24,000 genes; each probe was log-transformed, normalized, and standardized. The validated 20-item self-report multidimensional fatigue inventory questionnaire measured each patient's continuous fatigue score.

Methods: To predict fatigue, we used leave-one-out cross validation (CV). This means we built a Lasso regression model using GE probes from 43 of 44 subjects and used that model to predict fatigue for the remaining subject; this process was repeated 44 times until all subjects had a predicted fatigue score. We chose penalized Lasso regression because the 'penalty' performs variable selection and mitigates collinearity between the GE probes; the penalty was chosen by an extra layer of CV not described. We compared the predicted fatigue scores to the corresponding patient reported fatigue scores using R2 (higher values mean stronger correlation and better prediction).

Results: To test the approach, we allowed the Lasso regression to build a model based on all subjects and predict fatigue on all subjects. This prediction was expected to be high, and it was (R2=0.98). However, the approach was less successful predicting fatigue during the leave-one-out CV prediction (R2=0.15). The probes most influential predicting fatigue are linked to genes involved in tryptophan metabolism (precursor to the neurotransmitter serotonin), double strand break DNA repair, and transforming growth factor beta receptor signaling (inflammation cytokine).

Conclusions: Gene expression may predict fatigue in head and neck cancer patients, but there is room for improvement. The model suggests that a patient's personalized DNA repair process, metabolic and inflammatory response may play key roles in patient fatigue. Integrating more data focused on these biological processes, for instance patient metabolomics, may improve the model performance. Future efforts include collecting a larger sample, trying alternative machine learning methods, and testing the robustness of the model over time.

#4258

Magnesium intake and mortality due to liver cancer and other liver diseases: Results from the Third National Health and Nutrition Examination Survey Cohort.

Zhu Xiangzhu,1 Lijun Wu,2 Lei Fan,3 Edmond K Kabagambe,1 Yiqing Song,4 Menghua Tao,5 Xiaosong Zhong,6 Lifang Hou,7 Martha J. Shrubsole,1 Jie Liu,2 Qi Dai1. 1 _Vanderbilt University Medical Center, Nashville, TN;_ 2 _Fudan University, Shanghai, China;_ 3 _Shanghai University of Traditional Chinese Medicine, Shanghai, China;_ 4 _Indiana University, Indianapolis, IN;_ 5 _University of North Texas Health Science Center, Fort Worth, TX;_ 6 _Capital Medical University, Beijing, China;_ 7 _Northwestern University, Chicago, IL_.

People with fatty liver disease are at high risk of magnesium deficiency. Meanwhile, low magnesium status is linked to both chronic inflammation and insulin resistance. However, no study has investigated the association between intake of magnesium and risk of mortality due to liver cancer and other liver diseases. We evaluated the association between total magnesium intake and mortality due to liver cancer and other liver diseases in the Third National Health and Nutrition Examination Study (NHANES III) cohort, which included 13,504 participants who completed liver ultrasound examination for hepatic steatosis. Overall magnesium intake was associated with a reduced risk of mortality due to liver cancer and other liver disease at borderline significance (P=0.05). In multi-variable adjusted models, every 100 mg increase in intake of magnesium was associated with a 49% reduction in the risk for mortality due to liver cancer and other liver diseases. Although interactions between magnesium intake and alcohol use and hepatic steatosis at baseline were not significant (P>0.05), inverse associations between magnesium intake with liver cancer and other liver disease mortality were stronger among alcohol drinkers and those with hepatic steatosis. Our novel findings suggest higher intakes of magnesium may be associated with a reduced risk of mortality due to liver cancer and other liver disease particularly among alcohol drinkers and those with hepatic steatosis. Further studies are warranted to confirm the findings. 

## BIOINFORMATICS AND SYSTEMS BIOLOGY:

### Statistical Methods, Mathematical Modeling, and Molecular Modeling

#4260

Dynamic modeling of macrophage plasticity in bone metastatic prostate cancer.

Etienne A. Baratchart,1 Chen Hao Lo,2 Conor Lynch,1 David Basanta1. 1 _Moffitt Cancer Center, Tampa, FL;_ 2 _University of South Florida, Tampa, FL_.

Tumor-associated macrophages, particularly those with an anti-inflammatory phenotype, have long been implicated in the progression of primary solid malignancies, including prostate cancer. Metastatic prostate cancer typically manifests in the bone where it induces painful osteogenic lesions that are incurable. Bone is naturally rich in myeloid-derived macrophages whose temporal polarization into pro- (M1) and anti-inflammatory (M2) phenotypes is critical for regulating the bone repair program mediated by bone-resorbing osteoclasts and bone-building osteoblasts. However, the dynamics of macrophage polarization in the context of bone metastatic prostate cancer are underexplored and difficult to address with traditional biologic approaches. To address the role of macrophage polarization in the context of bone metastatic prostate cancer, we first investigated the macrophage polarization temporal dynamics in normal bones and analyzed macrophage plasticity in vivo subsequent to intratibial injury. Bone marrows were isolated at several time points and profiled by flow cytometry for pro- and anti-inflammatory monocyte macrophage content. Contralateral tibias were analyzed for bone volume, osteoblast and osteoclast numbers. We then designed a mathematical model describing the different cell population dynamics in the bones. Generation of the model required testing a number of assumptions regarding macrophage polarization behavior. For example, it is unknown whether M1 resolves at the initial stages of bone injury repair through death or by repolarizing into M2. For each aspect, competing mechanistic assumptions were proposed and simulated by sets of ODEs. The best-fitting assumptions for each aspect were integrated into a single comprehensive ODE model to fully describe the dynamics of the bone resident cell populations during bone remodeling. This experimentally validated model is now being used to address how bone resident cells respond to metastatic prostate cancer cells. In conclusion, we have generated an ODE model that describes macrophage polarization over time during bone repair and how pro- and anti-inflammatory macrophages interact with bone stromal cells. The mathematical model predictions are in agreement with our biologic experiments in vivo and will allow us to interrogate how macrophage polarization impacts the behavior of metastatic prostate cancer cells in the bone microenvironment.

#4261

Pathways of metastatic bladder cancer from a longitudinal patient data set.

Jeremy M. Mason, Zaki Hasnain, Gus Miranda, Karanvir Gill, Paul K. Newton, Inderbir S. Gill, Peter Kuhn. _University of Southern California, Los Angeles, CA_.

Bladder cancer (BCa), the 6th commonest cancer in the U.S., is highly lethal when metastatic. Sites and patterns of patient-specific metastatic spread are deemed random and unpredictable. Whether BCa metastatic patterns can be quantified and predicted more accurately is unknown. We used a prospective, longitudinal dataset of 3,505 BCa patients who underwent definitive treatment following diagnosis and were continuously enrolled (1971-2016; mean follow-up 6.13 years). Metastases developed in 30% (n=1,040) of patients, with 5-year survival rate of 18%, compared to 72% in those without metastases (n=2,465). The three commonest metastatic sites at time-of-first progression were pelvis (n=283; 27.2%), bone (n=274; 26.3%), and lung (n=247; 23.8%). We illustrate metastatic pathway progression as color-coded, circular, tree-ring diagrams (primary in the center; metastatic sites outwards). Markov chain modeling, denoting nodes as potential anatomic metastatic sites, indicated higher probability of spread from the bladder to pelvis (15.6%), bone (15.6%), and lung (13.9%), categorizing 4 of 10 anatomical sites as 'spreaders' and 2 as 'sponges'. We created a dynamical, data-visualization, web-platform that displays temporal, spatial and Markov modeling figures with predictive capability. For contrasting subgroups, this platform indicated differences in transition probabilities, rank-ordering of metastatic sites, and spreader/sponge node classification. Spatiotemporal patterns of BCa metastasis and sites of spread indicate underlying organotropic mechanisms in the prediction of response. This recognition opens the possibility of organ site-specific therapeutic targeting in the oligo-metastatic disease setting. In the precision medicine era, visualization of complex, time-resolved clinical data will enhance management of post-operative, metastatic BCa patients.

#4262

Mathematically universal and biologically consistent astrocytoma genotype encodes for transformation and predicts survival phenotype.

Katherine A. Aiello, Sri Priya Ponnapalli, Orly Alter. _University of Utah, Salt Lake City, UT_.

Recurring DNA alterations have been recognized as a hallmark of cancer for over a century, and observed in astrocytoma genomes for decades. Only recently, however, a copy-number genotype predictive of an astrocytoma survival phenotype was discovered, and only by using the generalized singular value decomposition (GSVD). We formulated the GSVD as a comparative spectral decomposition that can simultaneously identify the similar and dissimilar between two column-matched matrices, and, therefore, create a single coherent model from two datasets recording different aspects of a single phenomenon. In recent comparisons of microarray-profiled patient-matched glioblastoma (GBM) and, separately, lower-grade astrocytoma tumor and normal genomes, the GSVD invariably separated the tumor-exclusive genotype and phenotype from those that occur in the normal genomes and from experimental batch effects. The tumor-exclusive genotype invariably predicted the survival phenotype statistically better than any other indicator of astrocytoma.

Here we use the GSVD to compare whole-genome sequencing (WGS) read count profiles of patient-matched astrocytoma tumor and normal DNA. The GSVD uncovers a tumor-exclusive genome-wide pattern of copy-number alterations (CNAs). We find that, first, this WGS astrocytoma pattern is bounded by the microarray GBM and lower-grade astrocytoma patterns. Like the microarray patterns, the WGS pattern is correlated with an approximately one-year median survival time. By filling in gaps in the genome, the WGS pattern reveals that this biologically consistent genotype encodes for transformation via the Notch together with the Ras and Shh signaling pathways. Second, like the GSVDs of the microarray profiles, the GSVD of the WGS profiles separates the tumor-exclusive pattern from normal copy-number variations (CNVs) and from experimental sources of variation. These include the WGS technology-specific effects of guanine-cytosine (GC) content variations across the tumor and normal genomes, which are correlated with experimental batches. Third, by identifying the biologically consistent phenotype among the WGS-profiled tumors, the GBM pattern proves to be a technology-independent predictor of astrocytoma survival and responses to chemotherapy and radiation, statistically better than the patient's age and tumor's grade, the best other indicators of astrocytoma, and existing tests for MGMT promoter methylation and IDH1 mutation.

We conclude that by using the complex structure of the data, rather than simplifying them as is commonly done, comparative spectral decompositions, such as the GSVD, underlie a mathematically universal description of the genotype-phenotype relations in cancer that other methods miss.

#4263

A semi-mechanistic pharmacokinetic-pharmacodynamic (PK-PD) model of savolitinib (AZD6094/HMPL-504), a novel MET inhibitor, to explore extent and duration of target inhibition required for optimal efficacy in the EBC-1 mouse xenograft model.

Rhys D. Jones,1 Michael Grondine,2 Alexandra Borodovsky,2 Maryann San Martin,2 Michelle DuPont,2 Celina D'Cruz,2 Alwin Schuller,2 Ryan Henry,2 Evan Barry,2 Klas Petersson,3 Tarjinder Sahota,1 Ghada F. Ahmed1. 1 _AstraZeneca, Cambridge, United Kingdom;_ 2 _AstraZeneca, Boston, MA;_ 3 _qPharmetra LLC, Huddinge, Sweden_.

MET is a transmembrane tyrosine kinase receptor that is deregulated across multiple cancer types. Savolitinib is a selective small molecule inhibitor of MET being co-developed with Hutchison MediPharma for papillary renal cell carcinoma (PRCC) and non-small cell lung cancer (NSCLC). In preclinical xenograft models, savolitinib demonstrates rapid and extensive inhibition of phosphorylated-MET (pMET) with an EC50 of 0.35 ng/ml (CI 95% - 0.25 to 0.5), and anti-tumor activity in MET amplified models (RCC-43b & RCC-47 for PRCC; MKN-45, SNU-5 & Hs746T for gastric; EBC-1 for NSCLC). To evaluate the anti-tumor activity of savolitinib under a range of schedules, the EBC-1 xenografts were treated with savolitinib at 30 mg/kg daily (76% tumor growth inhibition (TGI)), every other day (43% TGI), or 4 on/3 off (39% TGI). Intermittent dosing was also explored at 100 mg/kg every other day (80% TGI), 4 on/3 off (67% TGI) and 2 on/5 off (46% TGI). To assess the effect of prolonged pMET inhibition, twice daily dosing at 15 mg/kg (83 % TGI) and 30 mg/kg (94 % TGI) and co-dosing savolitinib at 15 mg/kg (61 % regression) with the cytochrome P450 inhibitor, 1-aminobenzotriazole (ABT) were also tested. A population PK-pMET-TGI model was developed where savolitinib plasma concentration drives pMET inhibition, which in turn drives inhibition of tumor growth. The modelling objectives were to (1) determine whether a generalizable model can be applied to EBC-1, and data from other cell-lines investigated and (2) Determine the extent and duration of pMET inhibition that delivers optimal efficacy. An Emax model of pMET inhibition driving TGI was applied and an Emax was estimated as a factor of intrinsic tumor growth rate, where a value > 1 results in a shrinking tumor. It was found that a single Emax estimate of 3.2 (CI 95% - 2.8 to 3.6) could be applied to EBC-1 and models previously investigated. The pMET inhibition needed for 50% of maximum effect was estimated to be > 90 % confirming that tumor regression is achieved through continuous high levels of pMET inhibition. Estimating drug effect relative to tumor growth rate offers a novel way in which to apply a mathematical model with parameters of drug effect that are shared across different xenograft models. The developed model offers the potential to be translated to predict the expected tumor growth inhibition in humans under varying dose regimens by accounting for mouse-to-man differences in PK and tumor growth dynamics.

#4264

Mathematical modeling of differential effects of sunitinib on primary tumor and metastatic growth.

Chiara Nicolò,1 Michalis Mastri,2 Amanda Tracz,2 John M. Ebos,2 Sébastien Benzekry1. 1 _Inria team MONC, Institut de Mathematiques de Bordeaux, Bordeaux, France;_ 2 _Department of Cancer Genetics and Medicine, Roswell Park Cancer Institute, Buffalo, NY_.

Sunitinib is a drug with anti-angiogenic activity used in the treatment of patients with metastases from renal cell carcinoma or gastrointestinal tumors. However, despite clear efficacy in reducing established tumor growth, recent preclinical studies have shown limited, or even opposing, efficacies in preventing metastatic spread.

In this work, we evaluated a mathematical metastatic model to describe primary tumor and metastatic dynamics in response to sunitinib in a clinically relevant mouse model of spontaneous metastatic breast cancer that develops after surgical removal of an orthotopically implanted primary tumor. Mice received either vehicle or sunitinib in the neoadjuvant (presurgical) setting according to different schedules. The experimental dataset comprises measurements of primary tumor kinetics, metastatic burden and pre-surgical molecular and cellular biomarkers, including vascular cell Ki67 and CD31 expression, circulating tumor cells (CTCs) and myeloid derived suppressor cells (MDSCs). Estimation of the model's parameters was performed using a mixed-effects population approach. To describe tumor growth under sunitinib treatment, a simple ordinary differential equation model of tumor growth inhibition was used.

Population fits obtained modeling the effect of treatment only on primary tumor growth described well the experimental data of all the treated groups considered. On the contrary, simulations of treatment also on metastasis could not reproduce the behavior of the data. By inserting in the model the available biomarkers as covariates, through a Wald-test, measurements of Ki67+/CD31+, CTCs and granulocytic MDSCs were found significantly correlated with the model parameter expressing the metastatic aggressiveness of the tumor.

Together, these mathematical models confirm a differential effect of sunitinib on primary (localized) tumors compared to secondary (metastatic) disease. Our results suggest that Ki67+/CD31+, CTCs and MDSCs measurements might help in predicting metastatic potential and thus aid in predicting benefit in overall survival for preoperative antiangiogenic treatments.

#4265

Computational models of the role of base excision repair in sensitization of cancer cells to 5-fluorodeoxyuridine.

Mengdi Qian,1 Alexandru Almasan,2 Evren Gürkan-Çavusoglu1. 1 _Case Western Reserve Univ., Cleveland, OH;_ 2 _Cleveland Clinic, Cleveland, OH_.

The purpose of this study is to computationally analyze the effect of inhibition of base excision repair (BER) in the response of cancer cells to treatment with 5-fluorodeoxyuridine. 5-fluorouracil (5-FU) and its metabolite 5-fluorodeoxyuridine (5-FdU) are standard treatments for different solid tumors and especially for colon cancer. In the literature, it is shown that the depletion of uracil DNA glycosylase (UDG), one of the enzymes that initiate the BER pathway, enhanced the cytotoxicity of 5-FdU, making UDG a potential target to enhance efficacy of chemotherapeutic agents. It is also shown that the activity of UDG is significantly higher in colon cancer cells compared to normal tissue. We have developed a computational model of the BER pathway to capture the BER dynamics in colon cancer cell lines after treatment with 5-FdU. Our model is composed of a series of ordinary differential equations where the parameters are the kinetic rate constants and the enzyme concentrations. We have used the data from the literature to initially parameterize our models. We have then used least square estimation to find the parameters that will capture the BER time course for colon cancer cells after 5-FU treatment using the experimental data from the literature. We have performed sensitivity and identifiability analyses to determine the parameters that are reliably estimated. The experimental data from the literature showed that the depletion of UDG resulted in incorporation of uracil and 5-FU in the DNA. We have computationally replicated this effect by decreasing the protein concentration of UDG in the models and simulating the amount of remaining 5-FU in the DNA after BER with reduced activity has taken place. We have studied the depletion effect for different protein concentrations of UDG and the results have shown that the accumulation of 5-FU in these colon cancer cells correlate with the level of UDG. The suppression of UDG activity has the potential to enhance the outcome of treatment with 5FdU, and we will use the developed computational models to quantitatively optimize these effects in our future work.

#4266

Computational exploration of mechanistic determinants of ADC pharmacokinetics using QSP modeling strategies.

A. Katharina Wilkins, Andrew Matteson, Lore Gruenbaum, Jennifer Park, John M. Burke, Joshua F. Apgar. _Applied BioMath, LLC, Lincoln, MA_.

The pharmacokinetics of antibody drug conjugate (ADC) therapeutics typically show a discrepancy between the PK of total antibody (conjugated and unconjugated antibody) and that of conjugated antibody, carrying one or more payload molecules. This discrepancy is often attributed strictly to deconjugation (Kamath, 2014), however recent evidence suggests that the underlying mechanisms may be more complex. This work employs a computational quantitative systems pharmacology (QSP) approach that generates a hypotheses to better understand the impact of drug antibody ratio (DAR) and the resulting changes in molecular properties on overall PK and relative payload disposition as observed in preclinical and clinical studies. Our work establishes the benefit of using computational models to design novel ADCs and to optimize the discovery and development of existing ADCs to better enable clinical trial design.

References: Kamath, A. V., and Iyer, S. (2014). Preclinical Pharmacokinetic Considerations for the Development of Antibody Drug Conjugates. Pharmaceutical Research, 32(11), 3470-3479.

#4267

Patterns of DNA copy-number alterations revealed by the GSVD and tensor GSVD encode for cell transformation and predict survival and response to platinum in adenocarcinomas.

Theodore E. Schomay, Katherine A. Aiello, Cody A. Maughan, Sri Priya Ponnapalli, Heidi A. Hanson, Orly Alter. _University of Utah, Salt Lake City, UT_.

A quarter or more of lung, uterine, and ovarian adenocarcinoma (LUAD, USEC, and OV) tumors are resistant to platinum, the first-line systemic treatment for decades. Only recently, however, patterns of copy-number alterations (CNAs) were discovered that are predictive of OV survival, both in general and in response to platinum, and only by using a comparative spectral decomposition, the tensor generalized singular value decomposition (GSVD). We defined the tensor GSVD to extend the GSVD from two matrices to two tensors. Like the GSVD, the tensor GSVD can simultaneously identify the similar and dissimilar between two datasets recording different aspects of a single phenomenon. In a comparison of Agilent microarray platform-matched profiles of patient-matched normal and primary, mostly high-grade OV DNA, the tensor GSVD revealed the tumor-exclusive and platform-consistent patterns across the chromosome arms 7p and Xq and the combination of 6p+12p. No other diagnostic can distinguish a shorter from a longer survival in response to platinum throughout the course of an adenocarcinoma.

Here we show that the 6p+12p genotype predicts a survival phenotype in LUAD and USEC in addition to OV. We use the GSVD to compare whole-genome sequencing (WGS) and Affymetrix microarray profiles of patient-matched normal and primary LUAD and primary, mostly high-grade USEC and OV DNA. The GSVD reveals tumor-exclusive patterns of CNAs across the combination of the chromosome arms 6p+12p. We find that, first, like the Agilent OV pattern, the WGS LUAD and Affymetrix LUAD, USEC, and OV patterns identify a biologically consistent shorter survival phenotype, of approximately one and a half years median survival time in LUAD and USEC and three years in OV. Like the Agilent pattern, the WGS and Affymetrix patterns describe a biologically consistent genotype, where loss of the p21-encoding CDKN1A and p38-encoding MAPK14 on 6p, and gain of KRAS on 12p, together, but not separately, encode for human cell transformation. Second, by classifying the WGS and Affymetrix profiles, the Agilent pattern proves to be a technology-independent predictor of a LUAD, USEC, or OV patient's survival, both in general and in response to platinum. The pattern is independent of the tumor's stage, the best indicator of these adenocarcinomas at the time of initial diagnosis. Third, the pattern is a predictor of OV survival and response to platinum beyond the time of initial diagnosis and throughout the disease, even in patients experiencing remission after treatment of the primary tumor, and independent of the time to recurrence or progression.

We conclude that comparative spectral decompositions, such as the GSVD and tensor GSVD, underlie a mathematically universal description of the relations between a tumor's genome and a patient's survival and response to treatment, which other methods miss.

#4268

Magee Equations for predicting Oncotype DX recurrence risk: A further evaluation using a large independent data set.

Dooman Arefan,1 Rohit Bhargava,2 David Dabbs,2 Rachel C. Jankowitz,2 Shandong Wu1. 1 _University of Pittsburgh, Pittsburgh, PA;_ 2 _Magee-Womens Hospital of University of Pittsburgh Medical Center, Pittsburgh, PA_.

Purpose: Oncotype DX 21-gene assay is a costly clinical method to predict the risk of breast cancer recurrence in lymph node-negative and estrogen receptor (ER)-positive breast cancers. Magee Equations were linear regression models developed working on several standard clinically available histopathological markers for predicting the Oncotype DX risk score, with the intention to save expenses and time. Magee Equations were derived from 817 patients and initially evaluated on 255 separate patients, now publically accessible online to compute estimations of recurrence risk scores. The purpose of this study was to conduct a further validation of Magee Equations using a much larger independent dataset to examine its robustness in comparison to the initial evaluation. Methods and Materials: In a HIPAA-compliant and IRB-approved study, we collected 1,391 patients with invasive breast cancer at our institution and all those patients have Oncotype DX risk score available. This cohort is independent of all previous patient populations associated with the development and initial evaluation of Magee Equations. Out of the 1,391 patients, 1,210 have available the related histopathologic markers that had been used in Magee Equations, including ER status, progesterone receptor (PR) status, human epidermal growth factor receptor 2 (HER2) status, proliferation indices (Ki-67), Nottingham grade, and tumor size. We ran the three different forms of Magee Equations on all the 1,210 patients and compared the estimation output of each equation to the truth, i.e., the actual Oncotype DX risk score. The estimation results on the 1,210 patients were evaluated by two metrics: concordance and Pearson's correlation coefficients, and compared to the reported initial evaluation performance of Magee Equations on the 255 patients. Results: On the 1,210 cases, the concordance on the recurrence risk categories (i.e., low, intermediate, and high) between the actual Oncotype DX categories and the three Magee Equation-predicted categories was 61.9% (Eq. 1), 59.8% (Eq. 2), and 63.4% (Eq. 3), respectively, corresponding to the initial evaluation performance of 55.8%, 59.4%, and 54.4%. After eliminating the intermediate category, the concordance of the three equations increased to 99.0%, 98.3%, and 99.1%, respectively, corresponding to the initial performance of 100%, 98.6%, and 98.7%. Likewise, the Pearson's correlation on the continuous risk scores was 53.7%, 54.4%, and 56.2% for equations 1, 2, and 3, respectively, corresponding to the initial Pearson's correlation of 61.6%, 60.3%, and 59.4%. Conclusions: Magee Equations remain stable on 1,210 independent new patients, comparable to the initially reported evaluation performance. Clinical outcome studies are required to test the usefulness of Magee Equations as a stand-alone test.

#4269

Dynamic contrast-enhanced MSOT imaging to intratumoral pharmacokinetic modeling.

Ted G. Xiao,1 Abhilash Samykutty,1 Jared Weis,2 Scott Gayzik,2 Umit Topaloglu,2 Alexandra Thomas,1 Akiko Chiba,1 Lacey R. McNally1. 1 _Wake Forest Univ. Comp. Cancer Ctr., Winston Salem, NC;_ 2 _Wake Forest Univ. Comp. Cancer Ctr., Winston-Salem, NC_.

Introduction: Interactions between biological features and nanoparticles could affect tracer biodistribution, kinetics, clearance, and translocation. Because these effects cannot be simply extrapolated from in vitro studies, in vivo quantitative data are needed to determine the pharmacokinetic properties of tracers. Planar fluorescence or 2D fluorescent /bioluminescence imaging has the potential for the tracers to accumulate outside of the tumor and the potential of light scattering. MSOT allows for more precise determination of signal origin and intensity. HypoxiSense 680 is a tracer that targets the hypoxic tumor environment. Our objective is to assess the pharmacokinetics of HypoxiSense 680 in the context of an orthotopic breast tumor in vivo using MSOT. This study takes advantage of a novel contrast tracking imaging modality to study intratumoral pharmacokinetics in in vivo setting with a hypoxia-targeting tracer. Methods: Female, athymic mice were orthotopically implanted with MDA-MB-231 cells and tumors were allowed to grow to 4mm in diameter prior to the evaluation of the tracer. Mice were anesthetized with isoflurane and a mixture of 0.1L of O2 and 0.9L of medical air prior to the insertion of a tail vein catheter. The mouse was placed into the MSOT scanner for 10 minutes to equilibrate. Once the breast tumor was localized, images were acquired for 5 minutes to obtain a baseline measurement. Subsequently, 100μL of HypoxiSense 680 at the recommended dosage of 2nmol per mouse was injected over the course of 30s followed by a catheter flush with 100μL of saline solution. MSOT inVision 256-TF continuously measured optoacoustic signal within the mouse in vivo for a duration of 25 minutes. HypoxiSense 680 signal was isolated by spectral unmixing using backprojection algorithm. Results: A 2-compartmental PK model was constructed in MATLAB and curve-fitted against the animal data. There were two compartments to account for the contrast within the circulatory system and the tumor microenvironment. Rate constants of the model were determined by fitting the model curve to the experimental MSOT imaging data: kforward=2.76x10-4 sec-1, kreverse=4.73x10-4 sec-1, kelcentral=1.01x10-5 sec-1, kelperipheral=3.18x10-7 sec-1. The goodness of fit was evaluated (R=0.83, RMSE=0.0015). Conclusion: Optoacoustic imaging with MSOT provides live tracking of spectrally unique contrasts in vivo. This capability allows for a novel characterization method of intratumoral drug/contrast kinetics in vivo in mouse tumor models.

#4270

Success of T-cell immunotherapy predicted by a mathematical model of the adaptive immune system and evolving cancer cells.

Jason Thomas George, Herbert Levine. _Rice University, Houston, TX_.

It is now well-established that a patient's adaptive immune system plays an important role in identifying and eliminating cancer cells in much the same way that intracellular pathogens are cleared during infection. From a therapeutic standpoint, the adaptive immune system is unique in that it can co-evolve alongside a developing tumor. At present, the dynamics and survival probability of a mutating threat in the presence of an adaptive immune system has yet to be quantified. Here, we consider a foundational population model of a foreign invader faced with innate or adaptive immune pressure. We find that optimal growth strategies depend on whether or not the invading threat may mutate as well as the mode of immune detection. Our early model predicts that decreases in thymic output seen in advanced age may substantially affect the ability to recognize a growing population of cancer cells and recapitulates general features found in age-dependent AML incidence. This mathematical framework is able to predict the success of recent immunotherapeutic strategies, including personalized neoantigen vaccines. Our model serves as an attempt at modeling stochastic cancer evolution alongside an adaptive immune compartment.

#4271

Most expression quantitative trait loci discovered in tumors cannot be attributed to cancer cells.

Paul Geeleher,1 Fan Wang,1 Zheny Zhang,1 Robert L. Grossman,1 Cathal Seoighe,2 R. Stephanie Huang3. 1 _Univ. of Chicago, Chicago, IL;_ 2 _National University of Ireland, Galway, Ireland;_ 3 _University of Minnesota, Minneapolis, MN_.

Genome-wide association studies (GWAS) have identified hundreds of inherited genetic variants that increase cancer risk. However, most cancer risk variants are in non-coding regions of the genome and modulate risk by affecting gene regulation. Thus, determining how inherited genetic variation affects gene expression in cancer is critically important to understanding disease development. Consequently, by leveraging large genomics datasets like The Cancer Genome Atlas (TCGA), previous studies have examined the effect of inherited genetic variation on gene expression in tumors; an approach known as expression quantitative trait locus (eQTL) mapping. However, tumors are mixtures of both cancer and normal cells, for example, immune cells and stroma. No previous cancer eQTL study has accounted for this mixture of cell types, essentially having treated bulk tumor expression as representative of gene expression in cancer cells.

Building on research in deconvolution of mixtures of cell types, we have developed a new approach that can accurately account for the effect of tumor-infiltrating normal cells on cancer eQTLs. The approach involves first estimating the proportion of tumor-infiltrating normal cells (tumor purity) using a combined estimate from genomics data and H&E staining. Then, we developed a statistical model that can account for the effect of tumor purity on eQTLs by modeling the interaction of the tumor purity estimate and genotype. Intuitively, this works by determining how the magnitude of the association between gene expression and genotype changes as a function of tumor purity, then extrapolating this effect to 100% cancer cells. The model's accuracy was validated using simulated data and on expression profiles from purified cell types.

We used this model to map eQTLs in the TCGA breast cancer cohort. Remarkably, while 57,189 eQTLs were identified in TCGA breast cancer patients using a conventional model, only 8,833 (15.4%) could be attributed to cancer cells when tumor purity was accounted for. Analysis of the Genotype-Tissue Expression (GTEx) data provided evidence that almost 50% of tumor eQTLs inferred using a conventional approach affect gene expression in tumor-infiltrating immune cells and fibroblasts. We also investigated the eQTL profiles of cancer risk variants from a GWAS for breast cancer risk. Strikingly, for 33% of breast cancer risk variants identified as eQTLs using a conventional approach, we found strong evidence of an effect in tumor-infiltrating normal cells, but no evidence of an effect in cancer cells. This suggests cancer risk is mediated by the effect of inherited genetic variation on gene regulation in the cells of the tumor microenvironment, as well as in cancer and pre-cancer cells. Our findings profoundly challenge the current interpretation of inherited genetic regulation in cancer and should be considered in functional validation of results from all cancer risk GWAS.

#4272

A novel approach to differentiation of somatic vs. germline variants in liquid biopsies using a betabinomial model.

Tracy Nance,1 Elena Helman,1 Carlo Artieri,1 Jennifer Yen,1 Thomas P. Slavin,2 Darya Chudova,1 Richard B. Lanman,1 AmirAli Talasaz1. 1 _Guardant Health, Redwood City, CA;_ 2 _City of Hope, Duarte, CA_.

Background: A recent large study found that over 2% of advanced cancer patients have unidentified germline alterations found incidentally during next-generation sequencing (NGS) for targetable somatic alterations. However, tissue-based NGS cannot definitively distinguish germline from somatic mutations without comparison to normal tissue. Because somatic variants typically occur in plasma at allele fractions 1-2 orders of magnitude lower than germline, liquid biopsy often enables differentiation of incidental germline mutations. Not uncommonly, high tumor burden or allelic imbalance from copy number variation or loss of heterozygosity complicates simplistic differentiation based on assumption of germline 50% allele frequency. We developed a novel statistical method for discriminating between somatic and germline variants in deep sequencing data, and applied to data generated from the Guardant 360 targeted cfDNA assay. Our model dynamically incorporates prior knowledge of germline SNP variants and observed allele frequencies within a sample, and gains power in larger panels like the GuardantOmni 500-gene assay.

Methods: Our method uses common heterozygous SNPs to model local germline allele count behavior, and calls variants somatic if they deviate significantly from the observed germline mutant allele fraction (MAF). A betabinomial model is well suited to this problem, because it models both the mean and variance of mutant allele counts at common SNPs. This is important since simpler methods like fixed MAF cutoffs or Poisson models may not represent the variance in molecule counts appropriately.

Results: We evaluated our method against variants from 361 clinical samples that were manually annotated by reviewers with expertise in cancer genomics. Of the 11,679 heterozygous variants considered in these samples, 7,221 (62%) had two or more common SNPs in the same gene, so that a local germline heterozygous MAF could be estimated and the betabinomial model could be applied. Using the manual annotation as ground truth, we created ROC curves for a classifier based on the betabinomial model with varying p-value cutoffs, as well as a classifier based on MAF cutoffs. The AUC for the betabinomial model was 0.996, as compared with an AUC of 0.985 for MAF cutoffs. The ROC curve allowed us to choose a betabinomial p-value cutoff that maintained a similar level of specificity as a 15% MAF cutoff (99.2% for MAF cutoff, 99% for betabinomial), but with significantly increased sensitivity (89% for MAF cutoff, 97% for betabinomial).

Conclusions: We have developed a novel method for differentiation of somatic versus germline variants in a single plasma sample. Identifying incidental germline alterations in advanced cancer patients may identify new opportunities to apply targeted therapy such as PARP inhibitors for BRCA1/2, and may also serve to alert physicians and their patients to familial risk.

#4273

Identification of RNA-seq shape abnormality.

Hyo Young Choi, David N. Hayes, James S. Marron. _UNC Chapel Hill, Chapel Hill, NC_.

A substantial proportion of human genes differ in function through different forms of shape changes in read coverage of RNA-seq. For example, tumor-suppressor genes lose their function through various changes in expression such as aberrant splicing, frameshift indel, large deletions, or overexpression of noncoding RNAs. Although previous studies have examined mutational effect on such shape changes, it has been observed that a large fraction of shape aberration occurs in the absence of mutations. We hypothesize that read coverage shapes would show rich information on various forms of abnormality in RNA-seq data, which may have been missed in current mutation callers. We have developed a statistical method to systematically detect abnormal RNA-seq samples using read coverage data alone independently of mutational analysis. We model the underlying mechanism possibly generating aberrant shapes by multiple unknown mixture distributions, recasting the problem as high-dimensional latent variables framework. Based on this underlying mechanism, we normalize the read coverage to adjust different library sizes, extract the latent information in a robust way, and determine the cases that are strongly involved in abnormality. This approach allows us to detect not only local changes in expression such as alternative splicing events and frameshift indel but also landscape changes such as fusion and a wide range of deletions. This methodology can be applied genome-widely to detect key genes with strong shape aberrations, prioritizing genes for further investigation. We analyzed 522 TCGA head and neck squamous cell carcinomas RNA-seq tumor samples. At several known tumor-suppressor genes, we identified the cases with novel structural changes including alternative splicing, intragenic deletion, and fusion with/without mutations reported as well as the cases with no evidence of changes despite presence of mutations known for altering shapes. Notably, some of the identified shape changes in TP53 and CDKN2A were confirmed as being the outcomes of missing genetic variants near splice sites (exon-intron junctions). The genome-wide study with carefully chosen significance level provided a set of key genes with strong evidence of shape abnormality including TP53, CDKN2A, and FAT1 that are known for the most alternative splicing events. We also analyzed how often such shape changes arise with or without certain mutations in genome-wide scans. To conclude, our results provide a new statistical framework for various forms of RNA-seq shape changes and a tool for systematic discovery of such abnormal samples, and give insights into mutational effect on shape aberration.

#4274

Identifying the regulatory network structure of the genes involved in signaling pathways underlying ER-PR-HER2+ breast cancer using Bayesian modeling of genome-wide NRF1 DNA motif sequence-enriched genes.

Jairo D. Ramos, Jayanta Das, Changwon Yoo, Deoudutta Roy. _Florida International University, Miami, FL_.

Nuclear respiratory factor 1 (NRF1) [alpha-palindromic binding protein (α-PAL)] is a redox-sensitive transcription factor. NRF1 localizes to several thousand sites in the genome. Role of NRF1 has remained largely unexplored in breast cancer (BCa). We have recently shown that reactive oxygen species (ROS) contribute to estrogen-induced growth of BCa cells through an NRF1 signaling pathway. In this study, we used a systematic approach integrating ChIP DNA-seq computational analysis, NRF1 protein-DNA motif binding analysis, breast cancer signaling pathway analysis, and Bayesian modeling to discover genes involved in HER2+ (HER2+ER−PR−) breast cancer. First, we used ChIP-seq of normal human mammary epithelial cells (HMEC) and three breast cancer cell lines, HCC1954, MCF7 and T47D, to identify NRF1 regulated genes in breast cancer. We then identified the genome-wide NRF1-enriched DNA regions in estrogen- and progesterone receptors-negative and amplified HER2 (ER-/PR-/HER2+) - HCC1954 breast cancer cell line compared to normal human mammary epithelial cells (HMEC) to discover the NRF1 regulatory network. Analysis of NRF1 binding activity in breast cancer cell line HCC1954 compared to normal mammary epithelial cells (HMEC) shows that more than 7,000 genes have NRF1 binding activity in the region located +/- 2,000 bp from TSS and the top 2,000 correlated with breast cancer showed a significant difference in the average amount of binding protein. The majority (83%) of NRF1 target genes that may participate in the process of cells acquiring characteristics of malignancy present NRF1 binding sites in the regulatory region located +/- 2,000 bp from TSS. Finally we used RNA microarray data from TCGA to detect changes in NRF1 expression levels and to develop a probabilistic graphic model using Bayesian network analysis to investigate how the putative causal genes may interact in breast cancer. NRF1 expression was significantly higher in HER+ breast cancer samples compared to normal breast tissues. This is consistent with our experimental data, which also showed higher expression of NRF1 in HER2+ breast cancer brain metastasis samples. NRF1 binding sites are present in the regulatory region located +/- 2,000 bp from TSS in the majority of target genes (83%). NRF1 binding activity in the region located +/- 2,000 bp from TSS significantly correlated with breast cancer. Bayesian structural modeling revealed 27 NRF1 target genes are part of the hallmarks of breast cancer. NRF1 is a parent of GADD45A, WNT9A and SHC2. Likewise, SHC2 is parent of CDK4, which in turn is parent of BRCA1. In summary, these findings revealed novel roles of NRF1 and its regulatory network presumably involved in the development of HER2+ER−PR− breast tumor, one of the two most aggressive breast cancer subtypes with poor prognosis.

#4275

Analysis of gene and protein expression in lymphoma cell lines using multiple platforms.

Luciano Cascione,1 Marco Benvento,2 Eugenio Gaudio,3 Chiara Tarantelli,3 Filippo Spriano,3 Alberto Arribas,3 Andrea Rinaldi,3 Ivo Kwee,1 Jonh Luecke,4 Roger Geiger,2 Francesco Bertoni5. 1 _Università della Svizzera italiana (USI), Swiss Institute of Bioinformatics (SIB), Institute of Oncology Research (IOR), Bellinzona, Switzerland;_ 2 _Università della Svizzera italiana (USI), Institute for Research in Biomedicine (IRB), Bellinzona, Switzerland;_ 3 _Università della Svizzera italiana (USI), Institute of Oncology Research (IOR), Bellinzona, Switzerland;_ 4 _HTG Molecular Diagnostics, Inc., Tucson, AZ;_ 5 _Università della Svizzera italiana (USI), Oncology Institute of Southern Switzerland (IOSI), Institute of Oncology Research (IOR), Bellinzona, Switzerland_.

Introduction. Published studies on the correlation of mRNA and protein levels in complex samples are relatively limited, especially in lymphoma cells. Here, we present a large-scale analysis of gene expression and protein abundance across a panel of human lymphoma cell lines. We integrated mass spectrometry data with expression profiles obtained with a next generation sequencing (NGS) and a microarray based platform.

Methods. Transcriptome analysis of 51 cell lines derived from activated B cell (n=7) and germinal center B cell (n=15) diffuse large B cell lymphoma (DLBCL), Type 3 DLBCL (n=3), mantle cell lymphoma (n=10), marginal zone lymphoma (n=5), prolymphocytic leukemia (n=1), anaplastic large-cell lymphoma (n=7), primary mediastinal large b-cell lymphoma (n=1) were analyzed using the HTG EdgeSeq Oncology Biomarker Panel Assay, and the Illumina HumanHT 12 Expression BeadChips. Proteome analysis was performed by digesting each cell lysate with LysC and trypsin endoprotease. Subsequently, peptides were analyzed by LC-MS/MS with a QExactive HF mass spectrometer. The Pearson correlation was calculated between gene and protein measurements.

Results. High correlations (median rho 0.65, 95% CI 0.61-0.66) were observed between the gene expression profiles generated by the microarray and the NGS - based platforms (Illumina and HTG), with the latter demonstrating a broader dynamic range and ability in detecting low abundance transcripts. A comprehensive cell- and gene-specific analysis revealed that across cell lines, correlation between mRNA and protein expression was positive with a median correlation of 0.49 (95% CI 0.48-0.50) for the NGS assay, and of 0.43 (95% CI 0.42-0.44) for the microarray-based platform. We identified 577 (46% of common genes n=1253) and 3890 (51% of common genes n=7592) genes whose RNA and protein expression was significantly correlated across all cell lines for NGS and microarray, respectively. Most genes had high and concordant RNA-protein expression (rho values ranged from 0.34 to 0.91), showing that expression of target genes corresponded well to protein levels in lymphoma cell lines. In this group, we found representatives of essential cellular functions, such as cell cycle (CDK1 and CDC34), apoptosis (CASP8 and CD27), and the NFkB pathway (TBK1 and TNFAIP3). A low number of discordant genes (high expression-low protein level or low expression-high protein level) was identified (less than 1% of tested genes; 31 for microarray and 4 for NGS).

Conclusion. We observed a positive correlation between mRNAs and protein levels. In particular, we identified a set of known biomarkers and therapeutic genes whose expression were highly correlated with protein levels, implying that mRNA expression of those genes is sufficient to determine protein abundance. Moreover, we found few genes with discordant mRNA-protein ratios and further analyses are needed to better understand this phenomenon.

#4276

Analysis of sensitivity of genomic signatures of therapeutic responses of non-small cell lung cancer in patient-derived xenograft models.

Iman Tavassoly, Ravi Iyengar. _Mt. Sinai Icahn School of Medicine, New York, NY_.

Genomic alterations are important characteristics of therapeutic responses and therapeutic-resistance phenotype in cancer. These genomic alterations are complex and are determinants of aberrations in signaling pathways and their dynamics. A genomic signature includes a set of genomic alterations like gene expressions, mutations, copy number variations, etc. These genomic signatures are used as biomarkers for assigning treatment to individuals benefiting from a specific treatment plan and are validated using preclinical models such as patient-derived xenograft models (PDX models). We have proposed a methodology to optimize these genomic signatures in silico and evaluate their accuracy in vivo using PDX models of non-small cell lung cancer (NSCLC). Our integrative analysis has shown that different sizes and complexities of gene sets provide different layers of information applicable in designing therapeutic regimens. To eliminate sloppy signatures, it is possible to find optimized and sensitive genomic signatures for precision oncology.

#4277

Modeling selectin-coated microprobe adhesion to selectin ligands heterogeneously expressed on cancer tissues.

Eric Martin, Douglas Goetz, Monica Burdick. _Ohio University, Athens, OH_.

The transition to an era of functional glycomics has created a need for the development of novel techniques to assess complex glycoconjugate cellular adhesion molecules (e.g., selectin ligands) that may be involved in capturing and mediating the adhesion circulating tumor cells to distant sites. Cell line, knockout, and selectin antagonist evidence suggests that selectin/selectin-ligand interactions are involved in metastasis. However, identification and characterization of functional selectin ligand expression on cancer tissue (i.e., CTC origin site) remains challenging due to the inherent heterogeneity of tissues magnified by the "catch-slip" and stochastic nature of selectin/selectin-ligand bonds. We have developed a flow-based assay known as dynamic biochemical tissue analysis (DBTA) that involves the perfusion of selectin-coated microprobes over tissues at physiologic wall shear rates. Using DBTA, the expression of functional selectin ligands has been detected (i.e., specific binding and rolling of selectin-coated microprobes) on tissue from multiple solid tumors at primary and metastatic sites. Although DBTA provides evidence that multiple types of cancer can express complicated glycoconjugate structures (selectin ligands) that are able to bind to selectins in a shear-dependent manner, the challenge of the assay is in the analysis of the large amounts of image sequence data generated. This analysis is further complicated by the dynamic behavior of selectin-coated microprobe adhesion to selectin ligands expressed on a heterogeneous cancer tissue surface. To extract the biophysical underpinnings towards the development of this prospective in vitro diagnostic, a mathematical model was devised to refine the design space of this flow assay. The model is semi-empirical and consists of a coupled set of stochastic, 4D partial differential equations that account for the transport of probes to the tissue surface as well as the dynamics of probe adsorption to a heterogeneous tissue surface. Empirical data were obtained by processing image sequences with machine-learning, template-matching algorithms to conduct image segmentation, followed by probe tracking over time using a program written in Python. The empirical data displayed a stochastic adsorption profile and showed agreement with the model equations that were solved numerically using a finite difference scheme written in C++. Altogether, this approach is able to successfully bridge the gap between the seemingly random rolling interactions observed in the flow assay and the underlying physical mechanisms.

#4278

Somatic MAPK pathway mutations are associated with high mutational burden and good survival in head and neck squamous cell carcinoma (HNSCC).

Hoi-Lam Ngan,1 Andrew Y. Fong,2 Sharon Chan,3 Shing Chun Yu,1 Laura R. H. Ip,1 Jennifer R. Grandis,4 Vivian Lui1. 1 _The Chinese University of Hong Kong, Hong Kong, Hong Kong;_ 2 _Case Western Reserve University, Cleveland, OH;_ 3 _Imperial College London, London, United Kingdom;_ 4 _University of California, San Francisco, San Francisco, CA_.

Somatic mutations of the MAPK pathway occur in ~14% of head and neck squamous cell carcinoma (HNSCC; TCGA Provisional; www.cbioportal.org). Notably, mutations found in MAPK signaling components are apparent hotspot or activating mutations, potentially driving activation of the MAPK pathway. These mutations include HRAS p.G12V/D/A/C/S & p.G13V/R, MAPK1 p.E322K/*, BRAF p.D594N & p.V600E mutations. We found that this MAPK pathway mutated subset of HNSCC tumors have a higher mutational burden than MAPK-wildtype (WT) tumors. The average rate of non-synonymous mutations in MAPK pathway mutated tumors is 265.1±42.84 mutation/tumor vs 143.5±5.83 mutation/tumor in WT tumors in the TCGA cohort (P=0.0005). MAPK pathway mutations are not associated with HPV positivity (P=0.5815). Subsequent analysis shows that this subset of MAPK pathway-mutated tumors is unexpectedly associated with TP53 wildtype status (P=0.005584), as well as CASP8 mutations [29.3%; 27/92 cases vs. a 6.5% rate (27/418 cases) in the MAPK pathway WT group; P=9.91 x10-9]. Based on the current survival data of the TCGA Provisional cohort, it appears that patients whose tumors bearing HRAS, BRAF, MAPK1, MAP2K1, and RPS6KA1 mutations have a long median survival of 7.94 years (95.3 months; P=0.0165) vs. patients without these somatic mutations. Though TP53 WT status is a well-recognized biomarker for good prognosis in HNSCC, it only predicts with a median survival of ~45.8 months in this very same cohort. Thus, our findings suggest that MAPK pathway mutations may serve as potential genomic biomarkers for good prognosis in HNSCC. Recently, both HRAS and MAPK1 hotspot mutations have been shown to serve as potential neo-antigens in cervical cancer. Further investigation is needed to determine if these somatic MAPK pathway mutations are capable of mounting an anti-cancer immunity (via hotspot neo-antigens or neo-antigens due to higher mutational burden per se) in these patients, which may contribute to their improved survival. Acknowledgements: VWYL received funding supports from the Research Grant Council, Hong Kong (General Research Fund: #1711484, #17121616; Theme-based Research: T12-401/13-R) and the Hong Kong Cancer Fund, Hong Kong.

#4279

The genomic profile investigation of metastatic triple-negative breast cancer for precision medicine achievement.

Wooyeong Jang, Jihyun Kim, Hanna Yang, Youngmee Kwon, Keun Seok Lee, Sung Hoon Sim, Sun-Young Kong, Kyounghee An, In Hae Park, Charny Park. _National Cancer Center, Goyangsi Gyeonggi-do, Republic of Korea_.

Objective: The molecular profile of primary breast cancer has been studied associated with drug responses. Metastatic triple-negative breast cancer (mTNBC) is heterogeneous disease and there is no effective therapeutic target. We investigated the genomic profiles of mTNBC to find potential drug targets.

Methods: Metastatic breast cancer samples were collected for genomic analysis (fresh tissue, n=17; FFPE, n=28). We conducted both whole-exome sequencing and RNA-seq for these samples and validated genomic variants by Sanger sequencing. We built up pipelines for somatic mutation, copy number alteration, mRNA expression, and fusion gene analysis. Precise somatic mutations were additionally filtered out for FFPE without matched normal. In addition, we investigated the differences of genomic profiles of mTNBC with those of primary breast cancer from TCGA data.

Results: Most of somatic mutation profiles of mTNBC were similar with those of primary cancer. However, there were some specific alterations that were not found in primary tumor. TP53 (>40 %) was concordantly discovered with primary TNBC, but KDM6A (>20 %) was highly recurrent than other breast datasets. We ascertained the diversity of immune cell activity from mRNA expression analysis, and additional pathways also represented the variance within our mTNBC population, which was differentiated from those of primary breast cancer. In addition, we found two novel FGFR1/2 fusion in two cases and validated it by RT-PCR and Western blot.

Conclusion: We found specific genomic profiles of mTNBC that were distinct from those of primary tumor. Novel structural variants discovered in our dataset could be potential therapeutic targets for mTNBC patients.

#4280

Improving the sensitivity of wide targeted cancer gene panels via novel genome analysis tools.

Bahram G. Kermani,1 Evans L. Roberts,2 Theresa A. Boyle,2 Anthony M. Magliocco2. 1 _Crystal Genetics, Inc., Los Altos, CA;_ 2 _Moffitt Cancer Center, Tampa, FL_.

In the recent years, wide targeted gene panels have gained popularity, due to their ability to interrogate many genes at the same time. In cancer, particularly, there is a set of ~600 genes known as Cancer Gene Census for which mutations have been causally implicated. Many of the wide cancer panels, therefore, target a subset of these genes. As targeted panels widen, the average coverage of them reduces, if the total budget of the DNA reads remains constant. Therefore, wide panels usually have a more modest coverage, as compared to narrow panels. The reduction in coverage, along with the biases that exist in targeted enrichment approaches make a challenging environment for genome assembly pipelines. We argue that, in such systems, false negatives are prone to increase to non-trivial values, primarily due to: 1) inefficiencies in the enrichment methods, especially if they are hybridization-based; 2) sporadic insufficiency of coverage; 3) biases in the analysis pipelines; 4) filtering operations that take place, in attempt to reduce false positives. As much of the losses are due to the dependency of the assays and the pipelines to the reference genome, we have approached this issue by designing a tandem set of tools: 1) A hybrid-denovo genome assembly pipeline, which has significantly less bias to the reference genome, as compared to the conventional align-to-reference methods. This pipeline can be used in lieu of or in combination with the conventional pipelines; 2) An in-silico verification (ISV) tool that provides the experts with a multifaceted visual and textual information, to gauge the evidence (or lack thereof) for the variants of interest. While not meant to necessarily be a replacement to wet validation, ISV provides the ability to identify the errors that may otherwise seep through the system. In order to show feasibility of this approach, a small set of FFPE TruSight Tumor 170 samples including cases of lung and breast cancer was used. The comparisons were done for all RefSeq exonic bases on a hereditary cancer gene subset. Our preliminary analysis using this data shows that by using our proprietary genome analysis pipeline and ISV tool, the sensitivity of the reported variants could be improved from ~80% to ~98% or higher, without impairing the specificity. If we consider intronic bases which have reasonable coverage, including those involved in splicing, the sensitivity improvements could be even larger.

#4281

A family-wide computational analysis of the catalytic domains of the Never In Mitosis A-like kinase (NEK) proteins.

Carline A. Durmus, Shaneen Singh. _CUNY Brooklyn College, Brooklyn, NY_.

Members of the Never In Mitosis A-like kinase (NEK) proteins are overexpressed in leukemia, breast, ovarian, prostate, and cervical cancers. Overexpression of particular NEK proteins is also implicated in the increased resistance of certain cancer cells to premature senescence due to various cancer treatments. Depending on the cancer cell type, there are NEK members that, when inhibited, either increase the cancer cells' susceptibility to specific drugs or increase their resistance. Structural information and model of action of NEKs is limited to only two members of the family, even though the different members play an important role in human metabolism. The goal of this research is to perform a comprehensive computational analysis of the 11 members of the human NEK protein family. To provide detailed annotations of the NEK family members, the three-dimensional structures of the catalytic domains was modeled using various approaches including template-based modeling, threading, and ab inito techniques. The information gathered from the models was integrated with information obtained from sequence analysis, biophysical characterization including surface electrostatic profiles, and predicted molecular interactions involving NEK members and other proteins/ macromolecules to establish structure-function correlations. Here we present a detailed analysis of the catalytic domains of the 11 NEK proteins and propose interaction and binding partners for each of the individual members of the family. The role of NEKs in the cell cycle, numerous medical disorders, and as potential chemotherapeutic targets underscores the importance of understanding the structure-function relationship of this family of proteins and establishing their mechanism of action, including delineating the differences among the different members of the family, and our study is a step in that direction.

#4282

Deconvolution of plasma-derived exosomes for tracking and prediction of immunotherapy across multiple tissues.

Alvin Shi,1 Gyulnara Kasumova,2 Isabel Chien,1 Jessica Cintolo-Gonzalez,2 Dennie T. Frederick,2 Roman Alpatov,2 William A. Michaud,2 Deborah Plana,2 Ryan Corcoran,2 Keith Flaherty,2 Ryan Sullivan,2 Manolis Kellis,1 Genevieve Boland2. 1 _Massachusetts Institute of Technology, Cambridge, MA;_ 2 _Massachusetts General Hospital, Boston, MA_.

There is a critical need for robust and minimally invasive biomarkers for predicting and monitoring tumor progression and response to treatment. Transcriptomes of plasma-derived exosomes (PDEs) are suitable candidates to fulfill such a role, since they contain a subtranscriptome of their cell of origin, and, since nearly all cell types secrete exosomes, this allows for the potential monitoring of multiple cell types concurrently. However, a major issue preventing the widespread adoption of plasma-derived exosome as biomarkers is that observed plasma exosomes actually result from a mixture of exosomes from multiple cell types. This confounds detailed dissection and interpretation of putative plasma-derived exosomal biomarkers. To address this issue, we develop a two-component Bayesian deconvolution model to infer the tumor-derived and immune-derived exosomal contribution to the observed mixed plasma-derived exosome signal. Our model leverages transcriptomic information from 3 different sources: (1) paired patient bulk and plasma-derived exosomes, (2) paired cell-line tumor and cell-line tumor-derived exosomes, and (3) healthy control plasma-derived exosomes to learn gene-by-gene mixing fractions between tumor and immune components and the mapping from tumor to tumor-derived exosomes transcriptomic profiles. Using this information, we are able to further infer the patient-specific tumor-derived and immune-derived exosomal transcriptomic profiles for each gene. The outputs from our model enable us to derive tumor-specific and immune-specific exosomal biomarkers. We first show that our model is performant in an extensive set of in silico simulations. Next, we applied our model to transcriptomes collected prior to and during aPD1 immunotherapy treatment from a pilot cohort of N=44 patients (N=29 responders, N=15 nonresponders) with metastatic melanoma. Analysis of our deconvolved profiles yields novel and biologically informative immune-derived and tumor-derived exosomal biomarkers that predict immunotherapy success. Moreover, time-series analysis of the deconvolved profiles show that we are able to identify significantly different tumor and immune related genes whose time dynamics differ significantly between responders and nonresponders, suggesting that plasma-derived exosomes can enable longitudinal tracking of both immune and tumor components of immunotherapy response. Finally, we show that a more sophisticated extension of our deconvolution model is able to provide an estimate of global tumor fraction for each patient, potentially allowing us to infer tumor burden through plasma-derived exosomal transcriptomic signatures. Overall, our plasma-derived exosomal deconvolution model paves the way for more widespread usage of plasma-derived exosomes as a clinical monitoring prediction and monitoring tool.

#4283

**A structure network approach to predict the dynamics and structural stability effects of germline** PTEN **mutations associated with cancer versus autism phenotypes.**

Iris N. Smith, Stetson Thacker, Charis Eng. _Cleveland Clinic, Lerner Research Institute, Cleveland, OH_.

Individuals with germline mutations in the tumor suppressor gene phosphatase and tensin homolog (PTEN), irrespective of clinical presentation, are referred to as PTEN hamartoma tumor syndrome (PHTS). PHTS confers a high risk of breast, thyroid and other cancers. Yet, PTEN mutations are found in up to 20% of individuals with autism spectrum disorder (ASD) and macrocephaly. What remains unclear is why mutations in one gene can lead to seemingly disparate phenotypes. Thus, we sought to identify differences in cancer- vs. ASD-associated PTEN mutations by investigating putative structural effects. We utilized a computational theoretical approach combining protein structure network (PSN), normal mode analysis (NMA), and elastic network models (ENMs) to interrogate 6 PTEN mutations from patients with PHTS-associated cancer, 6 mutations from those with ASD only, 4 mutations shared across both phenotypes, and 1 mutation with both ASD and cancer. Combined with in silico prediction methods we calculated structural stability changes induced by each mutation. All 6 cancer-associated mutations showed decreases in structure stability and increased dynamics across domain interface. In contrast, 5 of 6 ASD-associated mutations showed only localized destabilization. We coupled ENM-NMA with PSN and found PTEN R130G (cancer only) and R173C (shared in patients with both ASD and cancer), play a role in inter-residue signal propagation, indicating both residue positions are crucial to structural stability. Our data suggest that ASD-associated mutations affect local structural dynamics, whereas cancer-associated mutations mediate long-range perturbations significantly altering structural stability and salient communication pathways. These approaches lend insight into altered structural effects of germline PTEN mutations associated with PTEN-ASD and PTEN-cancer, potentially aiding in identification of specific mutations that contribute to each phenotype. Moreover, this work may illuminate the shared and separate molecular features that contribute to autism or cancer – providing a deeper understanding of genotype-phenotype relationships for germline PTEN mutations. The ability to accurately predict PTEN-ASD may spare patients the need to undergo high-risk cancer surveillance that is now performed in all with PHTS.

#4285

Genomics-driven clustering of disease-related biomarkers identifies therapeutic options in myelodysplastic syndromes (MDS).

Leylah M. Drusbosky,1 Kimberly E. Hawkins,1 Amy Meacham,1 Elizabeth Wise,1 Neeraj Kumar Singh,2 Chandan Kumar,2 Sumanth M. Vasista,2 Rakhi P. Suseela,2 Taher Abbasi,3 Shireen Vali,3 Kaoru Tohyama,4 Maher Albitar,5 Peter P. Sayeski,1 Christopher R. Cogle1. 1 _University of Florida, Gainesville, FL;_ 2 _Cellworks Research India Pvt. Ltd, Bangalore, India;_ 3 _Cellworks Group Inc, San Jose, CA;_ 4 _Kawasaki Medical School, Kurashiki, Japan;_ 5 _Neogenomics, Aliso Viejo, CA_.

Hypomethylating agents (HMA) and lenalidomide (LEN) are approved and used in the treatment of patients (pts) with MDS, though these drugs fail in most pts. No method exists to predict drug response beyond associating single actionable mutations with a single drug's response. We hypothesized that MDS pts can be clustered by similarities in genomic/molecular profiles, & that each cluster may be assigned combos of FDA-approved drugs to target their unique biomarker profile. Bone marrow cells from 88 MDS pts & the MDS-L cell line were analyzed by cytogenetics & for mutations in 14 myeloid genes using NGS. 31 pts had sufficient data for analysis. 20 profiles had similar aberrations & were grouped. Genomic data from pts and MDS-L were entered into a computational biology modeling (CBM) software, which generates a disease-specific protein network map using PubMed to create digital models and identify characteristic biomarkers unique to each pt. An algorithm was created to cluster the models based on overlapping disease-specific biomarkers. Digital drug simulations (DDS) were conducted both on MDS-L & pt simulation models by quantitatively measuring drug effect on a cell growth score (CGS), a composite of cell proliferation, viability & apoptosis. DDS identified drugs by assessing their impact on disease-specific biomarkers and calculated CGSs. Predictions were validated using MTT. 14/31 MDS pt profiles, including the MDS-L cell line, clustered into 4 groups based on biomarker characteristics.MDS-L cells harbor NRAS (G12A) mutation,-7,-12,+1,+8,+19,+20 and +21. Genes associated with increased copy number (CN) include AURKA, IGFR, PAR5, MTOR, IL6, JAK3, MDM4, MYC, MCL1, COX2, PDE4A, and RCE1; genes associated with decreased CN include DUSP1, RASA1, NR3C1, IRF1, ETV6, and SHH. CBM identified active biomarkers in MDS-L cells (90RSK, MAPK7, AKT and BTK), validated by western blot. DDS predicted nelfinavir+celecoxib to be effective in MDS-L. MDS-L cells were treated with nelfinavir, celecoxib, and nelfinavir+celecoxib with increasing doses. Nelfinavir & celecoxib reduced MDS-L viability in a dose-dependent manner, while nelfinavir+celecoxib showed additive reduction of MDS-L viability. DDS was performed on each pt to predict response to HMA and LEN. Two of the clusters (n=2/cluster) were predicted to be non-responders to any SOC therapy. A third cluster (n=3) was predicted to respond to HMA, but not LEN, and the fourth cluster (n=6) showed varying or no response to either HMA/LEN. These results use a novel concept of using genomics & CBM to cluster profiles with overlapping disease-relevant biomarkers & similar drug response predictions. CBM can identify pt populations who may benefit from certain therapeutic regimens, improving response rates, & give insights into the mechanisms by which each drug impacts the MDS-specific biomarkers

#4286

Tissue-specific preference for ATM or ATR pathway activation determines DNA damage outcome and provides insights in cancer predisposition.

Marijn T. Van Jaarsveld,1 Difan Deng,1 Erik A. Wiemer,2 Zhike Zi1. 1 _Max Planck Inst. for Molecular Genetics, Berlin, Germany;_ 2 _Erasmus University Medical Center, Rotterdam, Netherlands_.

The DNA damage response (DDR) is an elaborate signaling network that protects against mutations and genomic instability. Despite its importance in preventing carcinogenesis, certain mutations in the DDR (e.g. BRCA1/2) predispose for specific cancers only. An explanation for this could be that the DDR is regulated differently across tissues; however, systematic analyses of the response to DNA damage in healthy human cells have not been carried out.

We analyzed proteomic changes in response to single- and double strand DNA damage in non-immortalized human primary cells and related them to cell outcome. Primary breast and lung cells were treated with cisplatin (single-strand damage) or doxorubicin (double-strand breaks) at doses that caused a similar amount of DNA damage. Cell outcomes that were studied were apoptosis and cell cycle progression. Protein dynamics of 366 proteins were assessed using protein arrays and quantitative western blots. Inhibitors and an ODE-based mathematical model were used to determine the network topology and link it to outcome.

Treatment of primary breast and lung epithelial cells with an equal amount of damage resulted in different viabilities. Primary mammary cells showed higher levels of apoptosis after single-strand damage, primary airway cells were more sensitive to double-strand breaks. The difference in outcome was reflected in distinct protein dynamics, including DNA repair pathways. Surprisingly, these differences originated at the earliest level of DNA damage detection by ATM and ATR. We demonstrated that differential activation of ATM and ATR pathways in breast and lung cells affects p53 and p21 dynamics and leads to distinct outcomes. We were able to identify key network nodes, providing new insights into the regulation of p53 and p21 and their relation to cell outcome. p53 and p21 dynamics are also linked to regulation of the cell cycle. In line with this, we found that cell cycle progression under normal conditions and after DNA damage is different. Importantly, we show that the double-strand break pathway plays a larger role in cell cycle control in breast cells than in lung cells. Since cell cycle checkpoints are a safeguard against genomic instability, differential regulation may affect cancer predisposition in a cell-type specific manner. Our work may provide an explanation for the fact that certain mutations in the DSB-repair pathway predispose for breast cancer, but not for lung cancer.

In summary, we show that primary breast and lung cells activate distinct programs in response to equal doses of DNA damage, resulting in different p53 and p21 dynamics and, consequently, diverse outcomes. In addition, this work sheds light on the tissue-specific cancer predisposition of mutated DNA repair genes and may be the basis for further studies.

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

### Checkpoints and Cell Cycle Progression

#4287

Whole-genome CRISPR/Cas9 screen of the CHK1 inhibitor prexasertib implicates FAM122A loss as a potential resistance mechanism.

Peter Deraska, Hunter Reavis, Shelby Labe, Alan D'Andrea, David Kozono. _Dana Farber Cancer Inst., Boston, MA_.

Introduction: Phase I clinical trials of the CHK1 inhibitor prexasertib have been completed in non-small cell lung cancer (NSCLC) and other cancers and several Phase I-II trials are currently recruiting. By inhibiting the G2/M and S-phase checkpoints, prexasertib has been shown to cause increased replication stress, DNA damage and premature entry into mitosis, resulting in mitotic catastrophe and cell death. While the inhibitor is showing therapeutic promise, acquired resistance is of concern. Little is known about how cancer cells may become resistant to CHK1 inhibitors. Understanding potential resistance mechanisms may help predict patient responses, design CHK1 therapy combinations and implement means of re-sensitization.

Materials and Methods: NSCLC cell lines A549 and NCI-H460 stably expressing the Cas9 endonuclease were obtained from the Genetic Perturbation Platform at the Broad Institute. Cells were infected with the Brunello v2 lentiviral sgRNA library and grown for 15 days in prexasertib-treated media. Cells were collected pre- and post-drug selection, genomic DNA was extracted, and sgRNA sequences were PCR amplified and Illumina sequenced. Top hits among knockouts that conferred drug resistance were identified by the STARS gene-ranking algorithm. Hit validation was performed with sgRNAs and pooled siRNAs (Qiagen). Prexasertib IC50s were assessed by colony formation and CellTiter-Glo cell viability assays. Cell cycle analysis was performed by propidium iodide flow cytometry. PP2A activity was measured using Ser/Thr phosphatase assay kits (Upstate Biotechnology). Dinaciclib and LB-100 were obtained from Selleck and Apexbio, respectively.

Results: CRISPR knockouts of several individual genes were found to promote resistance of both cell lines to prexasertib with false discovery rates < 0.01%. One top hit codes for a little-known but highly conserved protein FAM122A, which has been shown to inhibit PP2A, which dephosphorylates the Chk1 target protein CDC25C. SiRNA knockdown or sgRNA knockout of FAM122A conferred prexasertib resistance. Similarly, prexasertib-mediated G2/M accumulation was inhibited by FAM122A knockdown. FAM122A silencing increased PP2A activity. FAM122A knockdown mediated resistance was reversed with PP2A B subunit α siRNA knockdown or the PP2A inhibitor LB-100. Dinaciclib, an inhibitor of the CDC25C kinase CDK2, increased prexasertib resistance.

Conclusion: Although CHK1 inhibitors like prexasertib show promise, one mechanism of resistance may be through other cell-cycle regulating compensatory pathways. Identification of FAM122A loss as a potential mechanism for resistance suggests a predictive biomarker and means for re-sensitization of cancer cells to CHK1 inhibitors.

#4288

The cell cycle gene regulatory DREAM complex is disrupted by oncogenic B-Myb.

Audra N. Iness, Varsha Ananthapadmanabhan, Fatmata Sesay, Mikhail Dozmorov, Larisa Litovchick. _Virginia Commonwealth University, Richmond, VA_.

The oncogene MYBL2 (encoding B-Myb) is a poor prognostic biomarker in many cancers. B-Myb interacts with the MuvB core of five proteins (LIN9, LIN37, LIN52, LIN53/RBBP4, and LIN54) to form the MMB (Myb-MuvB) complex and promotes expression of late cell cycle genes necessary for progression through mitosis. Both MYBL2 amplification and over-expression are associated with deregulation of the cell cycle and increased cell proliferation. Alternatively, by interacting with E2F4-DP1 and p130/p107, the MuvB core becomes part of the DREAM complex (DP, RB-like, E2F, and MuvB). The DREAM complex opposes MMB by globally repressing cell cycle genes in G0/G1, maintaining the cell in a quiescent state. However, the specific mechanism by which B-Myb alters the cell cycle is not well understood. Herein, we show that B-Myb disrupts the DREAM complex by sequestration and stabilization of LIN52. Analysis of The Cancer Genome Atlas data revealed significant upregulation of DREAM and MMB target genes in breast and ovarian cancer with MYBL2 gain. Given that most of the DREAM target genes are not directly regulated by B-Myb, we investigated the effects of B-Myb on DREAM formation. We found that depletion of B-Myb results in increased DREAM formation in cancer cell lines, while its overexpression inhibits DREAM formation in non-transformed cells. Since the MuvB core subunit LIN52 is essential for assembly of both the DREAM and MMB complexes, we tested whether B-Myb disrupts DREAM by sequestering LIN52. Overexpression of LIN52 did not increase either DREAM or MMB formation, but instead increased the turnover rate of the endogenous LIN52 protein. Interestingly, co-expression of B-Myb increased the expression of both endogenous and overexpressed LIN52 while knockdown of B-Myb had an opposite effect. We found that regulation of LIN52 occurs at the protein level, and that activity of DYRK1A kinase, the enzyme that triggers DREAM complex formation by phosphorylating LIN52, is required for this regulation. These findings are the first to implicate B-Myb in the disassembly of the DREAM complex and offer further mechanistic insights for cancers with MYBL2 amplification. We conclude that B-Myb's oncogenic effects are not only secondary to increased mitotic gene expression by the MMB complex, but also broad disruption of cell cycle gene regulatory programs through compromised DREAM formation.

#4289

Involvement of the miR-6883 family and the circadian rhythm gene PER1 in the DNA damage response (DDR) and chemosensitivity.

Amriti R. Lulla, Avital Lev, Michael Slifker, David T. Dicker, Wafik S. El-Deiry. _Fox Chase Cancer Ctr., Philadelphia, PA_.

PER1 is a circadian clock protein known to play a role in the cell cycle by directly regulating levels of Wee1 and indirectly influencing levels of Cdc2 and Cyclin B1. PER1 interaction with CHK2 promotes apoptosis in response to DNA damaging agents and loss of PER1 promotes chemoresistance. Expression of PER1 is found to be lost in most CRC tumors and restoring it sensitizes CRC cell lines to DNA damaging chemotherapeutic agents. However, the regulation of PER1 and its mechanism of repression in CRC remains unknown. We recently discovered and characterized miR-6883-5p, which is an intronic miRNA in the PER1 gene. Since PER1 is lost in most CRC it is suggested that the expression of miR-6883-5p is also lost. We showed that restoring the expression of miR-6883-5p via mimics induces G1-arrest and subsequent cell death in CRC cell lines. Similar to the overexpression of PER1, expression of miR-6883-5p with mimics increased the sensitivity of CRC cell lines to chemotherapeutic agents such as irinotecan and 5-FU. miR-6883-5p is a direct negative regulator of CDK4 and CDK6 and the downstream G1-S transition via RB. Our ongoing studies are characterizing molecular targets of miR-6883-5p and its role in the DNA damage response (DDR). FOXM1 is a downstream effector of CDK4 that plays a role in the DDR. Downregulation of FOXM1 sensitizes cells to DNA damage and restores chemosensitivity. We are further evaluating the direct effects of miR-6883-5p on FOXM1. FOXM1 is a member of the forkhead box (FOX) transcription factor family, expressed exclusively in proliferating cells. Its upregulation in different tumor types, including CRC promotes cell proliferation, EMT and chemoresistance. Interestingly, FOXM1 is also a repressor of the clock gene CRY2. Here, we hypothesize that miR-6883-5p mediated targeting of FOXM1 is crucial for the DDR, and, further, like CRY2, FOXM1 might have a role in regulating PER1 and thus miR-6883-5p endogenous levels. We report that miR-6883-5p levels are increased following exposure of tumor cells to chemotherapeutic agents that cause DNA damage. We are further elucidating the role of FOXM1 in the induction of miR-6883-5p under the DDR, and propose that induction of miR-6883-5p may provide a novel mechanism for tumor growth arrest in chemotherapy-exposed cells.

#4290

Mad1 promotes tumor progression through destabilization of p53.

Jun Wan, Beth A. Weaver. _University of Wisconsin-Madison, Madison, WI_.

Mitotic arrest deficient 1 (Mad1) plays a well-characterized role in the mitotic checkpoint. However, interphase roles of Mad1 that do not impact mitotic checkpoint function are still largely uncharacterized. We have found that upregulation of Mad1, which is common in human breast cancer, results in decreased protein stability of the tumor suppressor p53 in multiple cell types. To gain mechanistic insight into this process, we first determined whether increasing expression of Mad1 alters its localization. Upregulated Mad1 localizes to puncta within interphase nuclei in both breast cancer tissue and cultured cells. We found that upregulated Mad1 localizes to ProMyelocytic Leukemia Nuclear Bodies (PML NBs), which have been implicated in the stabilization of p53. Immunoprecipitation results indicate that Mad1 and PML interact and that the C-terminal domain (CTD) of Mad1 and the N-terminal domain of PML are required for this interaction. Within the CTD of Mad1, the SUMO-Interacting Motif (SIM) is critical for the localization of Mad1 to PML NBs. MDM2 is an E3 ubiquitin ligase that targets p53 for degradation. In response to DNA damage, PML sequesters Mdm2 to the nucleolus, which stabilizes p53. In cells with elevated levels of Mad1, Mad1 interrupts the interaction between PML and MDM2. Mad1, rather than MDM2, is sequestered to the nucleolus after DNA damage, and Mad1 displaces MDM2 from PML. The displaced MDM2 is no longer sequestered in the nucleolus, freeing it to ubiquitinate p53, resulting in p53 destabilization. Upregulation of Mad1 promotes the growth of orthotopic mammary tumors. Mammary tumors with upregulated Mad1 show decreased levels of p53 and its downstream effector p21. Our results show that, in addition to causing a low rate of chromosome missegregation, Mad1 upregulation has an unexpected interphase role in promoting tumor formation and progression by destabilizing p53. Thus, Mad1 upregulation promotes tumors in through at least two distinct pathways.

#4291

Oncogenic ALK regulates cell cycle progression via CDK1: Implications for therapy.

Kathleen A. Scorsone, Sonal Gahlawat, Siddhartha Tyagi, Mayra C. Orellana, Thomas F. Westbrook, Ronald J. Bernardi. _Baylor College of Medicine, Houston, TX_.

Pharmacologic inhibition of oncogenic ALK can yield clinical responses in a variety of cancer types, including non-small cell lung cancer, anaplastic large-cell lymphoma, and neuroblastoma; however, de novo and acquired resistance remain major problems. As is seen with inhibition of other oncogenic receptor tyrosine kinases, ALK inhibition primarily induces G1 cell cycle arrest. However, we and others have also observed a role for receptor tyrosine kinases in regulating G2/M progression. For instance, oncogenic HER2 has been shown to directly phosphorylate tyrosine 15 of CDK1, the mitosis promoting factor. This tyrosine is also the well-known substrate of WEE1 and serves to inactivate CDK1 kinase activity as a critical component of the G2-M checkpoint. In the current study, we aimed to determine if oncogenic ALK functions in a similar capacity. We used recombinant proteins to demonstrate that ALK is also capable of directly phosphorylating tyrosine 15 of CDK1. Further, combined inhibition of ALK and WEE1 with crizotinib and AZD-1775 leads to greater reduction in pY15-CDK1 than either agent alone in ALK-amplified neuroblastoma cells. These findings suggest a model where oncogenic ALK and WEE1 provide redundant regulation of the G2-M checkpoint, which may be critical in cancer cells that have lost some of the usual complement of checkpoint controls, such as p53. Consequently, either ALK or WEE1 activity would be sufficient to maintain appropriate cell cycle regulation, while dual blockade would allow mitosis to proceed unchecked. In agreement with these predictions, we performed live cell imaging and observed that combined inhibition of ALK and WEE1 leads to aberrant mitotic progression and mitotic catastrophe. Further, this combination yields greater anti-proliferative activity than either agent alone in ALK-driven models. Thus, this therapeutic strategy holds great promise for ALK-driven cancers.

#4292

Negative regulation of oncogenes by a novel tumor suppressor microRNA in prostate cancer.

Nadeem Bhat,1 Altaf A. Dar,2 Sharanjot Saini,3 Varahram Shahryari,3 Priyanka Kulkarni,3 Pritha Dasgupta,3 Soichiro Yamamura,3 Yuichiro Tanaka,3 Taku Kato,3 Yutaka Hashimoto,3 Marisa Shiina,3 Z. Laura Tabatabai,3 Guoren Deng,3 Rajvir Dahiya,3 Shahana Majid3. 1 _UCSF & VA Medical Center, San Francisco, CA; _2 _CPMC, Research Institute, San Francisco, CA;_ 3 _VA Medical Center, San Francisco, CA_.

Background: MicroRNAs are small noncoding RNAs that regulate the expression of >60% of all human genes, either inhibiting target mRNA translation or inducing its degradation. MicroRNAs act as tumor suppressors or oncogenes in various cancers. The main objective of this study was to investigate the role of microRNA-588 (miR-588) in prostate cancer (PC).

Methods: The methods employed in this study include quantitative-real time PCR; western blot; fluorescence-activated cell sorting assays for cell cycle distribution and apoptosis; assays for cell viability, migration and invasiveness of prostate cancer cells. Luciferase reporter assays and in-vivo study in nude mice was also performed.

Results: The expression of miR-588 was significantly suppressed or silenced in PC tissue samples and cell lines when compared with normal tissues and a non-malignant cell line. Similar results were observed by analyzing the publicly available TCGA data sets for prostate adenocarcinoma. Functionally ectopic expression of miR-588 induced G0/G1 cell cycle arrest and apoptosis and suppressed cell proliferation. miR-588 exerted these functional effects by directly targeting the oncogenic Cyclin A2 that is involved in cancer cell cycle and proliferation. In silico algorithm showed a complimentary binding sequence in the 3'UTR of Cyclin A2 for miR-588. Over-expression of miR-588 significantly suppressed the luciferase activity of reporter plasmid containing the wild type 3'UTR sequences of cyclin A2 complementary to miR-588, which was abolished by mutations in these 3'UTR regions. miR-588 overexpression also significantly reduced the expression of cyclin A2 at both mRNA and protein levels. A significant decrease in the expression of various cell cycle pathway genes such as CycE1, MCM2, MCM4, CDC7 and CDT1 was also observed. These genes are involved in promoting cell cycle and proliferation and are overexpressed in prostate cancer. In vivo intracardiac implantation of PC3-MLuc-C6 prostate cancer cells constitutively expressing miR-588 showed a significant inhibition of the metastatic dissemination of these cells compared to the control miR expressing cells.

Conclusion: This is the first study demonstrating that miR-588 is significantly silenced and acts as a tumor suppressor in prostate cancer. Reconstitution of silenced miR-588 may contribute to novel therapeutic approaches for regulating prostate cancer.

#4293

Glycogen synthase kinase 3 b maintains mitotic arrest by regulating mitotic checkpoint complex levels.

Maisha S. Rashid, William R. Taylor. _University of Toledo, Toledo, OH_.

The cell cycle is regulated by checkpoints that ensure genomic integrity and proper cell division. We are investigating the role of a multifunctional ser/thr kinase, glycogen synthase kinase 3 (GSK3), in the regulation of the mitotic checkpoint. Our data show that spindle toxin-induced mitotic arrest is relieved in multiple cancer cell lines by the GSK3 inhibitors, SB 415286 (SB), RO-81220 (RO) and lithium chloride. Additionally, the mitotic index is reduced in CRISPR-generated GSK3β knockout human cells and GSK3β knockout mouse embryo fibroblasts compared to wild type cells in the presence of taxol. Mitotic arrest is dependent on the mitotic checkpoint complex (MCC), composed of Mad2, BubR1, Cdc20 and Bub1. GSK3 appears to target the MCC to regulate mitotic arrest. For example, Mad2, BubR1 and Bub1 localization at kinetochores and MCC assembly were both reduced by co-treatment of GSK3 inhibitors and spindle toxin compared to spindle toxin alone. These data imply that GSK3β plays a role in maintaining the mitotic checkpoint. The strength of the checkpoint was determined to act like a rheostat rather than a toggle-switch, as cells undergo mitotic slippage after prolonged arrest in spindle toxins. Our data indicates that GSK3 regulates the strength of the checkpoint, as illustrated by time lapse-imaging. GSK3-inhibition induced exit was observed to be dependent on how long the cells were arrested in mitosis prior to GSK3-inhibitor addition. GSK3 inhibition in taxol also decreases the amount of KnL1, part of the KMN network forming the kinetochore, at the centromeres. This indicates a regulatory role of GSK3 in kinetochore assembly and function. The Wnt-signaling pathway and the PI3K/Akt signaling pathway are known upstream regulators of GSK3β, negatively regulating GSK3β activity. Our data shows that inhibiting Wnt-signaling and PI3K/Akt-signaling in the presence of taxol, induces a longer mitotic arrest compared to taxol alone. This suggests that GSK3β is regulated upstream by the Wnt- and the PI3K/Akt signaling archs to control mitosis. Finally, our data implicated that de-acetylase sirtuin2 is a downstream target of GSK3 in its mitotic regulatory role. Our observations indicate a novel regulator of the checkpoint and novel insight in connecting growth-signaling pathways with mitosis.

#4294

SUMOylation promotes nuclear import and stabilization of polo-like kinase 1 to support its mitotic function.

Donghua Wen, Jianguo Wu, Lei Wang, Zheng Fu. _Virginia Commonwealth Univ., Richmond, VA_.

As a pivotal mitotic regulator, polo-like kinase 1 (PLK1) is under highly coordinated and multi-layered regulation. However, the regulatory pathways that control PLK1's activity and function have just begun to be elucidated. PLK1 has recently been shown to be functionally modulated by post-translational modifications (PTMs), including phosphorylation and ubiquitination. Herein, we report for the first time that a novel PTM, SUMOylation, plays an essential role in regulating PLK1's mitotic function. We found that Ubc9 was recruited to PLK1, upon initial phosphorylation and activation by CDK1/cyclin B. By in vivo and in vitro SUMOylation assays, PLK1 was identified as a physiologically relevant SUMO-targeted protein, preferentially modified by SUMO-1. We further showed that K492 on PLK1 was the lysine residue essential for SUMOylation. SUMOylation not only led to PLK1's nuclear import, but also significantly increased its protein stability by preventing the recruitment of APC/Ccdh1, the ubiquitin E3-ligase of PLK1. Resembling Ubc9 deficiency, cells expressing PLK1-K492R mutant exhibited mitotic aberrations characterized by prolonged mitotic progression, and misaligned and/or mis-segregated chromosomes, which could be rescued by reintroducing SUMOylation modification to PLK1. Collectively, our findings suggest that SUMOylation is another important regulatory mechanism governing PLK1's mitotic function to ensure normal mitotic progression and genomic integrity.

#4295

ASF1B promotes cell proliferation and regulates cell cycle in human benign prostate hyperplasia (BPH).

Nisha Dahiya Rani, Dr. Murali Ankem, Dr. Chendil Damodaran. _University of Louisville, Louisville, KY_.

Background

Benign Prostate Hyperplasia (BPH) is enlargement of the prostate, which occurs in 50% men above 50 years of age. Alpha-adrenergic blockers and 5-alpha-reductase inhibitors are the standard medical therapy for BPH. However, these treatments are not only partially effective but also cause significant toxicity in patients. Understanding the molecular pathogenesis of BPH may reveal novel preventive or therapeutic targets for BPH.

Methods

Normal Human Prostate Epithelial Cells (HPrEpC), RWPE-1 and Benign Prostate Hyperplasia (BPH) were utilized for global sequencing and molecular studies. Western blot analysis was performed to determine the expression of G0/G1 and S-phase and M phase regulators of the cell cycle in BPH and RWPE-1 lysates. Cell cycle and western blot analysis was performed at successive time-points post starvation to understand the dynamics of cell cycle progression in both the cell lines. Knock out experiments were conducted to understand the function ASF1B in normal and BPH cells.

Results

RNA sequencing analysis revealed that the level of ASF1b (Anti-Silencing Function 1B Histone Chaperone) was over 80 times upregulated in BPH cells as compared to healthy prostate epithelial cells. Induction of ASF1B is specific to BPH as confirmed at protein levels. Induction of ASF1B expression correlated in a time-dependent manner with the increased expression of G1 (cdk4, 6 and cyclin D), G1-S (cdk2) and S-phase (cdk2 and cyclin-A) regulators at both transcription and translation levels. The G1-S phase transcriptional enhancers such as E2-F1 were also upregulated with successive time-points. The cell cycle analysis at sequential time-points revealed that the BPH cells entry to S phase was achieved earlier and by higher fraction of cells than the healthy prostate epithelial cells. Knocking down ASF1B expression by siRNA inhibited the proliferation of BPH cells by 30%. The findings so far suggest that ASF1B might be related to hyper-proliferation in BPH cells. The results indicate that ASF1B might be a novel therapeutic target for preventing hyper-proliferation in BPH. Studies are ongoing to validate the expression of ASF1B in clinical samples along with screening of small molecules targeting ASF1B expression specifically. Current study may lead to identification of a novel preventive and/or therapeutic target and related treatment for BPH.

Conclusion

Our studies suggest that ASF1b is a G1 and S phase regulator which are critical phases for hyper-proliferative BPH cells.

#4296

Cofilin-1 is associated with replicative origins licensing through regulation of MCM2-7 helicase.

Yi-Jang Lee, Chun-Yuan Chang. _National Yang-Ming Univ., Taipei, Taiwan_.

G1 to S phase transition is strictly regulated to avoid uncontrolled growth in mammalian cells. Although DNA replication initiates in S phase, licensing of DNA replicative origins substantially begins from late M to G1 phase. Interference of replicative origins licensing will lead to genomic instability and cell death. In addition, integral actin organization is required for G1 phase to S phase transition. Whether actin architectures will influence the licensing of DNA replicative origins is unclear. Previously, we found that over-expression of cofilin-1, a member of actin depolymerizing factor (ADF)/cofilin family could destabilize actin cytoskeleton and cause G1 phase arrest. The microarray assay further showed that minichromosome maintenance complex 2-7 (MCM2-7) helicase involved in the establishment of licensing was regulated by cofilin-1. In this study, we aim to investigate whether cofilin-1 would affect the expression of MCM2-7 complexes and the initiation of replicative origins licensing. First, we exploited a double thymidine blockage assay to demonstrate that the cofilin-1 expression was down-regulated during G1 to S phase transition. Use of H1299/tet-on-cofilin-1 cells, we also found that doxycycline induced exogenous cofilin-1 could inhibit the expression of MCM2-7 complex in both dose and time-dependent manners. Chromatin fractionation for analyzing the replicative origin licensing was performed to confirm these observations. DNA fiber analysis for determining the interorigin distance (IOD) was applied to investigate the IOD after the overexpression of cofilin-1 in H1299/tet-on-cofilin-1 cells. Taken together, current results revealed a novel finding that cofilin-1 could regulate MCM2-7 complex for mediating the replicative origin licensing and cell cycle progression. Since MCM2-7 complex has been regarded a prognostic marker of several human cancers, this study may contribute to designing of new therapeutic strategy for cancer treatment.

#4298

Targeting the DNA damage checkpoint kinase Chk1 induces multiple pathways of cytotoxicity.

Nicholas Warren, Jennifer Ditano, Alan Eastman. _Dartmouth College, Lebanon, NH_.

DNA damaging agents are standard therapies for many cancers, but result in limited long-term clinical benefit. DNA damage checkpoints arrest DNA synthesis and provide time for repair and recovery. Using DNA checkpoint inhibitors to sensitize cancer cells to traditional chemotherapeutics is an emerging strategy. However, clinical trials involving Checkpoint Kinase 1 inhibitors (Chk1i) plus DNA-damaging chemotherapies have shown little clinical benefit. This may be due to sub-optimal scheduling of combinations. We have published that delaying Chk1i until 18 h after gemcitabine confers the greatest sensitization of cancer cells. We have now characterized the mechanisms by which the Chk1i, MK-8776, sensitizes human cancer cells to gemcitabine and SN38. SN38, the active metabolite of irinotecan, traps topoisomerase I on DNA resulting in S phase arrest. Chk1i re-activates replication and drives cells into mitosis before the damage from SN38 is repaired. Co-incubation with a CDK1/2 inhibitor surprisingly failed to prevent S phase progression, but did block mitotic entry and prevented further DNA damage. Conversely, the replication-associated kinase, CDC7, is essential for Chk1i-mediated S phase abrogation. CDC7 is presumably required to activate dormant origins of replication. Gemcitabine inhibits ribonucleotide reductase, thus preventing production of deoxyribonucleotides. Addition of Chk1i 18 h following gemcitabine causes aberrant activation of DNA helicases, but without deoxyribonucleotides, this results in excessive single-stranded DNA. Eventually, the level of single-stranded DNA exceeds cellular stores of the protective protein, RPA, which leads to nucleolytic cleavage and double-strand DNA breaks. Chk1i-mediated helicase activation is again dependent on CDC7, but insensitive to a CDK1/2 inhibitor. Concurrent incubation with gemcitabine plus MK8776 does not activate DNA helicases. These data provide a rationale for a delayed administration of Chk1i when patients are receiving gemcitabine. These investigations have detailed how Chk1i elicits distinct mechanisms of cytotoxicity depending on the context of DNA damage. Understanding of these mechanisms may help identify biomarkers to predict patient response. Furthermore, the clinical development of several Chk1i have been terminated due to unfavorable toxicity, but the cause of toxicity remains unknown. The work described here may help the clinical development by determining whether the observed toxicities are due to on- or off-targets.

#4299

Mutual influence of ROS, pH and CLIC1 membrane protein in the regulation of G1/S phase progression in human glioblastoma stem cells.

Ivan Verduci,1 Marta Peretti,1 Federica Maddalena Raciti,1 Valentina Carlini,1 Alessandra Patarozzi,2 Sara Barozzi,3 Massimiliano Garrè,3 Sarah Sertic,1 Alex Costa,1 Antonio Daga,4 Federica Barbieri,2 Tullio Florio,2 Michele Mazzanti1. 1 _Univ. of Milano, Milano, Italy;_ 2 _Univ. of Genova, Genova, Italy;_ 3 _Fondazione Istituto FIRC, Milano, Italy;_ 4 _Ospedale San Martino, Genova, Italy_.

Glioblastoma (GB) is the most lethal, aggressive and diffuse brain tumor. The main challenge of successful treatment is targeting the cancer stem cell (CSC) sub-population responsible for tumor origin, progression and recurrence. Chloride Intracellular Channel 1 (CLIC1), highly expressed in CSCs, is constitutively present in the plasma membrane where it is associated with chloride ion permeability. In vitro, CLIC1 inhibition leads to a significant arrest of GB CSCs in G1 phase of the cell cycle. Furthermore, CLIC1 knockdown impairs tumor growth in vivo. Here we demonstrate that CLIC1 membrane localization and function is specific for GB CSCs. Mesenchymal stem cells (MSCs) do not show CLIC1-associated chloride permeability and inhibition of CLIC1 protein function has no influence on MSC cell cycle progression. Investigation of the basic functions of GB CSCs reveals a constitutive state of oxidative stress and cytoplasmic alkalinization compared with MSCs. We now report that these three elements are temporally linked during CSC G1/S transition. Impeding CLIC1-mediated chloride current prevents both intracellular ROS accumulation and pH changes. CLIC1 membrane functional impairment results in GB CSCs resetting from an allostatic tumorigenic condition to a homeostatic steady state. In contrast, inhibiting NADPH oxidase and the NHE1 proton pump results in cell death of both GB CSCs and MSCs. Our results show that CLIC1 membrane protein is crucial and specific for GB CSC proliferation, and is a promising pharmacological target for successful brain tumor therapies.

This work was supported by grant n.16713, IG 2015 to MM Italian Association for Cancer Research (AIRC).

#4300

Cell cycle pathway gene regulation in glioblastoma multiforme (GBM) and GBM derived stem cells: Implicating Pentraxin 3 upregulation.

Umadevi V. Wesley, Paul Clark, Jacob Jaeger, John Kuo, Robert Dempsey. _University of Wisconsin, Madison, Madison, WI_.

Introduction: Glioblastoma Multiforme (GBM) is the most aggressive type of brain cancer and progresses at a rapid rate. The major obstacle in the treatment of GBM is selective survival of a subpopulation of GBM derived stem cells (GSC) which are highly proliferative and resistant to drug induced apoptosis. The proteins involved in the regulation of the cell cycle impact survival potential of GBM and GSCs. Pentraxin 3 (PTX3), an angiogenic and survival factor plays a key role in cell proliferation, angiogenesis and invasion. However, little is known about the association of PTX3 and cell cycle regulation in GBM derived stem cells. Mehods: GBM patient tumor lines, and GSC derived from the same GBM lines were used. These cell lines were exposed to hypoxia and re-oxygenation. Cell cycle profiling and analysis were carried out using flow cytometry and FlowJo Single Cell Data Analysis Software. Xeno-transplanted mouse brain sections were used for PTX3 expression analysis by immunohistochemistry and immunofluorescence staining. Targeted proteomic approach was used to determine PTX3 levels in GBM and GSC. Alterations in the cell cycle regulatory gene expression in GSC and GBM tumor line in presence and absence of PTX3 was identified by transcriptome array analysis. Results: Our targeted proteomic approach using antibody array identified substantially higher levels of PTX3 in GSCs. GBM tumor lines showed higher percent of cells in G1 phase, where as GSCs showed increased number of cells in G2 and S-phase, both under normoxia and hypoxia. GSCs showed increased levels of GADD45A, known to protect GBM cells from apoptosis. Increased PTX3 expression correlated with increased cell cycle progression. Furthermore, exogenous PTX3 increased CyclinD1, a protein required for progression through the G1 phase of the cell cycle. Conclusion: GSC cells show cell cycle progression, presumably with an increased growth advantage. PTX3 expression is associated with GBM malignancy, and may contribute to GBM cell cycle progression and invasion. Overall, these results indicate that PTX3 and the cell cycle pathway are strongly associated with malignant GBM.

#4301

Mechanisms underlying the inhibitory effect of CRT0066101 on bladder cancer growth in vitro and in vivo.

Quentin Li, Iawen Hsu, Reema Railkar, Thomas Sanford, Piyush K. Agarwal. _NCI, NIH, Bethesda, MD_.

CRT0066101 is an inhibitor of protein kinase D with anti-tumor activity in several types of human carcinomas. However, the effect and mechanism of CRT0066101 in bladder cancer remain unknown. In this study, we show that CRT0066101 inhibited the proliferation and migration of bladder cancer cells, but had only a marginal effect in human uroepithelial cells, indicating a differential inhibitory effect on cell growth between bladder cancer and normal urothelium. We also demonstrate that CRT0066101 blocked bladder cancer growth in a xenograft flank mouse model. Our cell cycle analysis revealed that CRT0066101 arrested bladder tumor cells at the G2-M phase, the arrest being accompanied by decreases in the levels of cyclin B1 and CDK1 and increases in the levels of p27kip1 and phospho-CDK1 (Thr14/Tyr15). Moreover, CRT0066101 reduced the expression of Cdc25C, which dephosphorylates/activates CDK1, but enhanced the activity of the checkpoint kinase Chk1, which phosphorylates/ inactivates Cdc25C. Finally, CRT0066101 was found to elevate the levels of Myt1, Wee1, phospho-Cdc25C (Ser216), Gadd45α, and 14-3-3 proteins, all of which cause a reduction in CDK1-cyclin B1 activity. Together, these data suggest that CRT0066101 suppresses bladder cancer growth partly through modulating the cell cycle G2 checkpoint and inducing G2-M phase arrest, leading to the blockade of cell cycle progression.

#4302

Analysis of the dose and schedule dependence of tumor kill in nonclinical tumour models after treatment with the WEE1 inhibitor AZD1775.

James William Thomas Yates, Elaine Cadogan, Jennifer I. Hare, Adina M. Hughes, Urszula M. Polanska, Mark J. O'Connor, Susan E. Critchlow. _AstraZeneca R &D, Saffron Walden, United Kingdom_.

AZD1775 is a highly selective, small-molecule inhibitor of WEE1 being developed to treat patients with advanced solid tumors, as monotherapy and in combination with olaparib (Lynparza). Previously a mathematical model was developed using data from patient-derived explant (PDX) and xenografted models with a range of sensitivities to AZD1775. This mathematical model could describe the dose and schedule dependency of pharmacokinetics, pCDK1 reduction in tumor and anti-tumor activity. This was for a dose range of 30mg/kg-120mg/kg dosed p.o. on a range of schedules from 3 days on 4 days off to 5 days on 9 days off. The model was then used to rank the potential effectiveness of each dosing regimen by calculating the fraction of tumor killed per week at doses resulting in drug exposure comparable to that observed in the clinic. This calculation was performed by integrating over time the rate of tumor kill predicted by the model. This analysis was complimented with a log cell kill (LCK) analysis using post treatment regrowth data to estimate in a more empirical manner the fraction of tumor killed over the treatment period. Specifically, if TC and TT are the times it takes the controls and treated tumors to grow to a prescribed volume and DT is the doubling time of control tumors then LCK=(TT-TC)/(2.3xDT). The analysis demonstrated that across the data set there was a consistent trend of increased LCK with dose level and number of days dosing in a week. The LCK values for each regimen were normalized by the total number of doses administered, to give an LCK per dose. There appeared to be a consistent LCK per dose level across the dose range considered. Interestingly, there was a greater than linear increase of LCK with increasing dose level. This was consistent with the observation that higher doses with shorter durations of dosing, were at least as active as more chronically administered lower doses. In the TNBC HBCx17 (Xentech) model, over a four-week period 60mg/kg dosed 5 days per week results in an LCK of 0.5 (70% killed) whereas 90mg/kg dosed 3 days per week has an LCK of 0.75 (83% killed). The same relationship was derived from the model simulated fraction tumor kill: higher doses generated significantly larger proportions of tumor kill, thus requiring shorter periods of dosing for the same net effect. The analysis drew greater differentiation between regimen than could be achieved by a tumor growth inhibition (TGI) analysis: where regressions were observed there were a few percentage point differences in TGI between regimen, but up to an order of magnitude difference in LCK. By concentrating on predicting potential cell kill, regimens were identified that are more likely to lead to responses in the clinic. The insights from this analysis have informed recommended dose and schedule for subsequent efficacy expansions.

#4303

Modulation of protein interaction states through the cell cycle.

Lingyun Dai,1 Tianyun Zhao,1 Xavier Bisteau,2 Wendi Sun,1 Nayana Prabhu,1 Yan Ting Lim,1 Radoslaw Sobota,2 Philipp Kaldis,2 Pär Nordlund1. 1 _Nanyang Technological University, Singapore, Singapore;_ 2 _Institute of Molecular and Cell Biology, A*STAR, Singapore, Singapore_.

Global profiling of protein expression through the cell cycle has revealed subsets of periodically expressed proteins. However, expression levels alone only give a partial view of processes determining cellular events. The cell cycle progression events are to a large extent controlled by the dynamic biochemical interactions of proteins with physiological ligands such as other proteins, metabolites, lipids, nucleic acids or low molecular weight effectors. Using a proteome-wide implementation of the Cellular Thermal Shift Assay (CETSA) to study specific cell cycle phases in K562 cell, we uncover modulations of interaction states for more than 750 proteins along the cell cycle. Notably, many protein complexes are modulated in specific cell cycle phases, reflecting their roles in processes such as DNA replication, chromatin remodeling, transcription, translation, and nuclear membrane decomposition. Surprisingly, only small differences in interaction states were seen between G1 and G2 phases, suggesting similar hardwiring of biochemical processes in these two phases. The present work reveals novel molecular details of the cell cycle. CETSA, therefore emerge as a novel approach to discover cellular modulations during cancer development and therapy.

#4304

Development of a new nucleoside triphosphate transporter (NTP) technology for rapid DNA labeling that measures cell cycle progression in live cancer cells.

Nick Asbrock,1 Vi Chu,1 Kevin Su,1 Tomas Kraus2. 1 _MilliporeSigma, Temecula, CA;_ 2 _Academy of Sciences of the Czech Republic, Czech Republic_.

At its core, cancer is a disease of uncontrolled cell division. Mutations in different genes cause or accelerate cancer by increasing cell division rates or inhibiting cell proliferation control mechanisms such as apoptosis and tumor suppression. Traditional DNA incorporation cell proliferation assays that label nucleic acids (BrdU, EdU) have been popular but are limited by requiring cell fixation, high cost and lengthy protocols. Here, we have developed a new NTP transporter technology that allows the incorporation of Cyanine-3 fluorescently labeled dUTP's into DNA during the S-phase of the cell cycle to measure cell proliferation in live cancer cells (U2OS). Similar cell cycle profiles were produced using this new NTP transporter technology as analyzed by FACS analysis and live cell imaging vs. BrdU based cell proliferation assays. This technology will allow new insights into cancer cell proliferation and progression.

#4305

**TET increases p27** Kip1 **protein stability and p21** WAF1/Cip1 **promoter activity to block deregulated cell cycle progression in pancreatic cancer cells.**

Karnika Singh, Qin Dong, Sweaty Koul, Hari K. Koul. _LSU Health Sciences Center- Shreveport, Shreveport, LA_.

Introduction: Pancreatic cancer (PaCa) is the fourth cause of cancer related deaths in the United States with an overall five-year survival rate of less than five percent. The current standard treatment/s for PaCa are ineffective thus there is an urgent need for development of new therapeutic agents for its treatment. Owing to its mutational landscape, which includes mutated KRAS and p53 these cells have a deregulated G1 checkpoint of cell cycle and thus confer high proliferation rate and therapeutic resistance. Recently we observed that Tetrandrine derivative (TET), a bis-benzylisoquinoline alkaloid halts cell cycle progression in PaCa cells at G1/S boundary in vitro and in vivo. In the present study we explored potential mechanism of TET induced cell cycle arrest in two PaCa cells with either wild type or mutant KRAS, and mutant p53 by evaluating critical cell cycle regulatory proteins involved in G1/S transition. Method: PaCa cell lines: BxPC3 (PDAC) and MiaPaCa2 (Pancreatic Carcinoma) were used. Cells were treated with various concentrations of TET for different time points. Cell growth was checked using the MTT assay. Cell cycle was analyzed after PI staining via Flow cytometry. Changes in protein levels were assessed by Western Blot analysis. mRNA changes were evaluated by real time PCR. Promoter activity was checked by luciferase reporter assay. Knockdown was achieved by lentiviral transduction. Results: Treatment of PaCa cells with TET resulted in decreased levels of cyclin D1 and phospho-Rb, two critical proteins required for G1/S transition and increased levels of p21WAF1/Cip1 and p27Kip1 proteins. Real time PCR revealed that cyclin D1 mRNA was decreased and p21WAF1/Cip1 mRNA was increased while there was no significant change in p27Kip1 mRNA levels. The cycloheximide chase showed that TET treatment increases p27Kip1 protein levels by improving its protein stability (t1/2 6h vs 2h: TET treated vs control). Further studies into the mechanism revealed that treatment of PaCa cells with TET resulted in decreased protein levels of Skp2 E3 ligase which is specific for degrading p27Kip1 protein during the cell cycle. Moreover TET also increased the p21WAF1/Cip1 promoter activity accounting for increased mRNA levels of p21WAF1/Cip1. These data suggest a notable mechanism by which TET regulates expression of critical cell cycle regulators at both transcriptional and post-translational level. Such a mechanism would provide for cell cycle block in PaCa cells following TET treatment. Conclusion: TET halts deregulated cell cycle in PaCa cells in part by modulating p27Kip1 and p21WAF1/Cip1 protein levels. Moreover p27Kip1 is regulated post-translationally (via Skp2 regulation) and p21WAF1/Cip1 is regulated at transcriptional level. These results suggest that TET can not only halt deregulated cell cycle but also overcome the therapeutic resistance in PaCa cells.

#4306

Degradation of Cyclin D1 protein is mediated by multiple Cullin Ring-associated E3 ligases.

Yue Zhao,1 Di Chen,2 Ming Zhang3. 1 _Zhejiang Cancer Hospital, Hangzhou, China;_ 2 _Rush University Medical Center, Chicago, IL;_ 3 _School of Medicine, Johns Hopkins University, Baltimore, MD_.

Cyclin D1 is a critical member of cyclin protein family which regulates G1-S phase cell cycle progression through activation of cyclin dependent kinase 4 or 6 (CDK4/6) leading to subsequent phosphorylation of retinoblastoma protein (pRb). Since cyclin D1 controls normal cell cycle progression and its abnormal up-regulation has been linked to many types of human cancers, cyclin D1 expression is tightly under control at transcriptional and post-transcriptional levels. In this study, we found that cyclin D1 degradation is mediated by multiple cullin-associated E3 family members. Through screening a siRNAs library targeting 156 genes coding cullin-associated ubiquitin ligase subunits, we identified 24 proteins that significantly affect cyclin D1 activity. Among these proteins, Keap1 (associated with CUL3), DDB2 (associated with CUL4A/4B), WSB2 (associated with CUL2/5) and Rbx1 (interacting with the C-terminal domains of the cullins) were selected to further investigate their roles in cyclin D1 ubiquitination. These proteins interacted with cyclin D1 and mediated cyclin D1 proteolysis through the ubiquitin-proteasome pathway in a phosphorylation-dependent manner. Silencing of Keap1, DDB2, WSB2 or Rbx1 in human colon cancer cells resulted in a significant increase in endogenous cyclin D1 protein levels. We have further investigated the role of these proteins in regulation of normal cell cycle progression and demonstrated that the expression of these proteins induced G1 arrest and subsequently suppressed DNA synthesis and cell proliferation. Our results indicate that these cullin-associated ubiquitin ligases mediate cyclin D1 proteolysis and regulate cyclin D1 protein levels during normal cell cycle progression. Considering that loss-of-function mutations of these E3 ligases were found in a variety of cancers which are correlated with up-regulation of cyclin D1, our results also implicate the importance of this regulatory mechanism in tumorogenesis.

#4307

Molecular basis of cell cycle arrest induced by erufosine in metastatic breast cancer cells.

Asim Pervaiz,1 Muhammad Shoaib Akhtar,1 Saqib Mahmood,1 Osheen Sajjad,1 Saba Khaliq,1 Martin R. Berger2. 1 _Univ. of Health Sciences, Lahore, Pakistan;_ 2 _German Cancer Research Centre (DKFZ), Heidelberg, Germany_.

Introduction: Breast cancer, a major health care burden with limited curative options for advanced metastatic phase, demands a search for novel therapeutic compounds. Synthetic alkyl-phospholipids (ALPs) comprise a promising class of anti-cancer agents in this context, where 1st and 2nd generation compounds are already in clinical use to treat various malignancies. Erufosine is the latest (3rd) generation of ALPs and has shown promising anti-tumour activities against breast cancer with minimal toxicity towards GI tract, negligible haemolytic activity and a good pharmacological profile in laboratory settings. Previously, we highlighted the significant potential of erufosine to induce arrest in G2/M phase of cell cycle in breast cancer cells. Currently, we decipher the mechanistic basis of these observed effects at molecular levels.

Materials & Methods: Metastatic breast cancer cells (MDA-MB-231) were exposed to low (IC25, 12.3µM), medium (IC50, 19.5µM) and high (IC75, 30.2µM) concentrations of erufosine for 48h. Distribution of the cells in different phases of the cell cycle was determined by propidium iodide based labelling of the DNA and flow cytometry analysis. Expressional modulations in 84 cell cycle relevant genes in response to erufosine exposure (IC75, 48h) were studied by using a ready-made panel (Qiagen, Cat#330231) and real-time PCR methodology. Alterations in expressional profile of significantly altered genes (≥2fold) were used to design a signalling pathway with the help of Ingenuity Pathway Analysis at core facility of DKFZ, Heidelberg, Germany.

Results: Exposure with erufosine induced a significant arrest in the G2/M phase of cell cycle in MDA-MB-231 cells in a concentration dependant format. Significant alterations (≥2fold) were found in 39% (33/84) of the cell cycle relevant genes incorporated in the ready-made panel. Most of the altered genes (28/33) were down-regulated in response to erufosine exposure. Significantly altered genes belong to various groups like kinases (ATM, AURKA, B), phosphatases (CDC25A, C), tumour suppressors (BRCA1), anti-apoptotic genes ( BCL2, BIRC5), cyclins (CCNA2, CCNB1, CCNB2, CCND2, CCNF, CCNG2), their target kinases (CDKs), activators (CDC6, MK167), inhibitors (CDKN1A, 2A, CDKN3, CKS1B), regulators (CDC20, MKI67), facilitators of the cell cycle (MCM2, 3, 4) and transcription factors (CKS2, E2F1).

Conclusion: Erufosine is a significant cytostatic agent and induces a major halt in the G2/M phase of cell cycle in metastatic breast cancer cells. The compound imposes noteworthy alterations in various cell cycle relevant genes to cause these cytostatic effects. Erufosine, provided with further studies in laboratory (in vitro and in vivo) and clinical settings, could be a valuable therapeutic agent for metastatic breast cancer.

#4308

RBBP6 expressional effects on cell proliferation and apoptosis in breast cancer cell lines with distinct p53 statuses.

lesetja R. Motadi,1 pontsho Moela2. 1 _North west university, Mmabatho, South Africa;_ 2 _University of Pretoria, Pretoria, South Africa_.

Breast cancer incidence rate has increased beyond that of lung cancer, making it the most common malignancy among women. Breast tumour progression is partly as a result of p53 inactivation by overexpressed ubiquitous regulatory proteins that possess p53 binding domain. RBBP6 forms a member of such protein since it has an E3 ligase activity due to its RING finger-like domain. Moreover, its overexpression in several malignancies makes it a potential target in cancer management. However, it is not clearly defined whether RBBP6 effect on cell growth and apoptosis is cell line dependent, more especially in breast cancer cell lines that have distinct p53 expression profiles. The present study therefore aims at evaluating RBBP6 effects on cell growth and apoptosis in breast cancer cell lines with different p53 expression profiles. Following analysis at mRNA and protein levels in breast cancer tissue, RBBP6 expression was successfully manipulated using gene silencing and protein overexpression techniques in MCF-7 and MDA-MB-231 cell lines. Transfection efficiencies were confirmed using qPCR and western blot, followed by monitoring of cell proliferation in the presence of transfection using xCELLigence system. The cells were then co-treated with anti-cancer agents followed by apoptosis detection using confocal microscopy and flow cytometry, which was further confirmed by caspase 3/7 activity and quantification of apoptotic genes. RBBP6 was overexpressed in breast cancer tissues that were classified as stage 3 and 4 while in stage 1 it was expressed but at much lower levels. The wt. p53-expressing MCF-7 cell line was more susceptible to apoptosis induction as opposed to the mt. p53-expressing MDA-MB-231. RBBP6 silencing led to a significant accumulation of p53 expression in MCF-7 as compared to MDA-MB-231. Co-treatment with GABA and camptothecin seemed to sensitize the cells to apoptosis induction. These data suggest that RBBP6 silencing triggers significant levels of intrinsic apoptosis and over-expression appears to promote cell proliferation in wild type p53-expressing MCF-7 rather than in MDA-MB-231 cells. Insignificant changes in the expression of mt. TP53 in MDA-MB-231 cell line following RBBP6 silencing and over-expression suggests that RBBP6 is highly likely to interact with wt-p53 rather than mutated p53. In conclusion, the effect of RBBP6 on cell proliferation and apoptosis induction in breast cancer seem to be cell line dependent based on p53 status

#4309

SPCIP2 enhances breast cancer cell proliferation, cell cycle and tumor formation by expression and interaction with Y1.

Hyunjung Byun, Changhoon Lee. _Dongguk University, Goyang, Republic of Korea_.

SPCIP2 is a member of the serine/threonine protein kinase family. However, the working mechanism of SPCIP2 in cellular functions has not yet been properly understood. In this study, we found that SPCIP2 directly interacts with Y1, a multifunctional protein that can act as a regulator of transcription and translation. Furthermore, we determined the binding domains required for the interaction between SPCIP2 and Y1. The ectopic expression of SPCIP2 activates cell growth by inducing Y1. Binding activates is confirmed by SPCIP2-dependent increase of Y1 expression, as well as by the induced cell proliferation and tumor formation. Also, expression of siSPCIP2 and siY1 is found to induce cell cycle arrest and apoptotic activity in breast cancer cells. These insights provide evidence that SPCIP2 induces cell proliferation and tumor formation via expression and interaction with Y1.

#4310

Condensin II complex as a potential driver of cancer proliferation.

Ellen L. Nutter,1 Emily S. Weyburne,1 Jeffrey Thompson,2 Jennifer A. Doherty,3 Giovanni Bosco1. 1 _Dartmouth College, Hanover, NH;_ 2 _University of Kansas Medical Center, Kansas City, KS;_ 3 _Huntsman Cancer Institute, University of Utah, Salt Lake City, UT_.

BACKGROUND

The Condensin II complex's known function is in chromosomal segregation and assembly. Leiserson et al. demonstrated that Condensin II was enriched for mutations in a pan-cancer sweep, particularly lung cancer; one gene, NCAPH2, had a notable mutation cluster. Venet et al. created metaPCNA, a robust proliferation-based gene expression (GE) signature from 36 tissues, which includes 4 Condensin genes. This suggests that Condensin II expression is correlated with proliferation. We hypothesize that Condensin II influences cancer proliferation in two ways, and demonstrate evidence for the first: 1) rare stabilizing subunit mutations or 2) pairing of high Condensin II expression with a driver mutation that affects pathways controlling Condensin II activity.

METHODS

Using the TCGA lung adenocarcinoma (LUAD) gene expression dataset (n=571), we calculated the Condensin II index (mean expression of 5 genes) and the metaPCNA index (median expression of 131 genes). Pearson correlation between indices was calculated to assess information overlap between gene signatures. For outcome assessments, scores were split into tertile labels (high, middle, low). Linear-by-linear testing was assessed for score agreement. Unsupervised hierarchical clustering of the top 2,500 most variant genes with samples labeled by index tertile inclusion was completed for both indices.

After noting the similarity between indices, we tested Condensin II as a driver of proliferation by running overexpression assays using the rate limiting subunit of the complex, NCAPH2. Plasmids with NCAPH2, both wildtype and engineered to have the R551P mutation identified by Leiserson, were transfected into RPE1 cells, a telomere elongation immortalized cell line with a 'normal' proliferation profile. We had 3 conditions 1) myc-tagged control, 2) wild-type NCAPH2, and 3) R551P mutated NCAPH2, utilizing Incucyte imaging for 3 days after a 1-day rest to assess the proliferation of cells.

RESULTS

The Condensin II index and metaPCNA index were strongly associated (correlation = 0.85). Cluster analysis by either the metaPCNA or Condensin II index demonstrated that the lowest tertile tends to separate from the middle and highest tertile. Samples had a high overlap of tertile labels between the 2 indices (n labels matched =406, n not matched = 165, z= 18.57, p= <2.2*10-16).

A 72-hour time course of the 3 transfected cell lines demonstrated that overexpression of R551P mutated NCAPH2was associated with an increase in proliferation, while wild-type NCAPH2did not differ significantly from the myc-tagged control.

CONCLUSIONS

Condensin II index is a simple, 5-gene signature strongly associated with a known proliferation signature and tumor expression patterns. A potentially stabilizing mutation to NCAPH2, R551P, increases proliferation potential in cell line experimentation, but overexpression of wild-type NCAPH2does not. Condensin II may be a driver of cancer proliferation.

#4311

Rb may play a key role in green tea-induced growth inhibition of human myeloid leukemia cells.

Darrell Henry, Sebastien Brumaire, Xiaotang Hu. _Barry Univ., Miami Shores, FL_.

Although the benefits of green tea to human health including preventing cancer have been widely reported in recent years, the specific effect and action mechanisms of green tea on cancer cells are largely unclear. In this study, we presented evidence that green tea extract and its major chemical component, Epigallocatechin-3-gallate (EGCG), inhibit both suspension and adherence cancer cells through the regulation of pRb-CDK complexes. Addition of green tea extract to the culture of myeloid leukemia TF-1a and MV4-11 cells significantly inhibited their proliferation measured by XTT and BrdU assays. The inhibition is dose dependent with more than 35% inhibition being observed at a concentration of 4% (v/v) of the tea extracts derived from 8% (g/ml) green tealeaf in distilled water. The tea extract also markedly reduced the numbers of stem/progenitors derived from TF-1a, MV4-11, and Hep-G2 cells, determined by counting colony-forming cells in semi-solid clonal culture. EGCG showed a similar inhibitory effect on TF-1a, MV4-11, and Hep-G2 cells and their stem/progenitors. Next, we investigated what mechanism(s) is responsible for the green tea-induced growth inhibition of the cells. Surprised to us the expression of Rb was significantly upregualted with a maximal enhancement being observed when 0.00001% of EGCG (g/ml) or 4% (v/v) of green tea extracts were added to the cell culture. The upregulation of Rb was significantly blocked by pre-treatment with cycloheximade, a protein synthesis inhibitor. In contrast, green tea extract and EGCG downregulated dramatically phosphorylation of pRb. Addition of cycloheximade had no effect on the phosphorylation of Rb in the absence or presence of green tea extract or EGCG analyzed by Western blotting. We also found that green tea extract and EGCG had no significant effect on the expression of CDC2, CDK2, CDK4, CDK6, and CDK inhibitors, p21 and p27, but the formation of CDK4-pRb and CDK6-pRb complexes were significantly downregulated detected by immunoprecipitation. Taken together, our data suggest that green tea (the extract and EGCG) regulate Rb protein at both translation and post-translation levels. The upregulation of Rb synthesis, downregulation of Rb phosphorylation and CDK-Rb complexes all promote the binding of Rb to E2F transcription factors and inhibit the E2F-dependent transcription, leading to cell growth inhibition (supported by Barry Faculty Stimulating Grant 2017-2018).

#4312

3-Hydroxyterphenyllin induces S phase arrest in ovarian cancer cells.

Yi C. Chen. _Alderson-Broaddus Univ., Philippi, WV_.

Epithelial ovarian cancer is the fifth most common cause of cancer-related death among women in the United States. 3-Hydroxyterphenyllin is a secondary metabolite of Aspergillus fungi. In this study, we evaluated 3-hydroxyterphenyllin as a potential anti-cancer agent using human ovarian cancer cells. The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO2. The [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, flow cytometry and western blot analysis were used to confirm that 3-hydroxyterphenyllin exhibited a potent growth inhibitory effect against human ovarian cancer OVCAR-3 and A2780/CP70 cells. 3-hydroxyterphenyllin (2, 4, 8, 12 and 16 μM) reduced the viability of OVCAR-3 and A2780/CP70 cells in a concentration-dependent manner. 3-hydroxyterphenyllin (2, 4 and 8 μM) induced S phase cellular arrest for OVCAR-3 and A2780/CP70 cells by upregulating CyclinB1 and Cdc25A expression and inhibiting Cdk4, CyclinA2 and CyclinE1 expression. These results suggest that 3-hydroxyterphenyllin has the potential to fight ovarian cancer cell growth by preventing progression past the S phase of the cell cycle. 

### Epigenomics

#4313

Methylated BAF155 promotes cancer metastasis by cooperating with BRD4 to potentiate genes addicted to super-enhancers.

Eui-Jun Kim, Yidan Wang, Li Lu, Lixin Rui, Xuehua Zhong, Kari B. Wisinski, Wei Xu. _University of Wisconsin-Madison, Madison, WI_.

Triple-negative breast cancers (TNBC) lack the expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). TNBC is the most aggressive breast cancer subtype and is often associated with distant metastasis, poor prognosis, and lack of targeted treatment options. The failure to identify a few genes driving metastasis through sequencing of paired tumors at primary and metastatic sites implies that epigenetic mechanisms may play important roles in TNBC metastasis. Recently, we found that an epigenetic enzyme coactivator associated arginine methyltransferase 1 (CARM1) is highly expressed in TNBC and its high expression correlates with poor prognosis. Subsequently we identified BRG1-Associated Factor 155 (BAF155), a component of SWI/SNF chromatin remodeling complex, as a novel CARM1 substrate. Transcriptome analyses of BAF155 methylation competent and defective cell lines identified many genes involved in regulating cancer metastasis are methyl-BAF155 targets. Methylation of BAF155 was found to promote metastasis in the TNBC xenografted mouse models. We generated a methyl-BAF155 antibody that could specifically detect methylated BAF155 (me-BAF155) by IHC and chromatin immunoprecipitation followed by sequencing (ChIP-seq). We mapped global me-BAF155 genomic association sites using ChIP-seq and discovered that me-BAF155 was highly enriched in super-enhancers (SEs), regulatory elements on chromatin that are known to drive high transcription of many oncogenes. SEs are known to recruit BRD4, a BET family bromodomain protein. Indeed, global me-BAF155 binding peaks greatly overlap with those of BRD4 binding sites. Treatment of TNBC cells with either BRD4 inhibitor JQ1 or CARM1 inhibitor TP-064 significantly reduced the expression of me-BAF155 target genes. Together our data suggest that me-BAF155 is an important epigenetic target for treatment of cancer metastasis in TNBC cells.

#4314

Chromatin state alterations during melanoma progression.

Kunal Rai,1 Christopher Terranova,1 Mayinuer Maitituoheti,1 Ming Tang,1 Kadir Akdemir,1 Elias Orouji,1 Lynda Chin,2 Petko Fiziev,1 Mayura Dhamdhere,1 Neha S. Samant,1 Amikhsha Shah,1 Sneha Sharma,1 Jason Ernst1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _University of Texas System, Houston, TX_.

The extent and nature of epigenomic changes associated with melanoma progression is poorly understood. To determine chromatin state patterns of human metastatic melanomas, we defined epigenome of melanoma by profiling 6 histone modification marks (H3K4me, H3K27Ac, H3K4me3, H3K79me2, H3K27me3 and H3K9me3) in 48 metastatic melanoma tumors, cell lines and short-term cultures. Computation of chromatin states identified specific chromatin state aberrations in human tumors and their relationship with specific genotypes. Specifically, we defined patterns of enhancer elements, broad H3K4me3 domains and bivalent chromatin states enriched in specific genotypes of BRAF, NRAS, and WT groups of melanomas. Importantly, we identified switch from specific bivalent chromatin states in melanocytes to active states leading to upregulation of EMT genes in NRAS mutant melanomas. Similarly, we noted gains of broad H3K4me3 domains in particular classes of oncogenes in specific gentoypes as well as during transitions from melanocytes to melanomas. Finally, we identified clusters of enhancers that are specifically enriched in subset of melanoma samples defining intertumor epigenetic heterogeneity in melanoma tumors. In addition, to investigate chromatin state changes associated with early stages of melanoma progression, we systematically profiled 35 epigenetic modifications in a cell phenotypic model of non-tumorigenic and tumorigenic states (and verified in human nevi and melanoma samples). Specific chromatin state transitions featuring loss of histone acetylations and H3K4me2/3 were observed on specific promoter and enhancer elements targeting cancer-regulatory genes in important melanoma-driving cell signaling pathways. Intriguingly, only a small fraction of chromatin state transitions correlated with expected changes in gene expression patterns. Restoration of acetylation levels on deacetylated loci by histone deacetylase (HDAC) inhibitors selectively blocked excessive proliferation in tumorigenic cells and human melanoma cells, suggesting functional roles of observed chromatin state transitions in driving hyperproliferative phenotype. Overall, our study reveals principles of epigenetic reprogramming in melanoma progression stages and how they may establish transcriptional programs that impart aggressive characteristics to melanomas.

#4315

Colorectal cancer epigenomic landscape.

Elias Orouji, Ayush Raman, Ming Tang, Mayura Dhamdhere, Kunal Rai. _MD Anderson Cancer Center, Houston, TX_.

Cancer cells utilize genetic and epigenetic aberrations for their excessive growth. Although we have sufficient understanding of the genomic alterations in colorectal cancer, we have incomplete knowledge of epigenomic aberrations and their impact on tumor growth. In order to comprehensively define the epigenetic patterns specific to colorectal cancer, we generated profiles for 6 histone modification marks, including H3K4me1 (enhancer), H3K27Ac (active enhancer), H3K9me3 (heterochromatin), H3K27me3 (polycomb repression), H3K79me2 (transcription) and H3K4me3 (promoter), using a high-throughput ChIP-Seq methodology developed in house applicable to frozen tumors. Chromatin state transitions specifically pointed to drastic changes in enhancer patterns, consistent with some prior studies. Furthermore, using similarity network fusion (SNF) we identified the best singular mark and are currently developing a tool to identify combinatorial chromatin states that could most efficiently distinguish and eventually predict CRC from normal colon. In a more detailed investigation into patterns of active enhancers using normal colon, adenomas and colorectal cancers, we identified specific changes in enhancers from normal tissue to these neoplastic lesions. Importantly, we noted gains of enhancers in a large number of genomic loci in colon cancer compared to adjacent normal tissues. These enhancers are enriched in important signaling components including Notch, Wnt, and stem cell regulators. We specifically noted changes in enhancer regions in the proximity of ASCL2 (intestinal cancer stem cell gene), SALL4 (early pluripotency marker) as well as FZD10 and CTNNB1 (Wnt signaling). A preliminary Cas9-based deletion study suggested these enhancers to be functional in regulating gene expression, and currently its effect on cell proliferation and other cancer properties is being investigated. Further, we reasoned that blocking aberrantly gained enhancers using BRD inhibitor in combination with pathway inhibitors could be a useful strategy in this cancer. Our data supported this hypothesis, and we are currently performing a systematic study to identify most effective combinations. In summary, we have identified aberrant enhancer gains as a major feature of colorectal cancer and propose this to be utilized as a therapeutic approach.

#4316

Disruption and restoration of pioneer transcription factor hub function in bladder cancer.

Caroline Schuerger, Yogen Saunthararajah. _Cleveland Clinic Taussig Cancer Institute, Cleveland, OH_.

Urothelial bladder cancer is characterized by the failure to complete transitional epithelial-differentiation. The mechanisms underlying this failure are unknown. To address this gap in knowledge, we analyzed 2000 genes differentially expressed between normal bladder vs. bladder cancer (BLCA) (n=426, TCGA RNA sequencing). Remarkably, genes suppressed in BLCA are found in embryonic stem cells (ESC) in compacted chromatin (decreased H3K4me3, increased CpG methylation), while upregulated genes in BLCA are found in ESCs in open chromatin (H1 ESC Encode).This striking property of genes suppressed in BLCA suggested loss-of-function of pioneer transcription factor (TF) function in BLCA, as pioneer TFs access compacted chromatin to initiate the necessary remodeling for subsequent gene activation. The pioneer master TF hub containing FOXA1, GATA4/6 is highly expressed in normal bladder, and these master TFs were frequently haploinsufficient in BLCA, 32%, 56%, and 24% respectively. To better understand why target genes of FOXA1, the most expressed bladder master TF, are unremodeled and repressed, we pulled down endogenous FOXA1 from UM-UC-6 and -3 BLCA cells and used mass spectrometry and Western blot to analyze interacting coregulators, which execute the biochemical work of chromatin remodeling. These analyses revealed enrichment for corepressors, e.g., DNMT1, in the FOXA1 interactome over coactivators, e.g., ARID1A. Genetic analysis of BLCA show frequent mutation/deletion of coactivators: 43% bi-allelic ARID1A inactivation, while corepressors SMARCA5 and DNMT1 were amplified or unaltered to remain functional. Restoration of ARID1A into ARID1A haploinsufficient BLCA (UC-6) upregulated epithelial-differentiation genes and induced terminal epithelial-differentiation, downregulation of MYC and upregulation of p27 protein. To translate into therapy, we restored coactivator/corepressor balance by pharmacologic corepressor depletion: we used the clinical drug decitabine to deplete DNMT1, to compensate for genetic coactivator inactivation. Decitabine recapitulated the effects of ARID1A introduction, inducing terminal epithelial-differentiation of BLCA cells. This was also seen in vivo in a xenograft model of BLCA (UC-6). Decitabine combined with an inhibitor of its in vivo degradation, tetrahydrouridine, decreased tumor size significantly with vehicle tumor size 917.8 mm3 to treated tumor size 321.0 mm3 (p<0.01). In sum, genetic alterations in BLCA alter composition of a pioneer master TF hub to remove remodeling needed for transitional epithelial-differentiation. Dynamic coactivator/corepressor opposition in gene regulation, however, enables pharmacologic reduction of corepressors to compensate for genetic reduction of coactivators.

#4317

Cell-type specific epigenetic effects of tobacco smoking on the immune cell methylome.

Xuting Wang, Ma Wan, Brian D. Bennett, Gary S. Pittman, Michelle Campbell, Dan Su, Isabel J. Thompson, Suzanne N. Martos, Douglas A. Bell. _NIH-NIEHS, Research Triangle Park, NC_.

Exposure to tobacco smoke dramatically alters DNA methylation in blood cells and this immune cell modulation may mediate smoking-associated complex diseases. To identify cell-type specific, smoking-associated differentially methylated regions (SM-DMRs) in hematopoietic cells from smokers and non-smokers, we used Illumina 450/850K arrays, reduced representation (RRBS) and whole genome bisulfite sequencing (WGBS) to measure methylation on DNA extracted from whole blood and 7 leukocyte subtypes (CD4+, CD8+, CD14+ monocytes, CD15+ granulocytes, B cells, NK cells and CD34+ progenitor cells). SM-DMRs preferentially occurred at enhancers (H3K27ac), and/or CpG island shores and were enriched for transcription factor (TF) binding suggesting crosstalk between TFs, DNA methylation, and tobacco smoke exposure. SM-DMRs in or near genes AHRR, ALPPL2, and GFI1 were highly significant in CD34+, CD14+, CD15+, B cells and NK cells but were absent in CD4+ and CD8+ T cells. We also identified genomic regions with distinct cell-type and lineage-specific differences in smoking-associated methylation and gene expression. AHRR displayed increased levels of both noncoding RNA and mRNA gene expression only in CD14+ monocytes. The SM-DMR in GPR15 was highly significant in CD4+ and CD8+ T cells, B cells, and NK cells but absent in myeloid cells. To explore possible links between smoking, DNA methylation/gene expression and immune cell functions, we carried out pathway analysis on smoking-affected methylomes and transcriptomes in each cell type. The smokers' transcriptome of CD56+ NK cells showed perturbation of genes either altered in leukemias, linked with leukemia GWAS SNPs, or under various types of innate immune stimulation. In particular, the locus containing HLA-DQA1 and HLA-DQB1 is highly significantly linked with chronic lymphocytic leukemia and these genes were strongly down-regulated in NK cells of smokers. The NK cell methylome of smokers also displayed perturbation of genes altered in leukemia cell lines. To identify and characterize rare blood cells among cell-type subsets that have smoking-altered transcriptomes, we are carrying out single cell RNA-seq. These studies may reveal new biological connections between tobacco smoke exposure and smoking-associated disease etiology.

#4318

Identifying drivers of SMARCA4/BRG1-deficient SCCOHT tumorigenesis by integrative multi-omic analysis.

Krystal A. Orlando,1 Jesse R. Raab,1 Jessica D. Lang,2 William P. Hendricks,2 Yemin Wang,3 David G. Huntsman,3 Jeffrey M. Trent,2 Joel S. Parker,4 Bernard E. Weissman1. 1 _The University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 2 _Translational Genomics Research Institute (TGen), Phoenix, AZ;_ 3 _British Columbia Cancer Agency, Vancouver, British Columbia, Canada;_ 4 _UNC Lineberger Comprehensive Center, Chapel Hill, NC_.

Over 94% of small cell carcinomas of the ovary, hypercalcemic type (SCCOHT), a rare and aggressive form of ovarian cancer, have mutations and concomitant protein loss in SMARCA4 (BRG1), one of the two mutually exclusive ATPases of the SWI/SNF chromatin remodeling complex. SCCOHT tumors rarely have secondary mutations, making them an excellent model for understanding the role BRG1 and SWI/SNF complexes play in tumor suppression. We hypothesize that BRG1 loss drives SCCOHT tumorigenesis by altering chromatin accessibility and gene expression. We have previously shown that BRG1 re-expression in SCCOHT cell lines suppresses cell growth and induces an elongated, neuronal-like morphology. To identify the top genes and transcription factors driving SCCOHT tumorigenesis, we performed ATAC-seq and RNA-seq in a SCCOHT cell line +/- BRG1 re-expression. BRG1 re-expression increased overall chromatin accessibly, shown by an increase in the number of ATAC-seq peaks. ATAC peaks gained following BRG1 re-expression were enriched in transcription factor binding motifs from FOS/JUN/AP-1, TEAD, and SOX family members. RNA-seq analysis demonstrated that BRG1 re-expression upregulated more genes than those downregulated, consistent with the increase in ATAC-seq peaks. Preliminary pathway analysis identified gene enrichments in epithelial-mesenchymal transition pathway and extracellular matrix remodeling following BRG1 re-expression, consistent with the observed morphology change. Cell types enrichment analysis (xCell) identified a shift from a mesenchymal stem cell-like gene signature in the control SCCOHT cells to an epithelial cell-like gene signature following BRG1 re-expression, further suggesting a possible mesenchymal-epithelial transition in SCCOHT cells after BRG1 re-expression. Future studies include integration of the ATAC/RNA-seq data to further identify correlations between gene expression changes and enriched transcription factor binding motifs, integrative ChIP-seq analysis for BRG1 following re-expression, and proteomic analyses. These studies will uncover the key genes, proteins, and transcription factors affected by BRG1 loss in other adult cancers, provide insight into BRG1's role in SCCOHT tumorigenesis, and potentially yield novel therapeutic targets.

#4319

Epigenetic reprogramming associated with BRCA1 loss of function in ovarian cancer induces enhanced interferon gamma signaling.

Horacio Cardenas,1 Jessica Thomes Pepin,2 Guanglong Jiang,2 Salvatore Condello,1 Kenneth P. Nephew,3 Yunlong Liu,2 Daniela Matei1. 1 _Northwestern University, Chicago, IL;_ 2 _Indiana University, Indianapolis, IN;_ 3 _Indiana University, Bloomington, IN_.

Inactivating mutations of the breast cancer type 1 susceptibility protein (BRCA1) have been implicated in breast and ovarian cancer (OC) initiation. Having observed differential response of OC cells to histone deacetylase inhibitors (HDACi) dependent on the presence of a functional BRCA1, we mapped promoters marked by acetylation of lysine 9 or lysine 27 of histone H3 (H3K9ac, H3K27ac) by ChIP-sequencing, and measured gene expression by RNA-sequencing in the isogenic OC cell lines UWB1.289 carrying an inactivating mutation of BRCA1 (BRCA1-null) and UWB1.289 cells with restored BRCA1 gene (BRCA1+). Significant differences in promoter levels of H3K9 and H3K27 acetylation were associated with marked alteration of gene expression levels between BRCA-null and BRCA1+ cells and in response to the HDACi, entinostat. Pathway analyses revealed cellular movement, cellular development, cellular growth and proliferation, TGFβ1, TNF, and INF-γ among the top cellular/molecular functions and upstream regulators enriched by the absence of BRCA1 among H3K9ac differentially marked and differentially expressed genes. Importantly, the same pathways and regulators were enriched in response to entinostat in BRCA1+ cells, indicating that changes in gene expression affecting these functions and regulators in BRCA1-null cells depend on promoter-associated histone acetylation. Integrated analyses revealed that TGFβ1 and IFN-γ were also activated in BRCA1-null/or low vs. BRCA1-normal ovarian tumors in the TCGA database. IFN-γ target genes were upregulated at baseline and responded less to IFN-γ stimulation in BRCA1-null vs. BRCA1+ cells. Binding of HDAC1 to promoters of IFN-γ target genes was increased in BRCA1-null cells. Similarly, the BRCA1-null breast cancer cell line HCC1937 responded less to IFN-γ stimulation compared with HCC1937 cells in which BRCA1 was restored. The knockdown of STAT1, the main IFN-γ signaling transduction molecule, inhibited BRCA1+ cell proliferation, but did not affect BRCA1-null cells. We conclude that the transcriptome of BRCA1-null cells and tumors is altered through changes in histone acetylation affecting cancer-related pathways, including IFN-γ cellular responses. These changes could alter tumor progression in BRCA-null OC and impact response to immunotherapy.

#4320

p53 binding sites in normal and cancer cells are characterized by distinct chromatin context.

Feng Cui. _Rochester Inst. of Technology, Rochester, NY_.

The tumor suppressor protein p53 interacts with DNA in a sequence-dependent manner. Thousands of p53 binding sites have been mapped genome-wide in normal and cancer cells. However, the way p53 selectively binds its cognate sites in different types of cells is not fully understood. Here, we performed a comprehensive analysis of 25 published p53 cistromes and identified 3,551 and 6,039 "high-confidence" binding sites in normal and cancer cells, respectively. Our analysis revealed two distinct epigenetic features underlying p53-DNA interactions in vivo. First, p53 binding sites are associated with transcriptionally active histone marks (H3K4me3 and H3K36me3) in normal-cell chromatin, but with repressive histone marks (H3K27me3) in cancer-cell chromatin. Second, p53 binding sites in cancer cells are characterized by a lower level of DNA methylation than their counterparts in normal cells, probably related to global hypomethylation in cancers. Intriguingly, regardless of the cell type, p53 sites are highly enriched in the endogenous retroviral elements of the ERV1 family, highlighting the importance of this repeat family in shaping the transcriptional network of p53. Moreover, the p53 sites exhibit an unusual combination of chromatin patterns: high nucleosome occupancy and, at the same time, high sensitivity to DNase I. Our results suggest that p53 can access its target sites in a chromatin environment that is nonpermissive to most DNA-binding transcription factors, which may allow p53 to act as a pioneer transcription factor in the context of chromatin.

#4321

Up-regulation of METTL3 promoted m6A modification and epigenetic silencing of SOCS2 in human liver cancer.

Mengnuo Chen, Lai Wei, Cheuk-Ting Law, Felice H. Tsang, Jialing Shen, Carol L. Cheng, Long-Hin Tsang, Daniel W. Ho, David K. Chiu, Joyce M. Lee, Carmen C. Wong, Irene O. Ng, Chun-Ming Wong. _The University of Hong Kong, Hong Kong, Hong Kong_.

Epigenetic alteration is a common trait of human cancers. Chemical modifications on DNA and core histone proteins are the major mechanisms for epigenetic regulation. Recently, emerging evidence suggested that reversible chemical modifications on RNA also play a critical role in epigenetic control of gene expression. N6-methyladenosine (m6A) is the most abundant modification found in mammalian mRNA. m6A is involved in regulating mRNA stability, splicing, and translation. However, the implications of m6A modification in human carcinogenesis remain poorly understood. In this study, we investigated the expression of m6A methyltransferases and demethylases in human hepatocellular carcinoma (HCC). We found the major m6A methyltransferase METTL3 was remarkably up-regulated in HCC. High METTL3 expression was associated with poor overall and disease-free survival in HCC patients. Consistent with the METTL3 overexpression, we found that mRNA m6A level was significantly elevated in human HCC. To investigate the roles of METTL3 in human HCC, we employed lentiviral-based shRNA and CRISPR/Cas9 systems to inactivation METTL3 in HCC cell lines. We showed that knockdown and knockout of METTL3 drastically suppressed HCC proliferation and migration in vitro and abolished HCC tumorigenicity and lung metastasis in vivo. In contrast, overexpression of METTL3 by CRISPR/dCas9 SAM system promoted HCC growth. Using RNA-seq and m6A-Seq, we identified tumor suppressor gene SOCS2 as a novel target of METTL3. We detected m6A modification in SOCS2 mRNA and the m6A modification was diminished upon METTL3 knockdown. m6A modification promoted SOCS2 mRNA degradation. We demonstrated that knockdown of METTL3, mutation of m6A modification sites, and treatment of demethylating agent augmented SOCS2 mRNA expression in HCC cells. In addition, knockdown of YTHDF2, an m6A reader protein, also rescued SOCS2 mRNA expression in HCC cells. The above findings together demonstrated that up-regulation of METTL3 lead to m6A modification of SOCS2, which in turn promotes SOCS2 mRNA degradation through YTHDF2 dependent mechanism. Our findings provided a proof-of-concept model to demonstrate the importance of aberrant m6A modification in epigenetic silencing of tumor suppressor genes.

#4322

Atrx inactivation drives motility and dysregulates differentiation in glioma cells of origin through global epigenomic remodeling.

Carla Danussi,1 Promita Bose,2 Pedro Silberman,2 John S. Van Arnam,1 Mark Vitucci,3 Oliver Tang,2 Adriana Heguy,4 Timothy A. Chan,2 Erik P. Sulman,1 Frederick Lang,1 Chad J. Creighton,5 Benjamin Deneen,5 C Ryan Miller,3 David J. Picketts,6 Kasthuri Kannan,4 Jason T. Huse1. 1 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Memorial Sloan-Kettering Cancer Center, New York, NY;_ 3 _University of North Carolina School of Medicine, Chapel Hill, NC;_ 4 _New York University School of Medicine, New York, NY;_ 5 _Baylor College of Medicine, Houston, TX;_ 6 _University of Ottawa, Ottawa, Ontario, Canada_.

Comprehensive genomic profiling in cancer continues to reveal frequent alterations in epigenetic regulators, firmly implicating chromatin biology in the oncogenic process. For instance, genetic inactivation of the SWI/SNF chromatin regulator ATRX (α-thalassemia mental retardation X-linked) represents a defining molecular alteration in both adult and pediatric malignant glioma, and occurs frequently in other cancers as well. ATRX deficiency has been linked to a wide spectrum of physiological dysfunction, including aberrant gene regulation, abnormal telomere maintenance, genomic instability, and aneuploidy. However, the precise oncogenic mechanism(s) induced by ATRX deficiency remain unclear, particularly those involving epigenomic dysregulation. To model these events in putative glioma cells of origin, we inactivated Atrx in primary murine neuroepithelial progenitors (mNPCs). Atrx loss, especially when coupled with Tp53 inactivation, promoted mNPC motility while also modulating differentiation state and potential, effectively recapitulating characteristic disease phenotypes and molecular features. Moreover, these phenotypes correlated with altered gene expression profiles in functionally relevant molecular networks (e.g. cell differentiation and migration). Integrating these transcriptional changes with shifts in chromatin accessibility occurring with Atrx deficiency, along with genome-wide Atrx distribution as determined by ChIP-seq, revealed highly significant spatial correlations between differentially expressed genes, regions of altered chromatin compaction, and genomic sites normally occupied by Atrx. Finally, target genes mediating specific Atrx-deficient phenotypes in vitro exhibited similarly selective misexpression in ATRX-mutant human glioma tissues and cell lines. These findings demonstrate that, in appropriate cellular and molecular contexts, ATRX deficiency and its epigenomic sequelae are sufficient to induce disease-defining oncogenic phenotypes.

#4323

**Ancestral obesity and early life metabolic dysfunction accelerates acinar-to-ductal reprogramming and PanIN/PDAC development in the P48** Cre/+ **/Kras** G120/+ **mouse model.**

Raquel Santana, Johan Clarke, Ana Cristina Curi, Ali Baird, M Idalia Cruz, Bhaskar Kallakury, Sonia de Assis. _Georgetown University, Washington, DC_.

Pancreatic cancer is virtually an incurable malignancy, with overall 5-year survival rates of about 7%. Excess body weight and metabolic dysfunction are believed to account for up to 50% of all pancreatic cancers. Epidemiological studies suggest that timing of obesity—and underlying metabolic dysfunction—onset is important in determining pancreatic cancer rates: Early and young adult abdominal obesity is more strongly associated with this cancer than obesity that develops later in life. However, the mechanisms behind this association are not understood. Children of obese parents are more likely to develop overweight, insulin resistance and diabetes. Here, we evaluated the effects of paternal overweight in the susceptibility of pancreatic cancer in offspring using the P48Cre/+ /KrasG120/+ mouse model of pancreatic cancer. LSL-KrasG120/+ and P48Cre/+ male mice were fed either an obesity-inducing (OID) or control (CO) diet for 8 weeks from weaning to sexual maturity. At the end of this period, OID- and CO-fed male mice were housed with female mice, with free access to CO diet, for 3 days. Pregnant dams were kept on the CO diet during pregnancy and after giving birth. Pups were weaned from mothers at 21 days of age, fed a standard chow diet for the extent of the study and weighed weekly. While both female and male OID offspring had higher weight at birth (p=0.005), only OID females were heavier at weaning (p=0.02) compared to CO. Those gender-specific differences were also observed in metabolic parameters with OID male, but not female, offspring showing impaired glucose tolerance (p<0.05) compared to CO. Interestingly, OID offspring also had significantly higher rates of acinar-to-ductal reprogramming as assessed by the ratio of CPA1,+Sox9+ pancreatic cells. Accordingly, we also observed accelerated development of high grade PanIN at 2 and 6-8 months of age in OID offspring compared to CO of the P48Cre/+ /KrasG120/+ genotype (p=0.02; p=0.007). Additionally, we found that the expression of Tenascin C (TNC), an ECM glycoprotein shown to suppress apoptosis, was upregulated in OID offspring (p=0.03). In line with that, OID offspring displayed increased (p=0.03) collagen content and decreased apoptosis (p=0.02) in pancreatic lesions compared to CO. In conclusion, an ancestral history of overweight through the paternal lineage and ensuing metabolic dysfunction in offspring increases their susceptibility to pancreatic cancer development. This increase was associated with higher acinar-to-ductal reprogramming and TNC expression, and reduced apoptosis in pancreatic lesions. While the exact mechanisms still need to be elucidated, our study offers some insights on why early obesity and metabolic dysfunction are more strongly associated to pancreatic cancer in humans.

#4324

Systematic characterization of A-to-I RNA editing in cancer development.

Han Liang. _UT MD Anderson Cancer Ctr., Houston, TX_.

Adenosine (A) to inosine (I) RNA editing, mediated by ADAR enzymes, is a well-established RNA modification mechanism. Recent studies show that A-to-I RNA editing introduces a large number of nucleotide changes in both coding and noncoding regions in cancer transcriptomes. First, to elucidate the contribution of RNA editing to proteomic diversity in human cancer, we performed an integrated analysis of The Cancer Genome Atlas (TCGA) genomic data and proteomic data from the Clinical Proteomic Tumor Analysis Consortium (CPTAC). We detected a considerable contribution of RNA editing in terms of amino acids changes induced per tumor sample in breast cancer. We validated some of these editing events at both RNA and protein levels, and further experimentally demonstrated the functional effects of some edited protein (e.g., increased proliferation, migration, and invasion of cancer cells in vitro). Second, we systematically characterized the miRNA editing hotspots across 20 cancer types from TCGA miRNA sequencing data and independently validated the vast majority of them by perturbation experiments in several cancer cell lines. These miRNA editing events show extensive correlations with key clinical variables and other molecular drivers. Focusing on the RNA editing hotspot in miR-200b, a key tumor metastasis suppressor, we found that the miR-200b editing level correlates with patient prognosis opposite to that pattern for the wild-type miR-200b expression. We further experimentally showed that in contrast to wild-type miRNA, the edited miR-200b can promote cell invasion and migration through its impaired ability to inhibit ZEB1/ZEB2 and acquired concomitant ability to repress new targets including LIFR, a well-characterized metastasis suppressor. Our results highlight the contribution of A-to-I RNA editing in cancer development and suggest its translational potential as both biomarkers and therapeutic targets.

#4325

Cisplatin treatment of testicular cancer introduces long-term changes to the epigenome.

Trine B. Rounge,1 Cecilie Bucher-Johannessen,1 Christian M. Page,2 Trine B. Haugen,3 Sophie D. Fosså,2 Hege S. Haugnes,4 Tom Grotmol1. 1 _Cancer Registry of Norway, Oslo, Norway;_ 2 _Oslo University Hospital, Oslo, Norway;_ 3 _Oslo and Akershus University College of Applied Sciences, Oslo, Norway;_ 4 _University Hospital of North Norway, Tromsø, Norway_.

Testicular cancer (TC) survival rates have increased substantially over the last decades, largely due to the introduction of cisplatin (CP) chemotherapy. This treatment is, however, associated with increased risk of developing metabolic syndrome (MetS), defined according to the National Cholesterol Education Program. We aimed to investigate if CP treatment were associated with epigenetic changes, and whether these changes render TC survivors susceptible for developing MetS later in life.

We included 279 Norwegian TC survivors with and without CP treatment and MetS, matched on age at blood sampling (Table 1). The TC survivors were re-examined on average 16 years after the orchiectomy, and for some patients, CP treatment. Whole genome DNA methylation profiles were measured with MethylationEPIC BeadChip and analyzed with the R package minfi. We used a linear regression model adjusting for smoking, age and cell type composition to identify CP differentially methylated CpG sites and logistic regression adjusting for smoking and age to identify differentially methylated CpG sites associated with MetS. Gene enrichment analyses were based on Fisher's exact test using KEGG and Reactome pathways.

32 and 15 differentially methylated CpG sites were associated with CP treatment after adjusting for multiple testing with False Discovery Rate (FDR) and Bonferroni correction, respectively. The PTK6-RAS-MAPk pathway was significantly enriched with the FDR significant CpGs (p-value < 0.1). We could not identify FDR significant differentially methylated CpGs associated with MetS with our sample size.

In conclusion, our results suggest that CP treatment has long-term effects on the epigenome. Genes involved in double-strand break repair, consistent with the cytotoxicity of CP treatment, are among the differentially methylated CpGs. Our top list of differentially methylated CpGs associated with MetS (lowest unadjusted p-values), should be further explored in a larger sample set of TC survivors. | |  | |

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

Table 1: Overview of sample characteristics | CP+ MetS+ | CP- MetS+ | CP+ MetS- | CP- MetS-

N | 61 | 34 | 115 | 69

Mean age at surgery | 31 | 31 | 27 | 30

Mean age at sample collection | 49 | 48 | 44 | 43

Time between surgery and sample collection | 17 | 18 | 17 | 14

#4326

The chromatin context and consequence of cisplatin-adduct DNA damage.

John Gallon, Erick Loomis, Nicholas Martin, James Flanagan, Robert Brown. _Imperial College London, London, United Kingdom_.

Improvements in survival rates for ovarian cancer lag behind those of other cancers. The combination of paclitaxel and carboplatin, along with debulking surgery, form the primary mode of treatment, however, the majority of patients eventually develop resistance. Epigenetic mechanisms have been implicated in the emergence of drug resistance, with platinum based therapies shown to induce DNA methylation changes at relapse which are associated with overall survival. Using a next generation sequencing approach, including a novel NGS method to map platinum adducts, we sought to investigate the association between platinum-DNA adduct formation and changes to chromatin conformation in the emergence of resistance to platinum therapy. We found chromatin conformation changes at the transcription start sites (TSS) of genes differentially expressed between platinum sensitive and resistant cell lines (p<0.05, n=3 pairs). Furthermore, we found non-TSS associated chromatin conformation changes which distinguish sensitive from resistant lines, and occur primarily in the direction of reduced accessibility in resistant lines (FDR <0.05, log2FC >±1.5). Finally, we found that 2kb windows enriched for platinum-DNA adducts are significantly more accessible than an equal number of random windows on the same chromosome (p<0.05, 1000 permutations). Taken together, these data suggest that platinum treatment may induce epigenetic changes, which reduce chromatin accessibility, rendering cells less susceptible to the formation of platinum-DNA adducts in subsequent treatments, resulting in cells becoming less sensitive to the drug.

#4327

**The epigenetic effects of benzo[** a **]pyrene exposure.**

Francesca Galea,1 Paul A. White,2 Volker M. Arlt,3 Paolo Vineis,1 James M. Flanagan1. 1 _Imperial College London, London, United Kingdom;_ 2 _Health Canada, Ottawa, Ontario, Canada;_ 3 _King's College London, London, United Kingdom_.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental carcinogens formed from the incomplete combustion of organic materials. Exposure to PAHs can be linked to at least 9 different cancer types, including lung and breast cancer. PAH are genotoxic and can form DNA adducts which can either be repaired or lead to mutations. DNA repair can lead to aberrant DNA methylation, however, few studies have investigated the link between PAH exposure and DNA methylation modifications. Here, we have used a mouse model to investigate the epigenetic consequences of PAH exposure. Mice (n=3/group) were treated by oral gavage with 0, 25, 50 and 75 mg/kg bodyweight/day of benzo[a]pyrene (BaP) for 28 days. Lung DNA from these mice was used to prepare Reduced Representation Bisulphite Sequencing (RRBS) libraries which were then sequenced using Illumina HiSeq2500to an average depth of 25x. For each mouse, DNA methylation was averaged over 500 bp tiled windows and differentially methylated regions (DMRs) were identified by comparing the controls (untreated) with all BaP-exposed mice (treated) irrespective of dose. In the treated vs untreated we identified 1815 windows (>25% methylation difference, p<0.05). Of these, we found that DMRs were under-represented in 5' untranslated regions (UTR), promoter, long interspersed nuclear elements (LINEs) regions and CpG islands compared with the genomic distribution of windows. DMRs were found to occur significantly more frequently in transcription termination regions. In addition, we found significantly more hypomethylation events in exon, intergenic, intronic and promoter regions, long terminal repeats (LTR) and short interspersed nuclear elements (SINE) and more hypermethylation in LINEs. Pyrosequencing validation on a selection of these DMRs is ongoing, using DNA from the same mice used in RRBS, plus mice exposed to BaP and other PAHs in lung and other tissues. In summary, these results show that PAHs have an effect on DNA methylation, and that these changes happen mostly within the gene body or intergenically, with significantly less differences observed at traditional gene expression regulators such as promoters. This

implies that the DNA methylation changes effected by BaP exposure may serve another purpose other than gene expression regulation.

#4328

hTERT protein expression, hTERT methylation and HPV infection in uterine cervical cancer.

Pablo Moreno-Acosta,1 Mónica Molano,2 Nicolas Morales,1 Alfredo Romero-Rojas,1 Oscar Gamboa,1 Jinneth Acosta,3 Raynner Alvarez,1 Nicolas Magné,4 Nubia Muñoz1. 1 _Instituto Nacional de Cancerología, Bogotá, D.C., Colombia;_ 2 _The Royal Women's Hospital, Parkville, Victoria, Australia, Australia;_ 3 _Universidad Nacional de Colombia, Bogotá, D.C., Colombia;_ 4 _Institut de cancérologie de la Loire-Lucien Neuwirth, Saint-Priest en Jarez, France, France_.

The analysis of expression of the hTERT protein, the methylation status of the hTERT gene as the type of HPV infection, will contribute to deepen the role played by HPV infection in the expression and methylation of hTERT. The study included a group of one hundred fifty-five samples of women with squamous cell carcinomas of the uterine cervix in stage FIGO IIB-IIIB treated at the National Cancer Institute in Bogotá, Colombia. The expression of the hTERT protein was detected by immunohistochemistry both at the nuclear level and at the cytoplasmic level. In 79.7% of the cases, the protein was expressed at the nuclear level and 78% at the cytoplasmic level. HPV16, the most frequent viral type found, was detected by means of a GP5+/GP6+ mediated PCR- RLB in 84.4% and in 82.2% of the cases that expressed hTERT at the nuclear level and at the cytoplasmic level, respectively; with respect to HPV species, both at the nuclear level and at the cytoplasmic level, the species most frequently found was the alpha 9 species (HPVs 16, 31, 35, 52.58) followed by alpha 7 (HPVs 18, 45.59) and alpha 6 (HPVs 56, 66). In the multiple correspondence analyzes, a close relationship was observed between the cytoplasmic expression as nuclear of hTERT and the alpha species 9. A distant relationship was also observed between the expression of the protein and methylation, as well as between the non-methylation and the alpha 7 family of HPV; likewise, a distant relationship was observed between the non-methylation of hTERT and the alpha 9 species. The analyzes carried out show a possible relationship between the expression of the hTERT protein and the alpha 9 HPV species.

#4329

Enhancer mapping in triple-negative breast cancer as a tool for novel biomarker and oncogene discovery.

Ryan Raisner, Russ Bainer, Karen Gascoigne. _Genentech Inc, South San Francisco, CA_.

Breast cancer is the most commonly diagnosed cancer in women, affecting ~8% of women in the U.S. in their lifetime. Triple-negative breast cancer (TNBC) represents a heterogeneous class of aggressive cancers that comprise 15-20% of all breast cancers. TNBCs characteristically do not have significant levels of estrogen receptor (ER), progesterone receptor (PR), or overexpression of human epidermal growth factor 2 (HER-2). Consequently, there is a lack of proven drug therapies and biomarkers for TNBC. Here we have used ChIP-SEQ combined with RNA-SEQ to profile TNBC cell lines for their super-enhancer patterns. These noncoding, trans-acting elements control gene expression and play an essential role in the establishment and maintenance of cell states. It has been suggested that a wide range of cancers arise in part due to enhancer dysregulation, and key oncogenes genes coming under control of super-enhancers. This can occur through translocations, amplifications, or creation of de novo transcription factor binding sites. We hypothesize that mapping tumor-specific super-enhancer patterns in TNBC will identify novel tumor-specific gene-regulatory networks, and dysregulated genes that contribute uniquely to the development of tumors. We have conducted a CRISPR-mediated drop-out screen on a number of TNBC cell lines for genes that are controlled by cancer-specific super-enhancers with the hope of identifying novel oncogenic drivers.

#4330

**Pan-cancer analysis reveals m** 6 **A modification is functionally important in cancer.**

Zhixiang Zuo, Yubin Xie, Yueyuan Zheng, Peng Nie, Shuai Jiang, QI Zhao, Yanyan Miao, Jian Ren. _Sun Yat-sen University, Guangzhou, China_.

N6-Methyladenosine (m6A) is the most abundant post-transcriptional modification in mammalian RNA molecules and has a critical role in many diseases, including cancer. However, the systematic investigation of the role of m6A in cancer is still lacking. We conducted a systematic analysis of the m6A-associated somatic mutations and expression of m6A writers and erasers in 3,401 cancer genomes of 12 cancer types from The Cancer Genome Atlas (TCGA) to find a potential role of the m6A machinery in cancer development. We revealed 1,973 genes with recurrent m6A-associated somatic mutations across multiple cancer types. We identified many known cancer genes, such as TP53, CTNNB1 and CDKN2A, as well as many candidate genes whose cancer-specific roles are less understood. Strikingly, liver cancer had the highest frequency of m6A-associated somatic mutations among the 12 cancer types, and these mutations were significantly enriched in metabolic pathways, such as retinol metabolism. In liver cancer, the abnormalities in m6A "writers" and m6A modification sites were significantly correlated with worse overall survival, independent of other clinical characteristics (e.g., tumor stage and race).

Implications: We for the first time consider somatic mutations relevant to the m6A modification sites to identify "driver" genes, thereby uncovering additional potential cancer "drivers" with rare mutations. Our study highlights m6A modifications as an important epigenetic mechanism of cancer development.

### Genomic Profiling of Tumors 3

#4331

High-throughput whole-genome sequencing of formalin fixed, paraffin-embedded tissues from colorectal cancer patients.

Matthew Kubit,1 Christina Wood-Bouwens,1 Sue Grimes,2 John Bell,2 GiWon Shin,2 Billy Lau,2 Mickey Miller,3 Kenneth Day,3 Helaman Escobar,3 Hanlee Ji,1 Lincoln Nadauld,3 Paul Van Hummelen1. 1 _Stanford University School of Medicine, Stanford, CA;_ 2 _Stanford University, Stanford, CA;_ 3 _Intermountain Healthcare, St. George, UT_.

Formalin-fixed, paraffin-embedded (FFPE) storage is a universally-adopted, cost-effective and long-term solution for tissue storage. Given the abundance of FFPE clinical samples, they provide a good source of tissue for genome sequencing studies. However, the extraction of nucleic acids from FFPE tissues yield highly fragmented DNA of variable quality. The poor quality and low yield make it difficult to generate adequate sequencing libraries for genomic studies.

For a population cancer genome study, we optimized a procedure for whole-genome sequencing on FFPE tumor and normal tissue of colorectal cancer patients. This pilot study aimed to first identify changes in DNA copy number using low-pass sequencing, with the additional goal of later adding structural variant and gene fusion detection and single nucleotide variant analysis with deeper sequencing. We processed, sequenced and analyzed 193 patients. These FFPE samples had varying storage characteristics. We optimized the initial extraction quality thresholds, DNA extraction and library preparation to generate high quality sequencing data.

We identified an initial sample quality and library threshold to hold the best correlation for sequencing quality. Optimal yield and DNA size from extracted FFPE was achieved by using Promega's LEV DNA FFPE Extraction Kit. Library prep optimization involved using mechanical shearing of DNA to increase overall library size for structural variant detection as well as using unique dual index adapters to identify index swapping artifacts during sequencing. An additional cleanup was also performed post-fragmentation to increase overall library size. Overall, we successfully developed a robust protocol for conducting whole genome sequencing on FFPE tumor samples.

#4332

Meta-analysis of molecular features associated with aggressive prostate cancer.

James C. Costello, Rani Powers, Andrew Goodspeed, Scott D. Cramer. _Univ. of Colorado Anschutz Medical Campus, Aurora, CO_.

The TMPRSS2:ERG fusion is the most common genomic alteration in prostate cancer, occurring in 40-50% of primary tumors. The frequency of the ERG fusion remains between 40-50% in metastatic tumors, supporting previous findings that the fusion can drive tumor development, but is not sufficient to drive aggressive, lethal disease. Large -omic datasets collected over thousands of patient tumors, such as The Cancer Genome Atlas (TCGA), paired with outcomes present the raw data needed to associate genetic aberrations with markers of aggressive disease. Accordingly, we performed a meta-analysis of over 2,000 patient samples to identify combinations of -omic features associated with aggressive ERG fusion-positive and ERG fusion-negative prostate cancer. The data were collected from 8 independent, publicly available prostate cancer cohorts and cover range of disease phenotypes (e.g., neuroendocrine, castration resistance). Our analysis consisted of training univariate Cox regression models separately for single genomic or pairs of genomic features within each independent dataset. Based on the availability, we explored gene expression, mutation, and copy number alterations, all used to build separate models. We then combined models for a genomic feature across all datasets by weighting individual coefficients by the inverse of their squared error in a fixed effects model; final results are reported as corrected p-values. We compared and contrasted results from combined models within data type (i.e., only gene expression) to models integrating multiple data types. Interestingly, we find that the genomic features associated with aggressive disease differed based on ERG status. We report genomic loci that are altered in all patients, but associate with aggressive disease only in patients with an ERG fusion. We also report loci that co-occur or are mutually exclusive of the ERG fusion and associated with aggressiveness. For example, SPOP mutations are known to be mutually exclusive of ERG fusions. We report a finer stratification of patients with SPOP mutations based on biochemical relapse. Our results present a compressive meta-analysis of molecular features in prostate cancer and identify novel molecular subtypes associated with aggressive disease.

#4333

**High incidence of** ERBB2 **amplification associated with microsatellite stable status in Chinese colorectal cancer patients.**

Sen Zhang,1 Yun Guo,1 Jun Jia,2 Weibin Shu,3 Hui Li,1 Yu Wang,3 Wei Zhao,4 Zusen Wang,4 Xueqing Yao,5 Tao Xiang,6 Yong Li,7 Ting Deng,8 Weifeng Wang,9 Kai Wang10. 1 _First Affiliated Hospital of Guangxi Medical University, China;_ 2 _Dongguan People's Hospital, China;_ 3 _Shandong Provincial Hospital Affiliated to Shandong University, China;_ 4 _The Affiliated Hospital of Qingdao University, China;_ 5 _Guangdong General Hospital, China;_ 6 _The First Affiliated Hospital, School of Medicine, Zhejiang University, China;_ 7 _Guangdong Provincial Hospital, China;_ 8 _Tianjin Medical University Cancer Institute and Hospital, China;_ 9 _Origimed, Shanghai, China;_ 10 _OrigiMed; Zhejiang University International Hospital, China_.

Introduction: Amplification of receptor tyrosine-protein kinase erbB-2 gene (ERBB2), also known as HER2, can serve as an effective biomarker for either dual targeted therapy with trastuzumab plus lapatinib or pertuzumab plus trastuzumab in treating refractory metastatic colorectal cancer (CRC) as shown in HERACLES and MyPathway clinical trials. Next-generation sequencing (NGS) based comprehensive genomic profiling (CGP) has increasingly proven to be a valuable analysis and detection method that can make a significant impact on cancer treatment decisions.

Experimental Procedure: Comprehensive genomic profiling was performed with a 450-gene next-generation sequencing (NGS) panel on both FFPE tumor and matched blood samples from a cohort of 123 Chinese colorectal cancer patients comprising 76 males and 47 females (median age was 60). All classes of genomic alterations including single-nucleotide variations, short and long insertions and deletions, copy number variations, and gene fusions were detected. Tumor mutational burden (TMB) and microsatellite instability (MSI) status were also determined by algorithms-based NGS data.

Results: The most common altered genes in the 123 CRC Chinese patients were TP53 (71%), APC (70%) and KRAS (50%). 19 pts were detected by NGS as MSI-high with a median TMB value of 119 muts/Mb, which was significantly higher than in the MSS subgroup (104 pts) with a median TMB value of 10 muts/Mb (p-value was 1.5E-08). In total, 9% of the CRC pts harbored ERBB2 amplifications (11/123) by NGS panel, and it was much higher than 3-4% in Western populations. Interestingly, all 11 ERBB2 amplified cases were presented exclusively in the MSS subgroup (11/104, 11%), and were all BRAF wild-type. Five out of the 11 cases were KRAS wild-type. In addition, 9 confirmed somatic ERBB2 point mutations in the COSMIC database were detected in 7 CRC pts (7/123, 6%), including T166M, S280F, A324T, R678Q, V812I, L725S, V842I and H848Y. Four pts were MSI-high.

Conclusion: CGP reveals significantly higher prevalence of both amplifications and point mutations of ERBB2/HER2 in Chinese CRC patients, compared with Western population. It may identify a subgroup of CRC patients potentially benefiting from specific targeted therapies including anti-HER2 therapies. Moreover, the highly exclusive presence of ERBB2 amplifications with MSS status could provide these patients with broader precision treatment opportunity.

#4334

Linked read whole genome sequencing reveals pervasive chromosomal level instability and novel rearrangements in brain metastases from colorectal cancer.

Li C. Xia, John M. Bell, Christina Wood-Bouwens, Daniel A. King, GiWon Shin, Stephanie Greer, Ian D. Connolly, Melanie H. Gephart, Hanlee P. Ji. _Stanford University, Stanford, CA_.

Little is known about the genomic features of brain metastases from colorectal cancer (CRC) and the contributing genetic factors. We conducted an analysis of large-scale structural aberrations, including chromosome aneuploidies, with a method called linked-read whole genome sequencing and a new somatic rearrangement detection software tool (ZoomX) to better understand the genomic architecture of brain metastases from CRC. We resolved on average 145 distal inter- and intra-chromosomal somatic junctions per sample with their exact haplotype information and basepair level breakpoints. In addition, we used a new haplotyping method based on these imbalances to generate cancer chromosome haplotypes of up to 146 Mbp long. We identified extensive chromosomal-level instability (CIN) in these cancer genomes, with an average of 90 large-scale copy number aberrations per sample in sizes ranging from hundreds of Kbp to hundreds of Mbp. A substantial fraction of CIN is likely attributable to earlier chromothripsis events. For example, we identified multiple chromothripsis events disrupting the loci of known cancer genes such as TP53, an essential colon cancer driver. We identified novel rearrangements including an oncogenic gene fusion; e.g. SET/DPP10 among these metastatic samples. A majority of chromosome arms demonstrated an allelic imbalance across all of the samples. In summary, the analysis revealed pervasive chromosome-level genome instability as a potential contributor to devastating brain invasion by CRC. It also demonstrated the advantages of linked-read whole genome sequencing. This approach is cost-effective and represents a high-resolution tool to assess genome-wide rearrangements and to generate megabase-scale haplotypes.

#4335

**High incidence of** POLE **and** POLD1 **mutations in Chinese colorectal cancer patients identified by comprehensive genomic profiling.**

Yinbo Chen,1 Sen Zhang,2 Wei Huang,2 Jun Guo,3 Weidong Guo,4 Fabo Qiu,4 Meihai Deng,5 Jingyu Cao,4 Samuel J. Klempner,6 Shun Yao,7 Jicheng Yao,8 Shou Mu,8 Ming Yao,8 Kai Wang,9 Weifeng Wang8. 1 _ZheJiang Cancer Hospital, China;_ 2 _First Affiliated Hospital of Guangxi Medical University, China;_ 3 _Xingtai People's Hospital, China;_ 4 _The Affiliated Hospital of Qingdao University, China;_ 5 _The Third Affiliated Hospital, Sun Yat-Sen University, China;_ 6 _The Angeles Clinic and Research Institute;The Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, CA;_ 7 _Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Science, China;_ 8 _Origimed, China;_ 9 _Origimed; Zhejiang University International Hospital, China_.

Introduction: The proofreading 3'-5' exonuclease activities of DNA polymerase-ε (POLE) and δ (POLD1) are critical for preventing mutations. POLE and POLD1 mutations can lead to high tumor mutational burdens (TMB), and predict responsiveness to immune checkpoint inhibitors (ICPI) in the absence of microsatellite instability (MSI). We therefore analyzed the genomic alterations of POLE and POLD1 in Chinese colorectal cancer (CRC) patients by comprehensive genome profiling (CGP).

Method: A cohort of 123 Chinese CRC patients (pts) comprising 76 males and 47 females (median age of 60) with both FFPE tumor samples and matched peripheral blood normal control were analyzed. The majority (40%) of the patients were stage IV, 26% were stage III, 18% were stage I/II and the rest were unknown. CGP was performed with a 450-gene panel. In addition to the genomic alterations, TMB and MSI were calculated by next-generation sequencing-based algorithms.

Result: CGP revealed a typical CRC mutation landscape with TP53 in 71%, APC in 70% and KRAS in 50% of the 123 Chinese pts. Fifteen percent of the pts (19/123) were MSI-H. Median TMB of all CRC pts was 11 muts/Mb, and the MSI-H subgroup had significantly higher TMB value compared to MSS subgroup (119 vs 10 muts/Mb, p-value is 1.5E-08). In total, 10% of the pts (12/123) harbored POLE and/or POLD1 known somatic mutations in the COSMIC database, including 10 POLE mutations in 8 pts and 7 POLD1 mutations in 7 pts. The 12 pts had a median age of 60 yrs (range 32-81), and 5/12 were females. Eight out of the 12 pts were associated with MSI-H. Additionally, 21 novel somatic short variants of uncertain significance (VUS) in 8 pts were detected (8/123=6.5%), and 5 pts were associated with MSI-H. Eight out of 123 Chinese CRC pts (6.5%) had POLE known mutations, which is significantly higher than 0.7% in Western population (p=9e-9). Four pts had loss-of-function (LOF) mutations. Two cases were POLE V411L associated with younger ages and the other two were truncations. All four CRC patients possess very high TMB compared to the whole cohort (305 vs 11 muts/Mb) while three of them are microsatellite stable (MSS) at stage III and IV.

Conclusion: Our small cohort suggests a higher incidence of known POLE and POLD1 mutations in Chinese CRC patients and warrants further study. Our results highlight the clinical utility of comprehensive genome profiling in identifying likely responders to immune checkpoint inhibitors.

#4336

Integrative genomic and transcriptomic analysis of leiomyosarcoma.

Priya Chudasama,1 Sadaf Mughal,1 Mathijs Sanders,2 Daniel Hübschmann,1 Inn Chung,1 Aurélie Ernst,1 Bernd Kasper,3 Hans-Georg Kopp,4 Sebastian Bauer,5 Karsten Rippe,6 Benedikt Brors,1 Marcus Renner,7 Peter Hohenberger,1 Claudia Scholl,1 Stefan Fröhling1. 1 _German Cancer Research CTR., Heidelberg, Germany;_ 2 _Erasmus Medical Center, Rotterdam, Germany;_ 3 _Mannheim University Medical Center, Mannheim, Germany;_ 4 _Eberhard Karls University, Tübingen, Germany;_ 5 _West German Cancer Center, Essen, Germany;_ 6 _Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg, Germany;_ 7 _Institute of Pathology, Heidelberg University, Heidelberg, Germany_.

Leiomyosarcomas (LMS) are malignant tumors of smooth-muscle origin that occur across age groups. The mechanisms underlying LMS development, including clinically actionable genetic vulnerabilities, are largely unknown, and few therapeutic options exist for LMS patients. To detect somatic mutations, copy number alterations, and structural rearrangements, we performed whole-exome and transcriptome sequencing of 49 and 37 LMS tumors, respectively, and performed integrative analysis. Recurrence analysis identified TP53, RB1, and ATRX as significantly mutated genes and various other cancer-associated genes mutated at low frequency, indicating substantial mutational heterogeneity. Copy number analysis revealed widespread chromosomal gains and losses and highly rearranged genomes in all tumors. Additionally, chromothripsis and whole-genome duplication were detected in 35% and 51% of cases, respectively. Principle component analysis and unsupervised hierarchical clustering of transcriptome data revealed three distinct subgroups of patients. Furthermore, we detected multiple non-recurrent fusion transcripts resulting from chromosomal rearrangements, many of which were predicted to result in loss of TP53 and RB1 function. In-depth analysis of these loci revealed protein-damaging microdeletions, intragenic or distal inversions, and exon skipping events as additional, previously unrecognized mechanisms of TP53 and RB1 disruption. Integration of whole-exome and transcriptome data demonstrated biallelic disruption of TP53 and RB1 in 92% and 94% of cases, respectively, and tumors with wildtype RB1 displayed loss of CDKN2A expression, overexpression of CCND1, or mutation of MAX resulting in CDK4 and CCND2 overexpression as alternative mechanisms of RB1 suppression. We also detected alternative lengthening of telomeres (ALT) in 78% of cases, and identified recurrent alterations in telomere maintenance genes such as ATRX, RBL2, and SP100, providing novel insight into the genetic basis of this mechanism. Finally, most tumors displayed hallmarks of "BRCAness", including alterations in homologous recombination DNA repair genes and enrichment of specific mutational signatures, and cultured LMS cells were sensitive towards olaparib and cisplatin. This comprehensive genomic and transcriptomic analysis has unveiled that LMS is characterized by mutational heterogeneity, genomic instability, near-universal inactivation of TP53 and RB1, and frequent whole-genome duplication. Furthermore, we have established that most LMS tumors rely on ALT to escape replicative senescence, and identified recurrent alterations in a broad spectrum of telomere maintenance genes. Finally, our findings uncover "BRCAness" as potentially actionable feature of LMS tumors, and provide a rich resource for guiding future investigations into the mechanisms underlying LMS development and the design of novel therapeutic strategies.

#4337

Identification of novel fusion gene, PUM1-TRAF3, as potent biomarker in bile duct cancer.

Dawoon E. Jung, Kahee Kim, Chanyang Kim, Soo Been Park, Jung Hyun Jo, Hee Seung Lee, Si Young Song. _Yonsei Univ. College of Medicine, Seoul, Republic of Korea_.

Background: Gene fusion occurs when a part of one gene fuses with a part of another gene by genome rearrangement and may possess oncogenic properties. Bile duct cancer (BDC) are one of the most lethal malignancy, but lack of proper diagnostic or prognostic markers. Our aim was to investigate BDC specific fusion genes found in patient's specimens and its clinical significance. Method : We extracted total RNA from five BDC tissues and normal tissues from the same patients. RNA sequencing was performed and the result data was analyzed using ChimeraScan, Jaffa or FusionCatcher softwares to detect gene fusion. The expression of the detected novel fusion gene was confirmed by ddPCR using tissues from the operated BDC patients and its clinical significance was analyzed with medical information of the patients. Result : We found 52 BDC specific fusion genes by RNA sequencing and we identified novel BDC specific fusion gene, PUM1-TRAF3 by bioinformatics analysis. Total 55 patients with BDC who underwent curative surgery were evaluated to investigate clinical significance of PUM1-TRAF3. There were 28 patients (50.1%) who expressed PUM1-TRAF3 by ddPCR. Patients with PUM1-TRAF3 presented higher rate of female gender (57.1 vs. 25.9, p=0.019), advanced stage (25% vs. 3.7%, p=0.025) and recurrence rate (64.7 vs. 48.1%, p=0.080) compared to patients without PUM1-TRAF3. Disease free survival (DFS) was 12.1 vs. 29.8 (p=0.078) and overall survival was 26.1 vs. 36.6 (p=0.239) between the patients with PUM1-TRAF3 and without PUM1-TRAF3. In multivariated analysis by cox-regression, expression of PUM1-TRAF3 (HR 2.86, p=0.023), advanced stage (HR 2.69, p=0.041), higher CA19-9 (HR 1.00, p=0.011) and positive resection margin (HR 4.57, p=0.002) were significantly associated with short DFS.Conclusion : In this study, we identified novel BDC-specific fusion gene, PUM1-TRAF3. Expression of PUM1-TRAF3 was confirmed in patient's tissues and associated with short DFS in operated BDC patients. PUM1-TRAF3 can be a potent biomarker in BDC.

#4338

Introduction of CALR mutations to a human erythroleukemia cell line, HEL, by CRISPR/Cas9 systems.

Shunichiro Yasuda, Satoru Aoyama, Daisuke Watanabe, Norihiko Kawamata. _Tokyo Medical and Dental University, Tokyo, Japan_.

[Introduction]Mutations of the calreticulin gene (CALR) are frequently found in patients with myeloproliferative neoplasms (MPN). Five-nucleotide insertion (Ins5) and 52-nucleotide deletion (Del52) in the last exon of the CALR gene are common mutations, generating an altered CALR protein with a mutated peptide sequence at the carboxy end due to the frame-shifts. Cell-lines with such CALR mutations have not been reported yet. CRISPR/Cas9 system is the novel technique commonly used in genetic engineering. CRISPR/Cas9 technology allows us to "edit" genome in mammalian cells. We introduced 46-nucleotide deletion (46Del) in the last exon of the CALR gene in a human erythroleukemia cell line, HEL. This 46Del abnormality lead to generation of an altered CALR protein with the abnormal peptide sequence at the carboxy end as seen in MPN cells with 52Del or Ins5. We compared the CALR-engineered HEL cells with the parental cells in the presence/absence of a Jak2 kinase inhibitor, ruxolitinib (RUX).

[Methods and Results]To introduce 46Del abnormality, we have designed two guide RNAs. We co-transfected vectors encoding the Cas9 with a fluorescence protein as a marker as well as the two guide RNAs into HEL cells using nucleoporation. Fluorescence protein positive cells were isolated and the single cells were cultured in 96-well plates for 3 weeks. Genomic DNA of each the clone was extracted, and genomic status of CALR was examined by PCR/sequencing. We have isolated one clone with 46Del (HEL-46DEL), of which expression of abnormal transcripts was confirmed by RT-PCR/sequencing. We also confirmed expressions of abnormal CALR protein by western blot analysis. Since the JAK2-STAT5 pathway is known to be activated in MPN cells, phosphorylation status of STAT5 in HEL and HEL-DEL46 was examined, showing comparable levels in each the line. Proliferation of HEL and HEL-DEL46 was examined with XTT assay, showing that the two lines proliferated similarly. RUX inhibited proliferation of HEL cells as well as HEL-DEL46. HEL cells have a JAK2 mutation which causes constitutive activation of the JAK2-STAT5 pathway. Introduction of the Del46 mutation into HEL cells might not enhance activation of the pathway because the JAK2 mutation already activates the pathway at the maximum level.

#4339

Molecular subtypes and novel genetic mechanisms of primary and acquired anti-EGFR resistance in colorectal cancer in the Prospect C biomarker trial.

Khurum Hayat Khan,1 Andrew Woolston,2 Georgia Spain,2 Louise Barber,2 Yatish Patil,2 Beatrice Griffiths,2 Reyes GonzalezExposito,2 Sonia Mansukhani,2 Matthew Davies,2 Sheela Rao,3 David Watkins,3 Francesco Sclafani,3 Jana Suntharanathan,1 Clare Peckitt,1 Ruwaida Begum,1 Isma Rana,1 Janet Thomas,1 Jacqui Oates,3 Annette Bryant,1 Andrew Wotherspoon,1 Nicos Fotiadis,1 Nasir Khan,1 Sebastian Guettler,2 Katharina von Loga,2 Naureen Starling,3 Ian Chau,3 Anguraj Sadanandam,2 David Cunningham,1 Marco Gerlinger4. 1 _The Royal Marsden Hospital/Institute of Cancer Research, Sutton and London, United Kingdom;_ 2 _Institute of cancer Research, London, United Kingdom;_ 3 _The Royal Marsden Hospital, Sutton and London, United Kingdom;_ 4 _Royal Marsden Hospital/Institute of cancer Research, London, United Kingdom_.

Despite stratification of only RAS wt CRCs for anti-EGFR antibody (aEa) therapy, many patients (pts) do not benefit and the molecular resistance (res) landscape remains incompletely understood. In order to decipher novel res mechanisms, we treated 40 RAS wt CRCs with single-agent aEa in a prospective trial and applied exome- and RNA-Seq to biopsies (bx) taken at baseline (BL) and at progression (PD). Among 20 BL bx from tumors with primary progression, 7 showed BRAF V600E mutations (mut), 3 harbored noncanonical BRAF D594K and/or KRAS L19F/A18D mut or KRAS amplifications (amp), 1 had a MEK1 mut and 1 was ERBB2 amp. Inactivating mut of the NF1 gene, a negative regulator of RAS, were found as novel mechanisms of primary res in 2 pts. No genetic drivers of primary res could be identified in 6/20 bx. Using transcriptomic analysis, 78% of pts with prolonged clinical benefit (≥16wks) displayed the Transit-Amplifying (TA) molecular subtype (Sadanandam et al., 2013). Stem-like (SL), goblet and inflammatory subtypes predominated (75%) among primary progressors. This significant enrichment in aEa sensitive CRCs (p=0.017) validates the TA subtype as a predictive biomarker. Exome- and deep Seq of PD bx from 13 pts who acquired res after prolonged clinical benefit detected KRAS mut or amp in only 2 cases. The FGFR2 ligand FGF10 was amplified in 1 PD bx and was validated in vitro as a novel mechanism of acquired res. No mut/amp of genes to which aEa res is usually attributed (RAS/RAF, EGFR, MET, ERBB2, MEK1) were found in the remaining 10 PD bx. Ultra-sensitive circulating tumor DNA-Seq at PD in 7 of these pts detected EGFR exodomain and RAS mut, including parallel evolution of multiple mut, in 3 cases. However, comparison to truncal TP53/APC mut showed that these res drivers were confined to small subclones and could therefore not explain the bulk of res. Overall, aberrations of RAS/RAF-pathway members and regulators were less abundant as drivers of acquired aEa res in this prospective trial than in reported retrospective series. Hence, we suspected additional novel mechanisms of acquired res. Molecular subtype switching from the aEa sensitive TA subtype to the comparatively insensitive SL subtype occurred in paired BL/PD bx from 3/7 pts who progressed after prolonged aEa benefit without detectable genetic res drivers in PD bx. Subtype switching was not observed in any of 6 analyzed BL/PD pairs from primary progressors. Considering the strong association of aEa sensitivity with TA subtype at BL, these data suggest subtype switching from TA to SL as a novel mechanism of acquired aEa res. This prospective trial revealed novel genetic (NF1 mut, FGF10 amp) and likely transcriptomic (TA to SL subtype switching) mechanisms of aEa resistance in CRC. These results should enable more precise aEa therapy stratification and may open opportunities to prevent res through SL subtype targeting strategies.

#4340

Integrating whole genome and transcriptome analysis to inform treatment decisions in the metastatic cancer clinical setting.

Martin R. Jones, Yaoqing Shen, Erin Pleasance, Elisa Majounie, Laura Williamson, Eric Zhao, Eric Chuah, Karen L. Mungall, Andrew J. Mungall, Richard A. Moore, Yussanne Ma, Stephen Yip, Howard Lim, Daniel Renouf, Steven J. Jones, Janesssa Jaskin, Marco A. Marra. _BCGSC, Vancouver, British Columbia, Canada_.

Genomic analysis is an established clinical tool for positioning patients to specific treatment rationales. However, given the vast range of genomic complexity that is observed between tumors the extent to which genomic characterization must be applied in order to generate a true picture of the actionable and informative genomic events in an individual's tumour is still a matter for intense debate. Historically, cost and the complexity of the bioinformatics analysis needed has limited the adoption of comprehensive genomic characterization. However with the emerging clinical significance of genomic signatures, mutation burden and immunogenomic profiling, information that can best be recovered from comprehensive genomic analyses, a better understanding of the utility of extensive genomic characterization of tumours in a clinical setting is needed. For the past 6 years, the Personalized Oncogenomics Project (POG) at the BC Cancer Agency has used paired tumour/normal Whole Genome and Transcriptome Analysis (WGTA) to comprehensively characterize tumour samples from patients with incurable metastatic cancer to inform treatment planning for each patient within a clinically relevant timeframe. To date tumours from over 700 patients have been genomically profiled using this approach. These data represent a cohort of tumours from the post-treatment metastatic setting that have been comprehensively characterized at a whole genome level augmented by comprehensive clinical annotation, including information about the primary diagnosis, progression of the disease and treatment history, as well as follow-up information including the response of the tumour to genome analysis-informed therapy. We will discuss findings from this powerful genomic cohort focusing on aspects that are particularly well represented by using a WGTA approach including; mutation signatures of biological and clinical significance, integration of expression data to complement genomic findings and highlighting the discovery of novel NRG1 gene fusions as a demonstration of how using a comprehensive multi-omic approach for genomic analysis to infer the oncogenic impact of novel genomic events can impact patient care.

#4341

Large mutational spectrum of cutaneous melanoma in a Brazilian population: The experience of A.C. Camargo Cancer Center.

Mariana Petaccia de Macedo, Felipe Fidaldo, Clovis Lopes Pinto, João Pedreira Duprat, Maria Dirlei S.f Begnami, Dirce Maria Carraro, Isabela Werneck Cunha. _A.C. Camargo Cancer Center, Sao Paulo, Brazil_.

Introduction: Cutaneous melanoma (CM) is an aggressive type of malignancy, with elevated mortality rates. The Cancer Genome Atlas Network (TCGA) recently reported the molecular classification of CM and divided these lesions according to their somatic mutation profile in 4 categories: BRAF, NRAS or NF1 mutated, and also the triple-negative group. The aim of this study is to describe the spectrum of molecular alterations from a Brazilian population of CM patients.

Methods: DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tissue from primary and metastatic melanoma samples from A.C. Camargo Cancer Center molecular pathology section. Next-generation sequencing (Ion Torrent®) targeting a 14-gene panel was performed, targeting the following genes: BRAF (códon 600), NRAS (códons 12, 13, 61, 117 and 146) e KIT (éxon 11, 13, 17), KRAS, CTNNB1, IDH1, EGFR, HRAS, PDGFR-a, PIK3CA, RET, JAK2, MET, and CDH1.

Results: 131 cutaneous melanoma samples from different patients were sequenced. The majority of the samples (57%) showed mutation in at least one gene. BRAF was the most frequent detected mutation, found in 40% of the cases, and p.V600E, p.V600K and p.V600R represented 90%, 8% and 2% of BRAF mutations, respectively. NRAS mutation was detected in 10% of the cases, and among those 88% involved codon 61. KIT mutation was identified in 10% of the cases. Mutations in KRAS, CTNNB1, PDGFRa, PIK3CA, EGFR and HRAS were identified in 5%, 4%, 4%, 3%, 2% and 1% of the cases, respectively. No mutations in IDH1, JAK2, MET or RET was identified. Most of the mutated cases showed mutation in only one tested gene (71%), while 14%, 10% and 5% showed mutations in 2, 3 or 4 genes respectively. None of the cases showed simultaneous mutation involving combinations of BRAF, NRAS or KIT mutations.

Discussion and Conclusion: The BRAF, NRAS and KIT mutation frequency in this population with CM diagnosis is similar to the one described in the world literature for this disease, as well as the distribution of the most frequent mutated regions and specific types of mutations. These results add the mutational spectrum of CM of a large Brazilian cohort to the world literature. Treatment of CM with targeted therapies aiming the MAP-Kinase pathway, and especially blocking BRAF, are a reality in the clinical oncology routine for treating CM patients, but there are still many obstacles in order to fully understand the resistance mechanism and the possibility of drug combinations. Additionally, new targeted agents are under development, such as the ones for NRAS mutated cases, and in this way the understanding of the large mutational spectrum of melanoma is of extreme importance in the clinical scenario.

#4342

Inverted sinonasal papilloma and associated carcinoma-transcriptome analysis and out-hoxing developmental genes.

Achim H. Bell, Ehab Y. Hanna, Randal S. Weber, Victor G. Prieto, Diana Bell. _UT MD Anderson Cancer Ctr., Houston, TX_.

Introduction Sinonasal papillomas, a group of benign epithelial tumors of the sinonasal tract, are classified into 3 distinct histologic types: exophytic, endophytic and oncocytic . Associated clinical problems include a tendency towards local destruction, recurrence and malignant transformation. While controversy persists in determining the optimal therapeutic approach in a given patient, prognostic markers identifying patients who will benefit from more aggressive treatment and novel molecular targeted therapies are urgently needed. The majority of endophytic papillomas require a lengthy period for malignant transformation, but the trigger underlying the malignant changes remains obscure. The present study aimed to identify underlying mechanisms in the malignant transformation of sinonasal inverted papillomas using RNA seq.

Material and methods Samples from 36 consecutive patients formed the study material and were microdisected from FFPE. Pools of RNA were subjected to expression profiling using whole-transcriptome shotgun sequencing. For validation, differentially expressed candidates were analyzed by IHC.

Results The studied cohort showed a continuum, with 14 sinonasal papilloma (11 inverted type, 2 oncocytic and 1 exophytic type), 2 sinonasal papilloma with dysplastic changes, 3 carcinoma in situ and 17 invasive squamous cell carcinoma arising in a papillomatous background (majority being associated with inverted type papilloma). Differential gene expression profiles from 14 papillomas, 14 squamous carcinomas versus 4 normal sinonasal mucosa were generated and compared to each other. 1447 genes and noncoding transcripts were detected as differentially expressed with fold change >2 and false discovery rate < 0.035. These genes and transcripts were annotated and computationally analyzed using the Ingenuity Pathway Analysis program. The highest expressed genes and potential drivers were predominantly development- and differentiation-related genes. To validate the RNA-Seq results, we used IHC assessing the protein expression of 5 highly upregulated genes considered important. These candidate genes were HOXA9, EN1, DUX4, CA-IX, and CK5/6 with expressions at 30%, 50%, 60%, 60% and 95% respectively.

Conclusion We assume that sinonasal papillomas are mainly defined by high number of overexpressed developmental/homeobox genes, which provide the potential for transformation/ plasticity, along with differentiation and proliferation behavior of neoplastic cells. The data form the basis for understanding cell fate determination and cellular homeostasis in the normal sinonasal mucosa and the contribution of different mucosal components to the etiology/ molecular pathology of sinonasal dysplasia and carcinoma.

#4343

Molecular identification of bladder cancer gene expression subtypes.

In-Sun Chu,1 Bic-Na Song,1 Sun-Hee Leem2. 1 _Korean Bioinformation Ctr., Daejeon, Republic of Korea;_ 2 _Dong-A University, Busan, Republic of Korea_.

Bladder urothelial carcinoma (BLCA) comprises two main types: non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC). To discovery the subtype of BLCA, we used gene expression data from 165 patient cohort (102 NMIBC, 63 MIBC). Three other gene expression data sets including the Sweden 1 cohort (97 NMIBC, 45 MIBC), the Sweden 2 cohort (213 NMIBC, 93 MIBC), and 408 MIBC sample from The Cancer Genome Atlas (TCGA) and 476 sample (460 NMIBC, 16 MIBC, the multicenter European UROMOL consortium). For selection of subtype-specific gene set, we used significance analysis of microarrays (SAM). Then, subtype-specific genes were trained by prediction analysis for microarrays (PAM) to build a classifier. Clustering analyses were applied to gene expression data of training set identified four molecular subtypes with different clinical outcomes. The majority of patients with class 1 were the low-grade NMIBC and associated with the prognosis of NMIBC, for cancer-specific survival (P =0.003, log-rank test). Class 2 patients, characterized by FGFR3 overexpression and downregulated immune pathways, showed the intermediate prognosis. Class 3 tumors showed high expression of cell-cycle genes and downregulated notch signaling pathway. Furthermore, high-grade NMIBC were predominantly found in class 3, indicating that NMIBC patients classified as class 3 would be more likely to progress to MIBC. Class 4 tumors, mainly composed of MIBC, were characterized by high expression of immune-related genes which is positively associated with potential response to immunotherapy. We further validated the BLCA subtypes in four independent data sets (n = 1,471), including the RNA-seq data set, by applying the 786-gene classifier onto the data sets. As expected, the progression rate of NMIBC patients classified as class 3 was significantly increased compared with other three classes in the RNA-seq data set (P < 0.001, log-rank test).

#4344

Low mitochondrial copy number induces resistance to chemotherapy in esophageal cancer.

Moyuru Yamada, Koji Tanaka, Yasunori Masuike, Tomoki Makino, Tsuyoshi takahashi, Yukinori Kurokawa, Makoto Yamasaki, Kiyokazu Nakajima, Masaki Mori, Yuichiro Doki. _Osaka University, Japan_.

Introduction: Esophageal squamous cell carcinoma (ESCC) is the one of the deadliest gastrointestinal cancers. The important problem in treating ESCC is resistance to chemotherapy. A few previous reports showed that mitochondrial DNA (mtDNA) copy number was decreased in esophageal squamous cell carcinoma (ESCC). However, it is unclear whether alteration of mtDNA copy number affects the effect of chemotherapy for ESCC. In the present study, we investigated the relationship between mtDNA copy number and the effect of chemotherapy in ESCC.

Methods: To analyze the mtDNA copy number of surgically resected primary tumors from ESCC patients, qPCR for Cytochrome Oxidase I was performed. Human ESCC cells (TE8 and TE11) with decreased mtDNA copy number were established by knockdown of mitochondrial transcription factor A (TFAM). Those cells were treated with 5FU, cisplatin and docetaxel. The response of chemotherapy was accessed by cell viability assay (WST assay) and apoptosis assay (flow cytometry). To investigate the mechanism of resistance of chemotherapy, expression of EMT-related genes such as CDH1, CDH2, vimentin and zeb1 was measured by qPCR. Wound healing assay and invasion assay were performed to access the cell migration and invasion.

Results: Patients were categorized into higher and lower subgroups according to the median value of the mtDNA copy number. Lower mtDNA copy number group was significantly correlated with tumor depth and pathologic response of chemotherapy. ESCC patients with lower mtDNA copy number had significantly poorer 5-year recurrence-free and overall survival than higher group. By shRNA knockdown of TFAM gene expression, mtDNA copy number decreased to 40% in TE8 and 60% in TE11 compared with non-target control cells. Viability of mtDNA-depleted TE8 and TE11 treated with chemotherapy was higher than that of control cells. Apoptosis assay showed the percentages of apoptosis induced by chemotherapy in mtDNA-depleted TE8 and TE11 were lower than those of control cells. Invasion assay and wound healing migration assay showed that mtDNA-depleted TE8 and TE11 were significantly more invaded and migrated than the control-sh cells.

Conclusions: These results suggest that low mitochondrial copy number induces resistance to chemotherapy in esophageal cancer.

#4345

Molecular comparison of imatinib-naïve and resistant gastrointestinal stromal tumors: Differentially expressed microRNAs and mRNAs.

Azadeh Amirnasr,1 Caroline M. Gits,1 Patricia F. Kuijk,1 Marcel Smid,1 Maria Debiec-Rychter,2 Stefan Sleijfer,1 Erik A. Wiemer1. 1 _Erasmus Medical Center, Rotterdam, Netherlands;_ 2 _KU Leuven, Leuven, Belgium_.

Background and aim: Gastrointestinal stromal tumors (GISTs) are rare mesenchymal tumors of the gastrointestinal tract. Gain-of-function mutations in KIT or, less frequently, PDGFRA underlie the pathogenesis of the majority of these tumors. The introduction of imatinib mesylate, a selective tyrosine kinase inhibitor, has dramatically improved the outcome for GIST patients. However, half of the advanced patients treated with imatinib eventually progress and acquire resistance within two years of treatment, underscoring the need to get better insight into the resistance mechanisms. In this context we examined the miRNA and mRNA expression profiles in primary (imatinib-naïve) and imatinib resistant GIST samples.

Material and Methods: Fifty-three frozen GIST samples derived from various anatomical sites (small intestine, stomach, colon) harboring a KIT mutation including exon 9 (n=11), exon 11 (n=41) and exon 17 (n=1), were analyzed. Total RNA was isolated from imatinib naïve (IM-n; n=33) and resistant (IM-r; n=20) tumors. The resistant tumors bearing secondary mutations in exon 13 (n=3), exon 17 (n=6) or no secondary mutations (n=11). The miRNA expression profiles were determined using LNA™ oligonucleotide arrays (Exiqon) capable of detecting 725 human miRNAs. Furthermore, from a subset of samples (IM-n; n=14; IM-r; n=15) the mRNA expression profile was established using Affymetrix U133A oligonucleotide arrays. Using the mRNA expression data and the Biocarta and KEGG databases, global testing identified a number of biochemical pathways containing genes that are differentially expressed between the primary and progressive tumors.

Results: Thirty-five differentially expressed miRNAs (P<0.009, FDR<20%) between IM-n and IM-r GIST samples were identified. MiR-30c, miR-181a and miR-144 were among the most significantly different, with p values of 0.006, 0.006 and 0.003, respectively. Analysis of the mRNA data has revealed the up-regulation of genes (P <0002, FDR<30%) involved in cell cycle, DNA replication and cancer related pathways (i.e. proliferation, genomic instability, resistance to chemotherapy, evading apoptosis, etc.) in IM-r samples. To further elucidate the possible involvement of the deregulated miRNAs in the regulation of the indicated genes, the most significantly differentially expressed genes were correlated to miRNA expression. Negative correlations indicate miRNA mediated gene regulation, and were further examined in target prediction programs.

Conclusion: A molecular comparison between primary, IM-n and IM-r GIST samples revealed significant differentially expressed miRNAs and mRNAs. Bioinformatic analyses provided insight into biochemical pathways, and their putative regulation by miRNAs, that may contribute to imatinib resistance as observed in GIST patients.

#4346

Integrated whole genome profiling of the immune tumour interaction identifies predictive biomarkers of checkpoint inhibitor response in metastatic cancer.

Hillary Pearson, Laura Williamson, Erin Pleasance, Scott Brown, Emma Titmuss, Martin Jones, Stuart Zong, Payal Sipahimalani, Yussanne Ma, Steve Jones, Robert Holt, Marco Marra, Janessa Laskin. _BC Cancer, Vancouver, British Columbia, Canada_.

Immune checkpoint inhibitors are fast becoming a key therapy in the medical oncologist's toolbox across a diverse array of tumour types. The excitement surrounding the utility of these emerging agents is marred by the heterogeneous response observed among patients, highlighting the urgent need for clinically applicable predictive biomarkers. Significant progress has been made in identifying potentially relevant biomarkers including mutation burden and immune infiltration. However, it is still unclear which biomarkers are most predictive across various disease types, and how to integrate various markers of response and resistance for clinical interpretation. We hypothesized that comprehensive genomic profiling, combined with clinical data, would reveal differences in the immuno-oncologic phenotype that could be used as biomarkers for response to checkpoint inhibitor therapy. The Personalized OncoGenomics (POG) study at BC Cancer performs whole genome and transcriptome sequencing of metastatic disease across a diverse array of cancer types to comprehensively characterize cancers and inform clinical therapeutic decision-making. Here we performed in-depth genomic profiling of 64 POG patients who received immune checkpoint inhibitors encompassing multiple disease types including skin, lung, breast, pancreatic and colorectal cancers and sarcomas. Single nucleotide variants, structural variants, copy number alterations, and RNA expression derived from whole genomic and transcriptomic data were used to characterize genomic instability, neoantigen landscape, immune infiltration, and other potential biomarkers of clinical response to checkpoint inhibitors. This collection of putative immune-oncologic biomarkers were integrated into a multivariate model to stratify markers of the tumour immune response. Our analysis shows limited association between PD-L1 expression and checkpoint inhibitor response, consistent with the inadequate effectiveness of this as a universal marker, and shows stronger association between signatures of T cell infiltration based on RNA-Seq data. We also observed patients with low mutation burden that respond to checkpoint inhibitors and show high levels of immune infiltration, and cases with high mutation burden that harbour mechanisms of resistance including reduction in predicted neoantigen diversity. Additional mechanisms of therapeutic resistance observed in post treatment biopsies highlight disruption of antigen presentation and JAK1 mutations in resistant tumours. Our study helps to define which features distinguish patients most likely to respond to checkpoint inhibitors, and uses these features in selecting patients for immunotherapy treatment within the POG clinical trial.

#4347

Integrated single-cell DNA and RNA analysis of intratumoral heterogeneity and immune lineages in colorectal and gastric tumor biopsies.

Billy Lau,1 Noemi Andor,2 Anuja Sathe,2 Christina Wood-Bouwens,2 George Poultsides,2 Hanlee Ji2. 1 _Stanford Univ. School of Medicine, Palo Alto, CA;_ 2 _Stanford Univ. School of Medicine, Stanford, CA_.

Intratumoral heterogeneity and immune cellular infiltration are important indicators for both prognosis and prediction of treatment efficacy. Intratumoral heterogeneity, arising from the backdrop of ongoing mutation accrual and genome instability, drives Darwinian evolutionary selection for therapy-resistant cells. Infiltrating immune cell lineages are repressed in their function, thus enabling tumors to evade the immune system. Currently, conventional bulk sequencing does not resolve features from individual cancer cells and other cellular components present in a tumor. Thus, one loses critical granular information describing the overall complexity of the tumor and its local microenvironment. To achieve a complete analysis of intratumoral heterogeneity and immune response to a tumor, we developed and applied a strategy for parallel single cell DNA-Seq and single cell RNA-Seq on excised tumors - our approach used tumor biopsies obtained directly after surgery with immediate processing and dissociation into single cell suspensions for genomic analysis.

For thousands of cells, single-cell DNA-Seq analysis was used to generate genome wide copy number results and total quantification of chromosomal aneuploidy. With these results, we determined the intratumoral phylogenetic distributions at single cell resolution. Single-cell copy number variant detection performance in cancer cell lines was cross-validated with traditional whole genome sequencing and karyotyping from established cell lines. Remarkably, we distinguished between replicating and non-replicating cells - this was defined by discrete and localized fluctuations in read coverage across the genome. Our findings were confirmed by flow cytometry analysis.

Single-cell RNA-Seq provided an overview of the different subpopulations with distinct transcriptional phenotypes. We resolved multiple subsets of tumor epithelial cells reflecting transcriptional heterogeneity as well as stromal components including endothelial, muscle, fibroblast and immune cells. Among immune cells, we further resolved phenotypes including T, B, NK, dendritic cells and monocytes. We also detected a small number of cells with expression of markers of immune exhaustion. Overall, our study successfully demonstrated the feasibility of conducting integrated single-cell DNA-Seq and RNA-Seq on patient tumors. As supported by our results, single cell genomics offers an unprecedented perspective on the genomic and cellular complexities of primary tumor biopsies.

#4348

In depth molecular analysis of 183 primary DCIS lesions and 78 subsequent invasive breast cancers.

Lindy Visser, Marlous Hoogstraat, Asli Kucukosmanogl, Frank Nieboer, Michiel de Maaker, Petra Kristel, Lennart Mulder, Lotte Elshof, Flora van Leeuwen, Emiel Rutgers, Marjanka K. Schmidt, Esther Lips, Jelle Wesseling. _Netherlands Cancer Institute, Amsterdam, Netherlands_.

Background. Ductal carcinoma in situ (DCIS) is a potential precursor of invasive breast cancer (IBC). As we are currently unable to predict which DCIS lesions will progress to IBC, almost all women with DCIS are treated with surgical treatment, adjuvant therapy, or both. Yet, most DCIS lesions will never progress, implying overtreatment in many women. To increase our knowledge of DCIS and improve DCIS risk stratification, we performed a molecular characterization of a large series of DCIS lesions with long-term follow-up.

Patients and methods. We made use of a DCIS case-control series, nested in a nation-wide population-based cohort of Dutch women diagnosed with primary DCIS and treated with breast conserving surgery alone between 1989-2005. Mean follow-up time was 12.0 years. DNA and RNA were simultaneously extracted from FFPE laser-microdissected tissue fragments of 183 primary DCIS lesions (100 associated with subsequent invasive recurrences; and 83 which remained free of invasive recurrence during the same follow-up duration) and 78 matched subsequent ipsilateral IBCs. RNA sequencing (RNAseq) and low coverage DNA sequencing (CNVseq) were performed to identify differentially expressed and aberrated genes between DCIS with and without an invasive recurrence and to compare primary DCIS and its subsequent IBC.

Results. After exclusion of samples with poor data quality, 158 DCIS lesions and 72 IBCs (88%) were included for CNVseq analysis and 155 DCIS lesions and 68 IBCs (85%) were included in the RNAseq analysis. CNVseq analysis identified copy number aberrations of known breast cancer-related genes. Amplification of FGFR1, CCND1, ZNF217, and ERBB2 was found in 16, 19, 22, and 44 DCIS samples, respectively. Furthermore, loss of CBFB, CDKN2A, CDKN2B, TP53, and MAP2K4 was found in 10, 11, 11, 11, and 14 DCIS samples, respectively. Deletion of CBFB, TP53, and CDH1 was more often detected in DCIS with subsequent IBC as compared of DCIS without invasive recurrence. When comparing DCIS and subsequent IBC, aberrations of breast cancer-related genes were mostly comparable. RNAseq analysis revealed genes, differentially expressed between DCIS with and without subsequent IBC, which were enriched in immune-related pathways.

Conclusion. We performed the first large-scale analysis of primary DCIS lesions with and without subsequent invasive recurrences, with long-term follow-up. Our data show differences in aberrated genes between DCIS with and without subsequent IBC, whereas DCIS and subsequent IBC were more alike. Furthermore, our transcriptomic data suggest that immune-related pathways are involved in DCIS progression. More extensive data analysis will be presented at AACR 2018. The results of our unique study add to the current understanding of DCIS and allow optimized risk stratification of DCIS in the near future.

#4349

Higher level of tumor mutational burden and 11q13 amplification in Chinese hepatocellular carcinoma patients.

Xiaofeng Tang,1 Le Fan,1 Gang Chen,2 Weidong Guo,3 Fabo Qiu,3 Jun Wang,4 Jun Guo,5 Lijuan Chen,6 Peng Zhang,6 Weiwei Shi,6 Kai Wang,7 Weifeng Wang6. 1 _The First Affiliated Hospital, School of Medicine, Zhejiang University, China;_ 2 _The First PeopIe's Hospital of KunMing and Affiliated Calmette Hospital of Kunming Medical University, China;_ 3 _The Affiliated Hospital of Qingdao University, China;_ 4 _People's Hospital of Hunan Province, China;_ 5 _Xingtai People's hospital, China;_ 6 _Origimed, China;_ 7 _Origimed; Zhejiang University International Hospital, China_.

Introduction: The approval of immune checkpoint inhibitor (ICPI) for hepatocellular cancer (HCC) has changed the landscape of HCC treatment, which is the second most common and lethal malignancy in China. Several genomic features associated with ICPI efficacy in various types of cancers has been reported as potential predictive biomarkers. The application of comprehensive genomic profiling (CGP) on HCC might be useful in identifying ICPI responders or avoiding adverse effect. Experimental procedure: Comprehensive genomic profiling of 107 advanced HCC was performed on the FFPE tumor samples and matched blood by using a 450-gene next generation sequencing assay. The cohort of HCC patients comprises 86 males (median age = 56) and 21 females (median age = 52) whose gender disparity is in the normal range of HCC epidemiological statistic study from Asia-Pacific region. The ICPI therapy related genomic alterations such as tumor mutational burden (TMB) and 11q13 amplification were subsequently analyzed. Result: CGP revealed signature genomic alterations in HCC as previously reported but with exceptional higher frequency of TP53 mutations at 66.3% of the pts and relatively low rate of TERT variants at 32.7%. Higher level of TMB high values (>20 muts/Mb) was found in a significantly larger portion of the Chinese HCC patients (N=10, 9.3%), compared to only 1% in Western population (p-value 1.5E-05). The median TMB of the Chinese HCC cohort was 7.2 muts/Mb. In addition, 11q13 amplifications (FGF3, FGF4, FGF19 and CCND1) were detected in 6.5% of the Chinese HCC patients, which was associated with ICPI hyper-progression in a variety of cancers. The 7 patients with 11q13 amplifications had a median age of 65 yrs (range 52-78) which is significantly higher than the median age of the rest cohort at 55 yrs (range 28-79, p=0.02). The median TMB was 8 muts/Mb. Conclusion: Our preliminary results found that TMB high values were more commonly detected in Chinese HCC patients compared to Western population. It may indicate that more Chinese HCC patients could benefit from ICPI treatments. However, the high incidence of 11q13 amplifications related to hyper-progression response in the same cohort was observed. Therefore, the application of comprehensive genomic profiling companion diagnosis might be necessary in guiding the regimen and ICPI treatments.

#4350

Integrative and multiregional molecular analysis of localized, high grade prostate cancer treated with neoadjuvant androgen deprivation treatment.

Stephanie A. Wankowicz,1 Michaela Bowden,1 Rosina Lis,1 Claire Margolis,1 Dimitri Livitz,2 Ignaty Leshchiner,2 David Liu,1 Meng Xiao He,1 Zhenwei Zhang,1 Gad Getz,3 Mary-Ellen Taplin,1 Eliezer Van Allen1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Broad Institute, Cambridge, MA;_ 3 _Broad Institute, Boston, MA_.

Neoadjuvant intense androgen deprivation treatment (ADT) in localized, high risk prostate cancer (HG PCa) has varied degrees of response. While some patients have an outstanding response with minimal residual disease [MRD; ≤0.5mm of tumor at radical prostatectomy (RP)], others have no response but delay potential curative treatment. Previous studies have demonstrated that HG PCa is heterogenous, however previous studies have not investigated whether heterogeneity itself or specific genetic molecular features correlate with response to intense neoadjuvant ADT. Herein, we provide an integrated molecular analysis of the spatial heterogeneity of HG PCa treated with neoadjuvant ADT. We examined 53 pre-treatment biopsy samples (n=35 biopsies containing tumor, n=18 normal prostate tissue) from 14 patients with matched blood normals who were treated with an intense neoadjuvant regimen of lupron plus abiraterone/enzalutamide. Patients were stratified as exceptional responders (n=8; MRD at RP) or non-responders (n=6; pT3 or lymph node positive at RP). We performed whole exome and whole transcriptome sequencing on multiple, spatially heterogeneous biopsies within each patient to examine the role of heterogeneity in response to therapy. We called somatic and germline variants, inferred mutational clonality, phylogenetic relationships, called genetic fusion, and analyzed differentially expressed genes. After QC, we observed well known prostate driver mutations (SPOP, ATM, TP53, FOXA1, PTEN, and APC) across the entire cohort (n=14 patients). Within each individual, all driver mutations were clonal across tumor cores, but the proportion of all mutations that were always clonal varied widely (0.08-0.57), median 0.3). Among known drivers, SPOP mutations were only observed in exceptional responders (4/8 v. 0/6), and TP53 and PTEN mutations were only observed in non-responders (4/6 v. 0/8). Responders and non-responders did not differ by mutational or copy number burden. Mutational heterogeneity varied greatly between samples, however this did not split between responders and non-responders. In terms of gene expression, exceptional responders showed upregulation of androgen and estrogen response pathways, while non-responders had increased expression of E2F targets and cell cycle pathways. We confirmed that a subset of localized, HG PCa are molecularly heterogeneous in terms of mutations and expression. We are presently underpowered to explain the genomic differences between excpetional responders and non-responders. However, molecular heterogeneous and homogenous HG PCa tumors can respond to intense neoadjuvant ADT therapy. We are currently analyzing a larger cohort.

#4351

Intratumoral heterogeneity and process of evolution of intrahepatic cholangiocarcinoma.

Akihiro Kitagaw,1 Hisateru Komatsu,1 Yosuke Kuroda,1 Syuhei Ito,1 Takaaki Masuda,1 Hidetoshi Eguchi,1 Hidetoshi Eguchi,2 Yuichiro Doki,2 Masaki Mori,2 Koshi Mimori1. 1 _Kyusyu University Beppu Hospital, Beppu, Japan;_ 2 _Osaka University, Suita, Japan_.

Background: Intrahepatic cholangiocarcinoma(ICC) accounts for about 10% of all primary liver cancer. South Asia and a part of Chile, but in recent years it has been increasing in other areas including Europe and the United States. The only potentially curative treatment for patients who have a resectable tumor is surgery. Unfortunately, even after curative-intent surgery, the clinical outcomes of patients undergoing liver resection are disappointing, with a 5-year survival rate of 20% to 35%. Furthermore, the role of the other treatments, including systemic chemotherapy and radiotherapy, remain poorly defined and have been reported to have a favorable therapeutic effect. In this study, we reveals the intratumoral heterogeneity and process of evolution of intractable ICC without fully genomic understanding.

Material and method: Surgical specimens of 10 patients with resection of ICC in curative intention were acquired between 10/2014 and 5/2016. The average tumor size was 47 mm(range 30-80 mm). We obtained 57 samples at 3-9 lesions for each case, and performed whole exome sequencing using the next generation sequencer (HiSeq2500). Multiregional analysis was performed respectively on these cases, and we investigated intratumoral heterogeneity of ICC. Furthermore, we guessed the process of clonal evolution of ICC from the result of whole exome sequencing.

Result: We defined "Founder mutation" as only a mutation present in all samples in each case , and defined "Progressor mutation" as several mutations present in some samples in each case. Furthermore, we classified "Progressor mutation" as "Shared mutation" present in several samples in each case but not all and "Unique mutation" present in one sample in each case). In 10 cases, mean number of Founder, Unique and Shared mutation were 53%(range 24-74%), 18%(range 5-40%), and 29%(range 6-54%), respectively, and this indicated intratumoral heterogeneity in ICC. In addition, Founder mutation mainly including TP53, SMAD4, and NRAS were common in 2 cases, but no common Founder mutation was observed in over 3 cases, so this indicated intratumoral heterogeneity in ICC. This results leaded to the process of clonal evolution during tumor formation, describing the evolutionary trees.

Conclusion: In this study, multiregional analysis was performed on samples of ICC, and intratumoral heterogeneity and the process of clonal evolution were evaluated. This result confirms the resistance to current treatment and it seems that it is necessary to search for further therapeutic targets.

#4352

High caveolin-1 expression in African American women with early-stage triple-negative breast cancer.

Lisa L. Baumbach-Reardon,1 Julie Getz,2 Mary E. Ahearn,2 John Carpten,3 Mark Pegram4. 1 _University of Arizonia School of Medicne, Phoenix, Phoenix, AZ;_ 2 _TGen (The Translational Genomics Research Institute), Phoenix, AZ;_ 3 _University of Southern California (USC), Los Angeles, CA;_ 4 _Stanford University Medical School, Stanford, CA_.

In the U.S., the incidence rates of breast cancer (BC) among Caucasian (CA) women are lower than those of African American (AA); however, AA women have a significantly higher mortality rate. It is imperative that we continue to investigate the underlying molecular biology that may contribute to the cause of health disparities between AA and CA with TNBC. In this study, gene expression profiling, using the Almac BC DSA Research Tool, was performed on archived FFPE samples, obtained from CA and AA women diagnosed with early-stage (Node 0) TNBC. Unsupervised hierarchical clustering revealed a pattern of differential gene expression in the AA cohort compared to CA. Using a subtyping Tool for TNBC, we found a distinct distribution pattern of TNBC molecular subtypes in the AA cohort, which was very different than the CA cohort: basal-like (14%), immunomodulatory (43%) and mesenchymal (43%). Gene expression analyses, comparing the AA and CA cohort (fold change > 2.0, p-value <.05), resulted in 190 differentially expressed genes (DEG). Pathway enrichment analysis conducted in MetaCore GeneGo revealed that the DEGs were over-represented in cytoskeletal remodeling, cell adhesion, tight junctions, and immune response in the AA TNBC cohort. Furthermore, several genes in the Wnt/β-catenin pathways were overexpressed in the top 10 enrichment pathways. We validated our results using RT-qPCR and identified Caveolin-1 (CAV1) as being significantly expressed in the AA-TNBC cohort (p-value 1.22 x 10-05). An independent cohort of FFPE samples, from AA and CA women with early stage TNBC, was used to create a tissue microarray (TMA). Immunohistochemistry results showed no difference in subcelluar localization of Cav1 between the AA and CA cohorts; however, the AA cohort had significantly higher levels of Cav1 staining (p-value 0.04). Additionally, using RT-qPCR, we demonstrated that CAV1 mRNA was significantly higher in the AA TNBC cohort (p-value 0.48). Furthermore, endogenous Cav1 was shown to be highly expressed in a cell line panel of TNBC, in particular, those of the mesenchymal and basal-like molecular subtypes. Finally, using siRNA, we demonstrated that CAV1 silencing resulted in a significant decrease in cell proliferation for each of the TNBC cell lines while it showed no effect on the luminal ER+ cell lines. Our combined study results suggest that CAV1 overexpression may be a biologic contributor to the observed health disparity between AA women and CA diagnosed with early-stage TNBC.

#4353

Integrated molecular analysis of upper urinary tract urothelial carcinoma.

Yoichi Fujii,1 Yusuke Sato,2 Hiromichi Suzuki,1 Tetsuichi Yoshizato,1 Yusuke Shiozawa,2 Kenichi Yoshida,1 Yuichi Shiraishi,3 Tohru Nakagawa,2 Hiroaki Nishimatsu,4 Toshikazu Okaneya,5 Masashi Sanada,6 Hideki Makishima,1 Satoru Miyano,3 Haruki Kume,2 Seishi Ogawa1. 1 _Kyoto Univ. Graduate School of Medicine, Kyoto, Japan;_ 2 _The University of Tokyo Hospital, Tokyo, Japan;_ 3 _Institute of Medical Science, The University of Tokyo, Tokyo, Japan;_ 4 _The Fraternity Memorial Hospital, Tokyo, Japan;_ 5 _Toranomon Hospital, Tokyo, Japan;_ 6 _Nagoya Medical Center, Nagoya, Japan_.

Backgrounds

Upper urinary tract urothelial carcinoma (UTUC) is a rare cancer accounting for 10% of all urothelial malignancies, whose molecular pathology, however, is poorly understood. To clarify diagnostic and prognostic impact of molecular profiles, we comprehensively investigated genetic alterations in UTUC.

Materials & methods

Surgical specimens of UTUC and matched normal samples were obtained from 102 patients with various stages, and were subjected to whole exome/RNA sequencing and array-based methylation analysis.

Results

UTUC cases displayed as many as 5.3 mutations per megabase (Mb). Seven cases showed hypermutation (>20/Mb), 6 of which harbored biallelic defects of mismatch repair (MMR) genes, suggesting the presence of Lynch syndrome in these. Genetic alterations were most frequently observed in TERT promoter (51% of cases), followed by KMT2D (48%), FGFR3 (46%, including fusion genes), CDKN2A (homozygous deletion) (42%), TP53 (31%), and RAS family genes (HRAS/KRAS/NRAS) (21%). More than 95% of cases harbored either TP53, MDM2, FGFR3, or RAS alterations in almost mutually exclusive manner. Hierarchical clustering on the basis of commonly altered genes in 95 non-hypermutated cases identified 3 distinct subgroups. Group 1 cases were frequently affected by TP53/MDM2 alterations and complex copy number alterations (CNAs), characterized by high stage, high grade, and poor prognosis. In Group2, FGFR3 and CDKN2A alterations and simple CNAs were enriched, which were associated with low stage/grade and favorable prognosis. All cases in Group3 harbored RAS-family gene alterations, showing intermediate prognosis. In mutation signature analysis, we extracted age-related, APOBEC, Transcriptional coupled repair (TCR), and MMR associated signatures. TCR signature was more frequently found in patients with an ALDH2 polymorphism implicated in low alcohol metabolism (rs671, GG/AG). MMR signature was observed only in the hypermutated cases with MMR deficiency. Gene expression analysis using unsupervised clustering identified 3 clusters. Cluster 1 was characterized by high expression of late cell-cycle genes, basal markers, and notably, immune blockade targets, CD274 and PDCD1LG2. Cluster 1 also harbored frequent TP53 alterations. In contrast, cases in cluster 2 and 3 showed high expression of early cell cycle genes and luminal markers. Cluster 2 were associated with FGFR3 mutations (positive in 75%), while almost all (92%) of RAS-mutated cases were classified in cluster 3. Finally, hierarchical analysis of methylation data revealed that approximately 60% of examined samples showed CpG island methylator phenotype (CIMP), which was positive in all the groups defined by mutational landscape.

Conclusion

UTUC showed distinct genetic landscape, associated with various clinical features. Our findings of molecular characteristics in UTUC will contribute to the development of novel diagnostics and therapeutics.

#4354

Molecular signature of multiple myeloma progression through single-cell RNA-seq.

Jin Sung Jang. _Mayo Clinic, Rochester, MN_.

Multiple myeloma (MM) is a malignant plasma cell disorder with well-defined primary genetic/cytogenetic abnormalities. Investigating genetic heterogeneity in the MM cells, may have an impact on therapeutic approaches and assessment of response to treatment. Single cell genomic profiling technology provides an opportunity for us to identify genetic heterogeneity during the pathogenesis of MM progression and select the most appropriate clinical interventions. We performed single cell whole transcriptome analysis in 597 CD138 positive cells derived from 15 patients at different stages of MM progression. Using Coefficient of Variation (CV) approach, we identified 790 genes and defined four main groups reflective of increasing levels of aggressiveness at the single cell level. Differential gene expression and gene set enrichment analyses showed that protein homeostasis pathways, unfolded protein response and mTORC1 related gene sets were significantly enriched at the most aggressive level consistent with the MM cell progression and predicts the overall survival. Our results provide novel insights into the cellular heterogeneity within each MM patient and a foundation for therapeutic opportunities including proteasome inhibitors.

#4355

Identification and functional characterization of glioma structural variants.

Kevin C. Johnson,1 Floris P. Barthel,1 Ming Tang,2 Samirkumar Amin,1 Qianghu Wang,2 Erik P. Sulman,2 Kunal Rai,2 Roel G.W Verhaak1. 1 _The Jackson Laboratory for Genomic Medicine, Farmington, CT;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Adult diffuse gliomas are a diverse class of brain tumors that ultimately result in fatal malignant progression. The highly variable disease progression intervals and poor treatment response observed in glioma have been explained, in part, by the extent of heterogeneity within and across gliomas. Somatic structural variants such as deletions, duplications, insertions, inversions, translocations, and extrachromosomal DNA contribute to glioma heterogeneity by generating genomic instability, a hallmark of cancer genomes. While comprehensive approaches such as whole genome sequencing have begun to catalog the glioma structural variant landscape, the molecular mechanisms by which they contribute to tumor progression remains poorly understood. Here, we identified genomic rearrangements in the whole genomes of 147 gliomas and glioma cell lines. Among the whole-genome sequencing samples were 62 glioblastomas (TCGA), 52 low-grade gliomas (TCGA), and our own set of 33 patient-derived glioma sphere-forming cells. On average, glioblastomas demonstrated a greater frequency of high-confidence structural variants (162 per tumor) than low-grade gliomas (130 per tumor, P=0.02) with two glioblastomas displaying evidence of chromothripsis. Across the World Heath Organization's molecular classifications, the IDH-mutant 1p/19q co-deleted tumors demonstrated the lowest structural variant burden when compared with both IDH-wildtype and IDH-mutant non-co-deleted tumors. Structural variant frequencies were found to be similar between patient tumors and an independent set of patient-derived cell lines. Across all samples, a majority of the structural variant breakpoints were found in intronic (42%) and intergenic regions (55%) suggesting that rearrangements predominantly impact distal regulatory regions. To investigate the functional consequences of structural variants in these regions we integrated these samples with available RNA-sequencing and epigenetic data to characterize deregulated transcriptional circuits. Analyses examining the association between genomic rearrangements and chromatin states to determine potential mechanisms of oncogene activation in select tumor and cell line samples are now underway. Together, our study demonstrates the importance of integrating structural variant calls from whole genome sequencing with expression and epigenetic data to define functional genomic rearrangements.

#4356

SIRT2-deacetylase activity is a potential predictive and prognostic marker in lung cancer patients.

Yang Guo, Ha Yong Song, Kirtee Raparia, Athanassios Vassilopoulos. _Northwestern University, Chicago, IL_.

Background: SIRT2 has been described as a tumor suppressor, whereas our previous study using an LSL-KRASG12D mouse model showed that Sirt2 loss induced lung tumorigenesis in vivo through increased KRAS acetylation (K147) and activity. However, there is limited information about the clinical significance of SIRT2 expression and/or activity in lung cancer patients.

Purpose: The aim of this study was to investigate the associations between SIRT2 levels and activity with clinical-pathological characteristics and patient outcomes in lung cancer.

Materials and Methods: SIRT2 expression and activity was analyzed using a tissue microarray of 162 resected lung cancer samples from Northwestern Memorial Hospital. To assess SIRT2 expression, we used an antibody against SIRT2 for immunohistochemistry. To assess SIRT2 activity, we evaluated acetylated levels of two specific deacetylation targets, α-tubulin (K40) and KRAS (K147). For all tested markers, staining intensity of each tissue sample was scored as 0 (no signal), + 1 (weak), + 2 (distinct), + 3 (strong). The percentage of each positive staining was categorized as A (<25%), B (25~50%), C (>50%). Following statistical analysis, it was determined whether SIRT2 expression and activity were associated with several clinical parameters or outcomes. p < 0.05 was assumed statistically significant.

Results: Acetylated α-tubulin and acetylated KRAS in lung tumor samples were significantly correlated with each other (p<0.05), but not with SIRT2 expression. Of note, acetylated α-tubulin was significantly associated with TTF1 expression (p<0.05), staging (p<0.005), pleural metastasis (p<0.005), surgery (p<0.001), and chemotherapy (p<0.005). Acetylated KRAS had significant associations with surgery (p<0.005), lymph nodal status (p<0.05) and pleural metastasis (p<0.05). Kaplan-Meier analysis further indicated that both TTF1 and acetylated α-tubulin were independent predictors of patient survival (p=0.005 and p=0.028, respectively).

Conclusions: Our data implicated that SIRT2 deacetylation activity, but not SIRT2 expression levels, could represent an optimal predictive and prognostic factor in lung cancer. Based on this finding, strategies to increase SIRT2 activity might be favorable in clinical treatment protocols for lung cancer.

#4357

Comparing amplification of 12q13-q14 and 12q15 chromosomal regions across cancer types through genomic and transcriptome analysis.

Prasantha L. Vemu, Gregory E. Hoy, Wenyue Sun, Jack Shern, Javed Khan, Frederic G. Barr. _National Cancer Institute, Bethesda, MD_.

Gene amplification, or an increase in copy number of a confined region on the chromosome arm, has been identified as a critical genetic event that contributes to the development of various cancers. There is increased expression of certain genes within the amplified regions, which alters normal cell growth and survival pathways, and contributes to tumorigenesis. Although previous studies show that some regions are amplified in more than one cancer type, direct analyses comparing the size and gene composition of these amplified regions across tumor types have not been performed. In the current study, we used copy number and RNA sequencing data from our published data and The Cancer Genome Atlas (TCGA) to characterize the commonly affected 12q13-q14 and 12q15 chromosomal regions in rhabdomyosarcoma, glioblastoma multiforme, lung adenocarcinoma, and liposarcoma. Based on our analysis of copy number data from high-density single-nucleotide polymorphism arrays, we observed a 0.08 Mb common region of overlap of the 12q13-q14 amplicons and a 0.20 Mb common region of overlap of the 12q15 amplicons across these tumor types. Differential gene expression analysis between amplified and nonamplified samples showed that OS9, TSPAN31, CDK4, CYP27B1, METTL1, EEF1AKMT3, and TSFM were overexpressed by the 12q13-q14 amplicons. Similarly, MDM2 and CPM were overexpressed by the 12q15 chromosomal amplicons. In addition to the common region of overlap, regions of tumor-type specific amplification were also found in our analysis. The 12q13-q14 amplicon in fusion-positive rhabdomyosarcoma extended 0.48 Mb toward the centromere, while the 12q13-q14 amplicons in dedifferentiated liposarcoma and lung adenocarcinoma extended 0.56 Mb and 0.96 Mb, respectively, toward the telomere. For the 12q15 region, the amplicon in lung adenocarcinoma extended 1.3 Mb toward the centromere, while the amplicons in dedifferentiated liposarcoma and fusion-negative rhabdomyosarcoma extended 1.4 Mb and 1.6 Mb, respectively, toward the telomere. Gene expression analyses showed that some genes from these tumor-specific regions of amplification were preferentially overexpressed in the corresponding tumor types. For example, four genes from the fusion-positive rhabdomyosarcoma-specific 12q13-q14 amplicon (NEMP1, NAB2, SHMT2, and R3HDM2) and two genes from the lung adenocarcinoma-specific 12q15 amplicon (MDM1 and RAP1B) were specifically overexpressed in these two tumor types. Our findings indicate that, in addition to common regions of amplification across multiple tumor types, there are tumor-specific amplified regions and overexpressed genes that indicate the presence of unique features within each cancer category affecting these amplification events.

#4358

Landscape of allelic imbalance in pan-cancer adjacent normal tissue: Insights into field cancerization and cancer pathogenesis.

Yasminka A. Jakubek,1 Francis A. San Lucas,1 Smruthy Sivakumar,1 Richard G. Fowler,1 Humam Kadara,2 Paul Scheet1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _American University of Beirut, Lebanon_.

It has been observed that tumors and adjacent normal appearing tissues share molecular signatures. This phenomenon has been observed in tissues of the skin, colon, and lung among others, particularly for patterns of gene expression. A recent pan cancer study of gene expression in normal tissue adjacent to the tumor (NAT) has shown that NATs have unique expression profiles that are distinct from non-tumor-bearing tissues. In this work, we sought to further characterize the molecular profile of NAT samples across cancer types. We have previously applied our haplotype based statistical technique to survey allelic imbalance (AI) in putatively normal lung tissues adjacent to tumor. Using this method, we can detect changes indicative of chromosomal alteration (gain, loss and copy-neutral LOH) even when present at low mutant cell fractions (< 5%). We focused our analysis on a subset of TCGA patients with both a solid tumor and NAT sample (n = 1530). In addition, to serve as a control, we surveyed alterations in normal samples derived from blood (n = 7,796). We detected 218 (megabase scale) chromosomal alterations in 44 of the NAT samples. These somatic alterations were present at a higher rate in NAT (2.9%) compared to the control normal blood set (1.9%). NAT samples with detectable AI had an average of 5 AI events compared to an average of 2 events in blood samples. We did not detect AI in the blood samples from the 44 patients with AI in the NAT. The most common recurrent alterations in NAT were 8p23.1, 17q21.31 and 1q42.12-1q42.13; each found in 14% of NAT AI positive samples. Chromosomal alterations in NAT were present at different rates between cancer types. We detected AI in more than 10% of NATs from breast invasive carcinoma, sarcoma, and bladder urothelial carcinoma. Of the 218 AI events in NAT, 67% were detected in the matching tumor sample. Two cancers, kidney renal papillary cell carcinoma and thyroid carcinoma, had a positive correlation between presence of AI in NAT and AI burden in the matched tumor sample (P = 0.005 and 0.03 respectively). The most common mutations in the tumors of patients with chromosomal aberrations in NAT were TP53 and MUC16 found in 39% and 20%, of tumors respectively. Lung squamous carcinoma and ovarian serous cystadenocarcinoma patients with detectable AI in NAT had a worse survival rate (P = 0.0241 and 0.0439 respectively). Our survey of AI in NAT tissues provides further support for NAT tissues as a model for understanding cancer pathogenesis.

### GTPases and Their Regulators and Effectors

#4359

Several cancer types are associated with increased expression and activity of the ECT2 RhoGEF and decreased expression and activity of the DLC1 RhoGAP, leading to increased RhoA activity.

Dunrui Wang, Xiaolan Qian, Marian E. Durkin, Alexander G. Papageorge, Douglas R. Lowy. _National Cancer Institute, Bethesda, MD_.

RhoA, which is a member of the Ras family of small GTPases, whose proteins cycle between an active GTP-bound form and an inactive GDP-bound form, frequently has high activity in advanced cancer. The RhoA protein is activated by RhoGEFs, which catalyze replacement of bound-GDP with bound-GTP, and inactivated by RhoGAPs, which hydrolyze bound-GTP to bound-GDP. In this study, we have used the The Cancer Genome Atlas (TCGA) and the NCI Clinical Proteomic Tumor Analysis Consortium (CPTAC) databases to examine the relationship between a specific RhoGEF, ECT2, and a specific RhoGAP, DLC1, and experimentally tested the biological relevance of some key findings. Using the TCGA dabase, we have found increased expression of the ECT2 RhoGEF frequently occurs in synchrony with decreased expression of the DLC1 RhoGAP in several tumor types, including lung adenocarcinoma, lung squamous cell carcinoma, and hepatocellular cancer. In lung adenocarcinoma, the combination of high ECT2 expression and low DLC1 expression level is more common in poorly differentiated tumors than in well differentiated ones. Using other publicly available datasets from NCBI Gene Expression Omnibus (GEO), the expression DLC1 and ECT2 was found to be cell cycle related, with higher DLC1 being observed exclusively in the G0 phase, while ECT2 was higher during the cell cycle, relative to G0, and peaked in the G2/M phase. In CPTAC, phosphorylation of a specific ECT2 amino acid (S861) is correlated with a poor prognosis in breast cancer. Consistent with this observation, experimental mutation of ECT2 S861 to A861 results in an ECT2 gene with loss-of-function. Point mutation of ECT2 and DLC1 were found at overall frequencies of 1.7% and 4.7%, respectively, in the tumors of the TCGA database. These cancer-associated mutations occur along the entire length of the ECT2 and DLC1 proteins, rather than being located primarily in the Rho-GEF domain of ECT2 or the Rho-GAP domain of DLC1, and experimental analysis of a subset of these mutants indicates that most of the ECT2 mutants are gain-of-function, while most of the DLC1 mutants are loss-of-function. Our results imply that ECT2 and DLC1 frequently act in concert, but in opposite directions, in cancer to increase RhoA activity.

* Authors contributed equally to this abstract

#4360

Altering the regulation of KRAS GTPase cycle via Src and SHP2 creates a potential therapeutic vulnerability for pancreatic cancer.

Teklab Gebregiworgis,1 Christopher B. Marshall,1 Yoshihito Kano,2 Nikolina Radulovich,1 Ming-Sound Tsao,1 Michael Ohh,2 Mitsuhiko Ikura1. 1 _Princess Margaret Cancer Center, Toronto, Ontario, Canada;_ 2 _University of Toronto, Toronto, Ontario, Canada_.

More than 30% of all human cancers have Ras mutations and more than 95% of pancreatic cancers harbor KRAS mutations. However, therapeutically targeting cancers driven by oncogenic Ras mutations is still an ongoing investigation. KRas is a small GTPase found in either a GDP-bound inactive form or a GTP-loaded activated form. The activated form of KRas localizes on the membrane where it binds and activates downstream effector proteins such as Raf kinases. GDP-bound Ras can be activated by nucleotide exchange of GTP for GDP, a reaction that is catalyzed by guanine nucleotide exchange factors (GEFs). Ras is inactivated by hydrolysis of GTP, which is assisted by GTPase activating proteins (GAPs). It was recently reported that Ras can be tyrosyl phosphorylated by Src and dephosphorylated by SHP2 (PNAS 2014;111(36):E3785-94; Nat Commun 2015;6:8859). Here we have characterized the structural and functional alterations of Src-phosphorylated KRas to mechanistically explain the impact of tyrosyl phosphorylation on the GTPase cycle. Our NMR and MS analyses show that Src phosphorylates KRas at Tyr32 and Tyr64, which perturbs the chemical shifts of several residues in each of the two "switch" regions that mediate interactions with effectors and regulators. Using real-time NMR and biolayer interferometry (Octet) assays, we demonstrated the negative impact of KRas phosphorylation on the GTPase cycle as well as BRAF binding. Conversely, the SH2 domain containing inositol 5-phosphatase 2 (SHP2) dephosphorylates KRAS and reverses Src-induced phosphorylation. In vivo, either the pharmacologic inhibition or genetic ablation of SHP2 promotes cell death in several KRAS mutant cancers. Our findings reveal that altering the KRas GTPase cycle regulation via the balance of Src and SHIP2 activities may create a therapeutic vulnerability for pancreatic cancer.

#4361

Role of Rab GTPases and endosomal adaptor proteins in oncogenic RAS induced formation of perinuclear signaling complexes (PSCs).

Srikanta Basu, Sandip K. Basu, Jacqueline Salotti, Peter F. Johnson. _NIH, Frederick, MD_.

Oncogenic RAS signaling leads to neoplastic transformation in immortalized cells and oncogene induced senescence in primary cells. Our laboratory has found that in both primary and immortalized cells oncogenic RAS induces constitutive perinuclear re-localization of p-ERK, CK2, and the MAPK scaffolding protein, KSR1. KSR1 binds to p-ERK and CK2, forming assemblies that we call "perinuclear signaling complexes" (PSCs). We observe PSCs in all cancer cell lines tested and in KRasG12V-driven mouse lung tumors, indicating that PSC formation is an important and integral characteristic of tumor cells. Our results show that KSR1 is essential for PSCs, as RNAi-mediated depletion of KSR1 or knockout of KSR1 disrupted their formation. We also demonstrated a key role for endosomal trafficking in this localized RAS signaling, as pharmacological inhibition of endocytosis prevented PSC formation. Furthermore, Rab11A, a Rab GTPase involved in trafficking of recycling endosomes, is required for establishment of PSCs and co-localizes with a pool of cellular CK2 and KSR1, but not p-ERK. Moreover, Rab11A knockdown caused decreased proliferation and survival of A549 cells (human KRAS mutant non-small cell lung adenocarcinoma). Our results demonstrate that there may be at least two classes of PSC-containing endosomes, p-ERK bound endosomes and CK2 bound endosomes. We are currently attempting to identify Rab GTPases that may differentiate these two kinds of PSC containing endosomes. Jongsma et al. (Cell, 2016) have shown that endosomes associated with the ubiquitin adaptor proteins EPS15, Tollip or TAXBP1 localize to the perinuclear region via the signaling adaptor, SQSTM1 (p62), which is tethered to the ER. We found that Tollip knockdown in A549 cells disrupted the CK2 bound endosomes but not p-ERK bound endosomes. Tollip knockdown also decreased cell proliferation and cell survival, while increasing apoptosis and autophagy. Whether other adaptor proteins are involved in RAS-mediated PSC formation remains to be determined. Growth factors (GF) also transiently induced PSCs in normal cells with delayed kinetics (4-6 hr). Our findings indicate that transformed cells sustain this novel late phase of GF signaling, localizing RAS pathway kinases to a nuclear-proximal compartment to drive tumorigenesis. We propose that PSCs are hallmarks of cancer cells and may represent useful biomarkers for cancer diagnosis and therapeutic responses.

#4362

Identification of novel combinatorial synthetic lethal vulnerabilities in KRAS-driven lung cancer.

Kaja Kostyrko,1 Marcus R. Kelly,2 Kyuho Han,2 Edwin E. Jeng,2 David W. Morgens,2 Michael C. Bassik,2 Peter K. Jackson,2 Alejandro Sweet-Cordero1. 1 _UCSF, San Francisco, CA;_ 2 _Stanford, Stanford, CA_.

Lung cancer is the number one cause of cancer-related deaths worldwide. The most prevalent type of lung cancer is Non-Small Cell Lung Cancer (NSCLC). A significant number of patients with NSCLC carry oncogenic KRAS mutations. However, the efforts to target KRAS directly have thus far proven unsuccessful and tumors harboring mutations in this gene remain the most difficult to treat, highlighting the need for alternative approaches. One promising strategy is to target KRAS-dependent cancers through synthetic lethality. However, KRAS activates multiple effector pathways, suggesting that targeting one gene may not be sufficient to fully inhibit KRAS oncogenesis. Therefore, we propose that targeting combinations of genes that together are synthetic lethal with KRAS may constitute a better therapeutic strategy. Furthermore, we hypothesize that a targeted approach focused on the protein-protein interaction network proximal to KRAS may be more effective than the current emphasis on genome-wide screens. To discover novel, combinatorial KRAS synthetic lethal genes, we used affinity purification/mass spectrometry (AP/MS), to systematically identify KRAS interacting proteins and construct a detailed map of protein-protein interactions centered on KRAS. Based on this network we designed a CRISPR/Cas9 library targeting pairwise combinations of KRAS-interacting genes. Using this library we simultaneously knocked-out pairs of 119 genes in two KRAS-driven non-small cell lung cancer (NSCLC) cell lines (A549 and H23). Knock-out of many gene pairs synergistically impaired growth of these cells, while the knock-out of each of the genes alone had no or little effect. We chose 20 most promising targets for further screening in vitro and in vivo in a panel of 9 KRAS-mutant and KRAS wild type Cas9-expressing NSCLC cell lines. We also selected six gene pairs that had the most synergistic effect on growth in A549 and H23 cells for individual validation in Cas9-expressing NSCLC cell lines and normal human bronchial epithelial cells (HBECs). We found that the simultaneous knock-out of one pair of genes, Rap1GDS1 and RhoA, significantly decreased growth of KRAS-dependent NSCLC cells, while having a limited effect on KRAS-independent cells or HBECs. Moreover the knock-out of either of these genes alone had no effect on growth in any of the cell lines, suggesting that only the combination of these two genes is synthetically lethal with KRAS. We are currently performing further validation in organoid cultures and in vivo. Additional validation and human relevance will be determined using patient-derived xenografts (PDX).

#4363

Pharmacologic targeting of Gq reveals new pathways in uveal melanoma.

Michael D. Onken,1 Carol M. Makepeace,2 Shiqi Wang,2 Kevin M. Kaltenbronn,2 Stanley M. Kanai,2 Tom J. Broekelmann,2 John A. Cooper,1 Kendall J. Blumer1. 1 _Washington Univ. School of Medicine, Siteman Cancer Center, St. Louis, MO;_ 2 _Washington Univ. School of Medicine, St. Louis, MO_.

Constitutively active G protein α-subunits are oncogenic drivers of several cancers, most notably in uveal (eye) melanoma (UM), which have constitutively activating mutations in GNAQ(Gαq) or GNA11(Gα11) in over 90% of tumors. Therapeutic intervention by targeted inhibition of constitutively active Gα subunits in cancer has yet to be achieved. Here we show that constitutively active Gαq in UM cells can be targeted by the cyclic depsipeptide FR900359 (FR). FR inhibits GDP/GTP exchange allosterically to trap constitutively active Gαq in inactive GDP-bound Gαβγ heterotrimers, and allosteric inhibition of other Gα subunits can be achieved by introduction of an FR binding site. In UM cells driven by constitutively active Gαq, FR inhibits second messenger signaling, arrests proliferation and reinstates melanocytic differentiation. At higher doses, FR also induces apoptosis. FR has no effect on BRAF-driven UM cells. FR promotes UM cell differentiation by reactivating polycomb repressive complex 2 (PRC2)-mediated gene silencing, antagonized by a heretofore unrecognized effector system of constitutively active Gαq in UM. Constitutively active Gαq and PRC2 therefore provide important therapeutic targets for UM. Further, the development of FR analogs specific for other Gα subunit subtypes may provide novel therapeutic approaches for diseases driven by constitutively active Gα subunits or multiple G protein-coupled receptors where targeting a single receptor is ineffective.

#4364

R-Ras activation cooperates with BRAF mutation in melanoma tumorigenesis.

Kristen S. Hill, Evan R. Roberts, Xue Wang, Youngchul Kim, Jane Messina, Minjung Kim. _Moffitt Cancer Ctr., Tampa, FL_.

The Ras family of small GTP binding proteins are tightly regulated through a complex network of proteins. In melanoma the Ras signaling pathway is frequently activated by mutations in NRAS (20%), KRAS (2%) and HRAS (1%); alternatively, Ras can also be activated by the inactivation of Ras GTPase activating proteins (RasGAPs) such as NF1, RASA1, and RASA2. Recently, we observed that inactivation of RASA1 (RAS p21 protein activator 1, also called p120RasGAP) suppressed melanoma via its RasGAP activity toward the R-Ras (related RAS viral (r-ras) oncogene homolog) isoform and that R-Ras was required to promote anchorage-independent growth driven by RASA1 inactivation. Moreover, a low level of RASA1 mRNA expression is associated with decreased overall survival in melanoma patients with BRAF mutations. Based on these observations, we hypothesized that, although not mutated, R-Ras is activated in melanoma by inactivation of RasGAPs and that BRAF activation cooperates with this RasGAP/R-Ras pathway activation in melanoma tumorigenesis.

In this study, we addressed the importance of R-Ras, a previously less appreciated member of the Ras small GTPases family, in melanoma tumorigenesis. We observed frequent activation of R-Ras in BRAF mutant human melanoma cell lines and human melanoma specimens. In addition, RNAi-mediated knockdown of R-Ras suppressed anchorage-independent colony growth and tumor growth. Of the 3 major RAS effector pathways, specifically MEK/ERK, AKT and Ral-A, reduced R-Ras expression suppressed Ral-A activation, which may explain the mechanism of Ral-A activation in BRAF mutant melanoma. Interestingly, anchorage-independent growth driven by R-Ras activation downstream of RASA1 inactivation was suppressed by both genetic (siRNA targeting Ral-A) and pharmacological (Ral inhibitor BQU57) inhibition of Ral-A. To further investigate the impact of RASA1 loss, and thus R-Ras activation, on BRAF mutant melanoma development in vivo, we generated a Rasa1L/L; BRAFCA/CA; Tyr-CreERT2 mouse model in which treatment with 4OHT results in expression of constitutively activated mutant BRAF and deletion of Rasa1 in melanocytic lineage cells. Preliminary analysis shows hyperpigmentation of the ear, tail, and foot pad in Rasa1L/L BRAFCA/CA mice compared to Rasa1+/+ BRAFCA/CA littermates; as well as, the development of cutaneous melanoma in Rasa1 deficient mice. Tumors generated in this animal model will be analyzed to determine the extent of R-Ras and Ral-A activity in vivo. This study demonstrates the importance of the RASA1/R-Ras/Ral-A signaling pathway in BRAF mutant melanoma and supports the possible combinatorial treatment strategy targeting both the BRAF/MAPK and Ral signaling pathways.

#4365

Targeting the RhoA guanine nucleotide-exchange factor Syx for the treatment of glioblastoma.

Wan-Hsin Lin, Ryan Feathers, Laura Lewis-Tuffin, Panos Anastasiadis. _Mayo Clinic, Jacksonville, FL_.

The RhoA GTPase is thought to play a crucial role in cancer development. Its activation is tightly controlled by numerous guanine nucleotide-exchange factors (GEFs). We previously showed that Syx, a RhoA-GEF, promotes junctional integrity in endothelial cells and governs cell polarity and directed migration in glioblastoma (GBM) cells. GBM is a grade IV glioma with a median survival of approximately 14 months. Importantly, the gene locus of Syx (Plekhg5) is located within chromosome 1p36, a region deleted in oligodendroglioma (a type II/III glioma) and associated with better prognosis, suggesting a role of Syx in GBM aggressiveness. Here we show that Syx is a potential target for the treatment of GBM. Knockdown of Syx resulted in altered expression of cell cycle regulators, including cyclins A, B, and E, cyclin-dependent kinase inhibitors (CDIs), namely p21 and p27, and mitosis modulators CDC20 and survivin. These defects resulted in G2/M arrest and dysfunction of cell division, leading to improper chromosome segregation and DNA double-strand breaks. However, DNA damage response could not be activated effectively for DNA repair, leading to cellular apoptosis. Consistent with these findings, we used an orthotopic patient-derived xenograft GBM model to show that Syx silencing results in decreased tumor growth accompanied by increased animal survival. Agents that cause DNA damage and/or disruption of DNA repair pathways are often used for cancer therapy. We thus tested the possibility of targeting Syx for GBM treatment, and showed that Syx knockdown in combination with temozolomide, a first-line drug for GBM, synergistically inhibit cell growth. In summary, this is the first study showing the role of Syx in GBM cell growth and that targeting this signaling pathway can be beneficial for GBM therapy.

#4366

The small GTPase Rac1 controls the stability of the Yes-associated protein 1 (YAP1) independently of the LATS1/2 kinases and SCF-βTRCP ubiquitin ligase.

Chitra Palanivel, Bailey Gabler, Ying Yan, Surinder K. Batra, Michel M. Ouellette. _University of Nebraska Medical Center, Omaha, NE_.

Activating mutations in the K-Ras gene are the earliest and most common genetic alterations detected in human pancreatic cancer (PC) specimens. In genetically engineered mouse models of PC, oncogenic K-Ras drives the formation of PanIN precursor lesions and their progression to invasive PC. Oncogenic K-Ras has at least four effector pathways that play distinct roles in malignant transformation: MAPK pathway, PI3K pathway, Ral-GDS, and the Rac1 GTPase (Ras-related C3 botulinum toxin substrate 1). Among these effectors, the least well understood is the Rac1 pathway. Yet, this pathway has now been shown to be critical for Ras-driven PC development and malignant transformation. However, the specific mechanisms by which Rac1 promotes anchorage-independent growth and tumorigenicity are still unclear. In recent studies, we have discovered that the Rac1 pathway regulates the stability of the Yes-associated protein 1 (YAP1). YAP1 is a transcriptional co-activator that controls the transcription of genes involved in proliferation and survival. The stability, location, and activity of YAP are controlled by signaling pathways involved in sensing cell-cell and cell-matrix interactions, such as the Hippo and Wnt pathways. In mouse models of Ras-driven PC, YAP1 is required for the formation of PanIN precursor lesions and their progression to invasive PC. In our telomerase-immortalized human pancreatic ductal cells, overexpressed YAP alone is sufficient to allow anchorage-independent growth. Our results show that in PC treated with Rac1 inhibitors (NSC23766, EHT-1864, AZA1, and Rac1T17N), YAP1 is rapidly and almost completely degraded within 24 hours. Importantly, this degradation of YAP1 is blocked by the preincubation of the cells with proteasome inhibitor MG-132, thereby suggesting the involvement of an ubiquitin ligase complex. A phosphodegron activated by the LATS1/2 kinases is present in YAP1 that promotes the degradation of the protein by the SCF-βTRCP ubiquitin ligase complex. Yet, the knockdown of neither the LATS1/2 kinases nor βTRCP affected the degradation of YAP1 after Rac1 inhibition. The knockdown of Elongin B and C, which are part of a different ubiquitin ligase complex controlling YAP1, also yielded similar results. A mutant of YAP1 lacking its LATS1/2 phosphorylation sites was also tested (YAP1-5SA). After Rac1 inhibition, this mutant was degraded with similar kinetics as the wild-type YAP1 protein. These findings reveal the existence of a novel ubiquitin ligase complex regulating YAP1 stability, this time in response to the inhibition of the Rac1 pathway. The screening of a siRNA library has now been undertaken to identify components of the ubiquitin ligase involved. The proposed work is expected to shed light on novel mechanisms underlying the development of PC and of other Ras-driven malignancies.

#4367

The small GTPase ARF6 is necessary for melanomagenesis.

Lehi Acosta,1 Aaron Rogers,1 Jingfu Peng,1 Alan Mueller,2 Zongzhong Tong,2 Donghan Shin,1 Jae Hyuk Yoo,1 Dean Y. Li,1 Shannon J. Odelberg,1 Sheri L. Holmen,3 Allie H. Grossmann1. 1 _University of Utah, Salt Lake City, UT;_ 2 _Navigen, Inc., Salt Lake City, UT;_ 3 _Hunstman Cancer Institute, Salt Lake City, UT_.

ARF6 is a member of the adenosine diphosphate (ADP)-ribosylation factor (ARF) family of small GTPases and is part of the RAS superfamily. We have shown that pharmacologic inhibition of ARF6 can inhibit proliferation of uveal melanoma and invasion/metastasis of cutaneous melanoma. Most cutaneous melanomas are defined by mutually-exclusive driver mutations in BRAF, NRAS, or NF1. These subtypes share similar mechanisms of oncogenesis because they align along a continuum of RAS signaling. Mutations in the PI3K pathway occur in a fraction of all melanoma subtypes, indicating a selective advantage for PI3K/AKT activation beyond BRAF or RAS activation. We and others have reported ARF6-dependent ERK activation occurring upon either extracellular ligand stimulation or mutant oncoprotein signaling. Recently, ARF6 has also been shown to be upstream of PI3K signaling. Based on these findings, we hypothesized that ARF6 is critical for both the RAF and PI3K arms of the RAS pathway and that loss of ARF6 will impede tumor growth driven by these oncogenes. We tested this hypothesis using human melanoma cell lines and with an inducible, genetically engineered mouse model (GEMM) of melanoma. Our data reveal that pharmacologic inhibition of ARF6 can reduce proliferation of BRAF, NRAS and NF1-mutated melanoma cells. Furthermore, loss of Arf6 causes a reduction in mean tumor size and improves survival in BRAFV600E/CDKN2ANULL/PTENNULL/AKTE17K and BRAFV600E/CDKN2ANULL/PTENNULL mice. These results indicate that ARF6 is necessary for melanomagenesis and for oncogenic BRAF and/or PI3K signaling. Our data suggest that ARF6 may be an actionable node in the RAS pathway and that therapeutic targeting of ARF6 has the potential to improve current treatment methods for progressive disease.

#4368

PAK4-NAMPT dual inhibition as a feasible strategy for treatment of resistant pancreatic neuroendocrine tumors.

Gabriel Mpilla,1 Irfana Muqbil,2 Amro Aboukameel,1 Philip A. Philip,1 William Senapedis,3 Erkan Baloglu,3 Yosef Landesman,3 Michael Kauffman,3 Sharon Shacham,3 Ramzi M. Mohammad,1 Asfar S. Azmi1. 1 _Wayne State Univ. School of Medicine, Detroit, MI;_ 2 _University of Detroit Mercy, Detroit, MI;_ 3 _Karyopharm Therapeutics Inc, Newton, MA_.

Our studies in pancreatic neuroendocrine tumor (pNET) cell lines demonstrate hyper-activation of the Rho GTPase effector p21 activated kinase 4 (PAK4) and the nicotinamide adenine dinucleotide (NAD) salvage pathway rate limiting enzyme nicotinamide phosphoribosyltransferase (NAMPT). The PAK4 protein is known to regulate a myriad of signaling proteins in the mTOR pathway including mTORC1, mTORC2, PI3K, and IGF-1. Similarly, NAMPT is recognized to regulate mTOR through energy sensor protein, AMPK. In this study, pNET cell lines QGP-1 and Bon-1 were subjected to either PAK4 RNA interference (RNAi), PAK4-NAMPT dual inhibitors (the clinical compound KPT-9274 or an analog KPT-7523) or the PAK4 specific inhibitor, PF-3738309 and NAMPT specific inhibitor FK866 in the presence or absence of mTOR inhibitors (everolimus or INK128). Gene expression profiling and phospho-proteomic analyses were performed to capture molecular changes post single or combination treatments. The anti-tumor activity of KPT-9274-everolimus was evaluated in a subcutaneous xenograft mouse model derived from QGP-1 and Bon-1 cells. PAK4 RNAi suppressed proliferation and restored everolimus sensitivity in pNET cell lines. The dual inhibitors were effective in reducing proliferation and inducing apoptosis. KPT-9274 or KPT-7523 could synergistically enhance the anti-tumor activity of everolimus or INK128 in pNET cell lines [CI <1]. Molecular analysis of the combination treatment showed down-regulation of known everolimus resistance drivers such as mTORC1, mTORC2, PI3K, ERK, FAK, RICTOR, β-catenin and IGF-1. In addition, KPT-9274 and analogs suppressed the steady state level of NAD and ATP. Importantly, KPT-9274 given i.v. or orally at the maximum tolerated dose (140 mg/kg, once/day for 5 days/week for 4 weeks) dramatically inhibited the growth of QGP-1 and Bon-1 tumors. This is the first study demonstrating the role of PAK4 and NAMPT in pNETs. KPT-9274 is currently in a Phase I trial of patients with advance solid malignancies or NHL (NCT02702492). Our pre-clinical work establishes a solid rationale for a Phase II clinical study of KPT-9274 and an mTOR inhibitor combination for the treatment of difficult to treat pNETs.

#4369

Functional role of RhoGEF FGD4 in promotion of aggressive prostate cancer.

Alexia Bossan,1 Richard Ottman,1 Domenico Coppola,2 Ratna Chakrabarti1. 1 _Univ. of Central Florida, Orlando, FL;_ 2 _Moffitt Cancer Center, Tampa, FL_.

Hyperactivation of Rho family of GTPases including Rac, Cdc42 and Rho, as a result of deregulation of Rho guanine nucleotide exchange factors (GEF) is commonly noted in various cancers including prostate cancer. A number of GEFs are overexpressed in prostate and breast cancer cells and tissues, and show an association with poor prognosis. Earlier, we showed that a prolonged treatment with androgen receptor antagonist Casodex resulted in up regulation of a GEF FGD4, traditionally associated with hereditary motor and sensory neuropathies including Charcot-Marie-Tooth disease. FGD4 is a Cdc42 specific GEF and contains an actin binding domain FAB, in addition to the conserved FYVE domain, a DH domain for catalytic activity and two PH domains for targeting proteins to the membranes through phosphoinositide binding. Our study revealed that Casodex treatment of prostate cancer cells lead to down regulation of a microRNA cluster miR-17-92a that targets FGD4. Here we show that FGD4 is upregulated in cancerous prostate tissues compared to benign prostatic hyperplasia and PIN. FGD4 protein expression and altered localization gradually increase with increasing tumor grade and aggressiveness. Inhibition of FGD4 expression using siRNAs, reduced cell proliferation, resulted in a G2/M arrest, inhibited cell migration and reduced Cdc42 activity. Inhibition of FGD4 also improved sensitivity of androgen sensitive LNCaP cells to Casodex and PC-3 cells to the microtubule stabilizing drug docetaxel. Our data demonstrate a tumor promoting function of FGD4 in prostate cancer cells and that inhibition of FGD4 expression has a therapeutic benefit for both androgen-dependent and -independent prostate cancer cells.

#4370

Regulation of AGO2-KRAS interaction through epidermal growth factor receptor.

Ronald F. Siebenaler, Sunita Shankar, Jean C. Tien, Vijaya L. Dommeti, Malay Mody, Chandan Kumar-Sinha, Arul M. Chinnaiyan. _University of Michigan, Ann Arbor, MI_.

The RAS gene family is among the most commonly mutated genes within cancer. While much research has elucidated its major functions and downstream pathways, little progress has been made in successfully targeting RAS mutations. We recently identified Argonaute 2 (AGO2) of the RNA-induced silencing complex (RISC) as a novel partner of the Switch II domain of KRAS. We have found that stable knockdown of AGO2 in KRAS-dependent cell lines lead to a decrease in KRAS protein expression with a subsequent decrease in cellular proliferation. In addition, we observed a decrease in microRNA unwinding in the presence of mutant KRAS, suggesting this interaction inhibits the endogenous RNAi function of AGO2. Despite this clear connection between KRAS and AGO2 in KRAS mediated oncogenesis, the precise function of this interaction remains unclear in both normal and cancer biology.

In order to identify endogenous regulators of AGO2-RAS, we investigated the ability of EGFR signaling to modulate the AGO2-RAS interaction. We established two mouse embryonic fibroblast cell lines (NIH-3T3 and MEF) with complete knockout of AGO2. When compared to normal control cells, we found that knockout of AGO2 resulted in an increase in WT RAS-GTP activation levels, phosphorylation of Y1068-EGFR, and MAPK/ERK and PI3K/AKT signaling. Rescue of AGO2 knockout resulted in a return to normal levels of active RAS-GTP, pEGFR, and downstream signaling.

Recent studies have described EGFR phosphorylation of AGO2 under hypoxic cell conditions, resulting in the inhibition of AGO2 association with RISC members. In order to better characterize the relationship between EGFR-AGO2-RAS, we found that overnight serum starvation followed by stimulation with EGF led to a decrease in AGO2-RAS co-IP in WT KRAS cells. Blocking AGO2 phosphorylation with a Y393F mutant of AGO2 prevented AGO2-RAS dissociation following EGFR stimulation. While WT KRAS cell lines displayed regulation of AGO2-RAS via EGFR, the phosphorylation of AGO2Y393 was unable to disrupt the interaction of AGO2 with mutant KRAS following stimulation with EGF. Together these results suggest a unique EGFR-AGO2-RAS signaling axis, and its dysregulation by mutant KRAS could increase oncogenic growth through promotion of AGO2-RAS interaction in cancer.

#4371

**Store-operated Ca** 2+ **entry and** NFATC1 **-activation control oncogenic signaling in B-cell transformation and leukemogenesis.**

Kohei Kume,1 Liting Chen,2 Jaewoong Lee,1 Markus Müschen1. 1 _Beckman Research Institute of City of Hope, Duarte, CA;_ 2 _University of California San Francisco, San Francisco, CA_.

Background: Nuclear factor of activated T cells (NFATC1) regulates activation and differentiation of B and T cells. Activation of NFATC1 requires sustained Ca2+ influx by store-operated Ca2+ entry (SOCE). We previously showed that oncogenic signaling from the BCR-ABL1 kinase in Ph+ ALL results in autonomous oscillatory Ca2+ signaling. However, the significance of Ca2+ oscillations remained unclear. Here we examined the function of Orai1 and Stim1, the central mediators of SOCE, and the role of Ca2+ oscillations in a BCR-ABL1-driven B-cell leukemia model.

Results: To investigate the consequence of inducible deletion of SOCE-effector genes, Orai1, Stim1, we generated BCR-ABL1- and CreERT2-transduced Orai1f/f and Stim1/2f/f B-cell leukemia. Both Orai1 and Stim1 deletion impaired SOCE and disrupted the patterns of autonomous Ca2+ oscillations, compared with that of empty vector controls. To determine whether the impaired Ca2+ signaling associated with cell survival and proliferation, we next analyzed their growth and colony-forming capacity. As expected, both Orai1- and Stim1-deleted B-cell leukemia exhibited growth disadvantage and reduced colony-forming capacity, compared with empty vector controls. Flow cytometry studies of Annexin V revealed that both Orai1 and Stim1 deletion induced apoptosis in the BCR-ABL1-transformed cells. Studying NFATc1 activity by Western blot, as reflected by nuclear localization, in B-cell leukemia before and after Orai1 and Stim1 deletion revealed that Cre-mediated deletion of Orai1 and Stim1 strongly reduced nuclear accumulation of NFATc1, with slight increases in the cytoplasmic fraction. To examine the consequences of NFATc1 inhibition, we generated BCR-ABL1- and CreERT2-transduced Nfatc1f/f B-cell leukemia and observed its effect on cell growth. As expected, inducible ablation of Nfatc1 resulted in rapid depletion of B-cell leukemia from the cell culture compared to empty vector controls. Importantly, a NFAT-calcineurin association inhibitor, INCA-6, displayed growth-inhibitory effects in all six patient-derived xenografts of B cell ALL tested, including samples from patients who relapsed after initially successful chemotherapy. These results suggest that the Ca2+-NFATC1 signal may suppress apoptosis and promote drug resistance and relapse in human B-cell leukemia.

Conclusions: Taken together, our findings showed that SOCE has a critical role in NFATC1 activation and survival of BCR-ABL1-driven pre-B ALL cells. Orai1- and Stim1-mediated autonomous Ca2+ oscillatory signal is required for BCR-ABL1-dependent proliferation. This signal may bypass NFATC1 activation, which suppresses apoptosis and enhances proliferation.

#4372

SET/PP2A/SHP-1/Lyn oncogenic signaling contributes to tumor aggressiveness in diffuse large B cell lymphoma.

Ji-Lin Chen,1 Wen-Chun Tsai,1 Tien-Yun Lan,1 Chun-Teng Huang,2 Pei-Yi Chu,3 Chia-Han Lee,1 Ka-Yi Lau,1 Wan-Lun Wang,1 Kuen-Feng Chen,4 Chung-Wai Shiau,5 Chun-Yu Liu1. 1 _Taipei Veterans General Hospital, Taipei, Taiwan;_ 2 _Yang-Ming Branch of Taipei City Hospital, Taipei, Taiwan;_ 3 _Show Chwan Memorial Hospital, Taipei, Taiwan;_ 4 _National Taiwan University Hospital, Taipei, Taiwan;_ 5 _National Yang-Ming University, Taipei, Taiwan_.

Purpose:

Diffuse large B-cell lymphoma (DLBCL) is aggressive non-Hodgkin lymphoma. It is a heterogeneous disease and is classified as germinal center B-cell like (GCB) and activated B-cell like (ABC) subtypes according to gene-expression profiling. The oncogenic pathways in DLBCL are potential targets for new therapies. In present study, the role and regulation of SET/PP2A/SHP-1/Lyn axis in DLBCL were investigated.

Experimental design: U2932, OCL-Ly3, OCI-Ly7, SU-DHL-6 and DB DLBCL cell lines were used for this study. TD-19, a novel SET/PP2A protein-protein interaction inhibitor, was used to address the molecular events of SET signaling. Cell viability was measured by MTT assay. The apoptotic cells were examined by PI/Annexin V staining and Western blotting. SHP-1, Lyn and pLyn were analyzed by immunohistochemistry on lymphoma tissue microarrays from DLBCL patients.

Results:

We first examined the effects of two different SET inhibitors, TD-19 that is a SET/PP2A protein protein interaction inhibitor, and FTY720 which is a sphingosine analogue that targets SET, on DLBCL cells. TD-19 and FTY720 significantly decreased cell viability and induced apoptosis. SET inhibition activated PP2A and SHP-1 by reducing pPP2AY307 and pSHP-1S591 and inactivated Lyn by decreasing pLynY396. Overexpression of SET rescued these molecular events, and promoted cell growth and migration. Interestingly, we observed the level of SET was declined by TD-19 and TFY720 treatment. We hypothesized Lyn might up-regulate SET expression. Overexpression of Lyn increased SET level, and was accompanied with phosphorylation of PP2A (pPP2AY307) and SHP-1 (pSHP-1S591). Lyn elevated cell growth and migration and suppressed PP2A and SHP-1 activities. Moreover, exogenous SET restored the FTY720- and TD-19-suppressed pSHP-1 and pLyn. Immunohistochemically, high SHP-1 level was linked to low pLyn level of patient with DLBCL and vice versa.

Conclusion: This study established the existence of positive feedback of SET/PP2A/SHP-1/Lyn axis in DLBCL cells, and targeting SET could disrupt this regulation.

#4373

AR signaling in concert with PP2A/B regulates YAP1 expression in prostate cancer cells.

Bekir Cinar,1 Carlos S. Moreno2. 1 _Clark Atlanta University, Atlanta, GA;_ 2 _Emory University, Atlanta, GA_.

The transcriptional coactivator YAP1 is a key nuclear effector of the Hippo pathway. STK4/MST1 and LATS1/2 are the core kinase components of the Hippo pathway, which restricts organ size and prevents tumorigenesis by attenuating cell proliferation and inducing cell death. Previously, we have reported that STK4/Hippo-YAP1 signaling could play a critical role in prostate cancer progression and therapeutic relapse. Here, we investigated the effects of androgen hormone signaling on YAP1 expression and post-transcriptional modifications in prostate cancer cells. We demonstrated that androgen exposure reduced Ser127 phosphorylation on YAP1 in a time-dependent manner in the castration-sensitive prostate cancer cell line, LNCaP, but without altering the levels of YAP1-Ser127 phosphorylation in the C4-2 cell line, a castration-resistant subline of LNCaP cells. As demonstrated by imaging and cell fractionation methods, androgen exposure promoted the nuclear accumulation of YAP1 in LNCaP; however, regardless of androgen exposure, YAP1 was accumulated in the nucleus of C4-2 cells. In addition, we demonstrated that androgen exposure reduced YAP1-Ser127 phosphorylation that was induced by okadaic acid, a potent activator of Ser/Thr phosphatases PP1 and PP2A. Also, we demonstrated that androgen exposure increased PP2A/B protein expression. Moreover, reduction of phospho-Ser127-YAP1 was correlated with increases in total YAP1 protein, which coincided with androgen receptor (AR) nuclear accumulation by androgen. Consistent with these observation, the genetic (siRNA) or the pharmacologic (enzalutamide) inhibition of AR signaling attenuated the expression of YAP1 protein. Furthermore, our analysis of the publicly available TCGA (The Cancer Genome Atlas) set indicated that YAP1 and AR mRNA expressions were positively correlated in prostate clinical samples. These observations suggest that YAP1 is a direct target of androgen hormone signaling, implicating that the AR-PP2A/B-YAP1 axis is a viable cancer drug target.

#4374

Non-canonical activation of β-catenin by PRL-3 phosphatase in acute myeloid leukemia.

Phyllis SY Chong,1 Jianbiao Zhou,1 Jing Yuan Chooi,1 Wee Joo Chng,1 Jayantha Gunaratne,2 Qi Zeng2. 1 _Cancer Science Institute of Singapore, Singapore, Singapore;_ 2 _IMCB, Singapore, Singapore_.

Aberrant activation of Wnt/β-catenin signaling pathway is essential for the development of AML; however, the mechanistic basis for the dysregulation is unclear. PRL-3 is an oncogenic phosphatase implicated in the development of LSCs. Here, we identified Leo1 as a direct and specific substrate of PRL-3. Serine-dephosphorylated form of Leo1 binds directly to β-catenin, promoting the nuclear accumulation of β-catenin and transactivation of TCF/LEF downstream target genes such as cyclin D1 and c-myc. Importantly, overexpression of PRL-3 in AML cells displayed enhanced sensitivity towards β-catenin inhibition in vitro and in vivo, suggesting that these cells are addicted to β-catenin signaling. Altogether, our study revealed a novel regulatory role of PRL-3 in the sustenance of aberrant β-catenin signaling in AML. PRL-3 may serve as a biomarker to select for the subset of AML patients subset who are likely to benefit from treatment with β-catenin inhibitors. Our study presents a new avenue of cancer inhibition driven by PRL-3 overexpression or β-catenin hyperactivation.

### Immunologic and Other Cancer Cell Death

#4375

High hydrostatic pressure modified potato patatin induces apoptosis like cell death in human colorectal adenocarcinoma (HT-29) cell line.

Rizwan Elahi, Tai Hua MU. _Chinese Academy of Agricultural Sciences, Beijing, Pakistan_.

The current study assessed the anticancer activity of HHP-induced potato patatin on human colorectal adenocarcinoma (HT 29) cell line in vitro. Cell proliferation, apoptosis, necrosis and autophagy were investigated. HHP treatment inhibited HT-29 cell line proliferation in a dose a dependent manner. IC50 values showed antiproliferative activity (59.1%) at 4 mg mL-1 for HHP-induced patatin at 450 MPa. Flow cytometric analysis indicated that HHP-induced patatin at 450 MPa was the potent inducer of in vitro apoptotic cell death and showed significant (p<0.05) accumulation in S-phase followed by patatin pressurized at 550 MPa. Surface charge of patatin decreased with the increase in pressurization up to 350 MPa level, while at 450 MPa zeta potential value increased to (-13 mV) and (-11 mV) respectively. Overall, results indicated that 450 MPa patatin is a strong inhibitor of HT-29 and a potent inducer of apoptotic cell death. Thus, HHP treatment is an effective method for structural modification of patatin which ultimately enhance the anticancer potential against colorectal cancer cells.

#4376

**Tumor treating fields (TTFields) decrease proliferation of patient-derived lung cancer brain metastasis cells** in vitro **.**

Sharon K. Michelhaugh, Sam Kiousis, Neil V. Klinger, Sandeep Mittal. _Wayne State University, Detroit, MI_.

Up to 40% of all non-small cell lung cancer (NSCLC) patients will develop brain metastases during the course of their disease, with ~20-25% of patients presenting with brain metastases at the time of their initial lung cancer diagnosis. For the latter patient population, median survival is a dismal 8 months. Systemic therapies are typically ineffective at treating the brain metastases, even in cases when the extracranial disease responds to the therapy. Clinical trials utilizing TTFields therapy in patients with lung cancer brain metastases are underway. For these trials, TTFields are delivered at 150 kHz, as that is the frequency that has been determined for the treatment of primary lung cancer. In contrast, for the FDA-approved treatment of the most common malignant primary brain tumor, glioblastoma, TTFields are delivered at 200 kHz. Therefore, in this study, a patient-derived lung cancer brain metastatic cell line was assessed to compare the response to in vitro TTFields application at either 150 or 200 kHz compared to untreated control cells. Methods: Use of patient tumor tissue for this study was approved by the Wayne State University Institutional Review Board and written informed consent was obtained from the patient. An adenocarcinoma lung cancer brain metastasis (from a patient that presented with the brain metastasis at the time of diagnosis) was collected immediately following microsurgical resection. A single-cell suspension from the tumor tissue was prepared by enzymatic and mechanical disruption. After 7 passages in vitro, cells were plated on plastic coverslips (10,000 each) in DMEM/F12 media supplemented with 10% fetal bovine serum. TTFields were applied at ~1.6 V/cm at 150 or 200 kHz (groups n=4-5) with one control group. Culture media was replaced every day. After 10 days of in vitro TTFields application, cell proliferation was assessed with the XTT assay. Control vs. TTFields-treated groups were compared with two-tailed t-tests. Results: The 10-day TTFields treatment resulted in significantly reduced cellular proliferation in the cells that received 150 kHz TTFields (0.220±0.006; mean±SEM) compared to the control cells (0.250±0.009; p=0.0237) with no statistical difference between the control and the cells that received 200 kHz TTFields (0.230±0.008; p>0.05). Conclusions: Application of TTFields in vitro at 150 kHz reduced cellular proliferation in patient-derived lung cancer brain metastasis cells, with no reduction of proliferation after application of TTFields at 200 kHz. This is the first report on the in vitro effects of TTFields in a patient-derived NSCLC brain metastasis cell line.

#4377

The role of Src in TRAIL signaling in non-small cell lung cancer cells.

Margot de Looff, Steven de Jong, Frank A.E. Kruyt. _UMCG, Groningen, Netherlands_.

Introduction: Non-small cell lung cancer (NSCLC) is the most prevalent form of lung cancer and accounts for most cancer-related deaths worldwide. Despite progress in the treatment of subgroups of patients with targeted therapies, better treatments are required to improve overall prognosis. Agents that target the tumor-necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL) receptors (TRAIL-R1 and -R2) are able to trigger selective apoptosis in tumor cells, however showed discouraging activity in clinical studies. Resistance to TRAIL-induced apoptosis and non-canonical pro-tumorigenic signaling hampers therapeutic efficacy. Previously, we found that TRAIL induces migration and invasion via RIPK1/Src/STAT3 pathway in apoptosis resistant NSCLC cells. Here we aim to analyze in more detail the role of Src in TRAIL non-canonical signaling and apoptosis resistance.

Methods: Cytotoxicity to rhTRAIL was assessed by MTT assays. Src was chemically inhibited or genetically ablated by short hairpin(sh)RNA or CRISPR/CAS9. Src phosphorylation was studied by western blotting. Protein interactions were examined by co-immunoprecipitation (IP) and subsequent western blotting. Src interacting proteins were examined by co-IP experiments in conjunction with LC-mass spectrometric (MS) analyses.

Results: The function of Src in TRAIL signaling was examined in TRAIL resistant A549 and sensitive H460 NSCLC cells. rhTRAIL treatment revealed distinct Src phosphorylation patterns, indicating that Src is differentially activated by TRAIL. Subsequently, co-IP experiments were performed showing that in A549 cells Src interacts with RIPK1 and Caspase-8 upon TRAIL treatment, but not in H460 cells. Next, we explored the possible role of Src in regulating TRAIL-induced apoptosis by chemical modulation of Src or by gene silencing or genetic knockout of Src. We found no role for Src in regulating sensitivity or resistance to TRAIL-induced apoptosis. To further investigate possible biological consequences of TRAIL-dependent Src activation we performed co-IP coupled with LC-MS analysis. Abundant differences were found in the Src interactome of A549 and H460 cells and in absence and presence of TRAIL. Various proteins known to be involved in tumor signaling were identified to be in complex with Src, including components of the RAF/MEK/ERK, Wnt and SMAD3 signaling pathways. Currently, mechanistic and validation studies are in progress to elucidate the role of these proteins.

Conclusions: Src has no role in controlling sensitivity or resistance to TRAIL-induced apoptosis in the examined NSCLC cells. On the contrary, the Src interactome showed the activation of various pro-tumorigenic pathways by TRAIL. We anticipate that a deeper knowledge of TRAIL signaling will lead to novel therapeutic strategies to improve TRAIL-receptor targeted therapy. Supported by a grant from the Dutch Cancer Society (KWF 2011-5211).

#4378

Novel CARP-1- H2AX interaction regulates apoptosis in breast cancer cells.

Sreeja C. Sekhar, Vino T. Cheriyan, Arun K. Rishi. _Barbara Ann Karmanos Cancer Inst., Detroit, MI_.

CCAR1/CARP-1 is a peri-nuclear phosphoprotein that regulates apoptosis signaling by breast cancer therapeutic Doxorubicin and a novel class of CARP-1 Functional Mimetic (CFM) compounds. Prior reports have established that Doxorubicin exposure causes DNA damage in cells. Phosphorylation of histone 2AX (often referred as γH2AX) protein is involved in regulating DNA damage repair and apoptosis signaling. Although, currently it is unclear whether CFMs also induce DNA damage, we found that Doxorubicin or CFM-4.16 treatments caused elevated CARP-1 and γH2AX in HBC cells. In fact, a robust nuclear or peri-nuclear co-localization of CARP-1 and γH2AX occurred in HBC cells undergoing apoptosis. Knock-down of CARP-1 abolished increase of CARP-1 and γH2AX, their co-localization, and apoptosis in CFM-4.16 or Doxorubicin-treated HBC cells. We found that CARP-1 directly binds with H2AX, and the H2AX-interacting epitope is located within CARP-1 amino acids 600 to 652. We generated HBC cells that have stable expression of myc-tagged, wild-type CARP-1 (1-1150) or CARP-1 (1-1150; Δ600-652) mutant. Further analyses revealed that CARP-1 (1-1150), but not CARP-1 (1-1150; Δ600-652) mutant, interacted with H2AX. Moreover, HBC cells expressing CARP-1 (1-1150; Δ600-652) mutant were resistant to apoptosis by Doxorubicin or CFM-4.16, and had diminished levels of γH2AX, when compared with their counterparts expressing wild-type CARP-1. We have earlier reported involvement of the Stress-Activated Protein Kinases (SAPKs) such as p38 and JNKs in apoptosis by Doxorubicin or CFMs. Since the PI3K family of serine-threonine kinases ATM/ATR, as well as JNK SAPKs, phosphorylate serine-139 of H2AX following DNA damage, our current studies are investigating whether H2AX binding with CARP-1 serves to facilitate phosphorylation of CARP-1 and H2AX proteins for transduction of apoptosis signaling.

#4379

Oncolytic adenovirus type 5 induces a novel form of programmed necrosis.

Melanie Weigert,1 Alex Binks,1 Suzanne Dowson,1 Elaine Leung,1 Dimitris Athineos,2 Xinzi Yu,1 Margaret Mullin,1 Josephine Walton,1 Clare Orange,1 Darren Ennis,1 Karen Blyth,2 Stephen Tait,2 Iain McNeish1. 1 _University of Glasgow, Glasgow, United Kingdom;_ 2 _Cancer Research UK Beatson Institute, Glasgow, United Kingdom_.

Introduction

Oncolytic viruses are a promising new cancer therapy, since they can infect cancer cells, selectively multiply within them and induce direct cytotoxicity, leading to the release of mature viral particles that can infect other neighboring cancer cells. However, the mechanisms by which oncolytic adenoviruses induce cell death remains uncertain. It was long thought that DNA viruses induce apoptosis, but there is now evidence that cell death induced by adenovirus, vaccinia and HSV-1 displays features strongly resembling a form of programmed necrosis.

Methods and Results

In order to investigate the role of necrosis in cell death as a result of oncolytic adenovirus infection, cancer cells were infected with the E1A CR2-deleted adenoviral mutant dl922-947, which specifically replicates in cells that have abnormalities in the pRB-pathway. We specifically sought to investigate the role of the core necrotic proteins RIPK1, RIPK3 and MLKL in adenovirus cytotoxicity.

Electron microscopy indicated that dl922-947 infection induces key morphological changes similar to necrotic death induced by TSZ (TNF-α, Smac-mimetic, Z.Vad.fmk) treatment. Using specific inhibitors of programmed necrosis (necrostatin-1, necrosulfonamide, GSK'840B and GSK'843A) as well as RNAi-mediated knockdown of RIPK1, RIPK3 and MLKL, we showed that adenovirus-infected cancer cells undergo RIPK3-dependent necrosis. We further found that, while TNF-α-induced programmed necrosis relies on the (RHIM)-dependent interaction of RIPK1 and RIPK3, dl922-947-induced cell death is independent of TNF-α signalling, does not involve RIPK1 and does not rely on the presence of MLKL. Caspase-8 inhibition, however, induces RIPK3-dependent necrosis that significantly enhances dl922-947 cytotoxicity. Using CRISPR/Cas9 gene editing, we have demonstrated that this increase in cytotoxicity during caspase inhibition was MLKL dependent.

Using a RIPK3 overexpression model, we observed that the extent of adenovirus-induced cell death correlated with RIPK3 expression even in the absence of caspase inhibition. Using RIPK3 co-immunoprecipitation, we identified an interaction between RIPK3 and MLKL as well as an interaction between RIPK3 and adenoviral proteins. In vivo experiments using human xenografts showed that expression of RIPK3 significantly improved anti-tumor activity following intra-tumoral injection of dl922-947.

Conclusions

Our data suggest that adenovirus infection induces a novel form of programmed necrosis that differs from classical TSZ-induced necroptosis, but still relies on the kinase RIPK3. Unfortunately, many cancer cells do not express RIPK3 and can therefore not undergo programmed necrosis. The integration of human RIPK3 into an adenoviral vector offers a therapeutic window to eradicate cancer cells that are resistant to apoptosis.

#4380

Role of FAM3B/PANDER in inhibition of cell death and tumor growth in breast and prostate cancer.

Izabela D. Caldeira. _University of São Paulo, São Paulo, Brazil_.

FAM3B/PANDER is a novel cytokine-like protein that induces apoptosis in beta-cells and regulates the effects of insulin in peripheral tissues. Since previous data revealed that FAM3B can be expressed by prostate and breast tumors, we evaluated the role of this cytokine in prostate and breast tumor progression. FAM3B expression was compared by quantitative PCR in LnCAP, PC-3 and DU145 prostate tumor cell lines and MCF-7 and MDA-MB- 231 breast tumor cell lines. After treatment with either recombinant FAM3B protein or secreted FAM3B obtained from conditioned media (CM) derived from FAM3B-overexpressing 293T cells, the cell death and viability of DU145 and MDA-MB- 231 cell lines were evaluated. DU145 and MDA-MB- 231 cells overexpressing FAM3B protein were produced by lentiviral-mediated transduction of full-length FAM3B cDNA. Cell viability and apoptosis were analyzed in DU145-FAM3B and MDA-231-FAM3B cells after treatment with cell death inducers. Anchorage-independent growth and scratch wound assays were used to evaluate in vitro tumorigenicity and cell migration, respectively. In vivo tumorigenicity and invasiveness were evaluated by tumor xenograft growth in nude mice. We observed that FAM3B was highly expressed by hormone responsive cells (LnCAP and MCF-7) and low expressed in unresponsive hormone cells (PC-3, DU145 and MDA-MB- 231). Cell viability and survival of DU145 and MDA-MB- 231 cells increased after exogenous treatment with recombinant FAM3B protein or CM containing FAM3B-secreted protein. Overexpression of FAM3B in DU145 and MDA-MB- 231 cells promoted an inhibition of apoptosis triggered by TNF-alpha and staurosporine. Cell death inhibition was accompanied by increased gene expression of the anti-apoptotic Bcl-2 and Bcl-xL genes, and by slight decrease in expression of pro-apoptotic gene Bax and with a decrease of caspases proteolytic activities. When compared to control, cells overexpressing FAM3B displayed a decreased anchorage independent growth and increased motility in vitro, as well as an increased tumor growth in xenografted nude mice. In agreement Immunohistochemistry analysis from tumor xenografts revealed similar anti-apoptotic phenotype in FAM3B overexpression, with an increase of Bcl-2. The results showed that FAM3B was capable to activates pro-survival mechanisms with an increasing of tumor growth by, activation of Bcl-2 and Bcl-xl pathways, suggesting the role of this cytokine in breast and in prostate tumors progression.

#4381

Cordycepin promotes apoptosis in renal carcinoma cells by activating the MKK7-JNK signaling pathway through inhibition of cFLIPL expression.

Ik-Soon Jang,1 Hyun-Jin Jang,1 Eunbi Jo2. 1 _Korea Basic Science Inst., Daejon, Republic of Korea;_ 2 _Korea Basic Science Inst., Republic of Korea_.

Cellular FLICE inhibitory protein (c-FLIP) is a key anti-apoptotic regulator that associates with the signaling complex downstream of NF-κB, negatively interfering with apoptotic signaling. The role of c-FLIP downregulation by negative regulation of NF-κB signaling during apoptosis is poorly understood. Here, we demonstrate that NF-κB-mediated c-FLIPL negatively regulates the JNK signaling pathway, and that cordycepin treatment of human renal cancer cells leads to apoptosis induction through c-FLIPL inhibition. TNF-α-induced inflammatory microenvironments stimulated NF-κB signaling and the c-FLIP long form (c-FLIPL) in TK-10 cells. Specifically, cordycepin inhibited TNF-α-mediated NF-κB activation, which induced renal cancer cell apoptosis. Cordycepin downregulated GADD45B and c-FLIPL, but upregulated MKK7 and phospho-JNK, by preventing nuclear mobilization of NF-κB. Furthermore, siRNA-mediated knockdown of GADD45B in cordycepin-treated TK-10 cells considerably increased MKK7 compared to cordycepin alone. siRNA-mediated knockdown of cFLIPL prevented TNF-α-induced JNK inactivation, whereas c-FLIPL overexpression inhibited cordycepin-mediated JNK activation. The JNK inhibitor SP600125 strongly inhibited Bax expression. In nude mice, cordycepin significantly decreased tumor volume. Taken together, the results indicate that cordycepin inhibits TNF-α-mediated NF-κB/GADD45B signaling, which activates the MKK7-JNK signaling pathway through inhibition of c-FLIPL expression, thus inducing TK-10 cell apoptosis.

#4382

**Complement system mutational landscape reveals** C4BPA **mutations enhance apoptosis in an immune-independent manner.**

Monica M. Olcina,1 Nikolas G. Balanis,2 Ryan K. Kim,1 Michael J. Thompson,2 Thomas G. Graeber,2 Amato J. Giaccia1. 1 _Stanford University, Stanford, CA;_ 2 _University of California, Los Angeles, CA_.

The complement system is an important pathway in immunity. When dysregulated in the tumor microenvironment, complement is associated with suppression of antitumor immunity and tumorigenesis promotion. While complement has been reported to have important intracellular functions in immune cells, the role of intracellular complement in cancer has remained poorly understood to date. In this study we investigated the prevalence and significance of pathway-wide complement mutations as well as their role with respect to intracellular complement in cancer. We describe mutations in both individual genes as well as within functional groups that are significantly associated with altered survival outcomes. Analyzing mutations occurring across multiple TCGA cancer types highlighted the potential clinical significance of certain mutations that would otherwise not have surfaced at a single cancer level. As an example, we test the significance of mutations in complement regulator C4BPA in vitro and in vivo in a single cancer type. This approach allowed us to uncover a new immune-independent biologic function of the complement system with potential clinical implications for colorectal cancer patients. Specifically, we find that colorectal cancer cells with specific C4BPA mutations display increased oxaliplatin-induced intracellular C4BPA stabilization. By studying the mechanistic basis of this association we report novel crosstalk between intracellular complement and apoptosis signaling, occurring in an NFκB/RelA-dependent manner. Based on both experimental and patient outcome data, we therefore propose that assessing complement mutation status might facilitate patient stratification. In the case of C4BPA mutations, this would be particularly relevant to improve accuracy of prognosis assessment in stage II colorectal patients where TNM staging alone does not accurately predict outcome in patients who might benefit from adjuvant chemotherapy.

#4383

CD44s is a crucial ATG7 downstream regulator for formation of sphere, a key stem-like property, invasion and lung metastasis of human bladder cancer/BC cells.

Xiaohui Hua,1 Xun Che,1 Jiheng Xu,2 Chuanshu Huang,1 Grace Huang,3 Wei Dai1. 1 _New York Univ., Tuxedo Park, NY;_ 2 _Nelson Institute of Environmental Medicine, Tuxedo Park, NY;_ 3 _Northern Highlands Regoinal High School, Allendale, NJ_.

Over half a million US residents are suffering with bladder cancer (BC), which costs a total $4 billion in treatment annually. Although recent studies report that autophagy-related gene 7 (ATG7) is overexpressed in BCs, the regulatory effects of ATG7 on stem-like phenotypes and invasion have not been explored yet. Current studies demonstrated that the deficiency of ATG7 by its specific shRNA dramatically reduced Sphere formation and invasion in vitro, as well as lung metastasis in vivo in invasive BC cells. Further studies indicated that the knockdown of ATG7 attenuated the expression of CD44 and CD133, while ectopic introduction of CD44s, but not CD133, was capable of completely restoring sphere formation, invasion, and lung metastasis in T24T(shATG7) cells. Mechanistic studies revealed that ATG7 overexpression stabilized CD44s proteins accompanied with upregulating USP28 proteins. Upregulated USP28 was able to bind to CD44s and remove the ubiquitin group from CD44's protein, resulting in the stabilization of CD44s protein. Moreover, inhibition of ATG7 stabilized AUF1 protein and in turn reduced tet1 mRNA stability and expression, which was able to demethylate usp28 promoter, thereby reduced USP28 expression, finally promoting CD44s degradation. In addition, CD44s was defined to inhibit degradation of RhoGDIβ, which in turn promotes BC invasion. Our results demonstrate that CD44s is a key ATG7 downstream regulator of the sphere formation, invasion, and metastasis of BCs, providing significant insight into understanding the BC invasions, metastasis, and stem-like properties.

#4384

The differential induction of apoptosis in MCF-7 and MDA-MB-231 breast cancer cells in response to RBBP6 silencing is associated with p53 expression.

Pontsho Moela,1 Lesetja Raymond Motadi2. 1 _University of Pretoria, Pretoria, South Africa;_ 2 _North West University, Mahikeng, South Africa_.

Breast cancer remains a female-related health problem on a global scale, accounting for over a million newly estimated cases that are still on the rise. More than 50% cases of breast tumorigenesis are due to mutations or inactivation of p53, a tumor suppressor gene involved in the regulation of cell cycle and apoptosis. In breast cancer, the frequency of TP53 mutations varies greatly between the heterogeneous subtypes, with basal-like breast cancers having the highest frequency whereas the luminal subtypes have been shown to generally express wt. p53. Active p53 is responsible for sensing any cellular stress signal with a potential to damage DNA, thus deciding the cell's fate depending on whether the damage is reversible or not. Therefore cellular p53 levels remain in check by the MDM2 regulatory protein, which promotes the degradation of unwanted p53 via a negative feedback loop mechanism using its E3 ligase activity. However, this interaction between MDM2 and wild-type p53 is cancer promoting in the same way that E6 oncoprotein is capable of leading to the degradation of wild-type p53 in cervical cancer. Negative regulators of p53, therefore, threaten cancer repression and play a role in drug resistance among cancer patients. Of particular interest in this study is RBBP6, another negative regulator of p53 that uses the E3 ligase activity as well in order to promote p53 degradation. Previous studies have shown that silencing RBBP6 in breast cancer restores p53 followed by apoptosis induction. However, it is unclear whether the observed apoptosis is linked to the recovered p53. To test this hypothesis, we aim to analyze the differential susceptibilities of MCF-7 and MDA-MB-231 cells expressing wt. p53 and mt. p53, respectively, to RBBP6 silencing. Real-time cell analysis revealed a clear distinction between the growth of the two cell lines, with MCF-7 showing a significantly reduced proliferation in comparison to MDA-MB-231. Apoptosis induction was also high in MCF-7 AND lower is MDA-MB-231, as was further confirmed by the differential expression of pro-apoptotic genes between the two cell lines. These findings suggest that difference in p53 statuses of the two cell lines play a role in apoptosis induced by RBBP6 silencing. These findings are necessary for reaching a better understanding of molecular mechanisms that impact patient response and resistance to candidate therapeutics and may be helpful in developing more effective treatment strategies for different subtypes of breast cancer.

#4385

**p53 activation induces a targetable dependence on FLIP** L **.**

Alexander McIntyre, Andrea Lees, Fiammetta Falcone, Gemma Gregg, Sessler Tamas, Gerard Quinn, Nyree Crawford, Darragh McArt, Phillip Dunne, Mark Lawler, Longley B. Daniel, Simon S. McDade. _Queen's University Belfast, Belfast, United Kingdom_.

Background

The tumor suppressive functions of the p53 transcription factor are inactivated via mutations or suppressed through non-mutational mechanisms in almost all cancer cells. A better understanding of the mechanisms through which p53 differentially regulates cell cycle arrest and cell death is important to maximize benefits from wild-type p53-dependent therapeutic strategies

Methods

A panel of matched p53 wild-type and deficient colorectal cancer cell line models were studied, using Nutlin-3A and Oxaliplatin as direct and indirect p53 activating agents respectively. A number of molecular (Western blot, RT-PCR), phenotypic (cell death) and genomic analyses were used to investigate the importance of p53 and its downstream transcriptional programs.

Results

Here, we report that activation of pro-apoptotic p53 targets in colorectal cancer cells imposes a critical targetable dependence on the long splice form of the caspase-8 regulator FLIP (FLIPL) for survival. p53 binds the promoter of the FLIP gene (CFLAR) and upregulates FLIPL expression in response to the p53 agonist Nutlin-3A in a manner dependent on HDAC1/2/3 activity. As such, preventing FLIPL upregulation with the clinically relevant HDAC1/2/3-selective inhibitor Entinostat promotes apoptosis induction in response to Nutlin-3A (or p53-activating chemotherapy), which otherwise predominantly induces growth arrest despite upregulating a range of pro-apoptotic target genes. Cell death in response to Nutlin-3A in FLIPL-depleted cells is primarily mediated via caspase-8. However, in the absence of caspase-8, apoptosis is delayed, but not prevented and is mediated via caspase-10. Of note, the cell death induced in both caspase-8-proficient and -deficient cells is mediated via TRAIL-R2 in a ligand-independent manner.

Conclusion

In summary, this work has uncovered novel, clinically-relevant biology, in which p53-mediated upregulation of FLIPL primes cells for TRAIL-R2-mediated apoptosis and identifies FLIPL as a key target for overcoming resistance to p53-stabilising agents in p53 wild-type cancers. Moreover, we show the potential of combining Nutlin-3A (or other p53 activating chemotherapies) with the clinically relevant Class I HDAC inhibitor Entinostat for the treatment of p53 wild-type CRC, and identify FLIPL as a critical p53-induced signaling node, the inhibition of which is necessary to promote Nutlin-3A-induced apoptosis.

#4386

Regulation of FIP200 by Her2 and its role in Her2-driven mammary tumors.

Mingang Hao, Jun-Lin Guan. _University of Cincinnati, Cincinnati, OH_.

Autophagy has been shown to play both tumor promoting and suppressive roles in various cancers, although the underlying mechanisms are not well understood. Compared to other subtypes of breast cancer (BCa), Her2+ BCa displays a lower autophagy gene signature, suggesting a potentially tumor suppressive function of autophagy in this subtype of BCa. FIP200 is a component of the ULK1/ATG13/FIP200/Atg101 complex essential for autophagy induction in mammalian cells, and our previous studies showed that Fip200 deletion suppressed BCa in MMTV-PyMT mouse model. Indeed, analysis of TCGA database showed that higher FIP200 expression correlated with decreased survival of BCa patients including all subtypes. In contrast, in Her2+ BCa population specifically, higher expression of FIP200 correlated with better patient survival, suggesting a differential function of FIP200. To uncover the role of FIP200 and autophagy in Her2+ BCa, we studied Fip200 in MMTV-Neu mouse tumor cells that model Her2+ BCa. We found that inhibiting autophagy by deletion of Fip200 or Atg13 using Crispr-Cas9 led to increased migration and partial EMT for MMTV-Neu cells. Further, deletion of Fip200 or Atg13 as well as autophagy inhibition by Spautin1 significantly enhanced β-catenin/LEF1/TCF reporter activity, which may promote cell migration and EMT. These changes in Fip200-null tumor cells could be rescued by re-expression of ectopic FIP200, excluding any potential off-target effects of gene knockout. We also found that Her2 knockdown or lapatinib treatment of MMTV-Neu cells increased Fip200 expression and autophagy. Together, these results raised the interesting

possibility that Her2 may promote tumor development and/or progression at least in part by reducing FIP200 expression to inhibit autophagy, which might play a tumor suppressive role in Her2-driven BCa.

#4387

Triplet nucleotide repeat-based siRNAs are highly toxic to cancer cells.

Andrea E. Murmann,1 Will Putzbach,1 Quan Gao,1 Monal Patel,1 Elizabeth T. Bartom,1 Calvin Law,1 Bryan Bridgeman,1 Siquan Chen,2 Kaylin M. McMahon,1 Shad C. Thaxton,1 Marcus E. Peter1. 1 _Northwestern Univ., Chicago, IL;_ 2 _University of Chicago, Chicago, IL_.

Triplet nucleotide repeat (TNR) expansions in the genome cause a number of degenerative diseases. A prominent TNR expansion involves the triplet CAG in the huntingtin (HTT) gene responsible for Huntington's disease (HD). Pathology is caused by both protein and RNA generated from the TNR regions including small siRNA-sized repeat fragments. An inverse correlation between the length of the repeats in HTT and cancer incidence has been reported for HD patients. We now show that siRNAs based on the CAG TNR can be used to kill cancer cells by targeting genes containing reverse complimentary long TNRs in their open reading frames. Of the 60 siRNAs based on the different TNRs, the 6 members in the CAG/CUG family of related TNRs are the most toxic to both human and mouse cancer cells. siCAG/CUG-induced cell death occurs in vitro in all tested cancer cell lines and in a preclinical mouse model of ovarian cancer with no signs of toxicity to the mice. We propose to explore TNR-based siRNAs as a novel form of anticancer reagents.

#4388

Anti-cancer effects of oleuropein olive leaf extract in K562 leukemia cells.

Sophia Fagan, Natalie Fulco, Justin Gilbertson, Aaron R. Van Dyke, Shelley A. Phelan. _Fairfield Univ., Fairfield, CT_.

Olive leaves come from a Mediterranean plant that many believe to have positive effects on human health, including anti-cancer properties. Oleuropein is one of the two main phenolic compounds derived from olive leaves, and it has been shown to increase apoptosis in many types of cancer cells. Numerous studies have found that the polyphenols contained in olive leaves possess strong antioxidant properties, suggesting that the compound could be effective in anti-cancer treatments. The purpose of this study was to examine the effects of oleuropein extract on cell growth and viability in K562 human leukemia cells, and begin to investigate possible molecular mechanisms of action. We purified our own extract from olive leaves for these studies. Oleuropein was microwave extracted from olive leaf powder using methanol and water as solvent. The oleuropein was successfully identified using HPLC to characterize elution time in comparison to an authentic standard. HPLC was then used in purification and fractions were lyophilized. Isolated oleuropein was greater than 94% in comparison to the authentic standard. We next demonstrated that K562 cells treated with 50-150 ug/ml of oleuropein exhibited significant reduction in cell growth and cell viability after four days when compared to 70% ethanol-treated controls. This effect was markedly greater after 6 days . Current studies are being conducted to determine the most effective concentration and duration of treatment for oleuropein-induced cell death. We are also examining cellular cyytotoxicity and cell cycle progression in these experiments using LDH assay and cell cycle staging, and further studies are being conducted to examine inflammatory and antioxidant pathways for their involvement in the anti-cancer effect of the compound in these cells. Given the observation that many of the cytotoxic effects of oleuropein are cancer-cell specific, this research provides a promising new avenue for the use of natural products as anti-cancer agents.

#4389

**HEXIM1 is a negative regulator of** H. pylori **-induced BIRC3 enhancer RNA.**

Donald R. Sheppard. _University of Illinois at Champaign-Urbana, Urbana, IL_.

The bacterium Helicobacter pylori inhabits the human stomach and is responsible for a number of pathologies, including being strongly associated with the development of gastric adenocarcinoma. H. pylori inhibits apoptosis in gastric cells, but the mechanism is poorly understood. We investigated how H. pylori inhibits apoptosis using cultured G27-strain H. pylori to stimulate cancer cell lines, and apoptosis was induced with Raptinal. We found that H. pylori induces the transcription of the apoptosis-inhibiting protein cIAP2, and this induction is sufficient to inhibit apoptosis in stimulated cells. We found an enhancer region upstream of the gene that codes for cIAP2 (BIRC3). H. pylori induces expression of RNA from this enhancer, and RNAi treatment targeting this eRNA inhibited induction of the BIRC3 mRNA. Chromatin immunoprecipitation revealed that positive transcription elongation factor b (P-TEFb) is recruited to the enhancer following H. pylori stimulation, and RNA polymerase II is activated. We found that the negative regulator of transcription HEXIM1 inhibits P-TEFb recruitment and eRNA production. HEXIM1 also inhibits BIRC3 mRNA transcription, cIAP2 protein induction and H. pylori's anti-apoptotic effect. Together, these findings indicate that H. pylori inhibits apoptosis induction in cultured cells, and this inhibition is mediated by the induction of BIRC3/cIAP2. H. pylori stimulates the production of RNA from the enhancer region of BIRC3, which is required for the induction of the gene, and the eRNA production is regulated by HEXIM1 and P-TEFb.

#4390

Developing methods for investigating cold atmospheric plasma effects on cervical cancer.

Nicole J. Sova, Yonry Zhu, Quinn O. Mitchell, Ariel L. Lanier, David Burnette, Monica M. Burdick. _Ohio University, Athens, OH_.

Cold atmospheric plasma (CAP) has been suggested for use as a novel cancer therapeutic due to its reported ability to induce cell-specific death. Cervical cancer is the fourth most common cancer in women. Its prevalence and accessibility for topical treatment make cervical cancer an ideal target for CAP treatments. Despite its potential impact, effects of CAP in biological systems are not well understood. To better understand the viability of CAP as a therapy, this project investigated in vitro cellular responses induced by CAP treatment within cancerous cervical cells (SiHa) and non-cancerous cervical (Ect1/E6E7 and End1/E6E7) and vaginal cells (VK2/E6E7).

Plasma is a state of ionized gas, produced using a controlled electric field or extreme heat. CAP, however, is a subset of plasma that is produced near 1 atm and 40oC. This allows for CAP treatment of biological systems without heating-induced effects. A dielectric barrier discharge plasma pen was developed for this study. Plasma modeling was conducted in order to predict the reactive species present in the system. CAP treatments were performed on cells for 30 seconds using a helium carrier gas at a flowrate of 1 L/min and a DC power supply with input settings of 10kV, 1µs, and 0.1A.

Cell vitality assays demonstrated induced cell death following exposure to CAP treatment in both cancerous and noncancerous cervical cells as indicated by flow cytometric analysis and florescence microscopy. CAP treatment induced cell death in 76 ± 8% of treated cancerous, SiHa, cells. In comparison, noncancerous cell lines Ect1/E6E7, End1/E6E7, and VK2/E6E7 underwent 49 ± 8%, 42 ± 13%, and 51 ± 7% cell death, respectively. Flow cytometry characterization and wound healing assays indicate that CAP treatment does not significantly affect the expression of investigated cell surface adhesion molecules or cell migration. Cells were treated with helium carrier gas as the negative control and hydrogen peroxide as an induced death control.

Altered levels and locations of uptake of FITC and quantum dots following CAP treatment suggest modification of cell membranes as compared to carrier gas treatment controls. Adjustments in membrane permeability may be a mechanism through which CAP treatment increases exposure to apoptotic triggers.

In conclusion, comprehensive understanding of CAP effects on cancerous and non-cancerous cells could lead to the development of effective CAP-based therapies for cervical cancer.

#4391

CD95L mRNA is toxic to cancer cells.

William Putzbach,1 Ashley Haluck-Kangas,1 Quan Gao,1 Aishe A. Sarshad,2 Elizabeth Bartom,1 Austin Stults,1 Abdul S. Qadir,1 Denise M. Scholtens,1 Markus Hafner,2 Marcus Peter1. 1 _Northwestern University Feinberg School of Medicine, Chicago, IL;_ 2 _National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, MD_.

CD95/Fas ligand (CD95L) is best characterized for its role in activating extrinsic apoptosis through binding to its receptor, CD95. However, we recently reported that the CD95L mRNA is enriched in sequences that when converted to si- or shRNAs kill cancer cells through an RNAi-dependent mechanism. When loaded into the RNA Induced Silencing Complex (RISC), CD95L-derived small RNAs downregulate genes critical for cancer cell survival, eliciting a unique form of cell death we call Death Induced by Survival Gene Elimination (DISE). We now report that expression of full-length CD95L mRNA itself is toxic to cells. Acute expression of either wild-type CD95L mRNA in CD95-deficient cells or a CD95L mRNA with a premature stop codon in CD95 wild-type cells induces cell death. RNA-Seq analysis of small RNA reveals that CD95L mRNA is specifically processed into discrete clusters ~19-25 nucleotides long that map along the CD95L ORF. These clusters of CD95L sequences were bound by AGO proteins in cells about to die, suggesting that the mRNA may be killing cells through an RNAi-dependent mechanism. Generation of AGO-bound CD95L mRNA sequences does not appear to be dependent on the canonical miRNA biogenesis pathway, as clusters of CD95L mRNA were detectable in both Drosha and Dicer deficient HCT116 cells. We propose that CD95L mRNA is toxic to cells through processing and loading of small CD95L-derived RNA sequences into the RISC.

#4392

Novel combination of Di-indolylmethane (DIM) and dexamethasone induces apoptosis in myeloma cells by modulating the telomerase activity.

Raman Kumar,1 Nidhi Gupta,1 . Himani,2 Alpana Sharma1. 1 _All India Institute of Medical Sciences, New Delhi, India;_ 2 _North DMC Medical College & Hindu Rao Hospital, New Delhi, India_.

Background and Objectives Multiple myeloma (MM) is a B-cell malignancy involving excessive proliferation and accumulation of atypical plasma cells specifically in the bone marrow (BM) niche. Multiple primary and secondary genetic alterations play crucial role in deregulating the biology of myeloma cells giving them a malignant phenotype, hence generating a need for novel efficacious and safe therapeutic agents. Increased expression of telomerase and higher telomerase activity (RTA) has been found in majority of cancer but displays very low activity in majority of normal somatic cells. Therefore, this maiden attempt unveils the therapeutic potential of Di-indolylmethane alone or in combination with dexamethasone by targeting telomerase activity in myeloma cells. Methodology In this study, two myeloma cell lines (RPMI8226 & U266) were procured from ATCC (Manassas, VA). Di-indolylmethane (DIM) and dexamethasone (Dexa) were purchased from Sigma Aldrich (St. Louis, MO). In in-vitro study, IC50 concentration of DIM and Dexa on both cells was determined using MTT dye. Cell-cycle and apoptosis assay was performed to determine the anti-proliferative and apoptotic effects of DIM and Dexa alone or in combination. Further, caspase-3/7 activity and mitochondrial membrane potential (MMP) was determined to assessed pathway of apoptosis. Effects of single drug or combination on telomerase activity were also assessed in both myeloma cells. Results Results demonstrated that, DIM showed an IC50 conc. of 40μM in both myeloma cells while these cells were resistant to Dexa even at higher dose. In cell cycle assay, significant increase in sub-G0/G1 phase were observed with increasing dose of DIM while combination of DIM and Dexa significantly increased sub-G0/G1 cell populations in comparison to vehicle control. Similarly annexin-V/PI apoptosis assay also demonstrated increased apoptosis of both myeloma cells. Further, DIM alone or in combination with Dexa demonstrated drastic increased caspase-3/7 activity and significant decrease in MMP in both myeloma cells. Additionally, increasing dose of DIM significantly decreased telomerase activity and combination of DIM and Dexa was much more potent in decreasing telomerase activity in comparison to vehicle treatment and DIM alone. Conclusions In nutshell, this maiden attempt provided initial evidences of therapeutic potential of DIM as novel anticancer agent alone or in combination with standard chemotherapeutic agent Dexa. Further, novel combination of DIM and Dexa was much more potential in inducing apoptosis cell death and chemo-sensitizing myeloma cells than single drug treatment regime. Also intrinsic pathway of apoptosis was followed by DIM in inducing apoptosis in myeloma. Furthermore, decreased TA by DIM emphasized its potential as novel molecular therapeutic target which need further validation.

#4393

Electrochemotherapy with irreversible electroporation and FOLFIRINOX improves survival in murine pancreatic adenocarcinoma by increasing apoptosis and decreasing tumor cell proliferation.

Neal Bhutiani, Harshul Pandit, Qianqian Zheng, Suping Li, Yan Li, Robert C. Martin. _Univ. of Louisville, Louisville, KY_.

Introduction: Combining gemcitabine (gem) with irreversible electroporation (IRE) has been shown to result in increased tumor cell apoptosis in a murine model of pancreatic adenocarcinoma (PDAC). However, it remains unclear whether this effect can be augmented locally and systemically by utilizing more active chemotherapies. The objective of this study was to evaluate the efficacy of IRE+FOLFIRINOX (FOL: 5-FU, leucovorin, irinotecan, and oxaliplatin).

Methods: Athymic nude mice underwent intrapancreatic injection with S2-013 PDAC cells. After 7-10 days, tumors were confirmed with ultrasound and mice were treated with chemotherapy (gem or FOL), IRE, or chemotherapy+IRE. Four hours after last chemotherapy, pancreas was evaluated histologically. Levels of irinotecan and its active metabolite (SN-38) were evaluated in organs of mice treated with FOL and IRE+FOL. Experiments were repeated and survival analyses performed to evaluate long-term efficacy. Pancreas was harvested for histology and evaluation of apoptotic machinery, cell cycle proteins, and tumor cell proliferation.

Results: IRE+FOL (ECT) resulted in increased tumor cells apoptosis compared to gem, gem+IRE, or FOL (apoptotic index (AI) 34.6% vs. 3.8%, 7.3%, 5.8%, respectively, p<0.001). High performance liquid chromatography (HPLC) demonstrated a 1.5-2-fold increase in irinotecan and SN-38 in the pancreas and a 4-fold increase of both compounds in the liver in mice treated with ECT vs. FOL. ECT significantly improved overall survival when compared to mice treated with IRE or FOL (median 7 days ECT vs. 4 days IRE vs. 3 days FOL, p=0.03). Histology demonstrated increased tumor cell apoptosis at time of death (AI = 34.2% ECT vs. 13.2% IRE vs. 4.4% FOL, p=0.026). Western blot demonstrated an increase in active caspase 3 (0.14 ECT vs. 0.00 IRE vs. 0.03 FOL fold change/GAPDH, p=0.02). ECT resulted in lower cell proliferation compared to IRE or FOL (0.2% vs. 0.3% vs. 0.9, p<0.001).

Conclusions: ECT with IRE and FOL causes significant tumor cell apoptosis, decreases tumor cell proliferation, enhances systemic FOL exposure, and improves overall survival. Human Phase 1 trials have been initiated to evaluate safety and efficacy of this therapeutic modality.

#4394

Novel edible medicinal plant induced cytotoxic and apoptotic effects on ovarian cancer cells.

Ariane T. Mbemi. _Jackson State University, Jackson, MS_.

Ovarian cancer (OC) is the leading cause of death in gynecologic cancer and the fifth most common form of cancer diagnosed among women. Caucasians have a higher incidence and mortality rate compared to African-Americans. The low survival rate of ovarian cancer among patients is caused by the fact that ovary organs are located inside the pelvic region in the female reproductive system; many cases are diagnosed in advanced stage, when cancer has already metastasized beyond the ovaries. Consequently, there is an urgent need to prevent ovarian cancer at the early stage and provide effective treatment. Herbal medicine with lesser or no side effect is currently considered as an alternative cancer treatment because it possesses many bioactive compounds essential to destroy cancer cells. A preliminary study in our laboratory demonstrates that extracts of Vernonia species have antitumor activities against cancer cells. Therefore, our overall goal in this study was to test the therapeutic mechanisms of a Vernonia species (an edible medical plant) in the treatment of ovarian cancer. To reach our goal, we tested the cytototic and apoptotic effects of the Vernonia species on OVCAR-3 cells. Data generated from the trypan blue test and MTT assay demonstrated that extracts of Vernonia species exhibited significant growth-inhibitory effects on OVCAR-3 cells. Oxidative stress biomarkers showed slight decrease in the production of malondialdehyde (MDA) levels and an increase in catalase activity and glutathione peroxidase in treated cells compared to the control group. Further experiments, including cell morphology and apoptosis analysis, showed that VA caused growth-inhibitory effects on OVCAR-3 cells through cell cycle arrest at the Go/G1 checkpoint and activation of caspase-3 activity leading to apoptosis. Collectively, findings from this study provide convincing scientific evidence that the Vernonia species tested here may represent a potential anticancer candidate against ovarian cancer.

This research work was supported by a grant from the National Institutes of Health (Grant No. NIMHD-G12MD007581).

#4395

KP-1339 (IT-139) induces the hallmarks of immunogenic cell death in a colon cancer 3D model in vitro.

Debora Wernitznig,1 Giorgia Del Favero,1 Konstantinos Kiakos,1 Nathalie Harrer,2 Herwig Machat,2 Doris Marko,1 Michael Jakupec,1 Wolfgang Sommergruber,2 Bernhard K. Keppler1. 1 _University of Vienna, Vienna, Austria;_ 2 _Boehringer Ingelheim, Vienna, Austria_.

Chemotherapeutic drugs exert their activity by directly killing tumor cells through a variety of cellular targets eventually leading to apoptosis. Apoptosis is regarded as non-inflammatory and non-immunogenic, however, recent data confirm that the induction of damage-associated molecular patterns by dying cancer cells treated with certain chemotherapeutic agents, such as oxaliplatin, lead to their recognition by the immune system, and result in dendritic cell maturation and immune responses, a mechanism described as Immunogenic Cell Death (ICD). The main hallmarks of ICD are: Calreticulin (CRT) exposure on the cell membrane and release of high mobility group box-1 (HMGB-1) and ATP from dying cells. We have recently shown that the mode of action of KP-1339, a clinically investigated ruthenium-based metal complex, involves the enhancement of endoplasmic reticulum stress (ERS) and the induction of apoptosis-like events. We next investigated the ability of KP-1339 to trigger ICD. There is currently no reliable method to study ICD in vitro, and therefore we aimed at a more suitable model that better mimics the tumor microenvironment in comparison to 2D monolayer cultures. For this reason, ICD was assessed in human colorectal cancer HCT-116 3D multicellular spheroids in vitro. We demonstrate that spheroids treated with KP-1339 activate the PERK/Phospho-eIF-2alpha pathway of the ERS, a prerequisite for CRT exposure on the cell membrane. HMGB1 protein levels decrease considerably during a 72 hours drug treatment, as shown by Immunoblotting, suggesting the depletion of the protein. Additionally, treated spheroids released significantly more ATP than untreated controls. Immunofluorescence of cryosections of KP-1339 treated spheroids stained for CRT and HMGB1 confirmed the above observations and, clearly showed the translocation of CRT to the cell membrane.

In conclusion, we have established a 3D tumor spheroid model as an alternative to the 2D monolayer cultures to study ICD in vitro. This model more closely resembles the tumor microenvironment and recapitulates relevant aspects of the in vivo situation. We successfully employed this model to demonstrate that the clinically investigated compound KP-1339 triggers the main hallmarks of ICD, which suggests enhanced induction anti-tumor immunity in colorectal cancer.

#4396

Targeting survivin to overcome docetaxel resistance in prostate cancer.

Robert C. Peery. _Indiana University School of Medicine, Indianapolis, IN_.

Despite therapeutic advancements, castration-resistant prostate cancer (CRPC) remains the second most common cause of cancer-related mortality in men. Docetaxel is the first cytotoxic agent to show modest improvements in overall survival rate in patients with CRPC. Unfortunately, over half of these patients do not respond to treatment and ultimately all develop resistance. The mechanism mediating docetaxel resistance remains unknown. However, survivin, an inhibitor of apoptosis (IAP) family member, and known mediator of chemo-resistance has been previously associated with docetaxel resistance, as inhibition of survivin expression sensitized prostate cancer cells to docetaxel in vitro, and a small molecule inhibitor targeting survivin expression led to a significant regression in prostate cancer xenograft tumors when given in combination with docetaxel. However, how survivin may mediate docetaxel resistance in prostate cancer remains unknown. In this study, we tested the hypothesis that overexpression of nuclear survivin contributes to docetaxel resistance in prostate cancer cells. First, utilizing western blot to assess protein level and methylene blue assays to determine ability to proliferate under cytotoxic conditions, we found that survivin expression and docetaxel IC50 correlates strongly in five prostate cancer cell lines. Furthermore, using paired parental drug sensitive and stepwise selected docetaxel resistant cell lines, we determined that resistant cells overexpress survivin as compared to parental cells, and knockdown of survivin decreases resistance to docetaxel. Our studies suggest that survivin is likely implicated in CRPC and treatment with a direct survivin inhibitor may sensitize resistant cells to docetaxel. We are in the process of determining how this IAP may mechanistically mediate docetaxel resistance.

#4397

Tak-1 inhibition re-sensitizes cancer cells to TNFα.

Scott Scarneo, Madeline Sell, Juliane Totzke, David Carlson, Phillip Hughes, Tim Haystead. _Duke University, Durham, NC_.

Identifying new pharmaceutical targets in cancer has been a long lasting goal of academia. Complex genomic changes coupled with a cancer cell's ability to adapt make tumors particularly difficult to target. To this end, we have identified the TAK1 kinase in the TNFα pathway as a potential mediator of cell survival or apoptosis. Here we evaluate the apoptotic effects of a novel TAK1 inhibitor, Takinib. The selectivity of Takinib was established with an IC50 of 9nm for TAK1, followed by an IC50 for IRAK4 and IRAK1 at 120nm and 390nm respectively. Treatment of MDA-MB-231 cells with Takinib on its own is not sufficient to induce consequential cell death. However, combination therapy with TNFα induces caspase 3/7 activity and subsequent cell death in a dose-dependent manner. Genetic knock out of TAK1 expression in MDA-MB-231 cells with the CRISPR/Cas9 system significantly sensitized previous resistant cells to TNFα. Furthermore, we have shown that activation of human THP-1 cells with LPS is sufficient to produce TNFα levels necessary to facilitate Takinib mediated cell death, indicating that potential immunotherapy applications may be possible. Together these findings support the idea that TAK1 may be an effective target in the TNFα pathway, shifting the cellular mechanisms from pro survival to pro apoptosis.

#4398

In vitro Tumor Treating Fields (TTFields) alter proliferation and morphology of patient-derived high-grade meningioma cell lines.

Sharon K. Michelhaugh, Sam Kiousis, Sam A. Michelhaugh, Neil V. Klinger, Sandeep Mittal. _Wayne State University, Detroit, MI_.

Introduction: Meningiomas are the most common neoplasm of the central nervous system (CNS), accounting for 37% of primary CNS tumors. Though derived from the arachnoid cap cells within the meninges, high-grade meningiomas (WHO grades II and III) invade into the normal brain tissue, and have a high rate of recurrence. As there are no efficacious chemotherapies for these high-grade meningiomas, standard of care is currently limited to maximal surgical resection followed by radiotherapy, with a median overall survival of only 2.5-3 years. Clinical trials with TTFields therapy are under way for patients with high-grade meningiomas. In this study, in vitro experiments comparing TTFields to untreated controls were performed on two patient-derived anaplastic (WHO grade III) meningioma cell lines to determine the effects of TTFields on cell proliferation and clonogenicity.

Methods: Studies utilizing patient tumor specimens were approved by the Wayne State University Institutional Review Board and written informed consent was obtained from participants. Meningioma tumor specimens were collected immediately following microsurgical resection. Single-cell suspensions from the tumor tissues were prepared by enzymatic and mechanical disruption. Cells were plated on plastic coverslips (10,000 each) in DMEM/F12 media supplemented with 10% fetal bovine serum. TTFields were applied at ~1.6 V/cm at 100, 200, 300, or 400 kHz (groups n=4-5) with one control group. Culture media were replaced every day. After 12 days of treatment, cell proliferation was assessed with the XTT assay and cells were harvested and replated for clonogenic assays (500 cells/well), which were stained with crystal violet and counted with an automated colony counter. Harvested cells were also fixed and immunostained with the diagnostic meningioma cytoplasmic marker vimentin to observe the cell morphology in control vs. TTFields-treated cells. Control vs. TTFields-treated groups were compared by one- or two-tailed t-tests.

Results: The 12-day TTFields treatment significantly reduced cell proliferation, with the greatest decrease found in the 200 kHz group (0.508±0.21; mean±SEM) compared to the control (1.515±0.08; two-tailed t test p=0.0019). Similar results were found in the clonogenic assay, with control cell counts of 4219±793 compared to the 200kHz treated cells 1793±444 (one-tailed t test p=0.0279). In the vimentin-stained cells, the control cells displayed a typical meningioma spindle morphology, while the TTFields-treated cells demonstrated pleomorphic and irregular morphologies.

Conclusions: Application of TTFields in vitro reduced proliferation and clonogenicity and altered morphology of patient-derived anaplastic meningioma cell lines. This is the first report on the in vitro effects of TTFields on meningioma.

#4399

Entinostat sensitizes colorectal cancer cell lines to the IAP antagonist, ASTX660 by down-regulating FLIP expression.

Nyree T. Crawford,1 Katie Stott,1 Tomoko Smyth,2 John Lyons,2 Roberta Ferraldeschi,2 George Ward,2 Daniel B. Longley1. 1 _Queen's University Belfast, Belfast, United Kingdom;_ 2 _Astex Pharmaceuticals, Cambridge, United Kingdom_.

Introduction

Inhibitor of Apoptosis Proteins (IAPs) are anti-apoptotic proteins which suppress the activation of caspases. IAPs have been shown to be overexpressed in several cancers, including colorectal cancer (CRC) and correlate with poor prognosis. IAP antagonists, including SMAC mimetics, can convert TNFα from a pro-inflammatory and pro-survival signal into a potent pro-death signalling molecule. In the presence of IAP antagonists, TNFα signalling is driven to cell death induction through the formation of a complex containing FADD/procaspase 8 and RIPK1, termed complex-II. However, high expression of FLIP, an endogenous regulator of procaspase-8 processing and activation, can inhibit cell death signalling from this complex. We have previously shown that FLIP is frequently overexpressed in CRC, and we and others have demonstrated that histone deacetylase (HDAC) inhibition results in down-regulation of FLIP protein expression. The aims of this study were to assess the efficacy of a novel, non-peptidomimetic, cIAP and XIAP antagonist, ASTX660 (Astex Pharmaceuticals), as single agent and in combination with the class I HDAC inhibitor Entinostat in CRC cell lines.

Results

ASTX660 was found to be a potent on-target IAP antagonist in a panel of human and murine CRC cell lines, resulting in reduced cIAP1 expression and formation of the apoptosis-inducing complex II, containing RIPK1 and caspase-8, in the presence of TNFα. However, viability of CRC cell lines was unaffected by the ASTX660 treatment alone or in the presence of TNFα, added to mimic the pro-inflammatory microenvironment frequently associated with CRC. Downregulation of FLIP expression using siRNA in a panel of CRC cell lines significantly enhanced cell death induced by ASTX660 in the presence of TNFα. Entinostat down-regulated FLIP protein expression at clinically achievable concentrations and potentiated ASTX660-mediated cell death in the panel of CRC cell lines. This cell death was confirmed to be apoptotic in nature and, more specifically, caspase-8-dependent using CRISPR-caspase-8 knockout models. Moreover, the mode of combination effects was demonstrated to be FLIP-dependent using CRC models engineered to overexpress wild-type FLIP.

Conclusion

High levels of FLIP expression in pre-clinical models of CRC may contribute to resistance to single agent IAP antagonist treatment. Pre-treatment with the class I HDAC inhibitor Entinostat can sensitize CRC cell lines toASTX660 by down-regulating FLIP expression. HDAC inhibitors could be used in novel combinations with IAP antagonists to promote apoptosis in pro-inflammatory CRC. 

### MicroRNA Therapeutics

#4400

MicroRNA loaded circular dumbbell RNAs, a novel antiangiogenic approach.

Manu Gnanamony,1 Sajani S. lakka,2 Jaime Libes,1 Julian Lin,1 Pushpa A. Joseph,1 Christopher S. Gondi1. 1 _Univ. of Illinois College of Med. at Peoria, Peoria, IL;_ 2 _Univ. of Illinois College of Med. at Chicago, Chicago, IL_.

Angiogenesis is a tightly regulated biological process where new blood vessels are formed from pre-existing blood vessels. This process is also critical in diseases such as cancer; therefore, angiogenesis has been actively explored as a drug target for cancer therapy. Many patients treated with endogenous angiogenesis inhibitors show increased survival; however, eventual resistance to therapies is associated with disease progression and death. The future of effective anti-angiogenic therapy lies in the intelligent combination of multiple targeting agents with novel modes of delivery to maximize therapeutic effects. We therefore propose a novel and 'out of the box' approach that utilizes dumbbell-RNA (dbRNA) to target pathological angiogenesis by simultaneously targeting multiple molecules and processes that contribute to angiogenesis. Dumbbell RNAs are a stable and safe alternative to viral, non-viral, and naked plasmid-based gene-transfer systems. They integrate the advantages of durable expression that is achievable with integrating viral vectors and short-term effects triggered by naked RNA. In this study, we have successfully constructed a plasmid expressing miR-34a dbRNA using the permuted intron-exon (PIE) method. We have also modified and optimized a cost effective protocol to purifiy dbRNA from bacterial culture with high purity. To test the efficacy of miR-34a dbRNA, we used the pancreatic cancer cell line MIA-PaCa2. When transfected into MIA-PaCa2 cell line we observed increased expression of miR-34a. Further, miR-34a overexpression caused a 3- fold and 2.5-fold decrease in VEGFA and VEGFB RNA expression respectively. Functional validation on the effect of miR-34a on angiogenesis was performed on Human umbilical vein endothelial cells (HUVEC) using the tube formation assay, where miR-34a dbRNA showed significant decrease in tube formation when compared to cells treated with scrambled dbRNA. We will further validate these results in vivo using Zebrafish angiogenesis model. In conclusion, our study demonstrate for the first time a novel approach to block angiogenesis using miR-34a loaded dbRNA. Our data also show that this approach may be used in targeting multiple molecules/pathways, and when used in combination with existing therapies can improve the efficacy of current treatment modalities.

#4401

Integrated genomic and functional microRNA analysis identifies miR-30-5p as a tumor suppressor and novel therapeutic nanomedicine in head and neck cancer.

Anthony D. Saleh,1 Robert F. Place,1 Hui Cheng,2 Rita Das,2 Thomas M. Harris,3 Geoffrey Childs,3 Gordon A. Robertson,4 Kathleen F. Pirollo,5 Esther H. Chang,5 Zhong Chen,2 Carter Van Waes2. 1 _miRecule, Inc, Gaithersburg, MD;_ 2 _NIDCD/NIH, Bethesda, MD;_ 3 _Einstein University, New York, NY;_ 4 _Michael Smith Genome Sciences Centre, Vancouver, British Columbia, Canada;_ 5 _Georgetown University Medical Center, Washington, DC_.

We have developed an integrative approach to elucidate the functional role of deregulated microRNAs (miRs) and identify candidates for miR replacement therapy in cancer, applied here to head and neck squamous cell carcinomas (HNSCC). Our method involves integration of results from TCGA and other validation databases of genetic and expression data, including: miR expression, mRNA expression, copy number variation, DNA methylation. This data is then intersected with functional in vitro genome-wide miR screening for anti-proliferative miR mimics. We report that the miR-30-5p family is commonly repressed and correlates with overexpression of a broad network of proliferative and metastasis-related oncogenic mRNAs, including the growth receptors EGFR, MET and IGF1R. We show that re-expression of miR-30a-5p repressed this gene program, downstream signaling, proliferation, migration, and invasion in vitro. We have developed a chemically modified mimic of miR-30-5p with 50x improved stability in human serum, and 5X improvement in IC50 in vitro by XTT assay. We have formulated our novel miR-30a-5p mimic into Transferrin Receptor targeted nanoparticles which strongly inhibited HNSCC xenograft tumor growth, and regulated miR-30-5p family targets in vivo. Together with data linking decreased miR-30 family expression with DNA copy loss and promoter hypermethylation, and clinical disease-specific survival, for the first time, we have reported a more global picture of the function of this important tumor suppressor and identified a subset of patients that may benefit from miR replacement therapy with our novel miR-30-5p based nanomedicine.

This research was supported by NIDCD intramural projects ZIA-DC-000073, 74, and NCI grant U43CA22156701.

#4402

MiR-195 potentiates the efficacy of microtubule-targeting agents in non-small cell lung cancer.

Xiaojie Yu, Yiqiang Zhang, Xiuye Ma, Alexander Pertsemlidis. _UT Health at San Antonio, San Antonio, TX_.

Introduction: microRNAs (miRNAs) are a family of small noncoding RNAs (18-24 nt) that post-transcriptionally repress gene expression by direct binding to the 3' untranslated regions (UTRs) of their targets. By targeting cancer-related genes, miRNAs have been shown not only to regulate cancer growth/progression, but also to modulate the response of cancer cells to chemotherapy. Such miRNAs are potential candidates for therapeutic intervention. Aiming to identify functional miRNAs in non-small cell lung cancer (NSCLC), we performed a high-throughput screen and found that miR-195 inhibits the growth of NSCLC cells and sensitizes them to microtubule-targeting agents (MTAs), a family of chemotherapeutic drugs widely used for NSCLC treatment. The function and mechanism of miR-195 in NSCLC were demonstrated both in vitro and in vivo.

Results: We demonstrated that miR-195 synergizes with both a traditional MTA (paclitaxel) and a recently discovered one (eribulin) to repress the growth of NSCLC cells. Overexpression of miR-195 sensitizes NSCLC cells to paclitaxel and eribulin, while knockout of miR-195 confers resistance to paclitaxel and eribulin. Importantly, lung tumors with miR-195 overexpression are more sensitive to eribulin treatment than control tumors. Induced expression of miR-195 in lung tumors potentiates the efficacy of eribulin to repress tumor growth. Additionally, we showed that miR-195 directly targets CHEK1 to regulate the response of NSCLC cells to paclitaxel and eribulin. The direct and specific binding of miR-195 to the 3'UTR of CHEK1 was confirmed by luciferase reporter assay. Repression of CHEK1 using siRNAs and chemical inhibitor synergizes with paclitaxel and eribulin to repress the growth of NSCLC cells. Overexpression of CHEK1 contributes to the resistance to paclitaxel and eribulin in NSCLC cells. Analysis of TCGA data shows that CHEK1 is significantly upregulated in lung tumors compared to adjacent normal tissues and that its upregulation is associated with worse recurrence-free and overall survival.

Conclusions: We report the identification of miR-195 as a sensitizer to microtubule-targeting agents in NSCLC, mediated by its repression of CHEK1. Mouse xenografts with induced or constitutive overexpression of miR-195 show that tumors with high miR-195 expression are more sensitive to drug treatment and that induction of miR-195 potentiates the efficacy of eribulin in repressing tumor growth. These results highlight the possible application of miR-195 expression as a biomarker to predict patient response to MTAs and the potential for delivery of miR-195 mimic as an adjuvant to chemotherapy.

#4403

Development of 5-FU modified tumor suppressor miRNAs as a platform for miRNA based cancer therapeutics.

Andrew Fesler,1 Shixiang Guo,2 Zachary Ye,1 Matthew Godwin,1 Hyaizha Wang,2 Jingfang Ju1. 1 _Stony Brook Univ. Medical Ctr., Stony Brook, NY;_ 2 _Institute of Hepatopancreatobiliary, Southwest Hospital, Third Military Medical University, China_.

Despite extensive efforts to develop effective delivery technologies for nucleic acid based therapeutic agents, it remains a challenge to deliver therapeutic miRNAs to cancer cells. We have recently developed a novel approach to modify miRNAs by replacing uracil with 5-Fluorouracil (5-FU) in the guide strand of the miRNA molecule (e.g. miR-129, miR-15a). This modification combines the power of 5-FU with tumor suppressor miRNAs to create a potent, and multi-targeted therapeutic molecule without altering the function of the native miRNA. miRNA with this modification have a number of unique features such as retaining target specificity, vehicle free delivery, enhanced potency, and stability. To demonstrate the general applicability of this approach to other tumor suppressor miRNAs, in this study, we have screened a number of miRNA mimics incorporating this modification (e.g. miR-215, miR-140, miR-200c, miR-194, let-7g, miR-502, miR-506) in several major cancer types including colon cancer, pancreatic cancer, and breast cancer. Our results show that the 5-FU modified miRNAs are more potent (low nM range) at inhibiting cancer cell proliferation than the native miRNAs. More importantly, the miRNA mimics are able to eliminate chemoresistant cancer stem like cells. These modified mimics can be delivered into cancer cells without the aid of a delivery vehicle. The modification also did not alter target specificity of the miRNAs, a critical feature of these miRNA mimics. This work demonstrates the potential of this modification to be applied to a broad spectrum of miRNAs in different cancers, and represents a significant advancement in the development of therapeutic miRNAs for cancer treatment.

#4404

Identification of small molecules that inhibit miR-21 activity in breast cancer.

Michel F. Guiraldelli,1 Alexander O. Schwartz,1 Michelle A. Markus,2 Jun Jiang,3 Justin D. Boyd,2 Branko Radetich,1 William E. Monahan,1 Nanguneri Nirmala,1 Johan A. Pontin,1 Dalia Cohen1. 1 _The RNA Medicines Company, Bedford, MA;_ 2 _Beryllium Discovery, Bedford, MA;_ 3 _ARK Laboratory, Detroit, MI_.

In cancer, microRNA (miR) dysregulation can lead to tumor development, invasion and metastasis. miRs are small single-stranded non-coding RNAs that bind to target mRNAs and stop protein expression. miRs are transcribed as a long transcript that fold to form secondary hairpin structures called pri-miRs that are subsequently processed by the microprocessor complex (Drosha and DGCR8) resulting in the formation of the precursor miRs (pre-miRs). Pre-miRs are exported to the cytoplasm via Exportin 5 and RanGTP. In the cytoplasm, pre-miRs are cleaved by Dicer resulting in double stranded molecule. This miR duplex is assembled into the RNA-induced silencing complex (RISC) and targets specific mRNAs. In breast cancer, miR-21 expression levels are increased, and are correlated with tumor grade. The use of small molecules targeting the miR process to modulate levels of mature miR is a novel approach for developing therapeutics. Inactivation of any of the processing steps of miR would result in an inactive mature miR or lower significantly its level. Using a parallel screening approach employing cell biology, NMR and chemistry, we identified several small molecules that bind and inhibit the activity of miR-21. Further characterization of one of our hits in MCF-7 cells showed a dose dependent inhibition of miR-21 activity. A decrease in proliferation with a concomitant increase in apoptosis signal was observed in cells treated with the compound. Analysis of miR-21 processing species by qPCR in MCF-7 cells showed a decrease on pri, pre-and mature miR-21 relative to cells treated with vehicle only. Furthermore, the levels of PDCD4, a direct downstream effector of miR-21, increased upon treatment with the compound. NMR experiments showed that this compound binds to a specific sequence and that the flanking C-G bases are necessary for its binding. These results support the feasibility of targeting miRs by small molecule as a novel therapeutic modality.

#4405

Pharmacologic profile of INT-1B3: A novel synthetic microRNA 193a-3p mimic for therapeutic intervention in oncology.

Sanaz Yahyanejad, Thijs de Gunst, Iman Schultz, Harm den Boer, Monica Raimo, Bryony Telford, Rogier Vos, Laurens van Pinxteren, Roel Schaapveld, Michel Janicot. _InteRNA Technologies, Utrecht, Netherlands_.

MicroRNAs (miRNAs) are a family of small, non-coding RNAs and serve as small snippets of genetic material that regulate gene expression. As a result, miRNAs modulate a wide range of biological processes including; cell cycle control and apoptosis, cell signaling and differentiation, cell adhesion and motility. Thus, it is not surprising that their misregulation is linked to cancer initiation, treatment response, and metastasis. Due to the inherent ability of miRNAs to concurrently target multiple pathways, their therapeutic potential to be used as anti-cancer drugs is attractive. In this study, we are investigating the mode of action and development of a novel synthetic miRNA mimic (INT-1B3) as a therapeutic candidate for substitution and functional restoration of a depleted miRNA in variety of cancers. To identify miRNAs that control the survival of cancer cells, we performed a functional miRNA screening in which a wide range of relevant miRNAs were selected and miR-193a-3p was chosen as the potential candidate for further preclinical characterization. Compelling evidence demonstrate a tumor suppressor function for miR-193a-3p in many cancer types and link its reduced expression with cancer, metastasis and therapy resistance. Consistent with the described role for miR-193a-3p in literature, our cell-based assays conducted in various experimental tumor cell systems demonstrated efficient target engagement leading to reduced cell proliferation/survival, cell cycle arrest and induction of apoptosis, inhibition of cell motility as well as a potential immune-modulatory role (via adenosine-mediated tumor immune evasion). Furthermore, development of a novel lipid nanoparticle-based formulation as an efficient delivery system demonstrated a marked anti-tumor activity of INT-1B3 as single agent following systemic administration in tumor-bearing mice. Robust tumor growth inhibition in these experimental tumor models is correlated and consistent with efficient target engagement in tumor cells and relevant organs. We are currently investigating the in vitro and in vivo effect of INT-1B3 in variety of cancer types including hepatocellular carcinoma, melanoma and triple negative breast cancer. Our data thus far supports an anti-tumor role for INT-1B3 and encourages further preclinical and clinical investigation.

#4406

Canine osteosarcoma as a platform to investigate the therapeutic potential of a genetically engineered pre-microRNA prodrug in human osteosarcoma.

Fernando Alegre Guerra,1 Kellie Snider,1 Amanda Ormonde,1 Ai-Ming Yu,2 Luke Wittenburg1. 1 _UC Davis School of Veterinary Medicine, Davis, CA;_ 2 _UC Davis School of Medicine, Sacramento, CA_.

This study was undertaken to investigate the therapeutic effect of a genetically engineered, human pre-microRNA-34a prodrug in canine osteosarcoma (OS), validating a robust preclinical model for the development of this proposed agent in humans. Although osteosarcoma is the most prevalent primary bone tumor in children and young adults, the low overall incidence presents an obstacle to timely development and testing of new agents. However, the incidence of canine OS is nearly 12 times that in humans, providing a useful preclinical model with spontaneously occurring OS. Despite multimodal therapeutic protocols, up to 40% of human and 90% of dogs will not survive long-term. Therefore, there is a clear need to identify new therapeutic agents for OS treatment. MicroRNAs (miRs) are small, noncoding RNAs that control relevant cellular pathways in tumorigenesis. MiR-34a, a downstream component of the p53 tumor suppressor, is downregulated in OS tumors and its reduced expression linked to poor therapeutic response. Recent evidence in human OS cell lines has shown that a novel genetically engineered human tRNA/miR-34a prodrug has anti-tumor effects following intracellular processing into mature miR-34a. We transfected a panel of canine OS cell lines (D17, HMPOS, Abrams and Gracie) with the human tRNA/miR-34a prodrug and evaluated the downstream effects to validate canine OS as a preclinical model. We measured miR-34a levels following transfection using quantitative real-time PCR. Proliferation was assessed via bioreductive and clonogenic assays. Western blot and immunofluorescence microscopy were used to investigate miR-34a target protein expression. Caspase 3/7 activity and TUNEL staining were used to measure apoptotic effects. In vitro properties associated with metastatic ability were assessed via transwell migration and invasion assays using Boyden chambers. We found significant increases in mature miR-34a levels following transfection, indicating that canine cells effectively process the prodrug. Increased miR-34a was associated with reduced PDGFRα protein expression and dose- and time-dependent inhibition of proliferation. Compared to control and MSA-transfected cells, miR-34a significantly reduced clonogenic cell growth, increased caspase 3/7 activity, and increased TUNEL positivity. We also found a significant inhibition of canine OS cell migration and invasion following transfection with miR-34a prodrug. Taken together, our findings demonstrate the successful internalization and processing of human miR-34a prodrug by canine OS cells. The increased miR-34a levels produce the same anti-tumor effects seen in human OS cells. These results strongly supports the use of canine OS as a preclinical model for the development of this therapy in human OS.

#4407

Targeting BMP4 and miR-139-5p in EGFR-mutant non-small cell lung cancer cells.

Duc-Hiep Bach, Thi-Thu-Trang Luu, Dong-Hwa Kim, Yong Jin An, Hyen Joo Park, Sunghyouk Park, Sang Kook Lee. _Seoul National Univ. College of Pharmacy, Seoul, Republic of Korea_.

Non-small cell lung cancer (NSCLC) cells with harboring EGF receptor (EGFR) mutations are sensitive to EGFR tyrosine kinase inhibitors (EGFR-TKIs). Recent studies suggest that bone morphogenetic proteins (BMPs) and microRNAs (miRNAs) might act as oncogenes or suppressors in tumor microenvironment. We identified the potential roles of BMPs and miRNAs involved in EGFR TKIs resistance by analyzing datasets from a pair of NSCLC-sensitive and NSCLC-resistant cells. BMP4 was found to be significantly over-expressed in EGFR-TKI resistant cells and its underlying mechanism was highly associated with induction of energy metabolism for cancer cells through modulating Acyl-CoA synthetase long-chain family member 4. In addition, miR-139-5p was found to be importantly down-regulated in the resistant NSCLC cells. The combination of miR-139-5p and yuanhuadine, a natural product-derived antitumor agent, synergistically suppressed the BMP4 expression in the resistant cells. We furthermore confirmed that LDN-193189, a small molecule BMP receptor 1 inhibitor inhibited the tumor growth in a xenograft nude mouse model implanted with drug resistant cells. These findings suggest a role of BMP4-mediated tumorigenesis in the progression of acquired drug resistance in EGFR-mutant NSCLC cells.

Key words: microRNAs, bone morphogenetic proteins, drug resistance, metabolism, EGFR-TKIs, NSCLC.

Acknowledgements

This study was funded by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2015R1D1A1A02062012).

#4408

Development and validation of a serum miRNA biomarker panel for the detection of early stage lung cancer.

Lisha Ying,1 Ruiyang Zou,2 Lingbin Du,1 Lei Shi,1 Nan Zhang,1 Jiaoyue Jin,1 Junzhou Wu,1 Fanrong Zhang,1 Kaiyan Chen,1 Herbert Yu,3 Weimin Mao,1 Dan Su1. 1 _Zhejiang Cancer Hospital, Hangzhou, China;_ 2 _MiRXES Bio-Technology Co., Ltd., Hangzhou, China;_ 3 _University of Hawaii Cancer Center, Honolulu, HI_.

Lung cancer is the most prevalent cancer and leading cause of cancer death worldwide. Low-dose spiral computed tomography (LDCT) scan is being recommended as a screening test for smokers in the U.S. However, the poor specificity (73% specificity at 94% sensitivity) of LDCT has raised significant concern for its high chance of false positive (96% of LDCT positives are benign nodules). This study aims to develop a blood (serum) miRNA based molecular diagnostic test for the detection of early stage lung cancer. In the discovery phase, more than 400 miRNAs were profiled with MiRXES's qPCR based high throughput assay platform through a highly defined Chinese male smoker case-control cohort (n=424) where the cases were collected from Zhejiang Cancer Hospital and the controls were collected from the LDCT screening program in Zhejiang province, China. 29 miRNA biomarkers with p-value (FDR) < 0.01 and more than one z-score (standardized score) difference were identified and validated in another Chinese case-control cohort (n=432) collected from similar sources and a Caucasian case-control cohort (n=218) collected from EU and US. 18 out of the 29 miRNA biomarkers were validated in both cohorts (p-value < 0.01 and z-score >0.4). With multiple time of two-fold cross-validation, 5 miRNAs were found to be minimally required to form the biomarker panel for the accurate prediction of early stage lung cancer and the panel gives 0.984 (95% CI 0.973-0.991) AUC for the discovery cohort, 0.936 (95% CI 0.912-0.957) AUC for the Chinese validation cohort and 0.970 (95% CI 0.939-0.986) AUC for the Caucasian validation cohort. The 5-miRNA biomarker panel were then further validated in three additional Asian cohorts: a Chinese cohort collected from the similar sources (0.973, 95% CI 0.950-0.986, AUC), a Chinese cohort collected from independent sources (0.916, 95% CI 0.852-0.949, AUC) and a Singaporean cohort (Chinese, Malay and Indian population) (0.911, 95% CI 0.822-0.963, AUC).

#4409

Developing brain tumor-specific gold-liposomal nanocarriers for a noninvasive delivery of microRNA inhibitors.

Nilmary Grafals,1 Blanca I. Quiñoes-Diaz,1 Janixa Del Valle,2 Gabriel L. Barletta-Bonano,3 Pablo E. Vivas-Mejía1. 1 _University of Puerto Rico, Medical Sciences Campus, San Juan, PR;_ 2 _University of Puerto Rico, Rio Piedras Campus, San Juan, PR;_ 3 _University of Puerto Rico, Humacao Campus, Humacao, PR_.

MicroRNAs are non-coding RNA molecules that regulate more than 60% of the protein coding genes and their dysregulation has proven to be involved in the initiation, progression and maintenance of malignant brain tumors such as glioblastoma multiforme (GBM). Targeting microRNAs in GBM has shown therapeutic value, although their translation to the clinic have been halted due to their rapid renal clearance, short circulatory life and inability to reach the brain tumor after systemic administration. Over the last years, nanodelivery systems such as liposomes (Lip) and gold nanoparticles (AuNPs) have gained acceptance for the treatment of cancer. This project is focused on developing anti-miRNA gold carriers further encapsulated inside nanoliposomes that are labeled with blood brain barrier specific peptides (Apolipoprotein E and Rabies Virus Glycoprotein). The synthesis of these dual gold-liposomal particles has three important steps: (a) Peptide Pegylation, (b) anti-microRNA conjugation to gold nanoparticles and, (c) nanoparticle assembly. To achieve each of them the corresponding peptide was conjugated to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[methoxy(polyethylene glycol)]-Malemide (DSPE-PEG-Mal) by a thioester bond and incorporated into DOPC and cholesterol lipid mixture. Afterwards, anti-microRNAs were functionalized to 15 nm gold nanoparticles (AuNPs) to obtain RNAi-AuNPs. Finally, the nanoparticles of interest where produce: RNAi-AuNP-Lip, RNAi-AuNP-Lip-APOE and RNAi-AuNP-Lip-RVG. These nanoparticles where characterized by dynamic light scattering, zeta potential and encapsulation efficiency. Additionally, we determine their toxicity in U-87 GBM cell lines. Our results obtained show sizes that range from 30 nm, 60 nm and 95 nm for gold-RNAi, gold-liposomes and gold-liposome-peptides respectively. These particles are slightly negative and have encapsulation efficiencies that are greater than 50%. Cell viability analysis also show that gold nanoparticles carrying microRNA inhibitor negative controls are not toxic to GBM cell lines. Additional studies for cell internalization, reactive oxygen species generation and RNA delivery and release are ongoing now. Developing systemic viable microRNA based therapeutics its a big challenge, but they are crucial to assure the patient a non-invasive therapy that could improve their quality of life and their overall survival.

#4410

miRNA-34a sensitizes triple-negative breast cancer cells to paclitaxel reducing cancer stem cell population and lung colonization.

Montserrat Climent, Paola Bonetti, Francesco Nicassio. _Istituto Italiano di Tecnologia, Milano, Italy_.

Background: Cancer stem cells (CSCs) have inherent or developed drug resistance for which chemotherapy fails to eliminate all tumor cells, a property that is critically contributing to recurrence and metastasis. Here, we combine miR-34a, a well-known tumor suppressor miRNA that controls proliferation, apoptosis and self-renewal, with Paclitaxel (PTX), a commonly used first-line chemotherapy drug. Based on our preliminary observations (Bonetti et al., manuscript under revision), we hypothesized a combinatorial approach, coupling miRNA- and chemo- treatment, aimed at reducing CSCs in vitro and recurrence in vivo using triple-negative breast cancer (TNBC) as a model. Methodology: As a model, we exploited SUM159pt cells (TNBC mesenchymal-like, with CSC features) to mimic aggressive breast cancer, which harbor a vector for pLUC, which is induced by luciferin in vivo, to monitor tumor growth in real time. Furthermore, we used i) pSlik34a, with tunable expression of miR-34a by doxycycline; ii) treatment with PTX to select for resistant cells (RC); and combined the two, by treating RC with doxycycline. CSC content was evaluated by ALDH (Aldehyde Dehydrogenase) activity, sphere-forming efficiency (SFE) and qRT-PCR of known markers. A similar approach was followed for in vivo studies, with NSG female injected through tail vein injection with 500,000 SUM159/pLUC/pSlik34a cells, and separated into three groups: not treated (NT), PTX alone, and PTX+34a. Results: As observed for aggressive breast cancer, treatment of SUM159 with PTX generated resistant cells (RC) with increased CSC content (ALDH activity and spheres forming ability). When miR-34a was induced in the RC, cells changed morphology, slowed proliferation rate, and decreased CSC content (ALDH activity and spheres). Moreover, a second PTX treatment almost completely abolished the ALDH activity and SFE. The combined effect was evaluated in vivo, following by luminescence the growth of metastases after lung colonization, with PTX+34a mice reducing lung colonization than the PTX treatment or miR-34a treatment alone. Conclusions: Overall, these results indicate that miR-34a sensitize PTX-resistant cells reducing cancer stem-cell capacities, and in this preclinical setting limited the aggressive phenotype of breast cancer, including proliferation, drug resistance, relapse and metastasis causing in delay of lung colonization in vivo.

#4411

MiR-130b identified by TCGA miRNA-seq data can predict the radio-sensitivity for patients with HPV-negative oropharyngeal squamous cell carcinoma.

Hitoshi Inoue,1 Yasunao Kogashiwa,1 Hirasaki Masataka,1 Akihiro Sakai,2 Mitsuhiko Nakahira,1 Akihiko Okuda,1 Masashi Sugasawa1. 1 _Saitama Medical University International Medical Center, Saitama-Pref, Japan;_ 2 _Tokai University Medical Center, Knagawa-Pref, Japan_.

Background: Human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) are associated with an increased radio-sensitivity and a favorable outcome compared to HPV-negative OPSCC. To the contrary, HPV-negative patients lack validated prognostic biomarkers. When surgery is selected in the locally advanced HPV-negative OPSCC, the function after surgery may be impaired. Thus, if there is a biomarker to know whether it is radio-sensitive or not, it is useful for treatment selection. It is therefore important to define prognostic biomarkers in this patient population.

Method: miRNA sequence (miRNA-seq) and mRNA sequence (mRNA-seq) of the Cancer Genome Atlas (TCGA) data includes 520 HNSCC tumors. Of them, we used gene expression data in HPV-negative OPSCC, which have data on the effects of radiation. We compared the results of miRNA-Seq in two groups of radio-sensitive and radio-resistant, and miRNA whose expression was different between radio-sensitive and radio-resistant HPV-negative OPSCC was selected. To estimate the function of the miRNA, we also performed mRNA-seq analysis. Finally, we validated the miRNA related to the outcome of radiotherapy in our clinical cases.

Results: We found that miR-130b is highly expressed in radio-sensitive HPV-negative OPSCC by miRNA-seq. From the mRNA-seq, we identified genes with significantly different expression between radio-resistant and radio-sensitive HPV-negative OPSCC. Clustering analysis and Principal Component Analysis have shown the possibility of discriminating between radio-resistant and radio-sensitive in these gene groups. From the gene ontology (GO) analysis in up regulated genes, we identified GO terms. It is presumed that miR-130b targets several genes related to radio-sensitivity, so it is considered that reduced gene expression in radio-sensitive is related to increased expression of miR130b.

Subsequently, we evaluated the expression of miR-130b in our clinical cases. Values of miR130b > 5.372, determined by ROC curves analysis, were associated with significantly longer progression-free survival (p=0.02, log-rank test). Conclusion: The expression change of genes and high expression of miR-130b in radio-sensitive were found by miRNA-seq and mRNA-seq. Our result suggests that miR-130b has a potential to become a biomarker for the radio-sensitivity of HPV-negative OPSCC. The expression of miR-130b as a biomarker for HPV-negative OPSCC requires further evaluation.

#4412

Elevated miR-193a attenuates 5-fluorouricil metabolism enzyme CMPK1, resulting in therapeutic resistance and poor survival in gastric cancer.

Hao Chen,1 Wei Wang,1 Ben Liu,1 Lu Han,1 Xinlei Chu,1 Hong Zheng,1 Yanrui Zhao,1 Xiangchun Li,1 Lian li,1 Fengju Song,1 Wei Zhang,2 Kexin Chen1. 1 _Tianjin Medical University Cancer Institute and Hospital, Tianjin, China;_ 2 _Wake Forest Baptist Medical Center, Winston Salem, NC_.

Aim: 5-FU chemoresistance is a major challenge in gastric cancer patient's post-operation treatment. CMPK1 is a key metabolism enzyme associated with 5-FU phosphorylation activation, which is also functioned by epigenetic regulation-microRNA. We characterized the role of CMPK1 and potential regulated target-miR-193a in 5-FU chemotherapy response in gastric cancers.

Methods: The roles of miR-193a on prognosis were assessed in a cohort of gastric cancer samples (n=90, tested by microarray assay) and reconfirmed in expanded samples (n=385, tested by q-PCR assay) with Kaplan-Meier and log-rank analysis. We used luciferase and Western-blot assays to test the regulation effect of miR-193a on CMPK1, and in vitro experiments including comet assay and 3-D spheroid assays to study whether miR-193a is associated with 5-FU chemoresistance. All statistical tests were two-sided.

Results: MiR-193a high expression was associated with worse response to therapy and shorter progression-free survival in two cohorts (PFS: Primary microarray samples: [HR] = 1.28, 95% CI= 1.04 to 1.67, P = 0.005; Expanded samples: [HR] =1.08, 95% CI = 1.00 to 1.17, P = 0.050). In the expanded samples, patients received 5-FU-based therapeutic regimen which have high miR-193a expression was more likely to relapse (PFS<24 months VS PFS>60 months: 0.38±1.17 VS -0.89±1.41,p=0.001;24<PFS<60 months VS PFS>60 months: -0.20±1.59 VS -0.89±1.41,p=0.021 ). Comet assay showed that knock-down of CMPK1 impeded 5-FU caused DNA damage, induced a diminished cell cycle arrest and cell apoptosis. MiR-193a bound directly to the 3'-UTR of CMPK1 and downregulated CMPK1 expression in gastric cancer cells, showed parallel results about 5-FU sensitivity alike to CMPK1. Spheroid assays indicated that miR-193a-mediated attenuation of CMPK1 resulted in significant resistance of gastric cancer cells to 5-FU caused DNA damage and cell death (mean diameters of cell clones ± SD, miR-NC plus 5-FU vs miR-193a plus 5-FU: 78.5±14.8 VS 124.5±16.3, P<0.001; miR-NC plus 5-FU vs si-CMPK1 plus 5-FU: 78.5±14.8 VS 116.0±18.4, P=0.002 ), but not in cisplatin treatment (miR-NC plus 5-FU vs miR-193a plus 5-FU: 75.1±11.3 VS 82.2±12.7, P=0.274; miR-NC plus 5-FU vs si-CMPK1 plus 5-FU: 75.1±11.3 VS 82.0±17.4, P=0.316). Rescue experiments with augmented CMPK1 expression abolished the effect of miR-193a demonstrating the key function of this miRNA in this pathway.

Conclusions: The discovery of a miR-193a-CMPK1-5-FU damage axis supports the methods that combining low miR-193a expression with 5-FU agents may substantially benefit gastric cancer management. MiR-193a might be a valuable predictive biomarker for the chemotherapy response in gastric cancer patients, and provided a therapeutic drug proposal for neoadjuvant chemotherapy.

### Noncoding RNAs: From Biology to Therapy

#4413

**DNA methylation of miR-7 is a mechanism involved in platinum response through** MAFG **overexpression in cancer cells.**

Olga Vera,1 Julia Jimenez,1 Carlos Rodriguez-Antolin,1 Olga Pernia,1 Carmen Rodriguez,1 Javier Soto,1 Rocio Rosas,1 Isabel Esteban-Rodriguez,1 Rosario Perona,2 Javier de Castro,1 Inmaculada Ibáñez-de-Cáceres1. 1 _University Hospital La Paz, Madrid, Spain;_ 2 _Institute for Biomedical Research CSIC/UAM, Madrid, Spain_.

One of the major limitations associated with platinum use is the resistance that almost invariably develops in different tumor types. In the current study, we sought to identify epigenetically regulated microRNAs as novel biomarkers of platinum resistance in lung and ovarian cancers, the ones with highest ratios of associated chemo-resistance. Experimental procedures: We combined transcriptomic data from microRNA and mRNA under the influence of an epigenetic reactivation treatment in a panel of four paired cisplatin -sensitive and -resistant cell lines, followed by real-time expression and epigenetic validations for accurate candidate selection in 19 human cancer cell lines. To identify specific candidate genes under miRNA regulation, we assembled "in silico" miRNAs and mRNAs sequences by using ten different algorithms followed by qRT-PCR validation. Functional assays of site-directed mutagenesis and luciferase activity, miRNAs precursor overexpression, silencing by antago-miR and cell viability were performed to confirm their specificity in gene regulation. Results were further explored in 187 primary samples obtained from ovarian tumors and controls. Results: We identified 4 candidates, miR-7, miR-132, miR-335 and miR-148a, which deregulation seems to be a common event in the development of resistance to cisplatin in both tumor types. miR-7 presented specific methylation in resistant cell lines, and was associated with poorer prognosis in ovarian cancer patients. Our experimental results strongly support the direct regulation of MAFG through miR-7 and their involvement in the development of CDDP resistance in human tumor cells. Conclusion: The basal methylation status of miR-7 before treatment may be a potential clinical epigenetic biomarker, predictor of the chemotherapy outcome to CDDP in ovarian cancer patients. To the best of our knowledge, this is the first report linking the regulation of MAFG by miRNA-7 and its role in chemotherapy response to CDDP. Furthermore, this data highlights the possible role of MAFG as a novel therapeutic target for platinum resistant tumors.

#4414

Radiation-induced miR-5094 expression inhibits cell proliferation by targeting STAT5b.

Nan Ding,1 Junrui Hua,1 Jinpeng He,1 Wenjun Wei,1 Hailong Pei,1 Guangming Zhou,1 Jufang Wang,1 Altaf A. Wani,2 Qianzheng Zhu2. 1 _Institute of modern physics, Chinese Academy of Sciences, Lanzhou, China;_ 2 _The Ohio State University, Columbus, OH_.

MicroRNAs (miRNAs) are highly conserved small non-coding RNAs that alter protein expression and regulate multiple intracellular processes including cellular stress response. We previously uncovered several novel miRNAs, whose expressions are induced by X-rays irradiation in HeLa cells, using a miRNA array. Here, we examined the function of miR-5094 during cellular radiation response. With software predictions and confirmatory experiments, we identified the signal transduction and activator of transcription 5b (STAT5b), a known regulator of proliferation and survival, as a target of miR-5094. The study also showed that both miR-5094 induction by X-rays irradiation and miR-5094 mimics transfection decreased STAT5b expression, down-regulated STAT5b downstream genes, and consequently, reduced the cell survival and cell viability. Knockdown of miR-5094 by miRNA inhibitor after radiation partially reversed these suppressive effects. As STAT5b plays an important role in lymphocyte proliferation, we further confirmed the proliferation inhibitory effects of miR-5094/STAT5b pathway on Jurkat cells and human peripheral blood CD4\+ T cells during cell radiation response. In conclusion, our study provided the first evidence that radiation induces upregulation of miR-5094, which in turn inhibits cell proliferation via targeting STAT5b.

#4415

**Characterization of tumor-derived exosomes in** BRCA **-associated breast tumors.**

Vivian Y. Shin, Man-Ting Siu, Isabella Cheuk, Ava Kwong. _University of Hong Kong, Pokfulam, Hong Kong_.

Background: BRCA-mutated tumors are usually with higher grade than sporadic tumors and the biology is poorly understood. Exosomes are small membrane-derived vesicles that function to mediate cell-cell communication via the transfer of tumor-promoting microRNAs (miRNAs), RNAs and proteins. Interestingly, different spectrum of miRNAs is released by cancer cells to promote tumor growth and metastasis in breast cancer. However, the selectivity of miRNA released from tumor-derived exosomes and its relevance in cancer treatment has not been widely studied. Hence, this study is sought to characterize the role of exosomal-miRNA (exo-miR) for the development of BRCA-associated tumors. Methods: Plasma samples from patients with BRCA-positive and non-BRCA carriers were recruited and selected from the Hong Kong Hereditary Breast Cancer Family Registry. By using miRCURY LNA array, the exosomal-miRNA expressions in plasma of patient with BRCA-positive (n=4) and non-carriers (n=4), as well as healthy controls (n=4) were profiled. Selected miRNAs were further validated in pre- and post-operative plasma and paired primary tissues (n=40) using real-time RT-PCR. Functional study of miRNA was also carried out in the cell lines. Results: LNA array data showed that differential expressed BRCA-associated exo-miRNAs were identified. Upregulated miRs (miR-106a, miR-20a, miR-23a, miR-451 and miR-486) were validated in BRCA-positive, BRCA-negative and healthy controls by real-time RT-PCR. In addition, the expression levels of miR-106a, miR-451 and miR-486 were significantly lower in post-operative plasma of BRCA-carriers than non-carriers. Importantly, the expression of miR-451 was reduced after tumor resection in BRCA mutation carriers. In parallel, high expression of exo-miR-106a, miR-20a, miR-23a, miR-451 and miR-486 were also seen in breast cancer cell lines (MB-231 and MB-468) relative to normal breast cells (MCF-10A). We found that cells transfected with miR-451 inhibitor significantly reduce cell proliferation. Conclusions: These preliminary results reveal differential exo-miRNA profiles between BRCA carriers and non-carriers with breast cancer, and identify potential targets of dysregulated miRNAs which is crucial in disease progression of BRCA-associated breast cancers.

#4416

MiRNAs genes are regulated by methylation in papillary thyroid carcinomas.

Isabella Maria D. Ortiz,1 Mateus de Camargo Barros-Filho,1 Mariana B. dos Reis,2 Caroline Moraes Beltrami,1 Fabio Albuquerque Marchi,1 Hellen Kuasne,1 Clovis Pinto,1 Luiz Kowalski,1 Silvia Rogatto3. 1 _A.C.Camargo Cancer Center, São Paulo, Brazil;_ 2 _Vejle Hospital, Institute of Regional Health Research, University of Southern Denmark, Dinamarca, Denmark;_ 3 _Institute of Regional Health Research - University of Southern Denmark, Vejle, Denmark_.

Background: Papillary thyroid cancer (PTC) is the most common thyroid malignancy. DNA methylation associated with microRNAs (miRNA) regulation is an effective epigenetic mechanism for controlling gene expression. This mechanism is poorly explored in PTC. Patients and Methods: To investigate miRNAs genes regulated by methylation, global DNA methylation (Infinium® Human Methylation450 BeadChip, Illumina) analysis was performed in 50 matched PTC and normal tissues (NT) and compared with those deposited in The Cancer Genome Atlas (TCGA) (515 PTC and 56 NT). An integrative analysis was performed in 510 cases evaluated by whole methylation and miRNA expression analysis (TCGA). Using these findings, three miRNAs were investigated by RT-qPCR and pyrosequencing in PTC (N=103), normal thyroid tissues (NT, N=40) and benign thyroid lesions (BTL, N=32). In addition, seven target genes of these miRNAs were evaluated by RT-qPCR (44PTC, 30BTL and 28NT). Functional assays using three PTC cell lines (TPC1, K1 and BCPAP) were used to investigate the role of methylation in the miRNAs expression. Results: We found 50 differentially methylated probes of which 42 (27 microRNA genes) were confirmed in the TCGA database. The miRNA expression data from TCGA presented 67 differentially expressed miRNAs in PTC. The integrative analysis revealed three miRNAs (hsa-miR-146b-5p, hsa-miR-146b-3p and hsa-miR-21-5p) as candidates to be regulated by methylation. A significant hypomethylation pattern in PTC compared to NT and BTL (thyroid benign lesions) was found for these three miRNAs. Increased expression levels of hsa-miR-21-5p and hsa-miR-146b-5p were detected in PTC compared to NT and BTL. Combining these two miRNAs expression values, 45/47 PTC were distinguished from 66/67 non-malignant tissues (96% sensitivity and 99% specificity). Similarly, the association of methylation and expression data distinguished 41/45 PTC from 55/60 non-malignant tissues (91% sensitivity and 92% specificity) for MIR21 gene and 45/47 PTC from 61/61 NT/BTL (96% sensitivity and 100% specificity) for MIR146B gene. Seven target genes (MPPED2, STXBP5L, MRO, FHL1, FLRT1, DOK6 and MOB3B) of these miRNAs were significantly down regulated in PTC compared to NT and BTL. The BRAFV600E mutation was significantly associated to hypomethylation and overexpression of hsa-miR-21-5p and hsa-miR-146b-5p. Methylation and expression data in these three cell lines revealed that MIR146B is putatively regulated by methylation. Conclusion: We provide evidences that MIR21 and MIR146B genes are regulated by methylation having control in the expression of target genes associated with PTC. These miRNAs have a strong potential to be used as diagnostic markers in the clinical practice.

#4417

MicroRNA 181a-5p reprogrammed glucose and lipid metabolism in lung cancer though regulating SIRT1 and ACSL4.

Ji Woong Son, Seul-Gi Kim, In Beom Jeong, Sun Jung Kwon. _Konyang Univ., Daejeon, Republic of Korea_.

Changes in metabolism are key contributors to tumorigenesis and metastasis. Micro RNAs (miRNA) play critical roles in glucose and lipid metabolism. In previous studies, miRNA 181a-5p (miR-181a-5p) was shown to be a significant component in down regulation of lung cancer tissue and mesenchymal-like lung cancer cell lines. To define the role of endogenous miR-181a-5p in cancer metabolism, we performed assays for luciferase, Lactate Dehydrogenase (LDH), glucose uptake and mitochondrial ATP synthase inhibitor. It was demonstrated that miR-181a-5p inhibited aerobic glycolysis and lipid metabolism and overexpression decreased LDH and lipid content in lung cancer. Furthermore, the overexpression of miR-181a-5p diminished invasion and migration by inhibition of Sirtuin 1 (SIRT1) and acyl-CoA synthetase long-chain family member 4 (ACSL4). Together these findings suggest that miR-181a-5p might be involved with reprograming lung cancer metabolism.

#4418

Investigation of miR-205 expression and its methylation status in prostate cancer.

Seodhna M. Lynch,1 Karla M. O'Neill,2 Michael M. McKenna,3 Colum P. Walsh,4 William Watson,5 William M. Gallagher,1 Declan J. McKenna4. 1 _UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Ireland;_ 2 _School of Medicine, Dentistry and Biomedical Sciences, Centre for Experimental Medicine, Queen's University Belfast, N. Ireland, United Kingdom;_ 3 _Western Health & Social Care Trust, Altnagelvin Area Hospital, County Derry, N. Ireland, United Kingdom; _4 _Biomedical Sciences Research Institute, Ulster University, Coleraine, N. Ireland, United Kingdom;_ 5 _UCD School of Medicine, UCD Conway Institute, University College Dublin, Ireland_.

Introduction: In prostate cancer (PCa), abnormal expression of several microRNAs (miRNAs) has been previously reported. Increasing evidence shows that aberrant epigenetic regulation of miRNAs is a contributing factor to their altered expression in cancer. In this study, we investigated whether expression of miR-205 in PCa is related to the DNA methylation status of its promoter and locus region.

Methods: PCR analysis of miR-205 expression was performed in PCa cell lines and in clinical FFPE biopsy and prostatectomy specimens. CpG methylation analysis of the miR-205 promoter and miR-205 locus, was performed in PCa cell lines and in clinical prostate specimens via pyrosequencing. The effect on promoter and locus methylation status in cells treated with demethylating agents including 5-aza-2'deoxycytidine (decitabine), knockdown of DNA methyltransferase 1 (DNMT1) and knockdown of enhancer of zeste homolog 2 (EZH2) was also examined. Finally, the biological significance of miR-205 in PCa cells was assessed by a series of in vitro bioassays.

Results: miR-205 was shown to be significantly down-regulated across PCa cell lines. This correlates inversely with the methylation status of the miR-205 promoter and miR-205 locus, which is hypermethylated across this panel of PCa cell lines in both regions. Interestingly, a trend towards an inverse correlation was evident between miR-205 methylation, for both regions, and miR-205 expression levels in clinical prostatectomy biopsy specimens. Moreover, in PC3 cells, miR-205 expression was subsequently elevated by treatment with the demethylating agents including 5-aza-2 deoxycytidine and knockdown of DNMT1 and EZH2, suggesting its expression is regulated by methylation. miR-205 promoter and locus methylation status, following treatment with 5-aza-2 deoxycytidine in PC3 cells, showed no change in methylation levels in either region. Finally, in vitro over-expression of miR-205 in PC3 cells inhibited growth and clonogenic potential, as well as inducing apoptosis.

Conclusions: Preliminary findings to date provide evidence that miR-205 is abnormally expressed in PCa and appears to have a tumour suppressor role in PCa. This study investigated the role of DNA methylation in regulating miR-205 expression. It is evident that DNA methylation of the regions analysed may not be responsible for regulating miR-205 expression; thus, other regions within the promoter and locus region which have not been identified may be more important. Furthermore, histone modifications may have a role, in conjunction with DNA methylation, in regulating miR-205 expression. Ongoing and future work entails investigating the biological and prognostic value of miR-205 in PCa.

#4419

FOXP3 mediated transcriptional regulation of microRNAs in breast cancer.

Wei Liu, Song Gao, Xin Li, Fengpin Hu, Lizhong Wang. _University of Alabama at Birmingham, Birmingham, AL_.

MicroRNAs (miRs) have potential as non-invasive biomarkers, but their relevance as biomarkers is limited by inconsistent results, and their regulatory mechanisms remain elusive. In the present study, our results show that the levels of miR-200c and 141 in tumor cells and in circulation differ for mice and for humans between cases with metastatic breast cancer, cases with localized breast cancer, and healthy female controls. The levels of miR-200c and 141 are low in primary tumor cells but are high in the circulation of patients with metastatic breast cancer. Further, we suggest the potential cell origin of circulating miR-200c and 141 and describe their transcriptional regulation in cultured cells and during tumor progression in animal models of spontaneous breast cancer. These results could provide useful insights in early prediction of tumor metastasis and influence treatment strategies for patients at high risk of developing metastatic breast cancer.

#4420

MiR-182 suppresses invadopodia formation and metastasis in non-small cell lung cancer by targeting cortactin.

Yongwen Li,1 Hongbing Zhang,1 Hongyu Liu,1 Jun Chen2. 1 _Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin, China;_ 2 _Department of Lung Cancer Surgery, Tianjin Medical University General Hospital, Tianjin, China_.

Metastasis is the leading cause of cancer mortality and is a major hurdle for lung cancer treatment. Invadopodia, which are cancer-specific protrusive structures, play a crucial role in the metastatic cascade through degradation of the basement membrane and surrounding stroma, but the exact mechanism of regulation is not yet fully understood. In this study, we show that cortactin (CTTN), an important component of invadopodia, is upregulated in lung cancer tissues and is a marker of poorer prognosis in human NSCLC. Phorbol 12,13-dibutyrate and hepatocyte growth factor can induce CTTN expression and motility, as well as invadopodia formation in non-small cell lung cancer (NSCLC). MicroRNA-182 (miR-182) suppresses metastasis and invadopodia formation by targeting CTTN in NSCLC. qRT-PCR results showed that CTTN expression was inversely correlated with expression of miR-182. miR-182/CTTN suppresses invadopodia formation of lung cancer cells via suppression of the Cdc42/N-WASP pathway. Furthermore, miR-182 negatively regulates invadopodia function and suppresses extracellular matrix degradation in lung cancer cells by inhibiting CTTN. In conclusion, our results demonstrated that miR-182 targets CTTN in NSCLC and suppresses lung cancer invadopodia formation and thus suppresses lung cancer metastasis, suggesting a therapeutic application of miR-182 in NSCLC.

#4421

A novel long non-coding RNA competes with endogenous RNA and mediates prostate cancer progression.

Xiao-Lei Shi, Yu-Tian Xiao, Fei Liu, Yue Yang, Xin-Wen Nian, Ya-Sheng Zhu, Shancheng Ren, Yinghao Sun. _Shanghai Changhai Hospital, Second Military Medical University, Shanghai, China_.

Prostate cancer (PCa) remains the leading cancer among men worldwide. There is a critical need for new biomarkers to predict PCa progression and emerging mechanisms of resistance. Long non-coding RNAs(lncRNAs) play pivotal roles in a plethora of cellular processes. Understanding the molecular mechanisms of lncRNAs implicated in the progression of PCa could have an important impact on cancer detection, diagnosis and prognosis.

We previously identified 17 lncRNAs that were found to be differentially expressed in oriental PCa patients. Here, We focused on one uncharacterized lncRNA, which is highly expressed in locally advanced or metastatic PCa, and named it lncAPP. Silencing of lncAPP in PCa inhibitied proliferation and tumour invasion both in vitro and in vivo, resulting in a less aggressive phenotype. In addition, we found that expression of lncAPP in PCa correlated with epithelial-to-mesenchymal-transition(EMT). Using miRDB and Target Scan prediction algorithm, we identified a list of putative lncAPP-binding miRNAs. Specifically, using RNA immunoprecipitation assay and luciferase reporter assay, we demonstrated that lncAPP functions as a molecular sponge for miR218 to facilitate expression of ZEB2 and CDH2.

In conclusion, we discovered a novel lncRNA which is highly expressed in PCa patients of poor prognosis. The expression of lncAPP is associated with tumor progression. We conclude that lncAPP is an intriguing therapeutic target in PCa.

#4422

miR-125b **mediates oral cancer suppression by targeting the antioxidative gene** peroxiredoxin like 2A **(** PRXL2A **).**

Yi-Fen Chen,1 Yun-Yen Wei,1 Cheng-Chieh Yang,1 Chung-Ji Liu,2 Kuo-Wei Chang,1 Shu-Chun Lin1. 1 _National Yang-Ming Univ. School of Dentistry, Taipei, Taiwan;_ 2 _MacKay Memorial Hospital, Taipei, Taiwan_.

Oral squamous cell carcinoma (OSCC) is a globally prevalent malignancy. The molecular mechanisms of this cancer remain to be elucidated to acquire further interception. Peroxiredoxin like 2A (PRXL2A) was named FAM213A previously, has been reported an antioxidant protein that protects cells from oxidative stress. Our previous study has identified the association between PRXL2A up-regulation in OSCC and the worse prognosis of patients. MicroRNAs (miRNAs) are small non-coding RNAs involved in the modulation of physical or pathological properties. miR-125 family genes drive pluripotent regulation in a wide variety of cancers. In this study, we specified the oncogenic eligibility of PRXL2A and clarified miR-125b as its upstream regulator. Down-regulation of miR-125b was observed in OSCC tumors. Lower miR-125b expression in OSCC tissues defined the worse patient prognosis. Reporter assays validated that PRXL2A as a direct target of miR-125b. Exogenous miR-125b expression resulted in the decreased oxidative stress, increased drug sensitivity, and suppressor activities, which was consistent with the knockout of PRXL2A gene in OSCC cells. That the suppressor activities of miR-125b being rescued by PRXL2A denoted the existence of miR-125b-PRXL2A regulatory axis in OSCC pathogenesis. Knockout of PRXL2A drastically attenuated the tumorigenic potential of OSCC cells. Overall, this study proposes novel clues demonstrating that the down-regulation of miR-125b suppressor underlies the PRXL2A up-regulation in OSCC, and this protects tumor cells from oxidative stress leading to the worse patient survival.

#4423

MiR551b-mediated Oncostatin M signaling in breast cancer.

Anjali Geethadevi,1 Deepak Parashar,1 Miriam Ragle-Aure,2 Bindu Nair,1 Yiling Lu,3 Vessela N. Kristensen,4 Gordon B. Mills,3 Sunila Pradeep,1 Pradeep Chaluvally Raghavan1. 1 _Medical College of Wisconsin, Milwaukee, WI;_ 2 _Institute for Cancer Research, Oslo University Hospital, The Norwegian Radium Hospital, Oslo, Norway;_ 3 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 4 _Institute for Clinical Medicine, University of Oslo, Oslo, Norway_.

We have previously demonstrated that an oncogenic microRNA miR551b-3p (also known as miR551b) regulates STAT3 transcription thereby enhancing tumor growth and metastasis in ovarian cancer. Herein, we have identified that Oncostatin M (OSM) is upregulated through STAT3-driven transcription, which plays an important role in promoting migration and invasion of breast cancer cells. The purpose of this study was to evaluate the role of miR551b mediated STAT3 activation and the precise mechanism that regulates OSM-Receptor (OSMR) signaling for breast cancer growth and metastasis. MDA-MB231 cells were either overexpressed or silenced with miR551b and anti-miR551b respectively and the effects on proliferation, migration/invasion (Matrigel transwell assay), wound healing and expression of OSMR and its downstream targets (Western blotting, Quantitative RT-PCR) were assessed. Our data demonstrate that miR551b promoted survival, proliferation, invasion and migration and epithelial to mesenchymal transition (EMT) and spheroid formation of breast cancer cells. Our results also show that miR551b-mediated STAT3 activation upregulated the components of OSMR signaling through elevated expression of OSMR, Interleukin 31 receptor-alpha (IL31RA) and their ligands Oncostatin (OSM) and IL31 thereby inducing phosphorylation of STAT3 for tumor cell proliferation, migration and invasion. Conversely, anti-miR551b reduced the expression of OSMR, IL31R and their ligands OSM and IL31 as well as the phosphorylation of STAT3. Briefly, our results provide the first report indicating that miR551b regulates OSMR expression by activating STAT3 and thereby promoting tumor cell growth and metastasis in breast cancer cells through autocrine feed-forward signaling loop.

#4424

Micro-RNA mediated deregulation of hHB-EGF in ovarian cancer.

Md. Mustafizur Rahman,1 Razia Sultana,2 Ryo Iwamoto,2 Eisuke Mekada2. 1 _Khulna University, Khulna, Bangladesh;_ 2 _Osaka University, Osaka, Japan_.

Background: Aberrant expression of microRNAs (miRNAs) has been implicated in human carcinogenesis where they function as oncogenes or tumor suppressor genes. Overexpression of HB-EGF, member of the epidermal growth factor (EGF) family, plays pivotal role in ovarian carcinogenesis. However the molecular mechanisms underlying miRNA(s) mediated aberrant function of HB-EGF in ovarian carcinoma remain unresolved. Here, we investigated the miRNA(s) mediated deregulation of HB-EGF in human ovarian carcinogenesis.

Methods: Web-based bioinformatics algorithms were used to identify candidate miRNAs with putative binding sites on target hHB-EGF mRNA. Expression of miRNAs and mRNA were checked by real-time PCR whereas protein expression was investigated by western blotting assay. Luciferase reporter assay was carried out to examine miR-27a target on 3´UTR of hHB-EGF mRNA. Changes in the oncogenic effects of HB-EGF were studied after gain/loss -of-function of the miR-27a using soft agar colony formation assay and 3D collagen culture. In vivo tumor growth potential of miR-27a was measured in BALB/c nude mice.

Results: By using web-based bioinformatics algorithms 11 miRNAs ((miR-27a/b, -29a/b/c, -96, -128, -132, -182,-194, -212) having putative binding sites on the 3′UTR of hHB-EGF mRNA have been selected for further analysis. Among the putative candidates miR-27a showed maximum reciprocal expression with hHB-EGF and thus we selected miR-27a as promising candidate for hHB-EGF regulation. Overexpression of miR-27a significantly inhibited the hHB-EGF expression in SKOV3 cells by almost 50% and miR-27a inhibitors (anti-miR-27a) upregulated the expression of endogenous hHB-EGF mRNA whereas miR-27a overexpression decreased hHB-EGF protein level in MCAS cells suggesting that miR-27a modulate endogenous hHB-EGF expression both at mRNA and protein level. Luciferase reporter assay revealed that miR-27a directly targets 3´UTR of hHB-EGF mRNA at the predicted site. Ectopic expression of miR-27a suppresses in-vitro growth of SKOV3 ovarian cancer cells. In addition, in-vivo tumorigenecity of SKOV3 cells was also inhibited by miR-27a treatment.

Conclusion: The current study has identified novel miR-27a that negatively regulates hHB-EGF expression by directly targeting 3`UTR of its mRNA. This miR-27a might act as oncosupressor in human epithelial ovarian cancer.

#4425

PARP1 inhibition impedes Warburg effect in breast cancer cells through modulation of the miR-98/STAT3 signaling axis.

Nidhi Shukla. _CSIR-Central Drug Research Institute, Lucknow, India_.

Cancer cells have increased metabolic autonomy and rewired cellular metabolism in order to meet the bioenergetics, biosynthetic, and redox demands during tumorigenesis. They tend to depend on glycolysis rather than oxidative metabolism to meet their energy demand. Therefore, elucidation of the key regulators of metabolism could potentially aid in devising selective and promising anti-cancer therapies. MicroRNAs (miRNAs) have recently attained prominence as key regulators of cancer cell metabolism. MiRNAs are known to regulate metabolism in cancer cells either through the direct regulation of key metabolic enzymes, tumor suppressors and oncogenes or the indirect modulation of master transcription factors and signalling pathways. Therefore dysregulation of cellular metabolism by miRNAs contributes significantly to cancer progression. Current therapeutic strategies include: (a) targeting metabolic pathways that are strictly required for cancer cells, or (b) impairing an oncogenic signalling axis involved in cancer metabolic rewiring. The poly (ADP-ribose) polymerase (PARP) inhibitors, used in the treatment of DNA repair deficient tumours are also known to facilitate oxidative metabolism. However, the effect of PARP inhibition on glycolytic metabolism is yet to be elucidated. In the present study we aimed to identify novel miRNAs involved in the regulation of glycolytic metabolism after PARP1 inhibition. MiRNA microarray analysis revealed upregulation of the tumor suppressor miR-98 in PARP1 inhibitor treated MDA-MB-231 breast cancer cells. Further mechanistic studies confirmed miR-98 regulation of STAT3 expression and downregulation of key glycolytic enzymes HK2 and PKM2 leading to decreased Warburg effect. MiR-98 overexpression or PARP1 inhibition in combination with miR-98 antagomir showed downregulation of STAT3, HK2 and PKM2. Glucose consumption and HK2 activity were decreased upon PARP1 inhibition associated miR-98 expression. Collectively, these results showed that miR-98 is a critical regulator of PARP1 inhibition associated reduction of Warburg effect in breast cancer cells.

#4426

Automated miRNA purification from plasma, serum or exosomes.

Michelle Mandrekar, Jami English, Douglas Horejsh, Chris Moreland, Herly Karlen, Marjeta Urh. _Promega, Madison, WI_.

Introduction: MicroRNAs (miRNAs) are endogenous small noncoding RNAs 18-24 nucleotides in size that play important roles involving gene regulation associated with cancer, disease control and gene silencing. Because of the impact on disease progression, miRNA research is rapidly shifting towards biomarker discovery. Many of the commercially available miRNA purification methods involve organic extraction during preprocessing. Here, we describe a novel chemistry that enables RNA purification including smaller RNA such as miRNA. This chemistry offers significant advantages with a simple automated workflow, no organic extraction and minimal preprocessing.

Method: In this study we show successful purification of small RNA, including miRNA, from 100uL to 500uL liquid samples utilizing a novel chemistry on the Maxwell® RSC instrument, which can process between 1 and 16 samples. Proteinase K and Lysis Buffer C are added to the liquid samples and incubated before being transferred to the Maxwell cartridge for miRNA isolation. Sample types tested include plasma, serum and isolated exosomes. The miRNA were evaluated using RT-qPCR including miR-16, miR-21, and let-7a.

Results: The eluates had high levels of small RNA including miRNA at levels comparable to manual competitor methods that require organic extraction. Quantitative PCR showed that the eluates had little or no detectable DNA contamination. Integration of the chemistry onto this automated platform reduced hands-on time while maintaining high quality needed for use in downstream RT-qPCR amplification assays.

Summary: This novel chemistry and method enables purification of small RNA from a range of samples types and volumes. The purified RNA has little or no detectable DNA contamination. Phenol extraction is not required, improving safety and simplifying waste disposal. Also, the automation of this workflow decreases manual hands-on time, saving scientist time and reducing the risk of RNase contamination.

#4427

Preparation and mechanism of polyethyleneimine coated miRNA let-7a microspheres.

Honglin Lai, Weixiao An, Yangyang Zhang, Xiukun Lin, Shousong Cao. _Southwest Medical University, Luzhou, China_.

Recent evidence demonstrates that microRNAs (miRNAs) are associated with cancer development and have the potential to be used as anticancer agents. However, the application of miRNAs as clinical cancer drugs has been limited due to their unstability at room temperature. Moreover, miRNAs are negatively charged and excluded by cytomembrane, which has the same polarity. Therefore, miRNAs are difficult to enter cancer cells to exhibit their anticancer effects. In order to solve the problem, we changed the polarity of miRNA let-7a, a member of the second identified miRNA let-7 families, by coating with a bioavailable polymer, polyethylenimine (PEI) using nanotechnology, to alter the charge of miRNA let-7a for positive charge so easily connecting with the anionic residues of cancer cell surfaces. Therefore, miRNA let-7a could easily entered cells through endocytosis. In addition, we performed formaldehyde denaturing SDS-PAGE analysis to determine the stability of miRNA let-7a, and we found the stability of miRNA let-7a has been significantly increased due to microspheres formation. MiRNA let-7a microspheres have showed potent cytotoxicity against a variety of cancer cells including HCT-116, A875, A549, and 4T1 cells with the IC50 as 31.77, 54.34, 44.35, 43.18 pmol/l, respectively by CCK-8 kit analysis. We also found that miRNA let-7a microspheres are able to induce cell apoptosis by promoting the expression of apoptosis-related genes Caspase 3, Caspase 9 and PARP in HCT-116 and A875 cells. Our findings may provide that miRNA let-7a microspheres may be developed as a novel anticancer agents in cancer therapy.

#4429

FOXP3 transcriptional regulation of MicroRNAs in human breast cancer.

Baozhu Yi, Ming Li, Ying Liu, Runhua Liu. _Univ. of Alabama at Birmingham, Birmingham, AL_.

MicroRNA (miR)-155 has been found to be overexpressed in breast cancer cells, but its transcriptional regulation remains elusive. The role of miR-155 is also not determined in breast cancer development and progression. In this study, we identified a FOXP3-BRCA1-miR155 axis in breast cancer cells. MiR-155 is induced by FOXP3 through a transcriptional repression of BRCA1 in breast cancer cells. Likewise, overexpression of miR-155 was found in patients with early stage or triple negative breast cancer, but not in patients with high stage or metastatic breast cancer. Of note, expression of miR-155 in blood cells was higher in patients with localized but not metastatic tumors. In addition, although miR-155 in tumor cells is induced by FOXP3, free or exosomal miR-155 in culture medium was not changed with induction of FOXP3. There is a transcriptional axis of FOXP3-BRCA1-miR-155 in breast cancer cells. MiR-155 is a potential biomarker for early stage breast cancer. Plasma miR-155 appears to be related to that in blood cells but not in tumor cells.

#4430

Genistein regulates non-coding RNA HOTAIR and epithelial-to-mesenchyme transition in renal cancer cells.

Mitsuho I. Sumida, Yuichiro Tanaka, Rajvir Dahiya, Soichiro Yamamura. _UCSF VA Medical Ctr., San Francisco, CA_.

Renal cell carcinoma (RCC) is one of the most common malignancies. Despite the development of therapeutic regimens, the prognosis of patients with RCC is poorly understood. Genistein, a naturally occurring isoflavone, has been reported to have anti-cancer effects on a variety of cancer types. While several studies have shown that genistein inhibits kidney cancer progression in various in vitro and in vivo models, the basic molecular mechanisms of genistein action have not been investigated in kidney cancer. In malignancies, long non-coding RNAs (lncRNAs) are differentially expressed in various tissues and have important functions in cell proliferation, motility and apoptosis. HOX transcript antisense RNA (HOTAIR) is a lncRNA localized in the Homeobox C gene cluster on chromosome 12. HOTAIR is necessary to target the polycomb repressive complex 2 (PRC2) in trans to the HOXD locus and can interacts with (PRC2, which enhances H3K27 trimethylation and represses the expression of tumor suppressors. HOTAIR is highly expressed in various cancers and involved in their progression and metastasis. In our study, we investigated the molecular mechanisms of genistein action through a novel pathway that represses HOTAIR. We found that HOTAIR expression is higher in renal cancer cell lines compared to normal controls. Genistein treatment was found to significantly decrease HOTAIR expression in renal carcinoma cell lines (786-O and ACHN cells). Genistein treatment also reduced expression of epithelial-to-mesenchyme transition (EMT)-related proteins, causing reduced cell migration, invasion, and increased apoptosis. We performed RNA immunoprecipitation assays, and found that genistein inhibits HOTAIR binding to PRC2. One of the other EMT markers, a tight junction protein ZO-1, is upregulated by genistein. We further investigated whether genistein represses PRC2 recruitment to the ZO-1 promoter by inhibiting binding of HOTAIR to PRC2. In this study, we revealed detailed mechanism of antitumor action of genistein. These insights indicate that genistein is a potent therapeutic agent not only for renal cancer but also for a broad spectrum of cancers.

#4431

Phosphoproteomic analysis of miR-21 modulation in Th17 cells: Potential implications for multiple myeloma bone disease therapy.

Emanuela Altomare, Marco Rossi, Daniele Caracciolo, Nicola Amodio, Marco Gaspari, Domenico Taverna, Domenica Scumaci, Cirino Botta, Annamaria Gullà, Eugenio Morelli, Paola Critelli, Mariamena Arbitrio, Pierosandro Tagliaferri, Pierfrancesco Tassone. _UNICZ, Catanzaro, Italy_.

Th17 cells play a critical role in Multiple Myeloma (MM) microenvironment by triggering osteoclastogenic activity, that leads to severe skeleton damage. We have recently found that Th17 cells directly promote osteoclast (OCL) differentiation and function via expression of RANKL. It has been demonstrated that miRNAs are involved in the differentiation of Th17 cells. Murugaiyan et al. demonstrated that miR-21 promotes Th17 differentiation by targeting and depleting SMAD-7, a negative regulator of TGF-β signaling. Indeed, we have been able to negatively modulate Th17 differentiation by a specific inhibitor of miR-21 (mir21i). The overall effect of mir-21 inhibition is the suppression of OCL resorptive activity, thus attenuating Th17 mediated multiple myeloma bone disease (MMBD). However, the molecular mechanisms underlying these biological effects have not been clarified. On these bases, we aimed to identify proteins involved in the effect of miR21i on Th17 cells. Quantitative phosphoproteomics technologies were applied to study the effects of miR21i in Th17 cells. To unveil the molecular targets responsible for this interaction, Ingenuity's IPA® software was interrogated. This approach allowed to identify several unknown miR-21 target proteins and the related signal transduction pathway.To perfom global phosphoproteome analysis, we performed a mass spectrometry study of phosphopeptides on protein extracts from miR21i- treated Th17 and control Th17 cells, enriched using SCX-IMAC/TiO2. High-resolution LC-Ms/MS data were processed using Proteome Discoverer software. Among analyses of more than 1134 phosphorylation sites, 386 have shown significant changes, as compared to controls. Interestingly, IPA analysis showed a generally higher level of phosphoserine sites and top canonical pathways affected by miR-21 inhibition including PI3K/AKT, 14-3-3 and GRANZYME A signaling. Moreover IPA analysis disclosed RANKL among the top upstream proteins. The most up regulated protein was CYTIP and the ones with enhanced activity were PDCD4, STAT1, MINK1, FOXO1, while ITGAL showed reduced activity. To validate these data, we performed WB analysis and explored additional widely acknowledged Th17 regulators. Upon miR-21i treatment, the expression of PDCD4 and FOXO1 were significantly increased while lower levels of the master Th17 transcription factor, RORC, were observed. Furthermore, PIAS3 and TBX21 were significantly upregulated by mir21i. As PIAS3 and PDCD4 negatively regulate STAT3, we checked phospho-STAT3 expression and found that it was indeed downregulated, as expected. Overall, this study defines a phosphoproteomic signature of miR-21i treated Th17 cells, providing new insights into molecular impairment of Th17 differentiation by miR21i and suggest innovative molecular targeted approaches to treat MMBD

#4432

miR-338-5p modulates growth and chemoresistance of esophageal cancer cell via targeting FERRMT2.

Wen-chun Lin,1 Yao-chin Hsieh,1 Li-han Chen,1 Pei-wen Yang,2 Chi-Cheng Huang,3 Liang-chuan Lai,1 Jang-ming Lee,2 Eric Yao-yu Chuang,1 Mong-hsun Tsai1. 1 _National Taiwan University, Taipei, Taiwan;_ 2 _National Taiwan University Hospital, Taipei, Taiwan;_ 3 _Fu-Jen Catholic University Hospital, Taipei, Taiwan_.

Esophageal cancer is the 5th leading cause of cancer death among male population in Eastern Asia. The esophageal squamous cell carcinoma (ESCC) is the major type of esophageal cancer in Taiwan. Due to the poor overall 5-year survival rate, effective treatment strategies are required to improve the long-term survival rate. miRNAs are important gene regulators that may be dysregulated in many cancer types. The expression level of miR-338-5p was found to be downregulated in esophageal tumor tissues compared to the adjacent nontumor tissues. However, the functions and the potential target genes of miR-338-5p in ESCC are still unclear. In this study, we transfected miR-338-5p mimic into the ESCC cell line, CE-81T/VGH, which expresses low levels of miR-338-5p. The results showed that miR-338-5p mimic can inhibit cell proliferation, migration, and colony formation. In order to identify the target genes of miR-338-5p, microarray analysis was performed to select significantly downregulated genes in the miR-338-5p mimic transfected CE-81T/VGH cells. Moreover, miRSystem was used to predict the miR-338-5p putative target genes. By cross-comparing the results from the microarray and miRSystem analysis, FERMT2 was identified as the most likely candidate gene, and it was validated by real-time PCR. The function of FERMT2 was further studied in the CE-81T/VG cell. Silencing of FERMT2 can inhibit cell proliferation, migration, and colony formation in the ESCC cells. The luciferase activity assays results showed the direct binding between miR-338-5p and 3'UTR region of FERMT2. To further confirm that miR-338-5p can go through FERMT2 to regulate cell functions, the "rescue" experiments were performed. As the results, overexpression of FERMT2 after miR-338-5p mimic transfection could restore cell migration and cell proliferation. Moreover, it has been reported that FERMT2 can increase chemoresistance in glioma cells. Therefore, the effects of miR-338-5p and FERMT2 on drug resistance were studied, and the results showed that miR-338-5p mimic transfection or FERMT2 silencing can increase cisplatin-induced cytotoxicity in the CE-81T/VGH cells. These data indicated that miR-338-5p can inhibit cell proliferation, migration, colony formation, and enhance cisplatin sensitivity in the ESCC cells by targeting FERMT2 expression. Therefore, miR-338-5p plays an important role in the ESCC cells via repressing FERMT2 and it could be used as a new research target for cancer treatment.

#4433

**Upregulation of** BLU **tumor suppressor gene by miR-34a.**

Shinichiro Ohno, Yoshihiro Sambu, Keiki Oikawa, Yoshitaka Naito, Yuichirou Harada, Masahiko Kuroda. _Tokyo Medical University, Tokyo, Japan_.

miR-34a is down-regulated in several tumor tissues and well known as strong tumor suppresser miRNA. However, the mechanisms of tumor suppression by miR-34a remain unclear. To elucidate the mechanisms of tumor suppression by miR-34a, we performed microarray analysis of miR-34a transfected cells. Interestingly, we found that the number of up-regulated genes was larger than down-regulated genes by the transfection of miR-34a. The tumor suppresser BLU (ZMYND10) is strongly up-regulated by miR-34a that primarily expressed in the normal lung and down-regulated in non-small cell lung cancer (NSCLC). Therefore, we investigated the possibility that miR-34a inhibits lung cancer development through inducing BLU expression. It was recently reported that miRNA also regulate transcription in the nucleus through binding to the lncRNA from promoter locus. Because, the predicted two binding sites of miR-34a are located in the BLU promoter locus, we searched the lncRNA expressed from the BLU promoter locus. As a result, we identified the lncRNA from the BLU promoter and siRNA against the lncRNA also induced BLU expression. These results suggest that miR-34a induced tumor suppresser BLU through the binding to the promoter-associated lncRNA.

### Post-transcriptional and Translational Control of Cell Fate

#4434

Contribution of heterogenous ribonucleoprotein A18 in prostate cancer progression.

Elizabeth Tsuying Chang, Eun Yong Choi, Palak R. Parekh, France Carrier. _University of Maryland School of Medicine, Baltimore, MD_.

Prostate cancer is the third leading cause of cancer deaths among men in the United States, with recurrent metastasis being the predominant cause of mortality. In spite of the significant progress made in the past half century with hormonal therapy for metastatic prostate cancer, a majority eventually progresses to a lethal stage of the disease commonly known as castration-resistant prostate cancer. New treatment strategies are therefore needed. Previous work from our lab has identified the RNA-binding protein heterogenous ribonucleoprotein A18 (hnRNP A18) as a new regulator of protein translation in cancer cells. While its localization is predominantly nuclear, hnRNP A18 translocates out to the cytosol in response to cellular stress like hypoxia. Immunohistochemistry data from tissue microarrays reveals increased expression of hnRNP A18 in prostate hyperplasia, and even greater overexpression in prostate adenocarcinoma as compared to the surrounding normal tissue. Furthermore, a greater percentage of hnRNP A18 is seen to shift from the nucleus to the cytosol as the disease progresses. In the cytosol, hnRNP A18 recognizes and binds to a specific 51 nucleotide RNA motif in the 3' untranslated region of targeted transcripts, stabilizing them and enhancing their translation. Transcripts targeted by hnRNP A18 confer tumor growth advantages. The down-regulation of hnRNP A18 was seen to reduce cancer cell proliferation, migration, and invasion. Moreover, down-regulation of hnRNP A18 significantly reduces prostate cancer growth in a mouse tumor xenograft model. Taken together, our data suggests that hnRNP A18 could be a potentially new target to stop or limit prostate cancer progression.

#4435

Functional role of four-and-a-half LIM protein 2 in the alternative splicing of cancer hallmark genes of hepatocellular carcinoma.

Stephen Kwok-Wing Tsui, Cyanne Ye Cao. _Chinese University of Hong Kong, Shatin, Hong Kong_.

Four-and-a-half LIM protein 2 (FHL2) participates in various cellular processes, including regulation of cell survival, transcription and signal transduction. In this study, FHL2 specific antibody was used to immunoprecipitate endogenous FHL2 together with its binding partners from WRL68 and Hep3B liver cell lysates, followed by protein identification using mass spectrometry. The top significant functional clusters of identified proteins were related to the splicing process. To validating the pull down results, the interaction of FHL2 with ASF and U1A in WRL68 cell line was confirmed using co-immunoprecipitation. To study the possible effects of FHL2 on mRNA splicing, we knocked down FHL2 in WRL68, L02 and Hep3B cells and the transcriptomes of knocked-down cells were examined by RNA sequencing. We identified a global impact on regulating differential exon usage by knocking down FHL2. Subsequently, several alternatively spliced cancer hallmark genes were selected for validation using real-time PCR. We found that knocking down FHL2 could significantly promote the extra domain B (EDB) exon inclusion in fibronectin 1 (FN1) and the E3b exon inclusion in Rac1. In addition, knocking down FHL2 significantly decreased the percentage of the epithelial specific transcript CD44E while only very mildly decreased the standard CD44S transcript in total CD44 mRNA. Using an ab initio motif identification approach, the possible splicing factor binding motifs in the alternative spliced exons regulated by over-expressing and knocking down FHL2 were obtained. ASF was found to be the most common splicing regulatory protein. To further explore the functional relationship between FHL2 and ASF, we examined the effects of FHL2 and ASF on exon inclusion/skipping of endogenous EDB exon of FN1 in WRL68 cells. It was found that knocking down FHL2 using siRNA promotes inclusion of EDB while knocking down ASF using siRNA promotes skipping of EDB of FN1. When FHL2 and ASF were knocked down simultaneously, the effect of FHL2 on EDB exon skipping was abolished, implying that ASF mediates the effect of FHL2 on the EDB exon skipping. To explore the clinical relevance of this study, the expression of FHL2 and the EDB exon were determined in 40 paired HCC and normal tissues using real-time PCR. Results showed that the fold changes of EDB inclusion level and the FHL2 expression were significantly correlated. In summary, we have identified many alternative splicing-related protein interacting partners of FHL2 and the downregulated of FHL2 could affect the splicing pattern of many cancer hallmark genes, including CD44, Rac1 and FN1. Using FN1 as a model, we elucidated ASF as one of the mediators of the effect of FHL2 on alternative splicing. Taken together, FHL2 is probably involved in the pre-mRNA splicing in liver cells and modulate genes involved in cancer development through the perturbation of RNA splicing machinery.

#4436

Eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) is upregulated modulates prostate cancer growth and proliferation.

Praveen K. Jaiswal, Sweaty Koul, Prakash Srinivasan Timiri Shanmugam, Hari K. Koul. _LSU Health Sciences Center - Shreveport, Shreveport, LA_.

Introduction: cap-dependent translation is necessary due to high protein requirement in cancer cells. An interaction between EIF4E and EIF4G is crucial for the formation of the EIF4F complex and initiation of cap-dependent translation. In the current study, we analyzed Human prostate cancer tissue microarray (TMA) and mRNA expression data in clinical datasets, and prostate tumor tissue from TRAMP (Transgenic Adenocarcinoma of the Mouse Prostate) model. We also assessed the functional role of EIF4G1 in commonly used PCa cell lines.

Methods: Human prostate cancer tissue microarray was used to analyze the EIF4G1 level in patient samples. mRNA expression data for EIF4G1 was analyzed from TCGA and Trento/Cornell/Broad clinical data sets. PCa cells viz. LNCaP, C4-2b, 22Rv1, DU145, PC3 and normal human prostate cell line RWPE-1 were used. For an in-vivo model of PCa, we used TRAMP and wild-type mouse. Loss of function studies was performed by using siRNA/shRNA. Real-time PCR and Western Blot analysis were used to quantitate the relative mRNA and protein levels respectively. Analysis of polysome was performed by sucrose density gradient fractionation. Polysome-to-Monosome (P/M) ratios were determined for global translation activity. Cell cycle, cell proliferation, cell migration and Clonogenic activity were measured by standard methods.

Results: Results from TMA analysis showed that protein levels of EIF4G1 are high in PCa as compared to normal prostate tissue, and there is a graded increase in EIF4G1 as the disease progresses. Results of our analysis of EIF4G1 expression in TCGA database revealed that increased expression of EIF4G1 positively correlated with higher tumor grade and stages (Gleason Score). Available data from Trento/Cornell/Broad clinical dataset revealed that 43% of castration-resistant prostate cancer (CRPC) patients have EIF4G1 mRNA up-regulation. PCa cells express a significantly higher level of EIF4G1 as compared to normal prostate cells. Similarly, prostate tumor tissue from TRAMP tissue showed higher EIF4G1 expression as compared to normal wild-type prostate tissue. Silencing of EIF4G1 causes G0/G1 cell cycle delay and decreases Cyclin D1 and p-Rb levels. There is a shift in polysome (P) to monosome (M) ratio with the siEIF4G1 knockdown in LNCaP and C4-2b. Loss of function studied by knockdown of EIF4G1 showed impaired Clonogenic activity as well as cell proliferation. Real-time PCR data suggests that EIF4G1 knockdown in LNCaP & C4-2b decreases the level of EMT markers such as N-Cadherin, Vimentin & Zeb1 and limits the cell migration in C4-2b cells.

Conclusions: Taken all together, our data indicate that EIF4G1 may function as an oncoprotein and is a novel target for intervention in PCa and CRPC.

#4437

**Down-modulation of ADAR1-mediated** **GLI1 editing alters extracellular and immune response genes in multiple myeloma.**

Leslie A. Crews,1 Elisa Lazzari,1 Phoebe K. Mondala,1 Nathaniel Delos Santos,1 Amber Miller,2 Gabriel Pineda,1 Qingfei Jiang,1 Anusha-Preethi Ganesan,1 Christina Wu,1 Caitlin Costello,1 Mark Minden,3 Raffaella Chiaramonte,4 A. Keith Stewart,2 Catriona H. M. Jamieson1. 1 _UCSD Moores Cancer Ctr., La Jolla, CA;_ 2 _Mayo Clinic, Rochester, MN;_ 3 _Princess Margaret Hospital, Toronto, Ontario, Canada;_ 4 _University of Milan, Milan, Italy_.

Introduction: Representing 10% of hematologic malignancies, multiple myeloma (MM) is typified by clonal plasma cell proliferation in the bone marrow (BM) and may progress to therapy-resistant plasma cell leukemia (PCL). Despite many novel therapies, relapse rates remain high as a result of malignant regeneration (self-renewal) of MM cells in inflammatory microenvironments. In addition to recurrent DNA mutations and epigenetic deregulation, inflammatory cytokine-responsive adenosine deaminase associated with RNA (ADAR1)-mediated adenosine to inosine (A-to-I) RNA editing has emerged as a key driver of cancer relapse and progression. In MM, copy number amplification of chromosome 1q21, which contains both ADAR1 and interleukin-6 receptor (IL-6R) gene loci, portends a poor prognosis. Thus, we hypothesized that ADAR1 copy number amplification combined with inflammatory cytokine activation of ADAR1 stimulates malignant regeneration of MM and therapeutic resistance.

Methods and Results: Analysis of MMRF CoMMpass RNA sequencing (RNA-seq) data revealed that high ADAR1 expression (n=162 patients) correlated with significantly reduced progression-free and overall survival compared with a low ADAR1 subset (n=159 patients). In contrast to lentiviral ADAR1 shRNA knockdown and overexpression of an editase defective ADAR1 mutant (ADAR1E912A), lentiviral wild-type ADAR1 overexpression enhanced editing of GLI1, a Hedgehog (Hh) pathway transcriptional activator and self-renewal agonist. Editing of GLI1 transcripts enhanced GLI transcriptional activity in luciferase reporter assays, and promoted lenalidomide resistance in vitro. Finally, lentiviral shRNA ADAR1 knockdown reduced regeneration of high-risk MM in humanized serial transplantation mouse models, indicative of reduced malignant self-renewal capacity. Whole-transcriptome RNA-sequencing of primary samples after lentiviral shRNA knockdown of ADAR1 revealed specific modulation of extracellular and immune response genes, while overexpression of wild-type versus edited GLI1 elicited distinct gene expression changes in human myeloma cells analyzed using NanoString nCounter assays. These data demonstrate that ADAR1 promotes malignant self-renewal of MM and, if selectively inhibited, may prevent progression and relapse through modulation of extracellular and immune response genes.

Conclusions: Deregulated RNA editing, driven by aberrant ADAR1 activation, represents a unique source of transcriptomic and proteomic diversity, resulting in self-renewal of MM cells in inflammatory microenvironments. In summary, both genetic (1q21 amplification) and microenvironmental factors (inflammatory cytokines, IMiDs) combine to drive GLI1-dependent malignant regeneration in MM. Thus, ADAR1 represents both a vital prognostic biomarker and therapeutic target in MM.

#4438

NaBt can regulate the expression of COX-2 post-transcriptionally in the presence of chemically induced stress in colon epithelial cells.

Sreeparna Banerjee,1 Shabnam Enayat,1 Sinem Tuncer,1 Doğukan H. Ulgen,2 Aydan Torun1. 1 _Middle East Technical University, Ankara, Turkey;_ 2 _Oxford University, Oxford, United Kingdom_.

Sodium butyrate (NaBt) is a histone deacetylase inhibitor (HDACi) produced in the colon by commensal microbiota-mediated fermentation of dietary fibers. It is regarded to have tumor suppressive and anti-inflammatory effects. NaBt can regulate gene expression through chromatin remodeling and altered transcription. However, it has not been addressed adequately whether inflammatory genes may also be regulated by NaBt in a post transcriptional manner via AU rich elements (ARE) in their 3'UTR. Overexpression of Cyclooxygenase-2 (COX-2) is observed in chronic inflammatory diseases and colorectal cancer (CRC). The aim of this study was to explore whether NaBt regulates COX-2 expression via post-transcriptional mechanisms. Pre-confluent Caco-2 and HT-29 cells were treated with different concentrations (1-5mM) of NaBt for short (3-6h) and long (24-48h) time points. Gene expression was determined by qPCR or Western blot. mRNA stability was determined with an Actinomycin D (ActD) chase assay. 3'UTR activity was determined in an RPSM30 vector containing the ARE rich and corresponding mutated regions of the 3'UTR from COX-2. NaBt strongly reduced COX-2 mRNA and protein expression with both short and long-term treatments. This reduction was not through NF-κB, since short-term treatment of cells with NaBt could not alter NF-κB activity in a luciferase reporter assay. An Act D chase assay indicated that in the absence of new mRNA synthesis, NaBt treatment induced stability of COX-2 mRNA in Caco-2 cells while it enhanced COX-2 mRNA decay in HT-29 cells. However, we did not observe any change in the 3'UTR activity of COX-2 in the presence of NaBt alone, indicating that the presence of a stress factor (such as ActD) was necessary for mRNA stabilization. Treatment of Caco-2 cells with NaBt and ActD together resulted in enhanced activation of the stress-related protein kinase MAPKAPK2 (MK2) compared to ActD alone, which can induce mRNA stabilization through nucleo-cytoplasmic shuttling of the ARE binding protein HuR. This was also reflected in increased COX-2 mRNA and protein levels. In HT-29 cells, simultaneous (NaBt+ActD) treatment resulted in a decrease in p-MK2, but an increase in p-Chk2 compared to ActD alone. p-Chk2 can preserve Cdk1 in a hyperphosphorylated state where it may promote retention of HuR in the nucleus, facilitating COX2 mRNA degradation and lower protein levels. No change in p-Chk2 was observed in Caco-2 cells in a similar experimental set up. These findings suggest that in the presence of a stress-inducing agent like ActD, NaBt can alter mRNA stability of COX-2 in a contextual manner, dependent on upstream signaling. Thus, while the rate of COX-2 mRNA degradation in HT-29 was enhanced, it slows down the same in Caco-2 cells. Future studies will indicate how NaBt can affect the dichotomy in Chk2/MK2 activation in the presence of stress.

#4439

Genetic alterations in specific RNA processing genes is associated with poor patient outcome in prostate cancer.

Koichi Sugimoto,1 Marco A. De Velasco,1 Yurie Kura,1 Kazuko Sakai,1 Masahiro Nozawa,1 Kazuhiro Yoshimura,1 Kazuhiro Yoshikawa,2 Kazuto Nishio,1 Hirotsugu Uemura1. 1 _Kindai University Faculty of Medicine, Osaka-Sayama, Japan;_ 2 _Aichi Medical University, Osaka-Sayama, Japan_.

Altered expression of splicing factors has been implicated in human cancers but their role in disease progression is complex due to the magnitude of potential protein products. We previously showed that androgen withdrawal led to increased alternatively spliced products in mouse Pten-deficient prostate tumors. Here, we perform gene expression analysis this mouse prostate cancer model to identify candidate mRNA processing genes implicated in the progression to castration-resistant disease. Genome-wide analysis revealed a set of 43 unique RNA processing and splicing factor genes associated with the progression to castration-resistant disease. Expression analysis of this gene set was examined in a pan-cancer analysis of 30 TCGA cancer studies covering 26 human cancer types. Alterations of this gene set (z-score threshold variances >2) were found in 70.5% (6978/9896) of cases. The median percentage of affected cases across the studies ranged from 28.2-98.2% with a median of 78.3%. In prostate cancer patients (TCGA-Prostate Adenocarcinoma (PRAD)), 65.7% (327/497) of the cases had mRNA alterations of this gene set. Twenty-seven of the 30 studies had survival data, of these only three studies indicated poor disease-free survival (DSF) outcomes with mRNA alterations: TCGA-PRAD, P=0.00616; TCGA-Brain Lower Grade Glioma (BLGG), P=0.0088; TCGA-Thymoma, P=0.0265. In the TCGA-PRAD study, the median time to DSF was 81.2 months in altered cases vs. median not reached in non-altered cases. In three studies, favorable outcomes were seen in cases with alterations in the gene set: TCGA-Uveal melanoma, TCGA- Glioblastoma Multiforme and TCGA-Lung Squamous Cell Carcinoma. TCGA-BLGG was the only study to show a statistically significant association between gene set alterations and overall survival (OS), P=0.0206. In TCGA-PRAD, median OS was 115 months in altered cases vs. median not reached in non-altered cases P=0.262. We further analyzed the scope of molecular alterations of this gene set in prostate cancer patients, overall, 78.9% (393/498) of the cases had gene alterations consisting of mutations, amplifications, deletions, and mRNA downregulation in six (1.2%), two (0.4%), 49 (9.8%), 193 (38.8%), and 143 (28.7%) cases, respectively. Notably a strong association was revealed for poor OS for cases with mutations/copy number alterations (P=0.00621). These data suggest that molecular alterations in specific RNA processing genes can cooperate to promote specific cancer types and further studies will be required to fully understand the prostate cancer-driving events in this gene set.

#4440

Differential regulation of p63 isoforms by alternative polyadenylation.

Shakur Mohibi, Jin Zhang, Xinbin Chen. _University of California at Davis, Davis, CA_.

p63, a transcription factor belonging to the p53 family, plays vital roles in epidermal development and tumorigenesis. p63 expresses from two different promoters generating two classes of proteins, TAp63 and ∆Np63 that differ in their N-terminus. Moreover, alternative splicing of the TA and ∆N isoforms in the C-terminus creates additional isoforms (α-ε); thus generating five TA and five ∆N p63 isoforms. The factors that give rise to this repertoire of p63 isoforms are not very well understood. Alternative polyadenylation (APA) can generate multiple isoforms of a gene with alternative 3' ends based on the polyadenylation site selection during cleavage and polyadenylation of mRNA. APA can give rise to isoforms that differ in the coding region (CDS APA) or that have varying lengths of 3' UTR (3' UTR APA). Recently, PABPN1 protein that binds the 3' poly(A) tail of mRNAs has emerged as a key regulator of APA. Here, we show that PABPN1 differentially regulates p63α and p63γ isoforms by alternative polyadenylation. Specifically, knockdown of PABPN1 increases the levels of p63γ mRNA as well as protein by regulating CDS APA of p63 gene. Moreover, knockdown of PABPN1 also generates p63α mRNAs with varying 3' UTR lengths. Although the mRNA levels of p63α remain unchanged upon PABPN1 KD, p63α protein is significantly decreased, suggesting that PABPN1 also regulates p63α protein translation. Interestingly, we also show that PABPN1 is a p63α target and that p63 transcriptionally regulates PABPN1 by binding to its promoter forming a negative feedback loop. Taken together, our study identifies PABPN1 as a novel regulator of various p63 isoforms by APA and that p63 in turn regulates PABPN1 transcriptionally.

#4441

Functional and clinical implications of an INDEL within the HuR regulatory region of the mitotic kinase inhibitor WEE1.

Samantha Z. Brown,1 Avinoam Nevler,1 Henry T. Thomsett,1 Shruti Lal,1 Mahsa Zarei,1 Fernando Blanco,1 Joseph A. Cozzitorto,1 Alexis L. Norris-Kirby,2 Charles J. Yeo,1 Jordan M. Winter,1 James R. Eshleman,2 Jonathan R. Brody1. 1 _Thomas Jefferson University, Philadelphia, PA;_ 2 _Johns Hopkins University School of Medicine, Baltimore, MD_.

The RNA-binding protein Human Antigen R (HuR) is upregulated in pancreatic ductal adenocarcinoma (PDA), where it promotes tumorigenesis via its mRNA pro-survival targets. PDA cells exposed to DNA damage upregulate the mitotic inhibitor kinase, WEE1, in a HuR-dependent manner to induce cell cycle arrest and facilitate drug-resistance (1). Herein, we further evaluate a 56 base-pair (bp) region within WEE1's 3'UTR (labeled WEE1.3UTR) where HuR binds and stabilizes expression. Within this regulatory site, we observed that a 10-thymidine (T) track contains frequent polymorphisms (mean allele frequency 8.67%) of thymidine insertions (i.e, an INDEL). Using a combined approach of Sanger sequencing and a more sensitive capillary-electrophoresis (CE) assay, we screened this region in various cancer cell lines and patient samples. Results revealed three distinct alleles between individual cohorts: the wild-type (10-T, 56 bps), a 1-T insertion (11-T, 57 bps), and a 2-T insertion (12-T, 58 bps). Luciferase reporter constructs were subcloned with the HuR regulatory region embedded in the WEE1.3'UTR. In response to stress, constructs with the wild-type allele reported a higher level of expression compared to the 11-T and 12-T alleles (p<0.01). Complementary RNA-binding protein immunoprecipitation (RNP-IP) assays validated the enhanced binding of HuR to the wild-type construct as compared to the others. Collectively, these data suggest that HuR's regulation of WEE1 is impaired when 11-Ts or 12-Ts are present. Electrophoretic mobility shift assay (EMSA) experiments will investigate HuR's physical interaction by quantifying the relative affinity of the protein to each variant transcript, by itself and in direct competition with each other. To investigate the clinical implication of these findings, we sequenced (via Sanger and CE) a cohort of resected patient tumor samples (n=99), and found a significant enrichment for individuals homozygous for 12-T among those with a unique, Lynch-like familial history of cancer (odds ratio 2.4-6.9, p<0.05). We postulate that the addition of the INDEL in the WEE1.3UTR disrupts the association of HuR, and therefore, the functional upregulation of the WEE1 transcript in response to the stressful PDA microenvironment. Thus, a dysregulated G2/M checkpoint could result in accumulated DNA-damage and eventually promote PDA tumorigenesis. Paradoxically, the disruption of the HuR-WEE1 axis may render PDA cells more sensitive to genotoxic agents, thus providing a potential therapeutic window for patients screened for the polymorphism.

Reference: 1. Lal et al., Cancer Res 2014;74(4):1128-40.

#4442

Wilms' tumor 1-associating protein increases cancer cell proliferation and stem-cell like properties through RNA modification.

Sae Whan Park, Young Taek Oh, Jong Bae Park, Jong Heon Kim. _National Cancer Center (Korea), Goyang-si, Republic of Korea_.

Purpose: Wilms' tumor 1-associating protein (WTAP) which is known for its role in RNA methylation machinery, is highly upregulated in the necrotic region of glioblastoma multiforme (GBM). The highly heterogenic nature of GBM is associated with the necrotic region within the brain, where many types of cells such as the stromal cell, immune cells, and cancer stem cells co-localize. The complex cellular network within the necrotic region is believed to cause the resistance of the conventional chemotherapy and radiotherapy. Here we attempted to identify the role of WTAP in GBM and what kind of RNA modification is associated with the proliferation and stemness in GBM. Improvement of our understanding about post-transcriptional regulation in GBM will allow us to find out the key oncogenic switch in GBM, and development of therapeutics targeting this mechanism will provide better survival benefits.

Methods: 23 Patient-derived cell lines, two established GBM cell lines, and astrocyte was arrayed to find out the correlation between WTAP and cancer stem cells. shWTAP and WTAP overexpression vector were infected into patient-derived GBM cancer stem cell lines to test the effect of WTAP. Western blot and polymerase chain reaction analysis was performed to find out the change in genes which are related to cell proliferation and stemness. Limiting dilution assay was performed to validate the function of WTAP in sphere-forming ability. RNA sequencing was performed on shWTAP infected patient-derived cancer stem cell to identify the type of RNA modification made by knocking down WTAP.

Results: Knocking down of WTAP using shWTAP in patient-derived cancer stem cell lines, we were able to see the decrease in cell proliferation and stemness-related genes while overexpressing WTAP in these cells slightly increased cell proliferation and stemness. WTAP knockdown cells had a significant decrease in sphere-forming ability. RNA sequencing of shWTAP cells has revealed the increase in intron retention and the decrease in exon skipping, which shows that WTAP has a role in decreasing intron retention while increasing exon skipping.

Conclusion: We have revealed that WTAP has an important role in increasing proliferation and maintaining stemness in GBM. We have also identified that WTAP decreases intron retention and increasing exon skipping.

#4443

A post-transcriptional program of chemoresistance regulators in quiescent cancer cells.

Sooncheol Lee,1 Samuel S. Truesdell,1 Syed I. Bukhari,1 Myriam Boukhali,1 Dongjun Lee,1 Maria A. Mazzola,2 Radhika Raheja,2 Adam Langenbucher,1 Nicholas J. Haradhvala,1 Michael Lawrence,1 Roopali Gandhi,2 David A. Sweetser,1 Wilhelm Haas,1 Shobha Vasudevan1. 1 _Massachusetts General Hosp., Harvard Medical School, Boston, MA;_ 2 _Brigham & Women's Hosp., Harvard Medical School, Boston, MA_.

Quiescent (G0) cells are a clinically relevant fraction in several cancers, which include dormant cancer stem cells, and resist clinical therapy. G0 cells reveal extensive changes in gene expression at the protein and translation levels. We previously identified that the translation mechanism is altered in G0 cancer cells. MicroRNAs, noncoding RNAs that target distinct mRNAs to alter gene expression—were found to associate with an important RNA-binding protein, and enable specialized functions in G0—where they recruit non-canonical translation factors to regulate specific mRNA translation. We find that G0 leukemic cells show similar proteome and translatome to cells isolated post-chemotherapy. These data suggest that specialized post-transcriptional and translational mechanisms in G0 leukemic cells regulate a distinct translatome to mediate chemoresistance.

To understand the role of post-transcriptional and translational regulation in chemoresistance, we compared global RNA, translational and proteome profiling in chemoresistant G0 acute monocytic leukemic (AML) cells. We find that chemotherapy or G0 induction leads to DNA damage responsive ATM and stress signaling, which alter post-transcriptional and translational mechanisms. ATM and stress activated p38 MAPK/MK2 increase AU-rich-element (ARE) bearing pro-inflammatory cytokine and immune gene mRNAs by regulating a key ARE RNA binding protein, and by activating STAT1/interferon pathway to alter canonical translation. AREs are present on 3'UTRs of critical, tightly regulated oncogenes and cytokines to post-transcriptionally control their expression. These changes permit translation of ARE bearing pro-inflammatory cytokine TNFα, and immune and cell-migration modulators that promote survival. Co-inhibiting p38 MAPK and TNFα that promote anti-apoptosis—prior to or alongwith chemotherapy—decreases chemoresistance in AML cell lines, in vivo, and in patient samples, without affecting normal cells. These studies reveal a pro-inflammatory subpopulation in AML that mediates chemoresistance, enabled by DNA damage- and stress-regulated post-transcriptional and translational mechanisms that are mediated by AU-rich-elements and a critical ARE RNA binding protein. Disrupting ARE regulation reduces TNFα and chemoresistance, revealing AREs and an important ARE RNA binding protein as key regulators of inflammation-mediated chemoresistance. These studies reveal the significance of post-transcriptional regulation of inflammation/immune gene-mediated chemoresistance.

Correspondence: vasudevan.shobha@mgh.harvard.edu

#4444

The XPO1 inhibitor Selinexor attenuates global translation and enhances the radiosensitivity of glioblastoma cells grown in vitro and in vivo.

Amy Wahba, John W. O'Neill, Barbara H. Rath, Kevin Camphausen, Philip J. Tofilon. _National Cancer Institute, Bethesda, MD_.

Radiation therapy remains a primary treatment modality for glioblastoma (GBM). To identify novel targets for GBM radiosensitization, the radiation-induced translatome was defined for a series of glioblastoma stem-like cells (GSCs). Analysis of the radiation-induced translatome of GSCs identified an interacting network in which Exportin 1 (XPO1) serves as a major hub protein. XPO1 transports a variety of proteins and RNAs from the nucleus to the cytoplasm via the nuclear pore complex and is dysregulated in multiple types of cancers, including GBM. To determine whether XPO1 provides a target for GBM radiosensitization, we defined the effects of the clinically relevant XPO1-inhibitor Selinexor (KPT-330) on the radiosensitivity of GSC lines. Using clonogenic survival analysis, Selinexor was shown to enhance the in vitro radiosensitivity of GSCs but not normal fibroblast cell lines. Because XPO1 mediates the nuclear export of ribosomal subunits, we tested the effect of Selinexor on gene translation using polysomes profiles to calculate translational efficiency and O-propargyl-puromycin (OPP) incorporation to measure de novo protein synthesis. Selinexor significantly reduced translational efficiency in GSCs beginning at 1-6h after treatment, which was accompanied by a decrease in protein synthesis. Moreover, Selinexor exposure increased in 18S and 5S rRNA accumulation in the nucleus with corresponding decreases in the cytoplasm suggesting that the inhibition of rRNA export plays a role in the Selinexor-mediated decrease in protein synthesis. In contrast to the tumor cells, Selinexor had no effect on protein synthesis in normal fibroblast cell lines and astrocytes. To determine the effect of XPO1 inhibition on GBM cells in vivo, mice bearing orthotopic xenografts initiated from a GSC line (NSC11) were treated with Selinexor (20 mg/kg) and tumors were collected over 48 hours. Polysome profiles generated from these orthotopic xenografts indicated that Selinexor treatment resulted in a decrease in translational efficiency with the greatest effect at 24 hours after treatment. These results suggest that consistent with in vitro data, Selinexor inhibits global translation in GBM cells grown under orthotopic, in vivo conditions. To determine the effect of XPO1 inhibition on radioresponse in vivo, mice bearing orthotopic xenografts were treated with Selinexor in combination with fractionated radiation. Whereas Selinexor alone had no effect on survival, the XPO1 inhibitor enhanced radiation-induced prolongation of animal survival with an apparent dose enhancement factor of 2. These results suggest that Selinexor delivered in combination with radiotherapy may improve the effectiveness of GBM treatment.

#4445

Post-transcriptional inhibition of PTPN23 by SND1: Potential mechanism for hepatocellular carcinoma.

Nidhi H. Jariwala,1 Rachel G. Mendoza,1 Garcia Dawn,2 Lai Zhao,2 Mark A. Subler,1 Jolene J. Windle,1 Paul B. Fisher,1 Chen Yidong,2 Devanand Sarkar1. 1 _Virginia Commonwealth Univ., Richmond, VA;_ 2 _University of Texas Health Science Center, San Antonio, TX_.

Oncoprotein SND1 regulates gene expression at a post-transcriptional level in multiple cancers including hepatocellular carcinoma (HCC). Staphylococcal nuclease domains of SND1 function as an RNAse and tudor domain facilitates protein-oligonucleotide interaction. In the present study, we aimed to identify RNA interactome of SND1 to obtain better insights into gene regulation by SND1. RNA interactome was identified by immunoprecipitation of RNA using anti-SND1 antibody from human HCC cells followed by RNA-sequencing (RIP-Seq). With an adjusted p value of <0.01 and >2-fold enrichment over control, 282 mRNAs were identified to significantly associate with SND1 protein. We focused on the tumor suppressor PTPN23 because its regulation by SND1 and its role in HCC are not known. PTPN23 levels were downregulated in human HCC cells versus normal hepatocytes and in human HCC tissues versus normal adjacent liver as revealed by immunohistochemistry. In human HCC cells, knocking down SND1 increased and overexpression of SND1 decreased PTPN23 protein. RNA binding and degradation assays revealed that SND1 specifically binds to and degrades 3'-UTR of PTPN23 mRNA. Tetracycline-inducible PTPN23 overexpression in human HCC cells resulted in significant inhibition in proliferation, migration and invasion and in vivo tumorigenesis. PTPN23 induction caused inhibition in activation of c-Met, EGFR, Src and FAK, suggesting that as a phosphatase PTPN23 inhibits activation of these oncogenic kinases. The present study unravels a novel function of SND1 in targeting PTPN23 and identifies PTPN23 as a unique tumor suppressor for HCC. PTPN23 might function as a homeostatic regulator of multiple kinases restraining their activation.

#4447

Structural basis of human translation initiation factor eIF4G-enhanced helicase activity of eIF4A in tumorigenesis.

Puja Singh,1 Hanyong Chen,1 Young-In Chi,1 Junxuan Lu,2 Zigang Dong,1 Yibin Deng1. 1 _The University of Minnesota Hormel Institute, Austin, MN;_ 2 _College of Medicine, Pennsylvania State University, Hershey, PA_.

Dysreguated translation of tumor suppressor gene and/or oncogene mRNAs into proteins plays a crucial role in tumorigenesis. Translation initiation in mammalian cells is the rate-limiting step of mRNA translation/protein synthesis and primarily exerted through the eukaryotic translation initiation factor 4 F (eIF4F) complex consists of three subunits: eIF4E, which binds the cap of mRNA; eIF4A, an RNA helicase implicated in unwinding mRNA structure; and a scaffold protein eIF4G.The helicase activity of free eIF4A is low but it is dramatically stimulated by forming a complex with eIF4G although the molecular mechanisms are not completely understood. The crystal structure of the yeast eIF4A-eIF4G complex and the mapped interaction sites of eIF4AI-eIF4GII complex from nuclear magnetic resonance (NMR) studies provide insights into structural basis for eIF4G-enhanced eIF4A helicase activity. However, the atomic structure of human eIF4A-eIF4G complex and the biological function of protein-protein interaction for enhancing eIF4A helicase activity in tumor development have not been elucidated. Utilizing X-ray crystallography, we reported here the structure of the complex formed by human eIF4GI (residues 722-999) in a complex with human eIF4A1 (residues 20-406) at 2.8 Å resolution. eIF4A1 protein crystallized in an open, dumbbell-shape conformation with the linker between the two RecA-like domains. eIF4G1 contains 10 α-helices that form a right-handed solenoid and its convex surface interacts with both N- and C-terminal domains of eIF4A (eIF4A1-NTD and eIF4A1-CTD). A total buried surface area from both the proteins at the two interfaces measures ~2700 Å2, with an extensive contact area between the eIF4A1-CTD and the N-terminal portion of eIF4G1 and a secondary, much smaller interface involves eIF4A1-NTD and C-terminal portion of eIF4G1. Utilizing site-directed mutagenesis coupled with biochemical analyses, we identified R324 as a previously uncharacterized residue of eIF4A1 which when mutated completely abolished the interaction with eIF4G1. Biological studies in cancer cells showed that exogenous expression of mutant eIF4A1R324 in human prostate cancer cells significantly inhibited cell proliferation in vitro and attenuated tumor growth of xenografted human prostate cancer cells in immune deficient mice. Given that eIF4A1 helicase activity plays an important role in selectively controlling oncogenes mRNA translation in human cancer cells, our findings from structural and biological studies will lay the groundwork for the development of novel anti-cancer therapeutics targeting the eIF4A1-eIF4G1 interaction to block eIF4A1 helicase activity-driven oncogene protein synthesis and ultimately to inhibit tumor development.

#4448

Mutations in the center MATH domain of Speckle Type BTB/POZ Protein lead to enhanced proteinstability in prostate cancer.

Joshua Fried,1 Vinayak Khattar,1 Jinlu Ma,2 Qinghua Zeng,3 Wei Zhang,3 Bo Xu3. 1 _UAB, Birmingham, AL;_ 2 _Xian Jiaotong University, China;_ 3 _Southern Research, Birmingham, AL_.

The Speckle type Poz Protein (SPOP) is a tumor suppressor that is often mutated in prostate cancer. Mutation of SPOP is linked to genomic instability and it plays a critical role in prostate cancer initiation. SPOP mutations are found to be in the heterozygous state in patients. The relevance of these mutations on its protein expression has not been systematically investigated. Here we report that mutations in the center MATH domain of SPOP lead to enhanced protein stability in prostate cancer. Among the mutations, F102C, S119N, F125V and WI31G showed increased protein stability. Further we show that these mutations in the MATH domain increase half-life of SPOP protein. Interestingly, the mutant SPOPs enhanced the endogenous wild-type SPOP level. We further explored the structural insights of these SPOP mutants. We present a computational

model to predict how SPOP mutations potentially lead to conformational changes within the protein, promoting its stability. Finally, immunohistochemistry in clinical samples of prostate cancer augment our findings. Together, we highlight a subset of clinically relevant SPOP mutations that have impact on endogenous SPOP protein stability, allowing to further understand its role in prostate cancer initiation and evaluating of potentially altered therapeutic response in patients harboring these mutations.

#4449

Synthetic lethality between CSTF2 and CSTF2T in lung adenocarcinoma and melanoma cell lines.

Johannes Popow, Corinna Wieshofer, Andreas Schlattl, Simon Woehrle, Ralph Neumüller, Mark P. Petronczki, Jark Boettcher, Manfred Koegl, Mark Pearson. _Boehringer-Ingelheim RCV GmbH & Co KG, Vienna, Austria_.

Genomic instability is a hallmark of cancer and can result in the deletion of genes with no apparent or as yet unknown relevance to tumor survival and proliferation. Although the deletion of such genes may be tolerated due to functional compensation it, however, entails a cancer cell specific vulnerability as the tumor cell relies on the function of compensating redundant genes. Recurrent deletions of cleavage stimulation factor subunit 2 Tau (CSTF2T) have been observed in several tumor samples (TCGA). During meiosis CSTF2, a paralogue of CSTF2T, which resides on the X-chromosome, is transcriptionally silenced and the zygote depends on the activity of CSTF2T. As a result, CSTF2T knockout mice exhibit male sterility which corroborates that CSTF2 and CSTF2T form a functionally redundant pair of genes with an essential function (Dass et al. 2007). With the aim to apply these findings to tumor biology, we therefore tested whether tumor cells with homozygous CSTF2T deletions depend on CSTF2 using CRISPR-based growth competition assays and confirmed this concept in lung adenocarcinoma and melanoma cell line models. Our data establish synthetic lethality between CSTF2 and CSTF2T and suggest inhibition of CSTF2 in CSTF2T deficient tumors as a new therapeutic concept.

#4450

Integrative genomic and transcriptomic analysis of CHD family chromatin remodelers in human cancers: Initial evidence of an oncogenic role for CHD7.

Zeng-Quan Yang, Xiaofang Chu, hui Guo, Yuanyuan Jiang, Huimei Yu, Lanxin Liu, Wenqi Shan,. _Barbara Ann Karmanos Cancer Inst., Detroit, MI_.

Chromodomain helicase DNA binding proteins (CHDs) are characterized by N-terminal tandem chromodomains and a central adenosine triphosphate-dependent helicase domain. CHDs govern the cellular machinery's access to DNA, thereby playing critical roles in various cellular processes including transcription, proliferation, and DNA damage repair. Accumulating evidence demonstrates that mutation and dysregulation of CHDs are implicated in the pathogenesis of developmental disorders and cancer. However, we know little about genomic and transcriptomic alterations and the clinical significance of most CHDs in human cancer. We used TCGA and METABRIC datasets to perform integrated genomic and transcriptomic analyses of nine CHD genes in more than 10 000 primary cancer specimens from 32 tumor types, focusing on breast cancers. We identified associations among recurrent copy number alteration, gene expression, clinicopathological features, and patient survival. We found that CHD7 was the most commonly gained/amplified and mutated, whereas CHD3 was the most deleted across the majority of tumor types, including breast cancer. Overexpression of CHD7 was more prevalent in aggressive subtypes of breast cancer and was significantly correlated with high tumor grade and poor prognosis. CHD7 is required to maintain open, accessible chromatin, thus providing fine-tuning of transcriptional regulation of certain classes of genes. We found that CHD7 expression was positively correlated with a small subset of classical oncogenes, notably NRAS, in breast cancer. Knockdown of CHD7 inhibits cell proliferation and decreases gene expression of several CHD7 targets, including NRAS, in breast cancer cell lines. Thus, our results demonstrate the oncogenic potential of CHD7 and its association with poor prognostic parameters in human cancer.

#4451

Dynamic translational regulation of cancer-associated genes through mRNA structure.

Lela Lynn Lackey, Aaztli Coria, Alain Laederach. _Univ. of North Carolina at Chapel Hill, Chapel Hill, NC_.

Our goal is to understand how disrupting translational regulation leads to cancer by analyzing the extent to which RNA structure contributes to the translational efficiency of mRNAs in different phases of the cell cycle. We hypothesize that RNA structure is a dynamic projection of the cellular environment and actively contributes to gene regulation by combining sequence-based and environment-induced structures to influence post-transcriptional regulation. We focus on cell cycle regulation as a common, dynamic cellular process that is highly relevant to cancer development and progression. Additionally, during the cell cycle the translational efficiency of many different mRNAs have been shown to change from one phase to the next. We have determined the secondary structures of eight translationally regulated 5'UTRs using SHAPE-MaP (Selective 2' Hydroxyl Acylation by Primer Extension and Mutational Profiling). This chemical structure probing technique is unique in that it reads through chemical modifications during reverse transcription and can be coupled to next generation sequencing and incorporated into RNA structural modeling. Furthermore, we are able to probe these structures in vivo during different stages of the cell cycle. Our data show important conformational rearrangements in 5'UTRs between G1-phase cells and S-phase cells. These results suggest that mRNA structure is a dynamic attribute that plays a role in translational regulation. Many genes involved in cell cycle regulation are implicated in cancer development and progression. Our results confirm that RNA structure of 5' UTRs is an important component of translation regulation and ultimately controls protein expression levels.

#4452

Musashi-2 regulates EGFR/HER3 expression in NSCLC, cell proliferation and response to EGFR inhibitors in EGFR-mutant NSCLC.

Peter Makhov,1 Alexander Kudinov,2 Alexander Deneka,1 Brian L. Egleston,1 Emmanuelle Nicolas,1 Kathy Q. Cai,1 Rohan Brebion,3 Eleanor Avril,4 Mark Hitrik,1 Anna S. Nikonova,1 Ilya G. Serebriiskii,1 Vladimir Khazak,5 Hossein Borghaei,1 Erica A. Golemis,1 Yanis Boumber1. 1 _Fox Chase Cancer Center, Philadelphia, PA;_ 2 _University of New Mexico, Albuquerque, NM;_ 3 _Temple University, Philadelphia, PA;_ 4 _Brown University, Providence, RI;_ 5 _NexusPharma, Philadelphia, PA_.

Musashi-2 (MSI2) is an RNA-binding protein that regulates mRNA translation. We recently established that MSI2 is elevated in a subset of non-small cell lung cancer (NSCLC) tumors upon progression and drives NSCLC metastasis, in part based on activity supporting a TGF-beta/SMAD3/claudin signaling cascade. Here, reverse phase protein array (RPPA) analysis of MSI2-depleted versus control KrasLA1/+;P53R172HΔG/+ murine NSCLC cell lines identified a significant ~2.7-fold upregulation of HER3 (ERBB3) upon MSI2 depletion. Negative MSI2-dependent regulation of ERBB3 protein was confirmed in multiple NSCLC models, based on analysis of MSI2 depletion or overexpression. Further, MSI2 positively regulated expression of the epidermal growth factor receptor (EGFR) protein in the same models. Comparing EGFR and KRAS driven models, we found that MSI2 depletion significantly impairs cell proliferation only in EGFR-mutant NSCLC cell lines. Using RNA immunoprecipitation analysis coupled with qPCR, we show that MSI2 directly binds to EGFR and, to a lesser extent, to HER3 mRNA. MSI2 mRNA binding was approximately correlating with the presence of predicted MSI2 binding sites in corresponding mRNAs. NSCLC lung tissue microarray analysis revealed that MSI2 total positivity by H-score, 2+/3+ and 3+ positivity correlated with EGFR 3+ staining. Finally, EGFR inhibitors erlotinib and afatinib synergized with MSI2 depletion in EGFR mutant models, suggesting that therapeutic targeting of MSI2 could be of clinical value, especially in EGFR-mutant lung cancer.

#4453

Osteopontin-c mediated drug resistance in breast and ovarian carcinoma cells.

Mariana C. Brum,1 Isabella S. Guimaräes,2 Luciana Bueno Ferreira,1 Letícia B. Rangel,2 Raquel C. Maia,1 Gabriela N. de Moraes,1 Etel R. Gimba3. 1 _INCA, Rio de Janeiro, Brazil;_ 2 _Universidade Federal do Espírito Santo, Vitória, Brazil;_ 3 _UFF/INCA, Rio de Janeiro, Brazil_.

Osteopontin (OPN) has been described as an important gene product mediating resistance to chemotherapeutic drugs. However, the specific roles of each OPN splice variant in mediating chemoresistance should be further investigated. This work aimed to evaluate OPNc expression patterns and its correlations with resistance to doxorubicin (DOX) and cisplatin (CIS) in breast and ovarian tumor cell lines, respectively. We used one breast cancer cell line resistant to DOX (MCF-7 DoxR), and one ovarian cancer cell line resistant to CIS (ACRP) and their corresponding parental cell lines (MCF-7 and A2780). Knockdown of OPNc expression levels has been achieved by transfecting cell lines with specific anti-OPNc phosphothiotate modified DNA oligomers. Cytotoxicity assays have been carried out using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), crystal violet and clonogenic assays. Gene expression has been assessed by quantitative real time PCR. Our results showed that drug-resistant cells overexpress OPNc and P-glycoprotein (P-gp), when compared to their age matched parental cell lines. After transfecting both MCF-7 DoxR and ACRP resistant cell lines with the anti-OPNc oligomers, we found a significant knockdown of OPNc isoform expression, as well as the expression of P-gp. Moreover, these cells exhibited a significantly increased sensitivity to DOX and CIS, and inhibited cell growth and colony formation (p<0,05). Additionally, the epithelial-plasticity phenotype of both MCF-7 DoxR and ACRP resistant cell lines has been reversed, as well the expression of epithelial-mesenchymal transition (EMT)-related interleukins. Altogether, these data evidence that OPNc overexpression can mediate resistance to these chemotherapeutic drugs, possibly by modulating the expression of P-gp and the EMT phenotype. In summary, our data provide early insights on the role of OPNc isoform as a potential molecular target in cancer. Therapeutic strategies aiming to interfere with this isoform expression are likely to overcome resistance to chemotherapeutic agents.

#4454

Ecdysoneless, a novel regulator of cell cycle progression and oncogenesis.

Irfana Saleem, Sameer Mirza, Aniruddha Sarkar, Hamid Band, Vimla Band. _UNMC, Omaha, NE_.

Precisely regulated cell proliferation is essential for embryonic development and for homeostasis in adult organs and tissues, whereas uncontrolled cell proliferation is a hallmark of cancer. We have identified the human orthologue of Drosophila Ecdysoneless (ECD) as a critical regulator of cell cycle progression and oncogenesis. ECD is overexpressed in cancers of several organs, and its overexpression correlates with shorter survival in breast cancer patients. Overexpression of ECD leads to an oncogenic phenotype in immortal mammary epithelial cells. ECD deletion in breast cancer cells on the other hand decreased the in vitro oncogenic phenotype and the in vivo tumorigenesis.

Mechanistically, we showed that ECD associates with a protein complex called R2TP, composed of RUVBL1, RUVBL2, RPAP3, PIHID1 proteins, which is involved in several biological functions, including the dynamic assembly of protein-protein and protein-RNA complexes. In addition, affinity purification followed by Mass Spec analyses showed that ECD binds to several proteins, including PRPF8, that are components of critical protein complexes required for RNA splicing. Based on these findings, we hypothesize that ECD interacts with the R2TP complex and protein complexes that control key steps in the cellular mRNA processing. We hypothesize that these complexes are required for the overexpressed ECD to regulate mRNA processing to promote cell proliferation and cell survival in cancer cells. We will present data to document the ECD complexes required for cell cycle regulation and cell survival. Our studies reveal novel roles of ECD in cancer cells, and support the rationale for use of overexpression of ECD and its interacting proteins as molecular biomarkers of tumor progression as well as explore the targeting of these complexes to develop novel cancer therapy agents.

#4455

Fatty acid synthase-induced S6Kinase facilitates USP11-eIF4B complex formation for sustained oncogenic translation in DLBCL.

Bandish Kapadia,1 Nahid Nanaji,2 Kavita Bhalla,1 Binny Bhandary,1 Rena Lapidus,1 Afshin Beheshti,3 Andrew Evens,3 Ronald Gartenhaus1. 1 _University of Maryland, Baltimore, MD;_ 2 _Maryland Healthcare System, Baltimore, MD;_ 3 _Tufts Medical Center, Boston, MA_.

Fatty acid synthase (FASN) and protein translational machinery are two emergent therapeutic targets for cancer treatment, but their mutual dependency on each other for tumor propagation especially in diffuse B-cell large lymphoma (DLBCL) is still unknown. Therefore, we investigated the functional implication of enhanced FASN activity on protein translational machinery employing a panel of 7 DLCBL cell lines and 40 clinical samples. Inhibition of FASN activity by a specific inhibitor, C75 or shRNA mediated knockdown, suppressed de novo protein synthesis in SUDHL2, TMD8, Pfeiffer and HLY-1 (ABC-origin) cells, whereas SUDHL4, SUDHL6 and Toledo (GC-origin) cells showed minimal effect after C75 treatment or FASN knockdown. Assessing the core translational machinery, the eIF4B (eukaryotic initiation factor 4B) levels were depleted in a dose dependent manner of C75 treatment or shRNA mediated knock down of FASN in ABC-DLBCLs. Mechanistically, eIF4B undergoes ubiquitin mediated protein degradation upon FASN depletion. Depleting the expression of eIF4B significantly reduced the proliferative capacity of both FASN sensitive as well resistant DLBCL cells. Screening the interacting protein partners from available databases, we observed that USP11 interacts with and deubiquitinates eIF4B increasing its overall stability. Consistently, altering the expression of USP11 or its activity by either chemical inhibition or mutation significantly alters nascent peptide synthesis. Of functional significance, either down regulating USP11 or inhibition via small molecules significantly reduced the cell proliferation with increased apoptosis of both, FASN activity sensitive and resistant DLBCL cells. USP11 expression was also elevated in DLBCL tissues as evidenced by significantly higher staining. Significantly there is a strong correlation between USP11, FASN as well as eIF4B expression in DLBCL. Elucidating the basis for FASN not regulating GC DLBCL, we observed that sustained PI3K signalling in GC-DLBCL plays a key role in FASN dependent eIF4B stability. Detailed molecular analysis established that S6Kinase phosphorylates USP11 at Ser453, which regulates its interaction with, and stability of eIF4B thereby promoting proto-oncogene expression. These results together demonstrate that FASN activated S6Kinase induced eIF4B/USP11 axis is required for cell proliferation and survival by regulating the translation of proliferative and pro-survival mRNAs. Our findings support targeting the eIF4B/USP11 axis and provide a novel therapeutic option for patients with relapsed/refractory DLBCL.

#4456

Increased NTF2 levels in melanoma cell lines affect nuclear size and cancer cell characteristics.

Lidija D. Vukovic,1 Bradley A. Stohr,2 Dan L. Levy1. 1 _University of Wyoming, Laramie, WY;_ 2 _University of California, San Francisco, CA_.

Xenopus studies have implicated two nuclear transport factors in the regulation of nuclear size: NTF2 and importin α. Importin α, a positive regulator of nuclear size, has been suggested as a biomarker in breast cancer and non-small cell lung carcinoma. We have focused our studies on NTF2. In different staged melanoma cell lines and tissue samples, we observed an inverse correlation between NTF2 levels and nuclear size. Compared to cancer cells, normal melanocytes exhibited the smallest nuclei and highest NTF2 levels. Based on transient transfection experiments in HeLa cells, MRC5 cells, and different melanoma cell lines, we found that increasing NTF2 expression levels reduces nuclear size. The largest effect was observed in WM3211 melanoma cells in which NTF2 expression reduced nuclear cross-sectional area by 40%. We next generated a stably transfected NTF2-inducible version of the metastatic melanoma cell line WM983B. By altering the doxycycline concentration, we can titrate the level of NTF2 protein, and we found that 20 ng/ml of doxycycline increases NTF2 levels by 5-fold and decreases the nuclear cross-sectional area by about 10%. Doxycycline-treated cells exhibited 3-fold higher rates of apoptosis and a 40% reduction in the rate of cell migration in a wound-healing assay. In vivo experiments were performed on RAG2 and NSG mice where NTF2-inducible cells were injected intravenously in order to examine metastatic potential. The number of lung metastases was reduced by 7-fold in doxycycline-treated mice compared to those that were not treated, suggesting that NTF2 overexpression suppresses metastatic potential. We are now testing the hypothesis that altering nuclear size by NTF2 expression leads to chromatin remodeling and altered gene expression. We performed RNAseq on doxycycline-treated and non-treated cells. Differentially expressed genes include those involved in cell migration (e.g. PRKX, LAMA5, ADAMTS15, ANK3, CERS6) and cell survival (e.g. RAMP1, WNK3, BMP5, TP63). Performing DNA FISH will show if the nuclear positioning of these genes is changed when nuclear size is reduced. We are now planning to test a combination therapy in mice in which we reduce nuclear size and treat with the well-established melanoma drug vemurafenib. Most melanoma patients with BRAF mutations show a good response to this drug, however relapse often occurs with drug-resistant melanoma cells expressing high levels of the cancer stem cell markers JARID1B, CD271, and fibronectin. According to our RNAseq data, cells expressing higher levels of NTF2 show reduced gene expression of JARID1B and fibronectin. Thus increasing NTF2 expression in drug-resistant tumors may improve prognosis and survival time.

#4457

Determining the role of novel GLI1 splice variants in breast cancer.

Maria S. Dixon,1 Lhoucine Chdid,1 David Lamson,1 Michael Tarpley,1 Jodie M. Fleming,1 Jennifer A. Freedman,2 Gayathri R. Devi,2 Kevin P. Williams1. 1 _North Carolina Central University, Durham, NC;_ 2 _Duke University Medical Center, Durham, NC_.

The purpose of this study is to investigate the biological significance of novel GLI1 splice variants in breast cancer. 15-20% of breast cancers are triple negative/basal-like, are associated with poor clinical outcomes and show disproportionately higher prevalence in younger women of African descent. The Hedgehog (Hh)/GLI1 developmental pathway has emerged as a therapeutic target in many cancers including studies on breast cancer such as those from our lab. Overexpression of the main Hh transcriptional mediator GLI1 correlates with poor patient prognosis and relapse. The human GLI1 transcript undergoes alternative splicing producing two shorter isoforms, an N-terminal deletion variant (GLI1∆N) and a truncated GLI1 (tGLI1) which have been reported to have different patterns of tissue expression and function. tGLI1 has been identified as being highly expressed in several cancers, including in breast cancer. We undertook an in silico analysis of the NCBI database for evidence of additional human GLI1 transcripts and have identified novel GLI1 splice variants that we have subsequently shown to be highly expressed in breast cancer cell lines. Our objectives were to examine expression differences between GLI1 splice variants in basal-like and luminal breast cancer cell models, AA and White patient samples and to investigate the biological function of GLI1 splice variants in breast cancer. In our studies, we have utilized phenotypic and functional assays to examine the functional impact of these novel isoforms. Our preliminary data suggest that these newly identified GLI1 isoforms are expressed in basal-like breast cancers at substantially higher levels than GLI1, tGLI1 or GLI1∆N. Previous studies assessing GLI1 expression have not considered these new isoforms and the relative contribution of each to breast cancer aggressiveness and require further investigation.

#4458

Identification of splicing regulators using retroviral vectors carrying intron-containing expression cassettes.

Mukta Asnani. _The Children's Hospital of Philadelphia, Philadelphia, PA_.

Exon inclusion is controlled by both positive (e.g., SR proteins) and negative (e.g., hnRNPs) regulators; yet high-throughput methods to comprehensively identify all relevant RNA-binding proteins are currently lacking. Thus, our long-term goal is to develop a genome-wide CRISPR sgRNA library-based screen, where cells with desired phenotypes are identified by flow cytometry and analyzed as pools using next generation sequencing. To achieve this goal, we took advantage of previous work in our laboratory demonstrating expression of the Δex2 CD19 isoform in B-cell leukemias and lymphomas resistant to CD19-directed immunotherapy (Sotillo et al., 2015; Yu et al., 2016). Skipping of exon 2 results in the truncated protein which fails to localize to the plasma membrane, yielding cells that are CD19-negative by flow cytometry (Bagashev et al., 2015). To study splicing regulation of exon 2, we designed a series of retroviral vectors containing the entire CD19 open reading frame retaining intronic sequences flanking exon 2 (i.e., intron 1 and intron 2). It is known that the nuclear splicing of provirus containing introns completely removes intron from the packaged viral RNA genome (Li et al., 1998). However, when viruses were stably transducedto B-lymphoid cell lines rendered null for endogenous CD19, the retroviral long terminal repeat (LTR) promoter-driven CD19 transcription resulted in skipping of exon2. Further addition of cognate 5'UTR sequence downstream of the retroviral LTR did not promote inclusion of this alternative exon. Similarly, the retroviral based expression of CD19 gene containing intronic sequences flanking constitutive exon10 did not allow proper splicing of exon10 and instead resulted into transcript retaining both introns (9 and 10). Interestingly, the flanking intronic sequences of exon10 allowed precise splicing of upstream exon2 when the expression cassette contained both exon2 and exon10 flanked by their respective introns. Thus, downstream placement of constitutive exon10 and its surrounding introns affected the splicing of upstream alternative exon2. Subsequent introduction of sgRNA libraries into cells transduced with such intron-containing expression cassettes will allow us to identify both positive and negative regulators of CD19 exon2 inclusion and potentially - novel general regulators of splicing. Bagashev, A., Sotillo, E., Wu, G., and Thomas-Tikhonenko, A. (2015). The importance of CD19 exon 2 for surface localization: Closing the Ig-like loop. Blood 126, 3433. Sotillo, E., Barrett, D.M., Black, K.L., Bagashev, A., Oldridge, D., Lanauze, C., Gazzara, M.R., et al. (2015). Convergence of acquired mutations and alternative splicing of CD19 enables resistance to CART-19 immunotherapy. Cancer Discov 5, 1282-1295. Li, K., Garoff, H. (1998). Packaging of intron-containing genes into retrovirus vectors by alphavirus vectors. Proc Natl Acad Sci USA.

#4459

EWS-FLI1 orchestrates beta-catenin regulation by a mechanism involving post-translational KDM1A stabilization.

Jozef Ban, Dave N. Aryee, Anna M. Katschnig, Raphaela Schwentner, Maximilian O. Kauer, Heinrich Kovar. _St. Anna Children's Cancer Research Inst., Vienna, Austria_.

Beta-catenin (CTNNB) fulfills different functions depending on its subcellular localization. At the membrane it is involved in homotypic cell adhesion. In the nucleus it acts as a transcriptional co-activator, while cytoplasmic CTNNB sequesters and stabilizes the transcriptional co-activator TAZ and prohibits its nuclear translocation. The most extensively characterized CTNNB function is that of a nuclear effector of canonical Wnt signaling, binding and activating TCF/LEF family transcription factors and leading to upregulation of master regulators of epithelial-to-mesenchymal transition. In Ewing sarcoma (EwS), the presence of the driver fusion oncogene EWS-FLI1 renders canonical Wnt signaling unresponsive to Wnts. The exact mechanism of EWS-FLI1 mediated suppression of this signaling pathway is still elusive, but may involve downregulation of the beta-catenin partner transcription factor TCF7L2. In the absence of Wnt/CTNNB/TCF signaling we still find nuclear localization of CTNNB in EwS cells, and demonstrate sensitivity of the tumor cells to the CTNNB targeting cell-permeable oxazole compound iCRT3, suggesting an essential TCF independent role of beta-catenin for EwS survival. The regulation of CTNNB stability is complex and involves phosphorylation and the activity of a multi-protein cytoplasmic destruction complex containing Axin, APC , GSK3B and other proteins. Here, we identify involvement of lysine specific demethylase KDM1A (LSD1) in EWS-FLI1 dependent nuclear CTNNB stabilization. We describe a mechanism, by which EWS-FLI1 directly activates expression of the ubiquitin specific protease USP1 leading to KDM1A post-translational stabilization and ultimately CTNNB nuclear accumulation. Consequently, knockdown of EWS-FLI1 resulted in modulation of USP1, depletion of KDM1A protein, activation of the LSD1 target and negative Wnt signaling regulator DKK1, and cytoplasmic sequestration of CTNNB. Strikingly, in presence of EWS-FLI1, genetic or pharmacologic inhibition of either USP1 or LSD1, or forced DKK1 expression, led to CTNNB degradation. As we find upregulation of TAZ upon EWS-FLI1 knockdown and loss of CTNNB upon modulation of TAZ by siRNA, we speculate that TAZ may be involved in CTNNB cytoplasmic stabilization in absence of EWS-FLI1, while LSD1 is required for nuclear stabilization in presence of the fusion oncogene. These findings indicate complex CTNNB regulation in EwS, orchestrated by EWS-FLI1, and suggest USP1, KDM1A and CTNNB as potential therapeutic targets in EwS. 

### Tumor Suppressor Genes 1

#4460

BRCA1 **-associated R-loop accumulation at noncoding putative ERα enhancer area regulates expression of adjacent genes.**

Huai-Chin Chiang,1 Xiaowen Zhang,1 Chi Zhang,1 Xiayan Zhao,2 Jingwei Li,1 Yanfen Hu,1 Rong Li1. 1 _UT Health Science Ctr. at San Antonio, San Antonio, TX;_ 2 _The Second Xiangya Hospital of Central South University, Hunan, China_.

Most BRCA1-associated breast tumors are basal-like yet originate from luminal progenitor cells. BRCA1 is best known for its functions in double strand break (DSB) repair and resolution of DNA replication stress. However, it is unclear whether loss of these ubiquitously important functions of BRCA1 fully explains the cell lineage-specific increase in breast tumor development. By sorting and profiling lineage-specific cells from precancerous human breast tissue, I found that BRCA1 mutation carriers tend to have a luminal cell-specific accumulation of R-loop, a transcriptional byproduct of DNA-RNA hybrid structure important for transcriptional regulation and genomic instability. R-loops accumulate preferentially at enhancers and transcription start sites (TSS) of luminal fate-related genes. Using CRISPR-Cas9 system, I've deleted an ERα enhancer area with BRCA1-associated R-loops accumulation in MCF7 cells. The deletion of this non-coding putative ESR1 enhancer area reduced the gene expression level of the neighboring genes of the deleted region, including ERα, RMND1, and CCDC170. In addition, compare to the reduced expression level of ERα, RMND1, and CCDC170 in BRCA1 knockdown of the parental MCF7, knocking down BRCA1 in these deletion clones showed less reduction of these genes. On the other hand, overexpression RNASEH1, an enzyme that specifically removes R-loop, had little effect in these genes after BRCA1 knockdown. These results indicate BRCA1-associated R-loop accumulation at least partially affect gene transcription at these loci. To investigate whether BRCA1-assocaited R-loop accumulation directly contributes to luminal differentiation blockage, I overexpressed RNASEH1 in breast epithelial cells freshly isolated from BRCA1 mutation carriers, flow cytometry comparing different breast lineage reveal a luminal population shift toward more mature luminal. All in all, the results infer that BRCA1 promotes luminal epithelial differentiation by alleviating R-loop accumulation at enhancer of ERα. Aberration in luminal transcription could contribute to lineage-specific BRCA1-associated tumorigenesis.

#4461

Sirt6 deletion slows mouse mammary tumorigenesis.

Pamela Becherini,1 Irene Caffa,1 Silvia Ravera,1 Patrizia Damonte,1 Francesco Piacente,1 Michele Cea,1 Antonia Cagnetta,1 Fiammetta Monacelli,1 Raul Mostoslavsky,2 Santina Bruzzone,1 Alberto Ballestrero,1 Patrizio Odetti,1 Alessio Nencioni1. 1 _University of Genoa, Genoa, Italy;_ 2 _MGH Cancer Center, Boston, MA_.

Studies show that the NAD+ dependent histone deacetylase Sirt6 exerts tumor suppressive effects in different tissues and organs by mechanisms that include the dampening of the Warburg effect and of Myc activity. In breast cancer (BC), Sirt6 was recently shown to blunt PI3K signaling and to reduce cancer stem cell like capacity in tumors with mutated PI3K. Here we sought define the effects of Sirt6 overexpression and of Sirt6 silencing in breast cancer cell lines, as well as the impact of a heterozygous Sirt6 deletion on polyoma middle T antigen (PyMT)-induced mouse mammary tumorigenesis, with the expectation of an accelerated mammary tumor development in PyMT+/-;Sirt6+/- as compared to PyMT+/-;Sirt6+/+ mice.

SIRT6 was overexpressed in either wild type or catalytically inactive (H133Y) form, or silenced in BC cell lines and in non-tumorigenic MCF10A and HMLE cells, monitoring glycolytic enzyme activity (iHK, PFK, PK, LDH), lactate production, oxygen consumption rate, complex I, III, IV, and ATP synthase activity, and matrix metalloproteinase 9 (MMP9) expression. In vivo, we monitored the growth of MDA-MB-231 xenografts in which SIRT6 was silenced vs. control tumors. In addition, we crossed Sirt6+/- mice with MMTV-PyMT+/- mice (a kind gift of Dr. Thorsten Berger, University Health Network, Toronto, Canada) and comparatively monitored tumor latency and overall survival in PyMT+/-;Sirt6+/- vs. PyMT+/-;Sirt6+/+ mice.

In cultured breast cancer cell lines, overexpression of catalytically active SIRT6 (but not of a catalytically inactive enzyme) reduced the activity of glycolytic enzymes as well as lactate production, while boosting OXPHOS and the ATP/AMP ratio. Opposite effects were obtained by SIRT6 silencing. Curiously, no effect of SIRT6 overexpression or of its silencing on glycolysis or OXPHOS was observed in non-tumorigenic mammary epithelial cells. SIRT6 was found to regulate MMP9 expression. Namely, in MDA-MB-231, SIRT6 overexpression increased, while SIRT6 silencing reduced MMP production. In vivo, subcutaneous xenografts of SIRT6-silenced MDA-MB-231 cells were found to grow slower than control cells. PyMT+/-;Sirt6+/- mice exhibited a markedly increased tumor latency and an increased overall survival as compared to the control PyMT+/-;Sirt6+/+ mice. The anticancer effect of Sirt6 heterozygous deletion did not reflect reduced glucose levels in Sirt6+/- mice, as the latter had normal blood glucose levels.

In conclusion, our data show that reducing Sirt6 levels has antitumor effects in mouse breast cancer models, which would not be anticipated based on the available knowledge of Sirt6's biological role. In addition, by virtue of its ability to regulate MMP9 expression, SIRT6 could be a potential target for countering metastasis. Future studies should assess which molecular features predict the potential benefit of SIRT6 inhibition in breast cancer and test the anticancer activity of SIRT6 inhibitors.

#4462

RUNX1 global binding and gene regulation in mammary epithelial cells revealed novel Runx1 mediated cellular activities.

Deli Hong, Andrew J. Fritz, Coralee E. Tye, Natalie A. Page, Joseph R. Boyd, Jane B. Lian, Janet L. Stein, Gary S. Stein. _Univ. of Vermont, Burlington, VT_.

The transcription factor RUNX1 is one of the most frequently mutated genes in breast tumors. However, the role of RUNX1 in the mammary gland is understudied. Previously, our group established that RUNX1 has tumor suppressor activities in both normal mammary epithelial and breast cancer cells, including inhibition of epithelial to mesenchymal transition (EMT), migration, and invasion. To better understand the cellular consequences mediated by RUNX1 loss-of-function in mammary epithelial cells, we performed global gene expression profiling (RNA-seq) in Runx1-depleted MCF10A cells. Upon loss of RUNX1, the expression of 1806 genes was significantly altered (with 2-fold change cutoff). Pathway analysis on these differentially expressed genes revealed that RUNX1 is involved in many cellular activities, including lipid metabolism, cell growth and cell cycle control, which were not previously reported. Consistent with expression profiling results, flow cytometry analysis showed the mitotic population in RUNX1 depleted cells was significantly decreased, indicating RUNX1 is important for proper cell division. To investigate the mechanisms by which RUNX1 controls expression of those differentially expressed genes, RUNX1 genome-wide occupancy analysis (ChIP-seq) was performed in MCF10A cells. We found that although RUNX1 binding is enriched at transcription start sites (TSS), RUNX1 also binds to loci distal to TSSs and multiple other regulatory elements. Therefore, RUNX1 has the capability to utilize multiple mechanisms to control target gene expression. Our results highlight crucial roles for RUNX1 in controlling normal growth of mammary epithelial cells, consistent with its tumor suppressor activities. Loss of RUNX1 leads to improper gene regulation and cell cycle defects, which may contribute to tumor progression in breast cancer. These RUNX1-mediated mechanisms point to novel intervention strategies for early stage breast cancer.

#4463

Loss of APC mediates doxorubicin resistance in breast cancer cells.

Casey D. Stefanski,1 Kaitlyn Keffler,1 Stephanie McClintock,1 Jenifer R. Prosperi2. 1 _University of Notre Dame, Notre Dame, IN;_ 2 _Indiana University School of Medicine-South Bend, South Bend, IN_.

Resistance to chemotherapy is one of the leading causes of breast cancer related deaths. Understanding the molecular basis for chemoresistance is essential for the advancement of novel therapeutic approaches to ultimately improve patient outcomes. The Adenomatous Polyposis Coli (APC) tumor suppressor is either mutated or hypermethylated in up to 70% of sporadic breast cancer; however, little is known about how loss of APC contributes to chemoresistance. Using the ApcMin/+ mouse crossed to the Polyoma middle T antigen (PyMT) transgenic model, we demonstrated that APC loss decreased cisplatin and doxorubicin-induced apoptosis. We previously showed that loss of APC in MMTV-PyMT;ApcMin/+ cells induced STAT3-activation resulting in an enhanced tumor initiating cell (TIC) population and increased multidrug resistance protein 1 (MDR1) expression. Therefore, we hypothesized that APC loss increased MDR1 activity and decreased doxorubicin-mediated DNA damage, ultimately preventing cell death. Using calcein incorporation, we first demonstrated that APC loss increased MDR1 activity which was normalized to WT control levels by the MDR1 inhibitor, Valspodar. We next showed that MDR1 inhibition in MMTV-PyMT;ApcMin/+ cells could restore doxorubicin-mediated apoptosis to that of MMTV-PyMT;Apc+/+ treated cells. Due to STAT3 inducing MDR1 activity and TIC population, MDR1 inhibition in MMTV-PyMT;ApcMin/+ cells was also shown to reduce the TIC population. In addition, we are investigating the effect of APC loss on DNA damage repair pathways. Preliminary studies have shown decreased γH2AX in MMTV-PyMT;ApcMin/+ cells treated with doxorubicin, suggesting a decrease in DNA damage. Downstream signaling of γH2AX, measured by phosphorylation of ATM, also demonstrated decreased DNA damage with ongoing studies measuring phosphorylation of ATR. Due to APC binding to topoisomerase IIα (topo IIα), a target of doxorubicin, the activity of topo IIα was measured to address whether this decreased DNA damage was attributed to impaired topo IIα activity or decreased intracellular doxorubicin due to increased MDR1 efflux. Future studies will investigate whether MDR1 inhibition in combination with doxorubicin will reduce tumor burden in vivo. Taken together, APC loss mediates doxorubicin resistance via enhanced MDR1 activity and altered DNA damage repair pathways demonstrating the potential use of combination therapy to overcome resistance to chemotherapy.

#4464

CPEB-2 is a tumor-suppressor gene in human breast cancer.

Peeyush K. Lala,1 Joshua Tordjman,1 Mehdi Amiri,1 Asma Hasan,1 David Hess,1 Mousumi Majumder2. 1 _Western Univ. School of Medicine, London, Ontario, Canada;_ 2 _Brandon University, Brandon, Manitoba, Canada_.

We found that expression of cyclooxygenase (COX)-2 promotes breast cancer progression by multiple mechanisms, including stimulation of cancer cell proliferation, migration, invasiveness, epithelial-to-mesenchymal transition (EMT), angiogenesis, lymphangiogenesis and induction of stem-like cells (SLC). Through combined gene expression and microRNA microarray analyses of COX-2-transfected MCF-7 cells, we identified two oncogenic miRNAs, miR-526b and miR-655, up-regulated by COX-2. They collectively target 13 tumour suppressor-like genes down-regulated by COX-2, of which Cytoplasmic Polyadenylation Element-Binding Protein -2 (CPEB-2) was the single common target, whose function is unclear in breast cancer. CPEB-2 is known to downregulate mesenchymal transcription factor TWIST1 and translation of HIF1α. CPEB-2 has multiple isoforms (A-F), of which a high B: A isoform ratio was reported to confer anoikis-resistance, a metastatic phenotype, in breast cancer cells. We found that CPEB-2 expression was inversely correlated with COX-2 or the microRNAs miR-526b and miR-655 in breast cancer cell lines; it was very high in a non-tumorigenic mammary epithelial cell line MCF10A.To define its functional roles we knocked out CPEB-2 in MCF10A cells by CRISPR/Cas9-double nickase approach. CPEB-2-KO cells exhibited oncogenic phenotypes in vitro: increased proliferation, migration, invasion, EMT phenotype (decreased E-Cadherin, increased Vimentin, N-cadherin, SNAI1, and ZEB1) and SLC properties (increased spheroid formation and SLC-linked markers). P53 protein was found to be a novel translationally- regulated target of CPEB-2. CPEB-2KO cells, but not wild-type cells, produced lung colonies upon intravenous injection and orthotopic tumors (identified by HLA staining) upon implantation at mammary sites in NOD/SCID/IL2Rϒ-null mice. In human breast cancer tissues, compared to non-tumour breast tissues, isoform A/E of CPEB2 revealed a lower expression in HER2+ breast cancer samples, suggesting that CPEB-2A/E are the tumour-suppressor isoforms. These findings demonstrate that CPEB-2 is a tumour suppressor gene in breast cancer. (Supported by the OICR and the NSERC)

#4465

Variants of uncertain significance of the cancer-predisposing genes in two thousand Japanese whole-genome sequencing data.

Jun Yasuda,1 Keita Iida,1 Kazuki Kumada,1 Soichi Ogishima,1 Yusuke Shibuya,2 Hideki Tokunaga,2 Nobuo Yaegashi2. 1 _Tohoku University, Sendai, Japan;_ 2 _Tohoku University, Graduate School of Medicine, Sendai, Japan_.

Many of the cancer predisposing genes are known to be associated with juvenile cancer syndromes such as Li Fraumeni syndrome. The responsible variants of the juvenile cancer syndromes are usually very deleterious and show strong biological effects on the gene functions. It is not so easy to estimate the clinical significance of the moderately deleterious variants in those genes that may have intermediate effects on the hosts' cancer predisposition and these moderately deleterious (or hypomorphic) variants are now classified as variants of uncertain significance (VUSs). The insight that VUSs may have moderate but clinically significant effects on cancer susceptibility now attracts cancer geneticists. To elucidate the state of VUSs of 135 cancer predisposing genes in general population, we functionally annotate the single nucleotide variants (SNVs) in the genes from a 2049 whole genome sequence high confident dataset (2KJPN-HC) obtained from participants in a genome cohort by Tohoku Medical Megabank Organization. Frequency spectrum of the Combined Annotation-Dependent Depletion (CADD) scores of the 135 genes showed very similar pattern to the whole 2KJPN-HC and a weak negative correlation (r = -0.0579) is observed between allele frequencies and CADD scores for each SNV. Among the variants found in 2KJPN, some of the variants are considered as responsible for hereditary cancer syndromes. We used the CADD and Eigen, other scoring system for estimation of impact of the variants, for the known damaging variants in the BRCA1 and BRCA2 genes to narrow down the candidate VUSs that may have moderate risks for cancer predisposition and found one VUS that is identified as a moderate risk SNP for breast cancer in the other studies. Combination of CADD and Eigen will be useful to narrow down the candidate deleterious SNVs for cancer predisposition.

#4466

Adenomatous polyposis coli transcriptionally regulates epithelial membrane protein 2 to regulate apical-basal polarity.

Alyssa Lesko,1 Carolyn Ahlers,1 Anna T. Lyons,1 Jocelyn D. Gaona,1 Jenifer R. Prosperi2. 1 _University of Notre Dame, South Bend, IN;_ 2 _Indiana University School of Medicine-South Bend, South Bend, IN_.

Adenomatous Polyposis Coli (APC) is a well-known negative regulator of the Wnt pathway; however, it also regulates polarity proteins. Loss of apical-basal polarity disrupts several cellular processes including epithelial structure and intracellular signaling, and is an early marker for tumor development. We previously demonstrated that APC knockdown (APCKD) in Madin-Darby Canine Kidney (MDCK) cells altered cyst size and inverted polarity in 3D culture. Through microarray analysis we made the novel observation that APC loss increased Epithelial Membrane Protein 2 (EMP2) expression. Interestingly, EMP2 knockdown in APCKD cells decreased cyst size and restored apical polarity. These data suggest a previously unknown role for EMP2 in regulating polarity; however, how EMP2 influences APC-mediated polarity remains unknown. Therefore we hypothesize that APC transcriptionally regulates EMP2 to influence apical-basal polarity. In this study, we investigate transcriptional activation of EMP2 using bioinformatics, transcription factor DNA/protein arrays, luciferase reporters, and ChIP assays. EMP2 promoter screens (ConTra v3) identified binding sites for signal transducer and activator of transcription 1 and 3 (STAT1 and 3), and E2F transcription factor 1 (E2F1). Interestingly, APCKD cells exhibited increased expression of STAT1 and E2F1 in DNA/protein arrays compared to controls. Additionally, STAT3 activation was increased in APCKD cells in reporter assays. These studies identified STAT3, STAT1, and E2F1 as possible APC-mediated transcriptional regulators of EMP2. Current studies utilizing luciferase reporter assays aim to determine if E2F1 and STAT1 are transcriptionally active upon APC loss. ChIP assays are being utilized to determine if these candidate transcription factors can bind the canine EMP2 promoter. As CREB is a known transcriptional regulator of EMP2, it was used as a positive control for EMP2 promoter binding. However, no changes in CREB binding are expected between APCKD and MDCK cells, as DNA/protein arrays and luciferase reporter assays did not reveal changes in CREB expression or activity upon APC loss. In preliminary studies, E2F1 was found to weakly bind the canine EMP2 promoter, though this binding was not regulated by APC. Current studies seek to determine if STAT1 and STAT3 bind the EMP2 promoter. Reporter assays using mutant EMP2 promoter constructs are being used to assess if the candidate transcription factors drive EMP2 expression upon APC loss. Future studies will determine if candidate transcription factors influence APC-mediated polarity. Transcription factors will be inhibited in APCKD cells and activated in MDCK cells and EMP2 expression, apical polarity and cyst size will be evaluated in 3D culture. Understanding the interaction of APC and EMP2 and the influence on polarity will identify key players in APC disease progression.

#4467

The role of HOXA5 in the malignant progression of engineered isogenic MCF10A sublines harboring common breast cancer mutations.

Priya Pai, Guannan Wang, Saraswati Sukumar. _Johns Hopkins University, Baltimore, MD_.

Background and Aims: HOXA5 behaves as a tumor suppressor gene in breast cancer and regulates E-cadherin, CD24, progesterone receptor and p53. Retinoic acid activates transcription of HOXA5 by binding to RAR elements in the HOXA5 locus. The goal of this study was to establish whether the loss of HOXA5 cooperates with loss or mutations in common tumor suppressor genes and oncogenes in isogenic untransformed breast epithelial MCF10A sublines to induce tumorigenesis.

Materials and Methods: Isogenic MCF10A sublines with a single knock in mutation in p53 (R248W), or double knock in mutations in both HER2 (V777L) and PIK3CA (E545) generated by Cre recombinase mediated excision/insertion by the Park lab were used. Genomic DNA extraction followed by PCR, gel extraction and Sanger sequencing was used to confirm genotype. Western blotting and quantitative real time PCR was used to analyze gene expression. All-trans Retinal- ATAL (Sigma #R2500) (1 μM for 7 days) was used treat p53 R248W cell line. Colony formation assay was performed and assessed using 0.5% crystal violet staining. XTT assay was used for cell proliferation assays. Immunocytochemical images were taken. For xenograft studies, 2 X 106 cells in 80% growth factor reduced Matrigel and 20% PBS were implanted subcutaneously.

Results: Stable knock down (KD) of HOXA5 using shRNAs in the V777L DKI cell line (HER2 V777L and PIK3CA E545K) resulted in a more spindle like, elongated morphology. Elevated levels of mesenchymal markers N-cadherin, Slug and Vimentin, and reduced levels of epithelial marker E-cadherin, at both the protein and RNA levels were seen. Ectopic overexpression of HOXA5 in the p53 R248W cell line caused reduced proliferation, colony formation and wound healing relative to vector transfected cells. ATAL treatment of p53 R248W restored expression of HOXA5, E-Cadherin, progesterone receptor and other HOXA5 targets. Subcutaneous injection of V777L DKI SCR cells into NSG mice resulted in small growths of ductal structures with large lumens surrounded by a single layer of cells, while V777L DKI-HOXA5 knock down cells formed structures with smaller lumens surrounded by multiple layers of cells, resembling hyperplastic outgrowths.

Conclusions: Knock down of HOXA5 expression in the partially transformed V777L DKI cell line results in a more mesenchymal phenotype accompanied by an increase in EMT marker expression, and a transformation from normal ductal structures to hyperplastic nodules when implanted in NSG mice. Whether the hyperplastic nodules will progress to invasive cancer as the tumor grows is under study. Conversely, ectopic expression of HOXA5 in the p53 R248W cell line causes a reduction in functional properties associated with a transformed phenotype: proliferation, colony formation and wound healing.

#4468

Repression of CD44 expression by p53 in a breast cancer progression model.

Fang Liu,1 Zhengxue Liu,1 Guannan Wang,1 Tanima Roy-Kundu,1 Isao Matsuura2. 1 _Rutgers Univ., Piscataway, NJ;_ 2 _National Health Research Institutes, Taiwan_.

CD44 is an important molecule in breast cancer. Although CD44 lacks its own signaling domain, it can associate with and co-stimulate signaling by a number of growth factor receptors, such as Her2 and EGF receptor 1. Knockdown of CD44 significantly reduces mammary tumor-initiating frequency and tumor weight. Disruption of tumor cell surface CD44 function induces apoptosis in metastatic mammary carcinoma cells in vivo. Some studies showed that the breast cancer stem cells are enriched in the CD44highCD24low population. The tumor suppressor p53 is mutated or functionally inactivated in over 70% of cancer. In breast cancer, p53 is mutated in ~35% cases, and ~50% breast cancers contain MDM2 or MDMX overexpression. A previous study showed that p53 repressed the expression of CD44 and that this activity was essential for the tumor-suppressor function of p53. We find that p53 potently represses the expression of CD44 in normal human breast epithelial cells. Mutation of the p53 binding site on the CD44 promoter greatly reduces the ability of p53 to repress CD44 expression. We show that p53 repression of CD44 expression is progressively reduced in a breast cancer progression model. Our ongoing studies are elucidating the underlying mechanisms. Our findings will help for better understanding and treatment of breast cancer.

#4469

Generating stable cell lines with mCherry-BRCA2VUS in human BRCA2 knockout cells.

Iyani Kelly. _Clark Atlanta University, Kennesaw, GA_.

BRCA2 is a tumor suppressor gene expressed in most cell types and its protein product is responsible for a specialized DNA repair pathway called homologous recombination. Prior research on BRCA2 has revealed that loss-of-function truncating mutations lead to breast and ovarian cancers. Our current work has revealed that pathogenic missense mutations in BRCA2 lead to mis-localization of the protein to the cytoplasm, preventing repair of DNA damage in the nucleus. VUS (Variants of Uncertain Significance) account for approximately 10-20% of BRCA2 sequencing results with unknown cancer risk for patients. Most VUS are missense mutations changing a single amino acid in the full-length BRCA2 protein. The purpose of my research was to express wild type and VUS BRCA2 proteins fused to the mCherry fluorophore to visualize their localization in live cells. The BRCA2 DNA was cloned, along with mCherry fluorophore and an antibiotic resistance gene (G418), into a mammalian expression vector (phCMV1). Then, Lipofectamine 3000 was used to transfect the phCMV1 vector containing the BRCA2 DNA into human BRCA2 knockout cells. After a few weeks of selection in G418 media, maintenance, and expansion of single-cell derived clones, live images were taken of each stable cell line expressing the mCherry-BRCA2 fusion proteins. The resulting images revealed that the BRCA2 missense VUS mislocalized to the cytoplasm while wild type BRCA2 was localized to the nucleus as expected. This research contributes to the field by identifying how specific pathogenic patient mutations alter BRCA2 localization within live cells. Further studies will explore the cellular biology underlying the cause of the mislocalization to the cytoplasm.

#4470

**Loss of ARL6IP4 induces the epithelial mesenchymal transition in breast cancer metastasis by transcriptionally phosphorylating NF-** k **B.**

Mi Kyung Park,1 Ji Yun Jang,1 Hyun Jung Byun,2 Hyun Ji Kim,2 Ho Lee,1 Chang Hoon Lee2. 1 _National Cancer Center, Republic of Korea;_ 2 _Dongguk University, Republic of Korea_.

The epithelial mesenchymal transition EMT is the major mechanism involved in cancer metastasis. During EMT, epithelial cells lose cell-cell adhesions to gain mesenchymal-like properties, such as increased invasive abilities. The function of ARL6IP4 in the EMT of breast cancers has not yet been studied. Here we show that the ARL6IP4 controls EMT in breast cancer metastasis. we demonstrate that the role of ARL6IP4 by analysis of ARL6IP4 conditional knockout animals, various in vitro and in vivo models of EMT and metastasis, a mouse mammary tumor virus-polyoma middle T antigen (MMTV-PyMT) model of breast cancer metastasis. We identified that loss of ARL6IP4 phosphorylated of nuclear factor-κB (NF-κB) by increasing the expression of the SNAIL and ZEB1 EMT transcription factors (EMT-TFs). Taken together, our findings establish that ARL6IP4 is a potential tumor suppressor by regulation the phosphorylation of NF-κB.

#4471

Pax9 regulates squamous cell differentiation and alcohol-associated carcinogenesis in the oro-esophageal epithelium.

Zhaohui Xiong,1 Shuang Ren,1 Hao Chen,1 Yao Liu,1 Caizhi Huang,1 Yawan Lyvia Zhang,1 Joab Otieno Odera,1 Tong Chen,2 Ralf Kist,3 Heiko Peters,3 Katherine Garman,4 Zheng Sun,5 Xiaoxin Luke Chen1. 1 _BBRI/North Carolina Central University, Durham, NC;_ 2 _The Ohio State University, Columbus, OH;_ 3 _Newcastle University, United Kingdom;_ 4 _Duke University, Durham, NC;_ 5 _Beijing Stomatological Hospital, Beijing, China_.

Pax9 is a transcription factor of the Pax family characterized by a DNA-binding paired domain. Previous studies have suggested a potential role of Pax9 in squamous cell differentiation and carcinogenesis of oro-esophageal epithelium. However, its functional role in differentiation and carcinogenesis remains unclear. In this study, Pax9 deficiency in mouse esophagus promoted cell proliferation, delayed cell differentiation and altered the global gene expression profile. Ethanol exposure down-regulated Pax9 expression in human esophageal epithelial cells in vitro and mouse forestomach and tongue in vivo. We further showed that PAX9 was down-regulated in human oro-esophageal squamous cell carcinoma (OESCC), and its down-regulation was associated with alcohol drinking and promoter hypermethylation. Moreover, ad libitum feeding with an isocaloric Lieber-DeCarli liquid diet containing ethanol for 40 weeks or Pax9 deficiency promoted NMBA-induced squamous cell carcinogenesis in mouse tongue, esophagus, and forestomach. In conclusion, Pax9 regulates squamous cell differentiation in the oro-esophageal epithelium. Alcohol drinking and promoter hypermethylation are associated with PAX9 silencing in human OESCC. Pax9 down-regulation, at least in part, contributes to alcohol-associated oro-esophageal squamous cell carcinogenesis.

#4472

Downregulation of POTEG predicts poor prognosis in esophageal squamous cell carcinoma.

Yan Li. _Cancer Center, Sun Yat-sen University, Guangzhou City, China_.

POTE ankyrin domain family, member G (POTEG) belongs to POTE family. The POTE gene family encodes very closely related proteins that are highly expressed in prostate, ovary, testis, and placenta. Recent studies indicated that the POTE proteins have a pro-apoptotic function. To examine whether POTEG also underwent apoptosis in ESCC cancer cells and the functions of POTEG in the development of esophageal squamous cell carcinoma (ESCC), we determined the location and expression of POTEG in ESCC cancer cells. Clinical association studies determined that POTEG downregulation was associated with poor clinical outcomes. Ectopic re-expression of POTEG effectively suppressed the tumorigenesis and metastasis of ESCC cells in vitro and in vivo, including the inhibition of cell growth rate, foci formation, soft agar colony formation, migration, invasion and tumor formation in nude mice. Molecular analyses revealed that POTEG downregulated CDKs, leading to subsequent inhibition of Rb phosphorylation, and consequently arrested Cell Cycle at G1/S Checkpoint. Prospectively, POTEG can reduce the level of anti-apoptotic proteins, while promote the level of proapoptotic proteins and inducing apoptosis. On the other side, POTEG inhibit metastasis by reducing epithelial-mesenchymal transition in ESCCs. Taken together, our results revealed that POTEG has a pivotal function in ESCC pathogenesis, with possible use as a biomarker and intervention point for new therapeutic strategies.

#4473

Tumorigenesis by deregulation of CRAD-controlled cytoskeleton dynamics.

Youn-Sang Jung,1 Wenqi Wang,2 Sohee Jun,1 Jie Zhang,1 Mrianl Srivastava,1 Moon Jong Kim,1 Esther M. Lien,1 Joan Shang,1 Pierre D. McCrea,1 Songlin Zhang,3 Junjie Chen,1 Jae-Il Park1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _University of California Irvine, Irvine, CA;_ 3 _UT Health, McGovern Medical School, Houston, TX_.

Epithelial cell behavior and homeostasis are mainly modulated by the cytoskeleton and cell adhesion. However, much concerning epithelial cell deregulation in cancer remains unknown. We identified Cancer-related Regulator of Actin Dynamics (CRAD) as frequently mutated or transcriptionally downregulated in colorectal cancer. We found that CRAD is a capping protein inhibitor and stabilizes the cadherin-catenin-actin (CCA) complex. CRAD inactivation disrupts the CCA complex via inhibiting actin polymerization, liberating β-catenin and aberrantly activating Wnt pathway. In mice, CRAD knockout induces the loss of epithelial cell integrity and hyperactivates Wnt signaling, resulting in intestinal adenoma development. With APC mutation, CRAD knockout induces and accelerates mucinous and invasive adenoma development in the colorectum. These results define CRAD as a new tumor suppressor, of which inactivation deregulates the cytoskeleton and hyperactivates Wnt signaling, initiating mucinous intestinal tumorigenesis. Our study reveals the unexpected roles of an actin cytoskeletal regulator in maintaining epithelial cell integrity.

#4474

An intricate role of p53 and p21 in cellular alterations and drug penetration in spheroids of colorectal cancer cells.

Viswanath Das, Narendran Annadurai, Marián Hajdúch. _Institute of Molecular and Translational Medicine, Olomouc, Czech Republic_.

The tumor protein p53 and cyclin-dependent kinase inhibitor p21Waf1/Cip1 (p21) are two proteins with an intriguing and a paradoxical role in cancer. Through p53-dependent signals, p21 negatively controls cell cycle progression to protect cells against deleterious effects of DNA damage. Although p53 is a tumor suppressor protein, recent evidence shows that some patients with wild-type (wt) p53 develop aggressive and drug-resistant cancers. Similarly, recent studies also suggest the oncogenic role of p21. In a p53 deficient background, p21 results in the development of a subset of highly proliferative and resistant population of cancer cells.

In this study, we examined the role of p53 and p21 in inducing cellular alterations using a panel of colorectal carcinoma cell lines with different p53 and p21 background and spheroid and spheroid-derived cell cultures. First, our data show that despite the presence of proliferating cells, spheroid-derived cultures of HCT116/p53wt cells continue to display an elevated p21 level even after 6 days of culture as a monolayer. These p21-overexpressing proliferating cells show increased resistance to doxorubicin and paclitaxel in comparison to their parental monolayer counterpart. In contrast to HCT116/p53wt cells, DLD-1/p53mut cells did not show elevated p21 levels following reversal from spheroid to monolayer. The increased p21 level in spheroids and spheroid-derived cultures of HCT116/p53wt cells led us to examine the role of p53 and p21 in drug penetration. Spheroids of HCT116/p53wt cells were less permeable to doxorubicin and showed increased expression of intracellular junctional (IJ) proteins (adherens and tight junctions). Knocking-out p21 in HCT116/p53wt cells decreased the level of IJ proteins, resulting in an increased doxorubicin penetration. Re-expression of p21 in p21-knocked-out HCT116/p53wt cells restored IJ protein levels, reducing doxorubicin penetration and the subsequent effect of doxorubicin on spheroids. Interestingly, however, knocking-out p21 in DLD-1/p53mut cells neither altered intracellular junctions nor affected doxorubicin penetration. These data indicate that the effect of p21 on IJ proteins and drug penetration is pertinent only in cells with wt p53. Overall, the data from our study suggest an intricate interplay between wt p53 and p21 in inducing cellular alterations that affect the sensitivity of cells to anti-cancer drugs in spheroids.

#4475

Adenomatous polyposis coli like protein (APCLP) functions as a novel negative regulator of NF-kB signaling in colon cancer cells.

Matthew Martin, Rasika Mundade, Tao Lu. _Indiana University School of Medicine, Indianapolis, IN_.

Colon cancer (CRC) is the second leading cause of cancer related deaths in the United States. CRC is marked by aberrantly activated signaling of the nuclear factor of kappaB (NF-kB), a family of transcription factors which regulate wide varieties of cellular processes. Despite recent advances in comprehension of players of NF-kB signaling, deeper understanding of its regulation is imperative for the development of novel cancer therapeutics. Using the powerful validation-based insertional mutagenesis (VBIM) technique, we recently discovered APCLP as a novel negative regulator of NF-kB. The objective of this study is to elucidate the role APCLP in regulating NF-kB signaling in CRC at the molecular and biological levels and to understand the mechanism by which this regulation occurs. To determine the biological effect of APCLP on NF-kB signaling, we used lentiviral vectors to either overexpress or knockdown (shRNA) APCLP in human CRC cell lines (HT-29, HCT116, DLD-1). We show that overexpression of APCLP decreased the NF-kB activity, reduced cellular proliferation, migratory ability, as well as anchorage-independent growth of cells while knockdown of APCLP had an inverse effect. Furthermore, in vivo experiments in a xenograft mouse model confirmed that APCLP overexpression impeded whereas shRNA knockdown promoted tumor growth. To study the mechanism by which APCLP regulated NF-kB signaling, we conducted co-Immunoprecipitation experiments and confirmed that APCLP and the major subunit of NF-kB, p65, may complex or bind directly to each other. Studies are ongoing regarding the mechanism of interaction between APCLP and p65. In summary, discovery of APCLP and understanding of its molecular mechanism and biological function are significant because the knowledge acquired from this study could lead to utilization of APCLP as a potential biomarker and therapeutic target in CRC as well as other cancers that are driven by hyperactivated NF-kB.

#4476

Role of MAP-Kinase suppressor in IBD.

Sharad Khare,1 Qiong Zhang2. 1 _Truman VA/University of Missouri-Columbia, Columbia, MO;_ 2 _University of Missouri-Columbia, Columbia, MO_.

BACKGROUND & AIMS: Endogenous suppressor of Mitogen Activated Kinase Pathway (MAPK) are considered cancer suppressors. In this regard , Sprouty (SPRY) proteins that are endogenous suppressor of receptor tyrosine kinase signaling are reported to be tumor suppressors in various cancers, whereas we discovered that SPRY2 augments Epithelial Mesenchymal Transition (EMT) in colorectal cancer (CRC) [Oncogene, 2016 Jun 16; 35(24): 3151-62]. Significance of SPRY proteins in Inflammatory Bowel Disease (IBD) is not clearly understood.

MATERIALS & METHODS: Biopsies from Crohn's Colitis patients were assessed for SPRY, Beta-catenin, Occludin and ZO1 expression. Tight junction profiler was employed to assess expression of integrin proteins in CaCO2 colon cancer cell line. SPRY was suppressed in colon cancer cells by siRNA and CRISPR/Cas9 technology and changes were assessed by immunoblotting, real time PCR, phase-contrast and confocal microscopy. Spry1 and Spry2 floxed mice were utilized to dissect SPRY functions in mesenchymal cells.

RESULTS: SPRY, E-cadherin, Beta-catenin and Occludin mRNA transcripts were significantly different in IBD patients (p<0.05). No significant changes in ZO1 mRNA transcripts were noted. Suppression of SPRY significantly decreased expression of several integrins in CaCO2 cells (p<0.05) . Treatment of CaCO2 cells with Dextran Sodium Sulfate (DSS) significantly altered SPRY, Beta-catenin, Occludin and ZO1 expressions that was reversed by SPRY suppression. Spry1-/- and Spry2-/- double mutant murine embryonic fibroblasts (MEFs) demonstrated restoration of several epithelial markers which protected cells from DSS-induced changes.

CONCLUSIONS: SPRY expression in colonic epithelium regulates IBD. SPRY has prognostic value in IBD.

#4477

Downregulation of ABCG2 expression in colitis and colon cancer: Relevance to iron overload, hemochromatosis and p53, and therapeutic use of carbidopa to reverse the downregulation.

Bojana Ristic, Sathish Sivaprakasam, Jiro Ogura, Vadivel Ganapathy. _TTUHSC, Lubbock, TX_.

The efflux pump ABCG2 is a part of cellular defense. In intestine it is located at the luminal side, and effluxes exogenous/endogenous dysplasia-promoting agents. Since cancer cells exposed to chemotherapeutics overexpress ABCG2 to remove these drugs, ABCG2 is considered a multidrug-resistance protein. However, its physiologic protective role in cancer initiation/progression has not received much attention. In numerous studies, ABCG2 is present in colon at lower levels in patients with colitis and chemotherapy-naïve colon cancer than in controls. We therefore hypothesized that the physiologic role of ABCG2 is to suppress colonic inflammation and colon cancer. To test this, we monitored Abcg2 expression in the intestinal tract in experimental and genetic mouse models of colon cancer and colitis. In normal mice, Abcg2 was expressed throughout the intestinal tract, jejunum being the site with maximal expression. Colon was positive for expression, but the expression was lower than in jejunum. Compared to control colon, Abcg2 was present at lower levels in colonic polyps from experimental and genetic models of colon cancer; the same trend was seen in normal and cancer cell lines of colonic origin. In line with human colitis studies, colons from colitis mouse models had lower expression of Abcg2 compared to control colon. The association of excess iron to colon cancer is well known, but it is not known if excess iron has any role in tumor-associated downregulation of ABCG2 in colon. We used Hfe-/- mice as a model for hemochromatosis, a genetic iron-overload disease, and exploited the ability of Abcg2 to export uric acid as a measure of Abcg2 function. Colons from Hfe-/- mice had lower expression of Abcg2 and higher accumulation of uric acid. This decrease in Abcg2 function indicated that excess iron, directly or indirectly, dictates Abcg2 expression in colon. To better understand this phenomenon, colon cells were treated with ferric ammonium citrate as an iron source. This in vitro iron-overload model demonstrated nuclear depletion of the tumor suppressor p53, and at the same time a decrease in Abcg2 expression. In addition, Abcg2 mRNA was undetectable in p53-null mouse embryonic mouse fibroblasts, suggesting that Abcg2 is a p53 target. Additional in vitro studies included treatment of normal and cancer colon cells with Carbidopa, a drug which was recently shown as an aryl hydrocarbon receptor agonist. Our studies showed that Carbidopa is a potent iron chelator and an inducer of ABCG2 expression. In summary, ABCG2 is silenced in colon cancer and colitis, and the decreased expression of p53 under these conditions might be responsible for the decreased ABCG2 expression. Carbidopa could be used to reduce cellular levels of iron and to reverse iron-induced downregulation of ABCG2 as a prevention/treatment strategy for colitis and colon cancer.

#4478

The tumor suppressive function of HPP1 is mediated by its EGF-like domain.

Abul Elahi,1 Abidemi Ajidahun,1 Leah Hendrick,1 S. Mazher Husain,1 Irina Getun,1 Andreas Becker,2 Evan S. Glazer,1 David Shibata1. 1 _University of Tennessee Health Science Center, Memphis, TN;_ 2 _H. Lee Moffitt Cancer Center, Tampa, FL_.

Introduction:

HPP1 is a putative tumor suppressor gene that has had significant interest as a potential serum- and stool-based biomarker in colorectal cancer. It is an Epidermal Growth Factor (EGF)-like ligand that we have previously demonstrated to mediate its effects via an erbB4-JAK-STAT signaling cascade. HPP1 is a secreted transmembrane protein comprised of two follistatin modules, a cytosolic tail with a potential G - protein-activating motif and an EGF-like domain which differs from EGF by replacement of arginine (R) by a histidine (H) residue at the critical 299 position. The exact component of HPP1 responsible for its biologic function has yet to be elucidated. We sought to examine the specific role of the EGF-like domain and the contribution of the H299R alteration.

Methods:

Synthetic wild-type (WT; H299) and mutant (R299) peptides were prepared at our crystallographic core facility. The change in amino acid did not alter the predicted 3D structure of the peptide. A total of 50ng/ml of each peptide per well (6-well plate) were used to treat the HPP1 non-expressing HCT116 colon cancer cell line. Cells were starved for 24 hours with a peptide exposure time of 7 minutes. Using immunoblot analyses, we examined alterations in the expression and activation status of erbB4, and STAT family proteins.

Results:

Delivery of the WT peptide into the HCT116 cell line resulted in increased activation/phosphorylation of erbB4, STAT1 and STAT2 with a concomitant downregulation of STAT3 activation as compared to controls. These findings are identical to the signaling profile that we have previously described for full length HPP1 and required for its tumor suppressive activity. The mutant peptide did not result in any change in the phosphorylation status of erbB4, STAT1 or STAT2 but was associated with an increase in STAT3 activation as compared to controls.

Conclusion:

The EGF-like domain of HPP1 is the key moiety responsible for triggering its tumor suppressive erbB -STAT signaling cascade. Furthermore, the histidine residue at position 299 is critical and differentiates it from the oncogenic function of EGF. Our findings may have implications for the further understanding of EGF-related signaling in cancer progression.

#4479

Protein arginine methyltransferase PRMT6 regulates cancer stemness through CRAF methylation in hepatocellular carcinoma.

LH Chan,1 L Zhou,1 Kai Yu Ng,1 TL Wong,1 TK Lee,2 YP Ching,1 YF Yuan,3 D Xie,3 S Richard,4 MS Huen,1 XY Guan,1 S Ma1. 1 _The University of Hong Kong, Hong Kong, Hong Kong;_ 2 _The Hong Kong Polytechnic University, Hong Kong, Hong Kong;_ 3 _Sun Yat-Sen University Cancer Centre, Guangzhou, China;_ 4 _McGill University, Montreal, Quebec, Canada_.

Arginine methylation is a post-translational modification that plays pivotal roles in signal transduction and gene transcription during cell fate determination. We found protein methyltransferase 6 (PRMT6) to be frequently down-regulated in hepatocellular carcinoma (HCC) cells and its expression to negatively correlate with aggressive cancer features in HCC patients. Silencing of PRMT6 promoted the tumor-initiating, metastasis and therapy resistance potential of HCC cells. Consistently, loss of PRMT6 expression aggravated liver tumorigenesis in a DEN+CCL4 HCC induced PRMT6-/- mouse model. Integrated transcriptome and protein-protein interaction studies revealed an enrichment of genes implicated in RAS signaling and that PRMT6 interacted with CRAF, and likely other RAF family members, and their methylation at conserved arginine 100, negatively regulating its activity, and as a consequence resulting in enhanced MEK/ERK signaling. Our work uncovered a critical repressive function for PRMT6 in maintenance of HCC cells by regulating the MEK/ERK pathway via arginine methylation of RAF, providing a new avenue of molecular mechanism by which ERK mediated stemness in HCC cells are developed.

#4480

Irx1 gene knockout in zebrafish induces to multiorgan tumorigenesis through cell cycle aberration.

Inhye Jung,1 Dawoon E. Jung,1 DoHee Kim,1 Dong Hee Kim,1 Yong Yoon Chung,2 Seung Woo Park1. 1 _Yonsei University College of Medicine, Seoul, Republic of Korea;_ 2 _3SMTbio Co., Seoul, Republic of Korea_.

The Iroquois homeobox 1 (IRX1) belongs to Iroquois homeobox family and plays an important role during embryonic development. Recent studies have suggested that IRX1 acts as a tumor suppressor in gastric cancer and head and neck carcinoma. The zebrafish allows easy genetic modification at a lower cost, thus being a valuable model for cancer biology. For functional annotation we established homozygous knockout mutants of Irx1a and Irx1b and followed to evaluate long-term phenotypes. Homozygous knockout zebrafish for either Irx1a or Irx1b are vital and survived long enough to see long-term phenotypes. However, majority of the homozygotes for both Irx1a /b were severely malformed, could not survive longer than 6 month, and were not fertile. Surprisingly, hyperplasia and tumorigenesis developed in homozygous Irx1a mutants in multiple organs, including bile duct, intestine, kidney, ovary, and testis where irx1 genes are expressed in adults when evaluated by in situ hybridization. Bile duct hyperplasia or tumor was the most common phenotype, occurring in half and two thirds at 6 and 12 months, respectively. Irx1b mutants also developed similar tumorigenesis but showed less prevalent phenotypes than Irx1a mutants, suggesting functional preservation and redundancy. Analysis of differential genes (Wild type vs. Irx1a/b knockout) by cRNA microarray (Agilent Zebrafish v3 GE 4X44 Microarrays) revealed 687 up- and 963 downregulated genes by Irx1a/b knockout. Significant canonical pathways were cyclins and cell cycle regulation, mitotic roles of polo-like kinase, FXR/RXR activation, estrogen-mediated S-phase entry, and cell cycle control of chromosomal replication, suggesting aberrant cell cycle regulation as an important mechanism of tumorigenesis. Real-time RT PCR validated the differential expression of cell cycle-related genes, especially genes involved in G2/M phase progression. Differential genes included CCND1, ARAF, CDKN2C, and FGFR1 among the upregulated genes, and ANAPC7, CCNA2, CCNB1, 2, HAUS1, 3, 6, CDC7, CDC20, CDCA8, FBXO5, 32, 43 and PLK3 among the downregulated genes. Overexpression by transfection decreased cell proliferation in cholangiocarcinoma cells (SNU-1196 and HuCCT1) without altering sensitivity to H2O2-induced apoptosis on annexin V flow cytometry. Western blot revealed decreased expression of CCND1, CCNE, CCNA1, CCNB1 by IRX1 expression. Flow cytometry using propidium iodide and Alexa647-anti-pHH3 showed decreased mitotic fraction by IRX1 expression. On cell cycle synchronization and release using hydroxyuria and nocodazole showed slight delay in G1-S progression and marked delay in G2-M progression and entry into G1. In conclusion, this study identified Irx1 gene as an important tumor-suppressor gene at various organs, and knockout or Irx1 leads to tumorigenesis by altering cell cycle regulation, especially at G2/M phase.

#4481

Tumor suppressive role of aquaglyceroporin-3 and PTPN13 in muscle invasive bladder cancer.

Arpita A. Roy, Dinuka De Silva, Molly M. Lee, Donald P. Bottaro. _NCI-NIH, Bethesda, MD_.

High mortality rates in bladder cancer (BCa) and lack of effective systemic therapies warrant identifying novel oncogenic pathways and therapeutic strategies. The cell-cell adhesion protein E-cadherin (Cdh1) is an important component of the transitional epithelium of the bladder wall, the cellular origin of most BCa. Cdh1 loss is crucial in epithelial-to-mesenchymal transition (EMT) and subsequent tumor metastasis in multiple cancers, including BCa. Cdh1 reportedly accumulates at cell junctions with the transmembrane aquaglyceroporin-3 channel (AQP3). Reports suggest that AQP3 expression is reduced in BCa of higher grade and stage. Although AQP3 has not been implicated in BCa metastasis, these observations led us to hypothesize that loss of AQP3 contributes to Cdh1 loss, thereby promoting EMT and systemic BCa invasion. Our previous data suggested that AQP3 expression is lower in higher grades of BCa cell lines. Reduced levels of Cdh1 and increased levels of Cdh1 repressors TWIST1, SNAIL1 and ZEB1 were noted in higher grade BCa cells. Our results indicate that AQP3 co-localizes with Cdh1 in lower grade BCa cell lines. This co-localization is not noted in higher grade BCa cells. Silencing of AQP3 by a specific siRNA in lower grade BCa cell lines resulted in reduced Cdh1 localization on the cell membrane. It remains to be deciphered if there is a physical association between Cdh1 and AQP3 and if reduced expression of AQP3 in higher grade BCa cell lines contributes to the loss of Cdh1 and thus facilitate increased EMT progression.

Using tools at c-BioPortal to analyze The Cancer Genome Atlas (TCGA) BCa provisional dataset (n=413) revealed a direct correlation between AQP3 and tyrosine phosphatase PTPN13 expression, and similar to other cancers, PTPN13 deletion was associated with significantly reduced overall survival (p<0.04). It is known that PTPN13 negatively regulates Src activation. Furthermore, Src-mediated Cdh1 phosphorylation leading to Cdh1 degradation is known. We hypothesize that PTPN13 loss may promote BCa progression by increased Src-mediated Cdh1 phosphorylation resulting in Cdh1 degradation. Our data indicate that transcript and protein levels of PTPN13 are significantly lower in BCa cell lines derived from higher grade tumors relative to lower grade tumors. Silencing PTPN13 in low grade BCa cell lines significantly increased Src phosphorylation at Tyr416, the activation loop of the kinase domain. These results suggest that loss of PTPN13 in higher grade BCa cells result in increased Src-activation. PTPN13 has a novel tumor suppressive function in BCa: loss of PTPN13 results in sequential Src activation, Cdh1 degradation and increased EMT and metastatic potential of BCa cells. Further defining these mechanisms may help identify new strategies to effectively control BCa progression.

#4482

MTAP regulates cell malignancy and is novel prognostic factor for kidney cancer.

Carissa Huang, Jihao Xu, Yichin Wu, Robert H. Weiss, Ching-Hsien Chen. _University of California, Davis, Davis, CA_.

Renal cell carcinoma (RCC), the 6th most common cancer in the US, is now considered a metabolic disease, which is increasing in incidence and demonstrates resistance usually within 2 years to all available therapies. Given that patients with metastatic RCC have unusually poor prognosis, it is urgent to discover potential molecules for predicting malignant changes that will lead to RCC. Herein, we have identified S-methyl- 5'-thioadenosine phosphorylase (MTAP) and its substrate methylthioadenosine (MTA) as a possible biomarker for early detection of RCC. In a screen of patients with RCC, we found that low MTAP expression is accompanied by high MTA level in RCC specimens, whereas concomitant high level of MTAP and low MTA abundance are detected in adjacent normal kidney tissues. Datasets for RCC (n=538) from The Cancer Genome Atlas (TCGA) showed that the patients with low MTAP levels have a significantly shorter overall survival as compared to the high MTAP group. Immunohistochemistry staining of normal kidney tissues confirmed an increase of MTAP protein expression compared to RCC tissues, and MTAP gene expression is inversely proportional to tumor grade. Accumulation of the metabolite MTA, a major substrate of MTAP, was observed in high-grade tumors showing high malignant potential. MTAP-knockout RCC cells displayed an elongated, spindle-like morphology with extended pseudopodial branches. Genetic manipulation of MTAP studies demonstrated that MTAP expression inhibits epithelial-mesenchymal transition, invasion and migration of RCC cells. Loss of MTAP resulted in an activation of IGF1R signaling in RCC cells. Taken together, our findings indicate a major contribution of MTAP loss to kidney cancer cell malignancy and provide a viable biomarker for tumor detections.

#4483

**Functional implications of** PRDM16 **loss in kidney cancer.**

Anirban Kundu,1 Eun-Young Kho,1 Sandeep B. Shelar,1 Hyeyoung Nam,1 Garret Brinkley,1 Shimoga Darshan,1 Yawen Tang,1 Richard Kirkman,1 David K. Crossman,1 Sooryanarayana Varambally,1 Glenn C. Rowe,1 Shi Wei,1 Phillip Buckhaults,2 Sunil Sudarshan1. 1 _University of Alabama at Birmingham, Birmingham, AL;_ 2 _University of Texas Health Sciences Center, San Antonio, TX_.

The most common genetic aberration associated with inherited or sporadic renal cell carcinoma (RCC) is the loss or nonfunctional mutations in the von Hippel-Lindau (VHL) tumor suppressor gene. The mutations lead to the stabilization of hypoxia inducible factor (HIF-1α and 2α), thereby promoting the expression of HIF target genes. HIF target genes are reported to induce tumor phenotypes by altering cellular metabolism and upregulating the expression of protumorigenic/angiogenic factors as well. We report here tissue microarray data and histology-based analyses demonstrating that the gene PRDM16 (PRD1-BF1-RIZ1 homologous domain containing 16) is silenced in many RCC tumors regardless of their VHL status. Bioinformatics analysis demonstrates a role for promoter methylation in silencing PRDM16. We investigated the role of PRDM16 in the context of VHL loss in kidney cancer. PRDM16 is known to regulate brown fat mitochondrial metabolism by inducing expressions of peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PPARGC1A) and estrogen-related receptor-gamma (ESRRG). Altered cellular metabolism, in particular, decreased mitochondrial respiration is a hallmark of RCC, which is in attributed to VHL alterations. Analysis of TCGA data demonstrates that PRDM16 expression strongly correlates with PPARGC1A expression in RCC. Ectopic expression of PRDM16 in RCC cells can induce PPARGC-1α and ESRRG mRNA expression but fails to increase mitochondrial respiration. However, ectopic expression of PRDM16 in RCC cell lines can suppress both in vitro and in vivo tumor phenotypes. These data suggest that the tumor-suppressive activity of PRDM16 may be independent from its effects on mitochondrial metabolism. RNA-seq based analysis of a VHL mutated RCC line re-expressing PRDM16 demonstrates that a transcription suppressive role of PRDM16 predominates in RCC. PRDM16 suppresses genes involved in cell migration and proliferation. In particular, PRDM16 downregulates the expression of semaphorin 5B (SEMA5B). q-PCR analysis validates high-throughput data in a panel of RCC and embryonic kidney cell lines. SEMA5B knock down significantly decreases RCC proliferation supporting the protumorigenic function of SEMA5B in RCC. Given the central role of VHL loss in renal tumor initiation, we demonstrate that the VHL/HIF axis regulates the expression of SEMA5B in RCC. Collectively, these data suggest that transcription suppressive activity of PRDM16 and VHL controls expression of the protumorigenic factor SEMA5B and that epigenetic silencing of PRDM16 thus amplifies this consequence of VHL loss.

#4484

Synthetic essentiality of chromatin remodeling factor CHD1 in PTEN-deficient cancer.

Di Zhao. _UT MD Anderson Cancer Ctr., Houston, TX_.

Background: Prostate cancer (PCa) is the second leading cause of cancer death for men in the United States. Up to 70% of primary prostate tumors show loss of heterozygosity (LOH) at the PTEN locus, and loss of PTEN is a key initiation event in PCa development. Synthetic and collateral lethality have provided conceptual frameworks to identify cancer-specific vulnerabilities. Here, we explored an approach to identify potential synthetic lethal interactions through screening mutually exclusive deletion patterns in cancer genomes.

Methods: We sought to identify 'synthetic essential' genes, which might be occasionally deleted in some cancers but almost always retained in the context of a specific tumor suppressor deficiency, and posited that such synthetic essential genes would be therapeutic targets in cancers harboring specific tumor suppressor deficiencies.

Results: In addition to known synthetic lethal interactions, this approach uncovered the chromatin helicase DNA-binding factor CHD1 as a putative synthetic essential gene in PTEN-deleted cancers. In PTEN-deleted prostate and breast cancers, the functional analysis showed that CHD1 depletion profoundly and specifically suppressed cell proliferation, survival, and tumorigenic potential. Mechanistically, functional PTEN stimulates GSK3β-mediated phosphorylation of CHD1 degron domains, which promotes CHD1 degradation via β-TrCP-mediated ubiquitination-proteasome pathway. Conversely, PTEN deficiency results in CHD1 protein stabilization, which in turn engages the H3K4me3 mark to activate transcription of the pro-tumorigenic TNFα/NF-κB gene network. In addition, we found CHD1 depletion significantly inhibits the progression of Pten-deficient prostate cancer genetic engineered mouse model.

Conclusions: Together, this study identifies CHD1 as a novel downstream effector in PTEN pathway, and verifies CHD1 as a novel therapeutic target in PTEN deficient prostate cancer and breast cancer. Additionally, this study provides a framework for the discovery of trackable targets in cancers harboring specific tumor suppressor deficiencies.

#4485

VGLL3 is a novel outlier in African American prostate tumors and its expression is associated with a tumor suppressor phenotype in prostate cancer.

Hongjun Kang. _Northwestern University, Chicago, IL_.

Higher incidence, progression and mortality of prostate cancer (PCa) have been noted in African American (AA) men compared with Caucasian (CA) men. In fact AA race is an independent predictor of biochemical recurrence for all stage of PCa (Dr. Schaeffer's data analysis, n=1634 of AA patients). The etiology of PCa racial disparities is likely multi-factorial with increasing support of biologic contributors to these differences. By analyzing large cohort of PCa, we have demonstrated that AA PCa exhibits a unique spectrum of molecular subtypes. For example, AA men presented with PCa have a significant decrease in fusion proteins such as ERG, ETS and SPINK, with an enrichment of fusion unknown (FU; triple negative) cases. Given the increased mortality of PCa in AA populations and the enrichment of FU cases among AA tumors, there is an urgent need to define and explore individual outlier as race-based modifiers of disease aggressiveness. By utilizing a large multi-institutional cohort of prostate cancer cases of mixed racial composition, we have identified novel outliers enriched in AA among FU group by COPA outlier analysis.VGLL3 is one of the top outliers enriched in AA. VGLL3 positive cases are associated with low stage (less LNI, p=0.001; SVI, p<0.001; and EPE, p=0.009) and low grade disease (less Gleason >7, p=0.012) of higher protective phenotype of adverse cancer grade and stage. This preliminary work on VGLL3 expression suggests it may be associated with a tumor suppressor-like phenotype with reductions in expression with increasingly aggressive PCa. Studies of VGLL3 in cancer are limited and the role of VGLL3 in prostate has not been explored at all. The goal of this study is to investigate the role of VGLL3 as a novel outlier in AA prostate cancer that modulates disease aggressiveness. We found that mRNA expression levels of VGLL3 were lower in prostate cancer epithelial cells than in normal epithelial cells or stromal cells. By establishing and characterizing PCa models overexpressing VGLL3 in vitro and in vivo, our data demonstrate that VGLL3 significantly reduced migration and invasion of PCa cells and inhibited primary tumor cell growth. Our preliminary data also indicate VGLL3 inhibits PCa metastasis. Furthermore, VGLL3 seems to negatively affect both hippo and AR activity in PCa cells and these might be the mechanisms that VGLL3 inhibits tumor cell growth. These data is consistent with our clinical data suggesting tumor suppressive role of VGLL3 in advanced PCa.

#4486

**Reexpression of** LSAMP **, a gene frequently deleted in African American prostate cancers, alters adhesive qualities of prostate tumor cells and inhibits Akt, ERK1/2, and β-Catenin signaling axis.**

Kevin Babcock, Inger L. Rosner, Shiv Srivastava, Albert Dobi, Shyh-Han Tan. _Center for Prostate Disease Research, Uniformed Services University and the Walter Reed National Military Medical Center, Rockville, MD_.

INTRODUCTION: Although African American (AA) men have the highest prostate cancer (CaP) incidence and mortality rates in the US, most studies of CaP genome have been performed using tumor specimens from Caucasian Americans (CA). To address the underrepresentation of AA CaP in these studies, we performed whole genome sequencing, IHC and FISH validation on tissue microarrays, and analysis of TCGA data, on a total of 438 CaPs. We noted less frequent alterations of two common CaP driver genes, PTEN and ERG, in CaP genomes of AA compared to CA men, confirming earlier observations by others and us. We reported a higher frequency of a genomic deletion of the Limbic System Associated Membrane Protein (LSAMP) gene, located on 3q13.31 locus in AA CaP, which was also associated with rapid biochemical recurrence. A recent study of CaP genome in a Chinese cohort detected LSAMP alterations at a frequency similar to that in our AA cases. LSAMP loss in other cancers have been associated with aggressive disease. We hypothesize that the loss of LSAMP contributes to CaP progression. Here, we report further biological evaluations of LSAMP modulation in CaP cells.

METHODS: The copy number and expression of LSAMP in CaP cell lines were assessed. We established stable doxycycline inducible LSAMP expression in LNCaP and in MDA PCa 2b cells (AA patient derived cell line with monoallelic LSAMP deletion), and constitutive expression in DU145 cells. We assessed the effect of LSAMP expression on cell-cell adhesion by monitoring the binding of LSAMP expressing and control DU145 cells to mouse fibroblast (NIH-3T3), normal human prostate stroma (WPMY-1), human endothelial (HUVEC), and human bone marrow stroma (HS-5) cells. We analyzed LSAMP modulated signaling pathways in the inducible and constitutively LSAMP expressing cell lines by immunoblot and immunofluorescence assays.

RESULTS: Overexpression of LSAMP resulted in attenuated cell proliferation and increased adhesion of cells to extracellular matrix proteins. LSAMP expression in DU145 cells increased their adhesion to fibroblast and prostate stroma cell lines, and concomitantly decreased their adhesion to endothelial, and bone marrow stroma cells. LSAMP expression resulted in decreased expression of FGFR2, FGFR4, and EphA3 receptor tyrosine kinases, and decreased phosphorylation of their downstream targets in Akt and ERK1/2 signaling pathways, leading to the inactivation of β-catenin. LSAMP expression also alters the levels of some Integrins.

CONCLUSION: Cell biologic features of LSAMP reported here, along with observations of genomic inactivations of LSAMP in CaP support its role in CaP progression through altered cell adhesion. These data provide the first insight into LSAMP function in CaP, corroborating with studies that suggest bona fide tumor suppressor function for LSAMP in cancers.

### Tumor-Stroma and Cell-Cell interactions

#4487

Gene expression changes elicited by crosstalk between adipocytes and breast cancer cells.

Leann A. Lovejoy,1 Nicholas S. Costantino,1 Craig D. Shriver,2 Geogre Iida,1 Rachel E. Ellsworth3. 1 _Windber Research Institute, Windber, PA;_ 2 _Murtha Cancer Center, Bethesda, MD;_ 3 _Murtha Cancer Center, Windber, PA_.

Background: No longer considered simply an inert energy depot, adipose in the breast is increasingly recognized as an active endocrine tissue. Given the increasing rates of obesity in the US, choice of breast conserving surgery and use of autologous fat grafting, it is critical to understand how signals from adipocytes affect breast tumor cells and vice versa.

Methods: Human subcutaneous preadipocytes were co-cultured for 3 days. Triple negative cell lines MDA-MB-231, SUM-149 and HCC-38 were then added and grown for a total of 10 days, with exposure to mature adipocytes for 7 days. Adipocytes and tumors cells were lysed from the plate and total RNA was isolated. Libraries were prepared for RNAseq and sequenced using a HiSeq2000. Differentially expressed genes were detected using the edge R package in R (v 3.3.3).

Results: Co-culturing breast tumor cells demonstrated 110 differentially expressed genes compared to unexposed tumor cells including 6-fold lower expression of CLCA2 and 9-fold higher expression ZBTB16 in exposed tumor cells compared to controls. Pathway analysis demonstrated upregulation of 23 pathways such in the FoxO signaling and longevity regulating pathways and downregulation of tryptophan metabolism. In co-cultured adipocytes, 89 genes, including 9-fold higher expression of WNT7B and 13-fold lower expression of ADIPOQ. Pathways such as ECM-receptor interaction were upregulated and fat digestion and absorption were downregulated in co-cultured adipocytes.

Conclusions: Exposure of adipocytes to tumor cells alters pathways involved in lipolysis and fatty acid metabolism in the adipocytes which may serve as a fuel source for tumor cells. In conjunction, exposure to adipocytes alters gene expression profiles in tumor cells associated with proliferation, migration and metastasis. These data demonstrate that proximity of breast tumor cells to adipocytes elicit significant gene expression changes in both cell types which synergistically promote tumorigenesis. The views expressed in this abstract are those of the author and do not reflect the official policy of the Department of Army, Navy, Air Force, Department of Defense, or U.S. Government. The identification of specific products, scientific instrumentation, or organization is considered an integral part of the scientific endeavor and does not constitute endorsement or implied endorsement on the part of the author, DoD, or any component agency.

#4488

Osteosarcoma cells interact with bronchial cells in lung to drive production of pro-metastatic cytokines.

John M. Hinckley, Amy Gross, Ryan Roberts. _Nationwide Childrens Hospital, Columbus, OH_.

The survival rate for patients diagnosed with osteosarcoma (OS) drops from 70% to an abysmal 20% when diagnosed with lung metastasis. We have shown that metastatic colonization of lungs by OS cells depends on the production of IL6 and CXCL8 by tumor cells, which seems to increase within the metastatic niche of the lung. Identification of key tumor-host interactions that drive this phenomenon will facilitate the development of novel therapies aimed at prevention of metastases in patients diagnosed with OS. We hypothesized that OS cells interact with lung tissues in ways that establish paracrine signal amplification loops, where IL6 and CXCL8 stimulate lung-resident cells to produce secondary signaling molecules that, in turn, further stimulate tumor cell production of IL6 and CXCL8 in a feed-forward mechanism. Co-culture experiments have identified strong interactions between OS cells and primary cultures of bronchial epithelial cells and lung smooth muscle cells. Exposure of OS cells to conditioned supernatants from the primary cell cultures show that these interactions result from exchange of soluble mediators secreted by each cell type. Proteomic profiling of bronchial cell supernatants following incubation with OS conditioned media identified a number of intriguing candidate mediators, which might facilitate the growth and survival of OS cells within the microenviroment of the lung. Some of those same candidate mediators trigger expression of IL6 and CXCL8 in OS cell cultures. These results suggest a paracrine loop mechanism, where OS cells trigger a response from lung stromal cells that promotes survival and proliferation of the OS cells, establishing a favorable niche for the development of metastasis.

#4489

Effect of cancer-associated fibroblasts on the progression of malignant melanoma cells.

Heishiro Fujikawa, Masakazu Yashiro, Takaharu Hatano, Shusaku Maeda, Hisashi Motomura. _Osaka City University, Graduate school of Medicine, Osaka-shi, Japan_.

Introduction

Malignant solid tumor is composed of not only cancer cells but also various types of stromal cells, which include fibroblasts, vascular endothelial cells, and immune cells. Currently, it has been reported that fibroblasts in tumor stroma, namely cancer-associated fibroblasts (CAFs), might affect cancer progression. Some studies suggested that CAFs have the stimulation-activity of the malignant potential of melanoma cells, but some reports mentioned their inhibitory-activity of melanoma cells. In this study, we investigated the effects of CAFs on the proliferation and invasion ability of malignant melanoma cells.

Material and Method

We used 2 malignant melanoma cell lines, MMAc and COLO679. Three CAFs were established from sebaceous carcinoma or skin malignant melanoma. Serum-free conditioned medium (CAF-CM) from CAF was prepared as follows. CAF were seeded into 100 mm plastic dishes with 10 ml of DMEM containing 2% FCS. To obtain CAF-CM, CAF were washed by PBS and then incubated for 3 days with serum-free DMEM. The conditioned medium was collected as CAF-CM. Serum-free DMEM was used as the control. CAFs were used before the 15th passage in culture. The effect of CAF-CM on the proliferation of the melanoma cells was determined by cytometry assay using Coulter Counter. The effect of CAF-CM on the motility of the melanoma cells was examined by Matrigel-invasion assay.

Result

CAF-CM significantly increased the proliferation of melanoma cell, in compared the control. Under the condition of adding 25% of CAF-CM, the number of MMAc cells were increased about 300% after 72 hours of incubation, and 105% after 120 hours of incubation that of COLO679 increased about 56% after 72 hours of incubation, 130% after 120 hours of incubation. CAF-CM significantly increased the invasion of MMAc cells. The invasion of MMAc cells were increased about 200% after 72 hours of incubation following the addiction of 25% CAF-CM. In contrast, CAF-CM did not affect on the invasion of COLO679 cells.

Conclusion

CAFs are associated with the proliferation and invasion of malignant melanoma cells.

#4490

Autocrine and paracrine effects of miR-29 in hPSCs and PDAC cells.

Tricia Factora, Elise Briscoe, Jason Kwon, Janaiah Kota. _Indiana University School of Medicine, Indianapolis, IN_.

Pancreatic ductal adenocarcinoma (PDAC) continues to be one of the most lethal malignancies worldwide and responds poorly to current therapeutic modalities. Due to its late stage diagnosis and limited response to current treatments, it is imperative to develop novel and effective therapies for PDAC. Dense fibrotic stroma associated with PDAC abrogates drug perfusion into the tumor and activated pancreatic stellate cells (PSCs) are the major stromal cells responsible for stromal reaction. Furthermore, activated PSCs are known to secrete various factors causing autocrine effects to elicit sustained PSC activation and paracrine effects on cancer cells to promote tumor progression. Our previous work has shown that loss of miR-29 is a common phenomenon of both activated PSCs and pancreatic cancer cells, and its restored expression reduces stromal accumulation and cancer growth. In addition, conditioned media from miR-29 overexpressing PSCs reduces cancer cell viability. To extend our work on the role of miR-29 in tumor-stromal interactions, we examined how autocrine and paracrine signaling is affected by miR-29 overexpression in PSCs and cancer cells. We collected the conditioned media from miR-29a overexpressing human PSCs (hPSC-CM) or cancer cells (CC-CM) and cultured nascent PSCs or cancer cells to evaluate autocrine and paracrine effects. hPSCs and PDAC cells cultured in miR-29 hPSC-CM resulted in a significant reduction of cell viability. Likewise, miR-29 overexpressing CC-CM reduced the viability of hPSCs and cancer cells. Additionally, cancer cell migration was reduced when treated with miR-29 overexpressing hPSC- and CC-CM. Taken together, our results demonstrate that conditioned media collected from miR-29 overexpressing hPSCs and cancer cells reduces hPSC/cancer cell viability and cancer cell migration. Moreover, our findings highlight the important role of miR-29 in the autocrine/paracrine interactions between PSCs and cancer cells. In our future studies, we expect to identify the key factors that are regulated by miR-29 in tumor-stromal interactions.

#4491

The mesenchymal subtype biomarkers arise from stroma rather than cancer cells in ovarian cancer.

Qing Zhang, Kimberly R. Kalli, Chen Wang, William Cliby. _Mayo Clinic, Rochester, MN_.

Background: High Grade Serous Ovarian Cancer (HGSOC) is a highly aggressive form of ovarian cancer accounting for up to 70% of all cases. The Cancer Genome Atlas (TCGA) has further defined 4 molecular subtypes within HGSOC: differentiated (DIF), immunoreactive (IMM), mesenchymal (MES), and proliferative (PRO). The MES subtype is associated with the worst prognosis and the least likely to be completely resectable when compared with other subtypes. A recent study suggested the existence of a 5-th, so-called anti-mesenchymal (ANM), subtype which is characterized by down-regulation of genes typically upregulated in the MES subtype. Importantly, previous molecular subtype studies have been performed using tumor samples consisting of both cancer cells and stroma. However, the origin of MES biomarkers in OC is still largely unknown and several studies suggest that many of the MES biomarkers could arise from cancer-associated stroma rather than cancer cells. Methods: Specimens from 20 patients (MES subtype, n=15; ANM subtype, n=5) diagnosed with HGSOC between 2010 and 2013 were obtained from Pathology Research Core in Mayo Clinic. A total of 60 FFPE blocks from 1 primary tumor and 2 metastatic sites of each patient were used to make the slides. Five MES-subtype biomarkers, including ACTA2, FAP, POSTN, COL5A1, and VCAN, were selected for immunohistochemistry (IHC) to determine the protein localization and intensities. Two researchers scored tumor sections for intensity as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong), and for localization (stroma/tumor, cytoplasm/nuclear). Statistical analysis was performed using GraphPad Prism 7 software and statistical differences were evaluated using t-test. Results: All 5 proteins evaluated exclusively localized in stroma rather than cancer cells (p<0.001 for all 5 proteins). Only FAP showed significantly different staining intensity between MES and ANM subtype of OC patient samples (p= 0.0114). When comparing primary vs. metastatic sites, only COL5A1 showed significant difference (p=0.019). Conclusion: We report that 5 biomarkers traditionally associated with the MES subtype HGSOC arise from cancer-associated stroma instead of cancer cells. Considering the potential roles of these proteins in mediating HGSOC behavior, it may suggest "stroma targeting" as an alternative therapy to "cancer targeting" to improve OC patient outcomes. Although all 5 genes were reported as MES biomarkers in mRNA-based analysis, we found that FAP was the only protein differentially expressed between MES and ANM subtype suggesting it is a better characteristic biomarker for MES subtype. COL5A1 showed significance in staining between primary and metastatic cancer which may indicates the potential role of COL5A1 in metastasis in OC.

#4492

Aggressive breast cancer cells reduce CCL5 and Nodal expression in response to a human adipose-derived stromal cell microenvironment.

Daniel Berrebi, Abhishek Yadav, Eric Lundstrom, Nicole Werwie, Dominic Muttillo, Richard E. Seftor, Elisabeth A. Seftor, Linda Vona-Davis. _West Virginia University Cancer Institute, Morgantown, WV_.

Clinical studies indicate that obesity is correlated with poor outcomes in breast cancer patients. Obese individuals have a high proportion of adipose tissue, which is abundant in stem and progenitor cells. Human adipose-derived stromal cells (hADSCs) secrete several cytokines including CCL5, an inflammatory chemokine shown to increase tumor metastatic ability in vitro. We previously identified that CCL5 is secreted into the microenvironment when hADSCs are co-cultured with breast tumor cells. When the clinical relevance of CCL5 was assessed using cBioPortal, an open-source genomics database, we found that CCL5 mRNA overexpression was correlated with increased overall survival in breast cancer patients, which counters what we and others have observed in vitro. In this study, we compared the expression of CCL5 and Nodal, a potent embryonic morphogen re-expressed by aggressive cancer cells, in either hADSC or MDA-MB-231 breast cancer cells in response to three-dimensional collagen matrices conditioned by hADSCs or breast cancer cells. After preconditioning a three-dimensional collagen matrix with either hADSCs or MDA-MB-231 cells (and following removal of the cells), the denuded three-dimensional matrices were reseeded with the opposite cell type. We found that the expression of both CCL5 and Nodal was significantly reduced in the breast cancer cells plated on the hADSC-preconditioned matrix. However, hADSCs did not express Nodal or alter CCL5 protein expression when grown on a MDA-MB-231-preconditioned matrix. MDA-MB-231 cells plated onto a hADSC-preconditioned matrix appeared more mesenchymal and stem-like, while the adipose stem cells placed into a tumor-preconditioned matrix were induced into a more aggressive cancer cell phenotype by engaging in vasculogenic mimicry. Furthermore, we found there was no change in cell proliferation of any cells grown under these different conditions. These observations suggest a relationship between hADSCs and the breast tumor microenvironment where human adipose-derived stem cells appear to inhibit the expression of CCL5 and Nodal by the aggressive tumor cells without altering tumor cell proliferation.

#4493

p190B RhoGAP modifies growth factor signaling in breast cancer in a non-cell autonomous manner.

Eduardo Castro,1 Aaron Preston,1 Mykal Quinlan,2 Peter McHenry1. 1 _Southwestern Adventist University, Keene, TX;_ 2 _Loma Linda University, Loma Linda, CA_.

Estrogen mediates the growth and differentiation of breast epithelial cells and plays an important role in breast cancer progression. The upregulation of estrogen receptors has been linked to p190B RhoGAP in vivo. p190B regulates the activity of several Rho GTPases in vitro. To better understand the molecular mechanism of p190B's role in tumor progression, we sought to develop a cell culture model that would recapitulate the effects of p190B on various growth factor signaling pathways. Protein expression of p190B was knocked down using siRNA in MCF-7 breast cancer cells cultured in the absence of steroid hormones. Upon stimulation with estrogen, cell proliferation did not significantly differ between MCF-7 cells expressing reduced p190B and those expressing normal levels of p190B, suggesting that the effects of p190B on estrogen receptor expression are not cell autonomous and that additional components of the tumor microenvironment are required to recapitulate the in vivo findings.

#4494

HMGA1 drives tumor progression and recruits cancer-associated fibroblasts in pancreatic ductal adenocarcinoma.

Shuai Shuai, Lingling Xian, Tait Huso, Karen Reddy, Linda Resar. _Johns Hopkins Univ. School of Medicine, Baltimore, MD_.

Pancreatic ductal adenocarcinomas (PDACs) are highly lethal tumors for which there are no effective therapies. Emerging evidence suggests that the tumor stroma interacts with the cancer cells to induce cancer stem cell properties and drive tumor progression. In PDAC, the fibroblast stroma also provides a dense barrier preventing cytotoxic therapy from reaching PDAC cells. We previously discovered that high levels of High Mobility Group A1 (HMGA1) protein predict decreased survival in primary PDAC. Here, we report a novel role for HMGA1-FGF19 in mediating tumor-stromal interactions and tumor progression. Silencing HMGA1 in PDAC cell lines or low-passge, patient-derived cells abruptly halts proliferation. Spindle-shaped, mesenchymal cells became reprogrammed into cuboidal, more epithelial-like cells. Sensitivity to gemcitabine was enhanced and colony formation, migration, invasion, and three-dimensional (3D) sphere formation were all disrupted in cells with HMGA1 knock-down. Silencing HMGA1 also disrupted xenograft tumorigenesis and depleted cancer stem cells/tumor-initiator cells in limiting dilution tumorigenicity assays. To elucidate underlying molecular mechanisms mediating these striking phenotypes, we performed RNA-seq after silencing HMGA1 in invasive, highly metastatic, low-passage patient-derived PDAC cells (10.7). Among the genes regulated by HMGA1 were those encoding proteins involved in tumor-stromal signaling, including the fibroblast growth factor 19 (FGF19). The FGF19 gene is highly expressed in GI tumors (liver, colon, PDAC) and transgenic mice overexpressing Fgr15 (the murine homolog) in hepatocytes develop hepatocellular carcinoma (HCC). FGF19 also correlates with poor outcomes in human HCC and colon cancer, although it's role in PDAC was unknown. Here, we found that FGF19 expression is dependent upon HMGA1 in 3 different PDAC cell lines; silencing HMGA1 represses FGF19 in these cells. HMGA1 also binds directly to the FGF19 promoter at 2 predicted DNA binding sites as assessed by chromatin immunoprecipiation. To determine whether FGF19 plays a functional role in HMGA1-mediated tumor progression and cancer stem cell properties, we silenced FGF19 in PDAC cells. Similar to our results with HMGA1, silencing FGF19 impaired PDAC growth and 3D sphere formation in vitro. Because fibroblast growth factors interact with fibroblasts, we determined whether the HMGA1-FGF19 pathway was involved in tumor cell – stromal crosstalk. PDAC 10.7 cells recruit cancer-associated fibroblasts (CAFs) in a co-culture system, although this recruitment was abrogated when HMGA1 was silenced. CAF migration was also disrupted by anti-human FGF19 neutralizing antibodies. Together, these findings indicate that HMGA1 drive tumor progression and cancer stem cell properties through FGF19 and suggest that targeting the HMGA1-FGF19 pathway maybe efficaceous in PDAC.

#4495

A CD10+GPR77+ subset of cancer-associated fibroblasts promotes cancer formation and chemoresistance by sustaining cancer stem cells.

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

Carcinoma-associated fibroblasts (CAFs) are an abundant and heterogeneous population of stromal cells in the tumor microenvironment critically involved in cancer progression. However, the lack of effective tools to isolate CAF subsets has prevented a comprehensive functional characterization of CAFs as well as their therapeutic potentials. Here, we demonstrate that two cell-surface molecules, CD10 and GPR77, specifically define a new CAF subset correlated with chemoresistance and poor survival in multiple cohorts of breast and lung cancer patients. CD10+GPR77+ CAFs promote tumor formation and chemoresistance by providing a survival niche for the cancer stem cells (CSCs). Mechanistically the functions of the CAF subset are driven by persistent NF-κB activation via p65 phosphorylation and acetylation, which is maintained by complement signaling via GPR77, a C5a receptor. Furthermore, CD10+GPR77\+ CAFs are crucial for the successful engraftment of patient-derived xenografts (PDXs) in vivo and targeting these CAFs with a neutralizing anti-GPR77 antibody abolishes tumor formation and restores tumor chemosensitivity. This discovery represents the first time a functional CAF subset is identified using specific surface markers, which will greatly facilitate future investigations of the tumor microenvironment and suggests that targeting the CD10+GPR77\+ CAF subset could be an effective therapeutic strategy against CSCs-driven solid tumors.

#4496

Myofibroblast-secreted follistatin-like 1 promotes hepatocellular carcinoma formation and metastasis.

Natalie Tsz Wai Li,1 Man Tong,1 Lei Zhou,1 Wen Ning,2 Chung-Mau Lo,1 Kwan Man,1 Terence Kin-Wah Lee,3 Stephanie Kwai-Yee Ma1. 1 _University of Hong Kong, Hong Kong, Hong Kong;_ 2 _Nankai University, Tianjin, China;_ 3 _Hong Kong Polytechnic University, Hong Kong, Hong Kong_.

Hepatocellular carcinoma (HCC) develops in the setting of fibrosis/cirrhosis, a progressive process in which chronic inflammation and hepatocellular regeneration result in the malignant transformation of hepatocytes. Enrichment of stromal myofibroblasts in this pathological stage is commonly observed, which constitutes the bulk of the tumor microenvironment and contributes to disease progression. Cancer cells are regulated by signals generated intrinsically and extrinsically from the tumor microenvironment. Targeting critical factors involved in this process may offer important therapeutic opportunities. Follistatin-like 1 (FSTL1) has been widely reported as a pro-inflammatory mediator in different fibrosis-related and inflammatory diseases. Here, we report that FSTL1 is either absent or expressed at very low levels in HCC cells, but preferentially secreted from the myofibroblasts of peri-tumor liver and HCC tissue as well as in the liver following acute injury. FSTL1 was found to co-localize and positively correlate with α-SMA, marker of myofibroblasts, in both human HCC as well as in a DEN+CCl4-induced HCC mouse model. High FSTL1 expression in myofibroblast FAP+ cells (FAP+/FSTL1high) also correlated with advanced TNM stages as compared to HCC patients in the FAP+/FSTL1low subgroup. Functional investigations revealed that FSTL1 can induce HCC cell proliferation, metastasis and sorafenib resistance; while FSTL1 attenuation by neutralizing antibody elicited the opposite effects. These observations were further validated in HCC patient-derived organoid cultures in which FSTL1 can enhance the metastatic effect of the cells. Transcriptome sequencing analysis is now currently underway to delineate the underlying molecular mechanism by which myofibroblast-secreted FSTL1 drives carcinogenesis and tumor progression in HCC.

#4497

Delineating the effects of different levels of nitric oxide produced by "M1" stimulated macrophages on tumor rejection and response to therapy.

Veena Somasundaram, Debashree Basudhar, Erika M. Palmieri, Robert Y. Cheng, Lisa A. Ridnour, Daniel W. McVicar, David A. Wink. _National Cancer Institute, Frederick, MD_.

Nitric Oxide (NO) was historically known to be immunosuppressive in cancers by inhibiting T-cell function and endothelial activation. This is relevant however only in the aberrant, dysfunctional tumor vasculature. Recently, local low dose irradiation was found to cause the differentiation of NOS2+ macrophages in the tumor microenvironment, that then normalized the vasculature and allowed T-cell infiltration, subsequent tumor rejection and prolonged survival in mouse models of pancreatic cancer (Klug et al., 2013). It was later shown that high doses of NO inhibited normal vascularization while at low doses, NO induced the expression of endothelial cell adhesion molecules and favored T-cell infiltration (Sektioglu et al., 2016). Previously, we found that murine macrophages can be activated by different cytokines/LPS to produce different levels of NO in the extracellular space (Espey et al., 2000). In the current study, we used these cytokine/LPS combinations to stimulate macrophages to produce distinct NO levels and delineate their intracellular effects. The levels of NO in bulk solution in vitro was determined using various methods for measuring NOS2 protein and various products of the NO/O2 reaction using Griess assay and nitrosation of fluorescent dyes like diamino fluorescein (DAF) and diamino naphthalene (DAN). At the single cell level, microscopy and flowcytometry were employed to assess NOS2 protein levels. Status of oxidative phosphorylation (OxPhos) and glycolytic commitment in the stimulated macrophages was studied using the Seahorse bioanalyzer. The high NOS2 induction by IFNγ/LPS followed by IFNγ/TNFα and then IFNγ/IL1β as seen in the bulk environment was spatially and temporally regulated with IFNγ/LPS inducing NOS2 in as early as 4h earlier and in more number of cells while IFNγ/TNFα starts inducing NOS2 at 8h, in fewer cells and IFNγ/IL1β only induces very low NOS2 although in all the cells. These fluxes could be directly correlated to changes in bioenergetics with IFNγ/IL1β activating OxPhos above resting macrophages while IFNγ/LPS causes glycolytic commitment. The NO levels also regulate expression of cell adhesion molecules on endothelial cells. The NOS2 induction is spatially and temporally regulated and NO fluxes were also cell type and species specific although the trend of IFNγ/LPS > IFNγ/TNFα > IFNγ/IL1β is maintained across the macrophage types analyzed. We find that these different levels have distinct effects on macrophage metabolism and endothelial activation. Hence, we now have insights into which NO flux can help improve response to cancer immunotherapy versus cause immunosuppression.

#4498

Lysyl oxidase like-2 mediates tumor to stromal cell communication in oral cancer.

Faranak Mahjour, Philip C. Trackman. _Boston University Goldman School of Dental Medicine, Boston, MA_.

Introduction: The lysyl oxidase family consists of 5 members and oxidizes specific lysine residues in biosynthetic collagen and elastin maturation. Lysyl oxidase like-2 (LOXL2) is elevated in oral cancer and promotes metastasis and correlates with poor prognosis. The objective of this study is to determine the mechanism by which LOXL2 promotes the progression and invasiveness of oral squamous cell carcinoma.

Methods: In vitro: To investigate functions of LOXL2 in oral cancer, the effects of LOXL2 inhibitor (PXS-S1C) on human gingival fibroblasts treated with tumor cell conditioned medium (CM) were investigated. Cell proliferation assays, signaling arrays and western blots were used to evaluate the effect of PXS-S1C on CM-treated fibroblasts. The effect of PXS-S1C on cancer cells expression of LOXL2 and proliferation was determined. To find potential LOXL2 substrates in gingival fibroblasts treated with CM +/- PXS-S1C, carbonyl-containing proteins were purified by affinity chromatography and identified by MS and western blot. In vivo: The effect of PXS-S1C on cancer growth and metastasis in vivo was investigated using an orthotopic oral tongue cancer mouse model in both immunodeficient and immunocompetent mice. PXS-S1C at 10 mg/kg and 30 mg/kg was injected immediately following tumor cell injections. Tumors were monitored by caliper measurements, and by in vivo imaging (IVIS). The mice were sacrificed and their organs were subjected to immunohistochemistry staining for proliferation markers.

Results: PXS-S1C significantly inhibited gingival fibroblast proliferation triggered by tumor cell CM and attenuated phosphorylation of PDGFRβ Y771 and P-PDGFRβ Y857, but not PDGFRβ Y751 in response to CM. PXS-S1C inhibited ERK1/2-signaling in fibroblasts and not AKT in response to CM. PDGFR activation by oral tumor cells was mimicked by PDGF-AB, but not PDGF-BB. PXS-S1C decreased the expression of LOXL2 by 5-fold in HSC3 oral cancer cells in vitro, suggesting a positive autoregulatory loop. Assessing for direct LOXL2 substrates in fibroblasts with functional consequences identified both PDGFR and integrin αV. Caliper measurements, IVIS, and immunohistochemistry demonstrated that inhibition of LOXL2 significantly decreases oral cancer progression and metastasis in vivo. Mice without PXS-S1C treatment developed larger tongue volumes (p<0.05), and more mice developed larger lymph nodes (3 out 12) compared to the PXS-S1C-treated mice (1 out of 12). IVIS imaging revealed inhibition of metastasis by PXS-S1C treatment. The expression of proliferation marker (Ki-67) and LOXL2 was lower in tongue tumors treated with PXS-S1C (p<0.05).

Conclusions: LOXL2 secreted by cancer cells stimulates fibroblasts by enhancing PDGFR signaling and proliferation, and by stimulating αV integrin function and cell adhesion. Inhibition of LOXL2 could provide therapeutic strategies to address oral cancer.

#4499

Determining the role of discoidin domain receptors 1 and 2 in the pathogenesis of pancreatic ductal adenocarcinoma.

Jeanine Ruggeri,1 Christopher Halbrook,1 Anjum Sohail,2 Rafael Fridman,2 Howard Crawford1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _Wayne State University, Detroit, MI_.

Pancreatic ductal adenocarcinoma (PDA) is an aggressive and devastating cancer often characterized by an intense collagen-rich, fibrotic response. In the presence of an oncogenic Kras mutation, the pancreas undergoes an initial morphological event in which normal acinar cells transdifferentiate into a ductal-like phenotype in a process called acinar-ductal metaplasia (ADM). ADM advances into pre-cancerous lesions, followed by a subsequent replacement of epithelium with a collagen-dense stromal reaction as PDA progresses. Discoidin domain receptors, DDR1 and DDR2, are a unique family of tyrosine receptor kinases that bind to collagen and activate downstream cellular responses that affect cell proliferation, migration, and adhesion. Our preliminary data shows that during Kras-induced ADM in vitro DDR1 is downregulated and is accompanied by an upregulation of DDR2 expression. Immunohistochemistry on human tissue microarrays reveals DDR1 is expressed in normal epithelia and pre-neoplastic lesions, but downregulated during advanced PDA. Conversely, DDR2 is upregulated in metaplastic lesions and advanced carcinoma as well as in the stroma at all stages of progression, suggesting DDRs may play a differential role throughout the PDA.

DDR1 is expressed in epithelial cells and associated with disease states such as fibrosis and various cancers, including PDA. To study the role of DDR1 in pancreatitis, a known risk factor of PDA, we used DDR1-null mice (DDR1-/-) with our cerulein-induced pancreatitis protocol. DDR1 ablation induces tissue damage and impairs recovery from extended cerulein treatment. To understand the role of DDR1 in tumorigenesis, we mated DDR1-/- mice in the KrasG12D/+; Ptf1aCre/+ (KC model) of pancreatic neoplasia. The absence of DDR1 in the KC model does not inhibit nor delay tumorigenesis, however, pancreata are smaller with less differentiated morphology than wild-type KC animals. In contrast, DDR2 is expressed in mesenchymal cells and has been implicated in EMT, cell proliferation, tumor invasion, and required for metastasis in breast cancer, however, its significance in PDA remains unknown. Our preliminary data shows DDR2 co-stains with neuroendocrine positive cells within neoplastic lesions that are associated with poor patient survival. To further investigate the role of DDR2 in tumor progression we have successfully mated a conditional, global DDR2 knockout mouse (DDR2fl/fl; β-actinCreERT2) with our novel KrasFSF-G12D/+;p53FRT/+ ;Ptf1a-FlpO/+ (KPF) aggressive PDA mouse model. Collectively, the results from these studies will help determine the role of DDRs throughout the initiation and progression of PDA and help define a potential point of regulation at the interface between the epithelial-stromal interactions.

#4500

BET proteins regulate hyaluronan synthases in pancreatic ductal adenocarcinoma.

Krishan Kumar, Kazumi Ebine, Thao Pham, Meng Shang, Hidayatullah G. Munshi. _Northwestern Univ., Chicago, IL_.

Background: Pancreatic ductal adenocarcinoma (PDAC) is associated with an extensive desmoplasia which is composed of multiple extracellular matrix components, including collagen, fibronectin, laminin, and hyaluronan (HA). Inhibitors targeting bromodomain and extra-terminal (BETi) proteins have emerged as potential therapeutic agents and are in clinical trials for a range of malignancies. We have previously shown that BETi attenuate fibrosis in vivo by decreasing collagen expression; however, the effect of BETi on HA has not been previously evaluated. Increased HA, which is synthesized by HA synthases (HAS), is associated with poor outcome in PDAC. Targeting HA has been postulated to provide a potential therapeutic opportunity to overcome PDAC chemo-resistance. Preclinical data have demonstrated that enzymatic degradation of HA with PEGPH20 remodels the tumor microenvironment, decreases the interstitial pressure and enhance the drug entry in tumors. Several ongoing clinical trials are evaluating PEGPH20 in combination with chemotherapies. We now report that BET inhibitors markedly decrease expression of HAS2 and HAS3, which are overexpressed in PDAC.

Purpose: The aim of this study is to investigate the role of BET proteins in HA synthesis.

Methods: mRNA expression levels HAS2 and HAS3 were determined in a panel of 7 PDAC cell lines by quantitative real-time RT-PCR. Protein expression was determined by western blotting.

Results: We found that treatment with BET inhibitors decrease the expression of HAS2 and HAS3 in all cell lines. Furthermore, siRNA mediated knockdown of individual BET proteins demonstrated that HAS2 is primarily regulated by BRD2, while HAS3 is primarily regulated by BRD4.

Conclusion: Our results demonstrate that BET proteins play a role in HA synthesis by regulating expression of HAS2 and HAS3 enzymes.

#4501

Mammary adipocytes mediate cytokine production and malignant progression of ER-positive breast cancer through NF-kB activation.

Manuel Picon Ruiz, Cynthia Morata Tarifa, Rehana Qureshi, Hyunho Yoon, Miyoung Shin, Hetakshi Kurani, Joyce M. Slingerland. _University of Miami, Miami, FL_.

Obesity increases the adverse prognosis of all breast cancer subtypes at any age, and is also associated with an increased risk of developing estrogen receptor-positive (ER+) breast cancers after menopause. However, mechanisms thereof are not fully known. We have identified one mechanism whereby obesity contributes to tumor progression. Cancers contain a subset of self-renewing stem cells that mediate treatment resistance and metastasis. We showed prolonged co-culture of in vitro differentiated adipocytes together with breast cancer lines or cultured primary dissociated human breast cancer cells increases secretion of pro-inflammatory cytokines IL6, IL8, CCL2, CCL5 and IP10. Prolonged exposure to these fat cells or to each cytokine increased the proportion of both ER+ and ER- breast cancer cells that form mammospheres and express ALDH1 activity in vitro and that can initiate primary tumors and metastasis in vivo. Differentiated adipocyte or cytokine exposures activate Src, and Src family kinase activity induced Sox2, cMyc and Nanog upregulation and miR302b induction. miR302b upregulation is Sox2-dependent, promotes cytokine-driven sphere formation, and in turn, stimulates cMYC and SOX2 expression. Finally Src was not only activated by differentiated adipocyte or cytokine exposures, it was also required to sustain cytokine induction, since Src inhibitors decreased cytokine production after co-culture. Thus, cancer cell invasion into local fat would establish feed-forward loops to activate Src, maintain pro-inflammatory cytokine production and increase tumor initiating cell abundance, tumor growth and metastasis.

It is well known that obesity mediates a chronic inflammatory state through NF-κB pathway driven pro-inflammatory cytokine expression. Our ongoing work investigates the role of NF-kB pathway in the inflammatory tumor promoting effects of obesity. We have observed that cytokine induction in breast cancer cell exposed to isolated mammary breast adipocytes is estrogen:ER dependent, since this effect in ER+ breast cancer cell is much greater than that in ER- cancer cells; and since the aromatase inhibitor, letrozole, reduced co-culture induced cytokine induction. We found NF-kB inhibitors prevent co-culture mediated cytokine induction, and that adipocyte co-culture increased RelA (p65) levels and its nuclear localization. Following invasion into local obese fat, high aromatase levels would increase local estrogen to drive cytokine induction upon mature adipocyte:cancer contact, and increase ER+ cancer stem cells and metastasis.

#4502

Cxcl-12-cxcr4 inhibition enhances immune response in pancreatic ductal adenocarcinoma.

Bharti Garg, Anthony Ferrantella, Bhuwan Giri, Shrey Modi, Vrisketan Sethi, Saba Kurtom, Sulagna Banerjee, Ramakrishnan Sundaram, Eli Gilboa, Ashok Saluja, Vikas Dudeja. _University of Miami, Miami, FL_.

Introduction: Pancreatic ductal adenocarcinoma, one of the most aggressive malignancies and is refractory to Immunotherapy. This, in part, is mediated by the fibrotic stroma, creating an immune-suppressive microenvironment. CXCL12/SDF-1 has been shown to act as a chemo-repellant in several cancers. Therefore, we evaluated the role of stromal CXCL-12 in mediating an immunosuppressive phenotype within the pancreatic tumor.

Methods: Pancreatic cancer cells harvested from KPC, a genetically engineered mouse model for PDAC, were either injected or alone or co-injected with pancreatic stellate cells (PSCs) extracted from WT (C57/BL6) mice to induce tumors. SDF-1 expression levels were detected by IF and q-RT PCR in tumor sections. An SDF-1 inhibitor, AMD3100 (2mg/kg), or vehicle was administered i.p for 21 days in tumor bearing mice and tumor growth and immune infiltration were assessed as an end point by flow cytometry. To evaluate the T cell migration in vitro, PSCs were pre-treated with SDF-1 neutralizing antibody, educated with KPC cancer cells and further co-cultured with splenic CD3+ T cells extracted from KPC mice.

Results: In vitro results showed markedly increased secretion of SDF-1 by PSCs and neutralization of SDF-1 led to increased T-cell migration when cultured with cancer cells alone. We also evaluated the effect of inhibition of SDF-CXCR4 axis on tumor growth. Co-injection of KPC with WT PSC formed larger tumors with increased section of CXCL-12 as compared to when KPC cells were injected alone. Interestingly, Inhibition of SDF-1-CXCR4 interaction with AMD3100 led to reduced tumor growth with increased infiltration of activated CD8+ cytotoxic T cells as compared to the saline-treated mice.

Conclusion: Our results indicate that stromal cells contribute to tumor progression by blocking the infiltration of cytotoxic T cells via secretion of CXCL-12. Targeting CXCL-12 could pave the way for designing better therapeutic interventions against pancreatic cancer.

#4503

The paracrine role of p53 in skin.

Tamara Terzian, Nema Sobhani, Rohan Mylavarapu, Manale El Kharbili, Andrew Parker, Colleen Little, Ethan Krauspe, Neil Box. _Univ. of Colorado Denver, Aurora, CO_.

Exposure to ultraviolet radiation (UVR) in sunlight activates both defensive and repair pathways in the skin. As the major cell type affected by UVR, keratinocytes coordinate critical aspects of the skin response. Molecularly, the transcription factor p53 is induced by UVR in keratinocytes leading to their cell death by apoptosis (peeling). p53 also stimulates the expression of paracrine melanocyte growth factors involved in tanning defined as increase in melanocyte proliferation and melanin synthesis. Here, we investigate the less defined role of p53 in tanning. For this, we use a mouse model that has high p53 levels in keratinocytes and presents with elevated melanocyte number and melanin production. Interestingly, loss of p53 in keratinocytes completely abrogates the hyperpigmentation phenotype. These data demonstrate in a powerful way the paracrine role of p53 expressed in keratinocytes on melanocyte behavior and function. Moreover, it provides an excellent tool to examine molecular and cellular changes that accompany the melanocyte response to sunburn. Sunburn increases melanoma risk in epidemiological studies. To elucidate the role of the p53 response in melanoma, we treated mice with "sunburning" doses of UVB and followed melanocyte proliferation and the expression of keratinocyte-induced melanocyte growth factors. We present our latest findings reinforcing the signs of cross-talk between keratinocytes and melanocytes which support a paracrine role for p53 expressed in keratinocyte on promoting melanocyte proliferation through the release of paracrine factors.

#4504

Functional roles of KRT6-KRT14 fusion variant 7 in desmosome remodeling and cell spreading/migration

.

Jim J. Sheu,1 Brian Y. Kuo,1 Chia-Cheng Wu,1 Jacky Yang,1 Chih-Mei Chen2. 1 _National Sun Yatsen University, Kaohsiung, Taiwan;_ 2 _China Medical University Hospital, Kaohsiung, Taiwan_.

Keratins are intermediate filament (IF) proteins with major functions in controlling/maintaining cell morphology and stiffness. Recent studies indicated possible involvement of keratin IFs in cancer developments. In our previous study, we have identified the existence of keratin fusions between KRT6 and KRT14 genes in oral squamous cell carcinoma (OSCC), and patients with the history of chewing betel nut showed higher frequency to harbor such fusions in their tumor tissues. In this study, we studied bio-functional roles of a major keratin fusion isotype K6-K14/V7, which correlated with bigger tumor size, in cell migration and invasion. We established a tet-off inducible gene expression system in OSCC cells. After gene being turned on, K6-K14/V7 formed a peripheral-dominant network that subsequently up-regulated genes related to actin filament remodeling via mechano-transduction. Actin-associated protrusion at cell leading edge was also increased by K6-K14/V7 to enhance cell migration and invasion. Interestingly, the desmosome junction was found to be dramatically reduced, leading to loss of cell-cell contact and stemness formation via epithelial-mesenchymal transition (EMT). The K6-K14/V7-expressing cells showed reduced drug sensitivity toward 5-FU and cisplatin treatment, and formed more colonies in vitro and bigger tumors in vivo. This study uncovers new functions/definitions for keratin IF in cancer development, Mechanisms revealed by this study can help us to design new strategies for new drug development and clinical treatments.

#4505

Silencing connexin 43 induces cell cycle entry and invasion in non-neoplastic mammary epithelial cells.

Sabreen F. Fostok,1 Dana Bazzoun,1 Sophie A. Lelievre,2 Mirvat El-Sibai,3 Rabih S. Talhouk1. 1 _American University of Beirut, Beirut, Lebanon;_ 2 _Purdue University, West Lafayette, IN;_ 3 _Lebanese American University, Beirut, Lebanon_.

Studies demonstrated loss of connexin 43 (Cx43) expression in breast cancer cells in-vitro and in-vivo. Our previous studies have shown that silencing Cx43 in HMT-3522 S1 cells, non-neoplastic human mammary epithelial cells (S1 cells), altered apical polarity and mitotic spindle orientation under 3-dimensional (3-D) culture conditions. The purpose of this study is to investigate the effects of Cx43 loss on proliferation and invasion of non-neoplastic breast epithelium. Cx43 shRNA-transfected and control S1 cells were cultured under 2-D and 3-D conditions. Cx43-knockdown S1 cells exhibited enhanced proliferation both in 2-D and 3-D throughout the culture period, as shown by the increased cell counts and acinar size, respectively. In addition, cell cycle entry was enhanced, as indicated by the increased percentages of cells in S and G2/M phases and the reduced percentage of cells in G0/G1 phase. Cx43-silenced S1 cells upregulated the expression of proliferation genes, c-Myc and cyclin D1, in 2-D and 3-D, but maintained β-catenin levels. Furthermore, immunoprecipitation studies showed that Cx43 associated with β-catenin and ZO-2 in 2-D and 3-D cultures of S1 cells, and co-localized with β-catenin in 3-D, suggesting assembly of gap junction complexes in S1 cells. The loss of Cx43 caused mis-localization of β-catenin in 3-D cultures of S1 cells, suggesting involvement of the Wnt/β-catenin pathway downstream of Cx43 signaling. β-Catenin primarily exhibited apico-lateral distribution in S1 cells and re-localized to basolateral membrane domains in Cx43 knockdown cells. Silencing Cx43 enhanced migration of S1 cells by 40%, as shown by time-lapse imaging. In addition, Cx43-silenced S1 cells exhibited loss of the characteristic spheroid morphology and acquired a migratory phenotype in acini. Matrigel-invasion and MMP-9 activity were also enhanced. Cx43-silenced S1 cells were capable of invading the matrigel by two-fold compared to control cells. This was concomitant with enhanced expression and activity levels of Rho GTPases (RhoA, Rac1 and Cdc42), suggesting that Cx43 regulates non-canonical Wnt signaling, as a mechanism to induce migration and invasion. We propose a role for Cx43 loss in triggering proliferation and invasion events in non-neoplastic breast epithelium downstream of disrupted Wnt signaling.

#4506

Loss of SIRT1 alters the secretome of breast cancer cells by impairing lysosomal integrity.

Arash Latifkar, Lu Ling, Xiaoyu Zhang, Claudia Fischbach, Hening Lin, Richard Cerione, Marc Antonyak. _Cornell University, Ithaca, NY_.

Sirtuin 1 (SIRT1) is an NAD-dependent deacetylase implicated in processes ranging from lifespan promotion to tumor suppression. To probe its role as a tumor suppressor, we knocked down SIRT1 in breast cancer cells. This resulted in the decreased expression of one of the subunits comprising the multi-subunit vacuolar-type H+ ATPase (V-ATPase), which is responsible for the acidification of lysosomes and thus essential for proper lysosomal protein degradation. The impairment in lysosomal function led to a marked reduction in the number of multivesicular bodies (MVBs) targeted for lysosomal degradation, and a corresponding increase in MVBs fusing with the plasma membrane to release their content. Specifically, SIRT1 reduction increased the secretion of exosomes containing unique cargo and the release of soluble hydrolases that typically reside in the lysosome, producing a breast cancer cell secretome that strongly promotes cell invasion and survival.

#4507

C-X-C motif ligand 1 from melanoma cells downregulates the invasion of their metastatic melanoma cells.

Takaharu Hatano, Masakazu Yashiro, Hisashi Motomura, Heishiro Fujikawa, Shusaku Maeda. _Osaka City Univ. Grad. School of Medicine, Osaka, Japan_.

[Introduction]The prognosis of patients with malignant melanoma is extremely poor because of the frequent metastasis. We have occasionally experienced the clinical cases that malignant melanoma patients rapidly developed distant metastases after the surgical resection of primary cancer lesion. These cases suggested that a primary tumor may control a metastatic tumor. We hypothesized that primary melanoma cells might produce a factor(s) which regulate the progression of metastatic melanoma cells. We reported that the conditioned medium from B16 cells significantly (p=0.02) suppressed the invasion ability of B16/BL6 cells, which is a subline of B16 melanoma and shows high metastatic potential to lung at the previous meeting. Then we investigated the growth and invasion interaction between primary malignant melanoma cells and high metastatic melanoma cells at the molecular level. [Materials and methods]We used two mouse melanoma cell lines, B16 and B16/BL6, that latter of which is a subline of B16 melanoma and shows high metastatic potential to lung. We investigated the interaction between the parent B16 cells and daughter B16/BL6 cells by invasion assay, cell morphology, cytokine array, RT-PCR, and gelatin-zymography. [Results]The conditioned medium (CM) from B16 cells significantly (p=0.02) inhibited the invasion ability of B16/BL6 cells. The morphology of the B16/BL6 cells was changed by the CM from bipolar in B16 cells to a multipolar shape. The B16 cells produced high levels of C-X-C motif ligand 1 (CXCL1), CXCL10, and M-CSF compared to the B16/BL6 cells. CXCL1 significantly (p=0.01) decreased the invasion ability of B16/BL6 cells, but CXCL10 and M-CSF did not. The invasion-inhibitory activity of the CM from B16 cells was significantly (p=0.046) suppressed following the addition of a neutralizing anti-CXCL1 antibody. The CM of B16 and CXCL1 increased the E-cadherin mRNA level and MMP2 activity of B16/BL6 cells. [Conclusion]Primary melanoma cells might down-regulate the invasion activity of metastatic melanoma cells through CXCL1 signaling.

#4508

Glypican-3 (GPC3) signaling pathway involved in breast cancer progression.

Dolores Fernandez, Magali Cercato, Macarena Guereño, Maria G. Peters. _Angel H. Roffo Inst. of Oncology, Buenos Aires, Argentina_.

GPC3 is a proteoglycan downregulated in breast tumors. Employing LM3 murine mammary adenocarcinoma cell line (ER-, PR-), we have previously shown that GPC3 acts as a metastasis suppressor and induces a reversion of the epithelial to mesenchymal transition (EMT). GPC3 overexpressing LM3 cells presented an inhibition of canonical Wnt and Akt pathways. On the other hand, non-canonical Wnt (JNK) and p38 signaling were activated. However, the hierarchical sequence through which GPC3 modulates those pathways has not been determined yet.The aim of this work was to study the molecular mechanism involved in the GPC3 effect on breast tumor progression, focusing on canonical Wnt pathway. We investigated the receptor complex with which GPC3 is interacting and the relationships among the pathways modulated by this glypican. We determined by IP that GPC3 could physically interact with the Wnt receptor Fz. Our results suggested that GPC3 binds to Fz and blocks the binding of the canonical Wnt factors. We also confirmed by the determination of cytoplasmic β-Catenin levels through WB and their transcriptional activity employing gene reporter assay, that GPC3 is inhibiting this pathway. We showed by qPCR array that GPC3 also modulates canonical Wnt signaling in a genomic way. Of 84 examined genes, only Wnt5b (non-canonical factor) was upregulated in LM3-GPC3 cells, 66 genes were downregulated (several Fzds, LRP5/6, DVL1 and DVL2, Frat1, Ccnd1, Ccnd2, Ccnd3, Csnk1d, jun and myc, among others), and the expression of 17 genes was not modified (including several Wnt factors). Finally, LM3-GPC3 cells were treated with SB203589, SP600125 (p38 and JNK pharmacological inhibitors respectively), LiCl (canonical Wnt activator) or transduced with a constitutively active variant of Akt1 (CA Akt). The effect of the treatments on the other pathways activity was evaluated by WB. We showed that the Akt inhibition found in LM3-GPC3 cells is required for the activation of the non-canonical Wnt pathway, as well as for the inhibition of the canonical one, but it had no effects on p38 signaling. p38 activation was necessary for non-canonical Wnt upregulation and for Akt and canonical Wnt inhibition. Canonical Wnt inhibition was essential for Akt downregulation as well as for p38 and non-canonical Wnt signaling activation. We demonstrated that in addition to the significant role of JNK in the non-canonical Wnt pathway, it also regulates canonical signalling. Non-canonical Wnt activity also participates in p38 activation but has no effect on Akt activity.In sum, we have described the receptor complex with which GPC3 interacts, as well as the canonical Wnt signaling inhibition induced by GPC3 - both genomic and non-genomic - and finally several interactions among Wnt, Akt and p38 pathways. We believe that our results provide additional information regarding the molecular mechanism of GPC3 involved in metastasis suppression.

#4509

SATB2/β-catenin/TCF-LEF pathway induces cellular transformation by generating cancer stem cells in colorectal cancer.

Yiming Ma,1 Wei Yu,1 Sharmila Shankar,2 Rakesh K. Srivastava2. 1 _Kansas City VA Medical Center, Kansas City, MO;_ 2 _LSUHSC, New Orleans, LA_.

Recent studies have demonstrated the involvement of colorectal cancer (CRC) stem cells (CSC) in transformation, cancer progression and metastasis. The main goal of this paper was to examine the molecular mechanisms by which SATB2 induced malignant transformation of colorectal epithelial cells. SATB2 induced malignant transformation and these transformed cells gained the characteristics of CSCs by expressing stem cell markers (CD44, CD133, LGR5 and DCLK1) and transcription factors (c-Myc, Nanog and Sox2). Overexpression of SATB2 in normal colorectal epithelial cells increased cell motility, migration and invasion, which were associated with an increase in N-cadherin and Zeb1, and decrease in E-cadherin expression. SATB2 overexpression also upregulated XIAP and cyclin D1, suggesting its role in cell survival and cell cycle. Furthermore, the expression of SATB2 was positively correlated with β-catenin expression in CRC. In contrary, depletion of SATB2 inhibited cell proliferation, colony formation, cell motility and expression of β-catenin, Snail, Slug, Zeb1 and N-cadherin, and upregulated E-cadherin. Furthermore, SATB2 silencing inhibited the expression of stem cell markers, pluripotency maintaining transcription factors, cell cycle and cell proliferation/survival genes and TCF/LEF targets. Finally, β-catenin/TCF-LEF pathway mediated the biological effects of SATB2 in CSCs. These studies support the role of SATB2/β-catenin/TCF-LEF pathway in transformation and carcinogenesis.

#4510

SHH and GATA interplay: A potential therapeutic target for prostate cancer.

Rami Z. Morsi, Wassim Abou Kheir, Layal El-Khatib, Hiba Msheik, Georges Daoud. _American University of Beirut, Beirut, Lebanon_.

Background: Prostate cancer is the second leading cause of cancer deaths in males worldwide. Typically, prostate cancer cells depend on androgens, and many therapeutic strategies target receptors involved in androgen regulation. However, certain castration-resistant tumors of the prostate are resistant to androgen deprivation therapies. Moreover, advanced-stage prostate tumors can proliferate uncontrollably either due to androgens or due to alteration in other signaling pathways. For instance, the activity of the sonic hedgehog (SHH) pathway, which usually decreases during post-natal development, is increased during prostate cancer progression. Of interest, a recent study reported a novel role for GATA transcription factors as potential repressors of SHH signaling. Based on the above-mentioned findings, we hypothesize that the balance between GATA and SHH pathways is perturbed in prostate cancer leading to overactivation of SHH pathways and its downstream effector proteins. Methods: We screened human prostate cancer cell lines, DU145, 22Rv1, and PC3, as well as a human immortalized normal prostate epithelial cell line, RWPE-1, for baseline levels of expression of SHH downstream effector proteins via real-time PCR using specific primers. Subsequently, we treated the cell lines with bone morphogenetic protein 4 (BMP4)-conditioned media in order to induce the expression of GATA transcription factors, and then we measured the levels of expression for each of the SHH pathway effector proteins by real-time PCR. Results: We demonstrated that GATA can interfere with the expression of SHH and its effector proteins in the human prostate cancer cell lines. Upon treatment with BMP4, we noted that there was a decrease in expression of Gli1, Gli2, and Gli3, downstream effectors of SHH, suggesting an anti-proliferative response. Additionally, Patched1 expression was also attenuated after treatment with BMP4. Similarly, Sox9 expression was also abrogated. Conclusion: Our results demonstrate that GATA transcription factors can act as potential repressors of the SHH pathway in the context of prostate cancer in vitro by interfering with the expression of SHH downstream effector proteins.

#4511

The role of Wnt5a signaling pathway in epithelial ovarian cancer progression.

Marwa Asem,1 Allison Young,1 Carlysa Oyama,1 Rebecca Burkhalter,2 Steven Buechler,1 Daniel Miller,3 Sharon Stack1. 1 _University of Notre Dame, South Bend, IN;_ 2 _University of Kansas Medical Center, KS;_ 3 _The Johns Hopkins University School of Medicine, MD_.

Ovarian cancer (OvCa) is the most fatal gynecologic malignancy and the 5th leading cause of cancer death among U.S. women. The majority of women with OvCa (75%) have a very low survival rate (30%), as OvCa is usually diagnosed in late stages after development of intraperitoneal metastasis. Thus, it is indispensable to understand the molecular mechanisms that contribute to OvCa metastatic success, in order to design effective treatment strategies to improve the overall survival of women with OvCa. Wnt5a is a noncanonical Wnt ligand that binds to several cell membrane receptors and activates many downstream signaling pathways that are fundamental for normal developmental processes during embryogenesis. In the past decade, the aberrant activation or inhibition of Wnt5a signaling is emerging as an important event in cancer progression, exerting both oncogenic and tumor-suppressive effects. The role of Wnt5a in OvCa is controversial, as studies report conflicting data. In addition, mechanistic data regarding the contribution of Wnt5a to OvCa progression are largely unavailable. The main aim of this research is to obtain a molecular-level understanding of Wnt5a signaling in OvCa and to investigate its potential roles in influencing OvCa metastatic success. Our data show Wnt5a is prevalent in ascites samples from women in different stages with OvCa, suggesting a role for Wnt5a in promoting disease progression. Data obtained from TCGA (n=583) show high expression of Wnt5a in primary ovarian tumors. Furthermore, Wnt5a enhanced OvCa cells adhesion, migration and invasion in a panel of organotypic and ex vivo functional assays. This was combined with striking morphologic changes characteristic of an invasive phenotype in OvCa cells treated with recombinant Wnt5a protein and formation of tunneling nanotubes (TNT). Overall, our data suggest that Wnt5a plays an oncogenic role in epithelial ovarian cancer cells. More experiments exploring Wnt5a activated pathways and effects in epithelial ovarian cancer cells are under way.

#4512

Doublecortin-like kinase 1 (DCLK1) correlates with Notch pathway signaling, controls metastatic characteristics in head and neck cancer.

Esther Channah Broner,1 Tejaswini Subbannayya,2 Jayshree Advani,2 Alex Zhavoronkov,3 Artem Artemov,3 Ivan Ozerov,3 Ido Sloma,4 Harsha Gowda,2 David Sidransky,1 Eugene Izumchenko,1 Aditi Chatterjee2. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _Institute of Bioinformatics, Bangalore, India;_ 3 _Insilico Medicine, Inc, ETC, Johns Hopkins University, Baltimore, MD;_ 4 _Champions Oncology, Baltimore, MD_.

Despite advancements in the field, the 5 year survival rate of Head and Neck Squamous Cell Carcinoma (HNSCC) still hovers at 60%. In a quest for novel therapy targets for this disease, we performed an extensive and comprehensive quantitative phosphoproteomics scan on 10 HNSCC patient derived xenografts (PDXs) in order to find dysregulated phosphoproteins. DCLK1 is hyperphosphorylated but not over expressed in most of the HNSCC derived PDXs. DCLK1 has been shown to regulate epithelial-to-mesenchymal transition (EMT) as well as serving as a cancer stem cell marker in colon and pancreas cancer. DCLK1 is also associated with bad prognosis in colon, renal and oropharyngeal cancers. We performed a comprehensive transcriptome-based computational analysis on hundreds of HNSCC patients from TCGA and GEO databases. While our results confirm the role of DCLK1 in regulating EMT and focal adhesion kinase (FAK) mediated integrin signaling, we found that high DCLK1 expression also correlates with NOTCH pathway signaling signatures in HNSCC. We have selected 5 cell HNSCC cell lines (JHU-011, JHU-022, JHU-029, FaDu and Cal29) that express DCLK1, and inhibited DCLK1 with LRRK2-in1 inhibitor and siRNA. DCLK1 inhibition resulted in substantially decreased proliferation, migration, and colony formation. Furthermore, these effects paralleled downregulation NOTCH1 signaling in all cell lines tested. Overall, our results demonstrate the novel role of DCLK1 in regulating the NOTCH signaling network and suggest its potential as a therapeutic target in HNSCC.

#4513

Notch inhibitors relieve growth suppression by TGF-β and promote growth and metastasis of triple-negative breast cancers.

Pothana Saikumar, Prajjal K. Singha, Srilakshmi Pandeswara, Manjeri A. Venkatachalam. _UT Health Science Ctr. at San Antonio, San Antonio, TX_.

The paradoxical behavior of Notch1 and transforming growth factor-beta (TGF-β) is puzzling as they were shown to have both tumor-suppressive and tumor-promoting activities. We addressed the interaction between these two signaling pathways in cancer development and progression in a systematic way. Towards this goal, we utilized primary human mammary epithelial cells (HMEC) and various triple-negative breast cancer (TNBC) cells and examined the molecular crosstalk between TGF-β1 and Notch signaling pathways using biochemical, molecular and cell biology techniques. First, we observed lower expression of Notch1 in most of the TNBC cells compared to normal cells. Most of these TNBC cells also overexpressed TMEPAI (transmembrane prostate androgen induced), which we previously showed switches TGF-β from tumor-suppressive to a tumor-promoting role in breast cancer. Surprisingly, treatment of TNBC with Notch inhibitors increased their growth rate both in the absence and presence of TGF-β, while Notch inhibitors relieved normal cells from TGF-β mediated growth suppression. Both normal and TNBC cells showed increased growth in the presence of Notch inhibitors with decreased TGF-β signaling activity. In fact, normal cells that express very low levels of TMEPAI both at mRNA and protein levels, overexpressed TMEPAI in presence of various Notch inhibitors, suggesting that Notch1 negatively regulates TMEPAI expression. Correspondingly, knockdown of TMEPAI in TNBC cell lines showed increased expression of Notch1 and Jagged 1, suggesting a negative regulation of Notch1 by TMEPAI. In fact, TMEPAI, which binds to E3 ligases, promoted increased turnover of Notch1. In contrast to notch inhibitors, overexpression of active Notch1 in MDA-MB-231 cells inhibited tumor cell growth both in vitro and in vivo. Our results clearly suggest that a crosstalk between TGF-β and Notch1 signaling exists through TMEPAI. Importantly, these studies should be taken into consideration while contemplating the use of Notch inhibitors in triple-negative breast cancer therapy.

#4514

ASR458, a small molecule, targets Notch1 signaling in advanced colorectal cancer.

Balaji Chandrasekaran,1 Deeksha Pal,1 Ashish Tyagi,1 Srinivasa R Ramisetti,2 Arun K Sharma,2 Murali Ankem,1 chendil Damodaran1. 1 _University of Louisville, Louisville, KY;_ 2 _Pennsylvania State University, Hershey, PA_.

Cancer-related mortality rates are amongst the highest in colorectal cancer (CRC). Previously we have reported that Withaferin A (WA), a natural compound, induces apoptosis and inhibits AKT mediated pro-survival signaling resulting in growth inhibition both in vitro and in vivo models of colon cancer. Owing to the encouraging results with WA, we have developed a series of acetylated pro-drug conjugates of WA and our initial structure-activity relationship (SAR) studies led to the identification of a novel small molecule ASR-458 as the lead compound. Interestingly, ASR-458 has demonstrated better efficacy by inhibiting colon cancer cell growth at nano-molar concentration, even in AKT overexpressing (AKT/HCT) HCT-116 cells (IC50: 750nm). Induction of apoptosis was seen in both control vector and AKT overexpressed colon cancer cell lines. Induction of caspase mediated signaling confirmed the pro-apoptotic function of ASR-458 in colon cancer cells. Inhibition of pAKT (ser473) resulted in downregulation of Notch1 downstream signaling pathways (Presenilin 1, NFkB activation and BCl-2 expression) in AKT/HCT colon cancer cells. Epithelial to mesenchymal transition (EMT) is a major hallmark of metastatic colon cancer. ASR-458 also downregulated the expression of EMT markers snail as well as β-Catenin and significantly impaired invasive as well as the migratory efficiency of AKT/HCT cells. Our ongoing xenograft studies will validate the in vivo efficacy of ASR-458 (1mg/kg/BW by IP weekly 2 times for six weeks) will confirm the chemotherapeutic effect of the compound against colon cancer cells. Overall these results emphasize ASR-458 to be a promising small molecule as compared to WA. ASR-458 targets both AKT and Notch1 signaling pathways which remains an important target for the development of effective treatment of metastatic stage of CRC.

#4515

Lgr5 mediates positive B-cell selection and is critical for initiation and survival of B-cell malignancies.

Kadriye Nehir Cosgun,1 Anna Hecht,1 Xin Yang,2 Maurizio Mangolini,,3 Ali Aghajanirefah,1 Gang Xiao,1 Teresa Sadras,1 Zhengshan Chen,1 Lars Klemm,1 Huimin Geng,4 Chao Hong,4 Qingxiao Song,1 Deye Zeng,1 Hassan Jumaa,5 Defu Zeng,1 Hans Clevers,6 Markus Muschen1. 1 _Beckman Research Institute City of Hope, CA;_ 2 _University of California, CA;_ 3 _Cambridge Institute for Medical Research, Cambridge, United Kingdom;_ 4 _University of California, USA, CA;_ 5 _Ulm University, Ulm, Germany;_ 6 _Hubrecht Institute, Utrecht, Netherlands_.

Introduction: In B-cell leukemia and lymphoma, leukemia initiating cells occur at a high frequency (Rehe 2013), are phenotypically diverse (Aoki 2015) and can arise from any stage of B-cell development (Le Viseur 2008). Unlike stem cells, where self-renewal is regulated by a developmental hierarchy, in the B-cell lineage, positive selection events, i.e. induced by antigen-receptor (BCR) signaling dictates their ability to self-renew. Leucine-rich repeat containing G-protein coupled receptor 5 (Lgr5) is a Wnt target gene and through binding to its ligand R-spondin, Lgr5 modulates Wnt signaling strength. Lgr5 is widely used as stem cell marker in multiple epithelial tissues, however the role of Lgr5 in hematopoietic cells was not explored. Results: Upon successful completion of immunoglobulin V(D)J gene recombination and first encounter of antigen represent key events in the life of a B-cell that promote survival and positive selection. Here, we found that both events result in upregulation of Lgr5 expression in B cell precursors in the bone marrow and germinal center B cells. Likewise, engagement of BCR signaling on B-cell lymphomas and oncogenic BCR-signaling mimics in leukemia strongly increased LGR5 expression, which was sensitive to inhibition of SYK and BTK kinases in the BCR pathway.In patients with B-cell leukemia, higher than median mRNA levels of LGR5 at the time of diagnosis were associated with poor clinical outcome and higher likelihood of drug-resistance and relapse. Inducible ablation of Lgr5 during earliest stages of B-cell development resulted in a >100-fold reduction of absolute B-cell numbers. Studies in epithelial cells suggest a role of Lgr5 as potentiator of WNT-signaling. However, deletion of Lgr5 in B cells caused cell death in parallel with massive accumulation of nuclear β-catenin and increased expression of β -catenin target genes. Deletion of Lgr5 abolished colony forming capacity and reduced the ability of leukemia cells to initiate fatal disease in transplant recipients. Likewise, inducible activation of a gain-of-function mutant of β-catenin resulted in rapid cell death in normal and malignant B cells. Conclusion: Lgr5-expression and positive B-cell selection is induced by BCR-engagement by antigen or oncogenic mimics of BCR signaling in B-cell malignancies (e.g. transforming oncogenes that engage the BCR pathway). Unlike in epithelial cells, LGR5 expression in B cells restricts the levels of nuclear β-catenin and enables B-cell survival and transformation through negative regulation of Wnt-signaling. Targeting Lgr5 using a novel Lgr5-ADC seems promising to deplete B-cell leukemia- and lymphoma-initiating cells.

## CLINICAL RESEARCH:

### Diagnostic Biomarkers

#4516

A new MLH1 rabbit monoclonal antibody for immunohistochemical application.

Sean Cobb, Hongyang Pan, Qiong Yang, Jackie Chan, Julia Wu, Jason Law, Aihua Li. _Abcam, Burlingame, CA_.

Background: MLH1, a DNA mismatch repair gene, plays a critical role in colorectal cancer (CRC) development. Loss of nuclear MLH1 is attributed to hypermethylation of its promoter, occurring in 78% of microsatellite instability (MSI) which composes 15% of all CRC. Immunohistochemical (IHC) analysis of MLH1 in CRC is routinely performed in pathology laboratories, but remains challenging with the current selection of available MLH1 antibodies. Here we report a new MLH1 rabbit monoclonal antibody (RabMAb) developed with RabMAb® technology, designated as clone EP481.

Design: Sera collected from rabbits immunized with recombinant MLH1 protein were screened by ELISA, WB, and IHC. After fusion, antibody from a final hybridoma cell line was further characterized by extensive IHC testing using formalin-fixed paraffin-embedded human normal and tumor tissue microarrays (TMAs) comprised of 99 CRCs. EP481 performance was compared against MLH1 mouse monoclonal antibodies, G168-15, G168-728, and the current benchmark, ES05. Cases without tumor cell staining were considered negative; those also lacking stroma staining were excluded, as it could not be determined if there was germline mutation or poor tissue quality. Agreement was evaluated with Cohen's kappa.

Results: An 85 kDa MLH1 protein was detected by WB in SW480 cell lysate, but absent in the MLH1-negative cell line, HCT116. IHC analysis of EP481 was positive in normal epithelium and surrounding stroma, but negative in 10% of tumors. G168-728 showed nonspecific cytoplasmic staining and deviated from its counterparts. Concordance data (Table 1) demonstrates that EP481 is equivalent with ES05 and incongruous with G168-15.

Conclusion: The RabMAb MLH1 antibody, clone EP481, specifically labels normal cells and is absent in 10% of CRCs. This finding is consistent with both literature and the gold-standard anti-mouse clone, ES05. EP481 is a new alternative for MLH1 IHC and a promising tool to identify MSI in CRC.

Table 1. Concordance staining of MLH1 clone EP481 with clones ES05 and 168-15 on CRC TMA.

---

|

ES05 + | ES05 - | Total | 168-15 + | 168-15 - | Total

EP481 + | 74 | 1 | 75 | 66 | 9 | 75

EP481 - | 1 | 7 | 8 | 2 | 6 | 8

Total | 75 | 8 | 83 | 68 | 15 | 83

Cohen's kappa

Raters | Coefficient | Agreement

EP481/ES05 | 86% | Excellent

EP481/168-15 | 45% | Moderate

#4517

Characterization of a standardized immunohistochemical assay for detecting PD-L1 expression in non-small cell lung cancer using anti-PD-L1 clone 73-10.

Darlene Krohn, Mai Nguyen, Marko Srdanov, Christina Samathanam, Peng Duan, Monika Vilardo, Alexander Prenta, Sharika Vasudevan, Landry Nicholson-Legg, Debra Hanks, Aaron R. Ellison. _Dako North America, Inc., Carpinteria, CA_.

Background: Recent data have demonstrated the clinical efficacy of anti-PD-1/PD-L1 checkpoint inhibitors in non-small cell lung cancer (NSCLC), and approvals by regulatory authorities in first-line and second-line settings have followed. In selected treatment scenarios, expression of PD-L1 by immunohistochemistry (IHC) is considered predictive of patient outcome. A recent comparative study using the rabbit anti-PD-L1 monoclonal antibody clone 73-10 and other commercialized IHC assays demonstrated strong intra-assay reliability in NSCLC tumor cell scoring and inter-assay differences in the number of tumor cells detected by each assay (Blueprint 2 data presented at IASLC 2017). Here we report precision around a ≥ 1% tumor cell cutoff and PD-L1 expression in 428 procured NSCLC tissues using an immunohistochemical assay developed with this antibody clone.

Methods: PD-L1 expression was assessed using a standardized PD-L1 IHC assay. Scoring of tumor specimens was performed on a 0 - 3+ intensity scale, with membrane staining in ≥ 1% of tumor cells at ≥ 1+ intensity defining PD-L1 positivity. Assay precision around this cutoff was assessed in 28 specimens. PD-L1 expression was evaluated in 428 Stage IIIA, IIIB, and IV procured NSCLC tissues. Assay specificity was examined in 30 normal tissues.

Results: The PD-L1 IHC 73-10 assay showed > 95% negative, positive, and overall agreement in inter-day, inter-operator, intra-run, inter-instrument, and inter-lot conditions around the ≥ 1% tumor cell cutoff. The lower bound of the 95% confidence interval was > 90% for all agreement types and precision conditions. PD-L1 protein localization in normal tissues was consistent with known patterns. The 428 procured NSCLC samples had primarily squamous cell (39.5%), adenocarcinoma (43.7%) and a mixture of other less common (16.8%) histologic subtypes; 397 (92.8%) of samples were from primary and 31 (7.2%) were from metastatic tumors. Crisp membrane staining was observed in tumor cells. Other labeled cell types included macrophages, lymphocytes, endothelial cells, and fibroblasts that exhibited membrane and/or cytoplasmic staining. Of the 428 procured specimens, 270 (63.0%) were PD-L1-positive based on the ≥ 1% cutoff. Tissues of each NSCLC stage expressed PD-L1 in the full dynamic range from 0% - 100% positive.

Conclusions: These data provide insight into the prevalence of PD-L1 expression in procured NSCLC specimens when stained with PD-L1 IHC 73-10, and demonstrate that this assay is precise around a ≥ 1% tumor cell cutoff while exhibiting expected antigen specificity. PD-L1 positivity in the tumor stages examined using this assay is greater than in the literature, where prevalence using different antibodies has been reported at less than 50% in various stages.

#4518

Prevalence of high-grade prostatic adenocarcinoma in a black African population.

Emeka F. Nnakenyi, Ifeyinwa D. Nnakenyi, Okechukwu C. Okafor, Fred O. Ugwumba. _University of Nigeria Teaching Hospital, Enugu, Nigeria_.

INTRODUCTION:

Prostate cancer is the second most common type of cancer in men worldwide. Despite this, screening is currently disputed because studies in the western world have shown that more men die with the disease than from it. Black men have been documented to have a greater prevalence of prostate cancer which runs an aggressive course. Therefore, we aimed to determine the prevalence of high grade prostatic adenocarcinoma among Nigerian men.

METHOD:

This was a retrospective study of Nigerian men attending the urology clinic at University of Nigeria Teaching Hospital, Enugu, south-east Nigeria. Data on prostate histology were retrieved from the histopathology laboratory records for the last 5 years as thus: age, prostate diagnosis, gleason score. Histologic slides from core-needle biopsies had been reviewed by two Pathologists independently. If adenocarcinoma was diagnosed, Gleason system was used to grade the tumors, and a consensus was reached by both Pathologists. The data were analyzed with SPSS statistical software.

RESULTS:

Three hundred and forty-five (345) requests for histology on prostate biopsies were received, of which 118 (34.2%) were diagnosed with prostatic adenocarcinoma (PCa) at a mean age(sd) of 69(8.4) years. Minimum and maximum gleason scoring observed were 4 and 10.

|

---|---

Gleason system | No. of prostatic adenocarcinoma (%)

Gleason score 2 – 6 (well differentiated PCa with excellent prognosis) | 22 (18.6%)

Gleason score 7 (3+4 moderately differentiated PCa) | 19 (16.1%)

Gleason score 7 (4+3 moderate to poorly differentiated PCa) | 11 (9.4%)

Gleason score 8-10 (poorly differentiated with aggressive biologies) | 66 (55.9%)

CONCLUSION:

A high prevalence of high grade PCa was observed in this study, which are less likely to be cured. Therefore, early detection is crucial to prevent mortality. This may justify the need for prostate cancer screening in our African environment.

#4519

Preliminary data of a stool test for lung cancer from a prospective study.

Martin Tobi,1 Yosef Y. Tobi,2 Fadi Antaki,3 MaryAnn Rambus,3 Michael Lawson4. 1 _Saginaw VA Medical Ctr., Saginaw, MI;_ 2 _Detroit, Detroit, MI;_ 3 _Detroit VA Medical Ctr., Detroit, MI;_ 4 _Kaiser Permanente Medical Center, Sacramento, CA_.

Lung cancer is the biggest cancer killer Worldwide with a limited prognosis despite personalized treatment regimens. Low-dose CT scanning as a means to early diagnosis has been disappointing due to the high false positive rate. Other non-invasive means of testing need to be developed that offer both timely diagnosis and predict prognosis. Methods: In the course of stool testing in a large scale testing of patients at increased risk of CRC we were able to ascertain 55 patients documented to have prospectively been diagnosed with lung cancer. Stool and colonic effluents were tested for anti-adenoma antibody (Adnab-9) reactivity by ELISA and Western blot. Survival data were obtained where available. Results: Of 55 cancers 40% were squamous (SSC), 23.7 were adenocarcinoma, 14.6 small, 12.7 large cell and 9% were designated NSCLC. 49.1% of the lung cancer patients had adnab-9 testing. Overall 53.6% (14 of 26) had positive testing compared to 2 of 11 controls (18.2%) which bordered on significance (OR5(CI1-29)). Cancers with higher lethality were more likely to test positive (67% for both small and large cell lung cancers, 50% SCC and adenocarcinoma, 33% NSCLC). In the larger groups overall survival was worse in those testing positive (474 versus 844 days in SCC and 54 versus 749 days in adenocarcinoma patients). Most importantly the time from a positive test to the clinical diagnosis ranged from 2.72 years for small cell, 3.13 for adenocarcinoma, 5.07 for NSCLC, 6.07 for SSC, and 6.24 for large cell cancer). In excluded cases where lung cancer was believed to be metastatic 75% of cancers were positive. Conclusions: At a projected real world sensitivity of 0.53 and specificity of 0.82 and the ability to predate diagnosis by up to 4.7 years overall, this test could help direct lung cancer screening. In addition, the Adnab-9 testing selectively detects worse tumor types (67%) and those with worse prognosis amongst the more common, favorable phenotypes thus making early diagnosis possible in those patients who stand to benefit most from this strategy. Metastatic lung cancer also detected by the test should be identified by the follow-up imaging studies and therefore would not be considered to be a major pitfall.

#4520

Using SEREX approach to screen and identify the tumor-associated antigens in non-small cell lung cancer.

Lu Pei,1 Tingting Wang,2 Peng Wang,3 Hongchun Liu,4 Songyun Ouyang,2 Jianying Zhang,1 Liping Dai1. 1 _Zhengzhou University, Zhengzhou, Henan Province, China;_ 2 _First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, China;_ 3 _Zhengzhou Univ., Zhengzhou, Henan Province, China;_ 4 _Second Affiliated Hospital of Henan University of Traditional Chinese Medicine, Zhengzhou, Henan Province, China_.

Background: Lung cancer is the most common cause of cancer-related deaths worldwide and approximately 85% of lung cancer cases are non-small-cell lung cancer (NSCLC). The average survival and 5-year survival rates of NSCLC patients still remain poor. Autoantibodies against tumor-associated antigens (TAAs) have been shown to be relevant tumor markers. There is an urgent need to better understand the mechanism and to identify useful biomarkers for early immunodiagnosis of NSCLC.

Materials and methods: Serological analysis of recombinant tumor cDNA expression libraries (SEREX) was applied to screen the lung cancer tumor-associated antigens (TAAs) with sera from lung cancer patients. Oncomine online database was used to analyze multiple microarray datasets and to identify potential genes in NSCLC progression. Autoantibody responses to TAAs were evaluated by enzyme-linked immunosorbent assay (ELISA) in sera from 196 patients with NSCLC and 196 normal controls.

Result: Seventy positive clones had been screened by SEREX approach and 35 genes with known biological function were further identified by mass spectrometry analysis. Ten out of 35 genes were determined to be tested the corresponding autoantibodies since they were consistently over-expressed in NSCLC tissue in three mRNA arrays. The results of ELISA show that the level and positive frequency of the autoantibodies to TOP2A,ACTR3, RPS6KA5 and PSIP1 in sera from NSCLC patients were significantly higher than that in normal control (Ρ<0.05). The sensitivity of the four autoantibodies for diagnosis of NSCLC ranged from 17.4% to 32.1% with the specificity of 90%. The area under curve(AUC)of TOP2A,ACTR3, RPS6KA5 and PSIP1 was 0.758, 0.787, 0.707 and 0.668, respectively. The sensitivity of the panel of this four TAAs was 47.2% and the specificity was 81.5%.

Conclusions: TOP2A, ACTR3, PSIP1, RPS6KA5 were tumor-associated antigens and their autoantibodies might be potential biomarkers for immunodiagnosis of NSCLC.

#4521

Flow cytometric measurement of increased erythrocyte bound IgG: A potential screening method for colorectal cancer.

Akihito Kitao,1 Shinichiro Kawamoto,1 Keiji Kurata,1 Ikuyo Hayakawa,1 Hiroshi Matsuoka,2 Yasuo Sumi,2 Yoshihiro Kakeji,2 Toyomi Kamesaki,3 Hironobu Minami2. 1 _Kobe University Hospital, Kobe, Japan;_ 2 _Kobe University Hospital and Graduate School of Medicne, Kobe, Japan;_ 3 _Jichi Medical University, Shimotsuke, Japan_.

Introduction: Colorectal cancer (CRC) incidence is increasing worldwide. While screening for CRC has led to early detection, thus reducing mortality, checking for fecal occult blood, as the accepted screening method for CRC worldwide, has some shortcomings including false-negative results in early disease stages and/or advanced CRC of the right ascending colon. We have previously reported on the ectopic expression of Band3 (encoded by SLC4A1), an antigen abundant in the cytoplasmic membrane of erythrocytes, in a case of CRC with autoimmune hemolytic anemia (AIHA), and suggested that Band3 expression in CRC might stimulate production of the autoantibody that binds to the erythrocyte membrane. Besides, the amount of erythrocyte-bound IgG may be higher in CRC patients compared with healthy people, even though no symptoms or signs of AIHA are seen. We conducted a prospective observational study to compare the amount of erythrocyte-bound IgG, including anti-Band 3 antibody, between CRC patients and healthy controls.

Patients and Methods: From October 2016 to November 2017, 30 CRC patients and six healthy controls were enrolled. Direct Coombs test was carried out, using test tube and column methods, immediately after blood collection. Cell suspensions were incubated with either anti-human IgG antibody or a control isotype conjugated with FITC. Fluorescence was measured with a flow cytometer. Mean fluorescence intensity (MFI) was calculated as the difference in fluorescence intensity between anti-human IgG and control isotypes. Total serum IgG was collected using protein G sepharose. The amount of serum erythrocyte-bound IgG was quantified by ELISA using erythrocyte membrane protein coated 96-well plates and anti-human IgG antibody. Erythrocyte membrane protein was immunoprecipitated and subjected to immunoblotting using anti-Band3 IgG to confirm its proportion to total serum IgG.

Results: Clinical AIHA was not observed in any patient, but a direct Coombs test was positive in eight patients (one on the test tube method, five on the column method, and two patients on both methods). The median±SD MFI was 40±17.4 in patients with CRC and 23±11.6 in controls (p=0.005). Moreover, with the MFI cutoff value set at 31, ROC curve analysis showed the sensitivity, specificity, and area under the curve to be 100%, 70% and 0.867, respectively. In CRC patients, the median MFI was not affected by positive or negative results for fecal occult blood, involvement of the left or right colon, and/or disease stage. The serum erythrocyte-bound IgG and the proportion of anti-Band3 to total serum IgG was higher in CRC patients than in controls.

Conclusion: Whether flow cytometric measurement of total erythrocyte-bound IgG, including anti-Band 3 antibody, in CRC patients can be a useful screening tool warrants further evaluation in a larger cohort.

#4522

An optimized NGS workflow for detection of FLT3 internal tandem duplication (ITD) in AML samples.

Xiaodong Wang,1 Zhenyu Yan,1 Peng Fang,1 Weihua Liu,1 Scott Glynn,1 Jennifer Biroschak,1 Chad Galderisi,2 Cindy Spittle,2 Jin Li1. 1 _MolecularMD Corp., Cambridge, MA;_ 2 _MolecularMD Corp., Portland, OR_.

FLT3 ITD, a prognostic marker and drug target, is present in ~19% of AML patients. FLT3 ITD is a tandem repeat of the entire or partial exon 13-15 region. FLT3 ITDs range in size from 3bp to above 300bp and impact RTK signaling. Recent publications have shown that FLT3 ITD can be detected at single nucleotide resolution by NGS panels. However, detection of large FLT3 ITD and accurate reporting of ITD frequency remain challenging for NGS methods. False negative ITD results or inaccurate mutant/wild type allele ratio measurements could negatively alter treatment decisions for AML patients. The Illumina TruSight Myeloid panel (TMP) is a commercial NGS assay that covers 54 genes involved in myeloid malignancies. Although the TMP assay can detect SBS and small indel, medium to large size FLT3 ITD cannot be detected. Here we present an enhanced workflow to overcome these challenges. Positive controls, dynamic range, accuracy, and inter-run reproducibility were validated for the detection of SBS, indel, and FLT3 ITD using reference standards and DNA extracted from cell lines and AML patient EDTA blood and bone marrow aspirate samples. NGS libraries were prepared following Illumina's standard protocol. 275bp X2 sequencing instead of the standard 150bp X2 was run on MiSeq flowcell v3. Data analysis was performed using a MolecularMD custom pipeline. The analysis of FLT3 ITD was conducted using three methods including two off-the-shelf programs, Pindel and ITDseek, as well as Cigar, a MolecularMD developed ITD detection algorithm. FLT3 ITDs at 126 bp and 21 bp in PL-21 and MOLM-13 cell lines, respectively, were detected by TMP assay using the three algorithms. In contrast to ITDseek which only reports ITD sequence without frequency information, Pindel and Cigar report both ITD sequence and frequency. Compared to Pindel, ITD frequency reported by the custom algorithm is closer to that determined by the fragment analysis. Serial dilution of the 126bp ITD into wild type DNA showed the 0.55% ITD (from fragment analysis) can be reproducibly detected at ~0.6% by the Cigar algorithm. FLT3 ITDs ranging in size from 6bp to 300bp were detected in clinical AML samples using the enhanced TMP workflow and confirmed by a fragment analysis method. Limit of detection, reproducibility and accuracy of SBS and indel detection in other key genes by the TMP assay have also been validated. Low-level mutations, i.e. NRAS G12D at 1.7%, NRAS G13D at 2%, and IDH2 R140Q at 1.4%, were detected by TMP panel from AML samples and further confirmed with an independent assay. A long deletion (52bp) in exon 9 of CALR was also detected by the custom pipeline. In summary, the enhanced TMP workflow allows for accurate detection and reporting of SBS, indel and FLT3 ITDs including large duplications. Optimized detection of FLT3 ITDs will improve AML patient selection for targeted therapy.

#4523

Comparison of pre-analytical methods using liquid biopsy NSCLC samples for molecular diagnosis.

Feliciana Marinho, Michele Pereira, Cythia Machado, Maíra Freire, Elvis Mateo, Mariano Zalis, Priscila Cirillo. _Instituto Hermes Pardini, Vespasiano, Brazil_.

Introduction: Non-small cell lung cancer (NSCLC) represents approximately 85% of all lung cancer types with 5% of survival rate. The development of strategies to improve the treatment of these patients is important. EGFR druggable-specific mutations can be detected in plasma from circulating-free tumor DNA (cfDNA) in NSCLC patients through liquid biopsy. In plasma, cfDNA has short half-life then, it is necessary to ensure that sample collection and processing does not interfere in the yield and quality of the cfDNA.

Objective: The aim of this study was to compare three commercial blood collection tubes, acid nucleic acid purification kits and four different time-point of plasma processing after collection of NSCLC samples from different regions of Brazil.

Methods: Blood samples were collected from 10 NSCLC patients in EDTA, PAXgene and Streck tubes. EDTA samples were processed immediately after collection and plasma was frozen at -20°C. PAXgene and Streck samples were processed at room temperature within 24h, 72h or 168h after collection. Plasma were aliquoted (1mL) and frozen at -80°C before cfDNA extraction. cfDNA of all samples were extracted using QIAamp Circulating Nucleic Acid or QIAamp MinElute Virus Vacuum kits. cfDNA concentration and integrity were evaluated by fluorometry (Qubit) and Bioanalyzer (Agilent), respectively. In addition, patients also were evaluated by Cobas® EGFR Mutation Test v2 (Roche) and just T790M results were confirmed by Droplet Digital PCR (ddPCR). All samples were submitted to Next-Generation Sequencing (NGS) using Oncomine Lung cfDNA Assay (Thermo Fisher). Statistical analysis was performed by IBM SPSS Statistics v19.0 software.

Results: cfDNA concentration did not show strong variation regardless of the tubes collection and time-point processing (p=0.57) and no statistically significant differences were found between extraction kits (p=0.80). However, the cfDNA yield were 10x higher after virus (mean 0.49±0.10) compared to circulating (mean 0.03±0.01) kits. Bioanalyzer results showed cfDNA size among 100 and 200bp, but there was no observed difference between the cfDNA integrity regardless of all conditions. Cobas EGFR results showed 7 patients wild-type, 2 had exon 19 deletion (Del19) and 1 had G719X mutations. ddPCR confirmed the absence of T790M mutation among these patients in all cfDNA sample conditions. NGS data will be presented in the poster session.

Conclusion: Plasma collection using virus kit shown higher cfDNA yield, compared to circulating kit. Regardless of the tube and processing time, the cfDNA samples showed satisfactory yield and integrity for identification of EGFR mutations as observed by Cobas and ddPCR concordant results. NGS analysis will allow us to investigate whether the cfDNA quality could interfere in the amplification of the target regions (11 genes panel). This is a pilot study and will be replicated for a greater number of cases.

#4524

**Immunohistochemistry and alternative FISH testing in breast cancer with** HER2 **equivocal amplification.**

Sally Agersborg,1 Christopher Mixon,1 Thanh Nguyen,1 Sramila Aithal,2 Forrest Blocker,1 Lawrence Weiss,1 Robert Gasparini,1 Shiping Jiang,1 Wayne Chen,1 Gregory Hess,3 Maher Albitar1. 1 _NeoGenomics Inc., Aliso Viejo, CA;_ 2 _Cancer Treatment Centers of America, Philadelphia, PA;_ 3 _Symphony Health, Conshohocken, PA_.

PURPOSE

While HER2 testing is well established in directing appropriate treatment for breast cancer, a small percentage of cases show equivocal results by IHC and FISH. Alternative probes may be used in equivocal cases. We present a single community-based institution's experience in further evaluating these cases.

PATIENTS AND METHODS

Between 2014-2016, 4255 samples were submitted for HER2 amplification testing by alternative probes, TP53, RAI1, and RARA. Of patients tested by FISH, 505/3908 (12.9%) also had IHC data.

RESULTS

Most (73.9%) FISH equivocal cases remained equivocal after IHC testing. Duplicate testing by IHC using a different paraffin block resulted in poor reproducibility as 44% of IHC negative became equivocal and 40% of IHC equivocal became negative. Of the equivocal cases by IHC, 52.3% became positive by alternative FISH. However, 50.5% of cases equivocal by conventional FISH were classified as HER2 amplified by alternative probes. Most cases were positive by more than one probe; 78% of positive cases were positive by RAI1 and 73.9% by TP53. There was a significant difference between IHC and FISH alternative testing (P<0.0001) among the equivocal cases by conventional FISH testing, 44% of IHC negative cases became positive while 36% of the positive IHC cases became negative by alternative FISH testing. Available data showed that 41% of patients were treated with palbociclib and were positive by alternative FISH.

CONCLUSION

The prevalence of double HER2 equivocal cases and the discrepancy between IHC and alternative FISH testing suggest that FISH alternative testing using both RAI1 and TP53 probes is necessary for conclusive classification. Due to the significant difference between IHC repeat testing, reflexing on IHC alone in this group of cases may not be reproducible. Because almost half of FISH equivocal cases converted to HER2 amplified upon alternative testing, clinical studies to determine benefit of anti-HER2 therapy in these patients is urgently needed. | |  | |  | |  | |

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

Alternative FISH | IHC Score 0

Negative | IHC Score 1

Negative | IHC Score 2

Equivocal | IHC Score 3

Positive

|

No | % | No | % | No | % | No | %

Negative | 14 | 63.6 | 54 | 54.5 | 178 | 47.7 | 4 | 36.4

Positive | 8 | 36.4 | 45 | 45.5 | 195 | 52.3 | 7 | 63.6

Total | 22 | 100.0 | 99 | 100.0 | 373 | 100.0 | 11 | 100.0

#4525

Cancer/testis antigens: A biomarker panel for prostate cancer screening.

Luciane T. Kagohara,1 Prakash Kulkarni,2 Takumi Shiraishi,1 Robert Vessella,3 Robert W. Veltri,1 Elana J. Fertig1. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _University of Maryland, College Park, MD;_ 3 _University of Washington, Seatle, WA_.

The aim of the current study was to identify a panel of cancer/testis antigens (CTAs) with the potential to be used as a complementary test to the prostate-specific antigen (PSA) test for prostate cancer (PCa) screening. PSA test is capable of identifying men under risk of PCa before the presence of symptoms. However, this screening has been considered a controversy assessment since many men presenting benign lesions (benign prostate hyperplasia or other inflammatory conditions) can also present increased PSA levels. Hence, the great current dilemma in PCa screening is to develop a test that in combination with PSA assay could present higher accuracy for PCa early diagnosis. CTAs constitute an important class of potential cancer biomarkers that are poorly studied in PCa. CTAs are normally expressed in testis and germ cells and are aberrantly over-expressed in cancers. This unique pattern of expression makes these genes potential candidates as specific tumor biomarkers. CTA aberrant expression in malignant tumors is associated with phenotypic changes that confer the cancer cells essential advantages for proliferation and survival. Our hypothesis is that a panel of CTAs differentially expressed in PCa compared to normal prostate tissue would be useful to develop a diagnostic biomarker panel to be used in combination with the PSA test. We initially evaluated the expression of 22 CTAs, using the Nanostring approach, in localized and metastatic PCa to identify those genes associated with more aggressive tumors. After validation by qRT-PCR, we verified that 8 genes were differentially expressed between indolent and aggressive tumors. Using immunohistochemistry and a quantitative image analysis to measure protein expression, we evaluated CTA protein levels in PCa and adjacent normal tissue paired samples. The CTAs CEP55, NUF2, PAGE4, PBK, RQCD1, SPAG4, SSX2 and TTK presented increased protein levels in PCa when compared to normal prostate tissue from the same patient. Increased levels of PAGE4, PBK, RQCD1, SPAG4 and SSX2 were more frequent among patients with Gleason Score 4+3 or higher. In an attempt to identify a panel of CTAs with increased expression in PCa vs. prostate normal samples, we used multiple logistic regression analysis (MLR). MLR analysis showed that a panel that includes all 8 CTAs analyzed correctly classified 88.9% of the cases (AUC=0.96; sensitivity=88.5%; specificity=89.2%). For the MLR analysis, we considered the intensity and the frequency of the cancer cells with positive CTA expression. The intensity of all 8 CTAs was significant in the analysis, while the frequency of staining was only relevant for NUF2, PBK, SSX2 and TTK. Our findings suggest that our 8 CTAs panel represent a potential biomarker with high accuracy to discriminate normal prostate from PCa. Further studies to evaluate their expression in bodily fluids will determine their potential as a non-invasive PCa screening test to be used in combination with PSA.

#4526

Predicting response: PD-L1 biomarker testing by IHC and RNA-seq.

Jeffrey Conroy,1 Sarabjot Pabla,1 Mary Nesline,1 Sean Glenn,1 Blake Burgher,1 Maochun Qin,1 Jonathan Andreas,1 Vincent Giamo,1 Felicia L. Lenzo,1 Angela Omilian,2 Wiam Bshara,2 Antonios Papanicolau-Sengos,1 Yirong Wang,1 Marc Ernstoff,2 Mark Gardner,1 Carl Morrison1. 1 _OmniSeq, Inc., Buffalo, NY;_ 2 _Roswell Park Cancer Institute, Buffalo, NY_.

Background: Currently, four FDA-approved biomarker assays are available to screen for PD-L1 to enrich for patient response to checkpoint inhibitors (CPI). Each are immunohistochemical (IHC) assays that approximate the percentage of immune or tumor cells expressing PD-L1 using various antibodies, staining and scoring systems. Given this inherent variability, there are concerns whether any single PD-L1 IHC assay, or IHC in general, can be used as a companion or complimentary diagnostic. This is highlighted by the number of individuals, regardless of histology or antibody used, who score below the IHC scoring threshold but respond to PD-L1 inhibitors.

Methods: In this study, we compared PD-L1 protein expression (IHC) to PD-L1 gene expression (CD274) in 436 tumors. PD-L1 IHC was assessed in melanoma using the 28-8 antibody, with kidney, HNSCC, and lung cancer assessed with 22C3. All were scored as per published guidelines. CD274 gene expression was determined by targeted RNA-seq, with each sample's expression level compared and ranked to a reference population.

Results: ANOVA demonstrated a significant relationship between RNA-seq and IHC PD-L1 measurements (p.value < 2e-16). Tukey's HSD comparisons of mean TPS at <1%, 1-4%, and >5% demonstrate significant differences between the three groups that are consistent with gene expression rankings (p adj <0.002). Additionally, for metastatic melanomas with CPI response data, a strong association of objective response rate (ORR) to high RNA-seq expression exists, regardless of IHC result (Table 1). Conclusion: In 400+ tumors, PD-L1 demonstrates correlated mean expression values when assessing protein by IHC and gene expression by RNA-seq. For the CPI treated melanomas with outcomes, PD-L1 IHC ≥1% had a 56% ORR, which improved to >71% ORR when combined with high PD-L1 gene expression. With the need to better predict CPI response, this data suggests that combination PD-L1 testing is an improvement over the FDA approved IHC assays.

Table 1: ORR for 37 melanoma patients based on combination PD-L1 expression results

---

PD-L1 Method Combinations | CR | PR | SD | PD | ORR | 95% CI

RNAseq High + IHC ≥1% | 1 | 4 | 1 | 1 | 71.4% | 29.04%-96.33%

RNAseq High + IHC <1% | 1 | 2 | 0 | 1 | 75.0% | 19.41%-99.37%

RNAseq Low + IHC ≥1% | 1 | 0 | 1 | 0 | 50.0% | 1.26%-98.74%

RNAseq Low + IHC <1% | 0 | 5 | 4 | 15 | 20.8% | 7.13%-42.15%

#4527

Major pathologic response and biomarker predict survival in lung cancer patients receiving neoadjuvant chemotherapy.

Apar Pataer, Ruping Shao, Arlene M. Correa, Ignacio I. Wistuba, Stephen G. Swisher. _UT MD Anderson Cancer Ctr., Houston, TX_.

In a previous study, we determined that major pathologic response (MPR) as indicated by the percentage of residual viable tumor cells predicted overall survival (OS) in patients with non-small cell lung cancer (NSCLC) who received neoadjuvant chemotherapy. In this study, we assessed whether 2 gene and 5 protein biomarkers could predict MPR and OS in 98 NSCLC patients receiving neoadjuvant chemotherapy. We assessed whether gene mutation status or protein expression was associated with MPR or OS. We observed that KRAS mutation tended to be associated with OS (p = 0.06), but EGFR mutation was not associated with OS. We found that patients with high RAD51 expression levels had a poorer prognosis than did those with low RAD51 expression. We also observed that RAD51 expression was associated with MPR. MPR and RAD51 expression were associated with OS in univariate and multivariate analyses (p = 0.04 and p = 0.02, respectively). Combination of MPR with RAD51 is a significant predictor of prognosis in NSCLC patients who received neoadjuvant chemotherapy. No association of MPR or VEGFR2, EZH2, ERCC1, RAD51, or PKR expression with KRAS or EGFR mutation was found. We demonstrated that MPR or RAD51 expression was associated with OS in NSCLC patients receiving neoadjuvant chemotherapy. Prediction of a patient's prognosis could be improved by combined assessment of MPR and RAD51 expression.

#4528

RNAseq RNAaccess is the preferred method for expression profiling of low quality FFPE gastric cancer samples.

Emon Elboudwarej, Marianna Zavodovskaya, Xi Zhao, Luting Zhuo, Carrie Baker Brachmann, Jinfeng Liu. _Gilead Sciences, Inc., Foster City, CA_.

Clinical trial tissue samples are a valuable resource for biomarker discovery and are frequently collected as Formalin-Fixed, Paraffin-Embedded (FFPE) blocks. FFPE tissues pose challenges for next generation sequencing (NGS)-based expression analysis due to poor yield from highly fragmented RNA. The objective of the current study was to evaluate and compare RNAseq RNAaccess (RR) and Nanostring (NS) platforms for molecular profiling of highly fragmented gastric cancer FFPE tissue.

RR (TruSeq RNAaccess, Illumina) and NS (PanCancer immune) were performed according to manufacturer protocol using RNA extracted from sourced FFPE gastric cancer tissues (N=6) with a range of DV200 scores (6-34%) and case-matched fresh-frozen (FF) tissue (N=6). For NS, 200ng and 400ng RNA input levels were tested. Data quality from the 400ng samples proved to be superior, resulting in two more samples passing QC. A final sample size of 4 matched pairs was utilized for cross-platform comparisons. For RR, 100ng RNA was used. Pearson correlation coefficients were examined for the following comparisons: 1) FFPE vs. FF samples from RR, 2) FFPE from NS vs. FF from RR, and 3) FFPE NS vs. FFPE RR. Analyses were performed on log2 transformed expression data, for all available genes (N= 770 common genes from RR and NS) and an 18-gene IFNγ signature (IFNγ). All correlations were plotted against a range of DV200 scores from the FFPE samples in order to evaluate a potential tissue quality cutoff for RR, specifically at DV200 levels below Illumina guideline of 30.

All RR data samples yielded >80% exonic rate in uniquely mapped reads. Conversely, samples with DV200<14 analyzed by NS failed to pass QC due to low fraction of genes detected above background signal. Comparing FFPE to FF tissue from RR, samples with DV200>10 were highly correlated (r≥0.92), globally or for IFNγ. Comparisons of FF RR to FFPE NS were also well correlated (global median r=0.8; IFNγ median r=0.86). Similarly, expression of FFPE RR was well correlated with FFPE NS (global median r=0.81; IFNγ median r=0.89). Altogether, NS and RR data were highly comparable and minimally impacted by DV200 scores below 30.

RR generates gene expression profiles from FFPE tissue that are highly concordant with case-matched FF tissues. This study supports using RR for transcriptional profiling of poor quality gastric cancer FFPE samples. Samples below the Illumina suggested DV200 cutoff of 30 generated good quality RR and NS expression data. IFNγ scores derived from NS and RR from the same tissue were highly concordant. However, NS required higher RNA input level to compensate for low DV200 score and no acceptable expression data was generated on samples below DV200 14. This study will be expanded with data on FFPE samples with DV200<10 to identify a lower limit of RNA quality for RR.

#4529

Effect of preanalytic variables on established and emerging thrombosis-related biomarkers in an ethnically and racially diverse population of cancer patients and healthy subjects at a safety net hospital.

Morgan P. Thompson,1 Elizabeth R. Duffy,1 DJ Stearns-Kurosawa,2 Jasmin Bavarva,3 Shinichiro Kurosawa,2 Jiyoun Kim,2 Cheryl Spencer,1 Daniel Remick,2 Mark Sloan,1 Joel Henderson,1 Kerrie P. Nelson,2 Joseph Y. Tashjian,2 Yibing Wei,2 Rachana Agarwal,3 Michelle A. Berny-Lang,4 Chris Andry1. 1 _Boston Medical Center, Boston, MA;_ 2 _Boston University School of Medicine, Boston, MA;_ 3 _Leidos Biomedical Research, Inc, Frederick, MD;_ 4 _National Cancer Institute, Bethesda, MD_.

The link between cancer and thrombosis, especially venous thromboembolism, is well established and thrombotic risk is exacerbated by cancer treatments, such as surgery and chemotherapy. This ongoing study aims to determine the impact of pre-analytic variables (PAVs) on thrombosis biomarkers in a diverse cancer patient and non-cancer subject population at an urban safety net hospital.

Citrated blood from newly diagnosed, treatment-naïve patients of 11 cancer types or from non-cancer controls was processed to examine these variables: time to fractionation (2 and 4 hrs), plasma freeze-thaw cycles (2 and 3 cycles), and plasma delay to testing (24 or 72 hrs at 4oC). Regular processing (< 1 hour to centrifuge or 1 freeze-thaw cycle) served as the control sample for the variables. Current interim data presents biomarker data for D-dimer (DDE), Factor VIII activity (FVIII), soluble P-selectin (sP-Sel), prothrombin fragment 1+2 (F1+2), plasma DNA (DNA) and myeloperoxidase (MPO). Assays are performed in the hospital clinical lab (DDE, FVIII) or in our research lab following 30 detailed standard operating procedures (SOPs). Cancer patient demographics are 60% male, 40% Black/African-American, 38% Caucasian, 18% Hispanic, 3% Native American, and 1% Asian with an age range of 38-86 years. Non-cancer controls are 52% male, 59% Caucasian, 15% Black/African-American, 19% Asian, and 7% Hispanic with an age range of 23-64 years. Interim project data shows increased thrombosis biomarker levels in cancer subjects, except for sP-Sel and F1+2. Biomarker levels in cancer patients (n=9-52) were increased by approximately 300ng/ml DNA, 100ng/ml DDE, and 5ng/mL MPO with a trending increase in FVIII (~30%) when compared to non-cancer controls (n=17-22). Freeze-thaw of plasma had no effect, while a 2hr time to fractionation resulted in significantly increased MPO (~10ng/mL), FVIII (~14%) and F1+2 (~310 pg/mL). Delay to testing done for DNA and DDE showed with no apparent effect on biomarker levels after 24 or 72 hrs at 4oC. Current data show biomarker levels are impacted by presence of cancer rather than ethnicity of the patient.

Donor recruitment is ongoing with shifting strategies to meet recruitment goals of non-cancer donors for greater diversity and older age to more closely reflect our cancer patient population. Rigorous control of sample handling and assay performance using SOPs that are compatible with a hospital setting contribute to identifying PAVs that matter for design of generalizable procedures.

#4530

Single HER2-positive tumor cells are detected in initially HER2-negative breast carcinomas using the DEPArray™-HER2 FISH workflow.

Lisa Koenig,1 Amanda Gerber,2 Aditi Khurana,3 Farideh Z. Bischoff,2 Sabine Kasimir-Bauer1. 1 _Univ. Hospital of Essen, Essen, Germany;_ 2 _Menarini Silicon Biosystems, San Diego, CA;_ 3 _Research Dx, Irvine, CA_.

Background: In breast cancer (BC), HER2 expression does not only differ within various primary tumor (PT) areas, but also with respect to the expression on circulating tumor cells (CTCs). Despite clinical guidelines defined by CAP/ASCO, PT HER2 analysis displays limitations due to pre-analytical sampling, methodology and intra-tumor heterogeneity. Fluorescence in situ hybridization (FISH) is used for analyzing HER2/neu (ERBB2) gene amplification and for re-solving inconclusive HER2 immunohistochemistry results. In this study, we aimed to evaluate BC intra-tumor heterogeneity using the DEPArray™ - HER2 FISH workflow in BC patients characterized as i) PT HER2 neg / CTC HER2/neu pos (n=25).

Methods: 50 µm FFPE tumor curls (n=25) were cut, deparaffinized and underwent antigen retrieval. After dissociation into a single cell suspension, the cells were stained for cytokeratin (CK-AF488), vimentin (Vim-AF647) and DAPI to distinguish between stroma and tumor cells. These two cell populations were separated using the DEPArray™, an image-based single cell sorting system. Samples containing ~ 200 intact CK+/Vim-/DAPI+ tumor cells were deemed suitable for pure tumor cell recovery. Subsequently, HER2 FISH analysis was performed on the recovered single cell tumor sample for HER2 and chromosome 17 signal evaluations using a dual HER2/CC17 probe. The results obtained with the DEPArray™ - HER2 FISH workflow were then compared with the HER2 status obtained by routine pathology and correlated with the HER2 status on CTCs, enriched from 10 ml blood using positive immunomagnetic selection followed by RNA isolation and subsequent gene expression analysis by reverse transcription and Multiplex-PCR (AdnaTest, Qiagen).

Results: A concordant result for HER2 analysis comparing routine pathology and DEPArray™ - HER2 FISH analysis was found for 84% (21/25) of the patients. A discordant result was identified in 4/25 patients using the DEPArray™ - HER2 FISH workflow. 2/25 BC patients were proven to be HER2 positive in our approach, despite being tested HER2 negative in routine pathology. Two other patients showed an equivocal HER2 status in the DEPArray™ - HER2 FISH workflow, but a negative result in routine pathology. Consequently, four BC patients would have been eligible for anti-HER2 treatment.

Conclusion: The DEPArray™ system allows the recovery of a pure tumor cell fraction for subsequent HER2 FISH analysis and shows a high concordance with the HER2 status performed by routine pathology. However, the DEPArray™ - HER2 FISH workflow offers a more precise and accurate diagnostic test and could be used for BC patients at high risk, e.g. triple negative BC patients, patients with HER2/HER3 positive CTCs or in HER2 inconclusive BC cases to offer anti-HER2 treatment.

#4531

Development of a companion diagnostic assay for the detection of phosphatase and tensin (PTEN) protein loss and treatment with ipatasertib in metastatic castration-resistant prostatic cancer (mCRPC).

Crystal Stephens,1 Erica Harnish,1 Rebecca Bowermaster,1 Azita Djalilvand,1 Dustin Smith,2 Doris Kim,2 Steven Gendreau,2 Edmundo Del Valle1. 1 _Ventana Medical Systems, Tucson, AZ;_ 2 _Genentech, Inc., CA_.

Background: Ventana Medical Systems, Inc. is developing in collaboration with Genentech, Inc., an immunohistochemical (IHC) companion diagnostic to aid in selecting patients with metastatic castration-resistant prostate cancer (mCRPC) that may benefit from ipatasertib, an Akt inhibitor therapy. The phosphatase and tensin (PTEN) protein plays an important role in controlling cell survival and cell cycle progression as a negative regulator of the phosphoinositide 3-kinase/AKT pathway.

Methods: PTEN (SP218) rabbit monoclonal primary antibody was optimized for use with the VENTANA OptiView DAB IHC Detection Kit on the automated BenchMark ULTRA platform (PTEN (SP218) RxDx Assay). The PTEN (SP218) RxDx Assay was developed for use in formalin-fixed, paraffin-embedded tissue samples of mCRPC in a series of studies addressing sensitivity, specificity, robustness and precision. The scoring algorithm was defined using statistical analysis of clinical outcome data analysis, and the PTEN (SP218) RxDx Assay staining pattern and prevalence of PTEN loss of expression in a set of tissue samples. Inter-reader precision was established by 3 pathologists evaluating 90 mCRPC samples across the range of PTEN expression levels.

Results: The Ventana PTEN (SP218) RxDx Assay met all pre-defined acceptance criteria. mCRPC tissue samples are designated with PTEN-Loss status when ≥ 50% of viable malignant cells have no specific cytoplasmic staining with PTEN (SP218) in the presence of acceptable internal controls. Inter-reader precision in determining PTEN status resulted in agreement rates greater than 97%.

Conclusion: These results highlight the robustness and reproducibility of the Ventana PTEN (SP218) RxDx Assay. In the clinical outcome analysis, patients identified to have PTEN-loss status demonstrated clinically meaningful improvements when treated with ipatasertib. The clinical utility of the PTEN (SP218) RxDx assay will be further validated in additional patients in subsequent ipatasertib studies, including a Phase 3 study (IPATential150) beginning in 2017.

#4532

A free and easy to use morphological biomarker to serve as an internal challenge in the study of biomarkers by systematic tissue microarray analysis.

Leslie Dalton,1 Katharina Köhler,2 Anne-Kathrin Poßögel,2 Axel Niendorf2. 1 _St. David's South Austin Medical Center, Austin, TX;_ 2 _Pathologie Hamburg-West, Hamburg, Germany_.

Background: Tissue microarrays (TMA) and digital microscopy (DM) are modern era innovations used here to assess a potential "free" internal control for TMA studies. Morphological nuclear grading can be performed on very few cells as is inherent with TMA samples. DM allows for ease in "shipment" of TMA slides for rapid central review by an experienced morphologist. The TMA used for this analysis are part of a larger initiative with a special focus on cases, whose conventional prognostic parameters (i.e. no special type histology [NST], estrogen receptor positive [ER+], Nottingham grade 2, nodal negative [N0]) indicate neither a good nor bad clinical course and thus constitute a so called gray zone (GZ).

Methods: We compared three grading systems based on the analysis of H&E-stained TMA. Assigned to 267 ER+, Nottingham grade 2 (GZ-ER+) tumors, and 97 triple negative (TN) tumors was a 3-9 score (nuclear grade sum score - NGSS) summed from 1-3 scores for each of nucleoli, size nuclei, and variation size/shape. The Helpap nucleolar (HN) component was analyzed separately as was a traditional Nottingham nuclear pleomorphism score (PS).

Results: A highly significant uneven distribution of NGSS was found between GZ-ER+ and TN tumors (p<0.00001). 224 of 228 tumors (62% of all) NGSS < 6 were GZ-ER+, while 52 of 54 tumors (14% of all) NGSS 8-9 were TN. Mid-range NGSS (6, 7) had 41 of 82 GZ-ER+ (22% of all were mid-range). There is a clear-cut inverse relationship between NGSS and ER+ vs. ER- cases: ER+ cases (n=267) have a distribution of 48, 129, 47, 21, 20, 2 and 0 for NGSS grades 3 through 9, whereas ER- cases (n=97) exhibit 0, 2, 2, 15, 26, 27 and 25 in these respective categories. Receiver operator curve analysis showed area under curve of 0.95 in the ability for the 7 category NGSS to predict TN cancer, 0.91 for PS and 0.84 for HN. HN and PS suffered from frequent intermediate grade assignment (respectively 40 and 45%). Interestingly HN had 46% grade 1 tumors of which only 9 of 167 were TN. 23% of tumors were assigned PS=1 and none were TN.

Conclusion: NGSS can easily be performed on the same tiny samples which are being used to study a novel biomarker. In the appropriate research setting, the challenge for a biomarker would be to offer significant improvement over what is evident by "old fashioned" morphology. Here NGSS highlighted the morphological contrast between poor prognosis TN cancer and more favorable prognosis GZ-ER+ tumors. This was even though GZ-ER+ tumors had originally been assigned intermediate Nottingham grade. Furthermore, the question has to be raised as to whether or not NGSS might serve as a way to adjust PS and thereby contribute to stratification within the gray zone. This would reclassify more tumors into low grade which would have important clinical implications.

#4534

**Validation of highly sensitive TargetSelector** TM **ctDNA assays for** EGFR **,** BRAF **, and** KRAS **mutations.**

Shan-Fu Wu, Timothy T. Lu, Anh Pham, Jeffrey Chen, Tony Daher, Errin Samuelsz, Manisha Patel, Veena M. Singh, Lyle J. Arnold, Jason C. Poole. _Biocept, San Diego, CA_.

Background: Accurate detection of driver mutations in cancer patients is vital for targeted therapy. Compared to tissue biopsy, "liquid biopsy" offers a non-invasive and more systemic approach to identify tumor mutations by assessing circulating tumor DNA (ctDNA) released from tumor cells into peripheral blood. We have developed TargetSelectorTM Real-Time PCR based assays to detect low frequency mutant alleles in ctDNA. The TargetSelectorTM assay uses a proprietary blocker to suppress amplification of excess WT alleles released from normal cells, while allowing specific amplification of mutants. Here we focus on five important targets: EGFR (Del19, L858, and T790), BRAF (V600), and KRAS (G12/G13), which are relevant to lung cancer, melanoma, and colorectal cancer.

Methods: The TargetSelectorTM assay applies a specific blocker to cover variants on a short stretch of target DNA (up to 10 bp for nucleotide variants). For example, one KRAS exon 2 blocker covers all variants on both G12 and G13 positions. The TargetSelectorTM assays were first validated with cancer cell line DNA carrying mutation targets on QuantStudio 5 Real-Time PCR instruments (QS5). Sanger Sequencing was subsequently performed to confirm the mutation. Analytical validation was conducted by 3 independent operators using 5 instruments across 5 days in our CAP/CLIA certified laboratory. For ctDNA testing, whole blood samples were collected in CEE-SureTM Blood Collection tubes and DNA extraction from plasma was performed on the QIAsymphony.

Results: In total, we tested 3086 samples for EGFR, BRAF and KRAS TargetSelectorTM ctDNA assays, with EGFR WT assay as the background reference. All five ctDNA assays showed >99% analytical sensitivity and >99% analytical specificity. Based on practical and theoretical estimates, each ctDNA assay demonstrated single mutant copy detection sensitivity. In the presence of 14,000 copies of WT background, the sensitivity of our ctDNA assays are: EGFR Del19, 0.01%; EGFR L858R, 0.02%; EGFR T790M, 0.01%; BRAF V600E, 0.01%; KRAS G12C, 0.02%. The inter-assay and intra-assay analyses showed r2 >0.94, suggesting a consistent performance among operational variables. Samples tested from 20 healthy donors (100 tests in total) showed clinical specificity >99%. In the concordance study of 13 clinical samples (31 tests in total) between QS5 and ABI 7900HT platforms, TargetSelectorTM ctDNA assays with the QS5 identified the same plus additional mutations compared to the 7900HT.

Conclusions: TargetSelectorTM ctDNA assays were validated both analytically and clinically, showing single mutant copy detection and sensitivity at 0.02% or better in a background of excess WT DNA. Implementation of the QS5 qPCR platform into our TargetSelectorTM ctDNA assays leads to a higher sensitivity and faster turnaround time. These factors enable sensitive and efficient testing crucial for guiding treatment decisions and patient care.

#4535

Automated image analysis algorithm development for cell surface-binding therapeutic agent target & its comparison to manual pathology scoring.

Brittany Bahamon,1 Andrzej Cholewinski,2 Ruben Cardenes,2 Aleksandra Zuraw,2 Israel Barragan,2 Marise B. McNeeley,3 Ronald D. Luff,3 Adnan Abu-Yousif,1 Hadi Danaee1. 1 _Takeda Pharmaceuticals Inc, Cambridge, MA;_ 2 _Definiens Inc, Cambridge, MA;_ 3 _Quest Diagnostics, Teterboro, NJ_.

Standard pathology scoring of cell surface targets in tumor tissue is done routinely by semi-quantitative scoring and is usually performed by a pathologist assessing a given number of fields of view (FOVs) from an immunohistochemistry (IHC) stained slide. In clinical studies evaluating cell surface-binding therapeutic agents, a tissue based patient selection assay with pathology semi-quantitative scoring such as H-score or percent positive is frequently used to enroll patients. Image analysis has become a powerful tool to evaluate and quantify IHC assays including those used for patient enrollment. It allows one to leverage the power of computer software and machine learning approaches to develop algorithms for the detection of specific markers and to run these solutions across entire stained tumor sections. Here, we developed an automated image analysis solution using the Definiens Developer Software specifically to detect a cell surface target in human gastrointestinal tumors. This algorithm is able to detect and quantify apical/membrane staining of the target. We used an initial set of colorectal cancer (CRC) samples (n=30) stained for the target. Tumor and non-tumor compartments were identified using the algorithm; target expression was then assessed in the tumor compartment only. Multiple outputs were generated with the image analysis tool including area of apical stain over the total area of interior luminal border (H-scoreluminal), intensity of apical stain over total apical stain area (H-scoreapical) and apical stain over the total area of the tumor region (H-scoretotal tumor). H-scoreluminal was the output most similar to the semi-quantitative pathology scoring previously performed on these samples. H-scoretotal tumor was the only output that used the total area of the tumor in its composite score. The various image analysis parameters showed good correlation with each other. This use case is an example of applying digital pathology to an existing IHC assay to quantitatively assess marker expression across entire tumor sections. Implementation of quantitative image analysis allows clinical development programs including that assessing cell surface-binding therapeutic agents the ability to incorporate a variety of otherwise difficult-to-measure parameters such as total tumor area and other cellular parameters from an IHC stain into a more robust biomarker analysis package. In addition, this quantitative approach could help in establishing or fine tuning an IHC assay's cut off when used prospectively in clinical trials.

#4536

Rapid evaporative ionization mass spectrometry (REIMS) analysis of the mucosal lipidome has a high diagnostic accuracy for adenomas and early colorectal cancer.

Petra Paizs,1 Eftychios Manoli,1 Sam E. Mason,1 James L. Alexander,1 Zsolt Bodai,1 Emma White,1 Afeez Adebesin,1 Jonathan Hoare,2 Robert Goldin,1 Ara W. Darzi,1 James M. Kinross,1 Zoltan Takats1. 1 _Imperial College London, London, United Kingdom;_ 2 _Imperial College London NHS Trust, London, United Kingdom_.

Introduction

The aim of this study was to determine the diagnostic accuracy of REIMS for dysplasia in human adenomas and early colorectal cancer and to determine changes in mucosal lipid chemistry during cancer initiation. Moreover, we attempted to develop a proof of concept first in man study of a novel REIMS-based endoscope for in-vivo chemical phenotyping of adenomas based on the real time analysis of phosphatidic acid (PA) and phosphatidylethanolamine (PE) metabolism.

Experimental procedure

A prospective, observational cross-sectional cohort study was performed in patients undergoing elective resection for colorectal cancer or colonoscopy at Imperial College London NHS Trust. Two tissue types were obtained - early colorectal cancer (defined as T1 or T2 according to TNMv6 criteria) and adenomatous polyps. Patients with familial adenomatous polyposis, inflammatory bowel disease or hereditary non-polyposis colon cancer were excluded. Specimens were analyzed ex-vivo using a modified electro-surgical hand piece with the aerosol aspirated into a Xevo G2-S QTof mass spectrometer (Waters Corporation). Multivariate analyses were used to create models which underwent cross-validation using SIMCA (V14, Umetrics, Sweden). Characteristic lipid species were identified using univariate statistics in R Studio (V1.0.44) and Metlin. An endoscopic set up was created using a standard endoscope for proof of concept.

Summary of data

19 patients (12:7 F:M) were included with a median age of 74 (range 50-92). Six patients had T1/2 adenocarcinoma and 13 had adenomatous polyps. REIMS was able to accurately distinguish the presence of low grade dysplasia (LGD) vs high grade dysplasia (HGD) in adenomatous polyps (sensitivity 95.0%, specificity 100%). Phosphatidic acids (PA) 38:2 and 38:1 were more abundant in high vs low grade dysplasia (p=0.003). REIMS was also able to identify adenomatous polyps from early cancer (sensitivity 100%, specificity 96.3%). Phosphatidylethanolamine (PE) 36:1, phosphatidic acid (PA) 38:1, glucosylceramide (Glc-Cer) 30:1 and ceramide phosphoethanolamines (PE-Cer) d16:1(4E)/20:0 and d14:1(4E)/22:0 were associated with early cancer formation rather than adenomas (p<0.001, p<0.001, p=0.03, p=0.04 respectively). Endoscopic REIMS was successfully deployed in-vivo in six patients undergoing polypectomy. Spectra with good signal to noise ratio were acquired and the technology was able to correctly stratify adenomas.

Conclusion

Dysplastic adenomas exhibit mucosal lipid chemistry that is discrete from early colorectal cancer. PA, PE and Glc-Cer have potential use as diagnostic biomarkers. First in man studies of the endoscopic application of REIMS suggest it could serve as a real-time tool for early cancer detection based on in vivo analysis of the mucosal lipidome.

#4537

Cryogenic biopsy device for preservation of labile biomarkers.

Erez Nevo,1 Melinda G. Hollingshead,2 Jeevan P. Govindharajulu,3 Shoshan Nevo,1 Kay D. Gray,3 Ralph E. Parchment,3 James H. Doroshow,4 Apurva K. Srivastava3. 1 _Robin Medical, Inc., Baltimore, MD;_ 2 _National Cancer Institute, NIH, Frederick, MD;_ 3 _Frederick National Laboratory for Cancer Research, Frederick, MD;_ 4 _National Cancer Institute, NIH, Bethesda, MD_.

Background: Preanalytical factors such as the handling of biospecimens between resection and analysis can have a significant impact on assay results which may adversely affect patient care. Global phosphoprotein analysis indicates that a 5 minute delay in time-to-stabilization (ischemia time) can impact 20% of phospho-serine and 50% of phospho-tyrosine residues in kinases. Consequently, inadequate tumor biopsy processing is a significant impediment to the use of phosphorylated biomarkers in oncology. We aim to develop new biopsy devices and methodologies to stabilize highly dynamic biomarkers and minimize the influence of preanalytical factors.

Methods: A cryobiopsy device (CBD) was developed by integrating a 19G cryoablation needle with a 16G cutting cannula of a clinical biopsy device. The device enables in-situ freezing of the tissue sample before it is cut and removed from the tumor.

We measured the stabilization of a highly labile biomarker, phosphorylated-MET (pMET), to compare between percutaneous biopsy samples acquired by the CBD (zero ischemic time) and by a clinical grade 18G biopsy device (Temno) (10-20 second ischemic time).

The animal protocol was approved by the FNLCR's IACUC. Athymic nude mice (nu/nu NCr) were implanted with human gastric carcinoma cell line SNU-5. Tumors were biopsied 3 weeks after implantation (tumor size range 150-250mm3).

The needle was passed through the skin into the tumor, a tissue sample was collected, and the biopsy needle was retracted. The tissue sample of the Temno device was rapidly frozen in a cryovial that was precooled in liquid nitrogen [1]. The already frozen tissue sample of the CBD was rapidly transferred to a second precooled cryovial. The vials were sealed and returned to liquid nitrogen. Frozen specimens were stored at −80°C until processing. Each tumor was biopsied once and the animal was euthanized.

The methodology to determine the pMET/MET levels in the biopsy samples is detailed in [2].

Results: The pMET/MET ratio was not significantly different between samples acquired by the CBD and by the Temno device (mean±st dev):

pY1234/35-MET/MET ratio: CBD 0.75±0.11; Temno 0.71±0.10

pY1356-MET/MET ratio: CBD 0.56±0.08; Temno 0.50±0.06

pY1235-MET/MET ratio: CBD 0.90±0.15; Temno 0.83±0.14

Conclusions: Past studies showed that tissue levels of highly labile biomarkers like pMET can change significantly in 1-2 minutes from the time of tissue harvesting [2]. The current study demonstrates that rapid freezing of the tissue within less than 20 seconds from tissue harvesting maintains the in-vivo levels of pMET similarly to tissue freezing before tissue harvesting. This result suggests that devices that rapidly freeze tissue samples after removal from the body, which are more suitable for clinical use, may be sufficient for preservation and accurate quantitation of highly labile biomarkers.

1. PMID: 18980982

2. PMID: 27001313

Supported by NCI Contracts HHSN261200800001E and HHSN261201200050C

#4538

**LiquidGx** TM **qPCR, an extremely sensitive qPCR-based liquid biopsy platform.**

Yang Song, Danielle Quintanilha, Si Chen, Jun Huang. _Admera Health, South Plainfield, NJ_.

In recent years, circulating cell-free tumor DNA has emerged as an excellent source of biomarkers for cancer detection, diagnosis and monitoring. One great challenge of detecting cell-free tumor DNA molecules is that they are usually found in a large background of wild-type DNA; therefore, assays with high sensitivity and low limit of detection are required. We have developed an extremely sensitive qPCR-based liquid biopsy platform, LiquidGxTM qPCR, to detect and track key driver mutations from patient blood. The platform takes advantage of our patent-pending technology BESTTM (Blocker-based Enrichment System of Tumor DNA) to specifically enrich mutant alleles from as low as 0.01% to more than 90%. With this platform, we currently have developed a CLIA-approved non-small cell lung cancer panel (EGFR T790M/L858R/C797S/Exon19Del, KRAS G12X/G13CD, BRAF V600E, ALK-EML4 fusion), and a colorectal cancer recurrence monitoring panel (KRAS G12X/G13CD, NRAS G12DV/Q61KR, PIK3CA E542K/E545K/H1047R, BRAF V600E). Our validation data show that the NSCLC panel has superior performance, with a LOD as low as 0.005%, analytical sensitivity more than 95% and analytical specificity of 100%. We have used this panel to test samples from nearly 200 NSCLC patients, and the results are highly concordant with those obtained by error-reducing NGS-based tests, despite some samples only detected via LiquidGxTM qPCR due to superior LOD. Other features of LiquidGxTM qPCR include (1) simultaneous enrichment of multiple mutants in the same reaction; (2) cost-effective and no special equipment required; and (3) fast turnaround time that takes as little as 0.5 days to get the results. In summary, we have developed a potent liquid biopsy platform for highly sensitive genomic biomarker test. With the technology, we have developed two cancer panels and this platform is also available as an assay development service.

#4539

Examination of zone-based radiomic features for characterization of dominant intraprostatic lesions using MR multi-modal information.

Hassan Bagher-Ebadian, Branislava Janic, Chang Liu, Milan V. Pantelic, David Hearshen, Mohamed Elshaikh, Benjamin Movsas, Indrin J. Chetty, Ning Wen. _Henry Ford Health System, Detroit, MI_.

Purpose: We investigated radiomic features extracted from dominant intraprostatic lesions (DILs) of the peripheral zone (PZ) and central gland (CG) from MR multi-modal images of 20 patients with prostate cancer (PCa). Remaining prostate gland (RPG) were included in the analysis.

Material and Methods: 20 biopsy-proven PCa patients with no prior radiation treatment were studied. Axial T2 weighted images (T2WI) and diffusion weighted images (DWI) were acquired of the pelvis using a 3T MR scanner. ADC maps were constructed from DWIs. Region of interests delineating DILs and RPGs were contoured on each MR modality. 168 radiomic features were extracted from DIL and RPG volumes (15 pairs from PZ and 5 pairs from CG). Radiomic features were categorized into 8 different sets: Intensity Based Histogram (IBH, 9 features), Gray Level Run Length (GLRL, 7 features), Law's Textural Information (LAWS, 18 features), Discrete Orthonormal Stockwell Transform (DOST, 18 features), Local Binary Pattern (LBP, 6 features), 2D Wavelet Transform (2DWT, 48 features), 2D Gabor Filter (2DGF, 40 features), and Gray Level Co-Occurrence Matrix (GLCM, 22 features). ANOVA (with Bonferroni adjustment), overall mean percent difference (OMPD), and the Fisher combined probability were used to test the following 7 hypotheses: (1) DILs of PZ and CG from T2WI (2) DILs of PZ and CG from ADC (3) DILs and RPGs of PZ from T2WI (4) DILs and RPGs of PZ from ADC (5) DILs and RPGs of CG from T2WI (6) DILs and RPGs of CG from ADC (7) DILs of PZ for T2WI and ADC.

Results: Results imply that among 168 radiomics features, only 5 (DOST, and 2DGF, OMPD=107.8%), 2 (2DWT, and 2DGF, OMPD=141.7%), 13 (IBH, 2DWT, and GLCM,

OMPD=%226.6), 17 (IBH, 2DWT, and GLCM, OMPD=179.7%), 18 (IBH, 2DWT, and GLCM, OMPD=321.9%), 18 (IBH, 2DWT, and GLCM, OMPD=726.1%), and 74 (IBH, GLRL, LAWS, DOST, LBP, 2DWT, 2DGF, GLCM, OMPD=1564%) features are discriminant (pFisher <0.050 with Confidence Level of 95%) for hypotheses no. 1 through 7 respectively.

Conclusion and Discussion: Results for the discriminant features identified from hypotheses no. 1 and 2 can be used to construct a predictive model with a higher performance that benefits from the zone-based information (PZ and CG) as a-priori knowledge. As most of the discriminant features in tests no. 1 to 7 are primarily entropy-based, this suggests that potential feature-based biomarkers of DILs in PCa patients are more associated with spatial-locality, and frequency-based characteristics of MR images. The high value of the OMPD for test no. 7, strongly supports the use of the two MR modalities for increasing the information gain in perfecting predictive models for detection of DILs in PCa studies. The results of this pilot study, albeit subject to confirmation in a larger patient population, suggest a potential role for the use of zone-based radiomics information in models developed for detection of DILs in PCa patients.

#4540

Development of well characterized breast, lung, and brain cancer copy number variation reference materials.

Dana Ruminski Lowe, Jessica Dickens, Catherine Huang, Yves Konigshofer, Bharathi Anekella. _Seracare Life Sciences, Gaithersburg, MD_.

Introduction: Copy number variation (CNV), including gene amplification and deletion, can be a key driver of oncogenesis. Pathogenic CNVs are often associated with unfavorable prognosis and drug resistance, therefore detection of these types of genetic changes will be important for personalized treatment. Many tumor profiling workflows can detect CNVs in addition to somatic mutations, but well-characterized reference materials for CNVs are not widely available, which makes it difficult to assess accuracy and sensitivity. CNV content of cell lines can be variable and may change with passages, the genomic background may not be diploid, and germline and somatic variants may not be well characterized. Remnant patient samples often lack characterization and the volumes needed for such characterization and larger validation studies. Therefore, we developed and evaluated Seraseq CNV reference materials to serve as accuracy, precision, and limit-of-detection (LOD) controls for tumor profiling assay development and validation. By providing a consistent source of purified DNA with stable CNV content, these reference materials allow for repeatable assessment of clinical diagnostic assays targeting CNVs. Methods: Seraseq CNV reference materials were prepared by mixing genomic DNA with additional copies of target genes from the well-characterized cell line GM24385. Copies of ERBB2, FGFR3, and MYC were combined with genomic DNA to simulate CNVs found in breast cancer, while sequences for EGFR, MET, and MYCN were used to mimic lung and brain cancer CNVs. Three levels of amplification were targeted: +3, +6, and +12 additional copies. The copy number of each gene was precisely quantified using digital PCR and confirmed on several NGS-based assays internally and externally. Results & Conclusions: Testing revealed CNVs of each target gene were close to the expected copy number. Good correlation was observed between digital PCR data and NGS data generated internally with the Archer VariantPlex Solid Tumor assay. In addition, internal and external NGS data showed correlation and broad assay compatibility. While CNVs observed clinically can be upwards of 50 additional copies and more easily detectable, LOD controls such as those described here are imperative in evaluating assay performance. Collectively, the data demonstrate the utility of biosynthetic Seraseq CNV reference materials, which serve as superior alternatives to DNA from cell lines or patient-derived material.

#4541

MET **gene copy number gains evaluated by NGS is more predictive than other methods to enrich for papillary RCC patients sensitive to savolitinib, a selective MET inhibitor.**

Melanie M. Frigault,1 Sabina Signoretti,2 Aleksandra Markovets,3 Daniel Stetson,3 William Howat,4 Martine Roudier,4 Vincent Haddad,4 Brian Dougherty,3 Toni K. Choueiri,5 J. Carl Barrett3. 1 _Acerta Pharma, Redwood City, MA;_ 2 _Brigham and Women's Hospital, Boston, MA;_ 3 _AstraZeneca Pharmaceuticals LP, Waltham, MA;_ 4 _AstraZeneca Pharmaceuticals LP, Cambridge, United Kingdom;_ 5 _Dana-Farber Cancer Institute, Boston, MA_.

There is no approved therapy specifically for the treatment of papillary renal cell carcinoma (PRCC). Advances in molecular profiling of PRCC have identified a segment of PRCC with 10% MET mutation rates (Linehan et al 2016). Chromosome 7 gain is a hallmark of PRCC and thought to occur at 50% frequency in PRCC (Jiang et al 1998). We undertook a retrospective analysis of archival tumors to evaluate MET pathway aberrations for correlation with efficacy in a phase II study of savolitinib (volitinib, AZD6094, HMPL-504) in patients with PRCC (NCT02127710). Archival diagnostic tumor samples were mandated for central confirmation of PRCC diagnosis, histological subtyping, and for exploratory biomarker analysis. Eighty-four archival tumors were obtained and profiled using four methods: H&E stain for PRCC histological subtype, immunohistochemistry (IHC) for c-Met protein expression (Ventana, CONFIRM), fluorescent in situ hybridization (FISH) for MET gene amplification (Abbott, VYSIS), and Next Generation Sequencing (NGS) as an orthogonal method for confirmation of MET amplifications, detection of HGF gene amplifications, MET mutations, chromosome 7 ploidy, and other exploratory genomic biomarkers (Foundation Medicine Inc., T7 panel). Eight patients with a confirmed partial response (PR) to savolitinib were used to define MET-driven PRCC. Retrospective analysis demonstrated that only NGS, not IHC, FISH or histological subtype, identified all PRs. NGS detected any one of the following biomarkers alone or in combination in PRs: chromosome 7 copy number gain, MET or HGF gene focal amplification, MET kinase domain mutations. The frequency of these biomarkers in the phase II population were 31.5%, 10%, 1%, and 7.5%, respectively and captured all responders (18% ORR); however, 42% of non-responders (PD and SD) were also classified as MET-driven by NGS. Focal MET amplifications were confirmed with MET FISH testing, but FISH was unable to identify gains in chromosome 7. IHC was not as sensitive as NGS (57% vs 100%) but had a higher specificity. Molecular characterization of MET status was more predictive of response to savolitinib than a classification based on pathology.

#4542

**Analytical validation of the Signatera** TM **RUO assay, a highly sensitive patient-specific multiplex PCR NGS-based noninvasive cancer recurrence detection and therapy monitoring assay.**

Himanshu Sethi, Raheleh Salari, Samantha Navarro, Prashanthi Natarajan, Ramya Srinivasan, Scott Dashner, Tony Tin, Mustafa Balcioglu, Ryan Swenerton, Bernhard Zimmermann. _Natera, Inc., San Carlos, CA_.

Introduction: Liquid biopsy-based detection and monitoring of tumor-specific somatic mutations in cell-free DNA has great potential to improve patient care by detecting cancer early, assisting adjuvant therapy decision-making, monitoring residual disease, determining treatment effects, assessing relapse and the consequent need for follow-up intervention. We have developed a novel highly sensitive and specific approach for the detection and quantification of circulating tumor DNA (ctDNA) by tracking personalized cancer signatures in plasma. We recently described our ability to detect ctDNA presence longitudinally in patients with non-small cell lung cancer (NSCLC) and have validated this personalized approach for detection of tumor DNA in plasma, and made it available for research use only (RUO).

Summary of Experimental Procedures: This study describes results of the Signatera RUO assay's analytical validation test titrations of cancer cell lines and their matched normal counterparts. Multiplex-PCR (mPCR) assay pools specific to the tumor mutational signatures were derived from whole-exome data analysis of the tumor cell line and its corresponding matched normal cell line. Tumor DNA spike-in mixtures were amplified using the mPCR assays and then barcoded and sequenced at ultra-high depth on the Illumina HiSeq instrument (average > 100,000 reads per target). This study also demonstrates the ability to detect ctDNA using Signatera RUO assay's personalized somatic mutations in patients with epithelial and non-epithelial ovarian cancer.

Results/Data Summary: The Signatera RUO analytical validation results demonstrate a variant-level sensitivity of ~60% at spiked-in tumor DNA concentrations of 0.03% along with an assay specificity of 99.9% in wild-type DNA. By targeting 16 somatic mutations present in the tumor, an estimated LOD of 0.01% tumor DNA in a patient's plasma is achieved. The Signatera RUO assay also demonstrates the ability to detect ctDNA in plasma down to less than 0.1% VAF at baseline (prior to treatment) in epithelial and non-epithelial ovarian cancer.

Conclusion: The Signatera RUO assay provides a unique method to noninvasively detect ctDNA by assaying for personalized cancer signatures in plasma by ultra-high-depth sequencing of custom-made multiplex PCR assays, with high sensitivity and high specificity. Our study demonstrates the potential of this technology to improve the current standard of care by enabling early recurrence detection and the monitoring of treatment effectiveness across several tumor types.

#4543

Combined analysis of cell-free DNA methylation in plasma for early detection of NSCLC.

Li Cao,1 Hao Wang,2 Xu Dong,1 Li Zhong2. 1 _Hebei University College of Life Sciences, Baoding, China;_ 2 _Western University of Health Sciences, Pomona, CA_.

Introduction: Detection of cell-free DNA (cfDNA) methylations in plasma samples may provide diagnostic information for non-small lung cancer (NSCLC). Here, we evaluated 5 preselected genes for methylation analysis from patient and control plasma samples.

Method: Plasma samples were collected from 91 patients who were diagnosed with primary NSCLC with pathological or cytological confirmation. All the cases did not received any surgery or chemotherapy treatments when blood samples were collected. Meanwhile, 58 cases of healthy blood samples were collected as the control group. Methylation specific PCR (methylation specific-PCR, MS-PCR) was used to detect RUNX3, RASSF1A, 3-OST-2, DAPK, PTPRO genes methylation. Data was analyzed individually as well as jointly for the optimal diagnostic accuracy.

Results: The DNA methylation rates for the 5 genes RUNX3, RASSF1A, 3-OST-2, DAPK, PTPRO were 47.25% (43/91), 51.65% (47/91), 27.47% (25/91), 36.26% (33/91), and 41.76% (38/91) respectively in patient plasma samples. In the normal control group, only PTPRO gene methylation was detected with the methylation rate of 5.2% (3/58), and the rest of 4 genes were not detected. Methylation of RUNX3 gene was found to have higher frequencies in adenocarcinoma than other cell types of NSCLC (P < 0.05). DAPK gene methylation was found to be associated with the stages of lung cancer, and methylation rate was higher in stage III/IV than in stage I/II (P < 0.05). Combined analysis of the 5 genes methylation rates was achieved the sensitivity 86.81% (79/91) and the specificity 79.31% (46/58).

Conclusion: The combined DNA methylation analysis of RUNX3, RASSF1A, 3-OST-2, DAPK, and PTPRO genes with MS-PCR achieved a satisfied sensitivity and specificity in NSCLC diagnosis.

#4544

A validated diagnostic assay for identifying ovarian cancer patients with deleterious BRCA mutations and high genomic loss of heterozygosity (LOH).

James X. Sun,1 Kevin Lin,2 Yali Li,1 Kyle Gowen,1 Yuting He,1 Coren Milbury,1 Christine Burns,1 Jun Luo,1 Steve Roels,1 Murtaza Mehdi,1 John Truesdell,1 Pei Ma,1 Lakshman Ramamurthy,1 Christine Vietz,1 Jeri Beltman,2 Thomas Harding,2 Doron Lipson,1 Jeffrey Ross,1 Vincent Miller,1 Philip Stephens,1 Michael Doherty,1 Julia Elvin1. 1 _Foundation Medicine, Inc, Cambridge, MA;_ 2 _Clovis Oncology, CO_.

Introduction: In patients with advanced-stage ovarian cancer, FDA has recently granted approval of treatment with PARP inhibitors (PARPi) in patients harboring deleterious BRCA mutations (~25% of population). However, there is clear evidence of "BRCA-like" patients who respond to PARPi without BRCA mutations. To identify such patients, it has been shown that deficiency in homologous recombination repair leads to a common phenotype of genome-wide loss of heterozygosity (LOH). Depending on the cutoff employed, genomic LOH could identify more than twice the number of ovarian cancer patients who could benefit from PARPi than measuring BRCA alone. We present here an NGS-based platform developed and performed in compliance with FDA 21 CFR part 820. The assay provides a tumor measurement of BRCA1/2 (FDA-approved as FoundationFocus CDx BRCA) as well as genomic LOH, and is on the same platform as the comprehensive FoundationOne CDx, which interrogates 324 genes. Methods: DNA extracted from FFPE tumor tissue underwent whole-genome shotgun library construction and hybridization-based capture, followed by sequencing using Illumina HiSeq 4000. Sequence data were processed using a proprietary analysis pipeline designed to detect base substitutions, indels, copy number alterations (CNA), genomic rearrangements, microsatellite instability (MSI), and tumor mutational burden (TMB). A genome-wide LOH profile based on SNPs is measured as part of the CNA pipeline, and is summarized as the percentage of the tumor genome displaying LOH (scored from 0-100%), with ≥16% being considered LOH high based on clinical data derived from ARIEL2 Part 1, a phase II study of the PARPi rucaparib for the treatment of platinum-sensitive ovarian cancer (ARIEL2; NCT01891344) Results: For analytical validity, BRCA limit of detection (LoD) was at allele frequency 5.9% for substitutions and non-repetitive indels, and 30% tumor content for LOH. Overall percent agreement with comparator NGS assay was 97.3% for BRCA. No orthogonal platform concordance was established for LOH as no validated test exists. Within-assay reproducibility was measured with overall concordance of 100% for BRCA, and 98% for LOH. Conclusion: We developed a novel diagnostic assay (in compliance with FDA 21 CFR part 820) that can measure BRCA and genomic LOH simultaneously, and established robust analytical validation data.

#4545

A liquid biopsy for breast cancer diagnosis.

Jennifer Sims-Mourtada,1 Kimberly M. Arnold,1 Adam Marsh2. 1 _Helen F. Graham Cancer Ctr., Newark, DE;_ 2 _Genome Profiling, LLC, Newark, DE_.

Purpose: Although ductal carcinoma in situ (DCIS) is considered a precursor to invasive breast cancer, only 30-50% of DCIS lesions progress to invasive cancers. There has been considerable debate over the treatment of DCIS, with some groups recommending frequent monitoring of this population for progression instead of aggressive treatment. This approach would require either recurrent biopsy, an unattractive option for most women, or a useful diagnostic test capable of discriminating DCIS and invasive breast cancer. Here, we report a blood diagnostic that is capable of detecting and discriminating epigenetic biomarkers in breast lesions that are more likely to become either invasive or those that are more benign. Our approach is based on the premise that circulating lymphocytes undergo epigenetic changes upon exposure to the tumor microenvironment and these changes can be followed to track tumor progression from a non-invasive to an invasive disease state. To test the utility of these epigenetic biomarkers, a novel diagnostic model utilizing a combined machine learning approach integrated with the recombinatorial strategy of a genetic algorithm, was used to screen a large response space of 1,000's of CpG sites to winnow down to a small set of 40 CpG sites with the highest combined predictive power. Methods: Blood was obtained from women with histologically confirmed DCIS (n=14), histologically confirmed invasive ductal carcinoma (n=10) or women with no evidence of breast lesions on mammography (n=10). Lymphocytes were isolated using a modified Ficoll-Paque gradient, and DNA was extracted using standard commercial kits. Epigenetic profiling of DNA was performed using a highly sensitive and quantitative analytics platform, which utilizes methylation sensitive restriction endonucleases to detect changes in methylation of CpG sites from standard NGS data.Results: Non-metric multidimensional scaling ordination analysis of the CpG sites revealed highly distinct methylation patterns between Normal, DCIS and invasive samples. Using a Likelihood Ratio Test with defined ANOVA contrasts, over 14,000 significantly different methylated CpG sites were identified (p<0.05 after false discovery rate correction). A proprietary machine learning diagnostic model was employed to reduce this high-dimensional variable space to the most effective set of CpG sites. DCIS and invasive samples were discriminated in blinded tests (n=17) with 69% accuracy. Conclusions: Preliminary studies show strong ability of the identified DNA methylation metrics to detect and discriminate invasive and non-invasive breast lesions. The quantitative sensitivity and selectivity of this new diagnostic/prognostic blood test will be determined in larger patient cohorts. 

### Immune Checkpoints 4

#4546

The tumor inflammation signature is predictive of anti-PD1 treatment benefit in the CERTIM pan-cancer cohort.

diane damotte,1 jennifer arrondeau,2 pascaline boudou-rouquette,2 audrey lupo,3 jerome biton,3 hanane ouakrim,3 Marco Alifano,1 Gervais Claire,1 Audrey Bellesoeur,1 Nora Kramkimel,2 Angeline Duche,4 Franck Letourneur,4 Xing Ren,5 Pascale Morel,5 Alessandra Cesano,5 Sarah Warren,5 François Goldwasser,1 Karen Leroy1. 1 _University Paris Descartes APHP, Paris, France;_ 2 _APHP, Paris, France;_ 3 _INSERM U1138, Paris, France;_ 4 _INSERM, Paris, France;_ 5 _Nanostring, Seattle, WA_.

Purpose of the study : The 18-gene Tumor Inflammation Signature (TIS) (Ayers et al, J Clin Invest 2017; 127:2930) is a clinical research assay that enriches for clinical response to immune checkpoint blockade. In this work, the performance of the TIS was evaluated on standard tumor specimens for its ability to predict clinical benefit of immunotherapy in a cohort of cancer patients treated with PD1 checkpoint inhibitors in routine clinical care.

Experimental procedures : Consecutive metastatic cancer patients treated with anti-PD1 in the outpatient monocentric CERTIM cohort with available formalin fixed paraffin embedded tumor specimen were selected for analysis. RNA were extracted from tissue samples with High Pure FFPET RNA Isolation Kit (Roche) and 25-100 ng RNA was hybridized to a pilot version of the NanoString® PanCancer Immuno-oncology 360 Panel. The association between individual gene expression (GE), TIS and other GE signatures with outcome (response to therapy and overall survival) was assessed with logistic resgression model, LogRank tests and Cox model.

Results : A total of 69 patients with non-small cell lung carcinoma (NSCLC, n=42), small cell lung carcinoma (n=2), melanoma (n=9), renal cell carcinoma (n=10), urothelial carcinoma (n=5) or colorectal carcinoma (n=1), who had received nivolumab (n=59) or pembrolizumab (n=10) were analyzed. All samples but one SCLC could be successfully analyzed, demonstrating the feasibility of the testing even in small biopsy samples with little available RNA (>30 ng). In the whole cohort, clinical response (PR or CR versus SD or progression) was significantly associated with TIS score [OR=2.002, 95%CI=(1.123,3.977), p =0.029] as well as with the individual expression of PDL1, CTLA4, IDO1 or GE scores characterizing Th1, cytotoxic, NK cells infiltration, immuno-proteasome or antigen processing machinery. Receiver operating characteristics for response status demonstrated the high discriminatory ability of the TIS signature in this unselected series of patients (area under the ROC curve 0.71). Patients with a high TIS score (> 9.39) showed a longer OS (median OS 20.2 months) compared to those with lower scores (median OS 5.6 months, p value = 0.0017). A high TIS score was also significantly associated with clinical response when the analysis was limited to the NSCLC (n=42, [OR=3.156, 95%CI=(1.168,11.455), p-value=0.042] - or showed a strong trend in lung adenocarcinoma (n=29 [OR=11.251, 95%CI=(0.962,131.534), p-value=0.054] patients.

Conclusion : Due to costs and toxicity, it is mandatory to identify the patients who benefit of immune checkpoint inhibitors. Here we demonstrate that the Tumor Inflammation Signature, that reflects key processes involved in anti-tumor immune response, may be used in routine samples to predict clinical benefit and prolonged survival of cancer patients.

#4547

Characterization of the LAG-3 targeting antibody BI 754111 in monotherapy and in combination with the anti-PD-1 antibody BI 754091.

Markus Zettl,1 Melanie Wurm,1 Otmar Schaaf,1 Iñigo Tirapu,1 Sven Mostböck,1 Markus Reschke,1 Stephan-Michael Schmidbauer,1 Lee Frego,2 Ivo C. Lorenz,2 Michael Thibodeau,2 Diann Blanset,2 Elisa Oquendo Cifuentes,2 Jürgen Moll,1 Norbert Kraut,1 Eric Borges,3 Anne Vogt,1 Jonathon Sedgwick,2 Irene C. Waizenegger1. 1 _Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria; _2 _Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT;_ 3 _Boehringer Ingelheim Pharma GmbH & Co KG, Biberach, Germany_.

Lymphocyte activation gene-3 (LAG-3) is an inhibitory receptor involved in maintaining immunological tolerance via regulation of T-cell activation, proliferation and response. Ligand-mediated activation of LAG-3 negatively regulates T-cell activity that is thought to actively contribute to tumor immune evasion beyond the established programmed cell death-1 (PD-1) pathway.

BI 754111, a humanized IgG4 monoclonal antibody (mAb) with high affinity against LAG-3, blocks the interaction between LAG-3 and MHC II. BI 754111 was characterized in a panel of binding, blocking and functional cell-based assays; safety assessment was done in cynomolgus monkeys. BI 754111 is not mouse cross-reactive; therefore a surrogate mLAG-3 antibody was used for in vivo mouse efficacy studies. The ability of BI 754111 to stimulate cytokine production by exhausted human T cells in vitro was tested in an autologous assay system with antigen specific memory T cells being re-stimulated by antigen-pulsed dendritic cells in the presence of increasing amounts of BI 754111 or BI 754091 (anti-hPD-1 mAb) or a combination of increasing amounts of BI 754111 and a saturating dose of BI 754091. Under these assay conditions the majority of T cells co-expressed the exhaustion markers PD-1 and LAG-3 on the surface. At the end of the experiment supernatants were harvested and analyzed for IFNγ secretion as a measure of T-cell activation. Monotherapy of BI 754111 moderately increased IFNγ secretion (average of 1.8 fold) compared to BI 754091 monotherapy (6.9-fold average increase). Combining BI 754111 with BI 754091 was synergistic and led to a 13.2-fold increase in IFNγ secretion.

MC-38 tumor-bearing mice (C57BL/6NTac-PDCD1tm(PDCD1)Arte mice expressing parts of the human instead of the murine extracellular domain of PD-1) were used to determine the in vivo activity of the anti-LAG3 and anti-PD-1 combination. A mouse tool antibody against mLAG-3 (IgG1 D265A) and BI 905725 (a mouse IgG1 D265A version of BI 754091, anti-hPD-1 mAb) were tested at a dose of 10 mg/kg in a q3or4d schedule as monotherapy or in combination. No anti-tumor activity was observed under mLAG-3 mAb treatment, whereas treatment with anti-PD-1 resulted in a median TGI of 100% and 4 out of 10 tumors showed a complete response. The combination of anti-PD-1 and anti-LAG-3 resulted in a median TGI of 103% and doubled the number of complete responses (8/10).

BI 754111 binds to cynomolgus monkey LAG-3 with comparable affinity as to hLAG-3 thus allowing pharmacokinetic and toxicological assessment in this species. Repeated high doses of BI 754111 were well tolerated without adverse immune-related side effects.

The above results describe synergistic effects upon combination of PD-1 and LAG-3 treatment thus supporting the ongoing clinical trial in which BI 754111 is being tested in combination with BI 754091 (NCT03156114).

#4548

Host immunogenetics and hyperprogression under PD1/PD-L1 checkpoint inhibitors.

Sadal Refae, Nathalie Ebran, Jocelyn Gal, Josiane Otto, Damien Giacchero, Delphine Borchiellini, Joel Guigay, Frederique Peyrade, Gerard Milano, Esma Saada. _Centre Antoine Lacassagne, Nice, France_.

Background: Hyperprogressive disease (HPD) has been described in advanced solid tumors patients treated with anti PD1 anti PDL1 monotherapy (Champiat, Clin Can Res 2016; Saada-Bouzid, Ann Oncol, 2017). Our aim was to explore whether host constitutional variations in the nucleotide sequence of genes involved in immune response might be associated with the occurrence of HPD in patients treated with anti PD-(L)1.

Methods : From April to August 2017, 98 patients were treated in the Centre Antoine Lacassagne (Nice, France) with anti PD-(L)1. Potential SNPs for four candidate genes with a minor allele frequency≥ 5% in caucasians were selected according to genome browser data-base: PD1 (rs10204525, rs11568821, rs2227981), PD-L1 (rs2282055, rs2297136, rs2297137, rs4143815, rs10815225), IDO1 (rs3739319, rs3808606, rs373931, rs9657182) and VEGR2 (rs2305948, rs1870377, rs2071559). High through-output genotyping was done by MassARRAY (AGENA). HPD was defined as a TGKR (ratio of the slope of tumor growth before treatment and the slope of tumor growth on treatment) ≥ 2.

Results: Patients were treated for head and neck squamous cell carcinoma (16/98), non-small cell lung cancer (48/98), melanoma (13/98), renal cell carcinoma (14/98) and 7 others. Median age was 66 years old, 33 were male. All tested SNPs were in Hardy-Weinberg equilibrium. Responses were associated with G3-G4 toxicities (10% vs 42%, p=0.001). TGKR was assessable in 80 patients. HPD was observed in 11/80 (14 %) patients. HPD was significantly associated with age≥70y (6% vs 25%, p=0.02) and genotype for PD1 rs 2227981 (8/32=35% G/G vs 2/43=5% A/G or A/A, p=0.02), PDL1 rs 2282055 (1/37=3% T/T vs 10/43=23% T/G or G/G,p=0.02) and VEGFR2 rs1870377 (2/48=5% T/T vs 9/32=28% T/A or A/A, p=0.005). Multivariate analysis confirmed the association between HPD and VEGFR2 rs1870377 T/A or A/A (OR=15.3, p=0.007), PDL1 rs 2282055 T/G or G/G (OR=18.7, p=0.01) and age≥70y (OR=14.4, p=0.006).). Combining the three alleles at risk for HPD gave an OR =12.4 (p=0.001).

Conclusion: In patients treated with anti PD-(L)1, HPD was independently associated with older age and host related gene variations. Host related immunogenetics could become an integrative part of predictive factor for immunotherapy outcome.

#4549

Pemetrexed enhances anti-tumor efficacy of PD1 pathway blockade by promoting intra tumor immune response via immunogenic tumor cell death and T cell intrinsic mechanisms.

Ruslan Novosiadly,1 David Schaer,1 Nelusha Amaladas,1 Erik Rasmussen,1 Zhao Hai Lu,2 Andreas Sonyi,1 Carmine Carpenito,1 Yanxia Li,1 Shuang Luo,2 Andrew Capen,2 Catalina Meyer,2 Xiaodong Huang,1 Jason Manro,2 Gregory Donoho,2 Thompson Doman,2 Gerald Hall,1 Sandaruwan Geeganage,2 Michael Kalos1. 1 _Eli Llilly & Co, New York, NY; _2 _Eli Llilly & Co, Indianapolis, IN_.

Background: Recently, pemetrexed and carboplatin in combination with PD–1 antibody (pembrolizumab) demonstrated markedly improved clinical outcome in NSCLC patients (KEYNOTE–021G trial) suggesting a positive interaction between pemetrexed–based chemotherapy and immunotherapy. However, the role of pemetrexed in modulating antitumor immune response is largely unknown. The objective of this study was to characterize the effects of pemetrexed on intra–tumor immune response in monotherapy and combination with carboplatin or PD–1 pathway blockade in preclinical models.

Methods: Mice bearing syngeneic MC38 or Colon26 tumors were treated with pemetrexed with or without carboplatin or anti–mouse PD–L1 antibody (178G7). Immune cell subsets and immune–related changes in mouse tumor tissue and T cells were assessed by flow cytometry and gene expression analysis (Quantigene Plex and nanoString nCounter assays). Effects of pemetrexed on immunogenic cell death in tumor cells and mitochondrial respiration in T cells were evaluated by HMGB1 ELISA and Agilent Seahorse XF analysis, respectively.

Results: In MC38 tumors, pemetrexed monotherapy demonstrated a trend towards an increased frequency of intratumoral leukocytes and total and cycling (Ki67+) T cells accompanied by immune–related gene expression changes indicative of enhanced antigen presentation, T cell infiltration and/or activation and down–modulation of the myeloid cell compartment. Immune gene expression signature induced by pemetrexed was largely unaffected by carboplatin. In both MC38 and Colon26 tumor cells, in vitro treatment with pemetrexed induced a robust release of HMGB1 indicative of immunogenic cell death. Proliferation of primary human T cells stimulated with CD3/CD28 was inhibited by pemetrexed in a dose–dependent manner. At clinically relevant concentrations pemetrexed also enhanced T cell activation phenotype exemplified by an increased frequency of CD137+, GITR+ and PD–L1+ T cells as well as upregulation of multiple interferon gamma–induced genes, and increased mitochondrial respiration. In both MC38 and Colon26 models, treatment with pemetrexed and 178G7 demonstrated a combination benefit compared to either monotherapy, and nCounter profiling of Colon26 tumors followed by Ingenuity Pathway Analysis revealed that improved antigen presentation, enhanced T cell and cytokine signaling and an engagement of CD4+ T cell–mediated immunity might underlie this combination effect.

Conclusions: Pemetrexed promotes intra–tumor T cell–mediated immune response through immunogenic tumor cell death and increased activation and metabolic fitness of T cells leading to an enhanced anti–tumor efficacy in combination with PD–L1 antibody.

#4550

CD80 vIgD-Fc proteins combine checkpoint antagonism and costimulatory signaling for potent antitumor immunity.

Ryan Swanson, Mark F. Maurer, Chris L. Navas, Chelsea J. Gudgeon, Joseph L. Kuijper, Martin Wolfson, Katherine E. Lewis, Stacey R. Dillon, Steve D. Levin, Michael G. Kornacker. _Alpine Immune Sciences, Seattle, WA_.

Introduction: PD-1 pathway antagonists have revealed the importance of checkpoint pathways in regulating antitumor immunity, but an existing immune response is generally required for clinical efficacy. Specific T cell costimulation through CD28 is central to this process, but the CD28 ligands CD80 and CD86 are often poorly expressed in the tumor microenvironment, accounting for a second important mechanism of immune evasion by tumors. In contrast, PD-L1 expression has been found extensively in multiple tumor cell types. Therapeutics that combine PD-L1/PD-1 antagonism coupled with PD-L1 dependent CD28 agonism may therefore provide a more potent, yet safe immunotherapeutic approach.

Experimental Procedures: The variant Ig Domain (vIgD)TM platform has generated a diversity of human CD80 variants using yeast display affinity maturation and selections against all three CD80 counterstructures CD28, CTLA-4, and PD-L1. CD80 vIgDs were produced in a mammalian expression system as recombinant Fc fusion proteins (CD80 vIgD-Fc proteins) and their binding properties were quantified by flow cytometry. Functional activity was determined in vitro by assessing responses from human primary T cells or an IL-2-luciferase Jurkat T cell reporter line stimulated with PD-L1-expressing artificial antigen presenting cells (aAPCs). In vitro human T cell cytotoxicity assays with a human PD-L1-expressing tumor line were also performed. Antitumor activity was assessed in vivo with mice implanted with human PD-L1 transduced MC38 tumors.

Data Summary: The human CD80 IgV fragment was found to be optimal for high affinity PD-L1 and CD28 binding. A large panel of CD80 vIgD-Fc proteins demonstrated a range of binding towards CD28, PD-L1, and/or CTLA-4, and CD80 vIgD-Fc proteins with high affinity for PD-L1 antagonized the PD-L1/PD-1 interaction. Some CD80 vIgD-Fc proteins agonized CD28 in a PD-L1 dependent fashion with increased luciferase activity in the Jurkat reporter assay as well as increased cytokine production by primary human T cells when stimulated with a PD-L1 expressing aAPC in vitro. The same candidates also showed specific killing of human PD-L1 expressing tumor cells in vitro compared to the parental tumor line lacking PD-L1 expression. Importantly, selected CD80 vIgD-Fc proteins caused significant tumor reduction in the MC38 in vivo tumor model.

Conclusion: Engineered CD80 vIgD-Fc proteins that deliver a localized CD28 costimulatory signal to T cells while simultaneously antagonizing the inhibitory PD-L1/PD-1 pathway may provide a transformative mechanism of action to drive potent, tolerable antitumor immunity. Preclinical development of therapeutic candidates is under way.

#4551

Thyroid hormone induces PD-L1 expression in oral cancer cells.

Yu-Tang Chin,1 Kwan-Wei Su,2 Yee-Tang Lim,1 Ya-Jung Shih,1 Yi-Ru Chen,1 Sheng-Yang Lee,1 Paul J. Davis,3 Hung-Yun Lin,1 Earl Fu4. 1 _Taipei Medical UNIV., Taipei, Taiwan;_ 2 _Hsinchu MacKay Memorial Hospital, Hsinchu City, Taiwan;_ 3 _Albany College of Pharmacy and Health Sciences, Albany, NY;_ 4 _Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, New Taipei City, Taiwan_.

Oral cancer is a fatal disease, which accounts for the fourth highest incidence of malignancy in males and the seventh highest in the general population of Taiwan. Oral cancer is increasing in Taiwan. About 95% of oral cancer in Taiwan is oral squamous cell carcinoma (OSCC). With the development of cancer molecular biology and immunology, targeted therapy for immune checkpoints of programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) has shown enormous development prospects for treatment of head and neck cancer. The PD-1/PD-L1 checkpoint is a critical regulator of activated T cell-cancer cell interactions which defend tumor cells against immune surveillance. Thyroid hormone induces PD-L1 expression in human oral cancer cells. Human oral cancer OEC-M1 and SCC-25 cells were treated with different concentrations of T4 (10-8 to 10-6M) for 24 h and cells were harvested and total RNA was extracted. qPCR of PD-L1 revealed that PD-L1 mRNA was significantly induced by thyroid hormone on a concentration-dependent basis. Parallel studies were conducted to study the effect of thyroid hormone on PD-L1 protein accumulation. Cancer cells were treated with different concentrations of T4 for 24 h. Total proteins were extracted and western blot analysis of PD-L1 was conducted. Thyroid hormone-activated ERK1/2 and STAT3 were companied with PD-L1 expression. Inhibition of ERK1/2 and consequently STAT3 activation also blocked PD-L1 induced by thyroid hormone. Knockdown of PD-L1 expression by siRNA also inhibits thyroid hormone-induced proliferation of oral cancer cells which indicated that PD-L1 expression is involved in thyroid hormone-induced cancer growth via an ERK1/2-STAT3 signal transduction pathway in oral cancer cells.

#4552

Tumor-restricted immune modulation by multispecific molecules from the DARPin toolbox.

Ulrike Fiedler, Christian Reichen, Joanna Taylor, Patricia Schildknecht, Sophie Barsin, Clara Metz, Anja Schlegel, Simon Fontaine, Denis Villemagne, Julia Ahlskog, Yvonne Kaufmann, Alexander Link, Nicolo Rigamonti, Julia Hepp, Michael T. Stumpp. _Molecular Partners AG, Schlieren-Zurich, Switzerland_.

During the last years, immune-modulating drugs became an important cornerstone in the treatment of cancer patients. In particular, the PD1/PDL-1 and CTLA-4 antagonists revolutionized the field. However, only a limited number of patients benefit from these antagonistic molecules and more combination therapies are on the way to increase the number of patients benefiting from these novel therapies. Among the combinations, drugs that are T-cell or myeloid cell agonists belonging to the TNFR-superfamily show first promising clinical results. However, systemic immune activation bears the risk of severe side effects that will not allow using these powerful drugs at an effective dose. We have developed a new class of DARPin molecules that enable tumor-restricted immune cell activation of TNFR-superfamily agonists in the tumor only, thereby preventing systemic immune-activation. We generated DARPin molecules that bind with high affinity to TNFR-superfamily members (CD134, CD137 and CD40) and DARPin molecules that bind to tumor-specific antigens such as HER2 and EGFR or targets restricted to the tumor stroma compartment like FAP and extra-domain B of fibronectin (ED-B). Using these building blocks from our DARPin toolbox, we constructed a variety of multi-specific molecules consisting of a TNFR-superfamily receptor targeting DARPin molecule and a tumor-localizing DARPin molecule. In reporter cell assays the multi-specific DARPin molecules activate the respective TNF-superfamily receptor only in the presence of a cell expressing the tumor-localizer; e.g. the FAP-CD134 molecule activates CD134 only in the presence of a stromal cell expressing FAP and not in its absence. Moreover, activation of CD134 was dependent on the density of FAP expression on cells, showing that CD134 only becomes activated if a certain level of the tumor-localizing target is expressed. These finding were confirmed in experiments with primary immune cells where we see immune-cell activation only upon binding to the tumor-localizing target. This could be shown for multiple combinations (>8) and supports the concept that multi-specific DARPin molecules are powerful drug-candidates allowing tumor-restricted immune cell activation.

#4553

A novel bispecific antibody for EGFR-directed blockade of the PD-1/PD-L1 immune checkpoint.

Iris Koopmans,1 Douwe F. Samplonius,1 Robert J. van Ginkel,1 Peter J. Wierstra,2 Sandra Heskamp,2 Edwin Bremer,1 Wijnand Helfrich1. 1 _University Medical Center Groningen, Groningen, Netherlands;_ 2 _Radboud University Medical Center, Nijmegen, Netherlands_.

PD-L1-blocking antibodies produce significant clinical benefit in selected cancer patients by reactivating functionally-impaired anticancer T cells. However, their efficacy is potentially reduced by 'on-target/off-tumor' binding to PD-L1 widely expressed on normal cells. This lack of tumor selectivity potentially induces a generalized activation of all antigen-experienced T cells as is evidenced by frequent autoimmune-related adverse events during and after treatment. To address these issues, we constructed a tetravalent bispecific antibody (bsAb), designated PD-L1xEGFR, to direct PD-L1-blockade to EGFR-expressing cancer cells and thus reactivate anticancer T cells more selectively. The IC50 of PD-L1xEGFR for binding to EGFR+ cancer cells was ~140 fold lower compared to an analogous PD-L1-blocking bsAb of irrelevant target antigen specificity (PD-L1xMock). Importantly, treatment with PD-L1xEGFR selectively enhanced activation status, INF-γ production, and activity of anti-CD3xanti-EpCAM-redirected T cells in presence of EGFR+/EpCAM+, but not EGFR-/EpCAM+ carcinoma cells. Similarly, capacity of PD-L1xEGFR to promote proliferation and IFN-γ production by CMVpp65-specific CD8+ effector T cells was enhanced in the presence of EGFR+/CMVpp65+ cancer cells. In contrast, the clinically-used PD-L1-blocking antibody MEDI4736 (durvalumab) promoted T cell activation indiscriminate of EGFR expression on cancer cells. Additionally, in tumor-bearing mice 111In-PD-L1xEGFR showed a significantly higher tumor uptake compared to 111In-PD-L1xMock. In conclusion, PD-L1xEGFR blocks the PD-1/PD-L1 immune checkpoint in an EGFR-directed manner, which promotes a more selective reactivation of anticancer T cells. This novel targeted approach may be useful to enhance efficacy and safety of PD-1/PD-L1 checkpoint blockade in EGFR-overexpressing malignancies.

#4554

Assessment of the efficacy and safety of checkpoint inhibitors in the treatment of solid-tumor malignancies in a community cancer hospital.

Rachel Elsey,1 Nathan Smith,2 April Lick,2 Lauren Metzger,2 Casey Williams,1 Brian Leyland-Jones,1 Ping Ye1. 1 _Avera Cancer Institute, Sioux Falls, SD;_ 2 _South Dakota State University, Brookings, SD_.

Introduction: High dose chemotherapy, radiation, and/or surgical resection have historically been the standard therapy for patients with progressive malignancies. However, promising studies have shown immunotherapy may improve overall survival and reduce tumor burden in advanced disease. Checkpoint inhibitors, such as PD-1 inhibitors pembrolizumab and nivolumab and PD-L1 inhibitor atezolizumab, are approved for use in several solid tumors, but the response to these agents has been limited. This study was designed to identify predictors for the therapeutic response to immunotherapy agents in a community cancer hospital. Procedures: A retrospective review using the electronic medical record system was done to identify 43 patients treated with immunotherapy at Avera Cancer Institute. The inclusion criteria included patients who had solid organ malignancies, received checkpoint inhibitor-based immunotherapy between May 31, 2014 and May 31, 2017, and completed next generation sequencing of tumor DNA. The exclusion criteria included patients who were less than eighteen years of age, pregnant, or received care in an outreach clinic when records were not available. Data obtained from the records included age, sex, diagnosis, treatment history, immunotherapy agent used, start and end date of immunotherapy agent, clinical outcome, pathology results, adverse reactions, and next generation sequencing results of tumor DNA. The collected data was assessed for patient characteristics and biomarkers that predicted responses to immune checkpoint blockade in solid tumors. Disease progression was assessed in correspondence with the response evaluation criteria in solid tumors guidelines. Results: A total of 43 patients were included, 14 male and 29 female. The median patient age was 66 years old. The main malignancies included were, breast (11 patients), lung (10 patients), and ovarian (7 patients). On average, the patients were treated with three previous lines of therapy before receiving immunotherapy treatment. The most common immunotherapy agent was pembrolizumab, with 28 of the patients receiving this, followed by 14 patients treated with nivolumab and one treated with atezolizumab. The median time patients on immunotherapy was 118 days. Patients with longer duration of immunotherapy, fewer previous treatment lines, and higher tumor mutational burden (TMB) had the best response to checkpoint inhibitors. Conclusions: The predictive biomarkers found in this study can be used to assist in patient selection for immunotherapy in the clinical setting, including TMB and the number of lines of previous therapy. Further studies will continue to find predictors of non-response and hyperprogressors. We hope to further evaluate patients who experienced a durable response to create new hypotheses about indicators of response.

#4555

Potent Conditionally Active Biologic (CAB) PD-1 antibodies to reduce systemic toxicities associated with single agent and combination therapies.

Cathy Chang, Gerhard Frey, Leslie L. Sharp, Haizhen Liu, Jing Wang, Charles Xing, Safak Yalcin, Yong Ben, William J. Boyle, Jay M. Short. _BioAtla, San Diego, CA_.

PD-1 is an immune checkpoint inhibitor that limits T cell activity. The use of anti-PD-1 antagonist antibodies has demonstrated anti-tumor efficacy in mouse models and in a subset of human patients. However, blockade of PD-1 as a single agent and in combination with chemotherapy, targeted, and other immunomodulatory therapies has been limited clinically in part due to toxicities related to systemic immune-response related adverse events. In addition, there is increasing concern that systemic immune activation may lead to autoimmune-related adverse events in survivors.

The Conditionally Active Biologics (CAB) technology is a patented, proprietary platform that selects antibodies that reversibly bind to target antigen in the context of diseased tissues, but not normal tissues, by taking advantage of the unique cancer microenvironment that is produced largely as a result of glycolytic tumor metabolism including the Warburg effect. Using our CAB technology, we have identified anti-PD-1 Abs that reversibly bind human PD-1, and enhance T cell response to bacterial super-antigen under in vitro conditions that are present in the tumor microenvironment but not in normal tissues.

CAB-PD-1 Abs demonstrated anti-tumor efficacy, including tumor regression, against MC38 colorectal tumor in human PD-1 knock-in mouse model in which the mouse PD-1 gene has been replaced with human PD-1. The anti-tumor activity of CAB-PD-1 Abs was similar to a reference PD-1 Ab.

In conclusion, our data is consistent with our work on CAB-EGFR-ADC, CAB-AXL and CAB-ROR2-ADC antibodies, and suggests that anti-PD-1 Abs generated using the CAB technology provide potent, effective biologics with potential for increased therapeutic index. Specifically, the CAB-PD-1 is an excellent candidate for evaluation as a treatment for human cancers as a single agent or in combination with other anti-cancer therapies including immuno-oncology agents.

#4556

Phagocytosis response of macrophage to CD47 expression of tumor cells in anti-CD47 therapy.

Huajun Yang,1 Zhongliang Li,1 Phillip Wang,1 Beibei Tang,1 Qinyun Ma,2 Frank Xing,1 Qian Shi1. 1 _Crown Bioscience, Taicang, China;_ 2 _Huashan Hospital Affiliated to Fudan University, Shanghai, China_.

Macrophage as an important surveillance system is escaped by human cancer cells expressing CD47. To reinstate macrophage functionality in clearing tumor cells targeting CD47 and thereby blocking phagocytosis inhibitory CD47-SIRPalpha interaction has been an important strategy in immune-oncology therapy. We profiled a large panel of tumor cell lines, Crown's proprietary PDX models and clinical lung cancer tumors for CD47 expression. Although it has been reported as a ubiquitously expressed immune-escape marker in cancer cells, the expression levels of CD47 vary among different tumor cell lines and models when the cells were examined by flow cytometry. It is of interest that a portion of tumor cells in fact do not present CD47. To tackle the cause for the loss of CD47 expression, whole-exome sequencing was performed to examine the intactness of the gene in the CD47 loss models. The results will be presented. We next established a robust and reproducible in vitro assay platform to support anti-CD47 drug research. We applied a macrophage differentiation system using isolated CD14+ monocytes as starting material, and were able to achieve >80% of M1 and M2 populations, respectively, through the 6-day differentiation. Differentiated macrophage was co-cultured with target tumor cells labeled for flow cytometry analysis. In the presence of blocking anti-CD47 antibody BRICK126, steady and specific phagocytosis effect was observed when target tumor cells express high level of CD47 markers. BRIC126 antibody blocks the CD47-SIRPalpha interaction. In contrast, aCD274 antibody did not yield more than background phagocytosis effect in the same assay. To prove the CD47-specific effect, we constructed isogenic cell models from a couple of parental cell models in that CD47 gene is knocked out in one sub-cell line and overexpressed in another sub-cell line. The isogenic systems were then subject to the CD47 biology assay and more profound changes in phagocytosis responses were observed. Furthermore, the in vitro findings were validated in in vivo setting.

#4557

Dual targeting of innate and adaptive checkpoints on tumor cells limits immune evasion.

Yang-Xin Fu,1 Xiaojuan Liu2. 1 _UT Southwestern Medical Center, Dallas, TX;_ 2 _Institute of Biophysics, Beijing, China_.

CD47 on tumors inhibits phagocytosis, while PD-L1 limits T cell-mediated tumor killing. However, whether and how these two pathways coordinately evade immune responses are poorly understood. We reveal that both CD47 and PD-L1 on tumors coordinately evade innate and adaptive sensing. Targeted blockade of CD47 and PD-L1 on tumor cells with bispecific anti-PDL1-SIRPα showed better therapeutic efficacy than single blockades with reduced off-tumor targeting. Mechanistically, the dual-targeting approach significantly enhanced DNA sensing in dendritic cells (DC), DC cross-presentation and anti-tumor T cell response. Our data indicate that tumor cells evolve to utilize two-levels of checkpoints to evade anti-tumor immune responses, and tumor cell-specific dual-targeting of both innate and adaptive checkpoints represents a new strategy for increasing efficacy and reducing toxicity.

#4558

In vitro and in vivo characterization of the PD-1 targeting antibody BI 754091.

Markus Zettl,1 Melanie Wurm,1 Otmar Schaaf,1 Iñigo Tirapu,1 Sven Mostböck,1 Markus Reschke,1 Stephan-Michael Schmidbauer,1 Lee Frego,2 Ivo C. Lorenz,2 Michael Thibodeau,2 Diann Blanset,2 Elisa Oquendo Cifuentes,2 Jürgen Moll,1 Norbert Kraut,1 Eric Borges,3 Anne Vogt,1 Jonathon Sedgwick,2 Irene C. Waizenegger1. 1 _Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria; _2 _Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT;_ 3 _Boehringer Ingelheim Pharma GmbH & Co KG, Biberach, Germany_.

The programmed cell death-1 (PD-1) receptor provides inhibitory checkpoint signals to activated T cells upon binding to its ligands, PD-L1 and PD-L2, which are expressed on antigen-presenting cells and cancer cells leading to suppression of T-cell effector function and tumor immune evasion. Blockade of the PD-1 axis using either anti-PD-1 or anti-PD-L1 approved monoclonal antibodies (mAbs) results in improved T-cell effector function and anti-tumor immune responses. Durable tumor responses occur in 15-30% of cancer patients.

BI 754091, a humanized IgG4 mAb with high affinity against hPD-1 blocks the interaction between PD-1 and PD-L1 or PD-L2. BI 754091 was characterized in a panel of binding, blocking and functional cell-based assays. In addition, efficacy and safety was assessed in mice and in cynomolgus monkeys, respectively. The ability of BI 754091 to stimulate cytokine production in exhausted human T cells in vitro was tested in an autologous assay system with antigen-specific memory CD4+ T cells being re-stimulated by antigen-pulsed dendritic cells in the presence of BI 754091 or isotype control. Under these assay conditions the majority of T cells co-expressed the exhaustion markers PD-1 and LAG-3 on their surface. Furthermore, PD-L1 and PD-L2 were expressed on the dendritic cells. At the end of the experiment supernatants were harvested and analyzed for IFNγ secretion as a measure for T-cell activation. BI 754091 showed a potent dose dependent T-cell activation. The average fold increase of IFNγ was 7.9 as compared to isotype control, with an average EC50 of 0.9 nM.

The in vivo activity of BI 754091 was determined in MC-38 tumor-bearing mice, using a mouse strain where parts of the extracellular domain of murine PD-1 was replaced by the corresponding human PD-1 domain (C57BL/6NTac-PDCD1tm(PDCD1)Arte mice). A dose of 10 mg/kg BI 754091, given either as single treatment or in a twice weekly schedule, induced significant tumor growth inhibition (median TGI of 83% and 90%, respectively) and complete responses (CRs) in some tumors (3 CRs out of 10 and 2 CRs out of 10, respectively).

BI 754091 binds to PD-1 from cynomolgus monkeys with comparable affinities as to human PD-1, thus allowing pharmacokinetic and toxicological assessment in this species. Repeated high doses of BI 754091 were well tolerated without adverse immune-related effects.

BI 754091 is currently undergoing clinical investigations (NCT02952248).

#4559

KY-B602, a humanized anti-human OX40 antibody, enhances CD4+T cell activation and inhibits tumor growth in a syngeneic model.

feng hao, feng he, mingxuan ning, zhaoshuai bai, jinying ning. _KYinno biotechnology Co., Ltd, Beijing, China_.

OX40, a member of the tumor necrosis factor (TNF) receptor superfamily, is mainly expressed on activated T cells. As a secondary costimulatory molecule, activated OX40 can increase effector T-cell survival, proliferation, and memory. Anti-OX40 monoclonal antibodies have shown anti-tumor efficacy. Recent preclinical studies further support the potential synergistic effects of agonist OX40 immunotherapy in combination with cancer vaccination or in sequential co-administration with PD1 antibody to revert PD1 resistance. Several anti-OX40 agonistic monoclonal antibodies are currently tested in early phase cancer clinical trials either in monotherapy or in combination with other immune modulators. KY-B602 is a novel humanized IgG1 anti-OX40 antibody under pre-clinical development. It was generated from hybridoma and humanized by CDR grafting and structure simulation. KY-B602 specifically binds to the extracellular domain of human OX40 with high affinity (KD = 5.1 nM, determined by Biacore). FACS study showed KY-B602 also binds to both human and cynomolgus monkey OX40 over-expressed on 293T cells (EC50 =4.6nM, EC50=6.1nM, respectively). Another, in HT1080 cell reporter assay, KY-B602 significantly increased IL-8 secretion through OX40 pathway. In addition, KY-B602 dose-dependently augmented IL-2 and IFN-gamma secretion from CD4+T cells, that were pre-stimulated with IL-2 and PHA. However, in FACS competition assay, KY-B602 did not affect OX40L binding to OX40, suggesting that KY-B602 binds to a different epitope with that of OX40L. Moreover, KY-B602 inhibits in vivo tumor growth in MC38 syngeneic model. In summary, the current data of KY-B602 support its potency for cancer therapy.

#4560

A cost-effective and rapid assay for profiling of immunobiology and the development of predictive signatures for response to immunotherapies.

Peilin Chen, Sandy Spurgeon, Jing Wang, Michael Gonzales, Lianne McLean, Tom Goralski. _Fluidigm, South San Francisco, CA_.

Introduction: Gene expression profiling is an important tool to monitor the human immune response to different antitumor agents for drug development and to identify predictive signatures for response to immunotherapies. We have developed and analytically validated a gene expression assay panel, namely the AdvantaTM Immuno-Oncology Gene Expression Assay (AdvantaTM I/O GE Assay), on the Fluidigm BiomarkTM HD system, a microfluidic real-time PCR system, for investigation of antitumor agent effects on immune inhibitory pathways. The assay panel consists of 170 unique gene targets in categories such as T cell subset markers (e.g., CD4), cytokines and chemokines (e.g., IFNg), immune regulation (e.g., PD-L1, PD-L2) and immune cell-fate markers (e.g., NK) associated with immune-checkpoint inhibitory pathways for cancer treatments. Method: The Assay consists of Fluidigm Dynamic Array™ IFCs, an assay panel and RT-PCR reagents. The assay panel is composed of TaqMan™ gene expression assays provided in two 96-well plates: 96 assays on one and 79 assays on another with 17 unused assay wells available for customization. Each plate contains five housekeeping genes for assay normalization. This assay configuration allows customers to test 96 samples on two Fluidigm 96.96 Dynamic Array™ IFCs. Two primer pools for preamplification, and a positive control have been developed along with a 24.192 Dynamic Array™ IFC which enables 24 samples and the complete panel, 170 genes to be run on a single IFC. The assay has been analytically validated by a third party (Q2/EA) on the 96.96 IFC. This study is to verify the assay and kit reagents on the 24.192 IFC. A universal RNA was reversely transcribed into cDNA and pre-amplified with the preamp primer pools for all 170 genes. Three tissue cDNAs (lymph node, brain and Jurkat cells) and the positive control, which contains all 170 targets, were used to evaluate amplification efficiency, linearity and reproducibility. Each sample was tested in three replicates on 24.192 IFCs in six independent runs. Results: The results show an average amplification efficiency across all assays of 98.2% and linearity (RSQ) 0.999 for universal RNA. The efficiency and linearity for the positive control are 0.999 and 0.998, respectively. The within-run replicate correlation (RSQ) is 0.997 for the universal RNA, 0.992 for Jurkat cDNA, 0.986 for brain cDNA, and 0.990 for lymph node cDNA. The between-IFC replicate correlation (RSQ) is 0.981 for universal RNA, 0.982 for Jurkat cDNA, 0.972 for brain cDNA and 0.977 for lymph node cDNA. Gene expression signatures for individual tissues were observed. Conclusions: The AdvantaTM I/O GE Assay on 24.192 IFC and the Fluidigm BiomarkTM HD system provides high amplification efficiency, excellent linearity and reproducibility and reduces the cost of routine laboratory testing for both immune checkpoint research and drug development.

#4561

Preclinical characterization of GLS-010 (AB122): A fully humanized clinical-stage anti-PD-1 antibody.

Joanne B. Tan,1 Chris Chen,2 Kai Chen,3 Guochun Li,3 Jing Li,2 Jieying Liu,2 Hema Singh,1 Guoyong Wang,3 Baotian Yang,2 Kristin Zhang,1 Xiaoning Zhao,1 Yong Zheng2. 1 _Arcus Bioscienes, Hayward, CA;_ 2 _Wuxi AppTec, Shanghai, China;_ 3 _Harbin Gloria Pharmaceuticals, Beijing, China_.

INTRODUCTION: Exhausted T cells express high levels of several immune checkpoint proteins, including the programmed death-1 (PD-1) receptor. Preclinical and clinical data support the role of the PD-1/PD-L1 axis in promoting tumor evasion by curtailing immune responses. We present here the preclinical characterization of GLS-010 (AB122), a novel fully human anti-PD-1 monoclonal antibody currently in Phase 1.

METHODS: The affinity of GLS-010 (AB122) for human and cynomolgus monkey PD-1, its specificity for PD-1, and its ability to block the PD-1 interaction with PD-L1 and PD-L2 were measured by ELISA, flow cytometry and TCR-activated reporter gene assays. Functional assessment of GLS-010 (AB122) on immune responses (IFN-g, IL-2, and proliferation) was performed using mixed lymphocyte reaction (MLR) and re-stimulation with cytomegalovirus (CMV) pp665 peptide. The anti-tumor efficacy of GLS-010 (AB122) was evaluated using a mouse MC-38 tumor model grown in mice transgenic for human PD-1. For PK analysis, GLS-010 (AB122) was given in a single i.v. bolus to male and female cynomolgus monkeys at doses of 2, 6 and 18 mg/kg.

RESULTS: GLS-010 (AB122) is a fully human IgG4 monoclonal antibody that binds to human PD-1 (EC50 ~ 210 pM, ELISA; 770 pM, flow cytometry), cyno PD-1 (EC50 ~ 150 pM, ELISA), but not rat or mouse PD-1. Lack of GLS-010 (AB122) binding to other related members of the CD28 family, such as ICOS, CD28 and CTLA-4, confirms the specificity of the interaction. Functional studies showed that binding of GLS-010 (AB122) to cell-expressed hPD-1 inhibits the interaction with both hPD-L1 and hPD-L2 with an IC50 of 580 pM and 670 pM, respectively (by flow cytometry) and 2.2 nM and 5.8 nM, respectively (in reporter gene assay). Using allogeneic monocyte-derived dendritic cells, we showed a dose-dependent enhancement of IFN-g production and proliferation by CD4+ T cells, saturating at concentrations below 100 pM. Similar results were obtained in an antigen-specific T cell recall response assay using CMV-infected donors. GLS-010 (AB122) was very effective at blocking MC-38 tumor growth in hPD-1 transgenic mice. PK profiles following a single i.v. dose of GLS-010 (AB122) administered to male and female cynomolgus monkeys were dose-proportional and the rate of clearance was dose-independent.

CONCLUSIONS: GLS-010 (AB122) is a novel and selective antagonistic anti-hPD-1 antibody that potently blocks the interaction of human PD-1 with both PD-L1 and PD-L2. This blockade translates into potent enhancement of T cell activation in a variety of cell culture studies, which combined with its in vivo profile (in mice and monkeys) supports its ongoing clinical development in oncology.

#4562

Tumor B7-H3 (CD276) expression and survival of patients with pancreatic cancer.

Kentaro Inamura,1 Yusuke Yokouchi,1 Maki Kobayashi,1 Yosuke Inoue,2 Akio Saiura,2 Yutaka Takazawa,1 Yuichi Ishikawa1. 1 _The Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan;_ 2 _The Cancer Institute Hospital, Japanese Foundation for Cancer Research, Tokyo, Japan_.

Background: B7-H3 (CD276) belongs to a family of immune modulators that includes PD-L1 (B7-H1 or CD274), and has been associated with tumor immunosuppression. Although several studies have reported the association of B7-H3 expression with prognosis in pancreatic cancer, the results are conflicting and inconclusive. We therefore examined the association of tumor B7-H3 expression with prognosis of pancreatic cancer.

Methods: Using tissue microarrays of 150 consecutive cases of pancreatic cancer, we evaluated tumor B7-H3 expression by immunohistochemistry. We examined the prognostic association of B7-H3 expression, using the log-rank test. We also used Cox proportional hazards regression models to compute the mortality hazard ratio (HR), adjusted for covariates including pathological-stage, tumor differentiation, preoperative levels of CEA and CA19-9, and adjuvant chemotherapy.

Results: Tumor B7-H3 positivity (10% of tumor cells) was observed in 99 (66%) of 150 cases of pancreatic cancer. In a log-rank test, B7-H3 positivity was associated with higher pancreatic cancer-specific mortality (P = 0.0005). In a multivariable analysis, B7-H3 positivity was independently associated with higher pancreatic cancer-specific mortality (multivariable HR = 1.82; 95% confidence interval [CI] = 1.25-2.71; P = 0.0016). When we divided immunoreactivity of B7-H3 into four scores; i.e., score 0, negative; score 1, weak; score 2, moderate; score 3, strong, the immunoreactivity scores of B7-H3 were 51 (34%), 67 (45%), 20 (13%), 12 (8%) for score 0, 1, 2, 3, respectively. In a log-rank test, higher score of B7-H3 immunoreactivity was associated with higher pancreatic cancer-specific mortality (P = 0.0014). In a multivariable analysis, higher B7-H3 immunoreactivity was independently associated with higher pancreatic cancer-specific mortality (comparing score 3 versus score 0: multivariable HR = 2.78; 95% CI = 1.38-5.25; P for trend = 0.0011).

Conclusion: Tumor B7-H3 positivity and higher B7-H3 immunoreactivity are both independently associated with higher mortality of pancreatic cancer.

#4563

PD-L1 expression associated with treatment responses in colorectal cancer patients with XELOX/FOLFOX chemotherapy: Potential of checkpoint blockage and natural killer cell-based immunotherapy.

Elaine H.L. SIU,1 Simon S.M. NG,1 Anthony W.H. CHAN,2 James Y.W. LAU,1 Kwok Wai LO,2 Charing C.N. CHONG,1 Paul B.S. LAI,1 Stephen L. CHAN,3 Marcus H.N. LAW,1 Linda W.C. NG,1 S.T. CHEUNG1. 1 _Department of Surgery, The Chinese University of Hong Kong, Hong Kong;_ 2 _Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Hong Kong;_ 3 _Department of Clinical Oncology, The Chinese University of Hong Kong, Hong Kong_.

Chemo-resistance is a major obstacle in human cancer treatment. Immunotherapies including checkpoint blockage and cell-based therapy have been clinically validated for cancer treatment. The present study aimed to investigate the role of PD-L1 and potential of immunotherapy for chemo-resistant cancers. In a pilot study, we have examined the expression of PD-L1 in colorectal cancer (CRC) patients who received XELOX (capecitabine plus oxaliplatin) or FOLFOX (fluorouracil plus oxaliplatin) chemotherapy. Detection rate of PD-L1 expression by immunohistochemistry was higher in CRC patients who demonstrated poor response (5/10, 50%) compared to good responders (0/5, 0%) on XELOX/FOLFOX chemotherapy. Chemo-resistant CRC cell lines (HT29 and HCT116) were used to examine the role of PD-L1. Cells were plated out, selected under fluorouracil and oxaliplatin in serial, and referred as fluorouracil-oxaliplatin-resistant (FOR) cells. Half maximal inhibitory concentration (IC50) for respective drug was determined by MTT assay. HT29 FOR, compared to parental cells, showed chemo-resistance (359.4 and 10.1 fold increase to fluorouracil and oxaliplatin respectively) and similarly for HCT116 FOR (8.0 and 9.0 fold increase respectively). Hepatocellular carcinoma Hep3B and nasopharyngeal carcinoma C666-1 cells that have been selected for fluorouracil chemo-resistant (FR) were included. All the chemo-resistant cell lines showed increased expression of PD-L1 by real-time qPCR compared to their respective parental cells (HT29 FOR: 6.1 fold; HCT116 FOR: 1.9 fold; Hep3B FR: 3.1 fold; C666-1 FR: 4.4 fold). To explore the potential of immunotherapy, natural killer (NK) cells were isolated from peripheral blood PBMC of healthy donors. NK cells were expanded and activated ex vivo in stem cell growth medium supplemented with interleukins. NK co-culture assay was performed and apoptosis of cancer cells examined by flow cytometry. Parental cancer cells were sensitive to cytotoxic killing by NK cells compared to control no NK treatment (36.0% vs 2.8% apoptotic cells, P=0.001), and PD1 antibody (Nivolumab) treatment promoted NK-mediated cytotoxicity (52.9%, P=0.034). Notably, chemo-resistant cancer cells were similarly sensitive to cytotoxic killing by NK cells compared to control (27.1% vs 3.4%, P=0.002), and PD1 antibody (Nivolumab) treatment further enhanced NK-mediated cytotoxicity (52.8%, P=0.013). In summary, PD-L1 expression was associated with poor response for XELOX/FOLFOX chemotherapy in CRC patients. Chemo-resistant cancer cells showed elevated PD-L1 expression, nonetheless, susceptible to NK-mediated cytotoxicity and checkpoint blockage by PD1 antibody augmented the treatment response. Further studies are warranted to examine immunotherapy for chemo-resistant cancers.

### Immune Mechanisms Invoked by Therapies 2

#4564

The immune microenvironment in hormone receptor-positive breast cancer and treatment outcome following preoperative chemotherapy plus bevacizumab.

Adrienne G. Waks,1 Daniel G. Stover,2 William T. Barry,1 Deborah A. Dillon,1 Evisa Gjini,1 Scott J. Rodig,1 Jane E. Brock,1 Michele Baltay,1 Jennifer Savoie,1 Eric P. Winer,1 Ian E. Krop,1 Sara M. Tolaney1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Ohio State University Comprehensive Cancer Center, Columbus, OH_.

Background: Hormone receptor-positive (HR+) breast cancers (BC) have fewer tumor-infiltrating lymphocytes (TILs) and lower response rates to immune checkpoint inhibitors in early phase studies than other breast cancer subtypes. Immune biomarkers that accurately reflect the immune microenvironment have important clinical implications in HR+ BC patients. Prior evidence suggests that macrophage-related immune pathways may be relevant to the pathophysiology of HR+ BC.

Methods: Patients identified from a prospective trial of preoperative bevacizumab (preop bev) followed by bev with adriamycin/cyclophosphamide/paclitaxel dose-dense chemotherapy (chemo). Tumor samples were collected at diagnosis and surgery (pre-tx and post-tx). TILs and immunohistochemical staining for PD-L1, CD8, and CD68 were scored. Whole transcriptome sequencing (RNAseq) and Nanostring PanCancer Immune Profiling Panel were performed. Pathologic response at surgery was assessed by Miller-Payne (MP) and residual cancer burden (RCB) scores. An immune score was calculated for each pre-tx specimen by integrating 10 published immune signatures. Immune cell subsets were inferred from bulk transcriptional data using CIBERSORT.

Results: 55 patients had at least 1 evaluable specimen and were included for analysis. 18% of pre-tx tumors had 'high' (≥10%) TILs and 'high' TILs were associated with significantly higher immune signature score (p=0.004). Immune score correlated highly with proportion of CIBERSORT anti-tumor M1 macrophage and CD8 T-cell signatures (r>0.65 and p<0.001) and was significantly associated with RCB. Higher pre-tx TILs, tPD-L1, sPD-L1, CD8, and CD68 were associated with favorable RCB significantly associated with more favorable RCB after adjustment for tumor size and grade. Pathologic complete response occurred in 4 pts; all 4 had high pre-tx TILs, pre-tx tPD-L1, or both. Among patients with residual disease, there were significantly fewer TILs and CD8 cells after chemotherapy (Wilcoxon signed rank p=0.037 and p=0.002, respectively), however tPD-L1 and CD68 were not significantly different. Nanostring analyses demonstrated that chemokines and complement pathway components were among most significantly enriched post-tx relative to pre-tx.

Conclusions: Most HR+/HER2- breast tumors demonstrate low levels of anti-tumor immune activity; however, those with higher levels have a more favorable response to chemo plus bev. Assessment of immune activity based on RNA signatures is consistent with histology and immune-related protein expression. T-cell- and checkpoint-related biomarkers tend to decrease following preoperative chemo plus bev in HR+/HER2- breast cancer. Following treatment with chemotherapy/bevacizumab, we observe increased expression of chemokines and complement pathway genes.

#4565

Effective inhibition of metastasis in triple-negative breast cancer (TNBC) mouse model by combining immunogenic chemotherapy and immune checkpoint blockade.

Xiangliang Yuan,1 Yi Xiao,1 Xianghua Liu,1 Wenling Kuo,1 Hongzhong Li,1 Ping Li,1 Hideo Yagita,2 Dihua Yu1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Juntendo University School of Medicine, Tokyo, Japan_.

Triple-negative breast cancer (TNBC) has the worst outcome among all breast cancer subtypes because of its high rate of metastatic spread and recurrence. Standard first-line treatment for TNBC is chemotherapy (chemo) combinations. However, many (~50%) TNBC patients do not respond well to chemo-treatment, and new treatment options are needed. Immune checkpoint blockade (ICB) has led to a breakthrough in treating various types of cancers with durable responses in advanced cancer. However, its efficacy for treating TNBC is unsatisfactory, partly because breast cancers are not highly immunogenic. Additionally, the efficacy of ICB for inhibition of distant metastases has not been systematically examined. Here, we tested whether using an immunogenic chemotherapy - Doxorubicin or Doxisome (liposomal encapsulated formulation of Doxorubicin) may enhance the efficacy of anti-PD1 antibody (an ICB regimen), in the 4T1-TNBC mouse model, which is highly resistant to ICB. Also, we used the spontaneous metastatic models of TNBC in mice to test the therapeutic efficacy of neoadjuvant immunogenic chemo plus ICB in inhibition of metastasis after primary mammary tumor resection. Indeed, the combination of a low dosage of Doxo or Doxisome with anti-PD-1 antibody inhibited mammary tumor growth and improved the survival of mice bearing 4T1 mammary tumor significantly (P<0.05) better than Doxo or Doxisome or anti-PD-1 antibody alone. The antitumor response was triggered by direct immunogenic chemo drug actions on tumor cells, which induced infiltration of antigen presenting cells (e.g. DCs), and ultimately CD8+ T cell antitumor immunity. More strikingly, we found that neoadjuvant chemo plus ICB eliminated lung metastases following primary tumor resection significantly (P<0.05) more effective than single treatment with chemo or ICB. Flow cytometry analyses revealed highly significantly (P<0.05) increased antigen-specific T cells infiltration in lung metastases of mice who received the combination (Chemo plus ICB) treatment. These data provide a strong scientific rationale for several ongoing clinical trials combining immunogenic chemotherapy with anti-PD-1 checkpoint blockade therapy, which could be highly effective in treating metastatic breast cancer. The efficacy of combining immunogenic chemotherapy with anti-PD-1 checkpoint blockade therapy in inhibition of others ICB resistant solid tumors and metastasis should be further investigated.

#4566

PD-L1 and B7-H3 expression in serous carcinoma of endometrium.

Masato Nishimura, Ayuka Mineda, Akiko Abe, Minoru Irahara. _Tokushima University, Tokushima, Japan_.

Endometrial cancer keeps increasing in Japan explosively. In endometrial cancer, the prognosis of serous carcinoma is very poor, because of early progression to abdominal cavity. Remedy for endometrial cancer is limited, so new therapy has been required for a long time. Immunotherapy is very promising in various cancers recently. PD-L1 (programmed cell death ligand 1) and B7-H3 are factors of immunoresponse between cancer cells and lymphcyte. Purpose: The purpose of this study is to investing the expression of PD-L1 and B7-H3, moreover, relationship with clinicopathological factors in serous carcinoma of endometrium. We immunohistochemically evaluated PD-L1 and B7-H3 expression in 21 serous carcinoma. The result was graded as positive in case stained more than 10% area. Result: 2/21(9.5%) for PD-L1 and 10/21(47.6%) for B7-H3 were positive. There was no correlation with tumor diameter, lymphnode metastasis, advanced stage and recurrence in PD-L1, however, B7-H3 expression had trend node metastasis, 3/10 in positive cases vs 0/11 in negative cases (p=0.05). In 4 recurrent cases treated with taxane and carboplatin as second line therapy, one case with B7-H3 negative got complete remission, however 3 cases with B7-H3 positive got partial improvement once, but finally got progression. Conclusion: PD-L1 expression was limited in serous carcinoma, so immunotherapy targeted for PD-1 and PD-L1 may be limited. The expression of B7-H3 is related to chemotherapy resistance. B7-H3 may be a biomarker for chemotherapy and target of new immunotherapy for serous carcinoma of endometrium.

#4567

Immunomodulatory action of SGI-110 and combination with γδ T cell immunotherapy in ovarian cancer.

Marina Natoli, Anastasios Karadimitris, Sadaf Ghaem-Maghami. _Imperial College London, London, United Kingdom_.

Ovarian cancer (OC) is regarded as being an immunogenic cancer and strategies to enhance immune function in patients are thought to confer benefit and improve prognosis. Immunotherapy has been shown to be most promising when combined with other therapeutic approaches. Recent evidence in the field and preliminary data from our group suggest that treatment with epigenetic drugs, such as DNA methyltransferase inhibitors (DNMTi), can lead to improved immune activation in several cancers including ovarian.

The purpose of this study is to test the hypothesis that treatment with next generation hypomethylating agent SGI-110 can induce the re-expression of immunomodulatory genes and endogenous retroviruses (ERVs), which shifts OC cells towards a more immunogenic expression profile.

OC cell lines SKOV3, Kuramochi and OVSAHO were treated with low dose SGI-110 and assessed for increased immunogenicity. Expression of immune biomarkers and ERVs was assessed at different time points at mRNA levels by qPCR and protein levels by Flow Cytometry. Treated cell lines were functionally tested for increased immunogenicity by co-culture with γδ T cells, which were isolated from healthy donors and expanded by zoledronic acid selection. MTT assays were performed to assess cell viability and T cell killing following co-culture. IFNγ and Granzyme B release from T cells and chemokine release from tumor cells were assessed by ELISA.

Our results show up-regulation of key immunogenicity biomarkers and ERVs in our panel of OC cell lines, following treatment with SGI-110. Co-culturing SGI-110-treated cell lines with γδ T cells resulted in increased tumor cell killing and T cell activity.

We are now analysing RNA-sequencing data of SGI-110-treated OC cell lines in an effort to dissect the immunomodulatory mechanism of action of this novel hypomethylating agent. Moreover, we plan to test the in vivo feasibility of a SGI-110/γδ T cell combination therapy, which if successful might be further optimised for a clinical trial.

#4568

Reversal of immune tolerance and increased anti tumoral immune response in a mouse model of CNS B cell lymphoma after combined XPO1 and BCR inhibition.

Marta Crespo,1 Isabel Jiménez,1 Júlia Carabia,1 Sabela Bobillo,1 Pau Abrisqueta,1 Carles Palacio,1 Juan Camilo Nieto,1 Joan Boix,1 Cecilia del Carmen Carpio,1 Josep Castellví,2 Joan Seoane,1 Francesc Bosch1. 1 _Vall d'Hebron Institute of Oncology, Barcelona, Spain;_ 2 _Vall d'Hebron Research Institute, Barcelona, Spain_.

Primary CNS lymphoma (PCNSL) is an aggressive non-Hodgkin lymphoma usually classified as ABC-DLBCL. Selinexor inactivates XPO-1 protein and induces anti-tumor effects mainly due to forced nuclear retention of tumor suppressors. It has shown excellent brain penetrance and promising results in glioblastoma and can inhibit both BCR and NF-kB signaling in malignant B-cells. Herein, we assessed the role of XPO-1 inhibition in intracerebral xenograft murine models. Proliferation and survival analysis in DLBCL cell lines showed that cell of origin does not influence sensitivity to selinexor and a strong synergy with ibrutinib mainly in ABC-DLBCL. We then established an orthotopic xenograft model of PCNSL by stereotactic injection of OCI-Ly10 cells transfected with luciferase into the brain parenchyma of nude athymic mice. Mice were treated with 5mg/kg oral selinexor or vehicle three times a week and bioluminiscence was assessed twice a week. Selinexor significantly blocked tumoral growth with differences starting as soon as 12 days after treatment. Also, selinexor improved mice survival (median: 48 days vs. 34 days). Next, we evaluated the potential synergy between ibrutinib and selinexor in vivo. For that mice were assigned into one of the following groups: selinexor (5mg/kg twice a week), ibrutinib (25mg/kg daily), combination or vehicle. The combination of both drugs significantly increased survival compared to both treatments as single agent, whereas there was no significant difference between ibrutinib and selinexor alone. Tumoral growth was equally blocked by all treatments. We next evaluated the innate immune response to the lymphoma by flow cytometry. Remarkably, CNS lymphomas were infiltrated by PD-1 positive M2 macrophages, as well as NK cells. Treatment with selinexor was able to decrease the proportion of M2-protumoral macrophages by half whereas the combination induced a 3-fold reduction (V: 22.72%+/-1.6, S: 12.29%+/-1.38, C: 7.1%+/-1.19). As a consequence, the proportion of M1 macrophages increased (V: 70.22%+/-1.8, S: 82.4%+/-1.7, C: 86.59%+/-1.5). In turn, ibrutinib was the main responsible for downregulation of PD-L1 in malignant cells (V: 3.7%+/-0.68, I: 0.6%+/-0.19, C: 0.19%+/- 0.08). Only when both treatments were combined there was significant downregulation of PD1 expression in M2 (V: 37.6%+/-5.5, C: 24.61%+/- 2.2) and a downregulation in PD1/SIRPα double positive M2 (V: 14.4%+-/1.44, C: 9.16%+/- 0.78). Selinexor and combination were able to significantly increase the proportion of NK cells (V:4.8%+/-0.21, S:9.7%+/-1.16, C:11.54%+/-2.02) and downregulate their PD1 expression (V:8.2%+/-2.7, S:3.7%+/-0.9, C: 2.8%+/-1.06). These results provide pre-clinical evidence for the development of selinexor and ibrutinib combination as new therapeutic option for PCNSL or DLBCL with CNS involvement.

#4569

The CDK4/6 inhibitor abemaciclib synergizes with PD-L1 blockade to induce an immune inflamed tumor microenvironment through T cell and tumor cell intrinsic effects.

David Schaer,1 Richard Beckmann,2 Jack Dempsey,2 Lysiane Huber,2 Amelie Forest,1 Nelusha Amaladas,1 Ying Cindy Wang,1 Erik Rasmussen,1 Darin Chin,1 Yanxia Li,1 Andrew Capen,2 Marianne Deroose,1 Carmine Carpenito,1 Xueqian Gong,2 Kirk Staschke,2 Linda Chung,2 Farhana Merzoug,2 Trent Stewart,2 Lacey Litchfield,2 Philip Iversen,2 Sean Buchanan,2 Alfonso de Dios,2 Ruslan Novosiadly,1 Michael Kalos1. 1 _Eli Lilly, New York, NY;_ 2 _Eli Lilly, Indianapolis, IN_.

The approval of abemaciclib and additional cyclin dependent kinases 4 & 6 (CDK4/6) inhibitors for the treatment of HR+ breast cancer has provided new therapeutic options to patients. As CDK4/6 inhibitors become part of the standard of care, combination strategies leveraging abemaciclib together with immunotherapy may represent an opportunity to extend benefit to more patients and additional cancers. Accordingly, it is important to understand if and how a cell cycle inhibitor can be combined with immunotherapy. To investigate the immune combinatorial potential of abemaciclib, we studied the effects of treatment alone and in combination with PD-L1 blockade in immunocompetent murine syngeneic tumor models, and directly evaluated the tumor cell and immune cell intrinsic immunologic effect of abemaciclib in vitro. In vivo abemaciclib treatment of murine tumors (CT26, EMT6 and MC38) caused a dose-dependent delay in tumor growth and demonstrated the potential to induce complete tumor regression (CR ~10%). Combination with an anti-PD-L1 antibody after abemaciclib pretreatment greatly enhanced the anti-tumor response compared to abemaciclib and anti-PD-L1 monotherapies. Optimal combination therapy resulted in 50-60% CRs of mice in a setting where anti-PD-L1 monotherapy showed little or no efficacy (0% CRs). Mice maintaining CRs after cessation of combination therapy or abemaciclib monotherapy resisted later CT26 rechallenge, demonstrating the ability to generate immunological memory during abemaciclib therapy. Analysis of intra-tumor gene expression showed that abemaciclib monotherapy induced T cell activation and inflammation signatures. Combination therapy substantially enhanced this effect and was additionally associated with DC maturation, antigen presentation, cytokine signaling and helper T cell phenotype. Suppression of cell cycle genes indicative of inhibition of CDK4/6 was also more prominent during the combination therapy. In Jurkat and primary human T cells, treatment with abemaciclib in vitro resulted in a dose-dependent increase in NFAT activity upon TCR stimulation. This correlated with upregulation of both cell surface markers and genes associated with an enhanced T cell activation phenotype, while only modestly affecting T cell expansion. Abemaciclib also amplified expression of antigen presentation and other immune-related genes in human breast cancer cells. Although it was uncertain if agents that inhibit cell proliferation could be combined with immunotherapy, these preclinical results provide a strong rationale for combining abemaciclib with checkpoint immunotherapy to improve the anti-tumor efficacy. The T cell and tumor cell intrinsic effects, synergistic anti-tumor responses and intra-tumor immune activation, justify clinical investigation of this combination.

#4570

Telomerase-specific oncolytic virotherapy promotes therapeuticefficacy of PD-1 blockade in murine osteosarcoma.

Yusuke Mochizuki,1 Hiroshi Tazawa,1 Koji Demiya,1 Tadashi Komatsubara,1 Kazuhisa Sugiu,1 Joe Hasei,1 Toshiyuki Kunisada,1 Yasuo Urata,2 Toshifumi Ozaki,1 Toshiyoshi Fujiwara1. 1 _Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan;_ 2 _Oncolys BioPharma Inc., Tokyo, Japan_.

Background: Treatment with immune checkpoint inhibitor anti-programmed death protein 1 (PD-1) antibody has dramatically improved the clinical outcome in cancer patients. Then immunotherapy is the most exciting field for cancer treatment in recent years. However, PD-1 blockade therapy is limited to certain cancer types. Therefore, the enhancement of therapeutic efficacy in PD-1 blockade therapy is required. Recently, it has been suggested that the effect of PD-1 blockade therapy can be enhanced when combined with immunogenic antitumor therapy including chemotherapy and virotherapy. We recently developed a RGD fiber-modified telomerase-specific oncolytic adenovirus OBP-502, which can enter into tumor cells by binding to cell surface integrin and induce oncolytic cell death in a telomerase-dependent manner. In this study, we assessed the in vitro and in vivo antitumor efficacy of combination therapy with anti-PD-1 antibody and OBP-502 in murine osteosarcoma.

Methods: We used 2 murine osteosarcoma cell lines, K7M2 and NHOS. The expression of PD-L1, coxsackie and adenovirus receptor (CAR), and integrin on the cell surface was analyzed by flow cytometric analysis. The in vitro antitumor effect of OBP-502 was evaluated using a XTT assay. Virus-induced immunogenic cell death was assessed by analyzing the level of extracellular ATP and high-mobility group box protein B1 (HMGB1). To evaluate the therapeutic potential of anti-PD-1 antibody and OBP-502, we assessed the antitumor effect of combination therapy with anti-PD-1 antibody and OBP-502 using a subcutaneous K7M2 xenograft tumor model. Moreover, the number of tumor-infiltrating CD8+ and Foxp3+ T cells was analyzed by immunohistochemistry.

Results: K7M2 and NHOS cells showed the expression of PD-L1 and integrin, but not CAR, in flow cytometric analysis. OBP-502 efficiently suppressed the viability of K7M2 and NHOS cells in a dose-dependent manner. OBP-502 significantly increased the release of HMGB1 in K7M2 cells and the secretion of ATP in NHOS cells. Intratumoral injection of OBP-502 significantly induced the number of tumor-infiltrating CD8+ T cells and enhanced the suppression of tumor growth by PD-1 blockade in a subcutaneous K7M2 xenograft tumor model. Moreover, tumor free was observed in 6 out of 7 mice treated with anti-PD-1 antibody and OBP-502 even at 4 weeks after treatment.

Conclusion: These results suggest that telomerase-specific oncolytic virotherapy is a promising antitumor strategy to promote the therapeutic efficacy of PD-1 blockade therapy in osteosarcoma.

#4571

Activation of the cGAS-STING pathway by NBTXR3 nanoparticles exposed to radiotherapy.

Julie Marill, Audrey Darmon, Ping Zhang, Sébastien Paris. _Nanobiotix, Paris, France_.

Radiation therapy (RT) is one of the most used local treatment for many cancer types. In addition to DNA breaks and free radicals production leading to numerous cell damages and cancer cell destruction, many preclinical and clinical studies have demonstrated that RT acts as an efficient modulator of tumor immunogenicity. RT can set in motion a series of processes facilitating tumor recognition by the immune system, such as induction of the immunogenic cell death (ICD). Recent studies reported that RT could also activate the cGAS-STING pathway, which plays a fundamental role in the immune response to cytoplasmic DNA, by activation of the transcriptional factor IRF3, leading to expression of interferon β. Moreover, cGAS-STING activation appears to be an important component for tumor resident Antigen-Presenting Cells (APC) activation, a crucial step for induction of CD8+ T cell response against tumor derived antigens. Interestingly, recent preclinical data showed that STING agonist and RT could synergize to control local and distant tumors. NBTXR3 is composed hafnium oxide nanoparticles used for a single intra-tumor administration and activated by radiation therapy. The size, shape and surface of these nanoparticles have been designed to develop strong interactions with cancer cells - effective cell binding and uptake - and to persist within the tumor mass during the whole RT treatment. The high electron density of NBTXR3 is responsible for an increased probability of interaction with incoming ionizing radiations when compared to tumor tissues with low electron density. NBTXR3 increases energy dose deposition within the cancer cells which results in an enhanced tumor destruction when compared to RT alone. NBTXR3 is currently evaluated in clinical trials including soft tissue sarcoma (phase II/III), head and neck, prostate, liver and rectum cancers (phase I). Here, we explored the ability of RT-activated NBTXR3 to increase cGAS-STING pathway response, compared to RT alone. To achieve this goal, we used human colorectal cancer HCT116-Dual cells, which stably express a secreted luciferase under the control of a minimal promoter containing five IFN-stimulated response elements. This system allows the study of IRF3 transcriptional activity, by monitoring the chemiluminescence. A significant increase of luciferase activity (from 30% to >50% (n=5, p<0.05)) was observed for cells treated with NBTXR3 and irradiated (2Gy to 8Gy), when compared to RT alone. At equivalent dose of RT, NBTXR3 showed a significant increase of cGAS-STING pathway induction that could prime a more effective antitumor immune response.

#4572

The induction of PD-L1 positive immune cells and CD8-positive T lymphocytes by neoadjuvant chemoradiotherapy for rectal cancer.

Kimihiro Yamashita,1 Akio Nakagawa,1 Tomoko Tanaka,1 Akira Arimoto,1 Eiji Fukuoka,1 Yutaka Sugita,1 Junko Mukohyama,2 Piero Dalerba,2 Hiroshi Hasegawa,1 Takeru Matsuda,1 Satoshi Suzuki,1 Yoshihiro Kakeji1. 1 _Kobe University Graduate School of Medicine, Kobe, Japan;_ 2 _Columbia University, New York, NY_.

Background; Immunotherapy, particularly blockade of programmed cell death-1 (PD-1) has shown unprecedented success in treating various types of cancers. In recent clinical trial of PD-1 blockade, clinicians focused on the characteristics of metastatic colorectal cancer patients with MSI-H (microsatellite instability- high) tumor associated with somatic hypermutation and confirmed a benefit of this immunotherapy in the disease. On the other hand, for MSS patients who account for most of colorectal cancer patient, new immunological strategies are needed. From before, radiation therapy is expected as an approach to overcome this relative resistance across colorectal cancers. Some reports on the relationship between tumor immunological microenvirment and irradiation have also been reported. Based on these findings, we examined a tumor immunological microenvironment of rectal cancer performed by chemoradiotherapy with immunohistochemical analysis of biopsy and surgical specimens.

Methods; From January 2005 to December 2016, 77 lower rectal cancer cases with cT3-4 or cN (+) were surgically treated in our institution and enrolled and 68 were eligible for this study. Immunohistochemistry of PD-L1, CD8, and CD163 was performed on biopsy specimens and surgically resected specimens of all cases, and their changes of expression before and after treatment were examined. We also examined the relationship between the expression of these tumor immunity-related factors and clinicopathological factors and prognosis.

Results; There were 44 cases in which NACRT was performed (NA group) and 24 cases in which surgery alone was performed (S group). In the S group, there was no significant change in the infiltration of PD-L1 positive immune cells (PD-L1+IC) nor CD8 positive tumor-infiltrating lymphocytes (CD8+TIL) before and after treatment, but in the NA group, the infiltrations of them were significantly increased (p = 0.038 and p = 0.0027, respectively). Although no PD-L1 positive tumor cells (PD-L1+TC) were observed in the biopsy specimens, there were 5 cases in which PD-L1+TC were observed in the surgically resected specimens, and all of which were cases of NACRT group (not significant). In multivariate analysis, low infiltration of PD-L1+IC (p = 0.033) was revealed as poor prognostic factor in overall survival, and low infiltration of PD-L1+IC (p = 0.041) and CD8+TIL (p = 0.0094) were identified as poor prognostic factors in disease-free survival.

Conclusion; The infiltration of PD-L1+IC and CD8+TIL was induced by NACRT. The presence of PD-L1+IC in the tumor tissue was revealed to improve the prognosis of rectal cancer. In NACRT for rectal cancer, PD-1 inhibitor combination therapy should be introduced to tumors with PD-L1 positive IC.

#4573

Localized synchrotron radiation in mice induces persistent systemic genotoxic events mediated by the functional immune system.

Jessica Ventura,1 Pavel Lobachevsky,2 Jason Palazzolo,2 Helen Forrester,3 Nicole Haynes,2 Alesia Ivashkevich,4 Andrew Stevenson,5 Christopher Hall,5 Vassilis Gorgoulis,6 John Hamilton,7 Alexandros Georgakilas,8 Calr Sprung,3 Olga A. Martin2. 1 _Royal Women's Hospital, Melbourne, Australia;_ 2 _Peter MacCallum Cancer Ctr., Melbourne, Australia;_ 3 _Hudson Institute of Medical Research, Clayton, Australia;_ 4 _Canberra Hospital, Garran, Australia;_ 5 _Australian Synchrotron, Clayton, Australia;_ 6 _University of Athens, Athens, Greece;_ 7 _Royal Melbourne Hospital, Melbourne, Australia;_ 8 _Technical University of Athens, Athens, Greece_.

The discovery of the radiation-induced bystander effect has expanded knowledge of radiobiological mechanisms of ionizing radiation. A counterpart in vivo phenomenon is the radiation-induced abscopal effect (RIAE). It is not known how radiation settings affect non-targeted normal tissues and therefore the risk of adverse RIAE. At the Australian Synchrotron we examined systemic effects of microbeam radiotherapy (MRT) and broad beam (BB) configurations, in mice that were locally exposed to a very short pulse of a high dose-rate synchrotron beam (49 Gy/sec). We determined how radiation volume and dose impact the RIAE. C57BL/6 mice were irradiated with 10 or 40 Gy incident dose of MRT or BB in an 8x8, 8x1, or 2x2-mm area of the right hind leg. Blood samples, irradiated skin and a variety of normal unirradiated tissues were collected for DNA damage analysis of double-strand breaks (DSBs) quantified as gamma-H2AX foci and oxidatitive clustered DNA lesions (OCDL). OCDLs elevated in a wide variety of unirradiated normal tissues. In out-of-field duodenum, a trend for elevated apoptotic cells was observed, however DSBs elevated only after exposure to lower doses. These genotoxic events were accompanied by changes in concentrations of MDC, CCL2/MCP1, Eotaxin, IL-10, TIMP-1, VEGF, TGFβ-1 and TGFβ-2 plasma cytokines and by changes in frequencies of macrophages, neutrophils and T-lymphocytes in duodenum. Overall, systemic radiation responses were dose-independent (1). The MRT irradiations was repeated in immune-deficient mice: NSG, CCL2 knock-outs, and in C57BL/6 mice treated with anti-CSF1R antibody which effectively depletes macrophages. Strikingly, these effects and the abscopal innate and adaptive immune effector responses were completely or partially abrogated in the mice with various immune deficiencies, highlighting the role of the functional immune system in propagation of systemic genotoxic effects of radiation. These findings have implications for the planning of therapeutic and diagnostic radiation to reduce the risk of radiation-related adverse systemic effects. 1. Ventura et al, Cancer Research, e-pub (2017).

### Liquid Biopsy 4

#4574

Detection of minimal residual disease from less than one cell equivalent in liquid biopsy samples using the AVENIO ctDNA Surveillance Kit.

Amrita Pati, Alex Lovejoy, Paul W. Shi, Abraham Munoz, Vera Rapoport, Katrina Mayol, Seng Saelee, Janet Jin, Dan Klass. _Roche Sequencing Solutions, Pleasanton, CA_.

A key technical challenge in oncology is the detection of minimal residual disease (MRD, the presence of residual tumor in a subject post-treatment). Recent studies have shown poor prognostic outcomes for research subjects with ultra-low residual tumor burdens after treatment across a variety of cancer types (1-4). Since changes in ctDNA levels correlate with relative changes in tumor burden (2), in order to sensitively detect MRD, an assay must be able to detect circulating tumor DNA (ctDNA) 30X lower (5) than pre-treatment levels. The difficulty inherent in achieving this limit of detection (LOD) is compounded by the small amount of cell free DNA (cfDNA) derived from a typical 10ml blood draw (2). Detecting a single variant at sequencing depths commensurate with genotyping typically faces sensitivity limitations at low allele fractions (AFs), as the probability of observing even a single molecule with a specific variant are drastically low. To address this issue, we present here a method using the Roche AVENIO ctDNA Surveillance Kit (Research Use Only) that takes advantage of multiple variants to improve the LOD for ctDNA detection below the LOD for a single variant.

To assess MRD sensitivity at relevant allele fractions, cfDNA spikes were made mimicking ctDNA allele fractions ranging from 0.002% to 0.02%. Samples with different input masses (10-50 ng input) were run using the Surveillance Kit, which was designed for surveillance-centric research applications in lung cancer and colorectal cancer (CRC). Known variants were queried and a Monte-Carlo-based approach was used to determine a p-value for presence of ctDNA.

Here the results of three experiments to assess the performance of the monitoring algorithm are described. 1. Using healthy donor cfDNA samples, specificity analyses were performed with different p-value cutoffs, and a p-value of 0.04 was shown to give 95% specificity for lung cancer specific cancer variants. 2. To determine the expected variability within a given sample, 10 technical replicates of 2 distinct samples were assessed, showing the range of performance expected due simply to random sampling. 3. MRD sensitivities across different input masses, number of variants, and sequencing depths are shown across 3 distinct samples with variants at 0.002%, 0.004%, and 0.01%. With 30 ng of input, the Surveillance Kit achieves a MRD sensitivity of >99% with an AF of 0.002% and 34 known variants, and 90% MRD sensitivity at a 0.01% AF with 6 variants (the median expected number of variants per subject). Using the algorithm described, a retrospective clinical research study looking for MRD post-surgery in stage II/III CRC subjects (4) were analyzed. Subjects with ctDNA an average of 2 weeks after surgery recurred significantly sooner and ctDNA levels below 0.005% were enough to show prognostic significance.

#4575

Efficient and accurate NGS approach for liquid biopsy detection and monitoring.

Ke Tang, Christopher Kasbek, Yang Song, Qingxuan Song, Si Chen, Nouran Almalki, Xu Zhang, Jun T. Huang. _Admera Health, South Plainfield, NJ_.

Liquid biopsy detection using ctDNA has great potential in cancer diagnosis, monitoring, and predicting survival. However, detection sensitivity and specificity remains a major challenge at the current stage. Here we utilized targeted next-generation sequencing (NGS) approach to develop a noninvasive test to identify SNVs, indels, and fusions present at low frequencies in lung cancer patients. Target regions of interest were sequenced using tagging of individual molecules, followed by amplicon library generation and massive parallel sequencing using Illumina platform. The variants were called and annotated using our in-house AGIS (Actionable Genomic Interpretation System) bioinformatics pipeline. This CLIA-approved NGS panel (LiquidGx™ Lung) detects 112 variants in 10 genes (AKT1, ALK, BRAF, EGFR, ERBB2, KRAS, MET, PIK3CA, RET, and ROS1) with detection limit of 0.1%. Mutations are detected with 94% sensitivity and 99.8% specificity in a short turnaround time of 4-6 days. To assess the performance in a clinical setting, accurate sequencing was performed on 210 lung cancer patients; the frequency of mutations identified of each gene closely mirrored the published lung cancer tissue biopsy data. In summary, we have developed a potent liquid biopsy platform for highly sensitive genomic biomarker test. Our technology is also available as an assay development service and can be easily adaptable to the diagnosis and monitoring of other cancer types.

#4576

**Combination of a label independent CTC enrichment platform with fluorescence** in situ **hybridization for the molecular characterization of CTCs in non-small cell lung cancer patients.**

Aliki Ntzifa,1 Martha Zavridou,1 Pavlos Pollakis,2 Georgia Bardi,2 Vasilis Georgoulias,3 Athanasios Kotsakis,4 Evi Lianidou1. 1 _Univ. of Athens, Athens, Greece;_ 2 _BioAnalytica Genotype SA, Athens, Greece;_ 3 _Hellenic Oncology Research Group, Athens, Greece;_ 4 _Univ. of Crete, Greece_.

Background: Patients with EGFRmt non-small cell lung cancer (NSCLC) develop resistance to first (erlotinib/ gefitinib) and second (afatinib) generation EGFR tyrosine kinase inhibitors (EGFR TKIs). Circulating tumor cells (CTCs) may be used as a potential biological material to monitor genetic alterations during the clinical course of the disease. We used the ParsortixTM (ANGLE) system, a label-independent microfluidic-based technology platform, to enrich CTCs from whole blood of NSCLC patients to evaluate the presence of HER-2 and c-MET amplification using fluorescence in situ hybridization (FISH). Materials and methods: Peripheral blood (PB) (15mL) from 42 patients with EGFRmt and resistant to 1st and 2nd generation EGFR TKIs NSCLC was obtained before, during and after treatment with osimertinib. CTCs were isolated using the ParsortixTM system and were further analyzed for HER-2 and c-MET amplification by FISH. Results: PB samples were obtained before (n=42), after one cycle (n=12) and at PD (n=7) during treatment with Osimertinib. One patient had HER-2 amplified CTCs whereas in total, 5 patients had marginally HER-2-positive CTCs; HER-2-positive CTCs were observed in 1/42 baseline sample but in none post-1st cycle and PD samples. Similarly, c-MET amplification was detected in 3/42 patients at baseline whereas in total 4 patients had marginally c-MET-positive CTCs. Treatment with Osimertinib resulted in a decrease of the number of c-MET amplified CTCs in these patients. In all c-MET and HER-2 amplified CTCs there was no HER-2 or c-MET amplification in the primary tumor. c-MET amplification in the primary tumor was detected only in one patient, however this patient had no detectable CTCs. Conclusions: Our preliminary results indicate that in selected patients with EGFRmt NSCLC, detection of HER-2 or c-MET amplified CTCs may be correlated with tumor resistance to EGFR TKIs. The ParsortixTM system can be successfully combined with downstream FISH analysis for the molecular characterization of CTCs in NSCLC patients. More samples are currently being analyzed in order to reveal the implication of HER-2 and c-MET amplification in resistance to therapy with first, second and third generation EGFR TKIs.

#4577

ESR1 methylation: A liquid biopsy-based epigenetic assay for the follow up of patients with metastatic breast cancer receiving endocrine treatment.

Sofia Mastoraki,1 Areti Strati,1 Eleni Tzanikou,1 Maria Chimonidou,1 Eleni Politaki,2 Alexandra Voutsina,2 Amanda Psyrri,3 Vassilis Georgoulias,2 Evi S. Lianidou1. 1 _Analysis of Circulating Tumor Cells Lab, Univ. of Athens, Athens, Greece;_ 2 _University of Crete, Heraklion, Greece;_ 3 _Attikon University Hospital, Athens, Greece_.

Introduction: Liquid biopsy provides real-time monitoring of tumor evolution and response to therapy through analysis of CTCs and plasma-ctDNA. ESR1 epigenetic silencing potentially affects response to endocrine treatment. We evaluated ESR1 methylation in CTCs and paired plasma-ctDNA. We evaluated ESR1 methylation in CTCs and paired plasma-ctDNA as a potential biomarker for response to everolimus/exemestane treatment. Materials and methods: A highly sensitive and specific real-time MSP assay for ESR1 methylation was developed and validated in: a) 65 primary breast tumors (FFPEs), b) EpCAM+ CTC-fractions (122 patients and 30 healthy donors; HD), c) plasma-ctDNA (108 patients and 30HD), d) in CTCs (CellSearch®) and in paired plasma-ctDNA for 58 BrCa patients. ESR1 methylation status was investigated in CTCs isolated from serial peripheral blood samples of 19 patients with ER+/ HER2- advanced BrCa receiving everolimus/exemestane. Results: ESR1 methylation was detected in: a) 25/65(38.5%) FFPEs, b) EpCAM+ CTC-fractions: 26/112(23.3%) patients and 1/30(3.3%) HD, c) plasma-ctDNA: 8/108(7.4%) patients and 1/30(3.3%) HD. ESR1 methylation was highly concordant in 58 paired DNA samples, isolated from CTCs (CellSearch®) and corresponding plasma. In serial peripheral blood samples of patients treated with everolimus/exemestane, ESR1 methylation was observed in 10/36(27.8%) CTC-positive samples, and was associated with lack of response to treatment (p=0.023 Fisher's Exact Test). Conclusions: We report for the first time the detection of ESR1 methylation in CTCs and a high concordance with paired plasma-ctDNA. ESR1 methylation in CTCs was associated with lack of response to everolimus/exemestane regimen. ESR1 methylation should be further evaluated as a potential liquid biopsy-based biomarker.

#4578

**Development of a rapid clinical grade assay to detect and monitor secondary** KIT **mutations in circulating free DNA (cfDNA) for personalization of targeted therapy for gastrointestinal stromal tumors (GIST).**

Adrian Marino-Enriquez,1 Suzanne George,2 Julianna Supplee,2 Grace Heavey,2 Pasi A. Janne,2 Chandrajit Raut,1 Jonathan Fletcher,1 George D. Demetri,2 Cloud P. Paweletz,2 Yanan Kuang2. 1 _Brigham and Women's Hospital and Harvard Medical School, Boston, MA;_ 2 _Dana-Farber Cancer Institute, Boston, MA_.

Introduction: Genotype-directed therapies are revolutionizing care for gastrointestinal stromal tumors (GIST). Somatic mutations in KIT exons (ex) 11 and 9 induce constitutive kinase activity and account for ~95% of the primary oncogenic events in KIT-driven GIST; KIT mutations correlate with prognosis and predict clinical activity of tyrosine kinase inhibitors (TKI), such as imatinib. After a median of 24 months on imatinib, 85% of patients (pts) develop resistance and progress multifocally, mostly due to secondary KIT mutations in ex 13 and/or ex 17. We describe here a rapid, clinical grade droplet digital PCR (ddPCR) assay to detect and quantify KIT mutations associated with TKI resistance in cfDNA from plasma of metastatic (met) GIST pts. Unlike allele-specific ddPCR that requires splitting input DNA into many reactions, we designed pan-ex 17 KIT assay to capture the polyclonal mutational landscape of KIT in a single reaction.

Methods: Tumor and plasma samples and clinical data were collected from pts with met GIST. ddPCR assays were developed for the detection in cfDNA of all common secondary KIT mutations associated with imatinib resistance (V654A, T670I, A829P and multiple ex 17 mutations). The pan-ex 17 mutation drop-off assay uses a FAM-labeled mutant (mut) probe that spans sequences encoding amino acids 819~824, and a VIC-labeled reference probe that is shared by both wild-type (wt) and mut alleles. Common ex 17 mutations were detected via cluster separation. V654A, T670I, A829P are detected with allele-specific assays that use a FAM-labeled mut probe and a VIC-labeled wt probe. To demonstrate analytical sensitivity/specificity of each assay, ddPCR cycling conditions were optimized to yield the maximum fluorescence signal with minimal noise signal.

Results: The KIT ddPCR assays detect a mutation prevalence of 0.01 - 0.05%, with a sensitivity of 5 - 50 KIT mut copies in a background of 10,000 KIT wt copies, depending on the mutation assayed, with absolute specificity. Results were validated in a pilot observational cohort of pts who were treated with imatinib and progressed, for which matching plasma (1 ml) and tumor samples were analyzed to reveal identical mutations. In a separate cohort, ddPCR results were validated by sensitive plasma NGS.

Conclusions: We have developed rapid, highly sensitive, and 100% specific quantitative assays to enable plasma-based tumor genotyping for secondary KIT resistance mutations, which has been technically optimized for translation into clinical practice. Analysis of cfDNA by ddPCR can successfully detect KIT secondary mutations in met GIST pts. In an ongoing study, serial monitoring of KIT mutations in cfDNA from pts with advanced GIST should allow for early detection of secondary mutations and optimization of genotype-targeted therapeutic management.

#4579

The genetic heterogeneity and the molecular evolution of systemic metastatic castration resistant prostate cancer during therapy.

Rui PL Neves,1 Streit LRF Anna,1 Katharina Raba,1 Elina-Katharina Bongers,1 Bianca Behrens,1 Guus van Dalum,1 Penny Flohr,2 Joaquin Mateo,2 Semini Sumanasuriya,2 Mateus Crespo,2 Berni Ebbs,2 Gemma Fowler,2 Pasquale Rescigno,2 Suzanne Carreira,2 Maryou Lambros,2 Edoardo Petrini,3 Marianna Garonzi,3 Nicolò Manaresi,3 Johann de Bono,2 Nikolas H. Stoecklein1. 1 _University Hospital and Medical Faculty of the Heinrich-Heine University Düsseldorf, Duesseldorf, Germany;_ 2 _The Institute of Cancer Research, London, United Kingdom;_ 3 _Menarini Silicon Biosystems, Bologna, Italy_.

Background: Circulating tumour cells (CTCs) have a significant prognostic impact in metastatic castration-resistant prostate cancer (mCRPC) and provide direct access to the systemic disease. Here, we performed genome-wide copy number analysis of CellSearchTM detected CTCs to follow the genomic evolution of mCRPC during systemic therapy.

Methods: Blood samples were collected from 13 patients with mCRPC before, during and after two different targeted therapy regimes. Single CTCs were isolated from CellSearchTM cartridges using the DEPArray™ system and the MoFlo XDP cell sorter. Genomes of sorted single cells were amplified using Ampli1™ WGA kit. Amplification products were analysed by array-based comparative genomic hybridization (aCGH) and low-pass sequencing (LPS) using Ampli1™ LowPass kit to detect somatic chromosomal copy number aberrations (CNAs) and explore the degree of genomic heterogeneity.

Results: We analysed >300 CTCs for CNAs by aCGH and/or LPS. Although most CTCs displayed CNAs typical for mCRPC, we identified three genomic CTC-groups across all cells with the EpCAMpos/CKpos/DAPIpos/CD45neg phenotype: CTCs with typical mCRPC CNAs (Type A; 80%), extremely aberrant CTCs (Type B; 11%), CNA-negative/low CTCs (Type C; 9%). The occurrence of Type B and C was almost mutually exclusive. At baseline, we noted different levels of CTC-heterogeneity and different CTC-aberration levels between the different patients. Interestingly, CTCs of patients with a relevant decrease in CTC-count under PARP-inhibition (CTC-responder) displayed significantly elevated CNAs at baseline compared to CTC-non-responders. During therapy, we could observe clonal changes among all patients.

Conclusions: Our data show that CTC-analysis enables the dissection of clinical relevant intra-patient heterogeneity and clonal evolution under therapy in mCRPC patients. Comprehensive genomic monitoring during therapy might help to tailor therapies more effectively and may pinpoint to molecular mechanism of therapeutic resistance.

#4580

Changes in the efficacy of anti-EGFR antibody drugs by exosomes derived from colorectal cancer patients.

Rii Morimura,1 Kei Tsukamoto,1 Shinji Irie,2 Shiro Kitano,2 Eiji Shinozaki,3 Kensei Yamaguchi3. 1 _Toppan Printing Co., Ltd., Saitama, Japan;_ 2 _Osaka University, Osaka, Japan;_ 3 _Cancer Institute Hospital of Japanese Foundation for Cancer Research, Tokyo, Japan_.

Background: One of the biggest hurdles in cancer treatment is drug resistance. Exosomes are small membrane vesicles of endocytic origin, which contain mRNAs, DNA fragments, and proteins, and are released by many different cell types, including cancer cells. Several studies have shown exosome-mediated drug resistance mechanisms: drug export via the exosome pathway, neutralization of antibody-based drugs and exosome-mediated transfer of miRNAs. Here, we demonstrated the effect of exosomes derived from colorectal cancer (CRC) patients on the efficacy of anti-EGFR agent by in vitro cell-based assay.

Method: Exosomes were purified by ultracentrifugation from culture media of HCT116 cell line and the sera of CRC patients with stage IV pre- and post-treatment with cetuximab (Cmab). As a control, sera from healthy volunteers were used. The number of exosomes and amount of protein were determined by NanoSight and Qubit fluorometer, respectively. The isolated exosomes and 0.01 mg/mL and 0.1 mg/mL of Cmab were added to HT29 and NCI-H508, which stained with PKH dye previously. NCI-H508 is a Cmab super sensitive cell line. After culturing for 3 days, the cells were collected, stained with trypan blue, and the number of the remaining viable cancer cells was calculated with a cell counter. Cmab in the exosome fraction was quantified by ELISA.

Result: HT29 became drug ineffective depending on the number of exosomes produced by Cmab-insensitive HCT-116 harboring KRAS G13D mutation. In addition, exosomes in 9 of 18 cases of colorectal cancer patients changed NCI-H508 into a drug-ineffective state and also correlated with clinical information. In contrast, NCI-H508 was not converted into a drug-ineffective state by exosomes of healthy volunteers and Cmab responder patients. However, on the other hand, quantitative results by ELISA showed that Cmab in sera of some patients remained in the exosome fraction and inhibited the growth of NCI-H508. As a remarkable point, exosomes derived from healthy volunteers significantly promoted the efficacy of Cmab.

Conclusion: In this feasibility study, Cmab resistance in CRC patients may be reproducible by cell-based assay using exosomes. We believe that understanding this mechanism of action will contribute to drug discovery and personalized medicine in the future. Now, we are searching for biomarkers in these exosomes that change the efficacy of Cmab.

#4581

**Feasibility of an amplicon-based liquid biopsy for** ALK **and** ROS1 **fusions in advanced non-small cell lung cancer (NSCLC) patients.**

Laura Mezquita,1 Yuebi Hu,2 Karen Howarth,3 Cecile Jovelet,1 David Planchard,1 Ludovic Lacroix,1 Aurelie Swalduz,4 Sandra Ortiz-Cuaran,4 Virginie Avrillon,4 Vincent Plagnol,3 John Beeler,3 Katherine Baker-Neblett,3 Greg Jones,3 Nitzan Rosenfeld,3 Clive Morris,3 Emma Green,3 Edward S. Kim,5 Maurice Perol,4 Pierre Saintigny,4 Stephen V. Liu,6 Geoff R. Oxnard,2 Benjamin Besse1. 1 _Gustave Roussy, Paris, France;_ 2 _Dana Farber Cancer Institute, Boston, MA;_ 3 _Inivata, Cambridge, United Kingdom;_ 4 _Centre Leon Berard, Lyon, France;_ 5 _Levine Cancer Institute, Charlotte, NC;_ 6 _Georgetown University Medical Centre, Washington, DC_.

INTRODUCTION Circulating tumor DNA (ctDNA) is a surrogate material for somatic mutation detection, such as EGFR, BRAF or KRAS mutations in NSCLC patients, however the applicability of this technique for ALK and ROS1 fusion detection is poorly described. The aim of this combined analysis was to evaluate an amplicon-based ctDNA technology in a cohort of ALK and ROS1 positive NSCLC patients. METHODS ALK and ROS1 positive NSCLC patients were prospectively enrolled or retrospective specimens selected, to be included across 6 international centres. ALK/ROS1 positive status was determined by standard of care (FISH/IHC or NGS). The analysis of EML4-ALK fusions (variant 1,2,3) and ROS1 fusions (with partner genes CD74, SLC34A2, SDC4 and EZR) was performed using the InVision™ platform. RESULTS Patients included (n=65; 59 ALK, 6 ROS1): 35 (55%) females, 40 (63%) non-smokers, median age of 59 years, stage IV disease (88%) adenocarcinoma (97%). All patients were ALK/ROS positive by IHC (32), FISH (45) and/or tissue NGS (10). Samples (n=98) were collected across multiple timepoints. In total, 31 patients were fusion positive (25 ALK, 6 ROS1). Among treatment-naïve patients, sensitivity was 78% for ALK and 100% for ROS1. In contrast, fusions were detected in minority of samples (7/57) in patients responding to treatment. In patients with ctDNA positive ALK fusion (n=25): 8 patients (32%) presented the EML4-ALK variant 1; 2 (8%) the variant 2 and 15 (60%) the variant 3. In the ROS1 population (n=6), rearrangement with chromosome 5, resulting in CD74-ROS1 was seen in 4 of cases and translocation with chromosome 4 to generate SLC34A2-ROS1 occurred in 2 of cases. All patients had unique DNA fusion junctions identified and will be described, providing an insight at the DNA level of the molecular mechanisms leading to ALK and ROS1 driven lung cancers. Breakpoint microhomology was observed at the fusion junction in 50% of cases, ranging from 1 to 7bp. A 4bp insertion of non-templated DNA was seen in one case, with the remaining 47% of cases most likely the result of non-homologous end joining. Evidence for enrichment of microhomologous sequences and clustering of breaks within the introns will be investigated and reported at the conference. CONCLUSION The detection of ALK and ROS1 fusions using the amplicon-based NGS Invision liquid biopsy platform is feasible in routine clinical practice. Good sensitivity for clinically actionable ALK and ROS structural rearrangements in untreated advanced NSCLC patients was demonstrated.

#4582

Capturing T cell receptor clones from circulating free DNA in cancer patients with solid malignancies.

Lara McGrath,1 Elizabeth Maloney,1 Daniel Stetson,1 Tristan J. Lubinski,1 Rory Kirchner,2 Brian Dougherty,1 J. Carl Barrett1. 1 _AstraZeneca, Boston, MA;_ 2 _Harvard T.H. Chan School of Public Health, Boston, MA_.

Introduction Understanding the tumor microenvironment is challenging in situations where a tumor biopsy is unavailable. In these situations, liquid biopsies provide a less invasive alternative that can be collected at multiple timepoints throughout treatment. In particular, analyses of circulating free DNA (cfDNA) from plasma samples can distinguish genomic alterations from shed tumor DNA (ctDNA) within the cfDNA population. In this study, we aim to better understand the origin of cfDNA as well as its potential in understanding the intratumoral immune infiltration by developing and validating a strategy for identifying T cell receptor β (TCR β) rearrangements in the cfDNA from patients with solid malignancies. Methods We devised a target enrichment approach for high-throughput DNA sequencing that employs a hybridization capture strategy to bait germline variable (V) and joining (J) gene sequences in the TCR β loci. The target sequences are proximal to rearrangement sites flanking the CDR3, the principal site of contact for recognition specificity with foreign antigens presented by the major histocompatibility complex (MHC). We controlled for amplification bias by tagging DNA fragments with unique molecular indices (UMI). We validated our approach on dilutions of T cell lines in non-T cell lines, compared T cell levels determined from our method to those derived from flow cytometry, and compared data complexity at DNA input levels from 100 ng to 2 ng. Finally, we analyzed 27 pre-treatment plasma samples from cancer patients with solid malignanices, including quantification of total cfDNA concentrations, analyzing somatic genomic alterations using whole exome sequencing (WES), and employing our method for capture of TCR β rearrangements. Results Results indicate we are able to capture T cell rearrangements from cfDNA in cancer patient plasma. T cell rearrangements were identified in more than 25% of samples (7/27). Interestingly, the total cfDNA levels were not correlated to the number of T cell rearrangements detected, but all samples with detectable somatic alterations in the cfDNA (3/27) were also those with T cells identified. These preliminary results suggest a connection between apoptosis of lymphocytes and solid tumors that shed DNA into the bloodstream, and demonstrate potential utility as liquid biopsy for identifying tumor infiltrating lymphocytes.

#4583

Combined analysis of circulating tumor cells, fecal occult blood test and serum carcinoembryonic antigen for early detection and monitoring recurrence of patients with colorectal cancer.

Wei-Shan Hung,1 Wen-Sy Tsai,2 Tzu-Min Wang,1 Hsueh-Ling Hsu,1 Hsuan Liu,1 Chia-Yu Yang,1 Ching-Ping Tseng,1 Ju-Chien Cheng3. 1 _Chang Gung University, Taoyuan, Taiwan;_ 2 _Chang Gung Memorial Hospital, Taoyuan, Taiwan;_ 3 _China Medical University, Taichung, Taiwan_.

Current methods for clinical early detection and disease monitoring of patients with colorectal cancer (CRC) are immunochemical fecal occult blood test (iFOBT) and serum levels of carcinoembryonic antigen (CEA), which are known with low sensitivity. There is an unmet need to seek for additional molecular tools for early detection and disease monitoring of patients with CRC. Whether enumeration of circulating tumor cells (CTCs) supplements current clinical tests and facilitates early detection and monitoring disease progression was investigated. A total of 109 pre-operative patients with CRC (including early and advanced stages) and 65 non-cancerous controls were enrolled in this study. CTCs were enriched from the peripheral blood of these individuals by using the PowerMag negative selection system. Immunofluorescence staining of epithelial cell adhesion molecule (EpCAM), and podoplanin (PDPN, a marker related to poor cancer progression) was performed to define the two distinct subtypes of CTCs. Heterogeneous CTCs expressing PDPN or EpCAM are defined in the peripheral blood of patients with CRC. Regardless clinical stages in patients, the number of both EpCAM+-CTCs and PDPN+-CTCs explicitly differentiated non-cancerous controls and patients with CRC, and were also correlated with clinicopathological diagnosis. Receiver operating characteristic analysis demonstrated that the sensitivity and specificity of EpCAM+-CTCs was 86.32% and 78.46%, respectively, when the cutoff value was 23 EpCAM+-CTCs/mL. Using 7 PDPN+-CTCs/mL as the cutoff value, the sensitivity and specificity of PDPN+-CTCs was 77.98% and 75.38%, respectively. 84.2% and 80.3% of patients with serum CEA < 5 ng/mL were found to have higher EpCAM+-CTCs and PDPN+-CTCs counts, respectively. In addition, simultaneous measurements of iFOBT and CTCs could compromise the false-positive rate of iFOBT, while measurements of CEA and CTCs are applicable to monitor cancer recurrence after surgery. This study implicates that CTC testing supplements the current clinical methods to facilitate early detection and disease monitoring in CRC.

#4584

High-definition single-cell analysis of liquid and solid biopsies reveals heterogeneity of circulating tumor cells in metastatic colorectal cancer.

Anna S. Gerdtsson,1 Jana-Aletta Thiele,2 Randolph Schaffer,3 Kelly Bethel,3 Steven Curley,4 Heinz-Josef Lenz,5 Diana L. Hanna,5 Jorge Nieva,5 Anand Kolatkar,1 Mariam Rodriguez Lee,1 James Hicks,1 Peter Kuhn1. 1 _University of Southern California, Los Angeles, CA;_ 2 _Charles University, Plzen, Czech Republic;_ 3 _Scripps Clinic, La Jolla, CA;_ 4 _Baylor College of Medicine, Houston, TX;_ 5 _USC Norris Comprehensive Cancer Center, Los Angeles, CA_.

Treatment of cancer patients is transitioning to personalized strategies using targeted drugs with the required companion biomarkers. Many current biomarkers are tissue-based, requiring a solid biopsy and limiting sampling frequency. Given this, there is significant interest in determining whether noninvasive, blood-based liquid biopsies, utilizing measurements of factors that exist in the blood such as circulating tumor cells (CTCs), reflect solid tumors and can serve as substitutes for solid tumor biopsies. Colorectal cancer with hepatic metastases (mCRC) is one of very few scenarios where metastatic patients routinely undergo surgical resection, providing an opportunity for accessible tissue and simultaneous blood sampling. We collected blood and tumor touch preparations from patients undergoing resection of CRC with the purpose to determine the correlation of solid biopsies and the liquid phase of solid tumors in the mCRC setting. Samples were processed using the previously described HD-SCA (High-Definition Single-Cell Analysis) workflow for high-resolution imaging and characterization of tumor cells at a single-cell level. Briefly, tumor cells were identified by presence of nuclear stain, pan-cytokeratin, and absence of CD45. The colon-specific marker CDX2 was included as a fourth color for further characterization. Subsets of cells were subjected to copy number variation (CNV) profiling. The comparison of pre- and post-surgery blood samples showed that the concentration of CTCs decreased significantly after surgery. The concentration was markedly higher in patients with synchronous disease undergoing simultaneous resection of the primary tumor, indicating that the presence of CTCs is related to tumor burden. Principal component analysis demonstrated that the CTCs were more similar to the metastatic cells than to the primary colon cells. While protein levels varied between the liquid and solid biopsy cell populations, morphologic parameters including, e.g., cell and nuclear area and roundness, were similar in the two. The intrapatient analysis showed heterogeneous CTC populations, in general with a small subset of cells bearing characteristics more similar to the solid tumor cells than the bulk of CTCs. The presence of subpopulations was further indicated by the CNV analysis, which showed that only a minority of CTCs had genomic rearrangements and that, in contrast to the solid biopsy cells, the rearranged cells were largely nonclonal. Based on the high-content HD-SCA data, the CTCs were further grouped into different subtypes. In conclusion, we introduce an approach for high-content comparison of liquid and solid biopsies at a single-cell level. We show that the concentration of CTCs is related to tumor burden, and present a subclassification of rare circulating cells in mCRC.

#4585

Development of multi-marker capture and analysis of circulating tumor cells in renal cell carcinoma.

Rory M. Bade,1 Benjamin K. Gibbs,2 Jamie M. Sperger,1 Christos Kyriakopolous,1 Hamid Emamekhoo,1 Rana R. McKay,3 Toni K. Choueiri,4 Joshua M. Lang1. 1 _Carbone Cancer Center, University of Wisconsin-Madison, Madison, WI;_ 2 _University of Kansas Medical Center, Kansas City, KS;_ 3 _University of California, San Diego, San Diego, CA;_ 4 _Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA_.

Purpose: Circulating biomarkers are an emerging tool to monitor treatment response and the emergence of resistant phenotypes. However, studies of circulating biomarkers, including circulating tumor cells (CTCs), in patients with clear cell renal cell carcinoma (ccRCC) have been limited due to difficulty in biomarker identification. Platforms relying on EpCAM and cytokeratin to identify CTCs have been limited due to significant phenotypic and intrapatient heterogeneity in renal cancer. Carbonic anhydrase IX (CAIX) and XII (CAXII) are more broadly expressed in ccRCC and recently been shown to capture CTCs from patients with ccRCC. However, downregulation of these targets can also occur; EpCAM is also expressed on a subset of cells that could go undetected if only CAIX was used to capture these cells. The aim of this study is to optimize multi-marker capture and analysis of ccRCC CTCs using EpCAM, CAIX and CAXII for further molecular analysis.

Methods: We utilized the VERSA platform, an integrated CTC capture and analysis technology, to optimize capture of multiple ccRCC cell lines using antibodies to CAIX and/or EpCAM. To maximize the capture efficiency of ccRCC CTCs, we altered the magnetic particle type, antibody concentration, and tested both direct and indirect capture methods. Once an optimal method of capture was determined, we captured CTCs in an initial cohort of ten patients with ccRCC. CTCs were identified as cells that were captured by either CAIX or EpCAM, had an intact nucleus, were negative for CD45/CD34/CD66b, and positive for cytokeratin.

Results: Capture of cell lines show that a combined CAIX + EpCAM capture was more efficient than single antibody capture using either EpCAM or CAIX alone. The type of magnetic particle used in the assay also affected capture efficiency. Sera-Mag beads (GE Healthcare) captured significantly more cells than FlowComp Dynabeads (Life Technologies) (94% vs 76%). Further increases in efficiency were made by incubating with antibody prior to bead conjugation (indirect binding, 94%) when compared to incubating the cells with antibody-conjugated beads (direct binding, 98%). This may result from increased accessibility of free antibody to partially obstructed antigens that is unique to renal cell carcinoma. This optimized assay has now been applied to ccRCC patients and has identified CTCs in up to 90% of patients with metastatic disease.

Conclusions: We have increased the capture efficiency and identification of ccRCC cells by capturing with a combination of CAIX and EpCAM antibodies and optimizing bead and binding conditions. In doing so, we are able to identify and interrogate populations of CTCs that would be lost in capture methods that rely on EpCAM alone. These assays are now being utilized in multiple biomarker and therapeutic trials for patients with clear cell renal cell carcinoma.

#4586

Evaluation of available blood collection tubes for use in stabilizing concentrations of extracellular vesicles / exosomes and associated cell-free RNA.

Nicholas George. _Streck, La Vista, NE_.

Introduction: Liquid Biopsy has revolutionized the detection and the monitoring of cancer disease states. In addition to the widely utilized circulating cell-free DNA (ccfDNA), cell-free RNAs (cfRNA) and the extracellular vesicles (EV's) / exosomes they are stored in, is drawing significant interest in cancer diagnostics. Analysis of disease-specific EV's / exosomes or cfRNA requires that collected samples maintain draw-time concentrations of these analytes in order to remain relevant. Blood components, such as platelets, red, and white blood cells, are unstable when stored in normal blood collection tubes (BCT's), thus leading to increases of EVs and associated cfRNA concentration. We undertook an analysis of currently available commercial BCT's to determine whether any of these sufficiently stabilize EV / exosome and cfRNA concentration. Materials and Methods: Healthy consenting adult donors were drawn into blood collection tubes per approved protocol. Samples were either processed immediately (Day-0) or allowed to incubate at room temperature for the indicated times. Cleared plasma was isolated and subsequently frozen at -80oC until use. Assay of extracellular vesicle concentration utilized the Nanosight-300 (Malvern Instruments) per manufactures protocol. Isolation of cell-free RNA utilized the Circulating Nucleic Acid Isolation Kit (Qiagen) per manufacturer's protocol with the exception of the lysis step; this was extended from 30 minutes to 60 minutes. Digital PCR (ddPCR) utilized the Bio-Rad workflow (QX200 Droplet Generator and Reader). Conclusion: Stabilization of EV's and cfRNA was assayed for blood samples drawn into a variety of Streck nucleic acid stabilizing BCT's, non-Streck nucleic acid stabilizing BCT's, and a traditional K3EDTA BCT. A majority of the assayed tubes led to time-dependent increases in EV concentration with the most robust increases occurring between Day-3 and Day-7. Time-dependent increases in cfRNA concentration mirrored the increases observed in EV's, and based on ddPCR analysis; the majority of this increase was likely the result of erythrocyte membrane shedding. One type of Streck BCT was found to stabilize both EV and cfRNA concentration as a function of blood storage time. Incorporation of this tube into the Liquid Biopsy workflow will maximize assay target sensitivity by preventing the deleterious effects of excess cfRNA from non-specific release of EV's / exosomes.

#4587

ESR1 **methylation in primary tumors and paired circulating tumor DNA of patients with high-grade serous ovarian cancer.**

Lydia Giannopoulou,1 Sofia Mastoraki,1 Areti Strati,1 Issam Chebouti,2 Kitty Pavlakis,3 Sabine Kasimir-Bauer,2 Evi Lianidou1. 1 _Analysis of Circulating Tumor Cells Lab, University of Athens, Athens, Greece;_ 2 _University Hospital of Essen, University of Duisburg-Essen, Essen, Germany;_ 3 _IASO Women's Hospital, Marousi, Athens, Greece_.

Background: The estrogen receptor is highly expressed in epithelial ovarian cancer and represents the main target for endocrine therapy. The ESR1 gene is frequently methylated in many types of gynecological malignancies and previous studies have shown an inverse correlation between ESR1 methylation and gene expression. Few studies attempted to investigate the role of ESR1 methylation in ovarian cancer so far, and the clinical significance of ESR1 methylation status is not as yet clear.

Methods: The ESR1 methylation status was examined in primary tumors and corresponding circulating tumor DNA (ctDNA) samples of patients with high-grade serous ovarian cancer. For the detection of methylation we applied a novel highly specific and sensitive real-time methylation specific PCR (real-time MSP) assay. Two groups of primary tumor samples were recruited (training group, n=66 and validation group, n=61), along with the corresponding plasma samples (n=58) for the validation group. ESR1 methylation was also analyzed in a small group of 16 normal fallopian tube samples.

Results: ESR1 was found methylated in both groups of primary tumor samples and in the corresponding plasma samples of the validation group. More specifically, ESR1 methylation was detected in 32/66 (48.5%) and 17/61 (27.9%) primary tumor samples of the training and the validation group, respectively, and in 23/58 (39.7%) corresponding plasma samples. A significant agreement between ESR1 methylation in primary tumors and paired ctDNA was observed in 40/56 (71.4%) samples of the validation group (P=0.004, k=0.360). Almost all normal fallopian tube samples (15/16) were found methylated. Interestingly, the presence of ESR1 methylation in the primary tumor samples of the validation group was nearly significantly correlated (P=0.057) with a better overall survival.

Conclusions: We detected for the first time ESR1 methylation in ctDNA of patients with high-grade serous ovarian cancer. The agreement between ESR1 methylation in the primary tumors and paired ctDNA is statistically significant. Our results indicate a potential correlation between ESR1 methylation and better overall survival in high-grade serous ovarian cancer patients.

#4588

Prognostic association of plasma cell free DNA (cfDNA) copy number variation based algorithmic score with survival in metastatic castration resistant prostate cancer (mCRPC).

Manish Kohli,1 Meijun Du,2 Liang Wang,2 Chiang-Ching Huang3. 1 _Mayo Clinic, Rochester, MN;_ 2 _Medical College Wisconsin, WI;_ 3 _University of Wisconsin, Milwaukee, WI_.

Introduction: Survival in mCRPC stage is heterogeneous and in this stage no molecular predictors of survival are known. Elevated circulating tumor cell (CTC) counts (>5/7.5 ml blood CellSearch® assay) is the only FDA cleared prognostic biomarker in mCRPC stage that predicts poor prognosis but is not routinely performed in clinical practice. We evaluated a novel plasma cfDNA based molecular algorithmic score which is based on copy number variations (CNVs) in multiple candidate genes as a prognostic predictor of survival and also compared it to CTCs. Methods: We obtained biospecimens in a prospective cohort study (NCT#01953640) conducted in mCRPC patients who demonstrated progression on androgen deprivation therapy and were candidates to initiate pre-chemotherapy abiraterone acetate/prednisone (AA/P). Biospecimen collection was performed prior to initiating AA/P and included plasma and CTCs, both obtained at the same time. All samples underwent uniform processing. Cellsearch® assay was used for enumerating CTC counts. Plasma cfDNA was extracted using Qiagen whole blood kit and low pass sequencing was performed in the extracted plasma cfDNA and germline DNA. An algorithmic score based on CNVs in 13 candidate genes was developed after calculating an Absolute Log2 Ratio (ALR) for each pre-selected gene. The sum of ALRs for these 13 genes were combined to make a "Plasma Genome Abnormality" (PGA) score which was evaluated for association with the primary endpoint of overall survival (OS) using Hazard ratio (HR) with significance set at p ≤ 0.05. Association of concurrently collected CTC counts with OS was also determined. The PGA score based survival was adjusted for CTC counts in the final analysis. Results: Plasma and CTCs were available for analysis in 72/90 enrolled patients between 05/2013 and 09/2015 with the last enrolled patient having completed 23.5 months follow-up. 45/72 patients have died of cancer progression and the median OS of the cohort is 26.5 months (IQR: 19-46). The median plasma cfDNA amount obtained was 7.2 (Range: 1.6-70.4)ng/ml plasma and the median CTC count is 1/7.5ml blood (range: 0-308). The median PGA score was 1.108 (Range: 0.56-6.96) and the CNV based PGA score was observed to be associated with OS (HR: 1.63; 95% C.I: 1.144, 2.308); p-value= 0.0066). Association of elevated CTC counts (≥ 5/7.5 ml blood) with OS showed a statistically significant trend (HR: 1.92; CI: 0.99 - 3.7; p-value 0.051). After adjusting for CTC counts, an increased PGA score remained predictive of poor survival (HR: 1.52; 95% CI: (1.04, 2.21); p-value 0.028). Conclusions: In this cohort the PGA score, a molecular algorithm predictor based on plasma cfDNA copy number variations in candidate genes was a better prognostic marker of survival than CTC counts and potentially offers a biomarker tool which could be adopted for wider clinical application.

#4589

Expression of PD-L1 on circulating breast cancer cells: Correlation with clinicopathologic data and impact on prognosis.

William Jacot,1 Martine Mazel,2 Caroline Mollevi,1 Stéphane Pouderoux,1 Véronique D'Hondt,1 Laure Cayrefourcq,2 Angélique Ducteil,1 Marie Viala,1 Thierry Maudelonde,2 Séverine Guiu,1 Catherine Alix-Panabières2. 1 _ICM Val d'Aurelle-Paul Lamarque, Montpellier, France;_ 2 _University Medical Centre, Montpellier, France_.

Purpose

Immune therapies, including anti-PD-L1 therapies, are widely evaluated in the treatment of solid tumors, including metastatic breast cancer (MBC). While tumor PD-L1 expression has been largely reported, with conflicting results regarding its theranostic value, few data are available regarding the prognostic value of the determination of PD-L1 expression on circulating tumor cells (CTCs), allowing a minimally invasive determination of this biomarker of interest. We previously published a validated and robust method for PD-L1(+)-CTC detection. In this new report, we focused on the clinicopathological correlations and prognostic value of the presence of PD-L1(+)-CTCs in an expanded MBC cohort.

Methods

We investigated PD-L1(+)-CTCs as previously reported (Mazel et al., Molecular Oncology, 9 (2015); 1773-1782) using the CellSearch® system in 72 MBC patients from our previous report and a prospective dedicated biomarker study (NCT02866149).

Results

Median age was 65 years (range 35-87). 69.4% of tumors were classified ER(+)/HER2(-), 18.1% HER2(+) and 12.5% triple negative (TN). 40 patients (55.6%) were treated in ≥ fourth metastatic line. 58.3% of the patients presented with more than 2 metastatic sites. Archival tissue was available for 56 patients (77.8%) and 13 samples (23.2%) showed positive (≥1%) PD-L1 expression. Baseline CTCs were detected in 57 patients (79.2%), with 43 (59.7%) and 36 (50%) patients with ≥4 and ≥10 CTCs, respectively. PD-L1(+)-CTCs were detected in 26 (36.1%) cases. No statistically significant correlation was found between PD-L1 tumor tissue expression and the presence of PD-L1(+)-CTCs (p=0.694). At the time of analysis, with a median follow-up of 13 months, 55 patients (76.4%) had progressed and 33 (45.8%) died.

Median progression-free survival (PFS) was 5 months (95%CI 3.5-8.0). In univariate analysis, TN phenotype (p=0.020), more than 3 previous metastatic treatment lines (p<0.001) and detection of PD-L1(+)-CTCs (p=0.047) were significantly associated with PFS, while it was not the case for the CTC count (≥4 CTCs threshold) or the PD-L1 expression in the biopsy (p=0.100 and p=0.280).

Median overall survival (OS) was 20 months (95%CI 11.1-38.3). In univariate analysis, TN phenotype (p=0.007) and the CTC count, either at the ≥4 (p=0.024) or ≥10 (p=0.027) threshold were significantly associated with OS, while there was a trend for association with OS of PD-L1(+)-CTCs detection (p=0.078).

Conclusion

In this heterogeneous MBC cohort, PD-L1 expression on CTCs appears independent of PD-L1 expressed in the primary tumor site. MBC patients harboring PD-L1(+)-CTCs seems to have a worse PFS. Evaluation of the respective impact of PD-L1 expression in the tumor and by CTCs is needed in a population treated by anti-PD-1/PD-L1 antibodies in order to evaluate their respective predictive values. An update of the OS data is warranted.

#4590

Larger family sizes improve stochastic error correction during untargeted searches of very-low frequency variant alleles in circulating cell-free DNA (ccfDNA).

Preetida J. Bhetariya,1 David Nix,1 Sabine Hellwig,2 Gabor Marth,1 Mary Bronner,1 Hunter Underhill1. 1 _University of Utah, Salt Lake City, UT;_ 2 _ARUP Laboratories, Salt Lake City, UT_.

Detection of ccfDNA derived from non-metastatic solid tumors requires discrimination of very-low frequency true variants from NGS library preparation and sequencing noise that commonly occurs at a similar frequency (<1%). Correction of NGS-related errors is particularly important during searches of unknown tumor-associated variants with multi-gene panels. Unique molecular identifiers (UMIs) have been used during in silico error correction by molecular barcoding and performing a consensus base calling at each position from DNA labelled with the same UMI (i.e., PCR duplicates). In this study, we sought to determine the utility of UMIs for error correction of stochastic noise in ccfDNA. ccfDNA was isolated from the plasma of 11 healthy participants. An eight base-pair random barcode (UMI) was ligated prior to library amplification. Libraries were enriched using a custom capture probe set (128 genes; 128 kb) and then sequenced (HiSeq 2500). Reads were aligned, grouped based on UMI similarity, and a consensus read sequence was determined. The number of PCR duplicates that yielded a single consensus sequence was defined as family size (FS). For non-reference alleles detected at a variant allele frequency (VAF) ≥ 0.1% and ≤ 1.0%, there was a significant reduction in observed counts with increasingly larger FSs (Table 1). This effect was observed for both unique and common non-reference allele counts (Table 1). Of note, counts of unique non-reference alleles (stochastic noise) were consistently greater than the common non-reference alleles regardless of family size (Table 1). The use of larger family sizes reduced the stochastic noise that may confound detection of very-low frequency variants present in ccfDNA during searches for unknown tumor-associated variants. Strategies that generate larger family sizes or suppress early PCR errors may further improve NGS specificity to broaden liquid biopsy clinical applications.

Table 1. Mean (SD) Non-Reference Allele Counts with a VAF between 0.1% and 1.0% in Cell-Free DNA

---

|

FS >= 1 | FS >= 5 | FS >= 10 | FS >= 15 | FS >= 20 | ANOVA Results F(4,50) | P-value

All Variants | 5536 (1230) | 3988 (881) | 2621 (805) | 1028 (552) | 1028 (552) | 49.8 | <0.001

Unique Variants | 3597 (1107) | 2635 (766) | 1112 (526) | 709 (434) | 709 (434) | 27.6 | <0.001

Common Variants | 1939 (161) | 1353 (188) | 521 (141) | 318 (121) | 318 (121) | 186.1 | <0.001

#4591

Integration of genomic analysis and assessment of pre-analytic variables in the HD-SCA workflow: A technical validation study.

Stephanie Nicole Shishido,1 Mariam Rodriguez_Lee,1 Anand Kolatkar,1 Liya Xu,1 Sara Restrepo-Vassalli,1 Lisa Welter,1 Anders Carlsson,1 Emily Greenspan,2 Shelley Hwang,3 Kathryn Waitman,4 Jorge Nieva,1 Kelley Bethel,5 James Hicks,1 Peter Kuhn1. 1 _University of Southern California, Los Angeles, CA;_ 2 _Center for Strategic Scientific Initiatives (CSSI), National Cancer Institute, Bethesda, MD;_ 3 _Duke University Hospital, Durham, NC;_ 4 _Billings Clinic, Billings, MT;_ 5 _Scripps Clinic, La Jolla, CA_.

The liquid biopsy allows assessment of multiple biological analytes over time to provide dynamic temporal information with the potential for improving clinical management and guiding treatment decisions. While the promise of liquid biopsies for prediction and response monitoring are intensely investigated, the pre-analytic variables are of primary concern for its implementation in diagnostic clinical medicine, including such categories as collection method, shipping conditions, and sample storage. Here we utilize an integrated high-definition single cell analysis (HD-SCA) workflow for genomic analysis of rare cells and cfDNA from the liquid biopsy to characterize the effects of pre-analytical variation and reproducibility of data analysis from the same cohort of patients. The results presented here confirm consistent rare cell enumeration and morphometric characterization between 24h and 48h time to assay (TTA), with a high efficiency and capacity for both copy number variation (CNV) and single nucleotide variation (SNV) analysis at the single cell level. Additionally, the freezing process neither diminishes or increases the DNA quantity, nor does it affect the DNA quality for CNV or SNV analysis. The integration of genomic information is imperative to reveal essential disease-implicated mutational profiles, but effective molecular diagnostic tests require reproducible coverage over a broad dynamic range. The performance of the HD-SCA platform quantified here may be utilized as a guide for implementation into patient care and/or research biorepository processes.

#4592

Verification of a complete sample to insight liquid biopsy workflow: NGS of ccfDNA from stabilized blood.

Tomasz Krenz,1 Andrea Ullius,1 Ricardo Huebel,1 Thorsten Voss,1 Daniel Groelz,1 Eric Provencher,2 Timothy R. Buirkle2. 1 _PreAnalytiX GmbH, Hilden, Germany;_ 2 _BD, Franklin Lakes, NJ_.

Introduction:

Analysis of circulating cell-free DNA (ccfDNA) from blood in the fields of life science, clinical research and beyond is expanding and has become widely accepted. The complete workflow entails collection and stabilization of blood, transport, plasma processing, extraction of ccfDNA and downstream analysis. Considering the usual low sample input and required high sensitivity for quality control (QC) and variant detection, optimal outcomes require verification of the entire workflow.

In this verification study, human blood was collected, stabilized, and then processed to isolate ccfDNA from plasma using the PAXgene® Blood ccfDNA System for subsequent analysis by next-generation sequencing (NGS). To verify the workflow, several sample QC criteria were assessed at each step of the workflow, including ccfDNA yield, target enrichment, library preparation, target sequencing and analysis.

Methods:

Blood from 60 healthy consented donors was collected into PAXgene Blood ccfDNA Tubes* and stored for 7 days at room temperature (15–25°C) to simulate a typical processing delay in a routine setting. Automated ccfDNA extraction was performed on the QIAsymphony® SP instrument using the QIAsymphony PAXgene Blood ccfDNA Kit* and protocol. ccfDNA stability after blood storage was confirmed by qPCR. ccfDNA samples were sequenced on the QIAGEN GeneReader® NGS System, including the GeneRead™ QIAact Actionable Insights Tumor (AIT) Panel* for PCR target enrichment, library preparation on the QIAcube® instrument, QC with capillary electrophoresis, sequencing on the GeneReader instrument* and data management with the QIAGEN Clinical Insight (QCI™) Analyze* tool.

Results:

After blood storage for 7 days, ccfDNA yield was similar to ccfDNA yield observed directly after blood draw. All samples (60/60) passed the required QC criteria after target enrichment (amplicon size around 160 bp) and library preparation (amplicon size around 252 bp and absense of unspecific products <170 bp). Also, all GeneReader NGS acceptance criteria that defined "passed" or "out-of-spec" samples were passed after sequencing, including reads above average quality 25 with 90.16 ± 2.04% (acceptance criteria >80%), region of interest with coverage of bases >500x with 99.83 ± 0.47% (>90%) and region of interest with coverage of bases >200x with 99.99 ± 0.03% (>95%). Across all samples, 17 ± 5 different variants in 7 ± 1 different genes were identified with the AIT targeted panel.

Conclusions:

NGS is one of the most important applications for analysis of ccfDNA in research and clinical settings. This study verified that ccfDNA stabilized and extracted with the PAXgene Blood ccfDNA System is highly suitable for NGS applications, meeting quality control acceptance criteria for 100% of analyzed samples.

*For Research Use Only. Not for use in diagnostic procedures.

#4593

Genomic profiling of cell free DNA (cfDNA) from patients with inflammatory breast cancer (IBC).

Jianming Pei,1 Jacqueline Talarchek,1 Jennifer S. Winn,1 Katherine Alpaugh,1 Massimo Cristofanilli,2 Sandra V. Fernandez1. 1 _Fox Chase Cancer Center, Philadelphia, PA;_ 2 _Northwestern University, Chicago, IL_.

Inflammatory breast cancer (IBC) currently accounts for 2% to 6% of all breast cancer cases in the United States and up to 20% of all breast cancer cases globally. IBC exhibits distinctively aggressive clinical features compared to all breast cancers, and accounts for a disproportionally high mortality rate—15% of breast cancer-related deaths in U.S. In addition, the survival rates between stage-matched IBC and non-IBC differ drastically. Therefore, we are in need of better understanding the molecular abnormalities driving IBC aggressive phenotype. We performed a study to evaluate the genomic alterations in cell free DNA (cfDNA) from plasma from 13 IBC patients, including 9 with triple-negative disease. In 6 patients, mutation analyses were also studied in tumor samples (tumor tissue or tumor cells from pleural effusions). Mutation analysis was performed by next-generation sequencing (NGS) using a unique molecular identifiers (UMI) assay and a panel of 93 breast cancer-related genes (Qiagen). The data were analyzed using the Qiagen's GeneGlobe portal and Biomedical Genomics Workbench and interpretation was performed with Qiagen's QCI. Somatic mutations detected in cfDNA samples were seen in: TP53 (7/13), RB1 (2/13), GEN1 (2/13) and EP300 (2/13); additional somatic mutations were found in PIK3CA (1/13), ERBB2 (1/13), PALB2 (1/13) and MUC16 (1/13). In 4 patients with plasma and tumor samples taken at the same time of the disease progression, complete concordance was not found in the somatic mutations detected in cfDNA and tumor cells DNA. Interestingly, in 12 of 13 IBC patients some variants were observed with high variant allele frequencies (VAF), ~50% or ~100% at a total coverage depth ≥230 reads, in the following genes: BRCA2 (1/13), RAD51D (2/13), PALB2 (1/13), RAD51C (1/13), AR (1/13) and MUTYH (1/13), which were classified as pathogenic or likely pathogenic, and BARD1 (3/13), SYNE1 (2/13), KMT2C (2/13), BRIP1 (1/13), XRCC3 (1/13), RET (1/13), APC (1/13), RAD50 (1/13) and MUC16 (1/13), classified as variants of uncertain significance. Moreover, 11 of these patients had a family history of different cancers including breast, colon, prostate, stomach, bladder, cervical cancers, and melanoma and myeloma. In the 6 patients where cfDNA and tumor samples were available, mutations with high VAF were found in both samples (100% concordance), suggesting that variants with high VAF are germline variants. These results suggest that the described germline variants could increase the risk of IBC and somatic mutation information obtained from cfDNA is complementary to that obtained from tissue samples. Studies on more samples are in progress.

#4594

Detection of colorectal cancer related gene mutations from CTC and ctDNA.

Kohki Takeda, Takeshi Yamada, Michihiro Koizumi, Seiichi Shinji, Yasuyuki Yokoyama, Goro Takahashi, Masahiro Hotta, Takuma Iwai, Keisuke Hara, Hiroyasu Furuki, Eiji Uchida. _Nippon Medical School Hospital, Tokyo, Japan_.

[Background] It is well known that emerging mutations which is not found in the primary tumor exist in metastatic tumor and molecular therapy induce emerging mutation. Liquid biopsy, which includes circulating tumor cell (CTC) and circulating tumor DNA (ctDNA), may help detecting this spatial and temporal heterogeneity. We have reported that emerging KRAS mutation can be detected by using ctDNA (Yamada et al, Cancer Science 2016). However, mutation detection by using CTC has been difficult because enough amount of DNA cannot be extracted from CTC. Currently we have been able to collect more CTC than before, by using a new device which uses 3 antibodies (EpCAM, Her2, Trop2). In this study, we evaluated the potential to detect colorectal cancer (CRC) related gene mutations from CTC, and compared it with ctDNA.

[Methods] Cohort 1: This cohort included untreated CRC patients. Tumor tissue was collected from each patient, either by primary surgery or by colonoscopic biopsy. DNA was extracted from tumor tissue and was analyzed using Next Generation Sequencing (NGS). Ten mL of whole blood was also collected from the same patient. CTC, serum and white blood cell (WBC) was collected by using the CTC recovery machine (Ion Torrent Liquid Biopsy Instrument®). Cytokeratin positive, DAPI positive, CD45 negative cells were defined as CTC. DNA was extracted from each sample (CTC-DNA, ctDNA, WBC-DNA) and was analyzed using NGS. Cohort 2: This cohort included unresectable CRC patients with KRAS mutation in their primary tumor. All patients in this cohort were under treatment or after completion of chemotherapy. CTC and ctDNA was collected in the same method as cohort 1. KRAS mutations of CTC and ctDNA were detected by using digital PCR (dPCR).

[Results] Cohort 1: We enrolled 16 CRC patients (stage II: n=2, stage III: n=2, stage IV: n=12). A total of 30 somatic, hotspot mutations were detected from tumor tissue DNA. The median number of the detected mutation for each patient was 2 (0-4). The most frequent gene mutation was APC, followed by KRAS and TP53. In all patients, CTC was successfully collected. The median number of the CTC was 34 cells (5-94). However, only 6 somatic, mutations were detected from CTC-DNA. Conversely, 16 somatic mutations were detected from ctDNA. Cohort 2: We enrolled 14 stage IV CRC patients with KRAS mutation in their primary tumor. CTC was collected from 9 patients but not from 5 patients. In the 9 patients CTC was collected, the median number of the collected CTC was 26 cells (5-121). By dPCR, KRAS mutation was detected in 2 patients (2/14) from CTC-DNA, and 4 patients (4/14) from ctDNA.

[Conclusions] The new CTC capturing technology using 3 antibodies can improve detection rate and yield of CTC. However, in patients undergoing chemotherapy, the amount of CTC and ctDNA drastically reduces. At present, ctDNA is superior to CTC in potential to detect mutations, and dPCR is more sensitive than NGS to detect mutations.

#4595

The VAR2CSA malaria protein efficiently retrieves CTCs from a broad spectrum of cancers.

Mette Ø. Agerbæk,1 Sara R. Bang-Christensen,1 Ming-Hsin Yang,2 Thomas M. Clausen,1 Sisse B. Ditlev,1 Marina A. Pereira,1 Morten A. Nielsen,1 Swati Choudhary,1 Tobias Gustavsson,1 Poul H. Sorensen,3 Tim Meyer,4 David Propper,5 Jonathan Shamash,5 Thor G. Theander,1 Alexandra Aicher,2 Mads Daugaard,3 Christopher Heeschen,2 Ali Salanti1. 1 _University of Copenhagen, Copenhagen, Denmark;_ 2 _University of London, London, United Kingdom;_ 3 _University of British Columbia, Vancouver, British Columbia, Canada;_ 4 _University College London, London, United Kingdom;_ 5 _Barts Health NHS, London, United Kingdom_.

Metastasis is the leading cause of cancer-related deaths. The metastatic process requires malignant cells to break out of the primary tumor and travel to distant sites through the vasculatory system. Studies have shown that blood circulating tumor cells (CTCs) can be used for patient management as CTC enumeration provides a non-invasive tool for stratification and monitoring disease status. Furthermore, there is a great interest in fully characterizing this highly metastatic subfraction of the tumor cells. Various assays have been developed to enable magnetic enrichment of CTCs from patient blood. Most depend on antibodies targeting the epithelial marker, EpCAM, and are therefore limited to tumors of epithelial origin. Consequently, there is a need for technologies that enable broad capture of CTCs from all types of cancers.

We have recently shown that placental and cancer cells share a unique form of oncofetal chondroitin sulfate (ofCS), not present on normal tissue. We have further shown that ofCS is an acquired trait for cancer cells to enable migration and to potentiate the metastatic potential of cancer cells. Most importantly, we showed that VAR2CSA, a malaria protein involved in placental malaria, binds specifically to ofCS on a wide range of cancer patient-derived cell lines and tissues of both epithelial and mesenchymal origin. Since ofCS seems to be universally expressed across most cancer types and is heavily involved in cancer cell migration, we hypothesized that the recombinant VAR2CSA (rVAR2) protein could enable an efficient isolation of CTCs from blood samples from a broad spectrum of cancer patients.

Here, we show that we can use rVAR2-coated magnetic beads to capture ofCS-positive CTCs. The method was validated using blood samples spiked with cancer cells, followed by clinical feasibility testing in diverse types of cancers to demonstrate versatility. We show the presence of CTCs in blood from more than a hundred cancer patients having pancreatic, hepatic, lung, colorectal, melanoma or prostate cancer. CTCs were even found at very early stage disease. No CTCs were detected in samples from healthy subjects. This method not only enabled the efficient isolation of CTC from non-epithelial cancers, but also showed a dramatic increase in CTC capture compared to EpCAM-based CTC isolation. Furthermore, the method allows for downstream single cell analyses as well as for culturing of the CTCs.

This method could potentially prove beneficial in cancer diagnosis, patient stratification, monitoring response to therapy, and in future molecular characterization of this highly metastatic subpopulation of cancer cells.

#4596

Early noninvasive prediction of response to targeted therapy in non-small cell lung cancer.

Jillian A. Phallen,1 Alessandro Leal,1 Brian D. Woodward,2 Patrick M. Forde,1 Jarushka Naidoo,1 Kristen Marrone,1 Julie Brahmer,1 Jacob Fiksel,1 Doreen N. Palsgrove,1 Stephen Cristiano,1 Daniel Bruhm,1 Elizabeth Weihe,2 Vilmos Adleff,1 Parissa Keshavarzian,2 Valsamo Anagnostou,1 Robert B. Scharpf,1 Victor E. Velculescu,1 Hatim Husain2. 1 _Johns Hopkins Univ. School of Medicine, Baltimore, MD;_ 2 _University of California, San Diego, San Diego, CA_.

There is an unmet need for improved methods of rapidly identifying responses to targeted therapies. Liquid biopsy approaches have potential as early biomarkers of response based on noninvasive, real-time monitoring of disease burden. We have used the ultrasensitive targeted error correction sequencing (TEC-Seq) approach to analyze 58 cancer driver genes in patients with metastatic non-small cell lung cancer undergoing treatment with targeted tyrosine kinase inhibitors. As a proof-of-principle study, liquid biopsies were obtained from sixteen patients immediately prior to treatment, 6-22 days after treatment, and at serial timepoints until disease progression. Tumor derived alterations and copy number changes were directly detected in plasma and tracked over timepoints analyzed. Based on the dynamics of cell-free circulating tumor DNA (ctDNA) mutations identified, we developed a noninvasive measure of cell-free clonal tumor load (cfTL) to evaluate real-time response to treatment. These analyses revealed that patients with a radiographic response to therapy had a significant drop in cfTL from an average mutant allele fraction of 3.59% to 0.13% within 6-22 days (P < 0.05) as well as in the number of detectable mutations and aneuploidy scores, while radiographic non-responders had limited to no changes. Analyses of residual cfTL 6-22 days after treatment stratified patients into ctDNA responders and ctDNA non-responders. ctDNA responders had improved progression-free survival (12.4 vs 1.7 months, P < 0.001), which was detected on average 38 days earlier and was as predictive as CT imaging. These analyses provide an approach for rapid evaluation of response to targeted therapies and have important clinical implications for the management of cancer patients.

#4597

The Blood Profiling Atlas in Cancer: Accelerating progress through collaboration in liquid biopsy.

Jim Godsey. _Thermo Fisher Scientific, South San Francisco, CA_.

Liquid biopsies have the potential to improve early cancer detection, reduce the need for

invasive tumor biopsies, and improve personalized oncology treatment. To help drive

development within this space, the Blood Profiling Atlas in Cancer (BloodPAC) was

created as an initiative born out of the 2016 White House Cancer Moonshot. The

BloodPAC seeks to create an open database commons for liquid biopsies with the goal of

accelerating the development and approval of safe and effective blood profiling

diagnostic technologies for patient benefit.

BloodPAC was established as an independent not-for-profit in February 2017 and has

made exciting progress in advancing the liquid biopsy landscape. Today, BloodPAC is

comprised of over 30 member groups with a diverse range of stakeholders, representing

government agencies, academia, pharmaceutical, and diagnostic companies as well as

disease specific foundations, payers groups and patient advocates. BloodPAC members

actively contribute datasets that include sequencing data from circulating tumor cells,

circulating tumor DNA (ctDNA), and exosome assays, alongside relevant clinical data

into the BloodPAC Data Commons. The BloodPAC Data Commons helps facilitate the

aggregation and harmonization of submitted datasets, allowing researchers to generate

study cohorts size to make statistically significant inference in a scalable, reproducible,

and secure way. Establishing a comprehensive knowledge base through the BloodPAC

Data Commons provides an invaluable reference for anyone entering into the liquid

biopsy field, helps develop the evidence required to support clinical utility, assay

reproducibility, and provides support for FDA approval and payer reimbursement.

This presentation will discuss current progress made within the BloodPAC as an

innovative consortium along with the development of the BloodPAC Data Commons,

lessons learned, and upcoming next steps. The discussion will focus on examining the

BloodPAC consortium's strategy over the past year, the group's work in developing pre-

analytical standards, an overview of the contributed data, as well as the collaborative

process that has been established with the FDA and CAP to maximize the organization's

impact and effectiveness.

#4598

A comprehensive assessment of the impact of preanalytical variables on cell free DNA and circulating tumor cells in blood.

Anita Pottekat,1 Hatim T. Allawi,2 Genna T. Boragine,1 Michael W. Kaiser,2 Tamara Sander,2 Chateen Krueger,2 Janelle J. Bruinsma,2 Amber N. Murray1. 1 _Biomatrica, Inc., San Diego, CA;_ 2 _Exact Sciences Corp., Madison, WI_.

Liquid biopsy continues to gain traction as a minimally invasive method to monitor biomarkers associated with malignancy and metastasis. A simple blood draw can reveal key biomarkers such as circulating tumor cells (CTCs) and cell-free DNA (cfDNA). However, these analytes are present in low quantities and are prone to rapid degradation and contamination, which presents challenges for the detection of rare CTCs and cfDNA targets. Higher molecular weight genomic DNA from poorly stabilized leukocytes can contaminate plasma, compromising liquid biopsy assay results by increasing the total amount of extracted DNA while diluting rare cfDNA targets. Preservation of CTCs and cfDNA over time is thus essential for the accuracy of downstream tests, and preanalytical variables that impact plasma volume, cfDNA yield and quality, and cell recovery in ex vivo blood must be understood. Here, we identified several preanalytical variables, including type of blood collection tube, storage time and conditions post-phlebotomy, and centrifugation conditions for plasma fractionation. We then carried out a series of studies to better understand the interplay between these preanalytical variables and plasma recovery, cell recovery, and cfDNA preservation in healthy donor blood, contrived samples, and patient samples. We found that choice of blood collection tube, in particular, has a significant impact on preservation of plasma volume, CTCs, cfDNA over time, particularly under real-world stress conditions including temperature excursions, sample shipping, and storage time post-draw. The work presented here demonstrates the importance of assessing preanalytical variables for the preservation of cancer biomarkers in whole blood without compromising sample integrity. This will ultimately help researchers make informed decisions on how best to stabilize their analytes of interest without introducing un-intended variables due to blood tube selection, shipping and storage conditions, and plasma isolation conditions.

#4599

Detecting MSI phenotype in circulating blood DNA.

Marc-Henri Stern, Amanda B. Silveira, Ivan Bieche, Samia Melaabi, Luc Cabel, Bruno Buecher, Jean-Yves Pierga, Francois-Clement Bidard, Charlotte Proudhon. _Institut Curie, Paris, France_.

Background: Microsatellite instability (MSI) is a hypermutator phenotype occurring in gastrointestinal, endometrial and colorectal tumors, and more rarely, in urinary tract, ovarian, breast, prostate, lung, head and neck, liver and glioblastoma tumors. The diagnosis of MSI phenotype has recently emerged as the first pan-tumor biomarker likely to predict clinical benefit from immune-checkpoint blockade therapy, making its precise identification primordial for treatment decisions and disease monitoring. Current diagnosis of MSI is performed by multiplex PCR of five microsatellite markers, followed by capillary electrophoresis to detect shifts in allele size. An important limitation of this method is its low sensitivity preventing its use when tumor content is below 10%.

Methods: We designed ddPCR assays examining 3 microsatellites. Analytical performance was assessed in vitro. Clinical performance was evaluated in a series of FFPE and body fluid DNA samples obtained from patients with Stage IV colorectal or endometrial carcinomas, previously classified as MSI-H or MSS using the standard pentaplex PCR method. Mutant allele frequencies (MAF) quantified with the ddPCR MSI assay were also compared with the MAFs reported by ddPCR assays targeting specific BRAF or PIK3CA mutations.

Results: The ddPCR assays for the 3 microsatellites were found to have high specificity and reached a limit of detection of <0.1%. A perfect concordance with the MSI status determined by the pentaplex assay was observed in the 50 tumor samples tested (25 MSI-H and 25 MSS). Importantly, the ddPCR assays diagnosed the MSI phenotype in the plasma of all patients with MSI-H tumors, without false positives in MSS cases. Furthermore, an excellent correlation was observed in the 6 FFPE (R2:0.81) and 8 body fluid (R2:0.99) MSI-H samples were the MAFs measured by our assay could be compared with the ones obtained by ddPCR assays targeting specific mutations in BRAF or PIK3CA genes.

Conclusion: This technique allows a cost-effective, sensitive and large scale screening of the MSI phenotype, as well as the follow-up of MSI patients using liquid biopsies.

#4600

Biomarker analysis in circulating cell-free DNA in patients treated with sorafenib for advanced hepatocellular carcinoma.

Sook Ryun Park,1 Chung Ryul Oh,1 Sun-Young Kong,2 Min Kyeong Kim,3 Kyong-Ah Yoon,4 Eun-Hae Cho,5 Junnam Lee,5 Jihoon Kang,1 Baek-Yeol Ryoo1. 1 _Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea;_ 2 _National Cancer Center, Goyang, Republic of Korea;_ 3 _Graduate School of Cancer Science and Policy, Goyang, Republic of Korea;_ 4 _College of Veterinary Medicine, Konkuk University, Seoul, Republic of Korea;_ 5 _Genome Research Center, Green Cross Genome, Yongin, Republic of Korea_.

Purpose: We evaluated the potential role of Vascular Endothelial Growth Factor-A (VEGFA) amplification and genome-wide copy number variations (CNVs) using circulating cell-free DNA (cfDNA) as predictors of treatment outcome in hepatocellular carcinoma (HCC) patients treated with first-line sorafenib. Methods: Among 184 patients from a prospective biomarker cohort, who had started sorafenib between April 2015 and May 2016, 151 eligible patients were included in the analysis. Plasma cfDNA was extracted from peripheral blood in patients before starting sorafenib or healthy donors. Plasma VEGFA-to-EIF2C1 ratios (the VEGFA ratios) were determined using droplet digital polymerase chain reaction. We applied low depth whole genome sequencing in cfDNA to find CNVs and developed I-score to express genomic instability, which was defined as the sum of absolute Z-scores of sequenced reads on each chromosome. Results: The cfDNA concentrations were significantly higher in HCC patients than in healthy controls (0.71 vs. 0.34 ng/μL; p<0.0001). Patients who had progressive disease with sorafenib as best tumor response had significantly higher cfDNA levels (0.82 vs. 0.63 ng/μL; p=0.006) and I-scores (3,405 vs.1,024; p=0.0017) than those who achieved disease control. The disease control rate was 52.0% for the cfDNA-high group (above the median) vs. 75.0% for the cfDNA-low group (p=0.003), and 49.3% for the I-score-high group (above the median) vs. 77.6% for the I-score-low group (p=0.0003). The cfDNA-high group also had significantly worse TTP (median, 2.2 vs. 4.1 months; HR=1.71; p=0.002) and OS (median, 4.1 vs. 14.8 months; HR=3.50; p<0.0001) than the cfDNA-low group. Similarly, the I-score-high group had poorer TTP (median, 2.2 vs. 4.1 months; HR=2.09; p<0.0001) and OS (median, 4.6 vs. 14.8 months; HR=3.35; p<0.0001). In the multivariate analyses, cfDNA remained an independent prognostic factor for OS (p<0.0001), and I-score for both TTP (p=0.011) and OS (p=0.010). Although the VEGFA ratio was significantly higher in HCC patients than in healthy controls (2.50 vs. 2.17; p<0.0001), it was not significantly associated with treatment outcomes. Conclusions: Pretreatment cfDNA concentration and genome-wide CNVs in cfDNA are potential biomarkers predicting treatment outcomes in advanced HCC patients receiving first-line sorafenib.

#4601

Dissecting the spatial heterogeneity of circulating tumor cells reveals CCL5-Treg-mediated immune evasion in hepatocellular carcinoma.

Liang Wu,1 Miaomiao Jiang,2 Yunfan Sun,3 Xinrong Yang,3 Yong Hou,4 Jia Fan,3 Shiping Liu4. 1 _BGI Education Center, University of Chinese Academy of Sciences (UCAS); BGI-shenzhen, Shenzhen, China;_ 2 _School of Biological Science and Medical Engineering, Southeast University, Nanjing, China;_ 3 _Department of Liver Surgery, Liver Cancer Institute, Zhongshan Hospital, Fudan University; Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai, China;_ 4 _BGI-shenzhen, Shenzhen, China_.

The prognosis of hepatocellular carcinoma (HCC) is closely linked to the occurrence of recurrent, metastatic disease. Metastasis has been proposed to be initiated by circulating tumor cells (CTCs); however, the transcriptomic plasticity and adaptive mechanisms of CTCs in systemic circulation are not well defined. Here, we established single-cell RNA-sequencing profiles of 113 CTCs isolated from four key vascular sites along the dissemination route in ten HCC patients. In our study, we found that single CTCs displayed profound spatial transcriptional heterogeneity within the circulatory system, which is associated with increased transcriptional activity, cell cycle progression, and chemokine-induced immune escape programs. In particular, CTCs dynamically drove chemokine (C-C motif) ligand 5 (CCL5) expression over the course of circulation. We demonstrated that increased CCL5 expression in turn recruited regulatory T cells to facilitate immune evasion and metastatic progression, as rigorously validated by our in vitro and in vivo models. Finally, we identified an inherently dormant CTC subset with an epithelial-mesenchymal transition and immune-evasive phenotype, and demonstrated its potential prognostic implications. Collectively, our results reveal the previously unappreciated spatial heterogeneity of CTCs, and define its role in diverse regulatory programs and cellular states. Our study offers new insight into metastatic mechanisms, highlighting the potential utility of anti-CTC therapeutic strategies. 

### Molecular Classification of Tumors 2: Molecular Predictors of Response, Tumor Staging, and Correlation of Clinical and Molecular Markers

#4602

PTEN and ERG expression in MRI-ultrasound guided fusion biopsy correlated with radical prostatectomy findings in men with prostate cancer.

Andrew Erickson,1 Utku Lokman,2 Kanerva Lahdensuo,3 Sara Tornberg,3 Harri Visapää,3 Robin Bergroth,3 Henrikki Santti,3 Anssi Petas,3 Tuomas Mirtti,4 Antti Rannikko3. 1 _University of Helsinki, Helsinki, Finland;_ 2 _Helsinki University, Helsinki University Hospital, and Ankara Occupational and Environmental Diseases Hospital, Ankara, Turkey;_ 3 _Helsinki University and Helsinki University Hospital, Helsinki, Finland;_ 4 _University of Helsinki and Helsinki University Hospital, Helsinki, Finland_.

Introduction & Objectives

We previously compared ERG and PTEN protein expression in systematic biopsies with radical prostatectomy (RP) findings. MRI-Ultrasound (MRI-US) guided biopsies are expected to more accurately reflect the biological changes in the RP specimens. Here, we studied the correlation of PTEN and ERG status in MRI-US fusion biopsies (FBx) and RP specimens, and assessed the predictive value of FBx on disease stage and grade at RP.

Material & Methods

The study included patients who had undergone RP in Helsinki University Hospital between June 2015 and May 2017, and had preoperative FBx tissue available (n=110). Matched FBx specimens and tissue microarrays constructed from RP were immunostained with ERG and PTEN antibodies, and scored as either negative or positive. The RP TMA contained one to three clinically relevant cancer foci per patient.

Results

A high correlation of both PTEN and ERG status was observed between FBx and RP materials (Table 1). The correlation of PTEN status was particularly better compared with our previous findings in systematic 12-core biopsies (Lokman et al. 2017), where there was no significant concordance. High grade RP (grade group 4&5) correlated with ERG negativity in FBx (p=0.021), whereas PTEN was lost in high stage RP (pT≥3) to a higher extent than in low stage RP (pT2, p= n.s). Higher FBx grade group was associated with stage increase in RP (p=0.014).

Conclusions

FBx is feasible method to assess ERG and PTEN status correctly in RP specimens. More validation studies, accompanied with MRI features, are warranted. | |

|

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

Table 1A. Concordance of ERG IHC status in fusion biopsies (FBx) and radical prostatectomy (RP) specimens.

|

RP ERG Negative | RP ERG Positive | P-value

FBx ERG Negative | 54 (90.0) | 6 (10.0) | <0.001a

FBx ERG Positive | 3 (7.1) | 39 (92.9)

|

|  | |

Table 1B. Concordance of PTEN IHC (Any PTEN Loss) status in FBx and RP specimens.

|

RP Any PTEN Loss | RP PTEN Intact | P-value

FBx Any PTEN Loss | 14 (73.7) | 5 (26.3) | 0.002a

FBx PTEN Intact | 22 (32.4) | 46 (67.6)

|

|  | |

Table 1C. Concordance of PTEN IHC (Complete PTEN Loss) status in FBx and RP specimens.

|

RP Complete PTEN Loss | RP PTEN Intact | P-value

FBx Complete PTEN Loss | 6 (50.0) | 3 (50.0) | <0.001a

FBx PTEN Intact | 3 (4.0) | 72 (96.0)

|

aFisher''s exact test. All p-values two-tailed.

#4603

Clinical application and potential usefulness of targeted next-generation sequencing on resected pancreatic ductal adenocarcinoma.

Francesco Puleo,1 Rémy Nicolle,2 Yuna Blum,2 Jérôme Cros,3 Nabila Elarouci,2 Denis Franchimont,4 Jacques Devière,4 Aurélien de Reyniès,2 Pierre Laurent-Puig,5 Jean-Baptiste Bachet,6 Raphaël Maréchal,4 Jean-Luc Van Laethem4. 1 _Jules Bordet Institute, Bruxelles, Belgium;_ 2 _Ligue National Contre le Cancer, Paris, France;_ 3 _Hôpital Beaujon, Clichy, France;_ 4 _Erasme Hospital, Bruxelles, Belgium;_ 5 _Hôpital Européen Georges Pompidou, Paris, France;_ 6 _Hôpital Pitié-Salpêtrière, Paris, France_.

Introduction: Pancreatic ductal adenocarcinoma (PDA) carries a dismal prognosis. Virtually all PDA are characterized by mutation of four driver genes: KRAS, TP53, CDKN2A and SMAD4 and a long tail of rarely mutated genes. Application of targeted next-generation sequencing (NGS) has entered clinical routine for colon, lung and other cancers. Among patients with resected PDA, usefulness, applicability and prognostic significance of NGS results are still a matter of debate. Aims: To evaluate: 1) the alterations of the 4 main driver genes and patient outcomes after resection 2) the usefulness of targeted NGS in finding targetable alterations. Methods: We analyzed DNA alterations in FFPE tumors among 279 patients with curatively resected PDA who were treated at 4 Academic Hospitals (Franco-Belgian consortium). Sequencing libraries were prepared using a 50 genes panel (Ion AmpliSeqTM Cancer HotSpot Panel v2, Life Technologies). Sequencing was performed on an Ion ProtonTM System using an Ion PITM Sequencing 200 Kit and an Ion PITM Chip Kit v3 (Life Technologies). Associations of driver gene alterations with disease-free survival (DFS) and overall survival (OS) were evaluated using Cox proportional hazards regression with estimation of hazard ratios (HRs) and 95% CIs and adjusted for age, sex, tumor characteristics and institution. Results: Of the 279 patients analyzed, 163 (58.4%) were men and 116 (41.6%) were women, with a median age of 64.59 (36.9-87.5) years. KRAS, TP53, CDKN2A and SMAD 4 were mutated in 246, 193, 45 and 44 patients respectively. Patients with KRAS mutant tumors did not have worse DFS (median [95% CI], 12.2 [11.35-14.6] months) and OS (23.9 [21.1-30.1] months) compared to patients with KRAS wild-type tumors (DFS, 14.3 [9.64-19.4] months; OS, 31.8 [19.4-53.5] months). The mutational status of TP53 or SMAD4 was not associated with DFS or OS as well. CDKN2A mutations were associated with a lower OS (mutation: 20.5 [14.6-33.0] versus wild-type 26.5 [21.9-33.3] months OS, log-rank <1% HR 1.65 [1.124-2.436]) but not to a lower DFS. Patients had slightly worse DFS and OS if they had a greater number of altered driver genes. Compared with patients with 0 to 2 altered genes, those with 3 to 4 altered genes had worse DFS (HR, 1.377 [95% CI, 1-1.89; P=.05]) and OS (HR, 1.476 [95% CI, 1.04-2.09; P= .028]). For some patients (n=27, 9.68%), we found mutations that could be targeted (i.e PTEN, STK11, GNAS, PIK3CA, FLT3, BRAF, IDH1/2, RET, FGFR3, KIT, AKT1). Conclusions: Analysis of the four main driver gene alterations introduces weak prognostic information with little added clinical value. However, application of NGS in resected PDA is quite feasible and is able to find targetable mutations. KRAS and CDKN2A deserve future attention for targeting specific mutations

#4604

Long noncoding RNA Linc00162, as a novel prognostic marker, modulates cancer stem cell properties in triple-negative breast cancer.

Lichao Sun,1 ChangChing Lin,2 Joseph Burnett,2 Hebao Yuan,2 Duxin Sun2. 1 _Chinese Academy of Med. Sci. Cancer Inst., Beijing, China;_ 2 _University of Michigan, Ann Arbor, MI_.

Trastuzumab is an effective treatment for HER2+ breast cancer. However, trastuzumab resistance in HER2+ breast cancer may induce the epithelial-to-mesenchymal transition (EMT), transforming HER2+ to triple-negative breast cancer with cancer stem cell properties in some cancer cell populations. Long noncoding RNAs (lncRNAs) have been reported to be upregulated in many types of cancer tissues, and might play significant regulatory roles in cancer progression. However, the mechanisms of lncRNA in trastuzumab resistance still remain largely unknown. Here, we systematically analyzed the levels of LincRNA between BT474 (HER2+trastuzumab-sensitive) and the PTEN-deficient trastuzumab-resistant derivative (BT474-PTEN-LTT) by using RNA-sequencing, and identified that Linc00162 was significantly overexpressed in BT474 PTEN- LTT cells. In vitro functional assay indicated that downregulation of Linc00162 in BT474 PTEN-LTT cells completely inhibited the cell proliferation. To further evaluate the role of Linc00162 in BCSC self-renewal, we found that inhibition of Linc00162 led to a significant reduction in the number and the size of LTT cells sphere by using the mammosphere formation assay. Next, we examined Linc00162 expression levels in breast cancer tissues, and identified that Linc00162 was markedly increased. Clinicopathologic relevance analysis showed that Linc00162 expression was correlated with advanced clinical stage, lymph node metastasis and the depth of invasion. Furthermore, upregulation of Linc00162 was correlated with TRNB phenotype. In Kaplan-Meier survival analysis, high levels of Linc00162 was significantly associated with unfavorable survival in breast cancer patients. These findings suggest that Linc00162 can be used as a clinically relevant novel biomarker in breast cancer and may serve as a target for future drug development.

#4605

DICER1 **mutations are associated with diverse and frequently histologically mixed-type malignancies in young and adult patients.**

Zoran Gatalica, Michelle Ellis, Jeff Swensen. _Caris Life Sciences, Phoenix, AZ_.

Introduction: DICER1 is a ribonuclease III enzyme that processes pre-microRNAs into mature 20-23 nucleotide microRNAs required for their normal function. Heterozygous germline DICER1 loss-of-function mutations are associated with a cancer predisposition syndrome in which affected individuals present at a young age with rare neoplasms including pleuropulmonary blastoma (PPB), pineoblastoma, embryonal rhabdomyosarcoma (ERMS), Sertoli-Leydig cell tumors (SLCT) and thyroid hyperplasia. These tumors have secondary somatic missense mutations in the remaining DICER1 allele in exons 24 and 25 encoding the RNAse IIIb domain. Contribution of DICER1 somatic mutations to carcinogenesis in the adult population is largely unknown.

Methods: Caris Life Sciences (Phoenix, AZ) database of over 25,000 human cancers containing complete gene sequences of 592 genes (NGS) was searched for mutations in DICER1 gene. Histologic (H&E) slides were reviewed for morphologic characteristics of tumors associated with DICER1 mutations.

Results: 31 patients with biallelic pathogenic DICER1 mutations were identified (approximate frequency of 0.1% in the studied cohort of all cancer types). Female to male ratio was 30:1; age range was from 3 to 79 years of age. The most common primary site was in the endometrium/uterus (15), followed by ovary (8), lung (3), cervix, brain, kidney, retroperitoneum and thyroid (1 each). Majority of endometrial malignancies were observed in adult women (N=15; age 17-79 y. o.), of whom 7 presented with mixed epithelial-mesenchymal type malignancies (carcino- and adeno-sarcomas). Most common (7) ovarian malignancy was SLCT (with and without sarcomatoid elements). One male patient presented with epithelioid glioblastoma (48 y. o.). Three lung cancers seen in adults (39 and two 74 y. o.) were PPB, adenocarcinoma NOS and mucinous adenocarcinoma, respectively. Kidney neproblastoma and cervical ERMS with heterologous elements were observed in 3 and 50 y. o. females, respectively. Overall, DICER1 mutated tumors exhibited low mutational load with the exception of one endometrial adenocarcinoma associated with POLE/MSH6 mutations.

Conclusion: Biallelic somatic DICER1 mutations can be found in tumors of adult patients and are frequently associated with mixed-type malignancies. Sarcomatoid tumor elements were detected not just in endometrial malignancies, but in 2 SLCTs, cervical and thyroid malignancies. These findings are consistent with a recently reported role of DICER1 in epithelial-to-mesenchymal transition (EMT) in experimental cancers, and tumors with such histology should prompt investigations into DICER1 status.

#4606

**Subclassification of patients with lung adenocarcinoma harboring** EGFR **-activating mutations by gene expression profiling.**

Hirotsugu Kenmotsu,1 Masakuni Serizawa,2 Mitsuhiro Isaka,1 Hideaki Kojima,1 Shoji Takahashi,1 Akira Ono,1 Tateaki Naito,1 Haruyasu Murakami,1 Takeshi Nagashima,3 Shumpei Ohnami,2 Keiichi Ohshima,2 Kenichi Urakami,2 Masatoshi Kusuhara,2 Ken Yamaguchi,1 Takashi Sugino,1 Yasuhisa Ohde,1 Toshiaki Takahashi1. 1 _Shizuoka Cancer Center, Shizuoka, Japan;_ 2 _Shizuoka Cancer Center Research Institute, Shizuoka, Japan;_ 3 _SRL Inc., Japan_.

Background: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been effective in patients with lung adenocarcinoma (LUAD) harboring EGFR-activating mutations. However, the response to EGFR-TKIs in these patients is not uniform, indicating that patients with EGFR-activating mutations may be further stratified into some undefined molecular subgroups. Therefore, we aimed to identify these molecular subgroups in patients with LUAD harboring EGFR-activating mutations, by gene expression profiling (GEP) data.

Methods: Between January 2014 and April 2017, 294 patients with LUAD who underwent surgery at the Shizuoka Cancer Center were enrolled with informed consent. Whole-exome sequencing (WES) and GEP were conducted with an Ion Proton system and an Agilent SurePrint G3 Human Gene Expression 8×60K v2 Microarray, respectively. Corresponding peripheral blood samples were used to identify tumor-specific genetic alterations. Tumor-specific gene expression was identified by comparing expression profiles between tumor and adjacent normal tissues. Samples with estimated tumor purity <20% were excluded from the following analysis to avoid false negatives; the remaining 251 patients (85%) were included in the analysis. EGFR-activating mutations were defined based on OncoKB database. Statistical evaluation of GEP data was conducted using principal component analysis (PCA) and orthogonal projection to latent structures discriminant analysis (OPLS-DA).

Results: EGFR-activating mutations were identified in 98 patients (39%); the most frequently identified alteration was the deletion in exon 19 (47%, 46), followed by the L858R mutation (45%, 44). No significant differences in the expression profile were noted between patients with both major EGFR-activating mutations. Patients with EGFR-activating mutations were divided into two clusters (C1 and C2) by the evaluation of those expression profiles with PCA, and 10 and 88 patients were included in each cluster, respectively. As a result of OPLS-DA, the genes relevant to cell cycle progression, DNA repair and tumor immunity were selected, and were considered to have strongly contributed to the differences between two clusters. Associations between each cluster and clinicopathologic characteristics were evaluated by logistic regression analysis. Patients classified into C1 showed the significant association with poor differentiation, relapse and smoking history.

Conclusions: These results demonstrate that patients with EGFR-activating mutations can be further stratified into two additional molecular subgroups by assessing the expression level of genes above mentioned. To establish this expression profiling-based stratification as predictive marker of EGFR-TKIs, further clinical study to evaluate the association with the therapeutic response to EGFR-TKIs is needed.

#4607

G1/S transition-related gene mutations associated with survival of hepatocellular carcinoma patients.

Bingyuan Zhang,1 Tianqiang Song,2 Pingzhou Yang,3 Songzhu Yang,4 Chao Guo,3 Lei Li,5 Shuang Ren,6 Maolin Yan,7 Weiyu Hu,8 Honglin Guo,9 Yongjian Zhang,10 Yanlin Li,9 Weifeng Wang,9 Kai Wang,11 Ming Yao9. 1 _The Affiliated Hospital of Qingdao University, Shanghai, China;_ 2 _Tianjin Medical University Cancer Institute and Hospital, China;_ 3 _People's Hospital of Hunan Province, China;_ 4 _Yantai Mountain hospital, China;_ 5 _Shandong Cancer Hospital Affiliated to Shandong University, Shandong Academy of Medical Sciences, China;_ 6 _Affiliated Tumor Hospital of Guangxi Medical University. Nanning, China, China;_ 7 _Fujian provical hospital, China;_ 8 _The Affiliated Hospital of Qingdao University, China;_ 9 _OrigiMed Inc., Shanghai, China;_ 10 _Harbin Medical University Cancer Hospital, China;_ 11 _OrigiMed Inc.;Zhejiang University International Hospital, China_.

Background: Hepatocellular carcinoma (HCC) is one of the most common malignancies in China and has limited therapeutic options. Genomic alterations in HCC had been widely explored and some prognostic genomic alterations were identified. Different etiologic characteristics were discovered in Chinese HCC patients compared with Western population; however, the genomic profiles of Chinese patients had not been thoroughly elucidated.

Methods: FFPE tumor samples of 94 Chinese HCC patients, including 79 males and 15 females with a median age of 56, were collected for next-generation sequencing (NGS)-based 450 genes panel assay. Genomic alterations including single base substitution, short and long insertions/deletions, copy number variations, and gene rearrangement and fusions were assessed. Gene Ontology (GO) analysis was conducted with the genes whose variation rates were higher than 5% (N = 22). The TCGA HCC dataset with 198 patients was used to assess the prognostic value of the significantly enriched GO terms.

Results: The top 10 highly mutant genes in the 94 Chinese HCC cohort were TP53 (64.9%), TERT (31.9%), CTNNB1 (23.4%), AXIN1 (16.0%), STK24 (12.8%), RB1 (11.7%), CCND1 (9.6%), ARID1A (9.6%), LRPIB (8.5%) and ARID2 (8.5%). The Gene Ontology analysis revealed these gene alterations were significantly associated with pivotal signal pathways and cell proliferation including phosphatidylinositol phosphorylation, protein kinase B signaling, cell cycle arrest, and G1/S transition of mitotic cell cycle. The G1/S transition-related genes included TP53, TERT, RB1, CCND1 and CDKN2A. Moreover, the TCGA HCC patients with mutations of G1/S phase transition and mitotic cell cycle-related genes had a poor survival (p = 0.029).

Conclusions: Our study showed that Chinese HCC patients had different genomic alteration profile compared with Western population. The most frequent mutant gene in Chinese HCC was TP53 in 65% of the HCC patients, compared to 31% in the TCGA dataset (PMID:28622513). Genomic alterations in G1/S transition of mitotic cell cycle-related genes in Chinese HCC patients might be a negative prognostic factor. Our preliminary result suggested that functional analysis of HCC genomic alteration profile could have prognostic utility.

#4608

Immunologic and clinical implications of harboring mutations in mismatch repair (MMR) genes in non-small cell lung cancer (NSCLC).

Young Kwang Chae, Kyunghoon Rhee, Lee Chun Park, Anderson Cho, Taeyeong Ko, Sangmin Chang, Andrew Davis, Manali Bhave, Marcello Cruz, Wadw Iams. _Northwestern University Feinberg School of Medicine, Chicago, IL_.

MMR system is one of many cellular DNA repair mechanisms, verifying and maintaining repeat count of microsatellites during cell division. Mutations in MMR genes lead to increased genomic instability and tumor mutational burden (TMB). MMR gene mutation is known to occur in many solid tumors, including colorectal and endometrial tumors. Pembrolizumab, a PD-1 inhibitor, has recently been approved for MMR-deficient tumors regardless of their histologic origin. A minor portion of NSCLC has been reported to have traits of MMR deficiency. However, little is known regarding its biologic and clinical implications in NSCLC. Here we examined the association between the mutational status in MMR genes and change in TMB, immune cell landscape, and clinical outcome in NSCLC. We analyzed genomic, transcriptomic, and clinical data from The Cancer Genome Atlas (TCGA) database using cBioPortal of patients with lung adenocarcinoma (ADC, 522 samples) and squamous cell carcinoma (SQCC, 504 samples). We compared the group with at least one mutation (M) in 17 MMR genes (MLH1, MLH3, MSH2, MSH3, MSH4, MSH5, MSH6, PMS1, PMS2, PCNA, EXO1, POLD1, RFC1, RFC2, RFC3, RFC4, RFC5) with the group without mutation (N). The tumor immune landscape was analyzed from RNA-seq z-scores of 812 "immune metagene" signatures (Angelova M et al., 2015) which were used to predict immune infiltration of 31 distinct immune cells for each tumor sample. Among 522 samples of ADC, 9.2% (48 samples) of MMR gene mutations were found while among 504 samples of SQCC, 9.1% (46 samples) were detected. In ADC, M group did not have significant difference in TMB compared to N group (median mutation count M:206.5 N:150.5, t-test p=0.14). There was no significant difference in activated CD4 T-cell (M:31% N:30%, chi square test p=0.81), CD8 T-cell infiltration (M:35% N:26%, chi square test p=0.16) and overall survival (median M:33 N:50 months, log-rank test p=0.27). In SQCC, M group demonstrated significantly higher TMB (median mutation count M:268 N:206.5, t-test p=0.0001) compared to N group. However, activated CD4 T-cell (M:22% N:37%, chi square test p=0.04) infiltration was significantly low. CD8 T-cell infiltration (M:26% N:28%, chi square test p=0.79) did not show any significant difference. In terms of survival, although not statistically significant, there was a trend towards worse survival in M group compared to N group (median M:35 N:65 months, log-rank test p=0.07). This is the first comprehensive report describing the immunologic and clinical implication of MMR gene mutation in NSCLC. Interestingly, similar to colorectal cancer, in SQCC there was a positive correlation between MMR gene mutation and higher TMB. However, there was no clear association with activated CD8 T-cell infiltration nor any association with favorable prognosis.

#4609

High-grade TP53-mutated endometrial carcinomas have decreased NRF2 antioxidant activity.

Guillaume Beinse,1 Pierre-Alexandre Just,2 Bastien Rance,3 Brigitte Izac,4 Franck Letourneur,4 Nathaniel Edward Saidu,1 Sandrine Chouzenoux,1 François Goldwasser,1 Eric Pasmant,2 Frederic Batteux,1 Bruno Borghese,2 Karen Leroy,1 Jérome Alexandre1. 1 _Paris Descartes University, Sorbonne Paris Cité, INSERM U1016, Cochin Institute, CARPEM, PARIS, France;_ 2 _Cochin Hospital, PARIS, France;_ 3 _Georges Pompidou European Hospital, PARIS, France;_ 4 _INSERM U1016, Cochin Institute, GENOMIC plateform, PARIS, France_.

Background

NRF2 is a major transcription factor regulating the expression of antioxidative/detoxifying enzymes. Its activation, due to mutations or activation of signaling pathways, has been associated with chemoresistance and poor prognosis in several tumors. NRF2 status remains unclear in endometrial carcinoma (EC). We aimed to identify molecular alterations associated with NRF2 activation in the four molecular subgroups of EC described by the Cancer Genome Atlas (TCGA), and explored its impact on EC prognosis.

Methods

Ninety patients treated in Cochin Hospital (2012 to 2017) for EC were included. Formalin-fixed paraffin-embedded tissue samples were processed for histopathological analysis and DNA and RNA extractions. Next generation sequencing (NGS) of NFE2L2 (encoding NRF2), KEAP1 and CUL3 genes and a panel of 15 genes significantly mutated in EC was performed using AmpliseqTM panels on Ion TorrentTM PGM (Thermo Fisher). The nuclear expression of mismatch repair (MMR) and NRF2 proteins were analyzed by immunochemistry (IHC). NRF2 activity was assessed by NQO1, GCLC, and AKR1C3 mRNA expressions, normalized to MRPL19 and TBP housekeeping genes, using TaqManTM assays and quantitative RT-PCR. Clinical event of interest was event-free survival (EFS) (progression, relapse, or death).

Results

Tumors were classified according to NGS and IHC data as POLE exonuclease domain mutated (N=3, 3%), MMR-deficient (dMMR) (N=28, 31%), TP53 mutated (N=23, 25%), and MMR-proficient tumors (N=32, 36%). NRF2 nuclear immunostaining was not correlated to NRF2 activity. The 3 tumors with highest NRF2 target genes expression harbored known NRF2 pathway activating mutations (NFE2L2 p.W24G, KEAP1 p.R336*, KEAP1 p.D422N). In addition, 2 dMMR tumors showed intermediate/high (1st quartile) NRF2 target genes expression and low allele ratio NFE2L2 mutations, suggesting NRF2 subclonal activation. No correlation was observed between NRF2 activity and PI3K or KRAS pathways mutations. The TP53 subgroup showed a strikingly lower NQO1 expression compared to dMMR or pMMR tumors (ANOVA p<.05). This observation was confirmed by in silico analyses of publicly available TCGA data. NQO1 low expression was significantly associated with poor EFS, independently of tumor stage (Cox p<.01).

Conclusion

In contrast with previous reports based on IHC, NRF2 activation is a rare event in EC, associated with NFE2L2 or KEAP1 mutations. NQO1 downregulation in the TP53-mutated subgroup might be explained by the interaction of NRF2 with TP53 missense mutants, which has been shown to impair its anti-oxidant transcriptional activity while enhancing proteasome expression (Walerych et al. Nat Cell Biol 2016). Very low levels of NQO1 expression identifies a subset of EC with a poor prognosis, which might be sensitive to specific combination therapies (Liu et al. Nat Commun. 2017).

#4610

Inflammation-associated microsatellite alterations in colorectal cancers (CRCs): Definition, assay, prognostic value, and pathologic significance.

Minoru Koi,1 Yoshiki Okita,1 Stephanie Tseng-Rogenski,1 Koji Munakata,1 Chan Choi,2 Hyeong-Rok Kim,2 Erika Koeppe,1 Elena Stoffel,1 Joseph A. Galanko,3 Nikki McCoy,3 Temitope Keku,3 Takahito Kitajima,1 Takeshi Nagasaka,4 Yuji Toiyama,5 John M. Carethers1. 1 _Univ. of Michigan, Ann Arbor, MI;_ 2 _Chonnam University, Chonnam, Republic of Korea;_ 3 _Univ. of North Carolina, Chapel Hill, NC;_ 4 _Kawasaki Medical School, Kawasaki, Japan;_ 5 _Mie University, Mie, Japan_.

Background & Aims. Deficiency in the DNA mismatch repair (MMR) gene product MSH3 results in low levels of microsatellite instability (MSI-L) at dinucleotide loci and elevated microsatellite instability at selected tetranucleotide repeats (EMAST), but not at mononucleotide loci in CRC. We previously demonstrated that the pro-inflammatory cytokine interleukin 6 (IL6) and/or hydrogen peroxide triggers translocation of MSH3 from the nucleus to cytoplasm, resulting in MSI-L/EMAST (L/E). This is consistent with observed correlation between intraepithelial and nests of inflammatory cells within malignant epithelium that are the likely origin of IL6 and oxidative stress with the occurrence of L/E in CRCs, informing that L/E is a consequence of exposure to inflammation. Microbes such as Fusobacterium nucleatum may play a role in CRC initiation and progression, and may trigger inflammation. Clinically, L/E is associated with poor patient prognosis and CRC recurrence and/or metastasis. Threshold values for determining L/E range in the literature from 1 to 3 polymorphic markers frameshifted when compared to normal tissue. Our aim was to assess an optimal threshold value for determining L/E in CRCs.

Materials & Methods. For MSI-H, MSI-L and EMAST status, we developed a multiplex PCR-based assay consisting of 2 mono-, 5 di-, and 7 tetra-nucleotide microsatellite markers. MSI-H was defined when >2/7 mono- and/or di-nucleotide repeats exhibited frameshift mutations (FSM), and MSI-L when <2/7 mono- and/or di-nucleotide markers exhibited FSM. EMAST was defined when 1/7 (relaxed criteria) or >2/7 (stringent criteria) tetranucleotide markers showed FSM. Stage II/III CRCs (132 cases) from Korea were analyzed for the association between L/E and recurrence-free survival (RFS) using relaxed and stringent criteria for EMAST. A second CRC cohort (308 cases) was analyzed for MSI/EMAST and F. nucleatum infection.

Results. When EMAST was defined by relaxed criteria, P value for the association between L/E and RFS of 132 CRC patients was 0.415 while P value was 0.0038 when EMAST defined by stringent criteria was used. Similarly, F. nucleatum infection was associated with L/E by univariate analysis (P=0.027) but not by multivariate analysis (P=0.08) when EMAST was defined by relaxed criteria for 308 CRC cases. In contrast, this became significant in both uni- (P=0.014) and multi-variate analysis (P=0.024) when EMAST was defined by stringent criteria.

Conclusions. A clear distinction for pathological significance is detected between non-MSI-H, L/E CRCs (associated with inflammation) and non-L/E CRCs when EMAST is defined by <2/7 EMAST markers. This threshold may better define EMAST for these observed inflammation-associated microsatellite alterations.

#4611

Uterine leiomyoma driver events in uterine adenomyomas.

Tuomas A. Heikkinen,1 Anna Äyräväinen,2 Janne Hänninen,1 Terhi Ahvenainen,1 Ralf Bützow,2 Annukka Pasanen,2 Pia Vahteristo1. 1 _University of Helsinki, Helsinki, Finland;_ 2 _University of Helsinki and Helsinki University Hospital, Helsinki, Finland_.

Uterine adenomyosis is a condition in which ectopic endometrial glands are present in myometrial stroma surrounded by smooth muscle cell hyperplasia. Foci of adenomyosis growing as a tumor like mass are called adenomyomas. Uterine adenomyomas are common tumors and they share symptoms, including pelvic pain and abnormal bleeding, with uterine leiomyomas. The two tumor types are challenging to distinguish from one another and the diagnosis is usually confirmed only after surgery by pathological evaluation. The molecular background of uterine adenomyomas is not currently well known. In uterine leiomyomas, somatic mediator complex subunit 12 (MED12) mutations, high mobility group AT-hook (HMGA2) protein overexpression, and fumarate hydratase (FH) inactivation are well established as major mutually exclusive driver events covering 80-90% of the tumors. Here, we have analyzed the presence of these changes in a set of 21 uterine adenomyomas. Representative areas of formalin-fixed paraffin embedded archival uterine adenomyoma tissue samples were used to construct a tissue microarray. The HMGA2 overexpression and the FH inactivation were assessed using immunohistochemistry with anti HMGA and 2SC antibodies, respectively. DNA was extracted from the tumor samples to determine the MED12 mutation status by direct sequencing of exons 1 and 2 of the gene. MED12 c.131GA, p.G44D mutation was found in two adenomyoma samples out of 21 (9.5%). Strong positive staining of 2SC indicating FH inactivation was present in one sample which also showed reduced FH protein expression when validated with an independent method using anti-FH immunostaining. Sequencing revealed a frameshift mutation c.911delC, p.P304fs in exon 7 leading to a premature stop codon 25 codons later. The mutation was also found in a separate uterine leiomyoma of the same patient and both tumor samples mostly presented the mutant allele indicating loss of heterozygosity of the wild type allele. This, together with the patient's medical history of previous uterine leiomyomas, indicates the germline origin of the mutation and thus a hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome. No changes in HMGA2 expression were detected with all samples presenting normal expression levels. In conclusion, MED12 mutations are present in a subset of uterine adenomyomas. The mutation frequency of 9.5% that was observed here in our adenomyoma sample series is considerably lower than that of 70% in uterine leiomyomas. Our results also suggest that adenomyomas may be linked to HLRCC in which they have not been previously reported. The driver events behind uterine adenomyomas remain mostly unknown and further large-scale studies are warranted to clarify the spectrum of underlying mutations and molecular background of these common tumors.

#4612

SET overexpression is associated with recurrence-free survival in patients with primary breast cancer receiving adjuvant tamoxifen treatment.

Yu-Hsiang Huang,1 Pei-Yi Chu,2 Ji-Lin Chen,3 Chun-Teng Huang,4 Chia-Han Lee,3 Ka-Yi Lau,3 Wan-Lun Wang,3 Yu-Ling Wang,3 Pei-Ju Lien,3 Ling-Ming Tseng,3 Chun-Yu Liu3. 1 _National Yang-Ming University, Taipei, Taiwan;_ 2 _Show Chwan Memorial Hospital, Taipei, Taiwan;_ 3 _Taipei Veterans General Hospital, Taipei, Taiwan;_ 4 _Yang-Ming Branch of Taipei City Hospital, Taipei, Taiwan_.

Background:

Estrogen receptor (ER) positive breast cancer accounts for 70% of breast cancer. Tamoxifen, a selective ER modulator, remains an important hormone therapeutic agent for patients with ER positive breast cancer. A number of patients receiving adjuvant tamoxifen still experience recurrence in the long term. In current study we explored the clinical significance of four biomarkers including SET, CIP2A, PP2A and Akt in ER positive breast cancer patients receiving adjuvant tamoxifen.

Methods:

Specimens were from ER positive breast cancer patients treated with adjuvant tamoxifen for a median of duration of 54.8 months with documented outcomes. The median follow-up was 106 months. Immunohistochemical staining for SET, CIP2A, p-PP2A (Tyr 307), p-Akt were performed and a H-score was assigned to quantify protein expression. In silico analysis of gene expression was evaluated from the public database KM plotter (available at: http://kmplot.com/analysis/). Human ER positive breast cancer cell line MCF7 cells were used for in vitro studies. MTT assay, flow cytometry and Western blot were used to assess the cells properties. Estrogen response element (ERE)-dependent luciferase activity was assessed by co-transfection of SET-expressing or control plasmids and 3ERE bearing reporter plasmids into MCF7 cells and stimulated with estrogen.

Results:

In 218 primary ER positive breast cancer patients treated with adjuvant tamoxifen, 17 (7.8%) suffered from recurrence or metastasis. Higher expressions of SET and CIP2A by IHC analysis were associated with poor recurrence-free survival (RFS). Multivariate analysis revealed SET was independently correlated with worse RFS (Hazard ratio=3.72, 95% confidence interval 1.26-10.94, p=0.017). In silico, KM-plotter analysis revealed higher gene (mRNA) expressions of SET, PPP2CA and

Akt1 significantly correlated with worse RFS in breast cancer patients receiving adjuvant tamoxifen therapy. Because SET appeared to be the most prognostic for RFS among the four markers, we next explored the biological role in vitro. Tamoxifen exerted anti-proliferation and apoptotic effects in a dose-dependent manner of MCF7 cells. SET overexpression reduced tamoxifen-induced anti-proliferation in MCF-7 cells, in association with upregulated p-ER, suggesting that SET may affect ER pathway via the serine/threonine kinase PP2A. SET drove luciferase activity in an ERE-dependent manner, and also enhanced estrogen-promoted luciferase activity.

Conclusions:

Protein SET is a prognostic biomarker in ER positive breast cancer patients treated with tamoxifen and may contribute to tamoxifen resistance by modulating ER signaling pathway.

#4613

The difference of molecular signatures in left- and right-sided colorectal cancer Chinese patients.

Sen Zhang,1 Yun Guo,1 Weibin Shu,2 Hui Li,1 Yu Wang,2 Wei Zhao,3 Zusen Wang,3 Xueqing Yao,4 Tao Xiang,5 Yong Li,6 Ting Deng,7 Ruobing Ma,8 Qiang Cui,8 Lei Mei,8 Yang Lou,8 Shuo Mu,8 Zoltan Szallasi,9 Yongchang Zhang,10 Ming Yao,8 Kai Wang11. 1 _First Affiliated Hospital of Guangxi Medical University, China;_ 2 _Shandong Provincial Hospital Affiliated to Shandong University, China;_ 3 _The Affiliated Hospital of Qingdao University, China;_ 4 _Guangdong General Hospital, China;_ 5 _The First Affiliated Hospital, School of Medicine, Zhejiang University, China;_ 6 _Guangdong provincial hospital, China;_ 7 _Tianjin Medical University Cancer Institute and Hospital, China;_ 8 _OrigiMed Inc., Shanghai, China;_ 9 _9Boston Children's Hospital, MA;_ 10 _Hunan Cancer Hospital , China;_ 11 _OrigiMed Inc.;Zhejiang University International Hospital, China_.

Background: Recent reports have demonstrated inferior outcomes for patients with right-sided colorectal cancer (RCC) compared to left-sided (LCC) as well as differences in treatment response based on disease sidedness. Molecular analyses provide a partly biologic explanation for the association with anatomic location, but molecular variations between RCC and LCC remain undefined in Chinese patients.

Methods: Tumors with origins clearly defined as cecum to transverse colon for RCC, and descending colon to rectum for LCC. The FFPE tumor and matched blood samples of 61 Chinese colorectal cancer patients including 39 males (64%) and 22 females (36%) with a mean age of 56 years old (yrs) were subjected to comprehensive genomic profiling (CGP) assay consisting of 450 gene full exons and selected introns in 39 genes. We measured the genomic alterations and calculated tumor mutational burden (TMB) of total somatic substitutions and insertions/deletions per megabase after filtering known driver mutations. Chi-square tests were used for comparative analyses.

Results: Among 61 primary samples, 44 were LCC with a median age of 55 yrs and 17 were RCC with a median age of 58 yrs. Genomic alterations of BRAF, ARID1A, TERT, high TMB values and MSI-High were significantly more prevalent in RCC. BRAF was mutated in 25% of RCC compared to 2.3% in LCC (p=0.006). ARID1A and TERT were mutated in 37.5% and 25% of RCC respectively but none in LCC (p<0.05). The median TMB values were 18 vs. 7.2 muts/Mb for RCC vs. LCC (p<0.01). No significant difference was found in PIK3CA (37.5.4% vs. 18.6%), ERBB2 (6.2% vs. 9.3%) and SMAD4 (25% vs. 16.3%). MSI-high tumors were more likely to occur in RCC rather than LCC (30.8% vs. 7.3%; p = 0.07). Moreover, there were more DNA repair-related genes and MMR genes mutations in RCC, including BRCA2 (25% vs. 4.7%), FANCL (12.5% vs. 0%), PTEN (37% vs. 4.7%), MSH6 (25% vs. 2.3%), and PALB2 (12.5% vs. 2.3%). For LCC, there were more mutations of KRAS (53.5% vs. 43.7%), APC (67.4% vs. 56.2%) and TP53 (74.4% vs. 62.5%), though these differences did not rise to the level of statistical significance.

Conclusions: The molecular difference between RCC and LCC is probably related to the finding that the mutations of DNA repair and MMR genes were more commonly detected in the right-sided colorectal cancer. Higher TMB values and MSI-high status could predict a higher response rate to immune checkpoint inhibitors to RCC patients instead of LCC. A better understanding of anatomic location and disease biology may help to identify therapeutic targets and precision medicine.

#4614

Molecular subtypes in muscle invasive bladder cancer: Evaluation of clinical significance.

Daley S. Morera, Daniel Belew, Andre R. Jordan, Vinata B. Lokeshwar. _Medical College of Georgia at Augusta University, Augusta, GA_.

Introduction: Using RNA-seq (HiSeq) transcriptome data in The Cancer Genome Atlas (TCGA) bladder cancer (BCa) dataset two molecular subtypes, basal and luminal, were identified in muscle invasive BCa (MIBCa). Recurring reported markers of the basal subtype are KRT5, KRT6A, KRT14 high and KRT20, GATA3, UPK3A, FOXA1 low. The luminal subtype is the opposite pattern of expression. Double-Negative (DN) subtype was defined as no expression of the 7 markers. Luminal, basal and DN subtypes were reported as predictors of better, poor and worst prognosis, respectively, based on overall survival (OS). EGFR is defined as a squamous differentiation marker associated with poor prognosis. The objective was to assess the clinical significance of these subtypes in TCGA data with validation in datasets from Oncomine.

Methods: TCGA dataset containing 407 MIBCa patients was accessed through Xena Browser. The dataset included patient demographics, clinical parameters, OS, recurrence-free survival (RFS) and the transcript levels (log2(nor._count+1) of basal and luminal markers. All available follow-up data was included (27.9±28.49; max: 166 months). 25 BCa datasets (n=360 MIBCa patients) containing these same variables were accessed through Oncomine. High and low levels were stratified by median. Association of individual basal and luminal markers and the subtypes with clinical and outcome variables was analyzed by univariate and multivariate analyses. Kaplan-Meier analysis was performed to stratify patients into risk groups for OS and RFS.

Results: In TCGA data, neither basal nor luminal markers levels significantly correlated with metastasis or lymphovascular invasion (LVI); P = 0.2 to 0.9. Only KRT5 significantly but inversely correlated with lymph node (LN) positivity; P=0.011. EGFR levels did not correlate with metastasis, LVI, or LN positivity; P > 0.05. In TCGA dataset 77 (18.9%), 50 (12.3%) and 14 (3.4%) expressed basal, luminal and DN subtypes, respectively; 266 (66.4%) patients did not conform to any group. In univariate or multivariate analyses, the subtypes also did not correlate with metastasis, LVI or LN status. Only luminal subtype associated with better OS; P=0.003. However no subtype significantly correlated with RFS. In KM analysis no subtype stratified patients regarding RFS (P>0.2). Oncomine BCa datasets validated these results.

Conclusion: TCGA and Oncomine datasets show that the majority of MIBCa tissues express a mixed pattern of basal and luminal markers. Furthermore, basal, luminal or DN subtypes do not associate with clinical parameters or prognosis of MIBCa patients.

#4615

Phenotypic subtypes successfully stratify prognosis of patient with colorectal cancer: A step towards precision medicine.

Antonia Kathryn Roseweir,1 James H. Park,1 Sanne ten Hoorn,2 Arfon G. Powell,3 Campbell S. Roxburgh,1 Donald C. McMillan,1 Paul G. Horgan,1 Louis Vermeulen,2 Joanne Edwards1. 1 _University of Glasgow, Glasgow, United Kingdom;_ 2 _Academic Medical Center, Amsterdam, Netherlands;_ 3 _Cardiff University, Cardiff, United Kingdom_.

Background: There has been enormous effort to develop a prognostic genomic classification of colorectal cancer (CRC). As a result, mismatch repair (MMR) status was established as a prognostic marker in addition to TNM-staging. Phenotypic subtypes were recently proposed that stratified patient survival based on assessing the immune infiltrate (KM grade), tumour growth (Ki67) and stromal infiltrate (TSP). However their clinical utility had not been compared to other proposed classification systems including consensus molecular subtypes (CMS) or current prognostic markers including MMR status.

Methods: Three patient cohorts, a pilot cohort of 237 stage I-III CRC patients, a validation cohort of 879 stage I-III CRC patients, and the AMC-AJCCII-90 cohort with 81 stage II colon cancer patients were utilised to investigate associations between phenotypic subtypes, MMR status, CMS and patient survival.

Results: In the pilot cohort, phenotypic subtype stratified cancer-specific survival (P<0.001). In the validation cohort, phenotypic subtype stratified overall (p=0.003) and cancer-specific survival (CSS, p<0.001). This stratification of CSS was irrespective of adjuvant chemotherapy treatment (no chemotherapy - p<0.001 and chemotherapy received - p=0.006) and tumor location (right colon -p<0.001, left colon - p=0.007 and rectal p<0.001). However, MMR status only stratified CSS in the right colon (p=0.023) and was significantly associated with right-sided colon cancer (p<0.001) upon chi-squared analysis. Therefore further analysis was restricted to this subset of patient (n=380). The immune subtype had the highest MMR deficiency and the stromal subtype was mainly MMR competent (p=0.001). Furthermore, phenotypic subtype and not MMR status was independently prognostic in the full cohort (p<0.001) and right-sided colon cancer (p<0.001). In the AMC-AJCCII-90 cohort phenotypic subtypes aligned with CMS (p<0.001) and stratified overall survival better than CMS (p=0.125 v p=0.487 respectively). However, discordance was seen between CMS1 and the immune subtype, potentially due to differences in the site of inflammation assessed. To address this the KM grade was replaced with the immunoscore within the phenotypic subtypes and the pilot cohort assessed for survival. This method of subtyping stratified CSS (p<0.001) in a similar manner to the original (p<0.001), suggesting that the method of assessing the immune infiltrate is not influencial; therefore as KM grade is more clinically translatable this was chosen as the best method.

Conclusions: Phenotypic subtype is a more effective prognostic classification than CMS and MMR status. Phenotypic subtype is clinically relevant to all patients irrespective of site and adjuvant treatment. Phenotypic subtypes should be incorporated alongside MMR status as a clinical aid for the prognosis of patients with CRC.

#4616

Cancer-associated MUC1 epitope-recognizing antibody as a novel immunohistochemical marker for breast carcinoma.

Ai Shimizu,1 Kanako Hatanaka,1 Yutaka Hatanaka,1 Kentaro Naruchi,2 Masaharu Sato,2 Hiroshi Kase,2 Tomoko Mitsuhashi,1 Hiroko Yamashita,1 Yoshihiro Matsuno1. 1 _Hokkaido University Hospital, Sapporo, Japan;_ 2 _Medicinal Chemistry Pharmaceutical Company, Ltd, Sapporo, Japan_.

Background: MUC1 has been used in clinical practice as a serum tumor marker (CA15-3) for monitoring recurrence and response to the treatment of breast cancers. However, it is not so practical to use the conventional anti-MUC1 antibody (C-Ab) for the differential diagnoses among malignant and benign tumors, because of its limited specificity to cancer-associated sugar chain structures. Recently, a novel epitope-defined (ED) antibody, which specifically recognizes MUC1 with cancer-associated carbohydrate antigens including Tn and sialyl-T antigens, was developed by our collaborators. In the present work, we focused on this Tn antigen-recognizing ED antibody (Tn-ED Ab) for MUC1, and examined the potential utility of this novel antibody as an immunohistochemical diagnostic marker of the breast.Design: Tissue microarray (TMA) consisting of invasive lesions and their non-neoplastic counterparts were constructed from 169 lesions of 167 individuals with invasive breast carcinomas (including 2 bilateral cases) resected and diagnosed at Hokkaido University Hospital from 2000 to 2003. We compared MUC1 immunoreactivity of Tn-ED Ab (clone SN-102) to commercially available C-Ab (clone Ma552) using TMA to evaluate its diagnostic performance and to analyze the association with clinicopathologic factors. Results: In invasive cancer parts, the immunoreactive positivity of Tn-ED Ab was observed predominantly in the cytoplasm (146/169) rather than in the cell membrane (5/169). The positive reaction of C-Ab was found in the cytoplasm and the membrane as well (156/169 and 93/169). Tn-ED Ab showed a lower expression rate in non-neoplastic parts and a high specificity in invasive cancer parts. Sensitivity/specificity in invasive parts was 86%/92% for Tn-ED Ab and 92%/47% for C-Ab. Moreover, the invasive cancer group with Tn-ED-positivity was significantly correlated with their molecular subtypes, low Ki-67 indices, and high ages, compared to those with Tn-ED-negativity (p=0.008, p=0.010 and p=0.027, respectively). Conclusion: Compared to C-Ab, the newly-developed Tn-ED Ab for MUC 1 showed a higher specificity for invasive breast cancer with a low reactivity to non-neoplastic components. Future study plans are ongoing to show that this cancer-specific Tn-ED MUC1 Ab could be useful for differentiating malignant from benign intraductal breast lesions as well.

#4617

High expression of snail is associated with EZH2 expression and lymph node metastasis in large neuroendocrine lung cancer.

Yueh-Fu Fang,1 Min-Chia Lee2. 1 _Chang Gung Memorial Hospital, Taipei, Taiwan;_ 2 _National Central University, Taipei, Taiwan_.

Background Large neuroendocrine lung cancer was less than adenocarcinoma or squamous cell carcinoma of lung. EZH2 expression was high in small cell lung cancer with component of neuroendocrine. There was no study of EZH2 in large neuroendocrine lung cancer. We planned to identify the expression of EZH2 and EMT markers and the clinical outcome in large neuroendocrine lung cancer. Methods and Patient There were 25 resectable large neuroendocrine lung cancer patients in our hospital before 2014. Twenty-two patients' tissue was done for EZH2 and EMT markers. The EZH2-GSK3 and EMT signaling were performed in H1299 and H460 cancer cell lines. EZH2 inhibitor, Akt inhibitor and protease inhibitor was used to validate the cell signaling from EZH2 to EMT markers. Results There were 10 patients had high EZH2 and snail and 10 patients had low EZH2 and snail. The correlation between EZH2 and snail was very high. Snail expression showed high prevalence of lymph node metastasis but no EZH2. The overall survival and progression free survival showed no definite in EZH2 and other insomnia. The recurrence was also not significant difference. The cellular study showed the same result. Conclusion Over expression of snail showed high correlation with EZH2 and was a predictable marker.

#4618

Integrative molecular classification of extrahepatic cholangiocarcinoma.

Robert Montal,1 Wei Qiang Leow,2 Carla Montironi,1 Laia Bassaganyas,1 Agrin Moeini,1 Daniela Sia,2 Roser Pinyol,1 Laia Cabellos,1 Judit Peix,1 Miho Maeda,2 Carlos Villacorta,2 Parissa Tabrizian,2 Christine Sempoux,3 Beatriz Minguez,4 Tim Pawlik,5 Ismail Labgaa,3 Lewis Roberts,6 Manel Sole,1 Maria Isabel Fiel,2 Swan Thung,2 Sasan Roayaie,7 Augusto Villanueva,2 Myron Schwartz,2 Josep Maria Llovet1. 1 _Liver Cancer Translational Research Laboratory, Barcelona Clinic Liver Cancer (BCLC) Group, Liver Unit, IDIBAPS-Hospital Clínic, CIBERehd, University of Barcelona, Barcelona, Catalonia, Spain;_ 2 _Liver Cancer Program, Divisions of Liver Diseases and RM Transplant Institute, Tisch Cancer Institute, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY;_ 3 _Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland;_ 4 _Liver Unit, Hospital Vall d'Hebron, Barcelona, Catalonia, Spain;_ 5 _Department of Surgery, Division of Surgical Oncology, The Ohio State University Wexner Medical Center, Columbus, OH;_ 6 _Miles and Shirley Fiterman Center for Digestive Diseases, College of Medicine, Mayo Clinic, Rochester, MN;_ 7 _Department of Surgery, White Plains Hospital, White Plains, NY_.

Background and Aims: Cholangiocarcinoma (CCA) is the second most common primary hepatic malignancy. Based on its anatomical location, CCA can be divided into intrahepatic (iCCA) or extrahepatic (eCCA), with differences in etiology, pathogenesis and clinical management. Few studies have focused on the molecular profiling of eCCA as a single entity, even though it accounts for the most prevalent subtype. Thus, integrative genomic analysis of eCCA would provide critical understanding for the biological traits of this tumor.

Methods: 189 FFPE primary eCCA treated by resection were collected at seven international centers from 1995 to 2015. Median survival of the cohort was of 48.5mo. Whole gene-expression profiles were submitted to unsupervised clustering by NMF consensus. Clusters were characterized by Gene Set Enrichment Analysis and Ingenuity Pathway Analysis. Activation of signaling pathways (mTOR/pRPS6 and HER2) was assessed by immunohistochemistry (IHC). Molecular features were correlated with clinico-pathological data. Screening of most prevalent somatic mutations and copy number aberrations is ongoing.

Results: We have identified four distinct molecular subtypes of eCCA (cophenetic coefficient=0.995). Tumors classified within the metabolic class (18.7%) were enriched by gene signatures defining bile and fatty acid metabolism (p<0.001) and presented overexpression of classic hepatocyte markers, with HNF4A as the major activated upstream transcription factor (p<0.001). The proliferation class (22.5%) was associated with papillary histology (p=0.004) and presented enrichment of MYC (p<0.001), mTOR (p=0.018) and HER2 (p=0.024) signaling. Subclass mapping identified similarity with the iCCA proliferation subclass (p<0.001). The mesenchymal class (47.3%) was associated with signatures defining epithelial-mesenchymal transition (p<0.001) as well as stromal activation (p<0.001), which was in accordance with TGFB1 as the major activated upstream regulator (p<0.001) and the presence of higher desmoplasia at pathological analysis (p=0.046). Moreover, mesenchymal tumors were significantly associated with poor prognosis in terms of OS (33.2 vs 55.5mo, HR=2.02, p=0.022). Finally, tumors classified as immune class (11.5%) presented increased tumor-infiltrating lymphocytes (p=0.001) and were characterized by enrichment of IFNγ signaling (p<0.001).

Conclusions: Transcriptome-based subtyping of eCCA identifies four distinct molecular classes (metabolic, proliferation, mesenchymal and immune) that correlate with clinical-pathological characteristics. These findings enhance the opportunities for therapeutic development in this tumor with dismal prognosis and without approved molecular treatments.

#4619

Integrated genomic analysis of Hurthle cell carcinoma reveals TMEM233/PRKAB1 fusion as a novel oncogenic driver.

Yiyu Dong, Ian Ganly, Vladimir Makarov, Fengshen Kuo, Shyamprasad Deraje, Ed Reznik, Venkatraman Seshan, Gouri Nanjangud, Luc Morris, Nadeem Riaz, Eric Sherman, Ronald Ghossein, James Fagin, Timothy Chan. _Mem. Sloan Kettering Cancer Ctr., New York, NY_.

Background: Hurthle cell carcinoma (HCC) is an enigmatic malignancy of the thyroid that can behave in an aggressive fashion, sometimes lethal, yet its molecular foundations are poorly understood. Some HCC have a good prognosis (minimally invasive, HMIN) whereas others can be extremely aggressive (widely invasive, HWIDE), leading to metastasis and death. HCCs were not included in the TCGA thyroid cancer study, which focused solely on papillary thyroid carcinomas. To understand the development of HCC and unveil its molecular mechanism, we performed a comprehensive genomic characterization of 56 primary HCCs that span the spectrum of tumor behavior and investigated the role of TMEM233/PRKAB1 fusion as a critical driver of oncogenesis in HCC.

Methods: Tumor and matched normal specimens were obtained from 56 patients with primary HCC. Tumors were classified into minimally invasive ( n=24) or widely invasive subtype (n=32). Whole exome sequencing was used to identify somatic mutations. Copy number changes were identified using FACETS and validated by FISH. RNASeq was used to identify novel fusions genes and to identify differentially expressed genes. Genomic alterations associated with histological phenotype were identified. Genomic changes associated with recurrence and survival were identified by the Kaplan Meier method. Immortalized thyroid epithelial cell line, NTHY-ori 3.1 was used to express control and fusion gene for in vitro and in vivo experiments.

Results: We elucidate the mutational profile and driver mutations in HCC and reveal that they exhibit a diverse spectrum of recurrent mutations, most of which have not been previously associated with this cancer (EIF1AX, MADCAM1). Notably, HCC harbor an extremely high prevalence of disruptive mutations to both protein-coding and tRNA encoding regions of the mitochondrial genome. We reveal unique chromosomal landscapes that involve whole chromosomal duplications of chromosomes 5 and 7 and wide spread major loss of heterozygosity arising from haploidization and copy number neutral uniparental disomy. These chromosomal processes underlie genetic instability and are highly prevalent in aggressive forms of HCC. We also identify novel fusion genes such as TMEM233/PRKAB1 which expression resulted in a transformation, organoids formation and invasion phenotype in vitro and

tumorigenesis in vivo. These data suggested that TMEM233/PRKAB1 fusion plays as a critical driver and may serve as a therapeutic target for HCCs.

Conclusions: We performed integrated genomic analysis of hurthle cell carcinoma revealing novel oncogenic drivers, recurrent mitochondrial mutations and unique chromosomal landscapes, which will help guide development of new treatments for one of the most deadly types of thyroid cancer.

#4620

Expression of nestin affect resistance to chemotherapy and clinical outcome in small cell lung caner.

Kazuki Sone,1 Ken Maeno,1 Eiji Kunii,2 Osamu Takakuwa,1 Akira Takeuchi,1 Satoshi Fukuda,1 Takamitsu Asano,1 Yoshihiro Kanemitsu,1 Hirotsugu Ohkubo,1 Masaya Takemura,1 Yutaka Ito,1 Tetsuya Oguri,1 Akio Niimi1. 1 _Nagoya City University Graduate School of Medicine, Nagoya, Japan;_ 2 _Nagoya City West Medical Center, Nagoya, Japan_.

Background: Small cell lung cancer (SCLC) is an aggressive and highly metastatic lung cancer subtype. Nestin is a member of the intermediate filament family and serves as a potential proliferative and multipotency marker in neural progenitor and stem cell. Aberrant expression of nestin is linked to malignant characteristics and poor prognosis in different cancers, including non-small cell lung cancer. However, in SCLC, the association between nestin expression and clinicopathological feature or prognosis has remained unclear. In this study, we examined whether the expression of nestin affect malignant feature and clinical outcome in SCLC.

Material and methods: To assess the function of nestin, a short hairpin RNA (shRNA) targeting nestin and a forced expression vector of the nestin gene were transfected into SCLC cell lines (DMS53 and SKLC17). And three lung cancer cell lines (PC6, PC9 and PC14) and their cisplatin-resistant sub-lines were also analyzed. Nestin expression levels were assayed using Western blotting. Cell proliferation assay was performed using both nestin knock-down and overexpressed cell lines. Growth in vivo was assessed in SCID mice using a nestin knock-down cell line. Chemosensitivity was expressed as the drug concentration for IC50 in MTS cell proliferation assay. Then we retrospectively examined 84 SCLC patients. Correlations between immunohistochemical expression of nestin and various clinicopathological features were evaluated.

Result: In nestin knock-down and overexpression cell lines, nesin expression was related to cell proliferation in vitro and vivo and resistance to anticancer drugs (cisplatin, etoposide and SN-38). Nestin expression levels of three cisplatin-resistant cells were increased than their parent cell lines. In 84 SCLC patients (16 patients were surgically resected, and 68 patietns were biopsied), 24 patients had nestin positive tumor. Nestin positive ratio tended to be higher in operated patients than biopsied patients. Nestin expression was not associated with age, sex, smoking status, disease stage and serum pro-gastrin releasing peptide (ProGRP) levels. There were no significant differences between nestin positive and negative patients for response rate (RR), progression free survival (PFS) and overall survival (OS) in 1st line chemotherapy. However, positive expression of nestin was associated with shorter PFS in 2nd line chemotherapy (median PFS; nestin positive 81 days vs negative 117 days, P=0.03).

Conclusion: Nestin expression lead to resistance to chemotherapy and worse outcome in SCLC patients.

#4621

Risk stratification and biomarker discovery in HPV-positive oropharynx squamous cell carcinoma determined by HPV and human gene expression profile associations.

Frederico O. Gleber-Netto,1 Xiayu Rao,1 Kelly Erikson,1 Keiko Akagi,1 Faye M. Johnson,1 Jing Wang,1 Joseph Califano,2 Maura L. Gillison,1 Jeffrey N. Myers,1 Curtis R. Pickering1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _University of California San Diego, San Diego, CA_.

Determining which HPV+ oropharyngeal squamous cell carcinoma (OPSCC) patients will respond to therapy is of utmost important for implementation of treatment de-escalation to reduce morbidity or testing of novel therapeutic approaches. Due to the lack of reliable indicators of treatment response, we explored the gene expression profile of OPSCC in order to better understand the biology of these tumors and identify novel biomarkers. In this study, we investigated human genes associated with HPV transcription in 80 OPSCC samples from The Cancer Genome Atlas (TCGA). This exploratory analysis provided a list of 582 human genes associated with HPV biology. Hierarchical clustering analysis using the 582 human genes was used to separate HPV+ OPSCC in two groups with significantly distinct survival (log-rank test p < 0.001) and differential expression of HPV genes. A refinement of this analysis generated a prognostic gene expression signature for HPV+ OPSCC containing 41 human genes. These results were validated in two independent cohorts of HPV+ OPSCC (n=83 and n=47) and one independent cohort of cervical cancer (n=83). In all three validation cohorts our 41 gene expression signature was able to identify a group of HPV+ OPSCC patients with worse survival. Further refinement and validation of the signature is ongoing. In order to understand the biology related to poor outcome in HPV+ OPSCC, a whole transcriptome analysis between the two initial HPV+ OPSCC TCGA groups was performed. Pathway analysis identified cell metabolism, cell stress, and DNA damage related genes were highly associated with poor outcome. Similar patterns of gene expression were found in vitro in a panel of 10 HPV+ cell lines, and markers of the poor outcome were found to be associated with reduced radiation sensitivity in vitro. Our study has identified prognostic biomarkers in HPV+ OPSCC with potential clinical importance. These biomarkers may be useful for selection of low risk patients for treatment de-escalation or selection of high risk patients for novel therapeutic approaches. These biomarkers are also functionally linked to radiation sensitivity and may include new therapeutic targets.

#4622

Deciphering gene subsets associated with clinical outcomes of early stage breast carcinoma patients treated with Tamoxifen.

Zohair Riaz Hameed,1 Michael W. Daniels,2 D. Alan Kerr II,3 James L. Wittliff1. 1 _Univ. of Louisville, Louisville, KY;_ 2 _University of Colorado at Aurora, Louisville, CO;_ 3 _University of South Florida, Tampa, FL_.

NSABP Protocol B-09 reported primary breast carcinomas expressing estrogen (ER) and progestin receptors (PR) exhibited increased likelihood of response to Tamoxifen. However, some patients with early stage lesions relapse within 10 years. We identified a 5-gene model from 14 candidate expression profiles that improved differentiation of these patients (Kerr & Wittliff, Horm Canc 2:261-71, 2011). Goals are to examine relationships of clinical outcomes of patients whose carcinomas express individual genes and subsets of the models and externally validate their clinical utility. Procedures: De-identified primary breast carcinomas from 342 patients treated with Tamoxifen were used to assess clinical relevance of 14 candidate genes. Laser capture microdissection (LCM) was used previously to isolate carcinoma cells and assess 22,000 genes by microarray. qPCR validated expression of 14 candidate genes. Univariable Cox regression analyses were performed and Kaplan-Meier plots were constructed to assess relationships between either progression-free (PFS) or overall survival (OS) and expression of each gene using R Studio 1.0.143. Relationships of gene expression were segregated by ER/PR (quantified by EIA or radio-ligand binding) with clinical outcomes. SurvExpress evaluated performance externally of individual gene expression and signatures. Results: Univariable Cox regression of microarray results for 14 candidate genes from LCM-procured cancer cells of 247 biopsies without regard to ER/PR status indicated that RERG, ERRB4 and ESR1 were related to PFS while these 3 genes and EGFR and CAXII expression were associated with OS (adjusted p value < 0.30). Validation of individual gene expression of 274 intact tissue sections by qPCR revealed that 4 of these genes (exception of EGFR) as well as PGR, BCL2, SLC39A6, EDG-1, and CD34 were associated with PFS. Expression levels of each of these 9 candidate genes as well as PTGDS, SDF and NQO-1 were independently associated with OS. Kaplan-Meier plots assessed ability of each gene expression level to predict clinical outcomes (PFS/OS). Multivariate Cox regression, performed with backward conditional selection using either microarray data or qPCR results, revealed clinically relevant genes subsets for PFS and OS. SurvExpress, an online tool, was used to externally validate molecular signatures. Conclusions: We externally validated the 5-gene model composed of PGR, BCL2, ERBB4, RERG and CD34 that identified a subset of early stage, ER+ /PR+ breast cancers patients treated with Tamoxifen that exhibited high recurrence rates. ER, PR and HER2 proteins with gene expression were correlated with prediction of clinical outcomes. Clinically relevant molecular signatures identified gene candidates for development of companion diagnostics to their protein products to improve breast cancer management and prediction of risk of recurrence.

#4623

Meta-analysis of transcriptomic profiles identifies prognostic model for pancreatic ductal adenocarcinoma patients.

Vandana Sandhu,1 Knut Jorgen Labori,2 Ayelet Borgida,3 Ilinca Lungu,4 John Bartlett,4 Sara Hafezi-Bakhtiari,1 Rob Denroche,4 Gun Ho Jang,4 Danielle Pasternack,4 Faridah Mbaabali,4 Matthew Watson,4 Julie Wilson,4 Elin H. Kure,5 Steven Gallinger,6 Benjamin Haibe-Kains1. 1 _University Health Network, Toronto, Ontario, Canada;_ 2 _Institute for Cancer Research, Oslo University Hospital, Oslo, Norway;_ 3 _Mount Sinai Hospital, Toronto, Ontario, Canada;_ 4 _Ontario Institute of Cancer Research, Toronto, Ontario, Canada;_ 5 _Institute for Cancer Research, Oslo University Hospital, Toronto, Ontario, Canada;_ 6 _Ontario Institute for Cancer Research, Toronto, Ontario, Canada_.

Purpose

The main objective was to develop a robust molecular predictor model with high prognostic value across multiple independent cohorts of pancreatic ductal adenocarcinoma (PDAC) patients using a novel meta-analysis framework. The median 5-year overall survival (OS) of PDAC patient is <8%. Only 10-20% of patients are eligible for surgery, and of these, more than half will die within a year of surgery. The identification of a biologically relevant molecular predictor model is urgently needed to define strategies that may help patient selection at high risk of early death to inform treatment decisions.

Methods

We developed the Pancreatic Cancer Overall Survival Predictor (PCOSP), a new prognostic model built from a unique set 89 PDAC patients whose gene expressions have been profiled using both microarray and sequencing platforms. We used the recent binary gene pair method to create gene expression barcodes robust to biases arising from heterogeneous profiling platforms and batch effects. Leveraging the largest compendium of PDAC transcriptomic datasets to date, including 1,198 patients, we show that PCOSP is a robust single-sample predictor of early death (≤1 yr) after surgery in a subset of 823 validation samples with available trancriptomics and survival data.

Results

The PCOSP model was strongly and significantly prognostic overall with a meta-estimate of the area under the ROC curve of 0.70 (P=1.9e-18) and hazard ratio of 1.95 (P=2.6e-16)

for binary and survival predictions, respectively. The prognostic value of PCOSP was independent of clinicopathological parameters and molecular subtypes. The PCOSP model includes 2,619 gene pairs, with 1,070 unique genes. Over-representation analysis of these genes unveiled pathways associated with Hedgehog signalling, epithelial mesenchymal transition (EMT) and extracellular matrix (ECM) signalling at FDR<0.05.

Conclusions

This study reports a PCOSP model to predict post-operative OS independently of clinicopathological features. This may assist clinicians in making decisions that would ultimately improve the OS and facilitate decisions concerning a surgery-first versus a neoadjuvant approach. The functional analysis of the PCOSP genes may give further insight to tumor biology of short-term survival PDAC patients.

#4624

The sTRA glycan is complementary to CA19-9 as a serologic biomarker of pancreatic cancer.

Ying Liu,1 Daniel Barnett,1 Ben Staal,1 Ying Huang,2 Katie Partyka,1 Herbert Zeh,3 Aatur Singhi,3 Richard R. Drake,4 Randall E. Brand,3 Brian B. Haab1. 1 _Van Andel Research Institute, Grand Rapids, MI;_ 2 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 3 _University of Pittsburgh Medical Center, Pittsburgh, PA;_ 4 _Medical University of South Carolina, Charleston, SC_.

The CA19-9 biomarker is elevated in a substantial group of patients with pancreatic cancer, but not enough to be reliable for detection, diagnosis, or monitoring of the disease. We hypothesized that a glycan called sTRA complements CA19-9, and that the two biomarkers in combination provide added information over either biomarker individually. As an individual marker, the sTRA assay performed equivalently with CA19-9 for distinguishing cancers from benign pancreatic diseases, but in combination, CA19-9 and sTRA showed exclusive elevations in significantly different groups of patients. Using high-specificity cutoffs giving one false-positive detection in each marker, sTRA was elevated in 34/46 (74%) patients with low CA19-9 in stage III/IV cancers and 28/70 (40%) with low CA19-9 in stage I/II cancers. A three-marker panel consisting of CA19-9 and two versions of the sTRA assay gave 81% sensitivity and 88% specificity (84% accuracy) in a training set of 72 stage I-IV cancers and 75 benign disease controls, compared to 57% sensitivity and 92% specificity (75% accuracy) for CA19-9 alone. The application of the fixed three-marker classifier to a test set of 25 cancers and 25 benign controls gave 80% sensitivity and 84% specificity, compared to 56% sensitivity and 96% specificity for CA19-9 alone. In an analysis of patient-matched tumor tissue and plasma samples, we found that the tumors secreting both sTRA and CA19-9 into the blood tended to be conventional PDAC with well-to-moderate differentiation and heavy marker staining in the ductal lumens, but the tumors secreting only sTRA showed sTRA staining in glands with extremely large and unpolarized nuclei and vacuolated or foamy cytoplasm. Those without plasma elevations in either marker did not have glandular features of typical adenocarcinoma. Surprisingly, about half of the patients without plasma elevations of CA19-9 nevertheless showed strong tumor staining. Such tumors expressed CA19-9 mostly in poorly differentiated patches of cells with compact nuclei and cytoplasm. Thus, sTRA and CA19-9 are complementary serologic biomarkers that together could increase the diagnostic accuracy for PDAC and potentially identify distinct subgroups of tumors.

#4625

Tumor expression of PIM kinases in renal cell carcinoma and the association with disease progression.

Wei Luo,1 Yingyu Ma,1 Eduardo Cortes,1 Angela Omilian,1 Wiam Bshara,1 Gissou Azabdaftari,1 Brittany Bunch,1 Candace Johnson,1 Donald Trump2. 1 _Roswell Park Cancer Inst., Buffalo, NY;_ 2 _Inova Schar Cancer Inst, Falls Church, VA_.

Hypoxia-inducible factor (HIF) plays an important role in the tumorigenesis of renal cell carcinoma (RCC). Mutational inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene, which is the most frequent molecular event, has been implicated in HIF overexpression. Recent studies found that PIM kinases increase HIF expression in cancer. The PIM Kinases are a family of highly conserved serine/threonine kinases consisting of three isoforms (PIM1, PIM2, and PIM3). PIM kinases play multiple roles in promoting cell proliferation and survival and preventing apoptosis. The expression and the potential function of PIM kinases in RCC remain unclear. Thus, we examined the expression patterns and the clinical significance of the three individual PIM kinases in RCC. PIM expression was examined by immunohistochemical staining tumor tissue and adjacent non-tumor tissue samples from 218 patients with clear cell RCC (ccRCC), including 60 patients with metastatic ccRCC. The expression of each PIM kinase in primary and metastatic ccRCC tissues comparing with adjacent normal tissues was analyzed with variables including tumor stage, grade and survival. Effect of PIM kinase on cell growth in ACHN and 786-O RCC cell lines was examined by individual PIM knockdown with specific siRNA followed by in vitro proliferation assay. Effect of PIM kinase on RCC cell motility was examined by siRNA followed by in vitro Matrigel-based invasion assay. Expression of PIM1, PIM2 and PIM3 was detected in the majority of ccRCC samples (185, 85%), but localization of isoforms differed. PIM1 expression was higher in the nucleus than in cytoplasm, while the reverse was true for PIM 2 and 3. PIM2 and PIM3 expression was higher in cytoplasm than in the nucleus. Higher expression of PIM1 and PIM2 in tumor tissues was significantly associated with metastasis. Nuclear PIM2 was associated with poorly differentiated ccRCC cells. ccRCC patients with increased nuclear PIM2 expression had significantly poorer survival compared with patients with low nuclear PIM2. siRNA-PIM2, but not siRNA-PIM1 and siRNA-PIM3, significantly reduced proliferation of ACHN and 786-O cells. Further, knockdown of PIM2 by siRNA reduced the ability of cell invasion in 786-O cells. In conclusion, PIM kinases, especially PIM2, are associated with ccRCC progression, indicating that PIM kinases may serve as potential markers for metastatic ccRCC. Targeting PIM kinases may have a therapeutic potential in ccRCC. This work was supported by NIH/NCI grant CA067267, CA085142 (Johnson, CS) and National Cancer Institute (NCI) grant P30 CA016056 involving the use of Roswell Park Cancer Institute's Bioinformatics, Pathology Resource Network, Genomics, and Clinical Data Network Shared Resources.

#4626

A TRIM24-like mutational signature predicts worse relapse-free survival (RFS) and overall survival (OS) in patients (pts) with metaplastic breast cancer (MpBC).

Clinton Yam, Aundrietta Duncan, Jeffrey T. Chang, Michael Z. Gilcrease, Vrutant V. Shah, Michelle Barton, Stacy L. Moulder. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Background: TRIM24 is a multifunctional, epigenetic PHD/Bromodomain protein that negatively regulates p53, via an E3-ubiquitin ligase RING domain, and co-regulates transcription. Over-expression of TRIM24 in murine mammary epithelium results in the development of carcinosarcomas that bear close histologic resemblance to human MpBCs and are characterized by a distinct genomic signature. Here, we investigate the association between the presence of a TRIM24-like mutational signature and clinical outcomes in a cohort of human MpBCs.

Methods: 20 archived samples of human MpBC with annotated clinical data underwent whole exome sequencing. In parallel, we sequenced 7 spontaneously arising carcinosarcomas from a TRIM24 overexpressing mouse model. To compare the mutations with those seen in the human MpBCs, we grouped them into pathways and created a signature of pathway activation seen in TRIM24-driven tumors. Median RFS and OS were estimated using the Kaplan-Meier method and compared between pts with and without a TRIM24-like mutational signature.

Results: Baseline clinical characteristics are summarized in Table 1. Of the 20 MpBC pts, 5 had a TRIM24-like mutational signature and the remaining 15 pts did not. There were no statistically significant differences in baseline characteristics between the 2 groups. Median RFS for pts with a TRIM24-like mutational signature was 9 months (95% confidence interval [CI]: 8-undefined months). Median RFS for pts without a TRIM24-like mutational signature has not been reached (log-rank, p=0.0149). Median OS for pts with and without a TRIM24-like mutational signature was 17 months (95% CI: 10-undefined months) and 147 months (95% CI: 22-undefined months), respectively (log-rank, p=0.0402).

Conclusion: In this cohort, pts with MpBCs that exhibit a TRIM24-like mutational signature had worse RFS and OS. This finding should be confirmed in larger, prospective studies.

Table 1: Baseline Clinicopathological Characteristics by TRIM24 Status

---

|

TRIM24-like (n=5) | Non TRIM24-like (n=15) | p value

Age at diagnosis - Median (years, interquartile range) | 55 (50-61) | 56 (47-66) | 0.965

Clinical Tumor Size | |

|

Mean (cm, standard deviation) | 4.2 (1.5) | 3.4 (1.6) | 0.383

Clinical Nodal Status | |

|

Negative - n (%) | 3 (60) | 11 (73) | 0.570

Positive - n (%) | 2 (40) | 3 (20)

|

Unknown - n (%) | 0 | 1 (7)

|

ER/PR Status | |

|

Negative - n (%) | 5 (100) | 14 (93) | 1.000

Positive - n (%) | 0 | 1 (7)

|

HER2 Status | |

|

Negative - n (%) | 5 (100) | 15 (100) | NA

Positive - n (%) | 0 | 0

|

Histology | |

|

Spindle Cell - n (%) | 4 (80) | 7 (47) | 0.319

Matrix-producing - n (%) | 1 (20) | 8 (53)

|

#4627

Integrated multi-layer analysis reveals novel insights into the molecular landscape of colorectal cancer (CRC).

Manuela Salvucci,1 Liam Poynter,2 Reza Minerzami,2 Steven Carberry,1 Robert O'Byrne,1 Mattia Cremona,1 Bryan T. Hennessy,1 Kirill Veselkov,2 James Kinross,2 Jochen H. Prehn1. 1 _Royal College of Surgeons in Ireland, Dublin, Ireland;_ 2 _Imperial College London, London, United Kingdom_.

Metabolic reprogramming, rewired signalling cascades, altered microbiota composition and aberrations in the tumor micro-environment have been implicated in CRC progression and response to treatment. In this study, we integrated proteomics, metabolomics, gut microbiota and predictions from network-analysis in tumor and non-cancerous tissue to build a comprehensive atlas to decipher the associations among alterations that occur in the different molecular layers. We performed our molecular fingerprinting in fresh frozen tissue samples from both tumor (centre and/or junction) and matched mucosa (5 and/or 10 cm distance) prospectively collected from a cohort of n=53 stage 0-IV CRC patients. We quantified the protein expression of a panel of 86 cancer-related targets involved in DNA damage/repair mechanisms, cell cycle regulation, growth/angiogenesis signalling, apoptosis and bioenergetics by Reverse Phase Protein Array (RPPA) in n=157 samples (n=48 from tumor and n=109 from mucosa) from n=53 patients. RPPA profiling on both tumor and matched normal tissue was available for 34 out of 53 patients. We performed High Resolution Magic-Angle Spinning Nuclear Magnetic Resonance Spectroscopy (1H HR-MAS NMR) to profile metabolites in n=332 samples from n=52 patients (n=161 and n=171 from tumor and normal tissue, respectively). We characterized gut microbiota features from 16S rRNA sequencing in n=54 samples from n=18 patients. We assessed bioenergetic fitness using an ordinary-differential equations based model of core carbon metabolism that we are developing. The model can simulate the network dynamic and response to fuels (such as glucose, pyruvate and lactate) from extracellular milieu and their utilization and conversion via reactions in the cytoplasm and mitochondrial compartments. We used RPPA-based protein expression for HKII, TIGAR, PGYM, LDHA, MCT4, ATP5A as a proxy for enzymatic activities as case-specific inputs to the model. We identified distinct phenotypic differences when comparing tumors with normal tissue at each data type. Tumors showed higher protein levels for CHK1, GSK3B and LDHA; were enriched with fusobacteria; had increased metabolic levels of glycerphosphocholine, isoglutamine, phosphocholine, taurine and lactate; and were predicted by the mathematical model to have a more glycolytic phenotype. Normal mucosa had higher protein levels for GAB1 and elevated isobutyrate and lipids content. We further analysed the NMR spectra and using a genetic algorithm we identified 10 features (ppm levels) that could classify the tissue type with 88% cross-validated accuracy. We presented a unified analysis connecting dysregulations affecting the proteome, metabolome and microbiome. We provided insights into the heterogeneity of CRC which may lead to more specific molecular classifications and ultimately more targeted treatments.

### Novel Preclinical Therapies in Pediatric Solid Tumors

#4628

Tailoring personalized strategies for children with treatment-refractory liver cancer.

Stefano Cairo,1 Bénédicte Noblet,1 Samuel Rasmussen,2 Matthew N. Svalina,2 Katell Mevel,1 Enora Le Ven,1 Delphine Nicolle,1 Olivier Déas,1 Charlotte Mussini,3 Sophie Branchereau,3 Jean-Gabriel Judde,1 Charles Keller2. 1 _XenTech, Evry, France;_ 2 _Children's Cancer Therapy Development Institute, Beaverton, OR;_ 3 _Hôpital de Bicêtre, Le Kremlin Bicêtre, France_.

Tumour heterogeneity is probably the reason for different sensitivity to the same treatment in patients. Therefore, we urgently need to develop tools to help assign the right treatment to the right patient. This is particularly challenging for paediatric cancers. Hepatoblastoma (HB) is the most frequent malignant embryonal tumor of the liver in childhood. About 25% of HB patients will develop metastatic disease which is associated with a poor prognosis. Because of the small number of patients eligible for enrollment, clinical trials in most of cases could not provide clinicians with statistically significant results. In oncology, preclinical data are generally poorly predictive of treatment efficacy in patients, probably due to oversimplification of tumor biology and underrepresentation of the genetic tumor heterogeneity observed in patients. To face this issue, we have launched a program to generate patient-derived xenografts, the gold standard in preclinical oncology, from surgical tissue from HB patients after chemotherapy, at relapse and from local and distant metastases. Therefore, the models developed are representative of relapsing, metastatic HB. So far, a panel of 24 HB PDXs models has been established. From these models 10 cell lines could be established so far to be used for large-scale in vitro screening of anticancer compound libraries. The aim of this project is to combine in vitro and in vivo drug screening to provide strong preclinical rationale to the development of novel therapeutic options for children with HB, and to identify candidate biomarkers predictive of tumor response. In vitro screening of a HB PDX-derived cells with a library of 60 compounds corresponding to treatments already available in the clinic lead us to identify 4 compounds with very strong cytotoxic activity on HB cells. One of them, Volasertib, a PLK1 inhibitor, was selected to be tested in the HB PDX the cells were derived from. The in vivo study compared the effect of volasertib as single agent or in combination with irinotecan to irinotecan alone or in combination with temozolomide. The results showed that high dose volasertib induce tumor regression with efficacy comparable to irinotecan/temozolomide combination. Following these encouraging results, Volasertib was tested in the complete panel of HB PDX-derived cells, and the majority of them showed strong sensitivity to the drug. This sensitivity was associated with specific molecular alterations that might be used as biomarkers to predict tumor sensitivity. Overall, these results suggest that volasertib could be an effective second line treatment for children with recurrent HB, and that this platform could be a very important preclinical tool to foster translational research in HB.

#4629

Synergistic growth inhibitor effect on a patient derived NF1 pilocytic astrocytoma cell line with the dual mTORC1/2 inhibitor TAK228 and MEK inhibitor trametinib.

Antje Arnold, Ming Yuan, Fausto J. Rodriguez, Charles G. Eberhart, Eric H. Raabe. _Johns Hopkins University School of Medicine, Baltimore, MD_.

Pediatric low grade glioma (PLGG) is the most common brain tumor of childhood. If the tumor is located in a region of the brain that is not accessible for surgical resection, additional therapies are needed. We and others have identified mTORC and MEK-activation in PLGG. The dual mTORC1/2-inhibitor, TAK228, and the FDA approved MEK-inhibitor, trametinib, are promising candidates for targeted PLGG therapy. We hypothesized that TAK228 and trametinib would show synergistic effects in vitro and in vivo PLGG models. We treated in vitro four different patient-derived PLGG cell lines with TAK228, trametinib, or vehicle control: JHH_NF1_PA1 (NF1 mutation), BT66_SV40/hTERT (BRAFfusion), Res186 and Res259 (both cell lines with mTOR and MAPK activation). In vivo, BT40 (BRAFV600E) tumor cells were investigated with both agents in immunodeficient mice. Cell growth was investigated using MTS-assay compared to vehicle control. Activation of MAPK pathway was detected via Western Blot by phosphorylated ERK compared to total ERK, and β-actin. mTOR pathway was investigated with pAKT, pS6, and p4E-BP1 compared to the total protein amount and β-actin. In all of the cell lines, treatment with TAK228 or trametinib reduces cell growth and proliferation in a dose and time depended manner. We have found a robust synergistic (via Chou-Talalay method) effect for JHH_NF1_PA1, Res186, and Res259 cells in clinically relevant doses of both drugs (5nM, 10nM, 20nM). BT66_SV40/hTERT cells have a significant reduction in cell growth under TAK228 treatment after 4 days by up to 70% (p<0.001; via one-way ANOVA), but not under trametinib treatment. Interestingly for this cell line, the MAPK pathway was inactivated in all tested trametinib doses (≥1nM) and in combination treatment. In all cell lines trametinib treatment leads to a pERK inactivation at low nM levels. TAK228 leads to an inactivation of mTORC1 and mTORC2 in all four tested cells lines. In TAK228 treated cells, there was compensatory activation of pERK, which was reduced when trametinib was added. Apoptosis induction was verified through cleaved PARP via western blot and CC-3 via immunocytochemistry. The combination of TAK228 and trametinib increased apoptosis by up to 127% (p<0.001). After determination the optimal dosing schedule for TAK228 (1mg/kg/every other day), trametinib (3mg/kg/daily) and combination, BT40 transplanted nude mice are investigated for tumor size and survival. Our results show that PLGG-derived cell lines are sensitive to TAK228 and trametinib treatment. The increased MAP kinase activity we identified after TAK228 treatment, suggests a compensatory mechanism that may render these cells especially sensitive to treatment with both TORC1/2 and MEK inhibitors. The ongoing in vivo experimentation will provide a pre-clinical rationale for combination therapy of these agents in PLGG.

#4630

Characterizing oncogene addiction in alveolar rhabdomyosarcoma reveals novel strategies for combination therapy.

Johannes Ommer, Marco Wachtel, Beat W. Schaefer. _University Children's Hospital Zurich, Zurich, Switzerland_.

Rhabdomyosarcoma is the most common soft tissue sarcoma in children. The aggressive alveolar subtype (aRMS) is characterized by chromosomal translocations, most often a t(2;13) resulting in the expression of the oncogenic fusion protein PAX3-FOXO1 which is critical for tumorigenesis and cell survival. Our aim here was to identify a pharmacological combination therapy approach interfering with PAX3-FOXO1 biology at different levels.

Since loss of the fusion protein results in cell death, we first aimed to pharmacologically enhance this effect. To this end, we screened aRMS tumor cells with a library of 208 drugs while simultaneously silencing PAX3-FOXO1 by shRNA. This identified the BH3-mimetic ABT-263 to sensitize aRMS cells to cell death after PAX3-FOXO1 depletion. To further characterize the cell death mechanisms we used combined shRNA and CRISPR approaches to perform a BH3 protein profiling. In accordance with identification of ABT-263 we demonstrate that aRMS cells undergo intrinsic apoptosis in a NOXA-dependent manner upon depletion of PAX3-FOXO1.

In a parallel approach, and to identify drugs altering PAX3-FOXO1 protein stability, we screened the same drug library directly measuring fusion protein levels as read-out. This revealed that inhibition of Aurora kinase A negatively affects PAX3-FOXO1 protein levels.

Finally, using both aRMS cell lines and patient-derived xenografts we demonstrate that combination treatment of Aurora kinase A inhibitors together with ABT-263 synergistically induces cell death and greatly slows tumor growth in vitro and in vivo.

Taken together, these data show a novel functional interaction of Aurora kinase A and PAX3-FOXO1 and suggest new opportunities for targeted combination treatment of aRMS.

#4631

**Expression of ganglioside G** D2 **in Ewing sarcoma cells is not associated with specific functional characteristics or stem cell features.**

Sareetha Kailayangiri,1 Bianca Altvater,1 Silke Jamitzky,1 Stefanie Lesch,1 Jan-Henrik Mikesch,2 Sonja Schelhaas,3 Schaefers Michael,4 Jutta Meltzer,1 Nicole Farwick,1 Wolfgang Hartmann,5 Eva Wardelmann,5 Petra Fischer,5 Jendrik Hardes,6 Claudia Rossig1. 1 _Univ. Children's Hospital Münster, Department of Pediatric Hematology and Oncology, Muenster, Germany;_ 2 _Univ. Hospital Münster, Department of Medicine A, Muenster, Germany;_ 3 _European Institute for Molecular Imaging, Muenster, Germany;_ 4 _Univ. Hospital Münster, Department of Nuclear Medicine, Muenster, Germany;_ 5 _Univ. Hospital Münster, Gerhard Domagk Institute of Pathology, Muenster, Germany;_ 6 _Univ. Hospital Münster, Department of Orthopedic Surgery, Muenster, Germany_.

Safe and effective targeting of cancer with chimeric antigen receptor (CAR) engineered T cells relies on the presence of adequate tumor-associated surface antigens. We have found that Ewing sarcomas (EwS) express ganglioside GD2 and are recognized by T cells engineered with GD2-specific CARs. In an extended series of 97 EwS patient samples, 52 were GD2pos by immunohistochemistry. Based on reports that GD2 in breast cancer defines a malignant population with stem cell characteristics, we hypothesized that GD2 expression in EwS is associated with high capacity to self-renew and reinitiate tumor growth. Among 15 individual EwS cell lines with variable surface expression of GD2 by flow cytometry, GD2 expression levels were not associated with the capacities to proliferate and expand in vitro, form colonies in semi-solid medium, nor with their chemosensitivity, assessed by cell viabilities in the presence of increasing concentrations of the cytotoxic drug doxorubicin. Subpopulations with GD2hi phenotype selected from GD2pos and from GD2low EwS cell lines by cell sorting maintained their GD2hi and GD2low expression status during subsequent cultures over several weeks. The two subpopulations did not have different in vitro growth, colony-formation capacity, or chemosensitivity. In xenografting experiments, GD2hi and GD2low subpopulations of two EwS cell lines initiated tumors with comparable efficacies. To obtain direct evidence that GD2 surface expression is irrelevant for the biology of EwS cells, we performed genetic knockdown of the GD3S gene which drives biosynthesis of GD2 by CRISPR/Cas9 gene editing. GD3S gene editing resulted in effective elimination of GD2 surface expression in the GD2hi EwS cell lines TC-71, VH-64 and A4573. The knockdown did not affect the capacity of the cells to proliferate, form colonies in soft agar in vitro, nor their chemosensitivity compared to wild-type EwS cells from the individual cell lines. We conclude that GD2 expression in EwS cells, other than reported in breast cancer, is not associated with distinct functional features. Specifically, GD2 does not affect the growth characteristics, clono- and tumorigenicity and chemosensitivity of the tumor cells. Elimination of GD2pos subpopulations from heterogeneous tumors by CAR T cell targeting is therefore unlikely to eradicate the disease and will have to be combined with additional targeting strategies.

#4632

Decitabine for neuroblastoma cure: Target therapy induced ongoing acquisition of RD3 loss and reverts tumor evolution.

Aravindan Natarajan, Dinesh Babu Somasundaram, Sheeja Aravindan, Mohan Natarajan, Terence Herman. _University of Oklahoma Health Sciences Center, Oklahoma City, OK_.

Recently, we defined the novel tumor evolution stabilization role of Retinal degeneration protein 3 (RD3), regulating the metastatic state of neuroblastoma (NB) cells in vitro and their metastatic potential in vivo. Our earlier studies in various tumor models recognized the role of therapy resistant residual cells in tumor recurrence and relapse. Herein, we investigated the ongoing acquisition of RD3 transcriptional regulation in radiotherapy surviving NB cells and further identified the potential drug candidates that can reprogram and reinstate RD3 transcription in these cells. Human NB (SH-SY5Y, SK-N-AS) cells exposed to single dose (10, 20, 50cGY, 1, 2, 4Gy) or fractionated (FIR, 2Gy/Day for 5 days) irradiation resulted in significant transcriptional repression of RD3. Drug screening with a panel of drugs that are in current clinical use, those in pipelines or in developmental stages by exposing radioresistant cells identified many potential drug candidates that could effectively restore RD3 transcription in this setting. Evidently, we found RD3 promoter region hypermethylation drives its transcriptional regulation in therapy resistant cells. To that note, FDA approved hypomethylating agent decitabine significantly reinforced RD3 transcription in this setting and further promoted cellular differentiation and completely inhibited the formation of organized tumorosphere under stem-cell culture conditions. Together, these results demonstrate the ongoing acquisition of RD3 loss in therapy resistant cells and further recognized the mechanism of how RD3 is regulated in this setting. More importantly, this results of this study identified a drug candidate, that has been already in clinical use, that could be beneficial in mitigating therapy resistance and better clinical outcomes for the patients presented with high-risk advanced disease. Funding: NIH-COBRE-IP20GM103639-01, OUHSC-COM-Radiation Oncology Research Development Funds

#4633

Urinary biomarkers predict the presence of ependymoma non-invasively.

Julie Sesen, Gabrielle Luiselli, Alexander Moses-Gardner, Stephen Pineda, Micah Duggins-Warf, Maxwell Gruber, Maxwell Proctor, Edward R. Smith. _Children's Hospital Boston, Boston, MA_.

Objective: Pediatric brain tumors persist as a diagnostic and therapeutic challenge. The ability to identify a specific tumor diagnosis and non-invasively monitor the patient's response to therapy would serve as a major clinical advancement which could directly impact current practice. Knowing the tumor identity pre-operatively can influence surgical planning, so novel methods to improve upon existing imaging tools would help neurosurgeons treat children more effectively. In addition, post-operative follow-up of brain tumors currently relies on frequent imaging studies, which often requires the cost and risk of general anesthesia in children to facilitate scanning. Ependymoma is one of the most common pediatric brain tumors that can often mimics other pathologies on imaging reports, and typically requires extensive follow-up. Here, we present a novel method of utilizing non-invasive urinary biomarkers to diagnose pediatric ependymomas.

Methods: Urine was collected from 107 children with brain tumors, including a cohort with pathology-proven ependymoma (n = 19) and age- and sex-matched controls (n = 17). Using a previously validated screening panel of secreted proteins associated with tumor growth, including matrix metalloproteinases (MMPs), growth factors, and cytokines, putative biomarker levels were quantified using multiplex and ELISA assays in urine. Results were subjected to univariate and multivariate statistical analyses and compared between clinical cohorts. Once identified, further validation of biomarker expression was performed using in vitro analysis of primary tumor and control cell lines to link biomarker expression to source tissue.

Results: Evaluation of urinary biomarkers by multiplex and ELISA revealed that patients with ependymoma demonstrated significantly higher levels of urinary MMP3 and MMP9 as compared to healthy children. We identified clinically relevant cut-off values to identify the presence of tumor; >0.25 pg/µg for MMP-3 and >1.00 pg/µg for MMP-9. Multiplexing biomarkers markedly increased diagnostic accuracy. Urinary biomarker expression was correlated with in vitro cell lines, indicating that ependymoma tumor tissue is the source of the biomarkers.

Conclusions: We report for the first time the use of urinary proteins—MMP3 and MMP9— as biomarkers capable of identifying the presence of ependymoma non-invasively and with high accuracy. This study provides substantial in vitro and clinical data supporting the utility of urinary biomarkers as a novel tool to advance the diagnosis and treatment of pediatric brain tumors.

#4634

Mithramycin amplifies the imbalance between the BAF and PRC2 complexes to drive apoptosis in rhabdoid tumor.

Maggie H. Chasse,1 Elissa Boguslawski,1 Courtney E. Wernette,2 Susan Kitchen-Goosen,1 Patrick J. Grohar1. 1 _Van Andel Institute, Grand Rapids, MI;_ 2 _Ferris State University, Big Rapids, MI_.

BACKGROUND: Rhabdoid tumor (RT) is an aggressive pediatric malignancy defined by the genetic deletion of BAF47 (SMARCB1/SNF5/INI1), a subunit of the BAF chromatin remodeling complex. Loss of BAF47 leads to BAF complex redistribution and overexpression of EZH2, the catalytic subunit of the polycomb repressive complex 2 (PRC2) to favor a proliferative non-differentiated cellular state. Nevertheless, while EZH2 itself is an established, synthetically lethal therapeutic target, other vulnerabilities that result from this imbalance between the BAF and PRC2 complexes are relatively unexplored.

EXPERIMENTAL DESIGN: In a screen of multiple pediatric cancer cell lines, we identified a heightened sensitivity of RT cells to mithramycin (MMA), a small molecule transcription inhibitor. We hypothesized the hypersensitivity of rhabdoid tumor to mithramycin was due to the disruption of BAF and PRC2 dynamics. Here, we characterized the activity of mithramycin in three RT cell lines. To understand the effect of MMA, we performed in vitro qRT-PCR, immunoblot assays, and viability assays to characterize the expression and activity of the PRC2 complex, including a H3K27me3 histone demethylase. We further investigate the kinetics of cell viability and induction of apoptosis with live cell imaging. Finally, we demonstrate these findings in vivo using rhabdoid tumor xenograft models.

RESULTS: Mithramycin inhibited mRNA and protein expression of all four PRC2 subunits in a dose-dependent manner in two different rhabdoid tumor cell lines. Even though EZH2 is suppressed with MMA treatment, we found a striking increase in H3K27me3 that correlated with EZH2 trafficking into the nucleus. This amplification of H3K27me3 preceded suppression of cellular proliferation and induction of apoptosis suggesting H3K27me3 amplification may drive the apoptotic phenotype. In addition, knockdown of KDM6A potentiates the effects of MMA providing further evidence that KDM6A loss and H3K27me3 amplification lead to apoptosis. We are currently working on the mechanism of action for mithramycin in RT cells, including H3K27me3 ChIP-seq and RNA-seq to elucidate global transcription and chromatin changes. We have recapitulated these results in vivo with xenograft mouse models.

CONCLUSIONS: Mithramycin represses KDM6A, a histone demethylase, leading to apoptosis through the amplification of H3K27me3. Here, H3K27me3 amplification, in addition to depletion, is detrimental to rhabdoid tumor proliferation and progression. Overall, this study indicates KDM6A loss and H3K27me3 amplification are novel therapeutic vulnerabilities in rhabdoid tumor.

#4635

AKT/mTOR enhances chemosensitivity in human medulloblastoma cells through induction of necroptosis.

Aisha Naeem, Muhammad U. Choudhry, Varsha Harish, Olga Rodriguez, Chris Albanese. _Georgetown University, Washington DC, DC_.

Medulloblastoma (MB) accounts for almost 25% of child brain tumors. Despite recent advancements in determining genetic and molecular signatures of MB, predictive biomarkers of treatment outcomes remain to be identified. Using publically available databases, we identified p53 expression levels were much higher in desmoplastic and classic MB samples as compared to normal cerebellum. Moreover, many studies have identified p53 immuno-positivity is associated with significantly shorter survival in patients with MB and TP53 mutations were linked with chemotherapy and radiation therapy resistance. We previously reported that in MB cells with higher p53 levels [e.g. D556 (wild type p53) and DAOY (mutant p53)], chemical and genetic silencing of p53 significantly enhanced chemosensitivity and resulted in the more classical chemo-suppressive effect observed in MB cell line (D283) with much lower levels of wild type p53 than D556 or DAOY. Transcriptomic profiling of p53-suppressed D556 cells revealed that enhanced chemosensitivity was associated with the activation of mTOR, PI3K, ErbB2, p70S6k, Tec kinases and P2Y purinergic signaling, and with the inhibition of PTEN signaling and the cyclins. These findings raised the possibility that induction of Akt/mTOR may be involved in p53-mediated chemosensitivity in this subgroup of MB. Importantly, shRNA knockdown of mTOR rescued the D556 cells from the effects of p53 loss, restoring chemoresistance to vincristine and doxorubicin. Conversely, the overexpression of p110α to induce PI3-kinase-dependent signaling, resulted in a twofold increase in drug sensitivity in D556 cells while D283 cells were approximately twofold more chemoresistant. Furthermore, the mTOR inhibitor, RAD001, reversed the chemosensitivity induced by p110α in D556 cells. Using IPA analysis, we identified three potential routes of AKT/mTOR-mediated chemosensitivity in D556 cells; namely, ROS production, activation of phosphatases and/or induction of necroptosis assembly. While our analysis excluded the possibility of ROS-induced cell death, as ROS production was suppressed by p110α, the significant induction of necroptosis-associated RIP kinases and phosphatase PP2A was observed. These findings suggest that in MB cells with high basal p53 levels, p53 suppresses the AKT/mTOR pathway, enhancing chemoresistance and uncover unexpected roles for Akt and mTOR in inducing necroptosis to drive MB cell death. Collectively, our studies indicate that p53 immuno-positivity can serve as a diagnostic marker of de novo drug resistance and can potentially help define therapeutic strategies.

#4636

The antibody-drug conjugate D3-GPC2-PBD potently eradicates neuroblastoma patient-derived xenografts.

Kristopher R. Bosse,1 Zhongyu Zhu,2 Maria Lane,1 Daniel Martinez,1 John DeLong,1 Doncho V. Zhelev,3 Yang Feng,2 Yanping Wang,2 Jennifer Hwang,2 Dimiter S. Dimitrov,3 John Maris1. 1 _Children's Hospital of Philadelphia, Philadelphia, PA;_ 2 _National Cancer Institute, Frederick, MD;_ 3 _University of Pittsburgh School of Medicine, Pittsburgh, PA_.

Background: We recently developed an antibody-drug conjugate (ADC; D3-GPC2-PBD) for neuroblastoma linking a specific glypican 2 (GPC2) targeting antibody (D3) to the potent DNA damaging pyrrolobenzodiazepine (PBD) dimers (Cancer Cell, 2017).

Methods: To determine the spectrum of in vivo efficacy of the D3-GPC2-PBD ADC, we utilized a panel of highly annotated neuroblastoma patient-derived xenografts (PDXs) passaged in C.B-17 scid mice. Cohorts of mice (n = 9 - 10) were stratified to treatment with either vehicle or D3-GPC2-PBD at doses of 0.5 - 3 mg/kg given once or 1 mg/kg given twice weekly x 2 weeks when mean tumor volumes reached 200-300 mm3. Co-treatment with excess D3-GPC2-IgG1, the efficacy of free PBD dimers, re-treatment of the rare ADC treated tumors that regrew, treatment of larger PDXs, and the assessment of DNA damage and apoptosis by immunohistochemistry (IHC) were also explored. PDX GPC2 cell surface expression was quantified by flow cytometry. The binding of D3-GPC2-IgG1 to mouse Gpc2 was tested by flow cytometry and mouse normal tissue Gpc2 expression was profiled utilizing IHC.

Results: We first showed D3-GPC2-IgG1 binds avidly to mouse Gpc2 by flow cytometry and that murine and human GPC2 expression is similarly restricted in normal tissues by IHC. To date, the D3-GPC2-PBD ADC was tested in three neuroblastoma PDXs with a range of GPC2 expression (Molecules of Equivalent Soluble Fluorochrome [MESF]) and genomic characteristics: CHLA79 (GPC2 MESF = 825, MYCN non-amplified, ALK wild-type, TP53 wild-type), COG-N-421x (GPC2 MESF = 3301, MYCN amplified, ALK wild-type, TP53 wild-type), and SKNAS (GPC2 MESF = 672, MYCN non-amplified, ALK wild-type, TP53 mutated). Treatment with D3-GPC2-PBD resulted in complete and sustained tumor regression up to 100 days in 61% (17/28) of mice treated with 1 mg/kg ADC, 96% (27/28) of mice treated with 3 mg/kg ADC, and 95% (18/19) of mice treated with 1 mg/kg ADC given twice weekly x 2 weeks. Treatment of the CHLA79 PDX with an equivalent dose of free PBD dimers had no effect on tumor growth and co-treatment with excess D3-GPC2-IgG1 (50x) abrogated a significant amount of the ADC induced tumor regression. The rare 1 mg/kg ADC treated CHLA79 PDXs that began to regrow after 100 days (n = 2) showed complete responses to re-dosing of the ADC. Additionally, the D3-GPC2-PBD ADC dosed at 1 mg/kg ADC given twice weekly x 2 weeks induced complete regression of larger COG-N-421x PDXs (tumor volumes of 600-1000 mm3). ADC treated tumors showed significant upregulation of DNA damage (γH2AX) and apoptosis (cleaved caspase 3 and cleaved PARP) by IHC. ADC treatment was well tolerated with no discernible drug-related toxicities.

Conclusions: The D3-GPC2-PBD ADC is safe and potently efficacious in a genomically diverse panel of neuroblastoma PDXs with a range of GPC2 cell surface expression. These data support the development of a PBD dimer containing GPC2 directed ADC.

#4637

Tumor treating fields (TTFields) have antiproliferative effects on high-grade pediatric brain tumor cell lines.

Joshua Branter,1 Maria Estevez-Cebrero,1 Richard Grundy,1 Surajit Basu,2 Stuart Smith1. 1 _University of Nottingham, Nottingham, United Kingdom;_ 2 _Queen's Medical Centre, Nottingham, United Kingdom_.

Introduction Approximately 400 children in the UK are diagnosed with a CNS tumor each year, which represents a quarter of childhood cancer cases in the UK. The 5-year survival rate of paediatric brain tumor patients is approximately 75%, but survival remains much worse for some brain tumor patients, particularly high-grade gliomas. Pre-clinical and clinical studies have demonstrated efficacy of TTFields as a treatment for high-grade glioma leading to the approval of TTFields for adult Glioblastoma multiforme (GBM) patients. The lack of overlapping toxicities associated with TTFields has made electric field therapy an attractive treatment strategy for poor prognosis paediatric brain tumors.Methods The inovitro is the laboratory based testing system used to deliver TTFields to cell cultures. inovitro was used to deliver TTFields over 72 hours at a range of clinically relevant frequencies (100-400kHz) to our panel of paediatric GBM, Medulloblastoma and Ependymoma cell lines. The effects of TTFields on cell viability and cell cycle was assessed using Resazurin based metabolic viability tests and flow cytometry. The determined optimal frequency for each cell line was also combined with a mitotic inhibitor to explore synergistic effects. Gene expression changes of TTFields treated cells was investigated. Results TTFields treatment reduces cell viability of all cell lines tested within the frequency panel. The optimum frequencies were between 100 and 400kHz for all cell lines tested. TTFields treatment had effects on cell cycling with increased G2 accumulation of treated cells being observed. Combining TTFields with mitotic inhibitors increased efficacy in an additive fashion. Conclusions This preliminary study has demonstrated efficacy of TTFields on paediatric brain tumor cell lines. The optimal frequencies for all the paediatric GBM cell lines used did not match the 200kHz as previously reported for U-87 MG cells. This report warrants further study into the potential for TTFields therapy for paediatric brain tumor patients.

## CANCER CHEMISTRY:

### Drug Discovery Tools

#4638

Predictive computational approaches to guide the covalent targeting of cysteine residues in drug discovery.

Richard A. Ward, Richard Lonsdale. _AstraZeneca, Cambridge, United Kingdom_.

Targeted covalent inhibition is an established approach for increasing the potency and selectivity of potential drug candidates, and has also been used to identify tool compounds for target validation studies. It is evident that binding to reversible recognition elements within the pocket is essential for selective covalent inhibition, but this must also be achieved with the appropriate level of inherent reactivity of the covalent functionality and compatible geometry and reactivity of the target amino acid. We have assessed the ability of several experimental and computational approaches to predict the intrinsic reactivity of a range of cysteine targeting warheads, some of which can be used in prospective compound design. In addition, molecular dynamics (MD) simulations in combination with pKa prediction methods have been used to investigate the accessibility and potential reactivity of target cysteines towards covalent inhibition. As part of this work a number of case studies will be reported including retrospective examples from covalent drug discovery efforts along with prospective use towards novel targets. Finally, a dataset of osimertinib analogues along with other EGFR inhibitors of varying reactivity will be discussed to enable further insight into the impact of reversible and covalent interactions on inhibitor binding.

#4639

Spherical nucleic acids induce an antitumor immune response against triple-negative breast cancer.

Lisa E. Cole, Dai Horiuchi, Chad A. Mirkin. _Northwestern University, Evanston, IL_.

Triple-negative breast cancer (TNBC) represents a breast cancer subtype with some of the poorest outcomes and is defined as tumors that lack the functional expression of estrogen and progesterone receptors and human epidermal growth factor receptor 2. TNBC has a high rate of reccurrence and therefore new therapies are needed for this disease. Recent clinical data have identified a correlation between intratumoral T cells and overall survival, suggesting the use of immunotherapy as a viable option for treating TNBC tumors. Spherical nucleic acids (SNAs) are a new class of molecules that can modulate the immune system through molecular interactions with immune cells. Immune-stimulatory SNAs consist of a dense shell of nucleic acids having multiple cytosine (C) and guanine (G) nucleotide repeats (CpG) that are radially oriented and attached to a liposomal nanoparticle core. These CpG motifs are recognized by Toll-like receptor 9 (TLR9) and used to induce an innate immune response. TNBC-specific antigens were encapsulated within the liposomal core to direct the antitumor immune response against TNBC cells. The SNAs were tested in a 4T1 syngeneic tumor mouse model for their ability to activate the immune system and initiate an antitumor response against TNBC mammary tumors. The SNA architecture increased the co-delivery of the TLR9 activating nucleic acids and antigens into the same dendritic cell by 50% compared with delivering the nucleic acids and antigens as free entities. SNAs were retained in the tumor-draining lymph node 24 hours after a subcutaneous injection whereas the linear nucleic acids were cleared by 1 hour post-injection. Tumor-bearing mice were treated on day 6, 11, and 15 after tumor initiation with a 50 µM dose of SNAs and the primary tumor volume was measured until day 28. Treatment with SNAs enabled a 30% reduction in the final tumor volume compared with untreated tumor-bearing animals. Flow cytometric analysis of the primary tumors showed an increase in the intratumoral T-cell lymphocyte population and a decrease in the myeloid-derived suppressive cell (MDSC) population in the SNA treated group compared with the untreated group, suggesting treatment with immune-stimulatory SNAs can alter the immunosuppressive microenvironment of TNBC tumors. In addition, T cells from lymph nodes of the SNA treated group secreted more IFN-gamma after ex vivo stimulation with 4T1 lysates, indicating an antigen-specific response was generated with delivery of TNBC-specific antigens encapsulated within the SNA core. Lastly, treatment with SNAs greatly reduced the number of lung metastases in these animals. Future studies are aimed at probing how SNAs are reducing the metastatic burden and ways to increase potency against the primary tumor through potential combination therapies.

#4640

Sustained co-delivery of dendritic cells and oncolytic adenovirus co-expressing IL-12 and GM-CSF using biodegradable polymer matrix for cancer immunotherapy.

Eonju Oh,1 Jung-Eun Oh,1 JinWoo Hong,1 YoonHo Chung,2 Yunki Lee,3 Ki Dong Park,3 Sung Wan Kim,4 Chae-Ok Yun1. 1 _Hanyang Univ., Seoul, Republic of Korea;_ 2 _Washington University, MO;_ 3 _Ajou University, Republic of Korea;_ 4 _University of Utah, UT_.

Administration of dendritic cells (DCs) combined with oncolytic adenovirus (Ad) expressing antitumor cytokines induces a potent antitumor effect and antitumor immunity by ameliorating the immunosuppressive tumor microenvironment. However, this combination therapy has significant limitations due to rapid dissemination and inactivation of the therapeutics at the tumor site, necessitating multiple injections of both therapeutics. In this study, we investigated injectable and biodegradable gelatin-based hydrogel as a carrier for oncolytic Ad co-expressing interleukin (IL)-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (oAd) and DCs to elicit a potent antitumor effect with single administration. Gel matrix enabled sustained release of both oAd and DCs while preserving their biological activity over a considerable time period, leading to efficient retention of both therapeutics in tumor tissue. Further, tumors treated with oAd- and DC-loaded gel (oAd+DC/gel) showed a significantly greater expression level of IL-12, GM-CSF, and interferon-γ (IFN-γ) than either single treatment (oAd or DC) or oAd in combination with DC (oAd+DC), resulting in efficient activation of both endogenous and exogenous DCs, migration of DCs to draining lymph nodes, and tumor infiltration of CD4+ and CD8+ T cells. Moreover, oAd+DC/gel resulted in a significantly higher number of tumor-specific IFN-γ-secreting immune cells compared with oAd+DC. Lastly, oAd+DC/gel significantly attenuated tumor-mediated thymic atrophy, which is associated with immunosuppression in the tumor microenvironment, compared with oAd+DC. Taken together, these results demonstrate that gelatin gel-mediated co-delivery of oncolytic Ad and DCs might be a promising strategy to efficiently retain both therapeutics in tumor tissue and induce a potent antitumor immune response for an extended time period via a single administration.

#4641

Improved chemotherapeutic delivery to brain metastases with targeted nanoparticles in preclinical breast cancer brain metastasis models.

Emily A. Wyatt, Mark E. Davis. _California Institute Of Technology, Pasadena, CA_.

Brain metastases present an increasing challenge to long-term survival for HER2-amplified breast cancer patients. While HER2 targeted therapies effectively control systemic metastases, they are largely ineffective against brain metastases due to their inability to cross the blood-brain barrier. We have recently demonstrated that brain uptake of targeted nanoparticles can be increased by adding an acid-cleavable linkage between transferrin (Tf) and the nanoparticle core [1]. Our lab has also developed a method for assembling targeted nanoparticles by complexing diol-containing polymer-drug conjugates and boronic acid-containing targeting agents [2]. Here, we report the first efficacy results of targeted nanoparticles formed via this assembly strategy in a preclinical model of breast cancer brain metastasis. Female Rag2-/-;Il2rg-/- mice were intracranially injected with human HER2-amplified BT-474 breast cancer cells, and formation of brain tumors was monitored by MRI. Mice were randomized into four groups of six mice per group (targeted nanoparticle, non-targeted nanoparticle, free drug, and saline). Targeted and non-targeted nanoparticles were prepared by functionalizing nanoparticle cores consisting of a mucic acid polymer conjugate of camptothecin (CPT) with either Tf-PEG or methoxy-PEG, respectively, using boronic acid-diol chemistry. The different formulations were administered intravenously once per week for four weeks at a dose of 4 mg/kg (CPT basis), and tumor volume was measured weekly by MRI. Targeted nanoparticles significantly reduced tumor growth in mice bearing intracranially implanted tumors compared to saline, free drug, and non-targeted nanoparticle controls. However, free drug and non-targeted nanoparticles also reduced tumor growth compared to saline, albeit to a lesser extent. Taken together, the results are highly suggestive that there isn't a completely intact blood-brain or blood-tumor barrier, and furthermore that artificial transport pathways may be introduced following intracranial implantation. To overcome limitations of the intracranial injection model, female Rag2-/-;Il2rg-/- mice were intracardiacly or intravenously (tail vein) injected with human HER2-amplified BT-474 breast cancer cells, and formation of metastatic brain tumors was monitored by MRI. Unlike brain tumors from intracranially injected cells, brain tumors formed from intravenous cell injections were not affected by systemic doses of either lapatinib or trastuzumab. Current investigations involve collection of efficacy data for the different formulations in the intracardiac and intravenous models that may provide further insight into the blood-brain barrier integrity and nanoparticle delivery. [1] Clark, AJ, Davis, ME, PNAS, 112 (2015) 12486-12491. [2] Han, H, Davis, ME, Mol. Pharmaceutics, 10 (2013) 2558-2567.

#4642

Personalized cancer-specific protein corona affects the therapeutic impact of nanoparticles.

Claudia Corbo, Morteza Mahmoudi, Omid C. Farokhzad. _Brigham and Women's Hospital, Harvard Medical School, Boston, MA_.

Despite recent progress in nanomedicine, there is still a lack of efficient nanotherapeutics for cancer treatment. When nanoparticles (NPs) are injected, they circulate the bloodstream and interact with biomolecules, creating a biomolecular shell. This shell of biomolecules surrounding the NPs consists mostly of proteins and is referred to as the protein corona (PC). The PC composition depends on various factors, including, but not limited to i) surface properties of NPs (e.g. surface chemistry and charge), and ii) experimental parameters (e.g. incubation time, pH, temperature). The impact of these factors on the formation of the PC has been widely investigated and is nowadays well known. However, the effects of the biological milieu (e.g. patient's health status, plasma vs serum) on the PC formation and composition have been underexamined and still need to be deeply clarified. Recently, it has been demonstrated that the protein pattern constituting the PC of NPs exposed to plasma of cancer patients is different than that of the PC formed by exposure of NPs to healthy plasma. These variations can be miniscule but crucial in the fate of NPs. In fact, NPs surrounded by a PC lose their synthetic identity and acquire a new biological identity responsible for their biological destiny (cell targeting, accumulation, immune response). In this work, we have studied the PCs formed around NPs using plasmas of patients with one of eight cancers to gain insights into the cancer-related variations of the PC composition and into their potential effects on the therapeutic efficacy of NPs. Our results confirmed that the same NPs incubated with plasmas of patients affected by different tumors have distinct PCs. Overall, this is the first wide study unveiling the specific PCs formed around NPs using plasmas of eight cancers. This comprehensive report acts as a tool for researchers in nanomedicine to design personalized nanotherapeutics in a cancer-specific manner for clinical applications. Methods: Silica NPs were incubated with plasma of healthy subjects and cancer patients. NPs in the study were 100 nm in size and the PCs in each subject's plasma were characterized and compared using SDS-PAGE and LC-MS/MS. Plasmas of patients with one of 8 cancers have been employed: breast, rectum, lung, kidney, thyroid, uterine, bladder, ovary.

#4643

Drug-Target Explorer: An interactive tool for examining chemical-biological interactions.

Robert J. Allaway,1 Sara J. Gosline,1 Marco Nievo,2 Salvatore La Rosa,2 Annette Bakker,2 Justin Guinney1. 1 _Sage Bionetworks, Seattle, WA;_ 2 _Children's Tumor Foundation, New York, NY_.

Background: Phenotypic high-throughput screens are often utilized in modern oncology drug discovery pipelines. Such screens are conducted with an array of molecules (panels of oncology drugs, drugs for other indications, and compounds with possibly unknown bioactivity), ultimately measuring a biological change within a disease model. For example, a screen might test drugs in NF1 wild-type and mutant cancer cell lines to find molecules that are selectively toxic to NF1 mutant cells. While these screens can yield valuable hits, they also present the challenge of identifying the target(s) that mediate the effect seen in the screen for several reasons. For example, many drugs have a polypharmacologic target profile making it hard to identify which targets are biologically relevant for a given drug. This can cause a single drug to be treated as a specific inhibitor of different targets by different researchers. Furthermore, some libraries contain molecules for which drug-target studies have never been conducted. To address these challenges, we developed an application that enables exploration of the chemical-biological interaction space. Methods: Data from ChEMBL, PubChem, DrugBank, and the Drug-Gene Interaction Database (DGIdb) for over 7000 small molecules were downloaded. Drug-target interaction data points as well as evidence to the contrary were quantified for each interaction. In addition, when quantitative data were obtained, median IC50s were calculated. Each molecule was annotated with a name and chemical structure, and every target was annotated with protein & gene identifiers. To enable exploration of this database, an interactive web interface was developed using the R Shiny platform and cheminformatics R packages (rcdk, fingerprint, webchem).Results: The app enables the end user with a specific query molecule to search a database of experimentally-derived drug-target interactions. The database can be queried using drug names or structures. A similarity parameter allows the user to expand their search to other structurally related molecules. The structure of the query molecule is compared to every database molecule, and structurally similar molecules and targets are presented in interactive tabular and network-based forms for in-depth exploration. The app also performs enrichment analysis on the target lists, and allows the user to evaluate structure-activity relationships for drug response data using publicly-available in vitro datasets. Finally, if a user has a target of interest, they can search for molecules that bind that target and explore the resulting data interactively. Conclusions: This app provides a multifunctional platform for exploring chemical space as it relates to biological targets, and may be useful at several steps along the oncology drug development pipeline including target discovery, structure-activity relationship, and lead identification studies.

#4644

Patient-derived 3D tumor cultures for clinical diagnostics and pre-clinical drug development.

Thomas Dijkmans,1 Sander Basten,1 Bram Herpers,1 Kuan Yan,1 Torsten Giesemann,2 Julia Schueler,2 Willemijn Vader,3 Leo Price1. 1 _OcellO BV, Leiden, Netherlands;_ 2 _Charles River, Freiburg, Germany;_ 3 _Vitroscan BV, Leiden, Netherlands_.

Ex vivo cultures of patient-derived tumor enable functional testing of treatment options and optimization of pre-clinical drug development. Patient biopsies or tumor from patient-derived xenografts (PDX) mouse models are a valuable source of human tumor material that can be cultured in 3D for screening of drug efficacy, drug resistance and cellular processes such as proliferation, survival and invasion. A high throughput approach using 384 well plates enables evaluation of multiple drug treatments and dose ranges in parallel. After compound exposure, cultures are fixed and stained with cellular markers. 3D image stack acquisition is followed by ultra-high content multiparametric analysis using the OMiner platform to profile drug responses and quantify tumor spheroid volume, apoptosis and tumor invasion.

Dissected PDX material and tumor biopsy material, were used to establish 3D tumor cultures derived from various indications, including breast, ovarian, cervix, endometrium, stomach, pancreatic, colon, bladder and lung cancer. These were exposed to standard-of-care chemotherapeutics (e.g. 5-FU, taxanes, platinum compounds, anthracyclines, alkylating agents), small molecules (e.g. erlotinib, lapatinib, trametinib), targeted therapies (PARPi; niraparib, olaparib, rucaparib), antibodies (e.g. cetuximab, trastuzumab) and antibody-drug-conjugate (ADC, T-DM1) dose ranges. The tumor culture response was measured, generating dose-dependent profiles for relevant features.

Patient-derived 3D tumor cultures were tested with standard-of-care and novel therapeutic agents and high content analysis was used to evaluate drug sensitivity. This method enables both the in vitro selection of drug candidates in a pre-clinical setting as well as efficient selection of PDX models for in vivo follow-up in the same tumor. This highly translational in vitro-in vivo PDX pipeline is expected to reduce attrition and increase efficiency in early drug-discovery. Correlation of drug sensitivity in 3D cultures from fresh patient tumor biopsies, on the other hand, can be used for development of predictive diagnostics and also provides a unique source of patient material for drug discovery and development.

#4645

Identifying clinically viable targeted therapies for patients with triple negative breast cancer.

Ratika Kunder,1 Sara Fernandez Dunne,2 Matt Clutter,2 Marc L. Mendillo,1 Dai Horiuchi1. 1 _Northwestern Univ. Feinberg School of Medicine, Chicago, IL;_ 2 _Northwestern Univ., Evanston, IL_.

Triple-negative breast cancer (TNBC), which lacks functional expression of the estrogen and progesterone receptors, and which does not overexpress human epidermal growth factor receptor 2 (HER2), represents one of the breast cancer subtypes with the poorest clinical outcome. No molecularly targeted therapy has been FDA-approved for clinical use against this subtype due to lack of validated molecular targets. We previously found that the MYC oncoprotein was disproportionally activated in ~50% of TNBC compared with receptor-positive (RP) tumors. MYC is a pleiotropic transcription factor, and clinical development of rationally designed small molecule inhibitors that directly inhibit its activity has remained challenging. An alternative approach to selectively kill MYC-driven tumors is to inhibit proteins required for tumor viability that are not essential in healthy cells. This form of indirect treatment strategy has become known as the "synthetic lethal" approach. Our effort to identify such MYC-dependent synthetic lethality recently identified PIM1, a non-essential kinase. We found that PIM1 expression was elevated in TNBC clinical samples and was associated with poor clinical outcomes specifically in patients with receptor-negative tumors. Preclinical PIM inhibitors showed activity in human TNBC with elevated MYC expression in a number of preclinical models. However, we also observed that PIM inhibitors induced only modest tumor cell death and regression in patient-derived tumor xenograft models and a MYC-driven transgenic mouse model of TNBC, suggesting the need for combination therapies. We have conducted robust, targeted drug combination screens in chemo-resistant, MYC-overexpressing TNBC cell lines in Matrigel-assisted three-dimensional (3D) culture system, which has resulted in the identification of some clinically viable combination therapies based on PIM kinase inhibition. We will describe the results from the 3D Matrigel screens and the subsequent proof-of-concept and mechanism of drug action studies.

#4646

**Pharmacogenomic screen identifies** KMT2D **mutations as a biomarker of sensitivity to Aurora kinase inhibition in head and neck and cervical squamous cell carcinoma.**

Tuhina Mazumdar,1 Nene N. Kalu,2 Shaohua Peng,1 Pan Tong,1 Li Shen,1 Jing Wang,1 Jeffrey N. Myers,1 Curtis R. Pickering,1 David Brunell,3 Clifford C. Stephan,3 Faye M. Johnson1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Lonza Viral Therapy, Houston, TX;_ 3 _Texas A &M Institute of Biosciences and Technology, Houston, TX_.

Purpose. To address the unmet need for biomarker-driven, effective, targeted therapy for human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC) and cervical epithelial squamous cell carcinoma (CESC), we conducted a high-throughput drug screen (HTDS) using 1122 compounds in all readily available HPV-positive HNSCC and CESC cell lines and an equal number of matched HPV-negative lines.

Methods. Cells were incubated in drug concentrations ranging from 0.01 μM to 3.16 μM for 72 h, fixed and stained with DAPI, and counted. Of the 1122 analyzable compounds, 865 unique drugs were tested because of overlap. All drugs were assigned to one of 36 classes based on their primary targets. Drug concentrations resulting in a 50% reduction in cell proliferation (GI50) and the area under the dose response curve were calculated. Two biological replicates were performed for all cell lines on separate days and at least 1 week apart.

Results. The HTDS was conducted using 24 cell lines. We identified 493 highly effective compounds, which we defined as those with GI50 values less than 0.5 μM in 2 or more of the cell lines screened. The most effective drug classes were inhibitors of polo-like kinase, proteasomes, histone deacetylase, and Aurora kinases. Of the 19 Aurora kinase inhibitors tested, 18 were highly effective. We confirmed the efficacy of 3 Aurora kinase inhibitors using colony formation assays in 15 cell lines. Treatment with a dual Aurora A/B inhibitor, danusertib, led to G2M arrest and apoptosis in all 6 tested cell lines. Additionally, danusertib treatment decreased tumor size compared to controls in patient-derived xenograft mouse models of HNSCC. To identify biomarkers predicting response to Aurora kinase inhibitors, we tested for associations between mutations in the cell lines and sensitivity to the Aurora kinase inhibitors using whole exome mutation data for the 50 most common driver mutations in HNSCC. To validate our findings in an independent dataset, we queried the Genomics of Drug Sensitivity in Cancer database. In both data sets, cancer cell lines with KMT2D (MLL2) mutations were more sensitive to Aurora kinase inhibitors than cells without mutations. KMT2D mutations are inactivating; experiments to knock down KMT2D in wild-type cell lines and assess sensitivity to Aurora kinase inhibitors are ongoing.

Conclusions. We identified Aurora kinase inhibitors as effective and understudied drugs in HNSCC and CESC. These drugs cause apoptosis and cell cycle arrest in vitro and decrease tumor size in vivo. This is the first published study to demonstrate that mutations in KMT2D (MLL2), which are common in many cancers (16% HNSCC, 12% CESC), correlate with drug sensitivity in 2 independent data sets.

#4647

Identifying drug targets in sarcoma using machine learning and cell phenotype-based compound screening.

Eric J. Lachacz,1 Zhi Fen Wu,1 John L. Bixby,2 Vance P. Lemmon,2 Sofia D. Merajver,1 Hassan Al-Ali,2 Matthew B. Soellner1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _University of Miami, Miami, FL_.

High-throughput phenotypic screens that incorporate compound biochemical activity annotations are positioned for novel target discovery in cancer. We used machine learning approaches to correlate kinome-wide profiling data of a collection of kinase inhibitors with phenotypic cell proliferation data for the same compounds. We assembled a library of profiled kinase inhibitors with diverse chemotypes and kinome-wide selectivities and determined their anti-proliferative activities in a panel of sarcoma cell lines. Using a previously published machine learning algorithm, we related the compound inhibition profiles across 237 kinases to their abilities to inhibit cell proliferation. This identified Protein Kinase D (PRKD) as a putative novel target kinase selectively in synovial sarcoma cell lines, such that its inhibition leads to a decrease in proliferation in these cells. Next, we reasoned that our approach could be leveraged to identify targets that, when co-inhibited, induce a synergistic phenotype. This would enable rational design of drug combinations for further testing as opposed to the labor-intensive, random, pairwise testing commonly performed. To identify targets synergistic with PRKD, we performed a screen of a synovial sarcoma cell line in the presence of a selective PRKD inhibitor. Several kinases became prioritized as new targets in this "synergy screen." Combining selective inhibitors for each synergistic target, as defined by Chou-Talalay, (here, CDK and AKT) with PRKD inhibitors synergistically reduced synovial sarcoma cell proliferation. Conversely, combining PRKD inhibitors with a selective inhibitor of a kinase that was deprioritized in the synergy screen (p38 MAPK) resulted in non-synergistic or even antagonistic effects. Overall, our approach provides a promising framework to identify new targets of cancer subtypes and a novel methodology to identify new combinational strategies for treatment.

#4648

Using the IntelliCyt iQue Screener to simultaneously profile antibody dependent cell cytotoxicity and cytokine release of therapeutic antibodies.

John O'Rourke. _IntelliCyt, Albuquerque, NM_.

Therapeutic antibodies can mediate cancer cell killing through a variety of mechanisms including antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and complement dependent killing (CDC). ADCC is induced through the binding of the antibody Fc region to Fc receptors (FcRs) expressed on effector cells of the immune system. Granule exocytosis of perforin, granzyme B, the induction of Fas ligand (FasL) expression on immune cells and the release of cytokines are major mechanisms involved in targeted cancer cell killing.

Traditional approaches to screen for antibody Fc effector functions focus on ADCC using a homogenous live/dead readout, which greatly limits the contextual and correlative value of the screening data. To overcome this limitation, a multiplex screening assay profiling ADCC using multiple cell death endpoints, and quantitating secreted proteins /cytokines was developed and analyzed using Intellicyt's iQue Screener PLUS and integrated ForeCyt software. The iQue Screener Plus is a high throughput flow cytometry platform featuring 3 lasers with 13 fluorescent channels and can sample a 384-well plate in 20 minutes.

As a proof of concept, a small set of therapeutic antibodies directed against the same tumor antigen were used to induce immune cell-mediated killing of tumor cells using different effector to target cell ratios and a range of antibody concentrations. To demonstrate antibody specificity, antigen positive target cells, negative control target cells and effector immune cells were barcoded with different encoding dyes and included in the same well. At specific times, a small aliquot from each well was transferred to a new assay plate to run a multiplexed cell/bead mixture assay by the addition of a cocktail of fluorescent immunophenotyping antibodies, fluorescent dyes measuring unique apoptosis parameters, and a panel of QBeads for secreted protein detection. The samples were assayed on the IntelliCyt iQue Screener PLUS with simultaneous data analysis including multi-plate analysis for cell killing kinetics. Positive/negative target cells and immune cells were digitally segregated by differently encoded fluorescence. Time-dependent cell killing of antigen positive target cells were observed using the apoptosis markers mitochondria depolarization staining and cell membrane integrity, whereas little killing was measured in antigen negative cells. The antibody set showed a range of ADCC mediated killing and granule exocytosis/ cytokine release suggesting differences in Fc effector functions. These results highlight the streamlined workflow on IntelliCyt iQue Screener PLUS platform to profile therapeutic antibodies in a functional ADCC/cytokine release assay predicting their clinical efficacy.

#4649

Targeting the cancer cell lysosome with transformable self-assembling peptide nanoparticles.

Christopher M. Baehr,1 Lei Wang,2 Kit S. Lam1. 1 _The University of California, Davis, Davis, CA;_ 2 _National Center for Nanoscience and Technology, Beijing, China_.

Lysosome-mediated cell death has recently become of great interest to the cancer community as several compounds have been shown to preferentially induce cancer cell death via lysosomal membrane permeabilization (LMP) (e.g., oleocanthal, salinomycin, and TiO2 nanoparticles). Further, many cancer cell lines that are resistant to traditional cancer therapeutics are highly susceptible to lysosome-mediated cell death. Here, we develop transformable peptide nanoparticles (NP) designed to traffic to cancer cell lysosomes, form aggregates, and induce LMP. The peptide consists of three motifs: 1) a hydrophilic, cell penetrating, and pH-responsive poly-d-Arg motif, 2) a hydrogen-bonding motif derived from beta amyloid (KLVFF); and 3) hydrophobic bis(pyrene) that fluoresces upon aggregation. The peptides initially self-assemble into NPs (50nm via TEM) in PBS, but when exposed to a lysosomal pH of 4.5, form much larger aggregates. We show that these nanoaggregates colocalize with the lysosome, leading to cell death, suggesting the induction of LMP. Moreover, utilizing bis(pyrene) we show that while these aggregates form in the lung carcinoma cell line, A549, they do not form in 3T3 fibroblasts. Additionally, we demonstrate that these nanoaggregates are highly toxic to A549 cells with IC50 in the low micromolar range (~2μM). This offers a novel organelle-specific strategy to preferentially target and kill cancer cells.

#4650

Quantitative assessment of the intracellular uptake of chlorotoxin in a U87 human glioma mouse model for the targeted drug delivery system.

Alan D. Roberts,1 Joseph O. Johnson,2 Aleksandra Karolak,2 Norma Alcantar,3 Katarzyna A. Rejniak,2 Marzenna Wiranowska1. 1 _USF Morsani College of Medicine, Tampa, FL;_ 2 _Moffitt Cancer Center and Research Institute, Tampa, FL;_ 3 _USF College of Engineering, Tampa, FL_.

This study quantitatively evaluates the tumor cell-targeting capabilities and intracellular uptake pattern of chlorotoxin (CTX) when delivered intratumorally (IT) and intravenously (IV) using subcutaneous (SC) U87 human glioblastoma mouse model. CTX is a scorpion-derived polypeptide that selectively binds to tumor cells of neuroectodermal origin, such as glioma and a wide range of other tumor cells, but not normal cells. The quantification of CTX cellular uptake presented here will assist in the further development of our tumor-targeting drug delivery system (DDS) consisting of a chemotherapeutic encapsulated in nonionic surfactant vesicles embedded in a thermosensitive cross-linked chitosan hydrogel, which has affinity for MUC1 receptor overexpressed on many tumor cells, including low-differentiated glioma. CTX addition to the DDS will serve as a second tumor-targeting molecule. In this study, U87 human glioma cells were implanted SC in the flank of nude athymic/ncr/nus mice. When tumors reached approximately the volume of 0.25 cm3, mice were injected either IT or IV (tail vein) with 50 μl of 100 μg CTX or phosphate buffer saline (PBS) as a control. The tumors were harvested at 5 minutes post IT injection or 7 hours post IV injection, fixed, sectioned at 5 μm, and followed by either immunofluorescence or immunocytochemistry using anti-CTX primary antibody and AlexaFluor-594 conjugated secondary antibody or immunoperoxidase- AEC kit, and evaluated by confocal or scanning microscopy, respectively. CTX levels were compared between three different tumor regions (A, B, and C; A = injection site marked by methylene blue dye, C = most distant from injection site, B = equidistant between these two) after direct IT injection and compared to CTX levels in tumor tissues following IV injection. Quantitative evaluation of CTX levels was performed with ImageJ software, while further intracellular analysis of CTX presence utilized MATLAB system. CTX was found to be evenly distributed between cells within each region post IT injection, but decreased in intensity from the injection site (A) toward the tumor edge. With region A set at 100% CTX intensity, the levels decreased to 23.7% (in region B), and 2.1% (in region C), compared to 1.8% post IV injection. Our previous in vitro studies showed CTX localization near the nucleus in U87 glioma cells but not in normal human astrocytes. Current evaluation of tumor z-sections quantified levels of CTX inside U87 glioma cells in vivo. These results confirm the specificity of CTX uptake by U87 human glioma cells in vitro and in vivo with CTX tissue levels post IV injection, which is comparable to those post IT injection in the distant C tumor region. This proves CTX is an important second tumor-targeting molecule for our DDS system.

#4651

Development of a targeted NGS panel for solid tumor actionable gene targets using multiplex PCR-based enrichment in an integrated fluidic circuit.

Peilin Chen, Jaibiao Gong, David Wang, Devin Do, Lianne McLean, Tom Goralski. _Fluidigm, South San Francisco, CA_.

Introduction: Next-generation sequencing (NGS) has been being rapidly adopted in clinical research to align actionable variants in tumors to targeted therapies or clinical trials. Comprehensive NGS-based tumor profiling assays are an efficient and effective method to characterize a variety of clinically relevant somatic mutations. We have developed a targeted NGS cancer gene panel that focuses on actionable gene mutations and employs a NGS library preparation method based on multiplex PCR enrichment in an integrated fluidic circuit (IFC) and the automated JunoTM System. Methods: 53 actionable cancer genes were selected based on both clinical and research knowledge that covers 15 solid tumor types. Assays for the selected regions of the 53 genes were designed by an internal assay design pipeline. Multiplex PCR using target-specific primers is conducted in an LP—48.48 IFC on the Fluidigm Juno™ system, where up to 48 DNA samples can be processed simultaneously. A unique barcode is incorporated in one of the PCR primers to distinguish individual samples. To evaluate the assay performance, three sets of cell line gDNA samples were identified from commercial sources: set 1: 22 commercial reference samples, set 2: 12 samples for single nucleotide variants (SNVs) and indels, and set 3: 12 samples for copy number variations (CNVs). Each sample was tested on the IFC in 4 replicates. PCR products harvested from the IFC were pooled and purified with Agencourt® AMPure® XP magnetic beads. A second PCR step using a universal primer pair was performed to add sequencing adapters. After a second purification, the DNA library was sequenced on a NextSeq™ 500 system. The data analysis was performed by a service provider partner (GenomOncology). Results: A total of 1,508 primer pairs with an average insert size of 155 bp were selected to cover SNVs, indels, and CNVs. The assays were separated into 44 pools to minimize the interaction between assay primers and improve performance. The percentage of reads mapped to the genome was 98.9%, and the percentage of reads that mapped to amplicons was 96.8%. Mutation detection sensitivity was 4% variant allele frequency (VAF.) In 47 selected samples, the total SNVs, CNVs and indels represented are 182, 154 and 28, respectively. For SNVs, the positive percent agreement (PPA) is 1.0 and positive predictive value (PPV) is 0.974. For CNVs, the PPA is 1.0 and PPV is 0.969 and for indels, the PPA and PPV are 1.0 and 0.966, respectively. Overall concordance is 0.99. Conclusions: A targeted NGS cancer panel employing PCR-based enrichment on an IFC has been developed and yielded high quality of libraries generated on the JunoTM system for detecting SNVs, indels and CNV in solid tumor samples. This panel covers actionable targets in 53 cancer genes and utilizes a microfluidic device to provide a simple streamlined workflow for library preparation of up to 48 DNA samples per IFC.

#4652

Targeted nanomedicines improve therapeutic indices of RAF and MEK inhibitors.

Daniel A. Heller, Yosi Shamay. _Memorial Sloan Kettering Cancer Center, New York, NY_.

Potent kinase inhibitors have been developed to treat tumors, but they suppress signaling in normal cells so that their dosing and ability to inhibit the pathway are limited by toxicity, resulting in a narrow therapeutic index. We developed a novel drug delivery strategy which efficiently encapsulates and targets kinase inhibitors to the tumor microenvironment while obviating the sources of dose-limiting toxicities. The platform employs nanoparticle scaffolds that we modified to target caveolae and P-selectin, that can both be overexpressed on tumor vasculature. We assessed the targeted drug delivery properties of the nanoparticles, encapsulating the RAF inhibitor sorafenib and MEK inhibitor trametinib. Strikingly, we found selective tumor uptake and exceptional net anti-tumor efficacies in patient-derived xenografts and genetically modified mouse models. The nanomedicine strategy also prevented the inhibition of ERK phosphorylation in the skin, demonstrating that this targeting strategy may abrogate dose-limiting toxicities of kinase inhibitors to improve their therapeutic indices.

#4653

Intracellular Delivery of SOCS3 Suppresses Pancreatic Cancer Progression.

Kuysook Lee, Seulmee Shin, Yunseo Hwang, Youngsil Choi, Daewoong Jo. _Cellivery Therapeutics, Inc., seoul, Republic of Korea_.

Background: JAK/STAT signaling, regulated by SOCS family proteins, plays a role in the progression of the inflammation, growth and metastasis of pancreatic adenocarcinoma. SOCS3 suppresses JAK kinase activity and degrades the activated cytokine receptor complex, and its loss contributes to pancreatic cancer development. We have developed advanced macromolecule transduction domains (aMTDs) to deliver recombinant proteins into to cells and tissues. In principle, aMTD enabled SOCS3 could provide an effective treatment for pancreatic tumors that depends on JAK/STAT signaling for growth or survival.

Objective: The present study utilized aMTD sequences to develop improved cell-permeable (iCP-) SOCS3 as a protein-based biotherapeutic against pancreatic cancer.

Methods: Improved cell-permeable SOCS3 proteins, with and without a 12-amino acid aMTD sequence, were expressed at high yields in E. coli. The proteins also contained a solubilization domain (SD) to enhance the solubility of the recombinant proteins in physiological buffers.

Results: The iCP-SOCS3 exhibited high solubility, cell-/tissue-permeability and biological activity in pancreatic cancer cell lines (BxPC3, HPAC and PANC-1) with decreased endogenous SOCS3 levels, hyperactived JAK/STAT signaling and hypermethylated SOCS3 promoter region. The iCP-SOCS3 suppressed cancer-associated phenotypes (i.e., up to 90% inhibition of cell viability, 52% induction of apoptosis, 14.8% inhibition of cell cycle progression, 54% inhibition of migration and 61.5% inhibition of invasion) in pancreatic cancer cells dependent on elevated JAK/STAT signaling, whereas, these effects were not observed in non-cancer cells (H6c7, pancreatic duct epithelial cell) or with proteins lacking the aMTD sequence. In contrast, gemcitabine, a conventional pancreatic cancer therapeutic, showed undesirable cytotoxic effects including decreased cell viability and induced apoptosis in non-cancer cells. Furthermore, iCP-SOCS3 (30 mg/kg) induced tumor regression (-168% at day 18) in PANC-1 CDX model. In BxPC-3 orthotopic CDX model, iCP-SOCS3 suppressed tumor growth by 98% which was significantly higher compared to the suppressed tumor growth induced by gemcitabine (77%). Correlating to the tumor suppression, iCP-SOCS3 significantly decreased the secretion of TNF-α and the expression of p-STAT1, p-STAT3 and VEGF in tumor tissues.

Conclusion: These results provide further evidence that SOCS3 can function as a tumor suppressor and intracellular delivery of SOCS3 with iCP-SOCS3 may provide a novel protein therapy against pancreatic cancer.

#4654

A cell-based translocation assay system identifies active fractions from plant that inhibit growth of FGFR3 overactivation cancers.

Yi-Ching Lee, Yun-Wen Lin. _Academia Sinica, Taipei, Taiwan_.

Fibroblast growth factor receptor 3 (FGFR3) is one of four tyrosine kinase receptors that bind and respond to fibroblast growth factors (FGFs). Gain-of-function mutations in FGFR3 cause a group of skeletal dysplasias. The overactivation of FGFR3 in somatic cells is reported to be associated with human cancers, including multiple myeloma (MM), urothelial bladder carcinoma, cervical carcinoma, as well as benign seborrheic keratosis. Patients with FGFR3 overactivation have demonstrated an overall poor prognosis, and the treatment remains a substantial challenge. Several tyrosine kinase inhibitors (TKIs) have been proposed for treatment of FGFR3-activating cancers and some of them are currently in clinical trials. However, the side effects and a modest efficacy of the compounds towards FGFR3 promote the need to identify high-selectivity and high-potency specific FGFR3 inhibitors. We established a cell-based protein translocation assay system that can monitor FGFR3 activity and be used for high-throughput screening of complex mixtures. With this system we identified plant ethanol extract as a FGFR3 inhibitor and performed bioassay-guided fractionation to identify potent active fractions. The functionality of extract and active fractions was validated in vitro in FGFR3-activated primary multiple myeloma cells. The active fractions can preferentially inhibit the proliferation of MM with FGFR3 activation compared with MM without FGFR3 activation and inhibit the FGFR3 downstream signaling pathways. Furthermore, we demonstrated that the active fractions enhance FGFR3 degradation but not FGFR1, 2, 4 in a Western blot analysis. Our results demonstrate the applicability of this discovery approach. The identified plant extracts and active factions hold therapeutic potential for the treatment of FGFR3-activated skeletal dysplasias and cancers.

#4655

Local drug activation: Making cytotoxics safer for local tumors.

Sangeetha Srinivasan,1 Filemon Dela Cruz,2 Andrew Kung,2 Ervin Gaviria,2 Ethan Miller,1 Nathan Yee,1 Maksim Royzen,3 Jose Mejia-Oneto1. 1 _Shasqi, San Francisco, CA;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 3 _University of Albany, SUNY, Albany, NY_.

Only 1-2% of systemic chemotherapies actually reach a localized tumor while the rest cause myelosuppression and sometimes even cardiotoxicity. There is a critical need to deliver cytotoxic agents to the site of the tumor and reduce overall systemic toxicity. Shasqi's Therapy (Shasqi Tx) is a two-pronged approach based on bioorthogonal chemistry consisting of i) a drug-activating gel and ii) a systemic prodrug. The biocompatible gel, modified with tetrazine (Tz), is injected at tumor site. Then, a trans-cyclooctene (TCO)-modified prodrug of a cytotoxic agent such as doxorubicin (Dox), with attenuated activity, is given systemically. The prodrug concentrates at the tumor site through a covalent cycloaddition reaction between TCO and the Tz located at the gel. The active drug is spontaneously released over multiple days, providing sustained local delivery directly to the tumor compartment. Remaining inactive prodrug is rapidly cleared by systemic clearance routes, thereby minimizing off-target effects. The attenuation of drug activity allows us to maximize the total drug dose while reducing systemic side effects, as the active drug predominantly localizes to the gel site. To this effect, NSG-H mice were injected with Tz-modified alginate gel (TAG) and systemic TCO-doxorubicin (TCO-Dox; prodrug of Dox). The maximum tolerable dose (MTD) of TCO-Dox was >12x the single MTD of Dox (control). When given as 5 daily doses, TCO-Dox dosage could be increased to >38x of Dox MTD, without any adverse effects. Preliminary serum and tissue bioanalysis provides a basis for the improved safety of Shasqi Tx over traditional chemotherapy. TAG-injected BALB/c mice received TCO-Dox intravenously. In serum 5 minutes after injection more than >90% of the circulating anthracycline remained as intact prodrug while ~7% had been converted to Dox. Finally, we tested Shasqi Tx's safety profile in a pilot canine study with a local spontaneous adenocarcinoma. Standard Dox therapy led to disease progression (tumor growth) as well as a drastic drop in body weight. Then, multiple cycles of Shasqi Tx given at higher doses reduced tumor size and led to stable disease for more than 9 months, gain in body weight and without major side effects. Serial evaluations by echocardiogram did not reveal any signs of cardiotoxicity. Collectively, our preliminary toxicity studies suggest that Shasqi Tx enhances delivery of toxic drugs to a target site while limiting exposure in off-target tissues in small and large animals. In addition, the increase in therapeutic index allows greater doses of toxic drugs to be delivered safely.

#4656

**Development of an RNA-based cancer therapeutic targeting the** let-7 **-LIN28 interaction.**

Sunghyun Myoung,1 Sergey Savinov,2 Lan Chen,1 Gaurav Chopra,1 Larisa Avramova,1 James Welch,1 Bradley Loren,1 David Thompson,1 Andrea L. Kasinski1. 1 _Purdue University, West Lafayette, IN;_ 2 _University of Massachusetts, Amherst, MA_.

Based on the knowledge that microRNAs (miRNAs) are dysregulated in diseases such as cancer, various attempts have been explored to develop miRNA-based cancer therapeutics. Although many strategies have been used to restore the levels of therapeutically relevant microRNAs, clinical delivery of the processed or mature miRNA to cancer cells still remains a challenge. To overcome this challenge, innovative methods are being explored to increase the pools of tumor-suppressive miRNAs, such as enhancing miRNA biogenesis. This is especially true for the let-7 family of tumor-suppressive miRNAs, which has an additional layer of regulation over the canonical miRNA biogenesis pathway. In particular, the DROSHA and DICER cleavage steps are blocked when unprocessed let-7 is bound by the RNA-binding protein LIN28. LIN28 interacts specifically with unprocessed let-7 via a sequence-specific motif, GGAG, contained in most let-7 family members. Because previous attempts to restore mature let-7 levels through increasing let-7 pools have been quite successful at reducing tumor burden, identifying novel and clinically relevant ways to increase the level of this tumor-suppressive miRNA represents a critical need. In this study, we hypothesize that small-molecule inhibitors that disrupt the LIN28-let-7 interaction will lead to enhanced processing and increased levels of mature, tumor-suppressive let-7. Thus, an in vitro high-throughput fluorescence polarization screen has been conducted using His-tagged LIN28 and a Cy5 fluorophore-tagged let-7 RNA probe. The nine-nucleotide RNA probe was designed through in silico modeling to include the GGAG motif, which associates with LIN28 through the zinc knuckle domain of LIN28. Due to the size difference between unbound let-7 probe and LIN28-bound let-7 probe, a wide window of polarization values was achieved, resulting in a Z′ score of 0.61, indicating that the assay was robust to proceed with the screen. The screen was performed against 23,680 compounds, which consist of FDA-approved LOPAC and blood-brain barrier-permeable CNS library compounds. Eight compounds were identified as positive hits from the screen. These compounds are currently being characterized in secondary assays both in vitro and in cell culture. The significance of this research is that small-molecule inhibitors may provide novel therapeutic strategies to increase the pool of mature, tumor-suppressive let-7 in tumors with elevated LIN28.

#4657

Docetaxel nanoformulation reverts drug resistance in prostate cancer.

Prashanth K. B. Nagesh, Pallabita Chowdhury, Elham Hatami, Vivek K. Kashyap, Bilal B. Hafeez, Sheema Khan, Subhash C. Chauhan, Meena Jaggi, Murali M. Yallapu. _Univ. of Tennessee Health Science Ctr., Memphis, TN_.

Objectives: Docetaxel (Dtx) is the primary choice for the treatment of castrate-resistant prostate cancer (PrCa). Besides its therapeutic benefits, many men with castrate-resistant PCa fail to respond to treatment due to Dtx resistance. There are multiple signaling mechanisms that play pivotal role in the development of Dtx resistance. Seminal reports stated that Tau, highly soluble microtubule-associated protein play a crucial role in Dtx resistance development. Disruption Tau signaling affects its chemoresistance. To revert the drug-resistance and target specific pathway, development of novel and strategic therapies are much needed. In this line, we employed magnetic nanoparticle (MNP) loaded Dtx (MNP-Dtx) to revert the chemo resistance of Dtx in PrCa.

Methods: Docetaxel loaded magnetic nanoparticle (MNP-Dtx) formulation is composed of an iron oxide core coated with cyclodextrin (for drug loading) and F127 polymer (for particle stability and chemosensitization). Dtx-resistant PrCa cell line models (PC-3R and DU145R) were developed in our laboratory by repeatedly treating cancer cells in a dose escalation manner for over 24 months. Intracellular drug levels of MNP-Dtx in wild type and drug resistant cancer cells was determined by flow cytometry, TEM and HPLC methods. The superior in vitro anti-cancer activity of MNP-Dtx was examined by MTS, proliferation kinetics, colony formation, immunoblotting and qPCR studies in both wild type and drug resistant PrCa cell lines. The interaction between tau and tubulin studies were assessed through immunoprecipitation studies.

Results: MNP-Dtx formulation showed optimal particle size and zeta potential which can efficiently be internalized in PrCa/Dtx resistant PrCa cells. MNP-Dtx exhibited enhanced cellular uptake in drug resistant cell lines over wild type cells in a dose and time dependent manner, with 2 to 3 folds increase. Immunoblot and q PCR studies showed the expression of Tau, whole β tubulin, β tubulin III, β tubulin IV and MDR1 were found to be deregulated in both wild type and resistant cells. To delineate the mechanism of chemosensitization by MNP-Dtx on resistant cells, we performed immunoprecipitation studies, found the expression of Tau was significantly reduced during MNP-Dtx treatments w.r.t control and Dtx. HPLC studies have shown higher amounts of Dtx levels were detected in cells after MNP-Dtx treatments, annotating the sustain drug release ability of MNP nanoformulation. Further, these results were confirmed through tubulin polymerization studies.

Conclusion: Overall, sustained drug release of Dtx aided in high intracellular levels of drug, thus facilitating high probability of Dtx-tubulin interaction instead of Tau-tubulin interactions resulting in tubulin fibers stabilization. This promotes Dtx induced apoptosis in wild type and Dtx resistant PrCa cells. This novel therapeutic modality might be effective to treat patients with Dtx resistant PrCa.

#4658

Novel paclitaxel medical nanoparticle formulation for breast cancer.

Pallabita Chowdhury,1 Sumeet S. Chauhan,2 Prashanth K. Nagesh,1 Elham Hatami,1 Bilal B. Hafeez,1 Sheema Khan,1 Meena Jaggi,1 Murali M. Yallapu1. 1 _Univ. of Tennessee Health Science Ctr., Memphis, TN;_ 2 _Houston High School, Germantown, TN_.

Objectives: Paclitaxel (Ptxl) is currently the most common first-line therapeutic option for breast cancer (BrCa). However, adverse side effects and problems associated with chemo-resistance, limits its use in clinical settings. Nanoparticle mediated delivery can improve Ptxl delivery and its activity at the tumor site, and considered to be an attractive strategy for BrCa therapy. Therefore, we aim to generate a novel Ptx medical nanoparticle formulation (PMNPs) using a polyphenol as a carrier, and evaluate its efficacy against BrCa cells. Methods: Physico-chemical characterization of this PMNPs was performed using TEM, DLS, FT-IR, TGA, X-RD, and SAXS methods. Its internalization and cellular availability was examined using HPLC method. Cell proliferation and colony formation assays were utilized to evaluate therapeutic efficacy of this unique nanoformulation in clinically relevant cell line models (MCF7 and MDA-MB-231). Additionally, molecular effects of this formulation on apoptosis, anti-apoptosis, and drug resistant associated proteins were evaluated using immunoblotting assays. A tubulin stabilization study was performed using confocal immunofluorescence microscopy analysis. Results: The PMNPs formulation showed optimal particle size and zeta potential, which can be efficiently internalized in BrCa cells. PMNPs exhibited potent anti-cancer efficacy via induction of the expression of apoptosis associated proteins (Bax and Bad, cleaved PARP, and caspase 3) and downregulation of anti-apoptotic proteins (Bcl-2 and Bcl-xL) in BrCa ell lines. Further, the expression of chemoresistance-associated proteins were also decreased with the treatment of PMNPs.

Conclusion: PMNPs formulation can efficiently be internalized in BrCa cells and induce enhanced therapeutic potential of Ptxl. Development of a targeted drug delivery system would not only reduce the dose of paclitaxel required to kill cancer cells but also minimize long-term paclitaxel associated systemic toxicity and drug-resistance.

#4659

Intratumoral sirolimus injection for prostate cancer.

Aishwarya Kundu, Sri Mudumba. _Santen Inc, Emeryville, CA_.

A key unmet need in the field of prostate cancer treatment is the lack of targeted therapy for local control of tumor burden, that circumvents the systemic adverse events related to currently available systemic therapy. Our clinical stage, sterile injectable 2% sirolimus formulation acts by inhibiting the activity of mTOR, a central signaling molecule with multiple mechanism of actions. mTOR functions downstream of two of the key driver mutations associated with prostate cancer namely PTEN (30%) and AR (63%) and affects downstream processes like proliferation, survival, protein synthesis, cell growth, EMT transition regulating metastasis as well as angiogenesis. Additionally, a feedback loop also exists between mTOR and AR, which aids in the development of androgen resistance, resulting in hormone resistant prostate cancer. Therefore, inhibiting mTOR activity can not only inhibit tumor growth but can potentially prevent metastasis and development of resistance. However, all current mTOR inhibitors are administered systemically leading to unwanted systemic adverse events in addition to poor tissue availability, which prevents achievement of optimal efficacy of the molecule.

In our preclinical study, athymic nude mice were injected with prostate cancer line 22Rv1 and PC3 cells to generate xenografted prostate tumors. The mice were randomized into 4 groups and each tumor was injected with either vehicle control, weekly or biweekly intratumoral injection of 2% sirolimus, while an oral 0.1% Rapamycin group was fed the drug daily for a period of 1 month at the end of which, all the mice were sacrificed, and the residual tumors were sectioned and probed for various markers to determine the specificity and molecular mechanism of action of our formulation.

Unlike Rapamycin, injectable sirolimus forms a depot at the site of injection that results in increased tissue availability and sustained local delivery of the drug over time. The tumors in the injectable sirolimus weekly and biweekly treatment groups showed significant decrease in tumor burden compared to oral Rapamycin and vehicle treated groups in both 22Rv1 and PC3 cell generated tumors. Blood PSA in the injectable sirolimus treated groups also showed a faster decrease than treatment with Rapamycin. Immunohistochemistry showed a decrease in the phosphorylation of both mTOR targets, S6K and 4EBP with no change in the protein levels of mTOR confirming that injected sirolimus acts by inhibiting the mTOR activity. Proliferation marker Ki-67 showed a decrease while apoptosis marker cleaved caspase-3 showed an increase upon treatment with sirolimus substantiating the underlying molecular mechanism of the observed tumor shrinkage. Interestingly, anti-metastasis marker E-cadherin levels increased with sirolimus treatment as well.

These results show the clinical potential of local sirolimus injection for the treatment of prostate cancer and underscores the need for next stage human trials.

#4660

Tumor multicomponent targeting nanoparticle library for personalized cancer therapy & imaging.

Samaresh Sau, Hashem Alsaab, Katyayani Tatiparti, Ketki Bhise, Arun Rishi, Arun K. Iyer. _Wayne State University, Detroit, MI_.

The fundamental limitations of conventional cancer treatment have promoted the development and application of nanoengineering platforms for achieving effective patient outcomes. Over a time, considerable clinical success has been accomplished in nanotechnology field but there are many challenges limiting the progress of nanomedicine from bench to the beside. The highly heterogeneous tumor environment, an inadequate understanding of interactions between tumor biomarkers and nanomedicine, and reproducibility in synthesis required for clinical translation are some of the major challenges. Thus, there is an urgent need to redirect our focus of nano-drug carrier that can target multiple components of tumor including, tumor epithelial cells, tumor angiogenic vessels, tumor hypoxia and stromal cells. To achieve this, we developed a library of various tumor biomarker targeting ligands, and drug and imaging agents encapsulated in nanoparticles (NPs). Finally, the library of targeting ligands was conjugated with NPs using reagent free 'click' chemistry. This strategy will serve as an "off-the-shelf ready to configure drug formulation" that can be engineered based on a mix-and-match strategy for individual patient needs. It is envisaged that the patient can walk in the clinic and be given the targeted drug that is personalized to their need. For the proof-of-concept, we evaluated tumor selectivity of multiple cancer biomarker targeting oligo-micelles (OMs), iron oxide and lipid NPs in various patient derived tumor xenograft (PDx) models, such as non-small cell lung and triple negative breast cancer. We observed that the dual tumor hypoxia and cancer stem cell (CSC) targeting rod shaped OM has 8-fold higher tumor core penetration compared to non-targeted OM in these PDx tumors, as obtained from NIR-fluorescence imaging. This OM has significantly higher tumor vs. liver ratio and they follow tumor specific receptor competition. The tumor growth inhibition of drug loaded OM is superior compared to free drug. This approach demonstrated significant improvement of tumor stroma penetration with low healthy organ uptake of NPs portending more promising potentials for selective cancer treatment.

#4661

**Magnetic nanoparticles (MNPs) for cancer drug delivery: The value of** in vitro **modeling.**

Sebastian P. Pernal, Alexander J. Willis, Herbert H. Engelhard. _Univ. of Illinois at Chicago, Chicago, IL_.

BACKGROUND: Magnetic nanoparticles (MNPs) have attracted great interest for use as delivery vehicles for cancer and other diseases due to their ability to be functionalized and localized using external magnets. In the presence of a magnetic field, individual MNPs form aggregates, which in turn can be made to spin and surface walk, in response to rotation of the field. Here, we present data from the use of a in vitro model system, which is useful in mimicking the various conditions and distances that MNP-drug combinations may encounter in vivo. METHODS: A sterilizable acrylic tray was designed, which has 1/8th inch wide lanes and can be used with or without the addition of cultured cells. The tray is compatible with standard plate readers, and the lanes can be modified to mimic various conduits within the body. In studies described here, glioma cell lines and normal vascular endothelial cells were used in the tray. In order to test the effect of fluid viscosity on MNP velocity in response to the rotating magnetic field, lanes were filled with 1mL of PBS, culture medium, serum, or whole blood. Pre-magnetized and unmagnetized MNPs were aliquoted into the lanes at volumes from 10 - 100 uL. T-PA and trypan blue were used as a model drugs. Velocities of MNP aggregates were determined by videography at five different tray positions relative to the magnet: centered, offset, push, pull, and below. Particle adhesion to cells could be quantified using ImageJ. RESULTS: Using the model system, greater MNP velocities were achieved with pre-magnetization, and larger aliquots. For example, 100ul aliquots had a total average velocity 1.27± 0.21 times that of 20ul. MNP velocity was found to be inversely -related to fluid viscosity, but particles could be moved magnetically even through whole blood. Test drugs could be successfully delivered by convection, even without prior binding to MNPs. MNP velocity also varied according to magnet position, with speeds up to 0.75 +/- 0.05 cm/sec in the pull (fastest) position. In the offset position, a distance of 20 cm above the magnet produced the greatest velocity. MNPs moved more slowly over confluent monolayers of endothelial or glioma cells (with greater adhesion), but a mean velocity of 0.25 cm/sec +/- 0.03 cm was typically observed. CONCLUSIONS: In vitro modeling is extremely helpful in predicting the behavior of particles intended for clinical use in magnetically-enhanced drug delivery. The velocity and cell surface adhesion of MNP aggregates can be quantified, and the effect of factors - such as fluid viscosity, cell type, and position with respect to the magnet - analyzed in order to better understand the advantages and limitations of this technology.

#4662

Comparing 2D and 3D culture models for in vitro therapeutic screening of paclitaxel loaded micelles co-administered with iRGD peptide.

Nayela N. Chowdhury, Nandita G. Das, Sudip K. Das. _Butler University, Indianapolis, IN_.

Clinical efficacy of paclitaxel, a potent chemotherapeutic agent, is limited by its nonspecificity that leads to toxic side effects. Polymeric micellar formulations of paclitaxel were designed with the long term goals of decreasing toxicity by tumor targeting, increasing circulation time, and enhancing permeability and retention in solid tumors. Biodegradable amphiphilic block copolymers that self assemble into micelles were used to entrap hydrophobic paclitaxel in the core. Traditional 2D cell culture models are standard for cytotoxicity assays. However, they lack the diffusion gradient and cell to cell interactions present in solid tumors which 3D multicellular tumor spheroids are capable of providing. We evaluated the in vitro cytotoxicity of polymeric micelles entrapping paclitaxel in 3D multicellular tumor spheroids of triple negative breast cancer cells and compared it with the standard 2D model. We added the cell penetrating iRGD peptide with the expectation that the iRGD peptide will increase the cellular uptake of the paclitaxel micelles. Particle uptake is enhanced due to the interaction of the iRGD peptide with the highly expressed integrin receptor on the cell surface, followed by neuropilin receptor binding which mediates an active transport system for deep tumor permeation. Paclitaxel loaded micelles were prepared and the drug content was analyzed. The cytotoxicity of the micellar paclitaxel nanocarrier system was studied in both the 2D and 3D cell culture models. Micellar paclitaxel induced cytotoxicity was significantly higher than free paclitaxel for the same concentrations of paclitaxel. Furthermore, a synergistic growth inhibitory response was observed upon coadministration of iRGD compared to treatment with the paclitaxel loaded nanocarriers on their own. Significantly higher concentration of the drug was needed in the 3D cultures to achieve comparable levels of cytotoxicity to the 2D cell culture models. This suggested that multicellular tumor spheroids exhibit higher resistance to anticancer drugs, supporting the fact that 3D culture is the better model to study in vitro cytotoxicity and likely to produce biorelevant data.

#4663

Lysosomal targeting from a gelatin-doxorubicin conjugate produces evidence of both nuclear and lysosomal cytotoxcity pathways in mcf-7 breast cancer cells.

Mohammed Alvi, Bayan Eshmawi, Rachel Nicoletto, Hyun Kate Kim, Christopher Cammarata, Clyde Ofner. _Univ. of the Sciences in Philadelphia, Philadelphia, PA_.

Background: Intra-lysosomal targeting has been investigated for the treatment of cancer but details of the cytotoxicity pathways remain unclear. A high molecular weight gelatin - doxorubicin conjugate (GDox) has been synthesized to target acidic lysosomes for intra-lysosomal drug release. The purpose of this investigation was to explore GDox feasibility for drug delivery and to make a preliminary determination of a cytotoxic pathway. Methods: GDox was synthesized in formamide using the carbodiimide, EDC. Drug load was determined spectroscopically, drug release at pH 4.8 was determined by HPLC, and molecular weight and its reduction in FBS was determined by HPSEC. Cellular localization was determined for free Dox and GDox at 10 µM or its equivalent Dox concentration using fluorescence microscopy with appropriate subcellular stains. Three to four independent trials (n=3-4) were examined with 200 to 700 cells per time point. Cytotoxicity was determing by the MTT procedure (n=3). Viabilities, growth inhibition profiles and their IC50 values were calculated with GraphPad Prism 7. The nucleus content of free Dox or Dox released from GDox after incubation was determined by UHPLC using fluorescent detection (n=3-4). Results: A typical GDox contained 5.6% w/w Dox, had a molecular weight of ~ 160 kDa, and released 58+2.3% Dox at pH 4.8 representing lysosomal pH. After incubation in FBS at 37°C for 24 hr the low molecular weight species increased a relatively small 16%. Fluorescent images with LysoTracker Green confirmed GDox localization to the lysosome which was detectable by 2 hr but substantial in swollen lysosomes by 24 and 48 hrs. No released Dox was observed in the nucleus, but a slight red nucleus haze at later times suggested this possibility. Cell debri from GDox was not observed from free Dox. As expected, free Dox rapidly accumulated in the nucleus by 2 hr. Viability determinations at 0.1, 1, and 10 µM for 2 to 48 hr showed no effect from either agent before 24 hr. By 48 hr at 10 µM, the viability of Dox (30+12%) and GDox (36+13%) were indistinguishable (p=0.68). However, by 72 hr, IC50 values varied 10-fold at 0.094 and 0.96 µM, respectively. The nucleus drug content after incubation at 10 µM for 2 to 48 hr was 0.27+0.07 to 1.39+0.6 µg/106 cells (p&lt0.01) for free Dox, and 0.014+0.01 to 0.11+0.06 µg/106 cells (p&lt0.01) for released Dox. Conclusions: Taken together these results suggest good feasibility of GDox lysosomal targeting and Dox release in MCF7 breast cancer cells. Of note, after 48 hr incubation GDox produced the same cytotoxicity as the free drug but with 13-fold less Dox in the nucleus. This striking nucleus difference and the substantial lysosomal GDox content suggest a lysosomal pathway of GDox cytotoxicity in addition to a nuclear pathway. Support: NIH/NCI R15CA135421 and the Agnes Varis Trust for Women's Leadership and Health.

#4664

Enzymatically-responsive tumor-targeted mesoporous silica nanoparticle for identification of pancreatic cancer.

Kylie Nairon,1 Abhilash Samykutty,2 Molly W. McNally,2 Girish Mishra,2 William E. Grizzle,3 Lacey R. McNally2. 1 _Washington University, St. Louis, MO;_ 2 _Wake Forest Univ. Comp. Cancer Ctr., Winston-Salem, NC;_ 3 _University of Alabama Birmingham, Birmingham, AL_.

Purpose: Pancreatic cancer remains an unsolved health issue, with its rapid progression and resistance to modern therapy leading to poor prognoses for most patients. The prevalence of metastasis in pancreatic cancer makes complete tumor location and removal rare, and those who do have resectable disease have only a 20% 5-year survival rate. In recent years, nanoparticles have been explored as targeted delivery agents for chemotherapeutic drugs and imaging dyes, however few have achieved clinical success due to overestimation of biological phenomena and reliance on passive targeting systems. In this work, an enzyme-responsive nanoparticle has been developed for increased active pancreatic tumor targeting and specific release at biologically-significant enzyme concentrations.

Methods: Mesoporous silica nanoparticles were formed using a scaffold of hexadecyltrimethylammonium bromide (CTAB) at 80°C. The scaffold was removed to form wormhole-like pores using a series of dialysis procedures. Transmission electron microscopy (TEM) confirmed the 35 nm diameter and porous structure of the particles. The particles were then loaded with IR780 dye, and surfaces were functionalized with (3-aminopropyl) triethoxysilane (APTES). The loaded particles were then encapsulated with a combination of Type A and Type B gelatin, followed by a stabilizing polyvinylpyrrolidone (PVP) layer. Dynamic light scattering (DLS) and zeta potential were used to confirm coating. Coated particle samples were exposed to collagenase type IV (MMP-9) enzyme to test encapsulation efficiency and enzyme sensitivity. Enzyme-treated and intact samples were transferred to tissue phantoms, and Multispectral optoacoustic tomography (MSOT) was used for comparative analysis.

Results: TEM confirmed the formation of stable 35 nm silica nanoparticles with a wormhole-like pore structure. Zeta potential decrease from 55 mV to 5 mV and DLS particle diameter increase from 35 nm to 334.5 nm indicated binding of gelatin and PVP to particle surfaces. MSOT imaging showed 10 X increased signals from untreated nanoparticles as compared to enzyme-treated nanoparticles, indicating that dye molecules remained inside the pores of coated particles and were released when exposed to MMP-9.

Conclusion: Gelatin/PVP-coated mesoporous silica nanoparticles encapsulated dye and demonstrated MMP-9 activated dye release at biologically-relevant enzyme concentrations.

#4665

Neurotensin receptor-based targeted delivery of thymoquinone for non-small cell lung cancer.

Santosh Kumar Singh, James W. Lillard, Rajesh Singh. _Morehouse School of Medicine, Atlanta, GA_.

Non-small cell lung cancer (NSCLC) is a very recalcitrant cancer, accounting for 80% to 85% of all lung cancer diagnoses. About 40% of NSCLC are adenocarcinomas. Thus, there is an urgent need to identify targeted therapies, specifically for NSCLC. Although, multiple signaling molecules have now been identified within the last decade, among them Neurotensin receptor 1 (NTSR1) have afforded an improved understanding of the underlying pathology and significant heterogeneity of NSCLC. NTSR1 belongs to the G-protein coupled receptors (GPCR) superfamily that has been shown numerous oncogenic effects in tumor initiation, progression, proliferation, apoptosis, and metastasis. In the present study, we used a novel method for targeted delivery of Thymoquinone (TQ), a constituent of Nigella sativa at the desired location. We described a proprietary planetary ball milling (PBM) approach that uses a natural polysaccharide (starch; FDA approved) to create a drug-polysaccharide nanoparticle as a core that is subsequently coated with poly (ε-caprolactone) / poly (ethylene glycol) copolymer and conjugated with anti-NTSR1-mAb, which allows binding to high-affinity NTSR1 receptors presents on NSCLC. Our tissue microarray data show higher expression of NTSR1 in lung cancer patients, which were negligible in normal cells. These studies were further confirmed by targeting NTSR1 in lung cancer cell lines NCI-H1563, NCI-H1975 and MRC-5 using our novel anti-NTSR1 conjugated, and TQ encapsulated PBM nanoparticle and further, validated using flow cytometry, immunofluorescence, qRT-PCR and western blot techniques. Our result showed the PBM nanoparticles encapsulated with TQ rapidly internalized and depolymerized the microtubule network by inducing apoptosis. The up-regulation of pro-apoptotic (BAX, BID) and down-regulation of anti-apoptotic (BCL-2, MCL-1) makers to confirm the targeted delivery of PBM-NPs to the tumor cells. This study strongly suggests anti-NTSR1-mAb conjugated PBM-NPs has its application to drugs targeting for the treatment of NSCLC. In summary, this study combines innovative ideas with an innovative technology to create new therapeutics and a new strategy to improve the treatment of NSCLC and has the potential to create a new class of targeted treatments for advanced lung cancer.

#4666

An evaluation of breast cancer cellular membrane lipid-extracted nanoliposomes (CLENs) in relation to formulation design, stability, mechanism of cellular entry and cardioprotective function in vitro.

Hanan M. Alharbi, Robert B. Campbell. _MCPHS University, Worcester, MA_.

One of the longstanding issues limiting the use of chemotherapeutic agents is the lack of tumor specificity, which leads to systemic toxicity. Therefore, various targeted nanocarriers have been developed to concentrate the cytotoxic drug agents at tumor regions to avoid uptake by normal healthy tissues. Previous studies from our laboratory have demonstrated that cell membrane lipid-extracted nanoliposomes (CLENs) are capable of selective targeting compared to nanoliposomes consisting of mainly conventional lipid materials. Also, CLENs were relatively nontoxic when employed at concentrations traditionally used to evaluate nanoparticles in vitro. However, parameters such as lipid composition, size, and surface charge all have a direct impact on liposomal cellular uptake. In this study, we prepared different CLENs different molar ratios of natural and cellular-derived lipids extracted directly from breast cancer cells (4T1), cholesterol, and DPPE-PEG-5000. The formulations were used to investigate the mechanisms of CLENs uptake and determine the underlying mechanisms regulating cellular entry. In the study, CLENs were used to evaluate cytotoxicity and cellular uptake for both target and off-target cell populations. CLENs containing cholesterol and DPPE-PEG-5000 (70/25/5) were able to retain doxorubicin in relevant therapeutic concentrations. CLEN formulations were able to exert selective cytotoxic drug effects against different target breast cancer cells in vitro. 4T1 CLENs containing cholesterol and DPPE-PEG-5000 (70/25/5) demonstrated greater binding to 4T1 (target) cells compared to CLENs prepared using 4T1 lipid extracts alone. The binding was not only temperature- and time-dependent, but also composition- and cell type-dependent. Off-target effect studies showed that 4T1 CLENs were taken up minimally by off-target cells (including normal breast fibroblasts and normal myocytes), compared to controls. Our studies collectively support the use of lipid extracts derived from target cells for selective drug targeting and enhanced cytotoxicity. Future studies will explore underlying mechanisms of cellular entry and cardioprotective function.

#4667

The use of fragment-based screening approaches to improve the specificity of inhibitors targeting malignant melanoma.

Sreya Mukherjee, Shardell Spriggs, Paul Wilder, Wenbo Yu, Kristen Varney, Alexander D. MacKerell, David J. Weber. _University of Maryland, Baltimore, MD_.

S100B belongs to the S100 protein family of Ca2+-binding proteins. Prior studies have shown that S100B is a biological marker for malignant melanoma. Increases in S100B levels are predictive of disease progression, recurrence, and overall low survival in the patients. It was shown previously that S100B binds to p53 and down-regulates its tumor suppression activities including growth arrest and apoptosis. Chemical biology and structure-based drug design methods are underway to inhibit S100B and restore p53 function. However, highly specific S100B inhibitors are required, so S100B can be blocked in melanoma without impairing S100A1 function in skeletal and cardiac muscle. With this goal in mind, a fragment-based screening approach using NMR and computational approaches is being applied. Results to date indicate that these methods can be used to identify specific small molecule fragments that target S100B and not S100A1. The fragments that uniquely bind S100B will then be considered with structural data of existing S100B-SBiX complexes to improve their specificity for binding S100B (i.e. versus S100A1). The inhibitor design strategy will also include improving the stability of the compounds their cell permeability, and their overall drug-like properties. The most promising of these new inhibitors will ultimately be used to target high levels of S100B found in malignant and rescue p53-dependent tumor suppression pathways in vivo with the goal of them having therapeutic value for treating malignant melanoma. 

## IMMUNOLOGY:

### Adaptive Immunity in Tumors

#4668

Evidence for antigen-driven TCRB chain convergence in the tumor infiltrating T cell repertoire of 85 research subjects with melanoma.

Timothy J. Looney,1 Sean Glenn,2 Sarabjot Pabla,3 Jeff Conroy,2 Carl Morrison,2 Alice Zheng,1 Lauren Miller,4 Elizabeth Linch,4 Denise Topacio,4 Geoff Lowman,4 Fiona Hyland,1 Mark Anderson4. 1 _Thermo Fisher Scientific, South San Francisco, CA;_ 2 _OmniSeq, Inc, Buffalo, NY;_ 3 _OmniSeq, Inc, Buffalo, CA;_ 4 _Thermo Fisher Scientific, Carlsbad, CA_.

Introduction

T cell convergence refers to the phenomenon whereby antigen-driven selection enriches for T cell receptors having a shared antigen specificity but different amino acid or nucleotide sequence. T cell recruitment and expansion within the tumor microenvironment (TME) may be directed by responses to tumor neoantigen, suggesting that elevated T cell convergence could be a general feature of the tumor infiltrating T cell repertoire. Here we evaluate evidence for T cell convergence within tumor biopsy research samples from a set of 85 subjects with melanoma.

Methods

Total RNA from 85 tumor biopsy research samples (non-FFPE) was extracted for use in long-amplicon TCRB chain sequencing (mean amplicon length of 330bp covering CDR1, 2 and 3) via the Ion AmpliSeq Immune Repertoire Assay Plus, TCRB. To evaluate T cell convergence within each biopsy, we searched for instances where TCRB chains were identical in amino acid space (shared variable gene identity and CDR3 amino acid sequence) but had distinct nucleotide sequences owing to N-addition and exonucleotide chewback within the V-D and D-J junctions of the CDR3. To provide context, we evaluated evidence for T cell convergence with T cell repertoires derived from healthy donor peripheral blood leukocytes (PBL).

Results

Sequencing of melanoma biopsies yielded an average of 6029 clones per sample. 11 of 85 samples yielded fewer than 100 clones and were eliminated from downstream analysis. Convergent T cell receptors were identified in 68/74 (92%) of tumor infiltrating T cell repertoires having greater than 100 detected clones. The frequency of convergent rearrangements was approximately 50-fold greater in the melanoma-infiltrating T cell repertoire than healthy PBL samples (p<.001).

Conclusions

These data suggest that T cell convergence may be a common feature of the melanoma infiltrating T cell repertoire. Convergence was more frequently observed within the TME than T cell repertoires derived from healthy PBL, consistent with elevated antigen-driven T cell selection within the TME. The extent to which convergence is a feature of the TME in other cancers is not yet known. T cell receptor convergence may be driven by T cell responses to tumor neoantigen within the TME. In such case, in silico identification of convergent T cell receptors by long-amplicon sequencing may serve as an approach for rapid identification of antigen-specific T cell receptors for future therapeutic use.

For research use only.

#4669

**Role of CD8** + **tissue-resident memory T (T** RM **) cells in anti-tumor T-cell response and cancer immunotherapy.**

Fathia Mami-Chouaib. _Gustave-Roussy, Villejuif, France_.

Tissue-resident memory T (TRM) cells, very often defined by the expression of CD103 integrin, correspond to a new subset of long-lived memory T lymphocytes that reside in tissues and are highly protective during localized reinfections. CD8+ TRM cells also frequently reside in human epithelial tumors and play an essential role in anti-tumor T-cell responses. In this context, our previous results indicated that the interaction of CD103 integrin with its ligand, the epithelial cell marker E-cadherin, favors adhesion of TRM cells to their specific epithelial cancer cells and participate to the potentiation of T-cell receptor (TCR)-mediated cytotoxic activity and cytokine production. We also demonstrated that an enhanced CD103+ tumor-infiltrating lymphocytes (TIL) subset correlates with improved early-stage non-small cell lung carcinoma (NSCLC) patient survival and increased intraepithelial T-lymphocyte infiltration. Notably, TRM cells are enriched with tumor-specific T cells and express high levels of inhibitory receptors, including PD-1 and Tim-3, making them an attractive target for cancer immunotherapy, such as checkpoint blockade. Moreover, we provided evidence that CD103 is involved in T-cell recruitment within epithelial tumor islets and in local early T-cell signaling. Our more recent results indicated that integrin-linked kinase (ILK) and paxillin adaptor protein are directly involved in CD103 bidirectional signaling events and defined the CD103 cytoplasmic domain that controls TRM adhesion, migratory behavior and TCR-mediated T-cell functions. Data emphasizing the contribution of CD8+/CD103+ TRM cells in promoting intra-tumoral cytotoxic T lymphocyte (CTL) responses and in success of cancer immunotherapy will be discussed.

#4670

Evidence of neoantigen-reactive T cell response in a case of relapsing, mismatch-repair gene proficient, colorectal cancer.

Mélissa Mathieu,1 Alexandre Paradis,1 Sandy Pelletier,1 Steven Hébert,2 Kevin Boutin,1 Éric Audemard,3 Sylvie Mader,3 Claudia Kleinman,2 Simon Turcotte1. 1 _CRCHUM, Montreal, Quebec, Canada;_ 2 _McGill University and Lady Davis Research Institute, Montreal, Quebec, Canada;_ 3 _Institute for Research in Immunology and Cancer (IRIC), Montreal, Quebec, Canada_.

Whether the endogenous T cell reactivity to antigens derived from cancer mutations, called neoantigens (neoAgs), can be exploited for immunotherapy in patients with mismatch-repair (MMR) gene proficient metastatic colorectal cancer (CRC) is largely unknown. Here, we investigated the frequency of neoAg-reactive T cells longitudinally in a patient with relapsing CRC. Using whole exome and RNA sequencing, we identified 96 single nucleotide variant (SNV), 8 frameshift and 29 insertion/deletion mutations in a liver metastasis. Co-culture assays between in vitro expanded tumor infiltrating T lymphocytes (TILs) and autologous CD40-activated B cells loaded with peptides derived from SNVs led to the identification of two CD8+ T cell clones specific for PAMA733E, and respectively 1, 4 and 2 CD4+ T cell clones specific for PABPC1G563S, PDE4DIPR685S and TRPM4A480V. T cell clonality was confirmed by TCR Sanger sequencing. We assessed the in vivo frequency of these neoAg-reactive T cell clones by TCRβ chain deep sequencing (Adaptive Biotechnologies). The dominant T cell clone, reactive to PAMA733E, represented 5.2% of TILs in the primary tumor resected in 2010, 2.2% of TILs in the colonic recurrence resected in 2011, and 3.2% of TILs in the liver metastasis resected in 2012. The neoAg-reactive T cells were also detected in the peritumoral liver, but not into the distant normal liver. Along the disease course, most reactive T cell clones were not detected in the tumor draining lymph nodes or in the peripheral blood, at an average detection capacity of 1/3680 and 1/185,495 T cells respectively. When compared to the RNAseq data from 60 other CRC liver metastases, the relatively high level of transcripts related to immune cells, antigen processing and presentation, IFN-γ responsive genes, T-cell inhibitory and stimulatory receptors, cytokines, and chemokines observed in our patient metastasis suggested that there was an ongoing spontaneous immune response intratumoraly, co-existing with many immune-suppressive molecules. Our results support that neoAg-reactive T cells can be found in non-highly mutated, MMR proficient CRC tumors, at a much higher frequency than in the peripheral blood or the draining lymph nodes. As an adjuvant strategy to prevent recurrence, it may be possible in some patients to boost the immune response against a relevant neoAg expressed in the primary tumor. At the metastatic stage, a broader array of neoAgs may be targetable.

#4671

Rapid, no-wash measurement of immune checkpoint molecules expression induced by interaction with peripheral blood mononuclear cells in breast and cervical cancer cell models.

Jeanine M. Hinterneder,1 Jen Carlstrom,2 Adam Carlson,1 Dawn Nida1. 1 _PerkinElmer, Inc., Southborough, MA;_ 2 _PerkinElmer, Inc., Hopkinton, MA_.

The purpose of this study was to examine the pathways involved in the induction of immune checkpoint molecules in basal breast and cervical cancer cell lines by measuring classical biomarker expression using AlphaLISA no-wash homogeneous assays. Programmed cell death-ligand 1 (PD-L1) expression increases with tumor severity in basal-like breast cancer and is enhanced in intraepithelial neoplasia and cervical cancers. Basal-breast tumors can adapt to lymphocytic infiltration by responding to heightened concentrations of interferon gamma (IFN-y) secreted by Type 1 helper T cells with upregulation of PD-L1 protein allowing the tumors to evade immune targeting and reduce the immune response. We show here that human peripheral blood mononuclear cells (PBMCs) secrete IFN-y and other cytokines in response to stimulation with CD3/CD38 Dynabeads. We then examined modulation in expression of immune checkpoint molecules (PD-L1 and others) in HCC38 (basal breast cancer-derived) and HeLa (cervical cancer) cells in response to co-culturing with activated PBMCs, treatment with conditioned media collected from activated PBMCs, and direct treatment with recombinant IFN-y. As expected, IFN-y treatment induced dose-dependent upregulation of PD-L1 protein expression in both HCC38 and HeLa cell lines, whereas the effects of conditioned media and direct co-culturing with PBMCs resulted in more complex effects which are discussed. To determine if tumor cytoarchitecture influences responsiveness to the PBMCs, biomarker expression was measured from cultures grown in both traditional adherent monolayers and in 3D spheroid cultures using Ultra-Low Attachment (ULA) microplates. Cellular health and proliferation was assessed by measuring ATP concentration with ATPlite luminescence assays and cellular imaging with viability dyes. The upregulation of PD-L1 expression was observed to be largely independent of cellular proliferation and is further shown to be dependent on signaling through the JAK/STAT pathway by probing with AlphaLISA SureFire phosphorylation

assays. These data illustrate and address some of the challenges in developing a biologically relevant culture system for examining the complex mechanisms involved in tumor evasion of the innate immune response.

#4673

Selective delivery of tumor specific antigen to dendritic cells by mannose-labeled PLGA nanoparticles to induce immune response for cancer immunotherapy.

Yeongseon Byeon, Ji Eun Won, Ga Hee Kim, Min Gi Kim, Yeong Min Park, Hee Dong HAN. _Konkuk universty, Seoul, Republic of Korea_.

Objective: To overcome limitation of ex vivo manipulation against dendritic cells (DC) vaccines, we developed mannose-labeled poly(d,l-lactide-co-glycolide) nanoparticles (MN-PLGA-NPs). The MN-PLGA-NPs enabled selective delivery of tumor-specific antigen to mannose receptor overexpressed DCs without ex vivo manipulation.

Methods: Conjugation of mannose (MN) and polyvinyl alcohol (PVA) was analyzed by H-NMR. Size and zeta potential of the MN-PLGA-NPs were examined by dynamic light scattering using an electrophoretic light scattering photometer. Selective delivery efficiency of MN-PLGA-NPs to DCs was examined by confocal microscope and flow cytometry. Migration of DCs containing MN-PLGA-NPs was assessed flow cytometry. Therapeutic efficacy of MN-PLGA(OVA+poly I:C)-NPs was examined in EG7 tumor-bearing mouse models. Biological effect and activated CD8+ T cells in tumor tissue were confirmed by immunohistochemistry assay.

Results: Conjugation of MN and PVA was confirmed that the peaks for the CH2 of methylene group in PVA was observed at 1.48 ppm and 1.59 ppm, and CH of tetrahydropyran group in MN was observed at 3.40 ppm, 3.49 ppm, 3.76 ppm, and 5.41 ppm. Size and zeta potential of MN-PLGA-NPs were 200 nm formed spherical shape and -10 mV, respectively. Loading efficiency of OVA or poly I:C into MN-PLGA-NPs was 90 % or 45 %, individually. Intracellular uptake of MN-PLGA-NPs in DCs was increased through selective binding between MN and MN-receptor compared to PLGA-NPs. Additionally, DCs containing MN-PLGA-NPs exhibited efficient migration to popliteal lymph node.

Therapeutic efficacy of MN-PLGA-NPs showed significant inhibition of tumor growth in EG7 tumor-bearing mice compared to the control (p < 0.001) and PLGA-NPs (p < 0.05), and showed 100% of survival up to 60 days. Additionally, MN-PLGA-NPs treatment group showed a greater number of activated CD8+ T cells in tumor tissue compared to the other groups (p < 0.001), and prevention effect of tumor growth resulted in effectively suppressed compared to the control (p < 0.001) or PLGA-NPs (p < 0.001).

Conclusion: MN-PLGA-NPs is an attractive system that can directly activate DC in vivo without ex vivo manipulation, which overcome the limitation of existing DC vaccine.

Keyword: dendritic cells, selective delivery, PLGA nanoparticle, cancer immunotherapy.

#4674

IL-35 regulates anti-tumor immune response in pancreatic ductal adenocarcinoma.

Bhalchandra Mirlekar, Ryan Searcy, Yuliya Pylayeva-Gupta. _UNC Lineberger Comp. Cancer Ctr., Chapel Hill, NC_.

Despite advances in our understanding of the mutational landscape in pancreatic ductal adenocarcinoma (PDA), this devastating disease is now the third-leading cause of U.S. cancer-related deaths. While recent successes of cancer immunotherapy have generated considerable excitement, this form of treatment has been largely ineffective in patients with pancreatic cancer. A major barrier for immunotherapeutic approaches is marked immunosuppression within the PDA milieu. We have previously identified a novel role for IL-35 producing B regulatory cells in the pathogenesis of pancreatic cancer. However, little is known about the mechanisms behind IL-35 activity in cancer. The overarching goal of this project is to elucidate molecular and cellular mechanisms by which IL-35 facilitates the emergence of pancreatic cancer. Our results demonstrate that IL-35, but not IL-10, potentiates PDA growth. This correlates with induction of immunosuppressive T regulatory cells (Treg) and suppression of T effector cell (Teff) and cytotoxic CD8+ T cell activity, suggesting that IL-35 controls anti-tumor immune responses in PDA. Remarkably, while PDA is typically resistant to anti-PD1 immunotherapy, IL-35 deficiency converted PDA from immunologically 'cold' to 'hot' tumor, which allowed for a robust synergistic reduction in tumor growth in response to anti-PD-1 treatment. Insights gleaned from these and further mechanistic studies of IL-35 in PDA may be expeditiously translated into IL-35 targeted combination immunotherapy.

#4675

B-cell and T-cell repertoire sequencing enables somatic hypermutation and minimal residual disease assessment.

Chen Song, Pingfang Liu, Andrew Barry, Eileen T. Dimalanta, Fiona J. Stewart, Salvatore Russello, Theodore B. Davis. _New England Biolabs, Ipswich, MA_.

The study of complex immunological diseases has progressed through recent developments that enable the sequencing of the immune repertoire. Using this approach, the interrogation of disease progression is facilitated through analysis of millions of V(D)J combinations from both B-cell antibodies and T-cell Receptors (TCR). One major challenge of immune repertoire sequencing is to capture the structural and sequence complexities of antibody and TCR genes. We have developed and optimized a method for accurate sequencing of full-length immune gene repertoires of B-cells and T-cells. RNA extracted from PBMCs were used to generate immune sequencing libraries in duplicate from a single patient. Using a unique barcoding scheme specifically designed to discretely barcode each mRNA molecule with a unique molecule index (UMI), PCR copies of each mRNA fragment can be collapsed into a single consensus sequence, improving accuracy through the resolution of PCR bias and sequencing errors. Utilization of the UMIs enabled absolute quantification of input RNA molecules and accurate ranking of the antibody/TCR clone abundance. This approach was also applied to the analysis of tumor samples for abundance of expanded clones through clustering based on V gene, J gene and CDR3 similarity and ranking by mRNA abundance. The use of isotype-specific primers (IgM, IgD, IgG, IgA and IgE) enabled measurement of the heavy chain isotype proportions within the samples. Full-length heavy chain antibody sequences generated using this method were aligned to germline genes from reference databases, enabling quantitation of the mutation level of each antibody sequence, providing information on the overall maturity and mutational profile of the sample repertoire. Additionally, this approach was capable of detecting minimal residual diseases by sensitive quantification of TCR rearrangement. This novel method allows for exhaustive somatic mutation profiling across complete V, D and J segments and full isotype information analysis. The information obtained enables the possibility for synthesis and expression of complete antibody chains and T-cell receptor chains for downstream immunological assays.

#4676

Spontaneous regression of Merkel cell carcinoma is driven by adaptive immune activation and clonal T cell expansion.

Mairead Baker,1 John W. Roman,2 Alexandre Reuben,3 Natasha Hill,1 Courtney W. Hudgens,3 Michael Tetzlaff,3 Nicholas F. Logemann,2 Isaac Brownell1. 1 _National Institutes of Health, Bethesda, MD;_ 2 _Walter Reed National Military Medical Center, Bethesda, MD;_ 3 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin tumor. MCC is immunogenic and highly responsive to immune checkpoint inhibitors. On rare occasions, MCC undergoes spontaneous regression of both local and metastatic disease in the absence of treatment. With at least 45 cases reported in the literature, this phenomenon has been described in 1.7-3% of MCCs but its mechanism is poorly understood. There has been speculation that biopsy-induced antigen shedding and subsequent recruitment of a host immune response drives spontaneous tumor regression. Here, we demonstrate activation of an adaptive immune response in a case of spontaneous MCC regression. A 65 year-old man presented with a rapidly-growing 3.5 cm skin tumor confirmed by histopathologic and immunohistochemical analyses to be Merkel cell polyomavirus-positive MCC. Eight days following the diagnostic shave biopsy, the tumor had clinically regressed. Histological analysis of a wide local excision specimen obtained 45 days after the initial biopsy revealed small, residual tumor nests and a surrounding lymphohistiocytic inflammatory infiltrate consistent with a regressing tumor. To evaluate the immune response at baseline and in the regressing tumor, we performed T cell receptor (TCR) sequencing as a metric of antigen-specific lymphocyte activation, and quantitative immunohistochemical analyses to phenotype the immune response. Both the baseline and regressing tumor contained intratumoral inflammatory infiltrates which were sparse relative to peritumoral infiltrates. There were notable reductions in CD4+ T cells and CD68+ macrophages in the regressing tumor relative to the initial biopsy. TCR clonality was increased in the regressing tumor relative to the baseline lesion, including both increased representation of the most dominant T cell clone from the baseline biopsy as well as emergence of new high-frequency clones. Together, these results suggest that spontaneous MCC regression is driven by expansion of novel and pre-existing adaptive cellular immune responses. Consistent with this, we observed analogous results with immunohistochemical staining and TCR sequencing performed in an MCC tumor regressing during treatment with the PD-L1 inhibitor, avelumab. Although further studies are needed to determine how biopsy unmasks immune responses and to elucidate target antigens of the T cell clones driving spontaneous MCC regression, our observations demonstrate that comparable immune activation underlies both spontaneous tumor regression and MCC response to immunotherapy.

#4677

A new pipeline to predict and confirm tumor neo-antigens.

Nicholas M. Durham, Yelena Lazdun, Han Si, Todd Creasy, Brandon W. Higgs, Katie Streicher. _MedImmune, Gaithersburg, MD_.

Identification of tumor-specific neo-antigens is crucial to developing new immunotherapeutic approaches, e.g. personalized vaccines. While computer algorithms can predict Class I or II HLA specific neoantigens, experimental confirmation of these potentially immunogenic neoantigens remains challenging, primarily due to the complexity associated with understanding the MHC-antigen and T cell receptor interaction. Here, we developed a systematic pipeline to efficiently identify immunogenic neoantigens and evaluate their immunogenicity in healthy donors.

Whole-exome sequencing(WES) of a melanoma was used to identify non-synonymous mutations. MHC presentation was predicted with digital HLA typing and MHC class I allele-restricted binding affinity/stability algorithms (netMHCpan/netMHCstab). The tumor clonal architecture was identified for each peptide to prioritize clonal over subclonal neoantigens using BubbleTree (https://bioconductor.org/packages/release/bioc/html/BubbleTree.html). Immunogenicity testing was done using a novel in-vitro assay with blood from healthy HLA-A201 donors. Naïve CD8 T cells were stimulated with autologous dendritic cells pulsed initially with peptide pools followed by individual peptides. The presence of antigen-specific T cells was determined via MHC-multimer staining and function was assessed by intracellular staining and IFNy EliSpot.

Nonsynonymous somatic mutations (N = 205) were identified by WES from the melanoma tumor biopsy. From a library of mutated tumor-specific peptides (N = 7512), 18 candidates predicted to be Class I neo-antigens were selected for immunogenicity testing. A new in-vitro assay using blood from healthy HLA-A201 donors identified one peptide that expanded antigen-specific T cells. Analysis of this mutation showed a VAF of 29.3% and TCGA revealed that the mutation identified was unique. IFNy secretion indicated that these antigen-specific T cells produced 6-fold higher IFNy to the mutant peptide vs the wildtype peptide. Direct binding of peptide to HLA was confirmed in vitro. Interrogation of the peptide expanded T cells by TCRseq showed clonal T cell expansion in response to the peptide. Unique TCR sequences identified in these T cells were present in the original melanoma tumor biopsy but not in adjacent tissue, confirming that naïve T cell donors can be used to validate neoantigens identified in cancer patients. The same TCR sequences were also identified in the peptide pool stimulated cells, suggesting that deconvolution may not be necessary to monitor neoantigen responses.

Using a newly developed pipeline combining next generation sequencing, epitope and clonal prediction algorithms, as well as an in-vitro assay to screen and evaluate putative neoantigens as targets of antitumor immunity, we confirmed one predicted neo-antigen from a melanoma patient. This pipeline could allow for the efficient and rapid identification of personalized neoantigens.

#4678

High-throughput single-cell linking of antigen specificities with T cell receptor sequences using de novo generated DNA-linked MHC tetramers.

Ning Jiang,1 Shu-qi Zhang,1 Chenfeng He,1 Eric Sun,1 Mingliang Zhang,2 Weiping Jia3. 1 _University of Texas at Austin, Austin, TX;_ 2 _Shanghai Jiao Tong University Affiliated Sixth People's Hospital, China;_ 3 _Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China_.

Identification of correlated antigens recognized by T cell and its T cell receptor (TCR) sequences at single cell level is extremely useful to the understanding and treating immune-related diseases. Although mass cytometry, CyTOF, has pushed the number of antigenic peptides that can be interrogated at once to around 100, the destructive nature of CyTOF prohibits linking pMHCs recognized by a T cell with its TCR receptor sequences. In addition, the high cost associated with peptide synthesis process prevents the quick generation of a peptide library that can be tailored to any pathogens or diseases. Here we describe a new technology, TetATCR-Seq for Tetramer Associated TCR sequencing, that is capable of de novo generating a large amount of peptides and mapping antigen recognition with single cell TCR sequences by DNA barcoded tetramers. It evolves three major steps: 1) de novo peptide generation, 2) DNA barcoded tetramer generation, and 3) single cell sequencing of tetramer DNA barcode and TCR sequences. By spiking in 5 altered peptide ligands (APL) that are known to bind to a HCV T cell clone with other 90 irrelevant peptides, we showed that the binding of all these 5 APLs can be faithfully detected by sequencing while non-specific binding is minimum. Using a published set of cancer neo-antigen sequences, we showed that these peptides and their wild type counterparts can be quickly generated. Resulted tetramers can be used to clearly identify a population of neo-antigen only or wild type antigen only or double positive T cells. Subsequent single cell TCR sequencing showed that TCR paired α and β chain sequences can be obtained in more than 80% of single sorted T cells. Simultaneous tetramer DNA barcode sequencing showed that many T cells are found to cross-reactive to both neo-antigen and its wild type counterpart, suggesting the necessity of strict screening for neo-antigen only TCR in adoptive cell transfer therapy. In summary, the TetaTCR-seq tool we developed here enables one to quickly survey antigen repertoire again TCR repertoire at single cell level for a large number of peptides. This is an extremely useful tool in profiling T cell repertoire reactivity in infection and vaccination. It is also a useful tool to screen for T cell cross-reactivity and select neo-antigen specific TCR for cancer immunotherapy.

#4679

Rectal cancer-infiltrating T cells show clonal expansion associated with spatially restricted tolerogenic phenotypes.

Livius Penter,1 Kerstin Dietze,1 Felix Aigner,1 Lars Bullinger,1 Thomas Blankenstein,2 Leo Hansmann1. 1 _Charité - Universitätsmedizin Berlin, Berlin, Germany;_ 2 _Max-Delbrück-Center for Molecular Medicine, Berlin, Berlin, Germany_.

T cell infiltration correlates with prognosis and outcome in colorectal cancer regardless of the tumor stage. However, data on clonal expansion, phenotypes, functions, and spatial distribution of tumor-infiltrating T cells (TILs) are limited.

We hypothesized that subsets of clonally expanded rectal cancer-associated T cells are specifically recruited into the tumor and show characteristic phenotypes and functions.

Paired TILs and T cells from adjacent unaffected mucosa were isolated from five treatment-naïve rectal cancer patients. Although T cell immune phenotypes were heterogeneous, the frequencies of CD38+, PD-1+, and TIM-3+ cells were significantly higher in CD8+ TILs when compared to T cells from unaffected mucosa (p < 0.05). To track clonal expansion and phenotypes at the single cell level, we combined 13-parameter FACS single cell index sorting with next generation T cell receptor (TCR) and phenotype sequencing. Paired TCRαβ sequences were obtained from 1237 TILs (on average 412 TILs per patient) and 1257 T cells infiltrating the unaffected mucosa (on average 419 T cells per patient) from three selected patients. Clonal T cell expansion was not restricted to TILs and occurred predominantly in CD8+ T cells (p < 0.05). Expanded TIL clones were more frequently TIM-3+, PD-1+, and CD38+ (p < 0.05) and occupied characteristic phenotype compartments when compared to T cells from unaffected mucosa with t-stochastic neighbor embedding (t-SNE) visualization. FOXP3 expression was enriched in non-expanded TILs (p < 0.05).

Only 11 out of 179 TIL clones were also detectable in unaffected mucosa of the same patients and these overlapping clones were exclusively PD-1- TIM-3-.

In conclusion, rectal cancer is infiltrated by oligoclonal T cells. Selected clones were exclusively detected in TILs suggesting their expansion and recruitment driven by tumor-associated cues. Tolerance was induced i) in an antigen-specific manner by the expression of immune checkpoint molecules predominantly on clonally expanded TILs, and ii) in an antigen-independent manner by infiltrating polyclonal FOXP3+ regulatory T cells.

#4681

**CD8** + **elite controller responses for cancer immunotherapy.**

Mariela A. Moreno Ayala, Alexandra Tsitsiklis, Derek Bangs, Shiao W. Chan, Ellen A. Robey. _University of California, Berkeley, Berkeley, CA_.

Background. Cancer immunotherapy seeks to stimulate the immune system to induce tumor rejection and develop immunological memory that can inhibit tumor recurrence or metastasis. CD8+ T cells are a key immune population during tumor rejection. The CD8 T cell receptor (TCR) recognizes the antigen presented by the MHC-I, leading to the activation and proliferation of antigen-specific CD8+ T cells. MHC-I genes are highly polymorphic and some alleles have been associated with control of infectious diseases. In mice, the MHC (known as H-2) allele, Ld, is important during Toxoplasma gondii infection. This allele is able to bind a peptide derived from T. gondii, called HF10, inducing a strong and persistent CD8+ response that allows the survival of BALB-c mice (H2d, Ld+). A remarkable characteristic of this immune response, is that CD8+T cell do not become exhausted in spite of persistent presentation of the HF10 antigen. This response is reminiscent of the protective anti-HIV T cell response in elite controllers, including the association with unusual MHC-1 alleles which share related structural features with mouse MHC-1 Ld. These protective MHC-1 alleles appear to act as "specialists" that exhibit limited peptide binding, in contrast to "generalist" alleles, that exhibit higher promiscuity in peptide binding. We hypothesized that the factors that make HF10-Ld interaction effective in chronic infection will be also useful for tumor immunotherapy. Methods: We studied two different tumor models derived from BALB-c mice, CT26, a murine colon carcinoma and 4T1 which is a model for triple negative breast cancer. Cells were genetically engineered to express antigens with different MHCI allelic restrictions. Taking advantage that AH1 antigen is express by several tumor cell lines and is Ld restricted, we designed peptides with amino acid substitutions to improved Ld binding. Results: Our data shows that expression of HF10 in BALB/c-derived tumor cell lines leads to the appearance of specific intratumor CD8 T cells coupled with a strong reduction in tumor growth and increased mice survival. Moreover, specific amino acid substitutions in the preexisting tumor antigen AH1 can increase Ld affinity, evaluated by RMA-s assay. In mice bearing tumors, inoculation with these modified peptides can delay tumor growth and increase mice survival. Conclusions. Our results suggest that tumor by antigens presented by the MHC-1 specialist Ld, have the potential to generate exhaustion-resistant anti-tumor responses. We propose that therapeutically harnessing elite controller T cell responses can be important to target potent CD8 T cell responses in appropriate patients.

#4682

IRF8 controls T cell development and survival to regulate T cell antitumor activity.

John D. Klement,1 Amy V. Paschall,1 Mary A. Zimmerman,1 Mohammed L. Ibrahim,1 Priscilla S. Redd,1 Chunwan Lu,1 Hussein Sultan,1 Esteban Celis,1 Keiko Ozato,2 Kebin Liu1. 1 _Augusta University, Augusta, GA;_ 2 _NIH, Bethesda, MD_.

Interferon Regulatory Factor 8 (IRF8, or ICSBP1) is a member of the Interferon Regulatory transcription factor family, and functions as a key hematopoietic transcription factor. Loss of IRF8 leads to defective antigen-presenting cell activity, perturbations in B cell development and, in mouse models, an accumulation of CD11b+Gr1+ immature myeloid cells. However, the role of IRF8 in T cell development and antitumor activity remains unclear. Whole body and chimeric IRF8-knockout mice (IRF8-KO) demonstrate increased susceptibility to both allogenic transplant and carcinogen-induced tumor models. T cell function is crucial for the immune system's endogenous antitumor response. Analysis of the T cell compartment of IRF8-KO mice demonstrated a deficiency in both naïve T cell percentages and total number. Despite this peripheral decrease in T cell numbers, early T cell progenitors in both the bone marrow and thymus were significantly increased in IRF8-KO mice compared to wild-type. To further investigate the role of IRF8 in T cell development and survival, IRF8-KO:WT mixed chimera mice were generated by lethal irradiation of CD45.1+CD45.2+ recipient mice, followed by transfer of CD45.2+ IRF8-KO and CD45.1+ WT bone marrow (BM). Surprisingly, analysis of blood obtained from reconstituted mice demonstrated preferential engraftment and survival of T cells derived from WT, rather than IRF8-KO BM. This imbalanced phenotype was not rescued by increasing the proportion of IRF8-KO BM administered to mice, suggesting the effect was not due to failure of IRF8-KO BM engraftment. Furthermore, analysis of T cell populations in both primary (thymus) and secondary (spleen) lymphoid organs showed a progressive loss of IRF8-KO T cells during their maturation and development process, while WT T cells remained unaltered. Given that IRF8 has been shown in tumor cells to regulate a variety of pro- and anti-apoptotic molecules, we hypothesized that IRF8 controls the peripheral survival of T cells. To test this hypothesis, resting T cells were isolated from the spleen of mixed chimera mice and viability was measured by Annexin V/PI staining. Resting IRF8-KO cells, but not WT, demonstrated a pro-apoptotic phenotype, as shown by increased Annexin V staining. Accordingly, upon in vitro stimulation and activation, IRF8-KO T cells demonstrated increased apoptosis. Our data determine that IRF8 controls both T cell development and peripheral survival, and that loss of IRF8 impairs the T cell antitumor immune response.

#4683

Distinct subsets of dysfunctional CD8+ T cells underlie response to checkpoint blockade.

Brian C. Miller,1 Debattama R. Sen,1 Rose Al-Abosy,1 Kevin Bi,1 Kathleen B. Yates,1 Evisa Gjini,1 Kristen Felt,1 Robert T. Manguso,1 Scott J. Rodig,1 Arlene H. Sharpe,2 Nicholas Haining1. 1 _Dana-Farber Cancer Inst., Boston, MA;_ 2 _Harvard Medical School, Boston, MA_.

T-cell exhaustion is a heterogeneous state, with recent work in chronic viral infections revealing at least two subtypes with different functional properties: "stem-like" and "terminal" exhausted cells. Whether these two populations exist in tumors and have different roles to control tumor growth remains unknown. We performed single-cell RNA-seq on exhausted CD8+ T cells from chronic virally infected mice, generating unique transcriptional signatures of both exhausted populations. By transcriptional enrichment and flow cytometry, we discovered both the stem-like and terminal exhausted populations in the B16 mouse melanoma model and confirmed their presence in human melanoma samples with multiplex immunofluorescence. ATAC-seq revealed that these two subpopulations are defined by state-specific changes in chromatin accessibility. These states are shared between exhausted CD8+ T cells from both viral and tumor models, demonstrating a common epigenetic program of T-cell dysfunction. The two populations have different functional properties, with the stem-like cells having a more polyfunctional cytokine response whereas terminal cells are more cytotoxic. When transferred into new tumor-bearing mice, stem-like cells were better able to control tumor growth, suggesting superior long-term functionality. Checkpoint blockade with anti-PD-1 is known to increase CD8+ T cell numbers in the tumor microenvironment, but it is not known which subpopulation responds to therapy. Treatment with anti-PD-1 antibody resulted in proliferation of the stem-like population, which differentiate into the more cytotoxic terminal exhausted cells. We have shown that two subpopulations of exhausted CD8+ T cells exist in mouse and human tumors, and anti-PD-1 therapy activates the stem-like CD8+ T cells to promote tumor control.

#4684

NSD1 inactivation defines an immune cold, DNA hypomethylated subtype in squamous cell carcinoma.

Kevin Brennan,1 Olivier Gevaert,1 John B. Sunwoo,2 June Ho Shin2. 1 _Stanford University School of Medicine, Stanford, CA;_ 2 _Stanford University Department of Otolaryngology, Stanford, CA_.

Normal 0 false false false EN-US JA X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:Cambria; mso-ascii-font-family:Cambria; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Cambria; mso-hansi-theme-font:minor-latin;} Chromatin modifying enzymes are frequently mutated in cancer, resulting in widespread epigenetic deregulation. Recent reports indicate that inactivating mutations in the histone methyltransferase NSD1 define an intrinsic subtype of head and neck squamous cell carcinoma (HNSC) that features pronounced DNA hypomethylation. We have identified a similar hypomethylated subtype of lung squamous cell carcinoma (LUSC) that is enriched for both inactivating mutations and deletions in NSD1. We identified cancer DNA methylation subtypes by first applying the MethylMix algorithm to whole genome DNA methylation and gene expression data from The Cancer Genome Atlas to identify DNA methylation deregulated genes, followed by consensus clustering to patients based on their profiles of these deregulated genes. The 'NSD1 subtypes' of HNSC and LUSC are highly correlated at the DNA methylation and gene expression levels, featuring ectopic expression of developmental transcription factors and genes that are also hypomethylated in Sotos syndrome, a congenital disorder caused by germline NSD1 mutations. Further, the NSD1 subtype of HNSC displays an 'immune cold' phenotype characterized by low infiltration of tumor-associated leukocytes, particularly pro-inflammatory M1 macrophages and CD8+ T cells, as well as low expression of genes encoding the immunotherapy target PD-1 immune checkpoint receptor and its ligands. Using an in vivo model, we demonstrate that NSD1 inactivation results in reduced T cell infiltration into the tumor microenvironment, implicating NSD1 as a tumor cell-intrinsic driver of an immune cold phenotype. NSD1 inactivation therefore drives widespread oncogene expression across cancer sites, and has implications for immunotherapy. <!--EndFragment-->

#4685

**Phenotypic difference of CD103** + **tissue-resident memory T cells in various cancers.**

Hye Seon Park, Young-Ae Kim, Won Seon Bang, Heejae Lee, Miseon Lee, In Ah Park, In Hye Song, Sun-Hee Heo, Hyeonjin Lee, Hee Jin Lee, Gyungyub Gong. _University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea_.

Background: The presence and clinical importance of tissue-resident memory T cells (TRM) have been recently described in human lung, liver, and bladder cancer. However, the frequency and phenotypic characteristics of TRM in other tumors are largely unknown.

Methods: We analyzed single-cell populations of renal cell carcinoma (n=20), breast (n=16), colorectal (n=20), and stomach cancer (n=15) by dissociation of tumor tissue with collagenase/hyaluronidase. We investigated population of memory T cells and TRM using anti-CD45RO or anti-CD103 antibodies by flow cytometry.

Results: Colorectal cancer had higher level of CD4+ T cells (70.1%) among CD3+ T cells than other tumors (stomach cancer, 59.6%; renal cell carcinoma, 49.4%; breast cancer, 49.0%; p=0.001). In CD4+ T cells, the percentage of CD45RO+ memory T cells was higher in renal cell carcinoma (75.7%) and breast cancer (85.4%) than colorectal (36.9%) and stomach cancer (38.1%, p<0.001). About 4% of CD4+ T cells in renal cell carcinoma, colorectal and stomach cancer were TRM, while 14.3% of CD4+ T cells were TRM in breast cancer (p<0.001). The percentage of CD45RO+ memory T cells among CD4\+ TRM was about 60% in all tumors (p=0.164). In CD8+ T cells, the percentage of CD45RO+ memory T cells was higher in renal cell carcinoma (73.5%), breast (66.4%) and stomach cancer (54.4%) than colorectal cancer (33.0%, p<0.001). Stomach cancer had higher level of TRM (74.3%) among CD8\+ T cells than other tumors (colorectal cancer, 19.6%; renal cell carcinoma, 20.1%; breast cancer, 30.6%; p<0.001). The percentage of CD45RO\+ TRM in renal cell carcinoma (89.3%) and breast cancer (89.5%) was higher than colorectal (43.0%) and stomach cancer (51.5%, p<0.001).

Conclusions: The frequency and phenotypic characteristics of TRM in renal cell carcinoma, colorectal, stomach, and breast cancer were different. Further studies regarding functional difference and clinical significance of TRM in various tumors are warranted.Acknowledgement: This study was supported by Basic Science Research Programs through the National Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT & Future Planning, Republic of Korea (NRF-2017R1D1A1B03033104).

#4686

T cell repertoire evolution from the normal lung to invasive lung adenocarcinoma.

Runzhe Chen,1 Junya Fujimoto,1 Alexandre Reuben,1 Lisha Ying,2 Xin Hu,1 Chi-Wan Chow,1 Jaime Rodriguez Canales,1 Wenyong Sun,2 Jinlin Hu,2 Edwin R. Parra Cuentas,1 Carmen Behrens,1 Chang-Jiun Wu,1 Latasha Little,1 Curtis Gumbs,1 Diana Wiesnoski,1 Guangchun Han,1 Won-Chul Lee,1 Paul Scheet,1 Humam Kadara,1 Mara Antonoff,1 Ara A. Vaporciyan,1 Stephen Swisher,1 Jianhua Zhang,1 John Heymach,1 Waun Ki Hong,1 Ignacio Wistuba,1 Andrew Futreal,1 Dan Su,2 Jianjun Zhang1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Zhejiang Cancer Hospital, Hangzhou, China_.

Background: Our knowledge in early lung carcinogenesis is rudimentary. Atypical adenomatous hyperplasia (AAH) is the only preneoplasia type that has been recognized by histopathology and It has been postulated that AAH can progress to adenocarcinoma in situ (AIS), to minimally invasive adenocarcinoma (MIA), and finally to invasive adenocarcinoma (ADC). However, the definition of these lesions remains controversial due to limited supporting molecular evidence. Carcinogenesis results from accumulation of multiple genetic and epigenetic events, with selection for events conferring phenotypic advantage. Immune surveillance, particularly anti-tumor response from T cells is an important host protection process to inhibit carcinogenesis. However, the T cell repertoire landscape and its evolution during lung carcinogenesis have not been well defined. Methods: We performed T cell receptor (TCR) sequencing on multiple spatially separated regions (2-6 regions per lesion) from 23 AAH, 26 AIS, 54 MIA and 14 ADC lesions and paired histologically normal lung tissues from 52 patients clinically presenting with indeterminate pulmonary nodules. Forty-one patients had multifocal diseases and 23 patients carried more than one type of pathology. Results: Compared to pre-/micro-invasive neoplastic lesions (AAH, AIS, MIA) or invasive ADC, normal lung tissues demonstrated significantly less T cell infiltration (p<0.0001) but a significantly higher clonality (p<0.0001). Furthermore, T cell diversity increased and as did evenness in pre-/micro-invasive neoplastic lesions (AAH, AIS, MIA) (p<0.0001) and invasive ADC (p<0.005) accompanied with higher frequency of the top T cell clones observed in the normal lung (p<0.05). Interestingly, homology with normal lung T cell repertoire decreased in invasive ADC compared to pre-/micro-invasive neoplastic lesions (p<0.05). Distinct T cell repertoire overlap was also observed between pre-/micro-invasive neoplastic lesions and invasive ADC (p<0.01). Conclusions: Our preliminary analyses demonstrate the distinct T cell repertoire between the normal lung and pre-/micro-invasive neoplastic lesions and invasive ADC, and that immunosuppression of T cells may have occurred prior to the development of pre-neoplastic states and T cell repertoire becomes progressively suppressed with disease evolution. The distinct overlap of pre-/micro-invasive neoplastic lesions and invasive ADC suggests that with disease evolution, the immune microenvironment of invasive ADC increases in complexity. Integration of the molecular landscape with T cell and immune profiling across different stage lesions is underway.

#4687

Double positive (DP) CD4+CD8+ T cells with an exhausted phenotype in renal cell carcinoma (RCC) patients.

Laurence Menard, Paul Fischer, Bijal Kakrecha, Deborah Lee, Becky Penhallow, Nataly Manjarrez Orduno, Steven Nadler. _Bristol Myers Squibb, Princeton, NJ_.

Checkpoint inhibitors target the inhibitory receptors expressed by tumor infiltrating T cells in order to reinvigorate an anti-tumor immune response. Therefore, understanding T cell composition and phenotype in human tumors is crucial. We analyzed by flow cytometry tumor infiltrating lymphocytes from two independent cohorts of patients with different cancer types, including RCC, lung and colon cancer. T cells are usually either CD4+ or CD8+ with a small percentage of CD4+ CD8+ DP cells (<5%). Compared to several other cancer types, including lung and colorectal cancers, about a third of RCC patients showed an increased proportion of DP CD4+CD8+ T cells (>5%, reaching 30-50% of T cells in some patients). These DP T cells express high level of PD1 and TIM-3 that tend to correlate with higher expression of PD1 and TIM-3 in conventional CD8 T cells. DP T cells also express markers of antigen-experienced T cells such as CD38 and HLA-DR. These results suggest that double positive T cells might be exhausted tumor specific T cells with the potential to be reactivated by checkpoint inhibitors.

#4688

Single-cell discrimination of altered human T cell states in the bladder tumor microenvironment.

David Yoonsuk Oh,1 Serena S. Kwek,1 Serghei Mangul,2 Siddharth S. Raju,1 Sasha Targ,1 Arun Burra,1 Eric Chow,1 Dvir Aran,1 Sima Porten,1 Maxwell V. Meng,1 Terence W. Friedlander,1 Chun Jimmie Ye,1 Lawrence Fong1. 1 _University of California, San Francisco, San Francisco, CA;_ 2 _University of California, Los Angeles, Los Angeles, CA_.

Bladder cancer can be responsive to immunotherapies such as anti-PD-1 and anti-PD-L1 checkpoint inhibitors, but overall response rates are low. Tumor-resident T cells demonstrate considerable heterogeneity as far as antigenic repertoire and phenotype. Whether the bladder tumor environment is enriched for specific types of T cells with a particular functional profile and antigenic specificity, and whether these tumor-specific populations are associated with treatment or response to immunotherapy, remains unclear. We performed single-cell RNA sequencing of CD4+ and CD8+ T cells from localized human bladder tumors and paired adjacent non-malignant bladder. We also assessed resected bladder tumors treated with neoadjuvant chemotherapy or atezolizumab (anti-PD-L1) as part of an ongoing phase II trial. We find both known and unexpected CD4+ T cell functional populations that are specific to the tumor microenvironment, including regulatory T cells and a novel population of cytotoxic CD4+ T cells. Furthermore, by combining whole-transcriptome data with paired T cell receptor identification on single cells, we find that tumor-specific CD4+ populations are clonally expanded and utilize an antigenic repertoire which is distinct from uninvolved bladder. These findings provide evidence for specialization of both phenotype and antigenic specificity of CD4+ T cells in the bladder tumor environment, and indicate that a limited number of tumor-specific antigens may drive in situ expansion and differentiation of specialized CD4+ subsets whose function may be critical to tumor control.

#4689

Immune functions and regulation of follicular helper CD4+CXCR5+T cells in human breast cancer.

Mireille Langouo,1 Gregory Noël,1 Gert Van den Eynden,2 Alexandre De Wind,1 Soizic Garaud,1 Pushpamali De Silva,1 Cinzia Solinas,1 Anais Boisson,1 Celine Naveaux,1 Hugues Duvillier,1 Ligia Craciun,1 Denis Larsimont,1 Martine Piccart-Gebhart,1 Karen Willard-Gallo1. 1 _Institute Jules Bordet-ULB, Brussel, Belgium;_ 2 _ĢŻǺ Ħǿșpįțǻl Șįňț-Ǻųģųșțįňųș, Ǻňțẅěřp, Belgium_.

Introduction: High levels of tumor infiltrating lymphocytes (TIL) have been associated with good clinical outcomes in patients with HER2-positive (HER2+) and triple-negative (TN) breast cancer (BC) . Recently, we demonstrated that 60% of BC TIL are organized in tertiary lymphoid structures (TLS) located in the stroma. We further identified a CXCL13-producing CD4+ T follicular helper cell (Tfh) subpopulation and demonstrated that this chemokine, important for TLS formation, is associated with positive clinical outcomes in BC. The aim of the present study was to investigate how conventional CD4+ Tfh cells, expressing the CXCL13 receptor CXCR5, contribute to immune function and regulation in BC-associated TLS.

Methods: We prospectively collected fresh primary BC tissues and prepared enzyme-free homogenates to prepare tumor supernatant and TIL for flow cytometric analysis and sorting. Matching formalin-fixed paraffin-embedded (FFPE) were used for subsequent spatial analysis by dual IHC and confocal microscopy.

Results: Flow cytometric analyses show that ~15% CD4+, ~13% CD8+ and >95% B cell TIL express CXCR5 while confocal microscopy reveals that these CXCR5\+ TIL subpopulations co-localize in BC TLS and their presence is tightly correlated. BC Tfh TIL (compared to activated tonsillar Tfh) have a memory phenotype and express ICOS and PD-1 suggesting they are activated, but BCL6 is undetectable and CXCL13 positive cells are rare in only some tumors. RNA analysis detected high expression of IL-21, IL-10 and CXCL13 in Tfh TIL. Investigation into the role of Tfh cells in TLS functionality was accomplished via an in vitro assay where Tfh TIL were activated with allogeneic splenic B cells. These experiments reveal that only ICOS+PD-1+Tfh TIL from TN/HER2+ BC are capable of inducing IgM and IgG secretion by B cells. A comparison of ICOS+PD-1+ with ICOS-PD-1- Tfh TIL confirms an activated, functional nature for the former characterized by high levels of IL-21, IL-10 and CXCL13 mRNA expression. We also found specialized follicular regulatory T cells (Tfr), expressing CXCR5, CD25, demethylated Foxp3 and GARP, localized in TLS. To understand the regulation of Tfh function(s) by Tfr we quantified immunoglobulins in the primary tumor supernatant, finding a correlation between the Tfh/Tfr ratio (fresh tissues) and IgG and IgM production (supernatant) in TN/HER2+ BC. These data suggest that the balance between effector and regulatory Tfh influences B cell differentiation in TLS.

Conclusions: We show that it is possible to isolate functional Tfh TIL from fresh BC tissues, demonstrate their activation and functional state and link a Tfr presence with negative regulation of TLS function. These data begin to shed light on anti-tumor immune responses occurring in TLS at the tumor site, whose functional activities may have important treatment implications, particularly for immunotherapy in BC.

#4690

**FoxP3** + **T cells program/re-program the prostatic tumor microenvironment.**

Sanjay Kumar,1 James Stoke III,1 Shalie Malik,2 Udai P. Singh,3 Selvarangan Ponnazhagan,4 Upender Manne,4 Manoj K. Mishra1. 1 _Alabama State University, Montgomery, AL;_ 2 _University of Lucknow, Lucknow, India;_ 3 _University of South Carolina School of Medicine, Columbia, SC;_ 4 _University of Alabama at Birmingham, Birmingham, AL_.

Foxp3+ T cells play a critical role in maintaining immunological tolerance by regulating functions of other immune cells, thus preventing autoimmunity. Previous studies have demonstrated the importance of Foxp3⁺ T cells in the tumor, however, the role increasingly recruited FoxP3⁺ T cells in the programming of prostatic tumor microenvironment is not well illustrated. Therefore, in this study, we have investigated the role of Foxp3⁺ T cells in the prostatic tumor microenvironment with the notion that increased Foxp3⁺ T cells facilitate tumor growth. To accomplish this, we carried out tumor induction studies in C57/BL-6 mice using transgenic adenocarcinoma of mouse prostate (TRAMP cell lines: -C1, -C2 and -C3) as tumor models. Mice were administered with TRAMP (C1-3) cells using the Matrigel system. TRAMP-C3 cells are non-tumorigenic while TRAMP-C1 and TRAMP-C2 cells form tumors. Mice were sacrificed by cervical dislocation when tumor sizes reached ~18-20mm in diameter. To prepare single cell suspension, spleen and tumor were dissected out and cells were stained with various cell surface as well as intracellular immune markers to determine the number of Foxp3⁺ T cells and other immune cells in the prostatic tumor microenvironment. Our findings suggest that the high number of Foxp3⁺ T cells was found in the spleen and tumor of TRAMP-C1 and TRAMP-C2 mice than the spleen and tiny tumor of TRAMP-C3 mice. Furthermore, the number of cytotoxic T lymphocytes and NK cells were also observed to be low in the tumorigenic TRAMP-C1 and -C2 mice as compared to non-tumorigenic mice (TRAMP-C3). Therefore, our findings suggest that the number of Foxp3⁺ T cells may play a critical role in shaping the prostatic tumor microenvironment.

#4691

Role of select T helper cell subsets in the development of Epstein-Barr virus-driven lymphoproliferative disease.

Elshafa H. Ahmed, Claire Hale, Shelby Sloan, Charlene Mao, Xiaoli Zhang, H. Gulcin Ozer, Deepa Subramaniam, Frankie Jeney, Sarah Schlotter, Porsha Smith, Wing Chan, Palak Sekhri, Christoph Weigel, Christopher C. Oakes, Gregory K. Behbehani, Jianhua Yu, Mireia Guerau, Michael A. Caligiuri, Robert A. Baiocchi. _The Ohio State University, Columbus, OH_.

Background: Post-transplant lymphoproliferative disease (PTLD) is a significant and often fatal complication of organ transplantation, strongly associated with Epstein-Barr virus (EBV). Severe combined immune-deficient (SCID) mice engrafted with human mononuclear cells (PBMCs) from EBV+ donors spontaneously develop human B cell lymphoproliferative disease (LPD) that resembles PTLD. Approximately 20% of EBV+ donors reproducibly develop LPD in 100% of engrafted mice (high-incidence, HI donors), and ~20% of donors never develop EBV-LPD (no-incidence, NI donors) despite all donors being EBV+. This finding suggests that host factor(s) may predispose individuals to develop EBV-LPD. PBMC from HI donors depleted of CD3+/CD4+ T helper (Th) cells do not develop EBV-LPD, suggesting that Th cells function as potential drivers of EBV-LPD.

Methods: PBMCs from three HI and three NI donors were engrafted intraperitoneally into SCID mice. At week 4, spleens were harvested and human Th cells were sorted to purity (>95%). Total RNA was isolated from sorted Th cells and gene expression profiles were evaluated by RNA transcriptome analysis. Sorted Th cells from total PBMCs (resting) were used as baseline controls. Human CD45+ cells from mouse spleens were analyzed by mass cytometry (Cytometry Time of Flight, CyTOF) using a multiparametric antibody panel to identify various Th subsets. To identify specific Th subsets essential to the development of EBV-LPD, PBMCs from 3 HI donors were depleted of Th subsets: follicular Th cells (Tfh), regulatory T cells (Treg) or Tfh/Treg cells.

Results: RNA Transcriptome data were analyzed using R DESeq2 for differential expression and revealed significant differences in genes associated with Tfh and Treg cell differentiation and function. Th cells sorted from mice engrafted with PBMCs from HI donors expressed high levels of IFNɣ receptor, IL6R, MHC class II, FoxP3, PRMT2 and SYK genes. Applying the analysis software viSNE to CyTOF data revealed significant differences in frequency of B cells, Tfh and Treg subsets between HI and NI. Mice engrafted with Treg depleted or Tfh/Treg depleted PBMCs showed significantly improved survival compared to control nondepleted group (p-value: 0.0008, 0.0145, respectively). There was no difference in engraftment efficiency. The Tfh depleted cohort also had improved survival as compared to the control group; however, this result was not statistically significant (p-value: 0.0941).

Conclusions: Significant differences in gene expression and immunophenotypic profiles exist between HI and NI donors in this spontaneous model of EBV-LPD. Depletion of Treg and Tfh subsets from PBMCs prior to engraftment significantly enhanced survival. These data suggest that select Th subsets promote EBV-LPD and provide justification for examining strategies to identify patients at risk for developing EBV-LPD in the pretransplant setting.

#4692

High density of cytotoxic and regulatory T-lymphocytes in gastric cancers with microsatellite instability.

Hyoung-Il Kim,1 Su-Jin Shin,2 Sang Yong Kim,1 Yoon Young Choi,1 Taeil Son,1 Jae-Ho Cheong,1 Woo Jin Hyung,1 Sung Hoon Noh1. 1 _Yonsei University College of Medicine, Seoul, Republic of Korea;_ 2 _Hanyang University College of Medicine, Seoul, Republic of Korea_.

Introduction: MSI-high (MSI-H) colorectal cancer is known to be associated with increased tumor-infiltrating lymphocytes (TILs) and good prognosis. In gastric cancer, however, MSI status has rarely been evaluated in accordance with TILs. The objectives of our study were to clarify the relationships between MSI status and antitumor host immune response and to identify the impact of these factors on the prognosis of gastric cancer.

Methods: We evaluated 345 patients with gastric cancer who underwent gastrectomy with MSI typing. The numbers of TILs in tumor center was counted after immunohistochemical staining with anti-CD3, CD4, CD8, Foxp3, and granzyme B to quantify the subset of TILs. To evaluate systemic immune response, differential white blood cell count and prognostic nutritional index (PNI) were obtained.

Results: Of the 345 patients, 57 were MSI-H and 288 were non-MSI-H groups. MSI-H tumors carried significantly higher numbers of CD8+ T-cells, Foxp3+ T-cells, GZB+ T cells and higher ratio of Foxp3/CD4 and GZB/CD8. The prognostic effect of TILs was different in MSI-H and non-MSI-H groups. All subsets of TILs were not significant prognostic factor for recurrent-free survival (RFS) and overall survival (OS) in MSI-H group. However, in non-MSI-H group, multivariate analysis showed that stage, PNI and CD4+ T cells was independent prognostic factor for RFS; on the other hand, stage, PNI and ratio of Foxp3/CD4 was independent prognostic factor for OS.

Conclusions: The number of subset of TILs and the prognostic influence of systemic and local immune response were different between MSI-H and non-MSI-H tumors. The immunogenicity caused by microsatellite instability and subsequent host immune response is associated with cancer progression and patient prognosis of gastric cancer.

#4693

Characterization of the tumor immune microenvironment in head and neck squamous cell carcinoma (SCCHN).

Farah Succaria,1 Pia Kvistborg,2 Elizabeth L. Engle,1 Tracee L. McMiller,1 Elizabeth Thompson,1 Alan E. Berger,1 John Haanen,2 Suzanne L. Topalian,1 Janis M. Taube1. 1 _Johns Hopkins Bloomberg~Kimmel Institute for Cancer Immunotherapy, Baltimore, MD;_ 2 _Netherlands Cancer Institute, Amderstam, Netherlands_.

Approximately 20% of advanced SCCHNs respond to anti-PD-(L)1. We aimed to inform rational combination therapy development, which might have greater efficacy, by understanding the potential immune effects of the oncogenic human papillomavirus (HPV) in SCCHN, and by characterizing potentially targetable immune checkpoints in the tumor microenvironment (TME).

Immunohistochemistry (IHC) was performed on archival SCCHN specimens from 27 treatment-naive patients. HPV testing with p16 IHC was confirmed by HPV genotyping. IHC for PD-L1 assessed percentage of positive tumor cells, and whether PD-L1 was expressed in an adaptive or constitutive pattern (i.e., PD-L1+ tumor cells juxtaposed to TILs or in the absence of TILs, respectively). PD-L1 on infiltrating immune cells was scored separately. IHC for CD3, CD4, CD8, CD20, CD68, FoxP3, PD-1, PD-L2, LAG-3, TIM-3, GITR and IDO was quantified using Halo image analysis for density of positive cells.

Among 27 SCCHN specimens, 14 were HPV(-) and 13 were HPV+ (one HPV58, 12 HPV16). In 19 of 27 specimens (70%), ≥5% of tumor cells had PD-L1 cell surface expression by IHC, including 9 HPV+ and 10 HPV(-) cases. Among 23 tumors expressing any level of PD-L1 (≥1% tumor cells+), 12 displayed an adaptive PD-L1 expression pattern, 5 a constitutive pattern, and 6 were mixed. All tumors were infiltrated by immune cells (ICs). Notably, 26 of 27 specimens (96%) contained PD-L1+ immune cells (range 5-80% of ICs expressing PD-L1). The proportion of PD-L1+ ICs exceeded PD-L1+ tumor cells in 21/26 (81%) cases. Neither the proportion of PD-L1+ tumor cells nor infiltrating immune cells correlated with tumor viral status. When compared to HPV(-) tumors, HPV+ tumors contained significantly higher densities of CD3+, CD4+, CD8+, CD20+, and PD-1+ cells (p<0.02); there was a trend towards an increased density of FoxP3+ cells in HPV+ tumors. However, when comparing specimens in which tumor cells were PD-L1+ vs. (-), none of these markers were differentially expressed. This may reflect heterogeneous mechanisms driving constitutive vs. adaptive PD-L1 expression patterns in SCCHN. In addition to finding abundant IDO expression in these specimens (>500 IDO+ cells/mm2 in 17/27 specimens), IDO was expressed by tumor cells as well as ICs in 12/27 (44%) cases (range 5-95% tumor cells+).

In summary, we found that both HPV+ and (-) SCCHNs abundantly express PD-L1 on tumor and/or stromal cells, although HPV+ tumors are more heavily infiltrated by ICs. Multiple other immune checkpoints are expressed in these tumors, providing options for therapeutic co-targeting. Tumor DNA sequencing is in progress to explore the genetic basis for constitutive vs. adaptive PD-L1 expression. These studies are expected to provide a comprehensive portrait of the SCCHN TME, with implications for future immunotherapy development.

Funded by the Bristol-Myers Squibb International Immuno-Oncology Network and NCI R01 CA142779

#4694

The contribution of Epstein-Barr virus to the impaired T-cell responses in nasopharyngeal carcinoma.

Hui Min Lee,1 Tracey Haigh,2 George SW Tsao,3 Paul G. Murray,2 Graham Taylor,2 Ian C. Paterson,1 Lee Fah Yap1. 1 _University of Malaya, Kuala Lumpur, Malaysia;_ 2 _University of Birmingham, Birmingham, United Kingdom;_ 3 _University of Hong Kong, Hong Kong, Hong Kong_.

Undifferentiated nasopharyngeal carcinoma (NPC) is a geographically skewed disease in which more than 80% cases are from southern China and Southeast Asia. NPC is consistently associated with Epstein-Barr virus (EBV) infection and characterized by a prominent lymphocyte infiltration. The consistent expression of EBV proteins in NPC cells has led to several clinical trials of adoptive T-cell therapy or vaccination to boost the immune response to EBV antigens. However, these treatments yield relatively modest clinical benefit, which may be due to varying degrees of immunosuppression in the tumor microenvironment. The lack of model systems to analyze the ability of therapeutically relevant T cells to recognize and kill NPC cells represents a fundamental limitation in our attempts to harness the immune system for clinical benefit. We used three NPC cell lines as targets in T-cell assays, together with a panel of established HLA-matched EBV-specific CD8 and CD4 T-cell clones. T-cell activity and killing were measured by IFN-γ ELISA and outgrowth assays, respectively. We demonstrate, for the first time, that NPC cells can be recognized and killed by EBV-specific T-cell clones in vitro, which suggests that tumor-derived factors inhibit such responses in vivo. These observations are consistent with the previous reported functional inactivation of EBV-specific cytotoxic T lymphocytes (CTL) in NPC patients. We hypothesized that EBV infection of NPC cells induces the secretion of immunosuppressive factors. The effect of conditioned media derived from an EBV-infected premalignant nasopharyngeal cell line, NP460htert, on T-cell activity was examined by treating several clones of EBV-specific CTLs in vitro. The conditioned media inhibited the secretion of IFN-γ from peptide-stimulated CTLs, indicating that secreted factors that could suppress CTL functions are induced following EBV infection in NP460htert cells. RNA sequencing analyses on NP460hTert and three NPC cell lines following EBV infection revealed a significant enrichment of genes whose proteins are located at extracellular region/matrix, including a number of immunomodulators. Our results suggest that EBV infection might induce the production of soluble molecules, which in turn suppress the antitumor T-cell responses. In conclusion, we have developed a system with which to study NPC-specific immune responses and shown that soluble factors induced by EBV infection can suppress T-cell activity. These model systems will be critical in the future to examine the effects of checkpoint inhibitors, secreted factors from cancer-associated fibroblasts or NPC cells upon T-cell activity, which will ultimately enhance the success of EBV-targeted immunotherapy strategies.

#4695

**Murine tumor growth suppression through CD8** + **CTLs via activated DEC-205** + **dendritic cells by sequential administration of α-galactosylceramide in vivo.**

Hideki Kogo, Hidemi Takahashi, Eiji Uchida. _Nippon medical school, tokyo, Japan_.

Cancer immunotherapy is mediated by the effective priming and activation of tumor-specific class I major histocompatibility complex molecule-restricted CD8+ cytotoxic T lymphocytes. DEC-205+ dendritic cells can cross-present the epitopes of captured tumor antigens associated with class I MHC molecules alongside co-stimulatory molecules to prime and activate tumor-specific CD8+ CTLs. Immunosuppressive tolerogenic DCs with reduced co-stimulatory molecules may be a cause of insufficient CTL induction. Hepa1-6-1 cells were established from the mouse hepatoma cell line Hepa1-6; these cells grow continuously after subcutaneous implantation into syngeneic C57BL/6 mice and do not prime CD8+ CTLs. Here, we show that the growing of tumors was suppressed by activated CD8+ CTLs with tumor-specific cytotoxicity through the administration of the glycolipid α-galactosylceramide, which is a compound known to stimulate invariant natural killer T cells and selectively activate DEC-205+ DCs. Moreover, we demonstrated that sequential repetitive intraperitoneal inoculation of α-GalCer every 48 hour appeared to convert tolerogenic DEC-205\+ DCs into immunogenic DCs with a higher expression of co-stimulatory molecules and a greater cross-presentation capacity, which primed CTL precursors and induced tumor-specific CD8+ CTLs within the tumor environment without activating iNKT cells. These findings provide a new basis for cancer immunotherapy to convert tolerogenic DEC-205+ DCs within tumors into immunogenic DCs via the sequential administration of an immuno-potent lipid/glycolipid, and activated immunogenic DCs expressing sufficient suitable co-stimulatory molecules will prime and activate tumor-specific CD8+ CTLs within the tumor to control tumor growing.

#4697

Immune cell infiltration and its clinical correlations in paired castration-naive and castration-resistant prostate cancers.

Ying-Chun Shen, Chia-Tung Shun, Ching-Ping Yeh, Jhe-Cyuang Kuo, Yu-Chieh Tsai, Chung-Hsin Chen, Chao-Yuan Huang, Yeong-Shiau Pu. _National Taiwan University Hospital, Taipei, Taiwan_.

Ineffectiveness of immune checkpoint inhibitors in patients with castration- resistant prostate cancer (CRPC) suggests that CRPC may harbor a profound immunosuppressive tumor microenvironment. However, immune tumor microenvironment of CRPC has never been well investigated due to limited availability of castration-resistant tumor tissue. To characterize the changes in immune tumor microenvironment during CRPC progression and identify disease stage which is potentially favorable for immunotherapy, we studied the densities of tumor-infiltrating immune cells by using the immunohistochemical method and manual counting in paired castration-naive (CN) and castration-resistant (CR) tumors. We identified 11 metastatic prostate cancer patients who had received palliative transurethral resection of prostate both before androgen deprivation therapy (ADT) and at the time when castration resistance emerged from the hospital database during 2006.1~2017.3. We also used prostatic tissue from 5 patients with benign prostate hyperplasia as the normal prostate control. The median age at diagnosis of PC patients was 74.7 (range: 62-85) year-old. The numbers of tumors with Gleason score 7, 8, 9 and 10 were 1, 1, 6, and 2, respectively. The median PSA level at diagnosis was 96.6 (range: 3.3-847.7) ng/ml. The median overall survival after initiation of ADT was 48.8 months. The median density of CD4+ T cells (no./mm2) was 182.3 (45.0-363.5) for normal prostate, 160.9 (range: 12.6-240.6) for CNPC and 69.4 (range: 31-499.6) for CRPC (non-statistically significant; n.s.). The median density of CD8+ T cells (no./mm2) was 12.5 (range: 32.5-220.7) for normal prostate, 65.7 (range: 3.7-195.6) for CNPC and 53.8 (range: 4.4-190.4) for CRPC (n.s.). The median density of CD20+ B cells (no./mm2) was 132.1 (range: 54.6-514.4) for normal prostate, 112.2 (range: 3.7-298.9) for CNPC and 47.6 (range: 7.0-137.3) for CRPC (n.s.). The median density of CD68+ macrophages (no./mm2) was 31 (range: 0.4-248.7) for normal prostate, 77.5 (range: 3.7-403.7) for CNPC and 31.7 (range: 2.2-180.8) for CRPC (n.s.). The tumor-infiltrating immune cell densities in CNPC did not correlate with PSA level, Gleason score or ADT response. Only high B cell density in CNPC was prognostic for overall survival (median: 23.9 months for patients with high tumor-infiltrating B cell density vs. 64 months for patients with low tumor-infiltrating B cell density; P=0.0463). In conclusion, immune tumor microenvironment of CNPC and CRPC is very similar and is not different from normal prostate. It suggests that immune checkpoint inhibitors may not be effective against CNPC unless combining with other treatment, such as ADT or radiotherapy, which can reprogram the tumor microenvironment toward more immunosupportive.

### Immunomodulatory Agents and Interventions 2

#4698

ONCR-1, a novel herpes simplex virus expressing MMP9 and ULBP3 transgenes, evokes potent oncolysis and development of antitumor immune responses.

Alexandra Hicks, Paola Grandi, Michael Paglia, Jingzang Miu, Cecilia Kwong, Jacqueline Gursha, Michael Ball, Weiguo Yao, Daniel Wambua, Terry Farkaly, Kyle Grant, Laura Viggiano Salta, Lorenz Ponce, Joseph Glorioso, Christophe Queva, Mitchell Finer. _Oncorus, Cambridge, MA_.

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor associated with poor prognosis and resistance to current therapies. ONCR-1 is a novel oncolytic herpes simplex virus type-1 vector in development for the treatment of GBM. ONCR-1 utilizes a unique conditional-lethal strategy in which miR124 binding sites are inserted into the ICP4 locus to prevent viral replication in neuronal cells while preserving one copy of the γ34.5 gene and enabling potent cytotoxicity in tumor cells. ONCR-1 is armed with a matrix metalloproteinase 9 (MMP9) transgene to facilitate viral spread in the tumor extracellular matrix. Further, ONCR-1 expresses a UL16 binding protein 3 (ULBP3) transgene to activate NK and CD8+ T cells via the NKG2D receptor and promote antitumor immune responses. In human and murine tumor cells in vitro, ONCR-1 evoked potent cytotoxicity. ONCR-1 increased proteolytic MMP in cell supernatants and ULBP3 cell surface expression with no effect on related NKG2D ligands. In mice bearing subcutaneous or orthotopic human U251 GBM tumors, ONCR-1 administered intratumorally (3x105-3x106 PFU QDx1) inhibited tumor growth and prolonged survival. Ex vivo analysis of immune cells in subcutaneous tumors showed significant increases in virally evoked NKG2D+ NK cells with ONCR-1 treatment. In mice bearing dual flank subcutaneous syngeneic A20 tumors, ONCR-1 significantly inhibited tumor growth in injected tumors (1x105-3x105 PFU, Q2Dx3) and evoked potent systemic antitumor immune responses as evidenced by significant inhibition of distal noninjected left flank tumors. IFNγ production from isolated splenocytes cultured in the absence and presence of A20 tumor cells was significantly augmented in ONCR-1-treated animals, providing further evidence of an enhanced antitumor immune response. Moreover, in mice bearing subcutaneous A20 tumors in which ONCR-1 evoked complete tumor regression, complete protection from rechallenge with A20 tumor cells was observed. Depletion of NK cells or CD8+ T cells in mice resulted in inhibition of ONCR-1-mediated antitumor effects in both injected and noninjected murine A20 tumors, supporting an immune mechanism of action. Our preclinical studies demonstrate that ONCR-1 evokes tumor cell killing by direct oncolysis and by enhancing antitumor immune responses. ONCR-1 represents a novel clinical candidate for the treatment of GBM.

#4699

Identification of novel potential biomarkers of response to sunitinib in glioblastoma.

Abu Shadat Mohammod Noman,1 Bassam Abdulkarim,2 Siham Sabri2. 1 _McGill University, University of Chittagong, Montreal, Chittagong (Bangladesh), Quebec, Canada;_ 2 _McGill University & Research Institute of McGill University Health Centre, Montreal, Quebec, Canada_.

Introduction: Glioblastoma multiforme (GBM) is the most advanced and aggressive form of primary malignant brain tumors in adults. O6-Methylguanine methyltransferase (MGMT) is a DNA repair protein well known for its role in resistance to temozolomide used in standard treatment of patients newly diagnosed with GBM. Sunitinib is an oral multitargeting receptor tyrosine kinase (RTK) inhibitor with antiangiogenic and antiproliferative activities targeting several RTKs. Our group has previously shown the role of MGMT as a negative regulator of angiogenesis and invasion and the differential antiproliferative effect of sunitinib based on expression of MGMT in GBM. Recent gene expression profiling (GEP) studies showed that besides its effects on RTKs, sunitinib affects the expression level of other genes at the transcriptional level in different cancer types. We hypothesized that: (i) the mechanism of action of sunitinib may not be limited to direct inhibition of its known targets (RTKs) in GBM and (ii) expression of MGMT may affect response of GBM tumor cells to sunitinib. We performed GEP analysis to identify genes that could be potentially modulated in response to sunitinib in GBM cell lines isogenic for MGMT. Empty vector (EV)-transfected T98/EV cells with constitutive expression of MGMT and its knockdown counterpart T98/MGMT-shRNA were treated with sunitinib or vehicle control. Treatment of T98/EV and T98/MGMT-shRNA cell lines with sunitinib significantly changed the expression of over 2,000 gene transcripts (analysis performed at Genome Quebec Innovation center, McGill University). Lists of significant genes were fitted into previously known biochemical pathways, available among the canonical pathways from various databases provided by ConsensusPathDB (CPDB). Strikingly, immune system was among the top significantly enriched pathways (p-value<10-3), with some immune pathways not previously reported for response to sunitinib.

Conclusion: Our study opens new avenues to further understand the mechanisms of action of sunitinib, investigate its immunomodulatory effects, and identify biomarkers for selection of patients who might benefit from the combination of antiangiogenic and immunebased strategies in GBM.

Funding Acknowledgment: Cancer Research Society Operating Grant Competition #22716, and Higher Education Quality Enhancement Project (HEQEP)- Window -4 (Grant Id. CP-4023), University Grant Commission (UGC), Bangladesh.

#4700

Manipulating the breast tumor microenvironment with histone deacetylase inhibitors for more robust and durable T cell responses.

Tyler R. McCaw,1 Mingyong Liu,1 Mei Li,1 Dmytro Starenki,2 Sara J. Cooper,2 Rebecca C. Arend,1 Andres Forero,1 Donald J. Buchsbaum,1 Troy D. Randall1. 1 _University of Alabama at Birmingham, Birmingham, AL;_ 2 _HudsonAlpha Institute for Biotechnology, Huntsville, AL_.

Malignant cells harbor an imbalance in histone acetyltransferase and histone deacetylase (HDAC) activity, epigenetically contributing to altered cellular programs. HDAC inhibitors disrupt this balance to impact both cellular transcription and protein function, changing the phenotype of tumor cells as well as responding immune cells, including tumor-infiltrating T cells. We hypothesized that HDAC inhibition could be used to boost anti-tumor T cell responses through inducing expression of immunomodulatory proteins on tumor cells as well as directly altering the transcriptional programs of tumor-specific T cells. To test this, we treated two murine breast cancer models, TS/A and 4T1, with HDAC inhibitors representing class I specificity, entinostat, or pan-specificity, panobinostat, in vitro and in vivo. Culture of tumor cells with either inhibitor increased surface expression of molecules involved in T cell recognition and stimulation, including MHCI, MHCII, CD74, 41BB, CD40, and ICOSL as well as the T cell chemoattractant CXCL10. Treating tumor-bearing mice with HDAC inhibitor resulted in a significant reduction in tumor growth that was absolutely dependent on adaptive immunity. Using depleting antibodies, we next showed that IFNγ and CD8 T cells, but not CD4 T cells or B cells, are necessary for the anti-tumor effects of entinostat. Interestingly, tumor infiltration of CD4 T cells was reduced following treatment and their effector functions were largely unchanged. However, CD8 T cell infiltration was dramatically increased, as was their production of IFNγ, TNFα, and granzyme B even at later time points. This upregulation of effector function was paralleled by a significant increase in the transcription factor T-bet, while Eomes actually decreased over time, trends opposite those seen in CD8 T cells from vehicle treated tumors and that would suggest entinostat treatment can imprint T cells with a transcriptional program less susceptible to exhaustion. Strikingly, we also found that simply adjusting the timing of entinostat dosing relative to T cell activation could abolish anti-tumor effects or lead to rejection in nearly 50% of mice. These effects corresponded with a significant shift in the responding T cell repertoire. Collectively, our data suggests that HDAC inhibition has important effects on both tumor cells and T cells; specifically, altering tumor cell gene expression leads to a repolarization of the tumor microenvironment more favorable to anti-tumor immunity and reprogramming transcriptional profiles of activated T cells improves effector functions and reduces susceptibility to exhaustion. Thus, appropriately timed administration of HDAC inhibitors may synergistically potentiate current tumor immunotherapies, especially adoptive cellular transfer and T cell reinvigoration strategies.

#4701

Verteporfin inhibits surface PD-L1 expression in triple-negative breast cancer (TNBC) cells.

Fatema Khambati, Neha Jaiswal, Srikumar Chellappan, Hatem Soliman. _Moffitt Cancer Center, Tampa, FL_.

Background: Programmed death ligand 1 (PDL1) is commonly expressed on the surface of many tumor cells, including breast cancer. The activity of tumor-infiltrating lymphocytes (TIL) is inhibited by PDL1. High PD-L1 basal cells (particularly basal B) overexpress genes involved in invasion, motility, and chemoresistance (Soliman et al., 2014). Targeting and blocking PD-L1 may enhance eradication of aggressive breast cancer cells by the immune system. Verteporfin (VP), a photosensitizer used to treat macular degeneration, was found to inhibit PD-L1 expression in a high-throughput screen. Studies demonstrated VP inhibits YAP activation by disrupting YAP‐TEAD interactions and preventing YAP induced oncogenic growth (Zhang et al., 2016). Here we demonstrate the inhibitory effect of VP on PD-L1 expression in TNBC cell lines.

Methods: MDA-MB-231 cells co-cultured with human PBMCs were treated with VP (Sigma) in presence of concanavalin A (ConA) and analyzed using flow cytometry to study levels of CD8+IFNg+ cells. TNBC cell lines (MDA-MB-231, BT-20, HCC-1143, Hs-578T) were treated with VP at doses ranging from 1µM-10µM for 24 hrs. The cells were processed for flow cytometry, Western blot and RT-PCR to check PD-L1 in mechanistic studies looking at manipulation of YAP pathway genes.

Results: When MDA-MB-231 cells were co-cultured with normal human PBMCs in the presence of ConA, the CD8+IFNg+ stained cells were reduced compared to PBMC + ConA alone. Interestingly, in the group treated with VP, rescue of CD8+IFNg+ cells was observed. Moreover, MDA-MB-231, BT-20, HCC-1143 and Hs-578T TNBC cells treated with VP showed a significant dose-dependent inhibition of PD-L1 expression by flow cytometry. Western blot analysis also showed complete clearance of PD-L1 protein band with the lowest dose (1µM) used. However, RT-PCR analysis did not show a significant fold change in mRNA levels of PD-L1 in MDA-MB-231 treated cells. Surprisingly, mechanistic studies performed by silencing YAP1, E2F1, and TBK1 in MDA-MB-231, BT-20 and HS-578 T showed a decline in PD-L1 in E2F1 silenced cells, highlighting a plausible role of E2F1-PDL1 signaling axis. However, no change in PD-L1 expression was seen in cells silenced with YAP1 and TBK1. Further, chromatin-immunoprecipitation assay demonstrated E2F1 binding to PD-L1 promoter.

Conclusion: Our data so far demonstrate that verteporfin treatment leads to inhibition of PD-L1 in TNBC cell lines and improvement in CD8+IFNg+ cells, indicating that VP might have potential for treatment approaches. Our study warrants further attention towards understanding the mechanism of action of VP in inhibiting PD-L1 and the role of E2F1 in the process.

#4702

Upgrading cancer immunotherapy: Checkpoint blockade mAb-ODN conjugate.

Ana Paula Benaduce, Tal Gefen, Sergio Rodriguez, Brian Marples, Eli Gilboa, Adrian Ishkanian. _University of Miami, Miami, FL_.

Programmed death 1 (PD-1) and its ligand PD-L1 provide inhibitory signals that affect the balance of T cell activation, playing major roles in tumor immune escape. Inhibition of negative regulators of T cell activation effectively remove signals that restrain T cell proliferation and gain of effector function. Hence, disruption of the PD-1/PD-L1 signaling pathway by the use of monoclonal antibodies (mAbs) can improve tumor control by enhancement of the endogenous antitumor immune responses. Toll-like receptors (TLR) are a specialized set of pathogen recognition receptors found in innate and adaptive immune system cells. Activation of these receptors by danger-associated molecules such as bacterial DNA or RNA, initiate signaling cascades leading to protective immune responses. Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs can mimic bacterial DNA and therefore function as ligands for intracellular TLR9, prompting consequential activation of immune responses. The purpose of this study was to assess the feasibility of using PD-1/PD-L1 mAbs conjugated to TLR9 agonists (ODNs) as a novel strategy for tumor-targeting immune checkpoint signaling and activation of immune cells. Antibodies recognizing PD-1 and PDL-1 were covalently modified with a 21mer ssDNA oligonucleotide linker sequence that served as an anchor site for the ODNs synthesized with a 3' extension. Annealing of complementary strands formed mAb-ODN conjugates. Quantification of ODNs per PD-1 or PDL-1 mAbs was verified by flow cytometry. Functionality of PD-1 and PDL-1-ODN conjugates was analyzed in vitro using splenocyte cultures and cell proliferation was evaluated by incorporation of radiolabeled H3 Thymidine. In vivo experiments were carried out using Balb/c mice bearing 4T1 subcutaneous tumors. Flow cytometry analysis confirmed each modified mAb had 5 linker sequences available to bind ODNs, and also defined the binding capability of mAb portion of the conjugate to epitope target. Splenocytes proliferation at day 5 after treatment revealed that PD-1 and PDL-1 conjugated to ODNs have significantly increased proliferation in comparison to non-conjugated mAb and ODN controls. These results indicate that conjugation of mAb-ODN is feasible and that conjugation does not affect PD-1 and PDL-1 antibody functionality. Interestingly, in vivo experiments demonstrated PDL-1-ODN but not PD1-ODN conjugate affects 4T1 tumor growth. Use of this approach to improve the therapeutic index of immunotherapeutic mAbs requires further study and validation in different murine tumor models.

#4703

The inhibitor of apoptosis protein (IAP) antagonist Debio 1143 enhances the immune response to anti-PD1/L1 inhibitors in vitro and in vivo.

Antoine Attinger, Bruno Gavillet, Anne Vaslin Chessex, Norbert Wiedemann, Gregoire Vuagniaux. _Debiopharm International S.A., Lausanne, Switzerland_.

Background: Debio 1143 is an oral antagonist of IAPs, currently in clinical development, which sensitizes tumor cells to radiation- or chemotherapy-induced apoptosis. IAPs inhibitors modulate NF-κB signaling and drive the expression of genes relevant for inflammation and immunity. Here, we hypothesized that Debio 1143 could improve antitumor immunity by directly enhancing T-lymphocyte activation and by improving the effects of immune checkpoint inhibitors in vitro and in vivo.

Methods: Ex vivo human PBMC anti-CD3/CD28 stimulation and modified mixed-lymphocyte reaction assays were performed to evaluate the immunostimulatory potential of Debio 1143 alone or in combination with the anti-PD-1 antibody nivolumab (N= 5 donors). T cells' proliferation and activation were measured by flow cytometry and cytokine release was measured by ELISA. The antitumor activity of an anti-PD-L1 antibody (5 mg/kg BIW IP) was tested either alone or in combination with Debio 1143 (100 mg/kg QD1-5 PO) in MBT-2 immunocompetent mouse model of bladder cancer over 3 weeks (n=8 /group).

Results: Debio 1143 significantly enhanced CD4+ and CD8+ intracellular IFNγ expression in a concentration-dependent manner following anti-CD3/CD28 stimulation. This result was confirmed using a mixed-lymphocyte reaction assay, where Debio 1143 at concentrations achieved in clinical studies significantly increased IFNγ expression by activated CD4+ cells, and this effect was even further increased in presence of nivolumab. In MBT-2 tumor-bearing mice, the combination of Debio 1143 and anti-PD-L1 antibody significantly decreased tumor growth (P=0.001 using two-sided t-test) and increased survival, whereas monotherapies only displayed moderate activities (median survival time of 42 days vs. 33 or 28 days for Debio 1143 or anti-PD-L1 alone, respectively).

Conclusion: These data show a key mechanistic role for future combination therapy of the IAP antagonist Debio 1143 and immune-checkpoint agents in cancer patients. This synergy will be further explored in a phase-Ib dose-finding clinical study combining Debio 1143 and Avelumab (anti-PD-L1) in patients with advanced solid malignancies and non-small cell lung cancer (CT# 03270176).

#4704

Triple combination of IMO-2125, epacadostat and anti-PD-1 antibody demonstrates maximal antitumor efficacy and eradicates large established tumors in preclinical models.

Evren Kocabas Argon,1 Fugang Zhu,1 Sudhir Agrawal,1 Jonathan Yingling,2 Daqing Wang1. 1 _Idera Pharmaceuticals, Inc., Cambridge, MA;_ 2 _Idera Pharmaceuticals, Inc., Exton, PA_.

Immune checkpoint blockade has revolutionized cancer therapy; however, the majority of patients fails to demonstrate a durable response. This suboptimal outcome is due to multiple mechanisms that range from low immunogenicity to inadequate generation or activation of tumor-specific T cells. Combination therapies targeting multiple checkpoints are likely to overcome the inadequate antitumor immune responses by a single agent. Intratumoral injection of IMO-2125, a TLR 9 agonist, activates dendritic cells to present tumor specific antigens and enhances T-lymphocyte infiltration within injected tumors. These activities lead to the priming and trafficking of tumor-specific cytotoxic T-cells against metastatic tumors. Intratumoral IMO-2125 is a personalized cancer therapy that enhances antitumor activity by synergizing with checkpoint blockade to elicit a productive immune response in cancer patients.

In the present study, we evaluated a combination immunotherapy regimen that involves an immune activator, IMO-2125, and checkpoint inhibitors targeting two negative immune regulators, IDO-1 and PD-1, to eliminate large tumors in syngeneic tumor models. BALB/c mice were implanted with CT26.CL25 tumors in the right and left flanks. Treatment was initiated on Day 10 after tumor implantation when tumors were fully established (311 ± 89 mm3). IMO-2125 (2.5 mg/kg) was intratumorally (i.t) injected in the right flank tumor 2x/week for 2 weeks. Epacadostat (EPA, Incyte IDO-1 inhibitor, 100 mg/kg) was administered by intragastric administration 5x/week for 2 weeks. Anti-PD-1 mAb (clone RMP1-14, 10 mg/kg) was administered by intraperitoneal injection 3x/week for 2 weeks. Treatment with IMO-2125 i.t alone resulted in 73% and 59% growth reductions in the treated tumors and untreated distant tumors, respectively. EPA and anti-PD-1 monotherapy or the double combination therapy did not demonstrate significant antitumor activity. The addition of IMO-2125 to either EPA or anti-PD-1 led to 84% and 81% tumor reductions in the i.t. treated tumors, and 71% and 69% reductions in the untreated distant tumors, respectively. Triple combination therapy with IMO-2125, EPA and anti-PD-1 exhibited maximal antitumor activity, with 98% and 88% tumor growth reductions in the treated and distant tumors, respectively. All treatments were well tolerated. Similar antitumor activities were also observed in a murine peritoneal model with NSCLC 3LL-C75.

Combination of IMO-2125 with checkpoint inhibitors targeting IDO-1 and PD-1 induces maximal antitumor efficacy and eradicates large established tumors in preclinical models. A Phase 2 clinical trial of IMO-2125 in combination with ipilimumab in patients with metastatic melanoma is ongoing (NCT02644967) and a Phase 3 multi-center, global trial in the same population is initiating enrollment.

#4705

The novel oncolytic peptide, DTT-304, induces regression of colorectal cancer tumors in vivo.

Karianne G. Fleten,1 Johannes J. Eksteen,2 Brynjar Mauseth,3 Ketil Camilio,3 Øystein Rekdal,3 Meng Yu Wang,1 Gunhild M. Mælandsmo,1 Kjersti Flatmark1. 1 _Oslo University Hospital, Oslo, Norway;_ 2 _Norut, Tromsø, Norway;_ 3 _Lytix Biopharma, Oslo, Norway_.

Colorectal cancer (CRC) is the third most common cancer in the world, and approximately 50% of the patients develop liver metastasis, which is a major cause of disease mortality. New drugs and treatment options are therefore necessary to improve the survival of these patients. Lately, different types of immunotherapy, such as check point inhibitors, have shown great promise in the treatment of metastatic cancer, although for most patients with CRC the efficacy has been restricted to cases with microsatellite instable disease. Another novel immunotherapeutic strategy is oncolytic immunotherapy, which has been shown to cause immunogenic cancer cell death. Following cancer cell lysis, damage associated molecular patterns (DAMPs) are released, which are recognized by immune cell receptors. Pro-inflammatory responses will then be triggered to induce release of tumor specific antigens that are presented to the immune system, leading to tumor-specific immune responses.

In the present study, we have investigated the efficacy of a novel oncolytic peptide, DTT-304, using two mouse models. Subcutaneous tumors were established in Balb c mice using the cell line CT26 and in C57Bl6 mice using the cell line MC38, both murine CRC cell lines. In both models, intra-tumoral injection of the peptide induced tumor necrosis and complete regression a few days after initiation of treatment, thus showing the potential of this peptide for use in colorectal cancer.

In ongoing experiments we are investigating if the peptide has the potential to induce long lasting protective tumor-specific immune responses by re-challenging previously treated mice with the same cell line in a liver metastasis model. In addition, the possibility of treating liver tumors by direct peptide injection will be investigated.

#4706

**Extracellular polysaccharides extracted from** Parachlorella kessleri **enhance host antitumor immune responses and inhibit colon carcinoma growth in mice.**

Susumu Ishiguro, Deepthi Uppalapati, Zachary Goldsmith, Dana Robertson, Jacob Hodge, Hayley Holt, Arashi Nakashima, Katie Turner, Masaaki Tamura. _Kansas State Univ., Manhattan, KS_.

Natural products obtained from various resources are often found to be new sources of effective drugs for disease including cancer. The novel extracellular polysaccharides (exopolysaccharides) purified from Parachlorella kessleri (PCEPS), a generous gift by Panac ADVANCE Co. Ltd. (Tokyo, Japan), were evaluated on their antitumor and immunomodulatory effects in cell culture and mouse colon carcinoma peritoneal dissemination model. In two-dimensional cell culture, the PCEPS treatment inhibited cell growth of both murine and human colon carcinoma cells in a dose- and time-dependent manner. In contrast, the cell proliferation of mouse splenocytes (SPLs) and bone marrow cells (BMCs) were stimulated by the treatment with PCEPS. The treatment with PCEPS also increased specific subpopulations of the cells in BMCs: antigen presenting cells (CD19+ B cells, 33D1+ dendritic cells and CD68+ macrophage) and CD8+ cytotoxic T cells. In three-dimensional spheroid culture, spheroid growth of CT26 cells co-cultured with HL-60 human neutrophilic promyeloblasts and Jurkat cells (human T lymphoblasts), but not THP-1 human monocytes/macrophages, was significantly attenuated by PCEPS treatment. In transwell co-culture of CT26 and HL-60, THP-1 or Jurkat cells, mRNA level of NOX2 in HL-60 and THP-1 cells, and TNFα, IFNγ, IL-2 and granzyme B (GZMB) in Jurkat cells was significantly increased by PCEPS treatment. In a mouse CT26 colon carcinoma peritoneal dissemination model, intraperitoneal injection of PCEPS (10 mg/kg, started from 1 week after CT26 inoculation for 11 days, twice per week) significantly attenuated the growth of CT26 colon carcinoma in syngeneic mice by PCEPS treatment-associated apoptosis without any noticeable adverse effect. Analysis of subpopulation of intraperitoneally infiltrating immune cells collected from CT26 cell tumor-bearing mice found that PCEPS treatment increased CD4+ and CD8+ T lymphocytes, CD19+ B cell, and CD68+ macrophage populations. In contrast, 33D1+ dendritic cell and LY6G+ neutrophil populations were decreased as compared to PBS-treated group. The present study suggests that PCEPS inhibits colon carcinoma growth via direct cell growth inhibition and a stimulation of the host antitumor immune responses. Taken together, the current study suggests that exopolysaccharides derived from Parachlorella kessleri contain significant bioactive materials that inhibit colon carcinoma growth.

This study was supported by Kansas State University Johnson Cancer Research Center 2011JCRC-EQ (MT), 2014CRA (ZG and MT), 2015CRA (JH, HH, AN, and MT), 2016CRA (HH and MT), College of Veterinary Medicine Dean's funds 2010CVM-EQ (MT), and NIH grant P20 RR017686 (MT).

#4707

Pelareorep promotes the expression of a chemokine signature that predicts response to immunotherapy.

Grey A. Wilkinson,1 Aine Piar,2 Zoe Cesarz,2 Hue Tran,1 Romit Chakrabarty,1 Andres Gutierrez,1 Matt Coffey1. 1 _Oncolytics Biotech Inc., Calgary, Alberta, Canada;_ 2 _Almac Diagnostics, Durham, NC_.

Introduction: It has been proposed that the presence of inflamed tumor phenotypes, characterized by the presence of infiltrating lymphocytes and the expression of specific chemokines and cytokines, can predict response to immunotherapy and result in better patient outcomes [1, 2]. We hypothesized that pelareorep, an immuno-oncolytic virus (IOV), may elicit predictive proinflammatory gene signatures in select cancer cell lines permissive to viral infection.

Methods: Cell lines derived from non-small cell lung cancer (NSCLC, H522), colorectal cancer (CRC, SW-620), and hepatocellular carcinoma (HCC, SNU 387) were infected at an multiplicity of infection equal to 50. We examined changes in gene expression and conducted cell viability assays at 6, 12, and 18 hours post pelareorep infection (including a non-infected control). To monitor changes in gene expression we employed a custom 780-gene Pan Cancer Immune panel developed by nanoString Technologies and specifically monitored for changes in the expression of key interferon and NF-κB signalling genes, immune checkpoint ligands, and a 12-gene chemokine signature predictive of a positive response to immunotherapy identified by Messina et al. [2].

Results: All cell lines examined were susceptible to pelareorep induced cytopathic effect. Strikingly, principal component analysis revealed that the changes in gene expression were unique and different for each cell line. Of the cell lines examined, only HCC cells infected with pelareorep promoted an inflammatory signature, similar to the one used to predict response to immunotherapy in melanoma [2].

Conclusions: This study demonstrates that pelareorep can prime or promote a predictive inflamed tumor phenotype in HCC, which correlates with the innate response recently described in HCC- animal models treated with pelareorep [3]. The role of pelareorep in the treatment of hepatocellular carcinoma deserves further investigation, particularly in combination with other immunotherapies.

References: [1] Gajewski, T.F., The Next Hurdle in Cancer Immunotherapy: Overcoming the Non-T-Cell-Inflamed Tumor Microenvironment. Semin Oncol, 2015. 42(4): p. 663-71. [2] Messina, J.L., et al., 12-Chemokine gene signature identifies lymph node-like structures in melanoma: potential for patient selection for immunotherapy? Sci Rep, 2012. 2: p. 765. [3] Samson, A., et al., Oncolytic reovirus as a combined antiviral and anti-tumour agent for the treatment of liver cancer. Gut, 2016.

#4708

Imprime (beta-1,3/1,6 glucan) synergizes with a CD40 agonist to stimulate T cell-dependent antitumor activity in a poorly immunogenic model of pancreatic carcinoma.

Mingen Liu, Kathleen Graham, Anissa S. Chan, Nadine R. Ottoson, Nandita Bose, Gregory L. Beatty. _Univ. of Pennsylvania, Philadelphia, PA_.

Introduction: While some solid tumors show remarkable sensitivity to immunotherapy, pancreatic ductal adenocarcinoma (PDAC) has demonstrated impressive resistance. This poor responsiveness to immunotherapy may reflect multiple mechanisms necessary for generating productive T-cell immunosurveillance, including ineffective tumor antigen presentation by myeloid cells. Here, we hypothesized that combining two immune agonists capable of "licensing" myeloid cells with enhanced T-cell stimulatory properties would synergize to reverse the poorly immunogenic state of pancreatic cancer and, in doing so, drive productive T cell-dependent antitumor immunity.

Methods: C57BL/6 mice were challenged subcutaneously with a syngeneic PDAC cell line (7940B.PDA) derived from a tumor arising spontaneously in the KrasG12D/+; Trp53R172H/+; Pdx-1 Cre (KPC) mouse model. Mice with tumors measuring approximately 5 mm3 in diameter were treated with defined combinations of gemcitabine chemotherapy, a CD40 agonist (FGK45), and Imprime (a yeast β-1,3/1,6 glucan that coordinately activates innate and adaptive immune responses through pattern recognition receptors). CD4 and CD8 T cells were depleted in vivo using GK1.5 and 2.43 antibodies, respectively. Tumor growth curves and overall survival were determined.

Results: Monotherapy with Imprime or a CD40 agonist only modestly delayed tumor outgrowth compared to chemotherapy alone. Delivering chemotherapy 48 hours prior to Imprime administration produced a two-fold increase in the median overall survival, but responses were not durable and all mice relapsed. In contrast, concurrent single administration of Imprime and a CD40 agonist induced complete and durable regressions in 60% of mice. Tumor-free mice resisted tumor rechallenge and therapeutic efficacy was completely abrogated with CD4/CD8 cell depletion.

Conclusions: The combination of two myeloid-directed immune agonists (Imprime and a CD40 agonist) shows promising activity for stimulating T cell-dependent antitumor immunity against PDAC, a poorly immunogenic cancer.

#4709

Cell-specific gene program-based small-molecule immunomodulators targeting solid-tumor microenvironments.

Erin Kischuk, Joydeb Majumder, Jonathan A. Fine, Travis C. Lantz, Deepika Dhawan, Deborah W. Knapp, Timothy L. Ratliff, Gaurav Chopra. _Purdue University, West Lafayette, IN_.

Cancer immunotherapy includes promising strategies based on immune checkpoint blockade (e.g., anti-PD-1/PD-L1, anti-CTLA-4). A limitation of such therapies for solid tumors stems from other immune-suppressive mechanisms mediated by myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages. We hypothesize that molecules that target specific suppressive immune cells in the tumor microenvironment can reprogram the pro-tumor microenvironment towards antitumor immunity. Using our proteome-scale target/antitarget network-based lead optimization method (CANDESIGN), we designed and synthesized cell-specific nontoxic chemical libraries with modular functions for anticancer potency and immunomodulation. We used machine learning iteratively on experimental data to identify cell-specific target/anti-target networks (gene programs) as well as designed and synthesized compound libraries targeting cell-specific programs for desired cancer and immune cell function ex vivo. Specifically, we altered suppressive function of MDSCs by targeting upregulated genes in activated monocytic MDSCs in the tumor microenvironment compared to cells in spleen. We synthesized a potent nontoxic compound that specifically modulates activated MDSC function and corresponding changes in CD4+ and CD8+ T-cell activity in a mouse bladder cancer model. We observed a significant reduction in tumor mass following treatment by oral gavage. Interestingly, the bladder cancer cells used in our mouse model (as well as human and dog bladder cancer cells) are insensitive to our lead compound in vitro, suggesting in vivo antitumor activity via immunomodulation. Our lead decreased frequency of monocytic and granulocytic MDSCs in the ascites and the tumor. Moreover, a decreased frequency of MDSC expressing suppressive functional markers (e.g., Arg1, iNOS, PD-L1) and increased IFNγ+CD8\+ T cells were observed in the tumor microenvironment after compound treatment. Finally, we performed a small clinical trial on pet dogs with bladder tumors treated with our compound tablets for 4-5 weeks that resulted in ~50% reduction in tumor volume. We conclude that designing cell-specific compounds perturbing the tumor microenvironment to combat immune suppression gives a selective advantage to the immune system to combat solid tumors via single and combination drug/cell therapies.

#4710

The effect of Wnt inhibition combined with paclitaxel on tumor burden and CD8+ T cell infiltration in syngeneic murine models of ovarian cancer.

David W. Doo,1 Angelina I. Londono,1 Dylana J. Moore,1 Selene Meza-Perez,1 Ashwini A. Katre,1 Tyler R. McCaw,1 Haller J. Smith,1 Carol Y. Lin,1 Sara J. Cooper,2 J Michael Straughn,1 Donald J. Buchsbaum,1 Lyse A. Norian,1 Troy D. Randall,1 Rebecca C. Arend1. 1 _University of Alabama at Birmingham, Birmingham, AL;_ 2 _HudsonAlpha Institute for Biotechnology, Huntsville, AL_.

Objective: The Wnt/β-catenin pathway is a major signal transduction pathway involved in ovarian cancer (OVCA) metastasis and resistance to chemotherapy. This pathway downregulates protective immunity mediated by intra-tumoral CD8+T cells in other cancer types. WNT974 is a novel drug that inhibits the enzyme Porcupine, which controls Wnt protein secretion. Our objective was to measure the effect of WNT974 alone and in combination with dose dense paclitaxel (ddPac) on tumor growth and on infiltrating CD8+ T cells in two syngeneic OVCA mouse models.

Methods: C57BL/6 mice were injected subcutaneously (SC) (n=20) or intraperitoneally (IP) (n=24) with 7 x 106 ID8 mouse OVCA cells. Mice were treated with vehicle control, ddPac, WNT974, or the combination (combo). C57BL/6 TgMISIIR-Tag-Low transgenic mice were injected with 7 x 106 MOVCAR cells IP (n=8) and treated with vehicle control or combo. WNT974 was given by oral gavage twice a day (5mg/kg for 7 days then decreased to 2.5mg/kg twice a day for up to 4 weeks). Paclitaxel was given IP (5mg/kg) 3 days on and 3 days off for a total of 9 doses. SC tumors were measured with calipers twice per week. In the IP model, mice were sacrificed after 13 days of treatment and tumor weights and ascites volume were determined. Flow cytometry was used to evaluate the presence of CD8+ T cells in IP tumors.

Results: In the ID8 SC model, combo therapy reduced tumor size compared to vehicle control (18 vs. 40mm2, p<0.01) or ddPac alone (18 vs. 39mm2, p<0.01). In the ID8 IP model, tumor weights trended down after treatment (p=NS) and the percentage of intra-tumoral CD8+T cells increased after ddPac or combo treatment (control 4.9%; WNT974 8.3%, p=0.12; ddPac 12.3%, p=0.01; combo 14.9%, p<0.01). In the MOVCAR IP model, combo therapy reduced tumor weight (0.13 vs. 0.19g, p=0.05) and ascites volume (1.0 vs. 8.5 mL, p<0.05), and trended toward increased intra-tumoral CD8+ T cells (33.5 vs. 13.5%, p=0.13).

Conclusions: The combination of WNT974 and ddPac reduced tumor size and increased tumor infiltration of CD8+ T cells in syngeneic OVCA mouse models. This suggests that the improved response observed with the ddPac and WNT974 combination is in part due to upregulation of the intra-tumoral immune response. Further investigation of this pathway is warranted as an immune modulator and a potential therapeutic target in ovarian cancer.

#4711

Modulation of NFκ-B signaling to optimize antitumor characteristics in macrophages.

Esther Liu,1 Alyssa A. Hoover,1 Whitney R. Harris,1 Whitney Barham,2 Oleg Tikhomirov,1 Dineo Khabele,3 Zahra Mirafzahli,4 Todd D. Giorgio,1 Andrew J. Wilson,1 Fiona E. Yull1. 1 _Vanderbilt University, Nashville, TN;_ 2 _The Mayo Clinic, Rochester, MN;_ 3 _University of Kansas School of Medicine, Kansas City, KS;_ 4 _Encapsula Nanosciences, Nashville, TN_.

While it is known that nuclear factor κ-B (NF-κB) signaling within macrophages regulates expression of downstream targets that can mediate either pro- or antitumor functions, the mechanisms that define predominant characteristics are not well understood. As macrophages are present in the tumor microenvironment of diverse tumor types, their characteristics have the potential to either support or hinder tumor growth and metastatic spread. Therefore, a method to shift the balance of tumor associated macrophages from pro- to antitumor functions could potentially target tumor cells directly and also recruit and activate other immune cells, resulting in significant therapeutic benefit. Our studies are designed to gain an understanding of how to use modulation of NF-κB signaling as the "pinch point" to generate antitumor phenotypes and determine how to achieve this modulation in a patient. We are using immortalized bone marrow-derived macrophages or ex vivo tumor-associated macrophages in cell culture approaches and inducible transgenic mice in in vivo approaches to investigate the mechanisms by which NF-κB defines macrophage characteristics. Interactions with tumor cells are also being investigated using co-culture and murine models. Our data suggests that high levels of NFκ-B activity in macrophages induce both direct tumor cell killing and immune stimulating responses. We are testing two different translational approaches. The first is liposomal muramyl tripeptide ethanolamine that acts as an immune stimulant by increasing NF-κB activity in macrophages. The second is preferential delivery of siRNA against the inhibitor of NFκ-B (IκB-alpha) to tumor-associated macrophages using polymeric nanoparticles. We hope to better understand the mechanisms by which NFκ-B regulates macrophage functions to inform development of a novel macrophage-based immunotherapy that could be effective across a wide spectrum of solid tumors and metastatic disease.

#4712

AT1965, a novel B cell-activating immunotherapy, exerts potent anticancer activity.

Monideepa Roy,1 Vineeth Krishna,1 Aniruddha Sengupta,1 Sudip Roy,1 Ashish Kulkarni,2 Shiladitya Sengupta2. 1 _Akamara Therapeutics, Inc, India;_ 2 _Brigham and Women's Hospital, Harvard Medical School, Boston, MA_.

Emerging clinical evidence implicate B cells in long term survival in cancer. However, a therapeutic activation of a B cell response against cancer has not been explored. Here we describe the results with AT1965, which exerts a potent anticancer effect via the activation of B cells. AT1965 completely regressed aggressive 4T1 breast tumors established in balb/c mice. Immunophenotyping revealed that IGKC, a B cell marker, was the dominant immune biomarker that is over-expressed following AT1965 treatment. The lack of functional B cells in vivo, via the deletion of the JH gene results in the loss of heavy chain production, blocked this anti-cancer activity of AT1965. Activation of a B cell response is associated with long term memory, which should prevent relapse. To test this, tumor-bearing animals were treated with AT1965 to induce regression; Not a single animal relapsed, i.e. developed the tumor when re-challenged with the tumor thirty days after the primary tumors had regressed. T and B cells are natural partners in an immune response. We therefore tested the combination of a PD1 immune checkpoint inhibitor and AT1965 in an immunocompetent lung cancer model. Only 12.5% of the animals treated with AT1965 or the PD1-inhibitor exhibited a complete regression of lung cancer, while 37.5% exhibited delayed tumor progression. In contrast, 50% of the animals underwent complete tumor regression and 25% exhibited tumor growth inhibition when the two drugs were combined, consistent with the basic immunology premise that both B and T cells work together to mount a complete immune response. Our results indicate that B cell activation could emerge as the next paradigm in cancer immunotherapy.

#4713

**BET bromodomain inhibition synergizes with PD-1 blockade to facilitate anti-tumor response in** Kras **-mutant non-small cell lung cancer.**

Dennis O. Adeegbe,1 Shengwu Liu,2 Michaela Bowden,2 Peter S. Hammerman,3 James E. Bradner,3 Anil K. Rustgi,4 Adam J. Bass,2 Gordon J. Freeman,2 Huawei Chen,5 kwok-Kin Wong1. 1 _NYU Langone Medical Center, New York, NY;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _Novartis Institutes for Biomedical Research, Cambridge, MA;_ 4 _University of Pennsylvania Perelman School of Medicine, Philadelphia, PA;_ 5 _AstraZeneca, Waltham, MA_.

KRAS mutation is present in about 30% of human lung adenocarcinomas. While recent advances in targeted therapy have shown great promise, KRAS remains undruggable and concurrent alterations in tumor suppressors render KRAS mutant tumors even more resistant to existing therapies. Contributing to the refractoriness of KRAS mutant tumors harboring these co-mutations are immunosuppressive mechanisms such as increased presence of suppressive Tregs in tumors and elevated expression of the inhibitory receptor PD-1 on tumor-infiltrating T cells. BET bromodomain inhibitors demonstrate clinical benefit in hematologic malignancies, and prior reports demonstrate their Treg-disruptive effects in a NSCLC model. Targeting PD-1 inhibitory signals through anti-PD-1 antibody blockade has also shown substantial therapeutic impact in lung cancer although these outcomes are still limited to a minor pool of patients. We therefore hypothesized that the BET bromodomain inhibitor JQ1 would synergize with PD-1 blockade to promote robust anti-tumor response in lung cancer. In the present study, using Kras+/LSL-G12D; Trp53L/L (KP) mouse models of non-small cell lung cancer, we identified cooperative effects among JQ1 and anti-PD-1 that included reduced numbers of tumor-infiltrated Tregs and enhanced activation of tumor-infiltrating T cells, which exhibited a Th1 cytokine profile that favored their demonstrated improved effector function. Furthermore, lung-tumor-bearing mice under this combinatorial treatment regimen showed robust and long-lasting anti-tumor responses compared to either agent alone, culminating in substantial improvement in the survival of treated mice. Thus, combining BET bromodomain inhibition with immune checkpoint blockade offers a promising therapeutic approach for solid malignancies such as lung adenocarcinoma.

#4715

Mifepristone extends both length and quality of life in a patient with advanced non-small cell lung cancer that progressed despite chemotherapy and check-point inhibitors.

Jerome H. Check,1 Diane Check,2 Trina Poretta3. 1 _Cooper Medical School of Rowan University, Camden, NJ;_ 2 _Cooper Institute for Reproductive Endocrinology & Infertility, Mt. Laurel, NJ; _3 _Kennedy Health System, Cherry Hill, NJ_.

The US FDA approved an investigator initiated phase II 40 patient study to treat patients with stage IIIB or IV non-small cell lung cancer who have progressed despite a minimum of 2 rounds of chemo or immunotherapy with the progesterone receptor modulator mifepristone taken orally at 300mg/day (www.clinicaltrials.gov). The first patient in the study reported at the 2017 AACR meeting with stage IV lung cancer with brain metastasis has had non-progressive disease with excellent quality of life (ECOG-zero) after 24 months on mifepristone therapy. His lung cancer was PD-L1 negative, and thus he did not receive check-point inhibitors. We report, herein, the clinical outcome of the second patient with stage IV disease who not only progressed despite 3 chemotherapy regimens but also nivolumab (she was positive for the PD-L1 marker). The second patient in the study began 300mg/day oral mifepristone at age 68. Her stage IV lung cancer had progressed despite 4 cycles of carboplatin, permetrexed, and bevacizumab, 6 cycles of carboplatin and docetaxel, and 6 cycles of erlotinib. Finally she progressed and treatment was stopped after 11 cycles of nivolumab (PD-L1 marker present). After 1 year of oral mifepristone there have been no new metastatic lesions. She is ECOG 1 (related to chronic obstructive lung disease). Her cancer is stable with slight reduction in some metastatic lesions. More importantly she states she feels better than at any time since her initial diagnosis. The mechanism of action of mifepristone is hypothesized to be related to suppressing a key immunosuppressive protein that seems to be required for rapidly growing cells (e.g., possibly cancer stem cells), to proliferate known as the progesterone induced blocking factor (PIBF). This protein stablizes perforin granules in natural killer cells, thus obviating their mechanism for cytotoxicity. Mifepristone has been shown to suppress the intracytoplasmic immunosuppressive isoform in cancer cell line studies. Mifepristone can also diminish T-cell cytotoxicity. Progesterone receptor modulators may provide a new treatment option for advanced non-small cell lung cancer, even those positive for the PD-L1 marker that progresses despite treatment with chemotherapy and check-point inhibitors. Because intracytoplasmic PIBF is present in only mesenchymal, embryonic, trophoblast, or cancer cells suppressing this protein does not engender any health risks - oral mifepristone is well tolerated.

#4716

Targeting specific TGF-β isoforms in combination with radiation therapy leads to differential antitumor effects in mouse models of cancer.

Aditi Gupta,1 Sadna Budhu,1 Rachel Giese,1 Jacques van Snick,2 Catherine Uyttenhove,2 Gerd Ritter,3 Jedd Wolchok,1 Taha Merghoub1. 1 _Memorial Sloan-Kettering Cancer Center, New York, NY;_ 2 _Ludwig Institute for Cancer Research Ltd, Brussels, Belgium;_ 3 _Ludwig Institute for Cancer Research, New York, NY_.

Background: TGF-β is a pleotropic cytokine, which has emerged as a potential target in cancer treatment due to its dual role in tumorigenesis and homeostasis. There are three isoforms of TGF-β (TGF-β1, TGF-β2 and TGF-β3), which are secreted by immune and nonimmune cells as a latent complex. Depending on the local context, TGF-β adopts opposing roles in carcinogensis and in modulating the immune system. These dueling roles of TGF-β are dependent on its secretion and activation. Local radiation therapy (RT) can activate TGF-β via reactive oxygen species. Such TGF-β expression is linked to radioresistance and dose-limiting toxicities, reducing the effectiveness of RT. In these studies, we aim to characterize the effect of RT on the temporal and cell-specific expression patterns of TGF-β isoforms in mouse tumor models. This will inform treatment regimens combining isoform specific anti-TGF-β therapy with RT.

Methods: Fluorescence-activated cell sorting (FACS): C57BL/6 mice were implanted on the hind limb with B16-F10 melanoma cells. On day 10, tumors were irradiated locally with 15 Gy. Expression of TGF-β isoforms was measured at 1, 3 and 5 days post-RT by FACS. In vivo: C57BL/6 mice were implanted with tumors and irradiated as described. Mice were treated (10/group) with anti-TGF-β1, anti-TGF-β3 or a pan-TGF-β antibody beginning 1 day after RT given intraperitoneally (200 ug/mouse) every other day for 8 doses. Tumor growth and overall survival were monitored. A similar experiment was conducted in the 4T1 breast cancer model, in which mice were treated 1 day prior to radiation.

Results: FACS data indicated that TGF-β1 and TGF-β3 expression increases on most immune cells in the tumor 1 day after RT, decreases 3 days after RT and reaches a peak 5 days after RT. Preliminary in vivo studies demonstrate that both αTGF-β1 and αTGF-β3 as monotherapies have activity against B16 melanoma. In combination with RT, αTGF-β3 shows greater antitumor activity compared to αTGF-β1 in melanoma. Similar observations were obtained in a 4T1 breast model; however, αTGF-β3 alone and in combination with RT as well as αTGF-β1 + RT showed a significant delay against tumor growth. No significant differences in survival were seen in either tumor model.

Conclusions: TGF-β1 and TGF-β3 are expressed on numerous lymphoid and myeloid cells in B16 tumors and spleens. TGF-β isoform expression peaks 5 days post-RT. Anti-TGF-β therapy is effective in delaying tumor growth and may synergize with RT in certain cancers. This demonstrates rationale for the use of anti-TGF-β therapy to enhance the effectiveness of RT in cancer.

#4717

MAP kinase inhibition induces metabolic reprogramming in T-cells leading to induction of stem cell memory CD8 cells that enhance potency of adoptive cell therapy and anti-OX40 antibody.

Vivek Verma,1 Shamim Ahmad,1 Peng Zeng,1 Mikayel Mkrtichyan,1 Simon T. Barry,2 Paul D. Smith,2 Paulo C. Rodriguez,3 John E. Janik,4 Seema Gupta,1 Samir N. Khleif1. 1 _Georgia Cancer Center, Augusta University, Augusta, GA;_ 2 _AstraZeneca, Oncology iMED, Cheshire, United Kingdom;_ 3 _Moffitt Cancer Center, Tampa, FL;_ 4 _Incyte, DE_.

Adoptive T-cell therapy has demonstrated remarkable activity in refractory malignancies; however, the short survival of these cells in some patients results in treatment failure. CD8+ T-cells with self-renewable stem cell memory (TSCM) phenotype persist longer than the effector cells. Hence pharmacologic agents that maintain lymphocytes in TSCM phenotype may benefit the adoptive T cell-transfer strategies as well as combination immunotherapies. Small-molecule inhibitors of mitogen-activated protein kinase (MEKi) have been used for treatment of solid tumors. Here we report that inhibition of MEK enhances immune-driven antitumor response. We show that MEKi prevents the terminal differentiation and exhaustion of CD8+ T-cells in the tumor microenvironment. Interestingly, we further observed that MEKi leads to a significant increase in the CD62LhiCD44loSca1\+ cells with low mitochondrial potential that are defined as TSCM CD8+ cells. Furthermore, ex vivo MEKi-treated CD8+ cells demonstrated a significantly higher antitumor activity in tumor-bearing mice. These MEKi treated CD8+ T-cells have higher mitochondrial respiration, which is fueled by fatty acid oxidation and exhibit significantly enhanced spare respiratory capacity (SRC). In addition, MEKi-treated cells have higher metabolic fitness, making them resistant to terminal exhaustion upon cell activation. Since OX40 co-stimulatory signals potentiate the self-renewal and homeostatic capacity of memory T-cells, we treated the tumor-bearing mice with MEKi in combination with OX40 agonistic antibody. Combining MEKi with OX40 agonist antibody significantly enhanced anti-OX40 antitumor effect. These data highlight the ability of pharmacologic MEK inhibition in inducing TSCM, which can be used ex vivo to enhance the potency of T-cell therapies and in vivo in combination immune therapy.

#4718

ZSTK474, a pan-PI3K inhibitor, enhances the anti-tumor activity of PD-1 blockade by decreasing Treg cells and increasing memory T cells.

Sho Isoyama,1 Shigeyuki Mori,1 Daisuke Sugiyama,2 Hiroyoshi Nishikawa2. 1 _Zenyaku Kogyo CO.,LTD., Tokyo, Japan;_ 2 _National Cancer Center, kashiwa, Japan_.

The phosphoinositide 3-kinases (PI3Ks) transduce important signals involved in T-cell development and activation. Inhibition of the PI3Kδ isoform reportedly decreases regulatory T (Treg) cells and activates anti-tumor immunity in preclinical murine models. PI3K is therefore an attractive target for cancer immunotherapy. Here we explored whether ZSTK474, a selective PI3K inhibitor, which inhibits cancer proliferation by inhibition of activated PI3K in cancer cells, decreases Treg cells, resulting in activation of the anti-tumor immune responses leading to tumor growth inhibition in combination with anti-PD-1 antibody. In syngeneic murine models, ZSTK474 administration decreased Treg cell number in tumors and increased CD8+ T cells / Treg cells ratio, suggesting augmented anti-tumor immunity. ZSTK474 treatment with anti-PD-1 antibody showed far stronger tumor growth inhibition against NY-ESO-1 overexpressing CMS5a cells (CMS5a-NY-ESO-1) and increased tumor specific CD8+ T cells / Treg cells ratio in tumors compared with each monotherapy group. These anti-tumor effect by ZSTK474 was dependent on both CD8+ and CD4+ T cells because depletion of CD8+ or CD4+ T cells from tumor bearing mice abrogated the anti-tumor effect by ZSTK474. We then examined whether ZSTK474 inhibits human Treg cells. CD4+CD25+ Treg cells were isolated from human peripheral blood mononuclear cells (PBMCs) and stimulated with CD3/CD28 antibody and found that ZSTK474 selectively inhibited proliferation of human CD4+CD25+ Treg cells as compared to CD4+CD25- and CD8+ T cells. Furthermore, when PBMCs were stimulated with NY-ESO-1 peptide with/without ZSTK474, NY-ESO-1 specific CD8+ T cells were highly induced by adding ZSTK474. We next examined memory T cell responses in these mice. Re-challenge of CMS5a-NY-ESO-1 cells into tumor-free mice did not lead to tumor growth. Interestingly, when parental CMS5a cells were inoculated into the tumor-free mice, mice treated with anti-PD-1 antibody alone showed an aggressive tumor growth by CMS5a, whereas those treated with combination of anti-PD-1 antibody and ZSTK474 rejected the tumor. These results suggest that treatment with ZSTK474 may promote the development of memory responses against wide ranges of tumor antigens, probably due to changes of intracellular T-cell activation signal to memory formation. Actually, treatment with ZSTK474 increased frequency of memory precursor effector cell (MPEC) in tumor antigen-specific CD8+ T cells. These results suggest that ZSTK474 decreases Treg cells and activates anti-tumor immunity providing a rationale for the combination therapy of a PI3K inhibitor, ZSTK474 and PD-1 blockade.

#4719

Small-molecule antagonists of the Aryl Hydrocarbon Receptor (AhR) promote activation of human PBMCs in vitro and demonstrate significant impact on tumor growth and immune modulation in vivo.

James Joseph, Marcos Gonzalez-Lopez, Christina Galang, Candy Garcia, Hadia Lemar, Jing Lu, Kedar Vaidya, Marcus Fischer, Christian Frey, Muzaffar Alam, Bing Yao, Michael Dillon, Jeffrey H. Hager, Eleni Venetsanakos, Fred Aswad. _Ideaya Biosciences, South San Francisco, CA_.

Cancer cells utilize a variety of pathways to generate an immune permissive growth environment and evade the host anti-tumor immune response. One such pathway is production of T-cell inhibitory tryptophan (Trp) metabolites, kynurenine and kynurenic acid (KYN), via up-regulation of indoleamine 2,3-dioxygenase (IDO1) and tryptophan 2,3-dioxygenase (TDO2) and up-regulation of the KYN- receptor (aryl hydrocarbon receptor, AhR) in infiltrating lymphoid and myeloid cells. Through AhR activation, KYN both directly and indirectly, via stimulation of regulatory T-cells (Tregs), dendritic cells (DCs) and tumor-associated macrophages (TAM), modulates CD8+ and CD4+ T-cell function resulting in potent inhibition of anti-tumor immunity. Inhibition of AhR activation therefore represents a novel approach to blocking the immuno-inhibitory effects of Trp metabolites generated by endogenous IDO1 and TDO2. To thwart the effects of KYN and other immunosuppressive AhR agonist ligands, we have generated a series of small-molecule antagonists of the human and mouse AhR. To further analyze the effects of AhR inhibition on immuno-modulation and tumor growth, we selected from our series a highly potent antagonist of AhR (IDB-AHRi). In an XRE-dependent luciferase reporter system, IDB-AHRi blocked activation of AhR by 2,3,7,8-Tetrachlorodibenzodioxin (TCDD) with an IC50 of 111 and 3 nM in human HEPG2 and mouse HEPA1C1C7 cell lines respectively. IDB-AHRi inhibited translocation of cytoplasmic AhR to the nucleus and protected AhR from TCDD-induced degradation. When human peripheral blood mononuclear cells were treated with IDB-AHRi ex-vivo, expression of IFN-γ and TNF-α was significantly enhanced, an effect that could be reversed with the addition of TCDD. In vivo, IDB-AHRi dosed at 30 mg/kg (PO QD) in the CT26 colon carcinoma syngeneic mouse model resulted in a reduction of tumor growth, an increase in tumor-infiltrated CD45+CD8+Ki67high T-cells and a significant reduction in the frequency of tumor infiltrated FoxP3+CD4+ regulatory T-cells and tumor associated M2-like macrophages. In summary, this data demonstrates that blocking AhR with a highly potent antagonist can significantly enhance T-cell activity in vitro and drive anti-tumor immunity in vivo, resulting in reduced tumor growth.

#4720

TIGIT blockade enhances cytolytic function in antigen-specific CTLs in a manner non-redundant to PD1 blockade.

Deepali Malhotra, Gordon Moody, Michael Overstreet, John Mumm. _MedImmune, Gaithersburg, MD_.

Background Many mechanisms contribute to the dysfunction of CTLs in the tumor microenvironment, rendering these cells unable to kill. TIGIT is a co-inhibitory receptor on T cells that is notably expressed at high levels by antigen-specific PD1-expressing CTLs in tumors. The ligands for TIGIT, CD155 and CD112, are in turn expressed by tumor cells and tumor-infiltrating immune stroma. TIGIT-mediated suppression of T cell function results from T cell intrinsic competition with the co-stimulatory molecule CD226 for engagement of CD155 and CD112, or enhancement of cell intrinsic repressive function of TIGIT downstream of the T cell receptor (TCR). To dissect the release of TIGIT-mediated suppression of polyclonal antigen-specific CTLs, we 1) examined the expression of TIGIT, CD226, and their shared ligands CD112 and CD155 in primary human tumor disaggregates and 2) studied the functional consequences of TIGIT blockade in an assay in which human polyclonal antigen-specific CD8+ CTLs were expanded and co-cultured with human tumor cell lines that endogenously express their cognate antigen.

Methods Primary human tumor disaggregates were examined for expression of TIGIT, CD226, CD155 and CD112 by flow cytometry. To further examine the repressive function of TIGIT, CTLs from donor human PBMCs were expanded in the presence of a specific major histocompatibility class I (MHC I) peptide antigen and subsequently screened for enrichment of activation and exhaustion markers to confirm expansion. CTLs were then co-cultured with CD155+ and CD112+ tumor cell lines endogenously expressing the MHC I-binding peptide of interest, in the presence and absence of TIGIT or PD1 blocking antibodies. To further investigate the mechanism of cytolysis, inhibitors of TCR signaling, cytoskeletal rearrangement, and cytokine receptor signaling were investigated.

Summary of Results TIGIT blockade induced a rapid, potent, and reproducible cytolytic response in co-culture experiments. The maximal effect was similar to that observed with PD1 inhibition, and moreover the combination of PD1 and TIGIT blockade was greater than either monotherapy, implying non-redundancy of mechanism. Inhibitors of cytoskeletal function and signal transduction downstream of the TCR appeared to block TIGIT mediated cytotoxicity.

Conclusion TIGIT has a clearly defined role in the repression of cytotoxic function in antigen-specific CTLs, in a manner non-redundant with PD1. The enhancement in cytotoxicity subsequent to TIGIT blockade is dependent on cytoskeletal function as well as additional signal transduction downstream of TCR engagement. Strategies to enhance these signal transduction events may be of further interest to potentiate TIGIT blocking therapy.

#4721

Combining STING agonists with an anti-PD-1 antagonist results in marked antitumor activity in immune-excluded tumors.

Samanthi A. Perera, Johnny E. Kopinja, Yanhong Ma, Jason Laskey, Kalyan Chakravarthy, Yiping Chen, Long Cui, Jeremy Presland, Shuxia Zhao, Ellen Minnihan, Heidi Ferguson, Jennifer Piesvaux, Bo-Sheng Pan, Hyun Chong Woo, Ian Knemeyer, Saso Cemerski, Jared Cumming, Wesley Trotter, George H. Addona, Brian J. Long. _Merck & Co., Inc., Boston, MA_.

The innate immune agonist STING (STimulator of INterferon Genes) binds its natural ligand 2'3'-cGAMP (cyclic guanosine-adenosine monophosphate) and initiates type I interferon production. This promotes systemic antigen-specific CD8+ T-cell priming that eventually provides potent anti-tumor activity. To exploit this mechanism we synthesized a novel STING agonist, MSA-1, that activates both mouse and human STING with higher in vitro potency than cGAMP. MSA-1 was administered to immune-competent mice bearing MC38 syngeneic tumors to monitor pharmacodynamics, pharmacokinetics and in vivo efficacy. Intratumoral (IT) dosed MSA-1 demonstrated robust tumor and plasma cytokine upregulation and effective anti-tumor activity. The highest tolerated doses provided complete responses (CRs) in 100% of MC38 tumors. Tumor models such as CT26 and B16-F10 that are intrinsically resistant to single-agent therapy with a fully murinized mouse anti-PD-1 antibody (mDX400) also demonstrated long-term tumor regressions or CRs. Mechanistic studies in immune-deficient mice suggested that anti-tumor activity of IT dosed STING agonists are in part due to cytotoxicity and/or innate immune responses rather than development of robust adaptive anti-tumor immunity. To enhance the adaptive immune response, we combined MSA-1 with mDX400 in mouse syngeneic tumor models previously characterized to be unresponsive to anti-PD-1 blockade. This combination restored T-cell responses in both blood and tumors of the treated mice and provided long-lived immunologic memory in a majority of the animals. Taken together, these data strongly support the development of STING agonists in combination with Keytruda for patients with tumors that are partially responsive or non-responsive to single agent anti-PD-1 therapy.

#4722

4SC-202 increases immunogenicity of tumor cells, induces infiltration of tumor microenvironment with cytotoxic T cells, and primes tumors for combinations with different cancer immunotherapy approaches.

Svetlana Hamm, Tanja Wulff, Kerstin Kronthaler, Sabine Schrepfer, Ulrike Parnitzke, Anne Catherine Bretz, René Bartz. _4SC, Planegg-Martinsried, Germany_.

Various histone deacetylases (HDAC) inhibitors were described as beneficially affecting anti-tumoral immune response. Although different HDAC inhibitors were investigated in syngeneic tumor models, their mode of anti-tumoral action (MOA) is not yet fully understood. Here, we analyzed the anti-tumoral MOA of 4SC-202, an orally available clinical stage small molecule inhibitor targeting HDAC class I. To ensure the relevance for the clinical situation we used a clinically equivalent dosage regimen. 4SC-202's effect on expression of tumor-associated antigen (TAA) and MHC molecules was analyzed in vitro and in vivo. Anti-tumoral efficacy and the impact on tumor microenvironment (TME) were analyzed in syngeneic CT26 and C38 models. Transcriptome analysis was performed by RNA-Seq, and the composition of immune cell subpopulations was determined by flow cytometry and immunohistochemistry. 4SC-202 increased expression of TAA and MHC molecules on tumor cells in vitro and in vivo suggesting a beneficial effect on immunogenicity of tumor cells. 4SC-202 significantly inhibited growth of syngeneic tumors at a dose that was inefficacious in immunocompromised mice. IFN-γ and chemokine expression was increased, and pro-inflammatory IL-1β and IL-23 decreased in the TME of CT26 tumors following 4SC-202 treatment. Detailed analysis revealed that 4SC-202 increased the number of cytotoxic CD8+ T cells (CTLs) in the tumor core without affecting their number in blood. Since the abundance of T cells in the tumor is pre-requisite for the efficacy of immune checkpoint blockade as well as the agonistic 4-1BB antibody, combinations of 4SC-202 with anti-PD-1, and anti-4-1BB antibodies were tested in the C38 model. The response rate to the antibodies alone was low in this model reflecting the refractory clinical situation. 4SC-202 was able to control the tumor growth, but did not induce tumor regression, whereas combination therapies resulted in significantly longer survival and durable complete responses in up to 83% of animals for the combination with the anti-PD-1 antibody. 4SC-202 already demonstrated a favorable safety profile with a low rate of > grade 3 treatment-related adverse events (17%) in a phase I study in heavily pretreated hematological cancer patients. With the daily dosing for 14 consecutive days a disease control rate of 83%, 1 partial and 1 complete response could be achieved. Complimentary to these clinical data, 4SC-202's immune priming capacity offers further options for development in combination with various cancer immunotherapy approaches. Combination of 4SC-202 with PD-1 blockade is now under evaluation in a phase Ib/II clinical trial in advanced cutaneous melanoma patients refractory/non-responding to treatment with anti-PD-1 antibodies ('SENSITIZE', NCT03278665).

#4723

Targeting the IDO and TDO pathway through inhibition of the aryl hydrocarbon receptor.

Jeremy Tchaicha,1 Karen McGovern,1 Luis Felipe Campesato,2 Silvia Coma,1 Xiaoyan Michelle Zhang,1 Meghan Walsh,1 Jill Cavanaugh,1 Taha Merghoub,2 Jedd Wolchok,2 Mark Manfredi1. 1 _Kyn Therapeutics, Cambridge, MA;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Though checkpoint inhibitors have become the cornerstone of immune-based oncology therapy, the majority of cancer patients do not benefit from these agents. Resistance to checkpoint inhibitors is due in part to the presence of immunosuppressive molecules which prevent immune activation despite T cell checkpoint inhibition. One such immunosuppressive molecule is kynurenine. Kynurenine, metabolized from tryptophan by indoleamine-pyrrole 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase 2 (TDO2), binds to the Aryl hydrocarbon receptor (AHR) in multiple immune cell types which leads to a net immunosuppressive environment. IDO1 inhibitors combined with checkpoint inhibitors have led to substantial clinical benefit in early clinical trials, validating disruption of the kynurenine pathway as a therapeutic strategy and substantiating that metabolites within human cancers induce immune cell tolerance. Given that kynurenine can be synthesized by both IDO1 and TDO2 and that AHR is activated by endogenous ligands other than kynurenine, AHR inhibition provides a novel approach to reverse immunosuppression in a broad range of tumor types. We will present data demonstrating that AHR antagonism can affect multiple immune cell types within the tumor microenvironment, reverse immune suppression and lead to tumor growth inhibition in tumor models. In addition, AHR inhibition combined with checkpoint inhibitors leads to enhanced anti-tumor activity and improved survival in these models relative to either agent alone.

Kynurenine can lead to increased levels of many immunosuppressive cytokines including IL22 in human T-cells which is reversed with AHR inhibition. In addition, we have previously shown that kynurenine addition during T-cell maturation leads to a more quiescent T-effector cells while AHR antagonism during maturation maintains T-cells in a more naïve-like, T-central memory state. In vivo studies in the B16 and the B16-IDO overexpressing models demonstrate that kynurenine-mediated activation of the AHR pathway leads to faster tumor growth and a more immunosuppressive tumor microenvironment, with FOXP3 T-cells playing a major role. There are also more suppressive myeloid cells in the IDO overexpressing model. AHR antagonism inhibits growth in the B16-IDO model and reverses the immunosuppressive microenvironment as indicated by changes in T cell and myeloid cell populations as well as AHR-dependent gene expression. Similar activity is also seen in the CT26 model. In addition, enhanced tumor growth inhibition and survival benefit is seen in combination with checkpoint inhibitors.

Our data indicate that AHR is a viable target for reversing immune suppression in the tumor microenvironment and could have activity in many tumor types due to activation via kynurenine and multiple other potential ligands.

#4724

Immuno-modulatory activity of BPM31510 supports T cell viability, proliferation, and function while reversing early signs of exhaustion.

Maria Nastke,1 Shiva Kazerounian,1 Nidhi Gaur,1 Shyamali Jayashankar,1 David Linsenmayer,1 Carrie Spencer,1 Arun Nambiar,1 Aishwarya Sarma,1 Anne R. Diers,1 Stephane Gesta,1 Vivek Vishnudas,1 Vikas P. Sukhatme,2 Niven R. Narain,1 Rangaprasad Sarangarajan1. 1 _BERG, Framingham, MA;_ 2 _Emory University School of Medicine, Atlanta, MA_.

Regulation of mitochondrial metabolism is crucial to alter immune cell differentiation and function; therefore, therapeutic agents which regulate mitochondrial metabolism may have efficacy in immune-mediated tumor elimination. BPM31510 is a clinical stage, nanodispersion of ubidecarenone (coenzyme Q10), an electron transfer molecule in the mitochondrial electron transport chain required for oxidative phosphorylation. Here, we used BPM31510 to assess the role of CoQ10 in the regulation of T cell function. Healthy donor peripheral blood mononuclear cells (PBMCs) activated ex vivo with αCD3/CD28 beads were used as model system to test the effects of BPM31510 on viability and functionality of T cell subpopulations. In contrast to its ability to initiate regulated cell death in cancer cells, treatment of PBMCs with increasing concentrations of BPM31510 lead to an increased frequency of viable CD3+ cells. Further phenotypic analysis revealed that cytotoxic T cells (CD8+/CD3+) and T helper cells (CD4+/CD3+), as well as NKT cells (CD56+/CD3+), contributed to the observed increase in T cell frequency. Proliferation measurements by EdU-incorporation indicated enhanced cytotoxic T cell proliferation in BPM31510 treated PBMCs, and likewise, BPM31510 increased degranulation of activated cytotoxic T cells, as indicated by measurement of plasma membrane-exposed lysosomal-associated membrane protein 1 (CD107a). Consistent with the ex vivo observations, in vivo studies using the syngeneic MC38 murine tumor model demonstrated that BPM31510 administration resulted in a dose-dependent enhancement of the number of CD3+ cells in the tumor microenvironment with cytotoxic T cells (CD8+/CD3+) representing the largest population. Together, these data define a supportive effect of BPM31510 on T cell frequency, viability, and functionality. In addition, in ex vivo activated PBMCs, BPM31510 decreased the percentage of PD1+ T cells while simultaneously increasing the percentage of PD1- T cells in the population. Moreover, in the PD1+ T cell population, PD1 expression on the cell surface was increased while PD1- T cells experienced no change in cell surface expression of PD1. These data suggest BPM31510 treatment promotes highly functional cytotoxic T cells while cells with early signs of exhaustion are induced to follow the path of exhaustion and elimination. Collectively, these results define an immune-modulatory activity for BPM31510, particularly in the T cell compartment, in part, through regulation of T cell exhaustion; this may have important implications for the use of BPM31510 in 'immunologically cold' tumor types or in combination with immune checkpoint blockade strategies.

#4725

Blockade of cyclin D-CDK4/6 sensitizes immune-refractory tumor cells to tumor antigen-specific T cell-mediated cytotoxicity by repressing SCP3-NANOG axis.

Se Jin Oh, Tae Woo Kim. _Korea University, Seoul, Republic of Korea_.

Antitumor immune drives tumor cell to acquire immune-refractory phenotypes. Previously, we demonstrated tumor antigen-specific T cells edited them resistant to CTL killing and enriched NANOGhigh cancer stem cell-like (CSC-like) cells. In this study, in an effort to elucidate targetable pathways of immune-edited tumor cells with the multiaggressiveness, we show synaptonemal complex protein 3 (SCP3), a member of Cor1 family, which is overexpressed in the immune-edited cells, upregulates NANOG by hyperactivating cyclin D1-CDK4/6 axis. The SCP3-cyclin D1-CDK4/6 axis was preserved across various types of human cancer and, notably, negatively correlated with the progression-free survival of cervical cancer patients. Moreover, targeting CDK4/6 with its inhibitor, palbociclib, succeeded in reversing the multiaggressive phenotypes of immune-edited tumor cells, and led to long-term control of the disease. Collectively, our findings establish a firm molecular link of multiaggressiveness among SCP3, NANOG, cyclin D1, and CDK4/6, and identify CDK4/6 inhibitors as an actionable drug for controlling SCP3high immune-refractory cancer.

#4726

Novel intravenous injectable TLR7 agonist, DSP-0509, synergistically enhanced antitumor immune responses in combination with anti-PD-1 antibody.

Yosuke Ota, Takeshi Otsubo, Junya Koroki, Yuko Hirose, Erina Koga-Yamakawa, Masashi Murata, Masashi Goto, Yasushi Matsuki. _Sumitomo Dainippon Pharma Co., Ltd., Osaka, Japan_.

Toll-like receptors (TLRs) are a family of pattern-recognition receptors (PRR) that recognize pathogen-associated molecular patterns (PAMPs). TLR7 is mainly expressed in plasmacytoid dendritic cells (pDCs) and recognizes virus derived single-stranded RNA (ssRNA). TLR7 stimulation in pDCs induces type I interferon secretion, which results in innate immune activation. Clinical studies have shown that TLR7 agonist induced anti-tumor immune activation. Despite these signs of clinical activity, administration of TLR7 agonist is generally limited to topical application, due to the potential of immune related adverse effects. In this study, we present a novel small molecule TLR7 agonist DSP-0509 formulated for intravenous administration. TLR7 agonistic activity was evaluated by in vitro reporter assay systems and TLR7 knock out mice. Induction of interferon alpha (IFNα) and inflammatory cytokines in mice and human blood was measured as PD response with ELISA. Anti-tumor effect was evaluated in LM8-bearing syngeneic mouse model. Combination effect of DSP-0509 with anti-PD-1 antibody was evaluated in CT26-bearing mice, along with flow cytometric analysis of tumor infiltrating lymphocytes (TILs). DSP-0509 had agonistic activity on human TLR7 (EC50= 316 nM), but not on human TLR8 (EC50> 10 μM). DSP-0509 had high water solubility and rapid elimination from the body (T1/2:0.69h), partly explained by excretion via organic anion transporting peptide (OATP) transporters. Intravenous administration of DSP-0509 induced IFNα secretion in wild type mice, but not in TLR7 knock out mice. Minimum cytokine induction dose of DSP-0509 in human whole blood was lower than that of a well-known TLR7/8 agonist 852A. Intravenous administration of DSP-0509 suppressed the primary tumor growth and the number of lung metastatic nodules in LM8-bearing model. Combination of DSP-0509 with anti-PD-1 antibody significantly suppressed the tumor growth compared to treatments with each monotherapy (P<0.05). The ratio of CD8\+ T cells and effector memory T cell populations (CD8+CD62L-CD127+) in TILs and the surface expression of MHC class I molecule on tumor cells was significantly increased in the combination group (P<0.05). All mice responded to combination therapy rejected re-challenged tumor growth. The study showed that DSP-0509 is a novel TLR7 agonist with an intravenous injectable profile and rapid elimination from the body (T1/2: 0.69h). DSP-0509 showed anti-tumor effect against primary tumor growth and metastasis. In addition, anti-PD-1 antibody furthermore enhanced the effect of DSP-0509. We also reported that the combination of DSP-0509 with anti-PD-1 antibody significantly induced effector memory T cells (p<0.05 vs each monotherapy) and showed durable response. Further evaluations of DSP-0509 are warranted.

#4726A

The oxygen carrier omx restores antitumor immunity and cures tumors as a single agent or in combination with checkpoint inhibitors in an intracranial glioblastoma mouse model.

Natacha Le Moan,1 Philberta Leung,1 Sarah Ng,1 Tina Davis,1 Carol Liang,1 Jonathan Winger,1 Stephen P. Cary,1 Nicholas Butowski,2 Ana Krtolica1. 1 _Omniox Inc, San Carlos, CA;_ 2 _University of California San Francisco, San Francisco, CA_.

Background: Hypoxia, a common feature in solid tumors such as glioblastoma (GB), is associated with resistance to chemo- and radio-therapies and poor patient outcomes. In addition, hypoxia promotes the immune escape of tumors. Therefore, reversing tumor hypoxia to create an immunopermissive microenvironment can improve antitumor response, and combined with immunotherapy approaches such as checkpoint inhibitors (CPI), may increase therapeutic efficacy. OMX is an oxygen carrier well tolerated in small (rats and mice) and large (sheep and dogs) animals. Following intravenous administration, OMX extravasates through leaky tumor vasculature and efficiently accumulates in orthotopic rodent GB and spontaneous canine brain tumors. Consequently, OMX significantly reduces hypoxia and improves the efficacy of radiotherapy and CPI.

Methods: We used in vivo bioluminescence imaging of tumor, immunohistochemistry, flow cytometry, and cytokine multiplex assays to evaluate OMX's ability to immunosensitize the GL261 brain tumor microenvironment and promote tumor cures.

Results: A single dose of OMX in brain tumor-bearing mice reduces tumor hypoxia, enhances the recruitment and infiltration of tumor-specific CX3CR1+ CD8 T cells into the tumor (using the EphA2 as a GL261-specific tumor antigen), decreases Tregs and increases activation and proliferation of cytotoxic T lymphocytes (CTLs). Specifically, OMX increases the Teff/Treg ratio by ~3-fold, indicating a switch from an immunosuppressive to an immunopermissive microenvironment. Similarly, when combined with anti-PD-1, OMX decreases Tregs, increases CTL infiltration, proliferation and cytotoxic activity, and modulates IFNg and IFNg-inducible cytokines that polarize T cells towards a Th1 phenotype. Treatment with OMX alone resulted in a 55% tumor cure rate, comparable to anti-PD-1 treatment. Furthermore, in late-stage tumor-bearing mice, we observed a 40% tumor cure rate for the combination of OMX with anti-PD-1, while anti-PD-1 alone resulted only in 5% tumor cures. In symptomatic mice with very high tumor burden, in which the combination of anti-PD-1 with anti-CTLA4 does not provide tumor cures, the addition of OMX resulted in a 20% tumor cure rate. Following rechallenge with GL261 tumor cells injected on the other side of the brain, all mice treated with OMX alone or in combination with CPI survived, indicating the presence of long-term immunologic memory against glioma cells. The survival benefit observed with OMX could be predicted with an identified circulating chemokine biomarker signature (post-hoc test).

Conclusion: By delivering oxygen specifically to the hypoxic tumor microenvironment, OMX may restore anticancer immune responses in GB patients and synergize with radiotherapy and immunotherapy to enhance tumor control and improve patient outcomes. 

### New Immunosuppressive Mechanisms in Cancer

#4727

MicroRNA-155 as a regulator of NK cell mediated solid tumor rejection.

Aman Prasad,1 Wing Keung Chan2. 1 _The Ohio State University, Dublin, OH;_ 2 _The Ohio State University, Columbus, OH_.

Natural Killer (NK) cells are granular innate lymphocytes that orchestrate immune response by releasing interferon gamma (IFN-γ): a critical cytokine heavily involved in intercellular immune and inflammatory processes. Prior work from our lab has linked microRNA-155 (miR-155) to significant cell proliferation and NK cell mediated IFN-γ release. Previously, our lab has also shown that miR-155 can increase hematological malignancy rejection and NK cell cytotoxicity in T-cell lymphomas. However, it is unclear how miR-155 can affect NK cell cytotoxicity in solid tumor models. IFN-γ has been reported in the literature to directly induce apoptosis in glioblastoma solid tumors. We therefore hypothesize that miR-155 induction can increase NK cell mediated cytotoxicity and IFN-γ mediated apoptosis for solid tumor rejection. To test this, 10x106 GL261 cells were injected subcutaneously into the flanks of both wild type (WT) littermates and miR-155 transgenic (miR-155Tg) mice. CD3(-)NK1.1(+) effector splenic NK cells were isolated from WT and miR-155Tg mice for total RNA extraction and subsequent gene expression profiling on NK cell activation pathways. We found that tumor growth in WT mice was at least 2-fold greater than that of miR-155Tg mice (average 2cmx2cm vs 0.7cmx0.7cm, n=6). Tumor weights from WT and miR-155 mice were 1052 mg vs 340 mg, respectively, with an average of 2 mice per group. Furthermore, we have successfully created an IFN-γ receptor 1 (IFN-γR1) shRNA knock-down (KD) lentiviral construct in a stable glioblastoma cell line. IFN-γR1 knockdown was measured to be 90% as determined by real-time PCR analysis. Future experiments will be performed wherein IFN-γR1 KD glioblastoma cells will be injected in miR-155Tg and WT mice to evaluate tumor rejection. In summary, we demonstrate a model system to investigate the effect of miR-155 on solid tumor glioblastoma multiforme. Further characterization of gene expression profiles of purified NK cells from both WT and miR-155 mice will provide important insights on the mechanisms of NK cell medicated tumor rejection.

#4728

Iron regulation of innate immune pathogen-associated molecular pattern receptors.

Evan Stater. _Weill Cornell Medical College, New York, NY_.

Pathogen-associated molecular pattern (PAMP) receptors are key triggers of the pro-inflammatory immune effector functions of the cells of the innate immune system. Ectopic PAMP activation is a contributing factor to both cancer development and pathology, as well as host anti-tumor immune responses. Iron, an essential mineral in biological systems, has a similar dualistic role in cancer. We have shown that iron acts as a key modulator of intracellular signaling cascades downstream from PAMP activation. Cellular iron enrichment enhances the transcriptional activity of a pathogen-induced transcription factor downstream of PAMPs in an immortalized human macrophage cell line treated with a PAMP receptor-specific agonist ligand. Cell surface expression of the receptor was not affected by the iron enrichment; therefore, any modulation of receptor signaling by iron is likely downstream of the receptor. Furthermore, iron supplementation in vitro increases expression of immunity-related genes regulated by the pathogen-induced transcription factor; by contrast, treatment with iron-chelating reagents reduces expression of these genes. These results suggest that the iron content of the tumor microenvironment may have a regulatory role in pathological immunoregulation. In particular, the iron-retaining phenotype of many tumor cells may serve a novel pathological function, inhibiting PAMP-regulated anti-tumoral effector functions of tumor-infiltrating macrophages cells by iron deprivation.

#4729

The aryl hydrocarbon receptor directly regulates microRNA-29b to inhibit human natural killer cell development and function in acute myeloid leukemia.

Steven D. Scoville, Ansel Nalin, Luxi Chen, Li Chen, Kathleen McConnell, Susana Beceiro Casas, Abd Al-Rahman, Naima Hashi, Michael Zhang, Jennifer Saultz, Jianhua Yu, Aharon Freud, Michael Caligiuri, Bethany Mundy-Bosse. _The Ohio State University, Columbus, OH_.

Acute myeloid leukemia (AML) is a deadly disease in need of novel therapeutic avenues to improve outcomes. Natural killer (NK) cells have the potential to be curative in the setting of AML, although successful strategies to deploy NK cells in patients have been limited to date, likely due in part to cancer-derived immune suppression. Recently, we showed that NK cell development is impaired in the presence of AML. In our present study, we uncover a novel mechanism by which AML releases aryl hydrocarbon receptor (AHR) ligands that in turn activate the NK cells to induce expression of microRNA (miR)-29b, resulting in impaired NK cell development and function. This developmental block was dependent on miR-29b in NK cells, as miR-29b deficient NK cells were resistant to these changes in the presence of AML and miR-29b knockout mice engrafted with AML showed improved survival. In support of these findings in mice, AML patients with a transcriptional profile consistent with an activated AHR pathway had more aggressive disease and decreased survival (p=0.006). AML-derived AHR ligands impaired NK cell development by repressing Tbet and Eomes, two master regulators of late-stage NK cell maturation. We also show that agonist-bound AHR binds a dioxin-responsive element (DRE) located in the promoter of miR-29b to directly induce its transcription as demonstrated by luciferase constructs and anti-AHR ChIP-PCR in primary NK cells. Indeed, transwell co-cultures of AML and immature NK cells found increased miR-29b in NK cells and impaired development that was contact independent. These effects were reversed with addition of the AHR antagonist CH-223191. Finally, we show that AML cells pretreated with CH-223191 and then cultured with NK cells lead to more NK cell IFN-γ production and higher cytotoxicity against AML. Collectively, these data show that AML blasts secrete AHR ligands and AHR is a significant contributor to AML-induced immune suppression by upregulating miR-29b in NK cells and delaying their development, blunting their IFN-γ production and decreasing their cytotoxicity. Furthermore, we demonstrate that targeting AHR may be an effective novel therapeutic avenue because it promotes NK cell maturation while also enhancing NK cell-mediated activation and killing of AML cells.

#4730

Targeting glutamine metabolism enhances tumor specific immunity by inhibiting the generation and function of suppressive myeloid cells.

Min-Hee Oh,1 Im-Hong Sun,1 Liang Zhao,1 Im-Meng Sun,1 Wei Xu,1 Chirag Patel,1 Robert Leone,1 Ada J. Tam,1 Judd Englert,2 Pavel Majer,3 Rana Rais,4 Barbara Slusher,4 Maureen R. Horton,5 Jonathan D. Powell1. 1 _Bloomberg~Kimmel Institute for Cancer Immunotherapy; Johns Hopkins University School of Medicine, Baltimore, MD;_ 2 _MedImmune, LLC, MD;_ 3 _Institute of Organic Chemistry and Biochemistry ASCR, Prague, Czech Republic;_ 4 _Johns Hopkins Drug Discovery Program, Baltimore, MD;_ 5 _Johns Hopkins University School of Medicine, Baltimore, MD_.

In order to sustain their inexorable growth, tumors have specialized reprogrammed metabolism. This metabolism creates an acidic, hypoxic and nutrient-depleted tumor microenvironment (TME). Such an environment inhibits antitumor effector cells while promoting the differentiation and function of inhibitory cells such as T regulatory cells, myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM). We hypothesized that by targeting tumor metabolism we could alter the TME and "condition" tumors to be more susceptible to immunotherapy. To this end, along with the Johns Hopkins Drug Discovery Program we developed a novel prodrug of 6-diazo-5-oxo-l-norleucine, inhibitor of glutamine metabolism (JHU-083). Recently, it has been shown that M2 macrophages require glutamine metabolism for differentiation and function. In light of the similarities between M2 macrophages and suppressive myeloid cells, we hypothesized that JHU-083 might inhibit the generation and function of MDSCs and TAMs. We tested this hypothesis in the 4T1 breast cancer model and 3LL lung carcinoma model. These tumors are relatively resistant to immunotherapy and are characterized by increased generation of MDSCs and distant spontaneous metastasis. JHU-083 treated mice suppressed tumor growth compared to the vehicle treated group. Immunologically, we observed markedly reduced numbers of MDSCs in circulating blood within 3 days of drug treatment compared to vehicle group, leading to favorable CD8 to MDSCs ratios. Consistently, JHU083-treated group displayed significantly decreased percentages and numbers of MDSCs in the tumor, and increased tumor infiltrating CD8 cells. Interestingly, JHU-083 treatment induced TAM reprogramming. While the TAM from the vehicle treated group displayed increased M2 markers and arginase; the JHU-083 treated tumor-infiltrating cells showed increased TNF-a producing M1-like macrophage phenotypes compared to vehicle group. These TNF-a producing cells were negatively correlated with tumor sizes. Notably, JHU083 treatment not only controlled primary tumor growth but also drastically reduced spontaneous lung metastasis. The decrease of MDSCs infiltration in the lung were also observed in JHU083-treated group. Mechanistically, our data suggest that JHU-083 inhibits CSF2/CSF3 production and survival of the tumor itself as well as directly affects macrophage metabolism and signaling. Also, LC-MS based metabolites analysis from JHU-083 treated tumors revealed reduced kynurenine:tryptophan ratios compared to the control group, indicating the metabolic modulation of the tumor microenvironment. Overall, our data support a novel role for glutamine inhibitor, JHU-083, in enhancing tumor-specific Immunity by targeting suppressive myeloid cells.

#4731

Contribution of FcγRIIB to creating the suppressive tumor microenvironment.

Yuki Kasahara, Hidekazu Shirota, Chikashi Ishioka. _Tohoku University, Sendai, Japan_.

Various immune cells are recruited in tumor microenvironment. It is well established that cellular immune responses, such as cytotoxic or suppressive activities play an important role in regulating tumor growth and metastasis. However, contribution of humoral immune responses against tumor is incompletely understood. The Fc receptors constitute critical elements for activating or down-regulating immune responses through immunoglobulin immune complex. Here, we examined the potential role of inhibitory Fc receptor, Fc gamma RIIb in tumor immunity using mice model. Current findings indicate that tumor-specific antibodies are induced and control tumor immunity. Deletion of Fc gamma RIIb receptor significantly improves both cellular and humoral anti-tumor immunity, delays tumor growth. These findings indicate that spontaneous antibody against tumor create the suppressive tumor microenvironment through Fc gamma RIIb signaling, and suggests attractive therapeutic target for cancer immunotherapy.

#4732

Myeloid-derived suppressor cells potentiate colorectal carcinogenesis: Identification of a novel RIPK3-PGE2 circuit in tumor microenvironment.

Yongsheng Li, Guifang Yan, Huakan Zhao, Qi Zhang. _Inst. of Cancer, Xinqiao Hospital, Third Military Medical Univ., Chingqing, China_.

The role of receptor-interacting protein kinase 3 (RIPK3), a central factor mediating necroptosis, in tumor immunity is largely unknown. Here we show the significantly decreased RIPK3 in the tumor microenvironment of colorectal cancer (CRC) correlated with the accumulation of myeloid-derived suppressor cells (MDSCs). Deficiency of RIPK3 boosted the infiltration and immunosuppressive activity of MDSCs in vitro and in vivo. Mechanistically, the reduction of RIPK3 in MDSCs and colorectal cancer cells elicited NF-κB expression in the nuclei and the transcription of COX-2 which catalyses the synthesis of prostaglandin E2 (PGE2). PGE2 exacerbated the immunosuppressive activity of MDSCs, i.e. increased arginase 1 (Arg-1), blunted CD8+ T cell activation and accelerated tumor growth. Moreover, PGE2 inhibited RIPK3 expression while enhanced the expression of NF-κB and COX-2 in MDSCs and CRC cells. Inhibition of COX-2 or PGE2 receptors significantly reversed the immunosuppressive activity of MDSCs and dampened tumorigenesis. The patient databases also delineated the correlation of RIPK3 and COX-2 with CRC survival. Our findings demonstrate a novel signaling circuit by which RIPK3 and PGE2 regulate tumor immunity, and provide potential targets for the immunotherapy of CRC.

#4733

Gastric cancer cells derived exosomes can affect the immunomodulatory function of MSCs through constant activation of NF-kB by PKM2.

Junjie Gu,1 Zhao Sun,1 Chunmei Bai,1 Yang Zeng2. 1 _Chinese Academy of Medical Science Peking Union Medical College Hospital, Beijing, China;_ 2 _Massachusetts General Hospital, Boston, MA_.

Background: Bone marrow or adipose-derived MSCs homing to tumor tissues are educated by tumor cells and become tumor MSCs (T-MSCs), which is an important part of the tumor immune microenvironment(TME). T-MSCs can secrete large numbers of inflammatory factors and increase the proportion of inhibitory immune cells, which plays an important role in tumor progression. In our previous study, we found that gastric cancer cells-derived exosomes(GC-exosomes) can activate the NF-κB signaling pathway of naïve MSCs, and GC-exosomes treated MSCs(GC-exosomes-MSCs) had the similar function of T-MSCs. GO analysis found that the proteins in tumor exosomes were mainly associated with anaerobic metabolism. Therefore, we plan to investigate whether GC-exosomes can activate the NF-κB signaling pathway through changing the metabolic phenotype of MSCs, and further transform the immunomodulatory function of MSCs.

Methods: The tumor exosomes were isolated and purified by ultrafiltration. The effect of GC-exosomes on the metabolic phenotype of MSC was detected by Agilent Seahorse XF Cell Energy Phenotype Test. The Effect of GC-exosomes on the immunomodulatory function of MSCs was tested by Flow Cytometric Assay. Western blot and RT-PCR were used to analyze the expression of anaerobic metabolism related enzymes in T-MSCs. Immunoprecipitation and Duolink in situ fluorescence were used to analyze the relationship between anaerobic metabolism related proteins and immune signaling pathways.

Results: GC-exosomes-MSCs could then up-regulate the expression of IL-6,IL-8,CCR2, inhibit the proliferation of T cells,promote the activation of CD4+ T cells,and regulate the homing of macrophages. GC-exosomes could switch MSCs towards a more glycolytic phenotype (similar to Warburg effect in tumor cells).The expression of anaerobic metabolism-related protein the M2 isoform of pyruvate kinase(PKM2) and other enzymes were obviously up-regulated. PKM2 could then translocate to the nucleus of GC-exosome-MSCs (gradually increased from day 3). PKM2 could bind to the transcription factor p65 of NF-kB signaling pathway and constantly promote the activation of NF-kB signaling pathways.

Conclusions: Our study suggests that in MSCs gastric cancer cells derived exosomes can promote the expression and translocation to nucleus of PKM2,which binds to p65, and ultimately trigger the constant activation of NF-kB signaling pathway and transform the immunomodulatory function of MSCs.

#4734

Suppressive myeloid cells of the solid tumor microenvironment enhance regulatory T cell function and differentially affect CAR-T cell function.

Charlotte H. Rivas, Anna Cole, Cliona M. Rooney, Robin Parihar. _Texas Children's Hospital, Houston, TX_.

Immunotherapy with tumor-directed immune cells, such as chimeric antigen receptor-bearing T (CAR-T) cells, have shown great promise. Indeed, CAR-T cells directed against leukemia antigens such as CD19 have produced impressive complete responses even in relapsed disease patients. In contrast, CAR-T cells directed against solid tumor have produced very few durable clinical responses. This may be, in part, due to the suppressive immune microenvironment of solid tumors that inhibits the anti-tumor activity of endogenous and CAR-T cells. Myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and inhibitory macrophages (M2s) are recruited by tumor-derived factors, and contribute to the immunosuppressive tumor microenvironment (TME) by secreting suppressive cytokines, expressing inhibitory ligands, and promoting tumor neovascularization. However, the contribution of each of these components to suppressing engineered cellular therapies has not been well defined. In order to design more effective cellular therapies for solid tumors, it is imperative that we understand TME-mediated immune suppression of human CAR-T cells. Therefore, the objective of this study was to evaluate the individual contribution of MDSCs, Tregs, and M2s to the suppressive TME and their ability to modulate CAR-T function. We hypothesized that MDSCs, Tregs, and M2s would inhibit GD2.CAR-T homing and proliferation at tumor sites, leading to decreased activity and tumor progression. To understand the individual contributions of these suppressive cells to inhibition of CAR-T cells, we developed a unique TME model in which human neuroblastoma tumor cells were co-inoculated subcutaneously with MDSCs, Tregs, or M2s in immunocompromised mice and then treated with GD2 neuroblastoma antigen-specific CAR-T cells. We found that while all three cell types decreased GD2.CAR-T cell-mediated tumor control in vivo, only MDSCs and M2s inhibited CAR-T homing and expansion at tumor sites. To investigate cross-talk between these suppressive cells, we assessed the phenotype and suppressive activity of MDSCs, Tregs, and M2 on autologous CAR-T cells after exposure to the other suppressive cell types. We found that the suppressive function of M2s increased by 30% after exposure to Tregs. Similarly, Treg suppressive function increased by 26% after exposure to M2s. Both M2 and Treg suppressive functions were enhanced in the presence of MDSCs. Our results suggest a potential cross-talk between suppressive cells of the TME and distinct roles in important aspects of CAR-T cell function, including homing and killing. Future studies in our lab aim to further understand the differential roles of these cells in regulating CAR-T cell function within the TME that will inform new approaches to engineer effective adoptive T-cell therapies.

#4735

Role of myeloid derived suppressor cells in promoting ovarian cancer.

Ramandeep Rattan,1 Sharif Sakr,2 Raymond Quiles,1 Robert Morris,2 Thomas Buekers,1 Adnan Munkarah,1 Shailendra Giri1. 1 _Henry Ford Health System, Detroit, MI;_ 2 _Wayne State University, Detroit, MI_.

Myeloid Derived Suppressor Cells (MDSCs) are a heterogeneous population of immature myeloid cells that are increased in tumors and create an immunosuppressive environment by inhibiting the T-cell function. In addition, MDSCs promote angiogenesis, tumor invasion, and metastasis. Increased MDSC accumulation in epithelial ovarian cancer (EOC) has been associated with poor prognosis. Our study investigated whether depletion of MDSCs will influence EOC progression and enhance the therapeutic response of programmed death-1 (PD1) immunotherapy. The intraperitoneal ID8-luciferase syngeneic mouse epithelial ovarian cancer cell model in B6 mice was used for the study ID8 bearing mice exhibited significantly higher levels of MDSCs (CD11bGr1+) (p<0.001) in bone marrow, blood and spleen compared to mice with no tumors control groups, including its sub-populations; granulocytic and monocytic as the tumor progressed. Additionally, the MDSCs isolated from tumor-bearing mice exhibited higher ability to suppress T cell proliferation (p<0.01) and function as reflected by lower IFN-gamma production (p<0.01). Adoptive transfer of tumor educated MDSCs resulted in earlier and aggressive ovarian tumor progression. The adoptively transferred MDSCs (from 45.2 mice) were found to infiltrate in the ovarian tumors of the donor mice (45.1 mice). Once a week treatment of the ID8 tumor bearing mice with the anti-Gr1 specific monoclonal antibody (Ly6G/Ly6C mAb) that targets and depletes MDSCs, resulted in significant retardation in the progression of ovarian cancer in mice as reflected by luciferase based quantification of tumor images (p<0.01), decreased ascites volume (p<0.001) and tumor burden at diaphragm, peritoneal cavity and other organs. Ascitic fluid from tumor bearing mice treated with anti-Gr1 showed decreased MDSC counts associated with an increase in the number of IFN-gamma producing CD4 and CD8 T cells (p<0.01). Also, associated with MDSC depletion and tumor retardation was increase in T-cytotoxic and T-helper cells, and decreased tumor associated macrophages and T-regulatory cells. PD-1 immunotherapy response in ID8 tumors was enhanced in combination with MDSC depletion as seen from decreased tumor burden and restoration of anti-tumor response. Overall, our data suggests that targeting immunosuppression by reducing MDSCs can improve the anti-tumor immune response and aids in containing EOC progression and enhance immunotherapy response. Thus targeting of MDSCs represents a potential therapeutic modality in ovarian cancer and may be considered in combination with other immunotherapies.

#4736

Bone marrow microenvironment-induced miR-300 expression impairs natural killer cell proliferation and antitumor activity.

Rossana Trotta,1 Giovannino Silvestri,1 Lorenzo Stramucci,1 Martin Guimond,2 Guido Marcucci,3 Xiaoxuan Fan,1 Maria R. Baer,1 Danilo Perrotti1. 1 _Greenebaum Comprehensive Cancer Center, University of Maryland School of Medicine, Baltimore, MD;_ 2 _Maisonneuve-Rosemont Hospital Research Center, University of Montreal, Montreal, Quebec, Canada;_ 3 _Hematologic Malignancies and Stem Cell Transplantation Institute, City of Hope National Medical Center, Duarte, CA_.

Natural killer (NK) cells mediate immune responses against cancer; however, NK cell quantitative and functional defects are features of cancers including chronic myelogenous leukemia (CML). As increased numbers of activated NK cells were found in CML patients in treatment-free remission, the understanding of the molecular events inhibiting NK proliferation and function may lead to the development of NK cell-based therapies against drug-resistant cancer stem cells. Because altered miRNA expression and inactivation of the protein phosphatase 2A (PP2A) tumor suppressor are also features of cancer, and SET-dependent PP2A inhibition is essential for NK cell function, we hypothesized that increased expression of miR-300, a miRNA with antiproliferative activity, found inhibited in CML and targeting the PP2A inhibitor SET, accounts for impaired NK cell proliferation/activity. An initial analysis revealed that miR-300 levels were significantly higher in peripheral blood (PB) CD56+CD3- NK cells from CML patients at diagnosis compared to healthy individuals. As NK cell activity is regulated by the bone marrow microenvironment (BMM), we evaluated whether hypoxic conditions and/or cell-to-cell interaction influence NK cell proliferation and cytotoxic activity by modulating miR-300 intracellular levels. A marked and significant increase in miR-300 expression was detected in NK-92 and primary CD56+CD3- NK cells exposed to low O2 levels or cultured in the presence of conditioned medium (CM) or exosomes isolated from BM-derived primary mesenchymal stromal (MSC) and HS-5 MSC cells. As expected, increased miR-300 levels correlated with decreased SET levels and markedly reduced NK cell number. Interestingly, miR-300-induced growth inhibition was rescued in NK cells treated with CM/exosomes from HS-5 cells expressing an anti-miR-300 lentivirus. Notably, qRT-PCR indicated that miR-300 was contained in MSC exosomes. Functionally, exposure to MSCs (CM or exosomes) inhibited NK cell IL-12/IL-18-induced IFN-γ production and cytotoxic activity against K562 CML-BC cells in a miR-300-dependent manner. Accordingly, miR-300 lentiviruses and/or CpG-miR-300 oligonucleotides inhibited SET expression, reduced proliferation and suppressed spontaneous cytotoxicity of NK-92 and/or primary NK cells, likely through reactivation of PP2A. Because BM hypoxic conditions and MSCs significantly contribute to decreased NK cell number and cytotoxic activity through upregulation of miR-300, its genetic or pharmacologic inhibition may result in reactivation of NK cell activity against leukemic stem/progenitor cells.

This work is supported in part by NIH-NCI RO1CA163800.

#4738

IL-17 induces myeloid-related stimulating factors by stromal cells.

Gianluca Mucciolo,1 Roberta Curto,1 Claudia Curcio,1 Cecilia Roux,1 Luca Vannucci,2 Francesco Novelli,1 Paola Cappello1. 1 _Università degli Studi di Torino, Torino, Italy;_ 2 _Institute of Microbiology v.v.i., Czech Academy of Sciences, Prague, Czech Republic_.

Pancreatic ductal adenocarcinoma (PDA) is one of the most aggressive cancer with a 5-year survival rate of 8%. An hallmark of PDA is the massive desmoplasia and the complex stromal-tumor interaction, regulated by a pletory of secreted factors. The role of IL-17 and T-helper 17 in cancer progression or anti-tumour immunity remain controversial. To assess the role of this molecule, we crossed genetically engineered mice (GEM) characterized by a spontaneous PDA development, with IL-17 knockout mice (GEM/IL-17 KO). The absence of IL-17 correlated with an increased pancreatic fibrosis, evaluated by second harmonic generation approach. The transcripts and secretome of Cancer Associated Fibroblasts (CAFs), obtained from tumors arose in both WT and IL-17 KO GEM, have been evaluated. More than 60 genes were differently modulated in the CAFs from GEM/IL-17 KO mice. Among them, GM-CSF, G-CSF, M-CSF, CXCL1 and 2 where strongly up-regulated in GEM/WT compared to GEM/IL-17 KO mice and significantly inhibited by the addition of the recombinant cytokine. The absence of those factors paralleled an increased Th1-skewed cytokine pattern released by CAF that affected T cell infiltration and response. Taken together, these results suggest that IL-17 does affect the tumor progression by promoting a CAFs-mediated release of cytokines involved in myeloid cells recruitment and suppression of an anti-tumoral immune response. This feature defines IL-17 as a potential target to shape the tumor microenvironment and favour immunotherapy success.

#4739

Elucidation of immunosuppressive microenvironment in colorectal cancer.

Tetsuro Sasada, Junya Ohtake, Tetsuta Satoyoshi, Keisuke Kazama, Satoshi Wada, Erika Yada, Manabu Shiozawa. _Kanagawa Cancer Center, Yokohama, Kanagawa, Japan_.

Colorectal cancer (CRC) is one of the most increasing cancers worldwide, and novel treatment modalities remain to be developed for advanced CRC patients. In recent years, immune checkpoint inhibitors have demonstrated impressive clinical efficacy in various types of cancers, but limited clinical responses were reported in CRC, except for MSI-high or MMR-deficient tumors. It has thus been suggested that immune suppressive mechanisms other than immune checkpoints might contribute to tumor progression in CRC. In the current study, various types of immune cells were isolated from peripheral blood or resected tumor/normal tissues and regional lymph nodes in 25 primary CRC patients, and their phenotypes and functions were comprehensively assessed. Flow cytometry analysis demonstrated a significant increase of CD4+FoxP3+ T cells, which might represent regulatory T cells, in proximal lymph nodes (N1 region), compared to distal (N2 or N3 region) or unrelated lymph nodes. In addition, the functional analysis of T cells assessed by cytokine production after non-specific stimulation (PMA and A23187) showed a significant decrease in the frequencies of IFN γ-producing CD8+ T cells in N1 region. Notably, reduction of IFN γ-producing CD8+ T cells seemed to be associated with advanced stages of disease. These results indicate that the immunosuppressive microenvironment, particularly in regional lymph nodes, may be related to tumor progression in CRC. The current study suggests that monitoring of immunosuppressive microenvironment would be useful as a predictive biomarker for cancer progression and patient prognosis in CRC. Further studies remain to be performed to more thoroughly elucidate immune suppressive mechanisms, and thereby to develop a novel immunotherapeutic strategy in CRC.

#4740

CLEVER-1 as a marker to identify breast cancer patients under immunosuppression.

Reetta E. Virtakoivu,1 Pia Boström,2 Riitta Aaltonen,2 Ilkka Koskivuo,2 Sirpa Jalkanen,1 Maija Hollmén1. 1 _University of Turku, Turku, Finland;_ 2 _Turku University Hospital, Turku, Finland_.

TAMs are the main innate immune cells regulating tumor growth and metastasis. In tumors, TAMs exhibit an immunosuppressive M2 phenotype attenuating anti-tumor immune responses. CLEVER-1 is a multifunctional scavenger receptor specifically expressed on a subset of M2 macrophages and human monocytes. Interestingly, a subset of CLEVER-1+ monocytes and TAMs are increased in conditions where immunosuppressive functions prevail, like pregnancy and cancer. In addition, high CLEVER-1 mRNA levels associates with poorer overall survival of breast cancer patients. Therefore, we want to investigate can CLEVER-1 be used as a surrogate marker for identifying cancer patients under immunosuppression. Since we detected CLEVER-1+ TAMs in 40% of breast cancer patients, we will characterize whether the amount of CLEVER-1+ TAMs in tumor biopsy samples correlates with the number of CLEVER-1+ monocytes in breast cancer patient blood. We will collect blood, plasma, healthy breast tissue and tumor tissue from 200 newly diagnosed treatment naive breast cancer patients. The tissue samples and the blood from each patient are characterized by flow cytometry to identify the activation status of monocyte and macrophage populations in relation to CD4/CD8, Th1, Th2, Th17 and Treg populations. By now, we have received 25 patient samples from which 12 have been initially analyzed for CLEVER-1 expression in the peripheral blood together with other immune cell populations. Our preliminary data show that CLEVER-1 expression on peripheral blood monocytes is increased in breast cancer patients who have higher levels of Tregs. Additionally, the amount of CD4+ effector T cells (Th1 and Th17) was decreased among patients, whereas the amount of Th2 CD4+ cells was increased when comparing to the blood of healthy volunteers. The abnormal Th1/Th2 ratio is linked to cancer progression. The expected outcome is that CLEVER-1 could be used as a surrogate marker to identify patients under immune suppression. These patients could benefit from anti-CLEVER-1 therapy or other clinically available immunomodulatory drugs (e.g. anti-CTLA-4; ipilimumab, anti-PD1; nivolumab).

#4741

Monocytic myeloid derived suppressor cells (M-MDSC) from spleen are multipotent while tumor M-MDSC have limited plasticity.

Ryan D. Calvert,1 James C. Fleet,1 Ye Chen,1 Alex Pothen,1 Bartek Rajwa,1 Pierrick G. Fournier,2 Patricia Juarez,2 Theresa A. Guise,2 Timothy L. Ratliff,1 Ben D. Elzey1. 1 _Purdue Univ., West Lafayette, IN;_ 2 _Indiana University Purdue University, Indianapolis, IN_.

The tumor (Tu) microenvironment contains monocytic myeloid-derived suppressor cells (M-MDSC, CD11b+Ly6ChiLy6Glo) that suppress cytotoxic T cell-mediated immune surveillance. M-MDSC-like cells are also found in the spleen of Tu-bearing mice (Sp) and are often used as surrogates for Tu MDSC. We conducted a series of experiments to test if these two cell types are functionally similar. Sp- and Tu M-MDSC were isolated and used in a short-term T cell suppression assay (16 h) but only Tu M-MDSC could suppress T cell proliferation. Next, we examined the differences between the Sp and Tu by examining cells with M-MDSC or granulocytic-MDSC (G-MDSC; CD11b+Ly6CmedLy6Ghi) markers using Affymetrix Mouse Gene 1.0 ST microarrays. More than 5600 differentially expressed transcripts (1.5 fold, 5% FDR) were seen between the Sp and Tu within each MDSC subtype; 1451 of these transcripts were unique for the M-MDSC type. While others have shown that Sp M-MDSC can differentiate toward other end-stage myeloid cells, it is not clear whether this plasticity extends to Tu M-MDSC. Thus, we tested the ability of M-MDSC cells from Tu or Sp to differentiate into osteoclasts (Oc; RANKL and M-CSF) and immune cells found in the Tu microenvironment: G-MDSC (GM-CSF), dendritic cells (DC; GM-CSF and IL-4), or macrophages (Mφ; M-CSF). Upon treatment, Sp M-MDSC acquired the markers of Oc (RANK), G-MDSC (loss of Ly6C, gain of Ly6G), DC (CD11c), and Mφ (F4/80). In contrast, Tu M-MDSC could differentiate into Mφ, but not into Oc, DC or G-MDSC. When Tu M-MDSC were cultured with both tumor explant supernatant (TES, to mimic the Tu microenvironment) and differentiating agents their capacity to differentiate did not significantly change. However, TES-treated Sp M-MDSC became less responsive to signals for conversion to G-MDSC and DC and more responsive to the Mφ-inducing treatment. Our data suggest that Sp M-MDSC are immature, multipotent cells and addition of TES causes them to mature towards a less plastic Tu M-MDSC. Finally, we tested whether the functional differences among naïve bone marrow, Sp, and Tu cells were detectable from morphological (FSC-A, SSC-A) or cell marker data (CD11b, Ly6C, Ly6G) from flow cytometry. Singlet myeloid cells were selected via automated gating and continuous phenotype data was subjected to unsupervised K- means clustering to create unique templates of cell populations for each tissue site (FlowMatch). An n-1 cross-validation approach tested the accuracy of the classification and showed that the 3 templates correctly classified each sample to its tissue of origin. Thus, multidimensional flow cytometry data reflects the functional differences between CD11b+Ly6ChiLy6Glo cells from different tissues. The major finding of these experiments is that Tu M-MDSC are a more mature phenotype that are morphologically and functionally distinct from the M-MDSC precursors found in Sp.

#4742

Role of tumor-associated macrophages in esophageal cancer: PD-L1 expression and prognosis.

Taisuke Yagi, Yoshifumi Baba, Yuki Koga, Tomoyuki Uchihara, Yuki Kiyozumi, Hiroshi Sawayama, Yukiharu Hiyoshi, Masaaki Iwatsuki, Yuji Miyamoto, Naoya Yoshida, Hideo Baba. _Kumamoto university, Kumamoto, Japan_.

Introduction: It has become clear that macrophages are important cells forming cancer microenvironment and tumor-associated macrophages (TAM) promote their proliferation in various cancer types. In particularly, M2-type macrophages have been reported to promote the production of anti-inflammatory factors and the infiltration of regulatory T cells and suppress anti-tumor immunity, and new antitumor therapies that control tumor growth by controlling TAM have been proposed. In addition, "anti-PD-1 antibody" has attracted attention also in esophageal cancer, but the relationship between TAM and PD-L1 expression has not been clarified.

Methods: A retrospective study was performed on 301 patients who underwent esophagectomy for esophageal cancer between March 2005 and June 2013. We performed immunohistochemistry and evaluation of macrophage markers (CD163 and CD204) and PD-L1. In addition, we performed in vitro experiments to clarify the cellular malignancy and relationship with PD-L1 expression.

Results: Out of 301 cases of esophageal cancer, 156 cases showed CD163 high expression (51.8%) while 146 cases showed CD204 high expression (48.5%). We divided into three groups of TAM status based on CD204 and CD163 expression (CD163 and CD204 both positive: TAM status high (n=100), either one positive: moderate (n=102), both negative: low (n=99)). In TAM high group, there were more advanced cancers and adenocarcinoma cases (P<0.0001, P=0.042, respectively). Interestingly, TAM status correlated with PD-L1 expression (P=0.0005). We found that there was a significant difference in overall survival, cancer specific survival and disease-free survival among these three groups (log rank P = 0.0062, P= 0.0012, P= 0.0006, respectively), and TAM status correlated with poor prognosis. We performed double immunostaining of PD-L1 and CD163, there is a part where PD-L1 is expressed around TAM, suggesting that the expression of PD-L1 and the presence of TAM are correlated. We performed co-culture of esophageal cancer cell lines (TE-8 and KYSE30) and activated macrophage. In real time PCR PD-L1 expression were elevated at co-culture with activated macrophage. Fluorescence activated cell sorting (FACS) revealed PD-L1 expression positive cells were increased at co-culture with activated macrophage. In invasion assay, when co-cultured with activated macrophages, the number of infiltrating cells were significantly increasing (P=0.00021). In timelapse imaging showed increased cell activity by co-culture with activated macrophage.

Conclusion: This study suggested that TAM correlated with poor prognosis in patients with esophageal cancer. In addition, TAM correlated with invasive capacity and migratory capacity. Importantly, TAM correlated PD-L1 expression in esophageal cancer cell lines.

#4743

**Inhibition of the** Hif1a **-mediated checkpoint refuels NK activation in cancer.**

Jing Ni,1 Lea Bühler,1 Ana Stojanovic,1 Annette Arnold,1 Veronika Sexl,2 Adelheid Cerwenka3. 1 _German Cancer Research Center, Heidelberg, Germany;_ 2 _Institute of Pharmacology and Toxicology, University of Veterinary Medicine Vienna, Vienna, Austria;_ 3 _Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany_.

Solid tumors remain a major challenge for immunotherapy of cancer due to their immunosuppressive microenvironment. Hypoxia is a common feature of solid tumors and might affect immune cell activation within the tumor tissue. Upon hypoxia the transcription factor Hypoxia inducible factor 1a (HIF-1α) that affects many cellular processes including cell proliferation, survival and effector function becomes induced. Its impact on innate immune cells such as Natural Killer (NK) cells, however, is still incompletely understood. Natural Killer (NK) cells are considered as highly potent effector cells against tumors. Within the tumor tissue, however, NK effector function is often impaired. Our microarray analyses of tumor-infiltrated NK cells revealed upregulated levels of Hif1a. To determine the role of the HIF-1α in NK cell function, we generated the mouse strain Hif1aΔ/ΔNcr1-iCreTg in which HIF-1α is deficient in the NKp46+ lineage. Hif1a deficient NK cells showed greatly enhanced anti-tumor activity compared to wild-type NK cells, leading to a pronounced reduction of tumor growth. Tumor-infiltrated Hif1a deficient NK cells expressed lower levels of checkpoint molecules and show enhanced responses to tumor cells indicating a less exhausted phenotype. Hif1a deficient NK cells preferentially utilize oxidative phosphorylation (OXPHOS) than glycolysis to fuel their energy resulting in enhanced IFN-γ production in the hypoxic environment. In addition, using HIF-1α inhibitors we were able to greatly enhance human NK cell anti-tumor activity. Accordingly, our data reveal HIF-1α as a negative metabolic checkpoint for NK cells. Moreover, inhibition of HIF-1α holds high promise to refuel NK cell activation in cancer with a high potential for improving current strategies of NK cell-based cancer immunotherapy.

#4744

Disruption of Clever-1 in macrophages activates the innate immune response and mediates tumor rejection.

Miro Viitala, Reetta Virtakoivu, Sina Tadayon, Sirpa Jalkanen, Maija Hollmén. _University of Turku, Turku, Finland_.

Macrophages are considered the main obstacles for successful cancer treatment since they can dampen anti-tumor immune responses and promote tumor progression and resistance formation. Macrophages are highly eligible candidates for targeted therapies since these cells are abundantly present in various tumors, they are very plastic and can be converted into pro-inflammatory macrophages supporting T cell activation and tumor rejection. Clever-1 (also known as Stabilin-1) is a multifunctional molecule conferring scavenging ability on a subset of alternatively activated macrophages. The aim of this study is to uncover previously unknown functions of Clever-1 in the regulation of macrophage-mediated anti-tumor immune responses and develop anti-Clever-1 therapy into clinical use for those suffering from refractory, untreatable solid tumors.

By using in vivo tumor models and sophisticated immunological assays, our data show that mice carrying a macrophage-specific genetic deletion of Clever-1 (Lyz2-Cre/Stab1fl/fl) reject implanted Lewis lung carcinoma and EL4 lymphoma tumors. Macrophage Clever-1 deficiency induced proliferation of Ly6Chi monocytes in the tumor microenvironment but impaired their maturation and polarization into tumorigenic Ly6Clo macrophages. Tumors in Lyz2-Cre/Stab1fl/fl mice consisted almost completely of proliferating CD8 T cells with an exhausted PD-1+Lag3+ phenotype two weeks after implantation. Functionally, Clever-1 depleted macrophages showed enhanced pro-inflammatory responses following TLR activation with a shift in IL-12/IL-10 secretion levels. This was due to enhanced activation of mTORC1 and NFκB signaling, which resulted in increased secretion of TNFα in steady state and after LPS induction.

In conclusion, our data show an important function of Clever-1 in controlling macrophage mediated local immune responses. Moreover, our data supports Clever-1 targeting as a novel approach to increase host defenses against immune compromised tumors and promotes anti-Clever-1 immunotherapy into clinical trials where it may have benefits in comparison with currently available immune activating drugs.

#4745

Tim-3 and TIGIT mark NK and T cells susceptible to effector dysfunction in human bladder cancer.

Adam M. Farkas,1 Francois Audenet,2 Harry Anastos,1 William K. Oh,1 Matthew D. Galsky,1 John P. Sfakianos,1 Nina Bhardwaj1. 1 _Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY;_ 2 _Georges Pompidou European Hospital, Paris Descartes University, Paris, France_.

Purpose: To characterize the evolution of Natural Killer (NK) and T cell exhaustion in peripheral blood and tumor tissue of patients representing the spectrum of human bladder cancer (BC), and to identify molecules for which therapeutic modulation enhances the anti-tumor immune response.

Experimental Procedures: PBMC and freshly resected primary tumors were analyzed by FACS to determine phenotype. NK function was assessed via activation with IL-2 or IL-15 followed by co-culture with K562 cells to induce cytokine production and degranulation. For blockade studies, X-Tim-3 and X-TIGIT mAbs were added prior to K562 stimulation.

Results: T cells undergo a process of exhaustion in the context of chronic inflammatory pathologies such as cancer that is characterized by diminished effector functionality and subsequent tumor outgrowth. Inhibition of signaling through certain mediators of this process (e.g. CTLA-4 and PD-(L)1) have shown clinical benefit in subsets of patients with a wide array of malignancies. However, it is unknown whether NK exhaustion occurs, and what molecules define it. We found that NK undergo an analogous process of exhaustion based on analysis of PBMC and tumor from 59 individuals with non, and muscle-invasive BC. NK exhaustion is marked by significant up-regulation of Tim-3 and TIGIT in both PBMC and tumor. T cells demonstrated a similar expression pattern but with a lower frequency of positive cells. The magnitude of NK Tim-3 expression is a barometer of tumor invasiveness on cells in both PBMC and tumor tissue, while TIGIT is induced equivalently in BC patients. Importantly, both molecules are expressed at similar frequencies on NK isolated from blood or tumor, independent of the magnitude of overall expression, yet define NK with different functional potential. NK in PBMC from BC patients are functionally comparable to NK from healthy donors in their ability to produce IFNγ/TNFα and degranulate in response to target cells, while tumor NK are refractory to both stimuli. NK from tumor tissue are not terminally exhausted as effector functions are restored after "resting" ex vivo prior to stimulation. Ex vivo blockade of Tim-3, but not TIGIT, enhances effector function in peripheral NK and T cells from BC patients, but is ineffective for NK in tumor tissue, implicating suppressive factors specific to tumor in mediating NK dysfunction. Tim-3 blockade was most efficient in peripheral NK from BC patients that were activated with IL-15 versus IL-2, suggesting that local cytokine milieu can affect responsiveness to subsequent checkpoint inhibition.

Conclusions: NK and T cell acquisition of Tim-3 and TIGIT are indicators of BC and detectable in peripheral blood and tumor, but mark effector dysfunction only in tumor tissue. Blockade of Tim-3 enhances NK IFNγ/TNFα production in PBMC of BC patients and represents a new strategy to modulate innate, anti-tumor immunity.

#4746

Microbes in the tumor microenvironment: Bacterial influences on host immunity in colorectal cancer.

Christine M. Pierce,1 Bo-young Hong,2 Hannah J. Hoehn,1 Maria F. Gomez,1 Marilena Melas,3 Kevin McDonnell,3 Youngchul Kim,1 Erica Sodergren,2 George Weinstock,2 Hedy S. Rennert,4 Thomas Giordano,5 Joel Greenson,5 Gad Rennert,4 Stephen B. Gruber,3 Stephanie L. Schmit1. 1 _Moffitt Cancer Center, Tampa, FL;_ 2 _The Jackson Laboratory for Genomic Medicine, Farmington, CT;_ 3 _University of Southern California Norris Comprehensive Cancer Center, Los Angeles, CA;_ 4 _Clalit National Israeli Cancer Control Center, Haifa, Israel;_ 5 _University of Michigan School of Medicine, Ann Arbor, MI_.

Mounting evidence suggests that a prominent T cell response in the colorectal cancer (CRC) tumor microenvironment is a valuable prognostic indicator, independent of stage at diagnosis. However, the strength and quality of host immune responses are highly variable across patients, and the factors that influence this variability are not well understood. The unique microbial communities present in the CRC tumor microenvironment may contribute to the modulation of local host immune responses, but prior investigations have been limited and focused on candidate microbes. Here, we investigated the association between the CRC tumor bacterial microbiome and the quantity and clonality of the T cell infiltrate. This investigation included 51 CRC from the Molecular Epidemiology of Colorectal Cancer (MECC) study, a population-based case-control study in northern Israel. Genomic DNA was extracted from fresh frozen tumors using microbiome-optimized techniques and sequenced using a PCR amplicon-mediated workflow on the Illumina MiSeq. Sequences from the V1-V3 hypervariable regions of the bacterial 16S rRNA gene region were clustered into operational taxonomic units (OTUs), a proxy for species, and taxonomic identification was determined using the Ribosomal Database Project. In addition, T cell repertoires were measured using the DNA-based immunoSEQ assay (Adaptive Biotechnologies), and T cell fraction (i.e. proportion of rearranged T cells relative to all nucleated cells) and clonality (i.e. T cell receptor diversity) were calculated. Bacterial sequencing was successful in 48 (94%) of samples, with a total of 868 OTUs identified. Klebsiella and Escherichia/Shigella were the most prevalent genera, detected in approximately 90% of tumors, followed by Propionibacterium, Pelomonas, and Veillonella, with each detected in approximately 50% of tumors. Fusobacterium, a genus of oral anaerobic bacteria believed to accelerate tumor progression and enable immune evasion in CRC, was detected in 20% of tumors. When present, Bacteroides and Fusobacterium were the most abundant, comprising 10-15% of all bacteria within each tumor, on average. ImmunoSEQ analysis identified a mean productive T cell fraction of 0.08 (SD: 0.16) and a mean productive clonality of 0.14 (SD: 0.06). Analysis of molecular variance (AMOVA) of the Theta YC measure of dissimilarity showed a statistically significant difference in bacterial community structure (i.e. composition and relative abundance) between samples in the highest versus lower three quartiles of T cell fraction (P=0.03) and clonality (P=0.01). These preliminary findings suggest a potential relationship between microbial community structure and the quantity and diversity of the CRC T cell infiltrate. Additional analyses are underway to better understand this association as well as the independent contributions of the microbiome to CRC prognosis.

#4747

Neutrophils in gastric cancer tissue inhibit the proliferation of CD4+T cells and contribute to immunosuppression.

Soichiro Hiramatsu, Hiroaki Tanaka, Yoshihito Yamakoshi, Junya Nishimura, Tatsuro Tamura, Takahiro Toyokawa, Kazuya Muguruma, Masaichi Ohira. _Department of Surgical Oncology, Osaka City University Graduate School of Medicine, Osaka, Japan_.

[Background] Neutrophils are essential effector cells in the host's defence against invasive pathogens, On the other hand, neutrophils in the tumor-microenvironment are known to have potential to induce angiogenesis, lymphangiogenesis, and be an important factor in tumor progression. We have reported that neutrophils infiltrating in gastric cancer tissue (Tumor-Associated Neutrophil: TAN) correlated with lymph node metastasis, systemic inflammatory response and be a poor prognostic factor. So we hypothesized that neutrophils in gastric cancer tissue suppressed anti-tumor immune response, but their details are still unclear. [Aim] The aim of this study was to investigate the immunosuppressive ability of neutrophils in gastric cancer tissue and to explore the influence of neutrophils on the proliferation of CD4 (+) T cells. [Materials and Methods] We isolated neutrophils from blood samples taken from healthy donors and added tumor tissue culture supernatants (TTCS) purified from human scirrhous gastric cancer cell line (OCUM12) or purified supernatants from patients who underwent gastrectomy at our department. Then, we examined the expression of apoptotic cells, PDL-1, and HLA-DR on neutrophils with TTCS. We also observed the expression of PD-1, CD25 on T cells co-cultured with neutrophils added TTCS. Furthermore, CD4 T cell labeled Carboxyfluorescein diacetate succinimidyl ester (CFSE) were co-cultured with allogenic dendritic cells and neutrophils with TTCS, and the proliferation of CD4 (+) T cells were examined by flow cytometry. We also performed immunohistochemistry (IHC) staining using anti CD15 and anti PD-1 antibodies for 117 patients who underwent gastrectomy in our department from 2007 to 2013 to explore the positional relationship. [Results] Neutrophils with TTCS were upregulated the expression of PDL-1 than those without TTCS. We also found that the apoptotic cell ratio and the expression of HLA-DR on neutrophils with TTCS was decreasing. CD4 (+) T cells co-cultured with neutrophils added TTCS were suppressed their proliferation, and upregulated the expression of PD-1 and downregulated the expression of CD25. IHC showed that PD-1 positive cells formed clusters and TAN was infiltrating around the clusters. And a positive correlation was found between the number of TAN and PD-1 positive cells in the scatter plot. [Conclusion] Our findings suggested that neutrophils in gastric cancer tissue inhibited the proliferation of CD4 T cells and formed a local immunosuppressive environment through the PD1-PDL1 pathway.

#4748

Snail1 regulates the tumor-infiltrating regulatory T cell population in breast cancer through Pglyrp3.

Dongjiang Chen, Mathew Sebastian, Son Le, Nagheme Thomas, David D. Tran. _University of Florida, Gainesville, FL_.

Breast cancer is the second-leading cause of cancer deaths in women. Nearly 30% of breast cancer patients developed recurrent and/or metastatic disease despite several effective receptor-targeting treatments. Although immunotherapy, especially immune checkpoint inhibitors, has proven to be efficacious against many cancers, they have shown more limited activities against poorly immunogenic cancers such as breast cancer. The cause of this relative resistance to immunotherapy is unclear but is thought to be due in part to an enrichment of inhibitory immune cells such as regulatory T cells (Tregs) in the tumor microenvironment (TME). Therefore there is an urgent need to have a better understanding of the underlying genetic and epigenetic determinants of immunotherapeutic resistance in breast cancer to develop methods to overcome it. To that end, using a computational algorithm that we have recently developed, we have discovered a novel gene regulatory subnetwork downstream of the master epithelial mesenchymal transition (EMT) regulator Snail1 in mouse models of human breast cancer. In advanced breast cancer, Snail1 is thought to initiate the epithelial mesenchymal transition (EMT) to promote local invasion and metastatic spread, while also maintaining tumor-initiating cells. On the contrary, this new Snail1-induced subnetwork operates independently of and possibly in place of its EMT function during early tumorigenic changes, when it is required to maintain a powerful tissue surveillance program to curtail transformed cells' ability to progress to advanced cancer. Breast-restricted deletion of Snail1 early in the course of tumorigenesis of several mouse models of breast cancer results in accelerated growth of the primary tumor in an immunocompetent TME, while in an immunosuppressed TME lack of Snail1 significantly reduced growth. We found that Snail1 suppressed regulatory T cells likely by tumor-associated cytokine production, possibly through several key nodes including Hmga2 and Pglyrp3, to promote immune surveillance and clearance of cancer cells. Ongoing experiments are aimed at validating the criticality of this new pathway as individual nodes and as a whole and to evaluate its utility as a novel therapeutic target to improve breast cancer's response to immunotherapy.

#4749

VISTA immune checkpoint deregulation in human triple-negative breast cancer.

Joshua J. Gruber,1 Marisa M. Juntilla,1 Soo-Ryum Yang,1 Benjamin Geller,1 Natalie Jager,1 Chien-Yu Lin,1 Andrew M. Lipchik,1 Justin Chen,1 Ashwin Ram,1 Shaveta Vinayak,2 Melinda L. Telli,1 Robert B. West,1 James M. Ford,1 Michael P. Snyder1. 1 _Stanford Univ., Palo Alto, CA;_ 2 _Case Western Reserve University, Cleveland, OH_.

Immunotherapy for breast cancer has lagged behind the advances in other tumor types, suggesting the need for novel immune targets. VISTA/PD-1H is a checkpoint receptor expressed on immune cells that is an effective anti-cancer target in mouse models and now in early human clinical trials. We utilized an in vitro system to examine epigenetic changes arising early after depletion of breast cancer tumor suppressor proteins. This demonstrates that epigenetic alterations occur in normal human mammary cell lines lacking BRCA2 and result in regulation of VISTA through an NF-kB-dependent pathway. VISTA is strongly induced by growth factor limitation, an effect that is blunted by BRCA2-induced NF-kB activation and binding to a kB site in the VISTA promoter. This suggests that repression of VISTA may be an early consequence of BRCA2 depletion in pre-neoplastic tissues leading to immune infiltration. In contrast, in primary, untreated human triple-negative breast cancers, VISTA is over-expressed on tumor cells, independent of its presence on immune cells. VISTA positive TNBCs (14%) have significantly increased tumor infiltrating lymphocytes (TILs) whereas VISTA strong positive TNBCs (6%) have significantly decreased TILs and tumor PD-L1 staining, suggesting a higher level of immunosuppression. Gene expression and tumor genomics in TNBCs suggests that transcriptional networks to regulate physiologic immunoreceptor levels are corrupted during malignant transformation. In summary, this work identifies a BRCA2-loss-of-function signaling pathway that regulates an important immune checkpoint molecule via NF-kB activation in pre-neoplastic tissues and triple-negative breast cancers with important consequences for the immune microenvironment. We expect these observations to enhance immunotherapeutics for breast cancer.

#4750

The immunosuppressive tumor microenvironment (TME) in nasopharyngeal carcinoma: implications for immunotherapy.

Amy S. Duffield, Maria Libera Ascierto, Robert A. Anders, Janis M. Taube, Tracee L. McMiller, Elizabeth L. Engle, Alan K. Meeker, Alan E. Berger, Drew M. Pardoll, Richard F. Ambinder, Suzanne L. Topalian. _Johns Hopkins University, Baltimore, MD_.

Nasopharyngeal carcinoma (NPC) is an EBV-driven tumor that shows variable expression of PD-L1 and ~20% objective response rate to anti-PD-1 monotherapy. As novel immune checkpoint inhibitors are being developed, combination therapies may allow for more effective treatment of both newly diagnosed and relapsed NPC. We characterized the TME in 13 cases of EBV+ NPC from the Johns Hopkins Pathology archives (7 primary tumors, 6 metastases). EBV status was confirmed with EBER ISH. Immunohistochemistry (IHC) was conducted on all cases for CD3, CD4, CD8, CD20, FoxP3, PD-1, PD-L1, LAG-3, TIM-3, GITR, IDO, COX2, and pSTAT3. Gene expression profiling (GEP) was performed on 7 cases with sufficient material (4 primary lesions, 3 metastases), using multiplex qRT-PCR for a panel of 61 candidate immune-related genes (Duffield, Blood Advances 2017). The immunosuppressive ligand PD-L1 was expressed on tumor cells in 11/13 cases (mean 22% tumor cells+, range 0-57%), as well as on infiltrating macrophages. The NPC inflammatory infiltrate was diverse, including CD4+, CD8+, CD20+ and CD68+ cells, and showed variable expression of immune-regulatory molecules. In all 13 cases, lymphocytes expressing PD-1 (mean 36% positive, range 8-70%), LAG-3 (7%; 1-30%) and GITR (12%; 2-27%) were found. FoxP3+ and TIM-3+ lymphocytes were infrequent. IDO+ macrophages were also infrequent; however, 7/13 NPCs showed expression of the immunosuppressive metabolic enzyme IDO by a proportion of tumor cells. Compared to 12 EBV+ Hodgkin lymphomas (Duffield, Blood Advances 2017), EBV+ NPCs demonstrated a Th17 cytokine profile with overexpression of IL1A, IL17RC, IL23A, and IL23R. The generation of pathogenic Th17 responses requires phosphorylation of the STAT3 transcription factor, and IHC confirmed that a subset of inflammatory cells in all NPC cases expressed pSTAT3 (mean 10%; range 1-40%). Additionally, upregulated gene expression characterizing activated macrophages was found (IDO, IL1A, IL12A, LYZ, TLR3). Of note, several molecules upregulated in the NPC TME are capable of inducing PD-L1 expression on human monocytes in vitro, including IL-1A and IL-32-gamma (Taube, Clin Cancer Res 2015; Duffield, Blood Advances 2017). Importantly PTGS2 (COX2), with known proinflammatory and immunosuppressive properties, was over-expressed in NPCs along with the downstream modulator CXCL8 (IL-8); IHC revealed COX2 expression in tumor cells but not infiltrating immune cells. In summary, NPC is characterized by markers of an immunosuppressive TME, including immune checkpoints and metabolic modulators. While these findings should be explored in a larger cohort, they have potential implications for designing combination NPC treatment regimens with anti-PD-1, which might include inhibitors of LAG-3, IDO, IL-17/-23, COX2, and/or IL-8. Funded by the Bristol-Myers Squibb International Immuno-Oncology Network and NCI R01 CA142779

#4751

APOBEC3A inhibits tumor immune response independent of deamination in a novel genetic model of pancreatic cancer.

Sonja Woermann,1 Robert Cowan,1 Susan M. Ross,2 Andrew D. Rhim1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _University of Illinois-Chicago, Chicago, IL_.

Introduction: Apolipoprotein B mRNA Editing enzyme, Catalytic polypeptide 3A (A3A) is a C to U cytidine deaminase, preferentially deaminating the lagging strand of double-stranded DNA at specific sequence motifs. A3A is overexpressed across multiple tumor types, and in pancreatic ductal adenocarcinoma (PDA), high expression is associated with significantly decreased survival in early stage patients. Large scale sequencing studies of various human tumors (TCGA and ICGC), including PDA, have implicated A3A as a potential driver of tumor mutagenesis. However, the biological relevance of the deamination function of A3A has not yet been shown. Here, we utilize a novel genetically engineered mouse model to show that A3A supports the development of aggressive PDA independent of mutagenic capabilities.

Methods: As opposed to humans, mice only contain one APOBEC3 isoform which has limited to no deaminating activity on genomic DNA. Thus, to dissect the function of A3A on PDA initiation and progression, we made mice with a germline knockin for the coding sequence for human A3A and bred to a well-established GEMM of PDA to yield LSL-KrasG12D; p53fl/+; Pdx1-Cre; RosaLSL-YFP; A3A+/- (KPCY;A3A) mice and compared to KPCY mice. We genotyped tumors from both cohorts using VarScan after exome capture sequencing. Tumor and immune cells were FACS sorted comparative RNAseq performed. The immune contexture of murine and 155 untreated and treated human PDAs was analyzed by performing 7-color multiplex staining and immune cell spatial interaction analysis.

Results: All KPCY;A3A (n=11) mice developed tumors and expired significantly faster compared to KPCY (n=18) controls (3.9 v. 7.0 mo; p<0.01). One third of the KPCY; A3A mice developed macrometastatic disease compared to 3/12 in the KPCY control. Whole exome sequencing of tumors revealed no significant difference in the number of point mutations or neoantigen load between KPCY;A3A and KPCY tumors. However, histologically, KPCY;A3A tumors contained significantly more desmoplasia and altered immune cell infiltrates. Specifically, comparative RNAseq and IHC analyses revealed dramatic differences in the number and distribution of various T-cell and B-cell subsets in KPCY;A3A mice. Moreover, A3A expression led to abrogation of CD8+ T cell activation and proliferation. Concomitantly, a panel of immune checkpoint-related genes were upregulated in A3A high expressing tumors and cell lines compared to non-A3A expressing controls. This was confirmed in a panel of resected human PDA.

Conclusions: Counter to our hypothesis, A3A supports the initiation and growth of PDA in vivo through effects independent of deamination or mutagenesis of cancer cell genomes but rather indirectly through suppression of the tumor immune response. Future studies will address the precise mechanisms underlying A3A-mediated tumor immune evasion.

#4752

EBV associated tumors have increased regulatory T cell recruitment and are therefore a potential indication for treatment with potent and selective small molecule CCR4 antagonists.

Oezcan Talay, Aparna Jorapur, Scott Jacobson, Sachie Marubayashi, Lisa Marshall, Silpa Suthram, Omar Robles, John Ketcham, Maureen K. Reilly, Ashkaan Younai, Berenger Biannic, Dennis Hu, Minna Bui, Jacob Schwarz, Paul Kassner, Gene Cutler. _FLX Bio, South San Francisco, CA_.

We have performed experiments to test whether Epstein Barr Virus (EBV)-infected tumors are enhanced for regulatory T cell (Treg) infiltration and whether selective and potent CCR4 antagonists would be a particularly effective therapeutic in this class of indications. Treg cells, which contribute to an immune-suppressive tumor microenvironment (TME), are attracted to tumors via the recognition of CCL17 and CCL22 ligands by the CCR4 receptor. These chemokines have been shown to be expressed in cells infected by the Epstein Barr Virus (EBV) via the viral LMP1 gene. Tumor types which are frequently associated with EBV-infection include gastric adenocarcinoma (~10% positive), classical Hodgkin's Lymphoma (~50%), and nasopharyngeal carcinoma (~100%).

Analyzing RNA expression in EBV-associated tumors, we found strong expression of CCL17, CCL22, and FOXP3, a marker of Treg, when compared to EBV-negative tumors. In fact, NPC tumors show extremely high FOXP3 levels. To further test this link, we obtained EBV-associated tumor samples and performed RNA in situ hybridization (ISH) to measure co-expression of these genes. Strong co-localization, was indeed found, further supporting a link between EBV and Treg recruitment. To directly test whether EBV-positive tumors recruit Treg into tumors via CCL22/17 upregulation, we developed models in which mice were inoculated subcutaneously with EBV-positive cancer cell lines. These EBV-positive cancer cells were assessed for chemokine production in vitro and in vivo by ELISA. We assessed tumor-infiltrating lymphocytes (TILs) in established tumors, including Treg, CD4+ and CD8+ T cells as well as T cell activation markers. Treating these tumor-bearing mice with selective and potent CCR4 small-molecule antagonists alone or in combination with checkpoint-targeting antibodies allowed us to demonstrate meaningful antitumor responses in EBV-positive tumors. Together, these data suggest that EBV-positive tumors, such as gastric adenocarcinomas, Hodgkin's lymphomas, and nasopharyngeal carcinomas, are a class of indications of particular interest and potentially increased responsiveness to small-molecule CCR4 antagonists. These results are helping to inform the ongoing FLX475 trials currently in the clinic.

#4753

Bone Morphogenetic Protein 7 is a candidate promoter of cancer immune evasion.

Eric D. Routh,1 Julia Chifman,2 Ashok Pullikuth,1 Lance D. Miller1. 1 _Wake Forest University Health Sciences, Winston-Salem, NC;_ 2 _American University, DC_.

Tumors evade immune recognition and destruction by various established mechanisms, but less is known about how tumors short-circuit successful immune cell trafficking. To identify candidate proteins that inhibit immune trafficking, we developed a bioinformatics screen that utilizes an intratumoral cytotoxic T lymphocyte (CTL) gene signature that quantifies the relative abundance of tumor infiltrating CTLs. This gene signature was used as a stratification tool to examine differential gene expression between tumors of "low" versus "high" CTL status. We hypothesized that a subset of recurrently overexpressed genes in low-CTL tumors (i.e., immunologically "cold" tumors) may functionally act to limit lymphocyte infiltration into the tumor mass. A pan-TCGA analysis of 23 solid tumor types (n=8,875 tumors) revealed a number of recurrent genes highly significantly associated with low CTL infiltration. We found that 88.5% of tumor pairwise comparisons (among the 23 tumor types) showed significant overlap of top genes associated with low CTL status, consistent with the existence of conserved, tumor-agnostic mechanisms of immune evasion. From these analyses, the secreted TGFβ superfamily member, BMP7, emerged as a top-ranking candidate regulator of immune trafficking. To investigate the role of BMP7 in CTL tumor trafficking, an immunogenic variant of the 4T1 mouse breast cancer cell line, 4T1-S, was modified using CRISPR-Cas9 to inducibly express BMP7 from the ROSA26 locus. 4T1-S BMP7-inducible cells were orthotopically transplanted into Balb/C female mice and tumors were allowed to form with or without BMP7 induction. Mice were sacrificed at 2, 3, and 4 wks post-tumor inoculation, and tumors, tumor-draining LNs (TDLNs), and spleens were harvested. At both 2 wks and 3 wks, a BMP7-dependent reduction in tumor CTLs was observed by anti-CD8a IF microscopy (p=0.002 and p<0.001, respectively). Flow cytometric analysis confirmed the significant reduction in tumor-infiltrating CD8\+ CTLs in the BMP7-induced group at 3 wks (p=0.02). BMP7 expression did not alter tumor trafficking of natural killer (NK) cells. Flow analysis also showed CTL reduction in TDLNs at 2 wks (p=0.02), and NKs were reduced in the TDLNs at 3 wks (p=0.04). Spleen sizes were also significantly smaller at all time points, indicating a systemic immunomodulatory function of tumor-derived BMP7 (p=0.002, p<0.001, and p=0.002 at 2, 3, and 4 wks, respectively). This study provides a framework for discovering yet unappreciated mechanisms by which tumors evade the immune system, and presents first evidence that BMP7 expression by breast tumors can function to limit CTL trafficking.

#4754

An ancestral link between tumor immune microenvironment and tamoxifen resistance in breast cancer.

Leena A. Hilakivi-Clarke, Kerrie Bouker, Fabia Oliveira De Andrade, Lu Jin. _Georgetown University Lombardi Comp. Cancer Ctr., Washington, DC_.

Maternal exposures to endocrine disrupting chemicals (EDCs) are known to increase daughter's breast cancer risk in humans and animal models. We have found that these exposures also pre-program mammary tumors to exhibit increased resistance to antiestrogen therapy (Hilakivi-Clarke et al. JNCI 109, 2016). Consistent with the idea that in utero EDC exposures alter later disease susceptibility by epigenetic means, we discovered that adult treatment of in utero EDC exposed rats with valproic acid (VA, non-specific HDAC inhibitor) and hydralazine (H, non-specific DNMT inhibitor) prevented tamoxifen (TAM) resistance. In contrast, in the control offspring VA+H promoted TAM resistance. Here we investigated if maternal exposure to synthetic ethinyl estradiol (EE2) alters tumor immune environment in the offspring, and if treatment with VA+H reverses the changes. Pregnant Sprague Dawley rats received 0 or 0.1 ppm EE2 during gestation days 10-20. Estrogen receptor positive (ER+) mammary tumors in the offspring were induced with 7,12-dimethylbenz[a]anthracene, and when a tumor reached 13 mm in diameter, core needle biopsies were obtained. Thereafter, offspring were treated with 15 mg/kg TAM in diet, with or without 1.2 g/kg VA and 5 mg/kg H via drinking water. Antigen processing and activating proteins CD74 and HLA-DRB5 were significantly elevated in pre-treatment tumor biopsies from in utero EE2 exposed rats. Ingenuity pathway analysis of iTraq protein data from the biopsies indicated that T cell receptor and IL15 were upstream regulators of most of the signaling changes between EE2 and control offspring. Further, non-treated mammary tumors of EE2 offspring exhibited higher mRNA levels of PD-L1, PD-1, Foxp3 and Tgfβ1 than tumors in the control offspring. TAM treatment further upregulated Foxp3 and Tgfβ1 as well as CD8a and PD-1 in the EE2 offspring. However, VA+H reversed all these changes in TAM treated in utero EE2 exposed animals. In the control offspring, a combination of TAM and VA+H increased tumor PD-1, PD-L1, Foxp3 and TGFβ1 levels, compared with non-treated tumors. Since elevated levels of Foxp3 and TGFβ1 are indicative of immunosuppression, and PD-1 and its ligand PD-L1 induce effector T cell exhaustion, our data suggest that in utero EE2 exposure promotes an immunosuppressive tumor microenvironment that allows cancer cells to evade elimination by cytotoxic T lymphocytes, especially during TAM therapy. Our findings also indicate that adding HDAC + DNMT inhibitors to the treatment regimen prevents these changes, and thus possibly explains the ability of the combination treatment to prevent antiestrogen resistance in the in utero EE2 exposed rats. Future studies will determine if the combination therapy benefits ER+ breast cancer patients exhibiting high PD-1/PD-L1 expression in pre-treatment tumors, and/or if these patients should receive checkpoint inhibitors with endocrine therapy.

#4755

Role of PAK4 in cancer immune cell exclusion.

Gabriel Abril-Rodriguez, Catherine S. Grasso, Jesse M. Zaretsky, Beata Berent-Maoz, Siwen Hu-Lieskovan, Antoni Ribas. _University of California Los Angeles (UCLA) and Parker Institute for Cancer Immunotherapy, CA_.

Lack of T-cell infiltration is the main mechanism of primary resistance to checkpoint blockade therapies. Here, we performed a transcriptomic analysis of metastatic melanoma biopsies taken from patients treated with anti-PD1 (n=23) with biopsies pre- (n=17) and during treatment (n=22), and investigated cancer cell intrinsic mechanisms of immune evasion. We classified our samples based on their T-cell infiltration status using a validated set of immune genes for T-cell infiltration (Spranger et al., 2015), normalized within gene and scored the samples from 0 (least infiltrated) to 1 (most infiltrated). In order to identify genes that anti-correlate with infiltration, we performed differential gene expression analysis between non-infiltrated (n= 18) and infiltrated tumors (n=21) regardless clinical response and condition. The analysis resulted in total of 1904 genes enriched in the non-infiltrated group (log2foldchange > 1, q-value < 0.05 and infiltration threshold = 0.5). Since Wnt signaling pathway has been previously suggested as a potential mechanism of immune exclusion, we aimed to select targetable proteins implicated in β-catenin signaling. Interestingly, p21 (RAC1) Activated Kinase 4 (PAK4) was significantly over-expressed in the non-infiltrated group (log2folchange = 1.04, q-value = 1.08e-05). PAK4 is a kinase involved in cell migration, proliferation and apoptosis and it is known that it can directly phosphorylate β-catenin to promote Wnt signaling. In addition, PAK4 was found to be also significantly enriched in on-treatment samples of non-responders (n=7) compared to responders (n=7) (mean of non-responder = 3.68, mean of responders = 2.69, p-value = 0.007). We next aimed to determine genes negatively associated with infiltration using Pearson correlation coefficient. PAK4 was on the top 5 of genes negatively associated with CD8A (pcc = -0.56, p-value 1.8e-04), HLA-DMB (pcc = -0.64, p-value 8.4e-06), TNF (pcc = -0.66, p-value 4.8e-06) among other immune genes. Altogether, this data suggests that PAK4 have an active role in T-cell exclusion. In order to validate our finding, we analyzed 472 melanoma samples from the TCGA. We first performed RNA-seq deconvolution to estimate the infiltration status of each sample and then determined genes that anti-correlated with the different immune cell subtypes. In line with the hypothesis that PAK4 was causatively excluding T-cell from tumors, we found that PAK4 performed the strongest anti-correlation with dendritic cells (pcc= -0.45, p-value = 2.2e-16) and CD8+ T-cells (pcc = -0.22, p-value = 1.12e-06). PAK4 expression was enriched in TCGA non-infiltrated samples (p-value = 4.92e-09, infiltration threshold = 0.5). In summary, this data suggests that PAK4 might be driving T-cell exclusion and that inhibition of PAK4 kinase activity with small molecules could facilitate T-cell infiltration and synergize with current checkpoint blockade therapies.

#4756

Secretory IgM exacerbates tumor progression by inducing accumulations of myeloid-derived suppressor cells in mice.

Chih-Hang Tang,1 Shiun Chang,1 Ayumi Hashimoto,1 Yi-Ju Chen,1 Chang Won Kang,2 Anthony Mato,3 Juan Del Valle,2 Dmitry Gabrilovich,1 Chih-Chi Andrew Hu1. 1 _The Wistar Inst., Philadelphia, PA;_ 2 _University of South Florida, Tampa, FL;_ 3 _University of Pennsylvania, Philadelphia, PA_.

To explore the role of B cell receptor (BCR) in promoting malignant progression of chronic lymphocytic leukemia (CLL) in mice, we generate MD4/Eμ-TCL1 mice, whose B cells harbor a monoclonal BCR against hen egg lysozyme (HEL) and secrete IgM against HEL. MD4/Eμ-TCL1 mice survive significantly shorter than Eμ-TCL1 mice. While precancerous B cells in young MD4/Eμ-TCL1 mice recognize HEL, CLL cells developed in older MD4/Eμ-TCL1 mice fail to recognize HEL. Nevertheless, MD4/Eμ-TCL1 CLL cells can be activated by goat F(ab')2 anti-mouse IgM and respond by phosphorylation of Igα, Syk and ERK1/2, indicating reactivation of a parental Ig gene allele. MD4/Eμ-TCL1 CLL cells also produce large quantities of secretory IgM (sIgM), which does not react with HEL. Compared with age-matched Eμ-TCL1 mice, MD4/Eμ-TCL1 mice also generate a significantly increased population of CD11b+/Ly6G+ granulocytic cells in the peripheral blood, spleens and bone marrow. CD11b+/Ly6G+ granulocytic cells purified from spleens of MD4/Eμ-TCL1 mice can suppress CD3/CD28-mediated proliferation of CD8+ T cells and gp100-loaded class I MHC-mediated proliferation of CD8+ T cells from PMEL-1 mice, qualifying these cells as myeloid-derived suppressor cells (MDSCs). Increased MDSCs may account for significantly decreased T cells and poor prognosis in CLL-bearing MD4/Eμ-TCL1 mice. Because MD4/Eμ-TCL1 mouse CLL cells and human CLL cells can produce sIgM, we hypothesize that sIgM may account for the accumulation of MDSCs. To test this hypothesis, we cross μS-/- mice, which cannot produce sIgM, with Eμ-TCL1 mice. The μS-/-/Eμ-TCL1 mice indeed develop significantly lower numbers of MDSCs, and survive significantly longer than Eμ-TCL1 mice. We decide to target the synthesis of sIgM by deleting the function of XBP-1, because the synthesis of sIgM is tightly regulated by a mechanism called regulated IRE-1-dependent decay (RIDD), which is hyperactivated in B cells as a response to XBP-1 deficiency. We cross B cell-specific XBP-1KO mice with MD4/Eμ-TCL1 mice, and the resultant XBP-1KO/MD4/Eμ-TCL1 mice indeed produce significantly reduced amounts of sIgM and decreased numbers of MDSCs. In addition, μS-/- mice grafted with Lewis lung carcinoma exhibit reduced functions of MDSCs in suppressing T cells, resulting in significantly slower tumor growth. These results clearly demonstrate that sIgM produced by B cells can upregulate the immunosuppressive functions of MDSCs in tumor-bearing mice to aggravate cancer progression.

## REGULATORY SCIENCE AND POLICY:

### Regulatory Science and Science Health Policy

#4757

A clinically validated comprehensive companion diagnostic platform for care of patients with advanced cancer.

James X. Sun,1 Yali Li,1 Coren Milbury,1 Joel Skoletsky,1 Christine Burns,1 Wai-ki Yip,1 Jun Luo,1 Ninad Dewal,1 Adrienne Johnson,1 Kyle Gowen,1 Jing Tong,1 Yuting He,1 Jie He,1 Pei Ma,1 Jared White,1 Steve Roels,1 John Truesdell,1 Eric Peters,2 Houston Gilbert,2 Charlie Wu,2 Erica Schleifman,2 Johannes Noe,2 Carl Barrett,3 Kenneth Thress,3 Suzanne Jenkins,3 Julia Elvin,1 Geoff Otto,1 Doron Lipson,1 Jeffrey Ross,1 Vincent Miller,1 Philip Stephens,1 Michael Doherty,1 Christine Vietz1. 1 _Foundation Medicine, Inc, Cambridge, MA;_ 2 _Genentech, South San Francisco, CA;_ 3 _AstraZeneca, Waltham, MA_.

Introduction: Increase in targeted therapies has resulted in the need for a single assay capable of detecting diverse biomarkers indicated for these agents. Comprehensive genomic profiling (CGP) provides such a solution, but due to the complexity and number of assays available today, standardization of validation has become critically important. We present FoundationOne CDx, the first NGS-based comprehensive companion diagnostics (CDx) platform developed and performed in compliance with FDA 21 CFR part 820. The assay interrogates 324 genes, and has CDx indications in five tumor types associated with 17 targeted therapies (Table 1). The versatile assay design will facilitate streamlined development of future CDx indications.

Methods: DNA extracted from FFPE tumor tissue underwent whole-genome shotgun library construction and hybridization-based capture, followed by sequencing using Illumina HiSeq 4000. Sequence data were processed using a proprietary analysis pipeline designed to detect base substitutions, indels, copy number alterations, rearrangements, microsatellite instability (MSI), and tumor mutational burden (TMB).

Results: Clinical validity was established such that the concordance between CGP and approved CDx were statistically non-inferior to that of two runs of approved CDx. For analytical validity, limit of detection (LoD) was at allele frequency 4% for known substitutions and indels. LoD was 16% tumor content for copy number amplifications, 30% for homozygous deletions, 11% for rearrangements, 12% for MSI, and 20% for TMB. Concordance with an orthogonal NGS platform was 94.6% for substitutions and indels. Within-assay reproducibility had PPA 99.4%.

Conclusion: Rapid expansion of targeted therapies and CDx has necessitated a new approach and urgency to defining performance standards. We developed a comprehensive CDx assay and demonstrated clinical and analytical validity to support and accelerate using CGP for routine clinical care.

Table 1. Companion Diagnostic Indications

---

Indication | Biomarker | Therapy

Non-small cell lung cancer (NSCLC) | EGFR exon 19 deletions and EGFR exon 21 L858R alterations | afatinib, gefitinib, or erlotinib

|

EGFR exon 20 T790M alterations | osimertinib

|

ALK rearrangements | alectinib, crizotinib, or ceritinib

|

BRAF V600E | dabrafenib in combination with trametinib

Melanoma | BRAF V600E | dabrafenib, vemurafenib

|

BRAF V600E and V600K | trametinib, cobimetinib, in combination with vemurafenib

Breast cancer | ERBB2 (HER2) amplification | trastuzumab, ado-trastuzumab-emtansine, or pertuzumab

Colorectal cancer | KRAS wild-type (absence of mutations in codons 12 and 13) | cetuximab

|

KRAS and NRAS wild-type (absence of mutations in exons 2, 3, and 4) | panitumumab

Ovarian cancer | BRCA1/2 alterations | rucaparib

#4758

Use of highly multiplexed reference materials to facilitate validation of a clinical NGS tumor fusion RNA assay.

Catherine Huang,1 Subit Barua,2 Deepika Philkana,1 Russell Garlick,1 Bharathi Anekella,1 Helen Fernandes2. 1 _Seracare Life Sciences, Gaithersburg, MD;_ 2 _Columbia University Medical Center, New york, NY_.

Introduction: Next Generation Sequencing assays for detection of tumor RNA fusions must undergo rigorous validation before clinical implementation. Validations include assessment of the assay's accuracy, precision, reproducibility and limits of detection or reportable range. Obtaining samples with all needed variants is difficult and time consuming. When found, they are often in limited quantities such that repeated testing for precision and reproducibility studies is not possible. Sample heterogeneity or lack of characterization further complicates interpretation of results. This study demonstrates how highly characterized, uniformly manufactured reference materials can facilitate clinical NGS assay validation. Methods: An RNA panel representing decreasing levels of 16 different RNA fusions and exon skipping events compared to the total cellular RNA was generated. GM24385 cell line was engineered to contain the fusion RNAs then formalin fixed, and the total RNA was extracted using the Maxwell RSC FFPE RNA kit. The fusion RNA was serially diluted into total RNA from similarly processed non-engineered GM24385 cells to create a panel with decreasing levels of each fusion. The panel was characterized by fusion-specific, TaqMan digital PCR assays to obtain a "truth set" on which to compare NGS results. Each panel member was tested in triplicate on three separate days as part of a validation study. Results: Precision and reproducibility were assessed quantitatively through analysis of the NGS unique reads. The intra-run precision varied among fusion targets with many targets having %CV of less than 15%, while a few targets had significantly higher variability between replicates. The linearity of the assay was good, with R-squared value greater than 0.95. Lower limits of detection for each fusion target were estimated using the digital PCR data and were different for different fusion targets; for example, detection of EML4-ALK fusion was five-fold more sensitive than detection of CD74-ROS1 fusion. Conclusions: Manufactured reference materials can supplement validation studies and their uniformity and digital PCR characterization allows greater insight into assay performance such as limits of detection than use of remnant specimens alone. The sensitivity of the NGS panel used was not the same for different fusion targets and, and underscores the need for highly multiplexed reference materials and for labs to test all clinically important fusions during validation

#4759

Designing clinical trials in tumor indications with a positive signal in phase 1.

Cong Chen, Keaven Anderson, Devan V. Mehrotra, Eric H. Rubin, Archie Tse. _Merck & Co., Inc., Kenilworth, NJ_.

Increasingly, the conventional proof-of-concept randomized Phase 2 study is skipped in favor of directly going to Phase 3 after an experimental oncology drug has demonstrated promising anti-tumor activity in Phase 1 with a small sample size. This shift in the balance between certainty and speed is especially evident in the immune-oncology space where the tremendous success of immune checkpoint inhibitors has brought unprecedented competition in the field. The aggressive approach can be very risky no matter how promising an experimental drug appears in Phase 1. In this presentation, we introduce a novel adaptive design that mitigates the risk of late-stage programs. The proposed approach starts with a Phase 2 trial and adds an option in the design that allows the expansion of the Phase 2 trial into Phase 3 if the interim result based on the Phase 2 endpoint is promising. If the decision is to not expand, the study is kept as a Phase 2 trial and the primary analysis is conducted at the end of Phase 2. Otherwise, the study is expanded into a Phase 3 trial and the primary analysis of the study is conducted at the end of Phase 3, utilizing data from all enrolled patients including those already used for the decision making in the ongoing trial. The proposed approach is more efficient than the conventional approach that conducts and analyzes Phase 2 and Phase 3 trials sequentially, and is less risky than the contemporary approach of skipping Phase 2. Importantly, we will show that this design controls overall Type I error regardless of the expansion criterion. As a result, the study can still be considered positive even without expansion to Phase 3.

#4760

Joint modeling of longitudinal tumor dynamics and survival in non-small cell lung cancer (NSCLC) patients.

Xiao Tong,1 James Dunyak,1 Diansong Zhou,1 David Carlile,2 Helen Tomkinson,2 Gabriel Helmlinger,1 Nidal Al-Huniti,1 Hongmei Xu1. 1 _AstraZeneca, Waltham, MA;_ 2 _AstraZeneca, United Kingdom_.

Objectives: Tumor size dynamics and survival are traditionally analyzed in a 2-stage approach, without consideration of joint dependencies. The objective of this analysis was to develop a joint model which associates tumor size dynamics and progression-free survival (PFS), to predict time-to-progression. Methods: Phase 2 trial data from selumetinib (AZD6244; ARRY-142886, MEK inhibitor), in NSCLC patients (SELECT-2; NCT01750281) were used to develop a joint model for tumor size dynamics and PFS. The analysis was performed using JM package in R. Treatment arm, KRAS mutation and WHO performance status were evaluated as covariates. Model was evaluated by survival estimation based on early time (e.g., first 3 months) tumor size data. The final joint model based on SELECT-2 data was then used to predict PFS of selumetinib in SELECT-1 (NCT01933932) phase 3 trial. Results: The joint model developed on SELECT-2 data identified a significant association (p value <0.001) between the slope of the longitudinal tumor dynamic and PFS. WHO performance status was identified to be the only significant covariate. Furthermore, the model built on phase 2 data adequately predicted PFS of the SELECT-1 data, using SELECT-1 tumor size data within 4 months of treatment. Prediction confirmed no significant difference in PFS between active treatment arm and chemotherapy arm in SELECT-1 trial. Conclusions: Using selumetinib as an example, we showed that joint modeling of tumor size dynamics and PFS may provide a novel quantitative tool to predict long-term outcome in NSCLC based on early tumor size measurements.

#4761

Joint model of longitudinal tumor size and overall survival (OS) in non-small cell lung cancer (NSCLC): A Bayesian approach.

Nidal Al-Huniti, Hongmei Xu, Diansong Zhou, Helena Edlund, Sergey Aksenov, Gabriel Helmlinger, James Dunyak. _AstraZeneca, Waltham, MA_.

Objective: The methodology of joint modeling longitudinal tumor size and OS data is very promising and provides the opportunity of survival prediction in individual patients. However, current joint modeling software are restricted in terms of distributions/parameterizations available and non-linear mixed effects models are not supported. The objective of this work was to develop a joint model in Bayesian framework to relax these restrictions and enable investigation of additional (continuous and/or discrete) biomarkers. Methods: Longitudinal tumor size and OS data from gefitinib Phase 3 study in NSCLC ('IPASS', NCT00322452) was used in the modeling analysis. Joint model was developed in JM package in R and the Bayesian formulation of the joint modeling was implemented in STAN. Different parametrizations were investigated for both sub-models and the impact of lag-times was explored. The parameter estimates and model fit were compared between models by JM package and STAN. The final Bayesian joint model was validated on an independent dataset from a Phase 4 study ('IFUM', NCT01203917, EGFR positive patients only) by simulating individual survival based on their tumor size data and compared with observed survival. Results: The estimated parameter values were similar to those from the fit from JM package. The different parametrizations provided similar fits for the tumor size model, while the survival model was improved by using a b-spline model. The association parameters indicate a strong association between tumor size and survival and introduction of lag-times did not impact the association parameters to a great extent. Posterior predictive simulations of OS in phase 4 study were consistent with observed data. Conclusions: Bayesian formulation further improved the joint model of tumor size and OS which would enable investigation of additional biomarkers (e.g. new lesions).

#4762

Clinicopathologic findings of borderline resectable pancreatic cancer after neoadjuvant chemoradiotherapy.

Yoshiki Naito, Masamichi Nakayama, Hiroto Ishikawa, Kenjiro Takahashi, Toru Hisaka, Koji Okuda, Masaru Fukahori, Yusuke Ishida, Yoshinobu Okabe, Yutaro Mihara, Masahiko Tanigawa, Jun Akiba, Takuji Torimura, Hirohisa Yano. _Kurume University School of Medicine, Kurume, Japan_.

BACKGROUND: The resection rate and survival period of resectable pancreatic ductal adenocarcinoma (PDAC) have increased gradually. Unfortunately, only 15-20% of patients have resectable disease at diagnosis, with the remainder presenting with distant metastatic or locally advanced tumors. Borderline resectable PDAC (BR-PDAC) is defined as a tumor that involves the portal vein (PV) and/or superior mesenteric vein (SMV), and has low resectability. However, there are many practical advantages to neoadjuvant chemoradiotherapy (NAC-RT) for BR-PDAC. Although the clinical effects of NAC-RT in BR-PDAC have been discussed, only a few studies have reported pathological features. We herein studied the effect of NAC-RT on the histological features of BR-PDAC.

MATERIALS AND METHODS: Tissue samples of 14 patients with BR-PDAC undergoing NAC-RT (NAC-RT group; male: 7 patients, female: 7 patients; average age:64.8+8.1 years) and 78 patients with conventional PDAC (control group), resected at the Kurume University Hospital were obtained. The BR-PDACs had PV and/or SMV invasion at initial clinical diagnosis. All patients with BR-PDAC underwent NAC-CRT, comprising chemotherapy and external beam irradiation (50.4 Gy). The microvessel invasion (MVI), lymphovascular invasion (LVI), neural invasion (NI), portal vein invasion (PVI), extrapancreatic plexus invasion (PLI), and TNM classification in the surgical specimens were examined. All statistical analyses were performed using StatMate IV (ATMS, Tokyo, Japan). All statistical tests were two-tailed, and P < 0.05 was considered statistically significant.

RESULTS: The distribution of stages in the NAC-RT group was as follows: stage IA (n = 5, 36%), IIA (n = 6, 43%), IIB (n = 3, 21%). The pathological grade was determined to be G1 in 9 cases (64%), G2 in 4 cases (28%) and G3 in 1 case (8%). The difference in average tumor size between the NAC-RT and control groups was significant (P> 0.001). Lymph node metastases were significantly infrequently observed in the NAC-RT group compared with the control (P=0.009). The NAC-RT group had a significant lower MVI (29%, 4/14) and LVI (57%, 8/14) than the control group (MVI: P<0.001, LVI: P=0.011). However, there was no significant difference in NI (P=0.129). Ten patients without PVI and /or PLI in the NAC-RT group (71%) had significantly better prognosis than those in the control group (P=0.029), although there was no significant difference in overall survival rates between both groups (P=0.100).

CONCLUSIONS: NAC-RT might be more advantageous by inhibiting local invasion, hematogenous and lymphatic metastasis, and could contribute to a better prognosis in patients with BR-PDAC, partly due to the prevention of PVI and/or PLI in pancreatic cancer.

#4763

NCI's Expert-Vetted Biospecimen Evidence-Based Practices (BEBP): A resource for harmonization of biospecimen collection procedures.

Esmeralda Casas-Silva, Lori D. Campbell, Kelly Engel, Sarah Greytak, Ping Guan, Helen Moore. _National Cancer Institute, Rockville, MD_.

High quality human biospecimens are essential to clinical diagnosis and treatment and serve as a vital foundation for medical research. Substantial variation in procedures for biospecimen collection, processing, and storage exists within and between institutions. Such variation can have a substantial effect on the quality of biospecimens and the accuracy of downstream analytical results, and is of particular concern at a time of high demand for biospecimens for large-scale studies. Recognizing the need for harmonization of biospecimen practices, the National Cancer Institute (NCI)'s Biorepositories and Biospecimen Research Branch (BBRB) began developing a series of documents known as Biospecimen Evidence-Based Practices (BEBPs). The first of NCI's BEBPs, "Snap-Freezing of Post-surgical Tissue Biospecimens" (https://biospecimens.cancer.gov/global/pdfs/NCI_BEBP_Snap-freezing_of_Post-surgical_Tissue_Biospecimens.pdf), was released in 2014.

BBRB will soon release two new expert-reviewed BEBPs. The first expert-vetted BEBP, "Formalin-Fixation and Paraffin Processing of Tissue Biospecimens", is a fit-for-purpose guide for formalin fixation, paraffin impregnation, and embedding of human tissues that was reviewed by more than 40 experts as part of a BBRB-sponsored workshop. The second, entitled "DNA and RNA Extraction from Formalin-fixed Paraffin-embedded Tissue Biospecimens", categorizes steps in the extraction of DNA and RNA that may affect the quality of nucleic acids obtained from FFPE specimens and was reviewed by five experts. Each BEBP has been developed in close partnership with scientists having state-of-the art biospecimen science expertise and a strong publication record. BEBP documents contain step-by-step recommendations and procedural guidelines as well as a summary of supporting literature and expert-derived evidence. Development of each BEBP employs a stringent and thorough analysis of literature indexed in the National Library of Medicine's PubMed database and curated in NCI's publicly accessible Biospecimen Research Database (BRD; http://biospecimens.cancer.gov/brd), a free database of peer-reviewed biospecimen science articles. The database also houses over 400 SOPs searchable by numerous parameters including specimen type, preservation, diagnosis, analyte, pre-analytical factors, and technology platform. Given the nuanced requirements for each individual step in biospecimen collection, NCI's BEBPs serve as a framework to inform the development of Standard Operating Procedures (SOPs) by individual institutions while providing room for flexibility and adaptation. NCI's BBRB continues to add to its BEBP series to improve the reproducibility of molecular data derived from biospecimens. Experts interested in contributing to future BEBPs are encouraged to contact BBRB at ncibbrb@nih.gov.

#4764

An analysis of factors associated with oncology clinical trial activation at University of Illinois Cancer Center Oncology Clinical Trials Office.

Mary A. Otoo, Michelle Uriostigue Preza, Margaret Gavor, Darlene Kitterman, Oana Danciu. _UIC, Chicago, IL_.

Background & Objective

The time from conception to the initiation of clinical trials has been described as costly and time-consuming because of administrative processes (Martinez, D. A. et al). The long clinical trial activation period results in delay in data and affects evidenced-based medical practice. Long clinical trial activation times may be associated with a variety of factors. The objective of this research is to identify and assess factors that impact clinical trials activation periods at the University of Illinois Cancer Center Oncology Clinical Trials Office (UICC OCTO). Methods A protocol search for UICC OCTO managed studies was generated in OnCore. The search was limited to trials that were activated from January 1st, 2016 through November 30, 2017. Time to activation was defined as the time of protocol submission to the Protocol Review Committee (PRC) to the time the study was opened to accrual. Studies which were PRC approved in 2016 but attained Open to Accrual status in 2017 was counted as part of 2016 studies. Time to activation differences between 2016 and 2017 studies was assessed by IIT, Sponsor Type, Institutional Review Board (IRB) Review Type and Study Type. Results A sample of 22 studies (Calendar Year 2016, 12; Calendar Year 2017, 10) was identified. The average activation time was 206.8 days for calendar year 2016 and 78.2 days for calendar year 2017. Results indicate that the activation time generally decreased in 2017 regardless of Sponsor Type, IRB Review Type or Study Type but not whether the study was interventional or not. The average time to activation for interventional studies decreased in 2017 but remained similar for non-interventional studies. Notably, average activation time was longer for non -IIT and Industry studies for both years. Conclusions Clinical trial activation times at the UICC OCTO decreased from 2016 to 2017. The mix of study Sponsor Type, IRB Review Type or Study Type between the two years was similar, and the decrease between the years appears to be independent of these variables. Therefore, it can be inferred that the decrease in activation time was due to interventions made to the process between the two years. These interventions included increase in staff, reorganization in staff duties including moving all activation oversight activities to a new start-up coordinator position, and process changes implemented to increase efficiency. These results should take into consideration that 50 % of studies submitted to the PRC in 2017 will roll over into 2018 for activation, and are therefore not included in the 2017 sample. The data will be reanalyzed at a later time to capture the activation time for all studies started in 2017. In addition, activation time will continue to be tracked to see the effects of additional process changes.

#4765

The impact of the Medicare Part D donut hole closure on use and out-of-pocket costs for aromatase inhibitors.

Ilana Graetz, Janeane N. Anderson, Cameron M. Kaplan. _University of Tennessee Health Science Center, Memphis, TN_.

Objective: Nonadherence to adjuvant hormonal therapy among women with breast cancer increases with higher out-of-pocket costs, particularly among older women. In 2006, Medicare Part D was introduced to provide prescription drug coverage for Medicare beneficiaries, but it had a large coverage gap, also known as "donut hole." In 2011, a provision of the Affordable Care Act was implemented to slowly eliminate the coverage gap, starting with a 50% reduction in brand-name drug pricing within the coverage gap. Also in 2011, several generic aromatase inhibitor (AI) medications became newly available to patients, further lowering their prescription drug costs. We examined whether the initial donut hole closure affected use of AIs and related out-of-pocket (OOP) costs.

Methods: We used a 5% sample of Medicare claims data for 2010 and 2011 (the first year of the donut hole closure) and included women ages 65 and older with a breast cancer diagnosis who filled one or more prescriptions for an AI in the first quarter of 2010 and had at least one AI fill in 2011. We used a difference-in-difference (DID) linear model to compare changes in the number of AI fills and OOP costs per fill between the standard population of individuals affected by the coverage gap to individuals who did not face the coverage gap because of low-income subsidies (LIS), adjusting for sociodemographic factors (e.g., race, age, dual status).

Results: Overall, 4,054 women filled an AI prescription in the first quarter of 2010 and had one or more fills in 2011; 37.59% received LIS, and thus were unaffected by the donut hole closure in 2011. After the initial donut hole closure, non-LIS beneficiaries had more AI fills (9.53 in 2011 versus 9.04 in 2010), and lower OOP costs per fill ($33.23 vs. $93.50). Conversely, LIS recipients filled fewer AIs in 2011 compared to 2010 (9.87 vs. 10.26) and had a smaller decrease in their OOP costs per fill ($2.96 vs. $4.40). The adjusted DID model estimates that after the initial prescription coverage gap closure, non-LIS Medicare beneficiaries had an additional 1.6 AI fills and $53.32 lower OOP costs per fill compared to LIS recipients (both p<0.001).

Conclusion: The first year of the closure of the prescription drug coverage gap led to greater use of AIs and lower OOP costs for women with breast cancer affected by the policy. Whereas treatment nonadherence among women prescribed AIs is associated with increased risk of recurrence and mortality, findings from this analysis suggest that the donut hole closure and concurrent introduction of generic AIs to the market may increase treatment adherence and improve health outcomes for older women with breast cancer.

#4766

The Global ROS1 Initiative: A patient-researcher partnership generating open-source, oncogene-driven cancer models and data.

Janet Freeman-Daily,1 Lisa Goldman,2 Tori Tomalia,3 The ROS1ders,4 Christine M. Lovly,5 Manali I. Patel,6 Alice T. Shaw,7 Bonne J. Addario,8 Guneet Walia,8 Steven W. Young,9 Robert C. Doebele10. 1 _ROS1der patient/activist, Federal Way, WA;_ 2 _ROS1der patient/activist, CA;_ 3 _ROS1der patient/activist, MI;_ 4 _ROS1der patients/activists, CA;_ 5 _Vanderbilt-Ingram Cancer Center, Nashville, TN;_ 6 _Stanford University, Palo Alto, CA;_ 7 _Massachusetts General Hospital, Boston, MA;_ 8 _Bonnie J. Addario Lung Cancer Foundation, San Carlos, CA;_ 9 _Addario Lung Cancer Medical Institute, San Carlos, CA;_ 10 _University of Colorado Denver, Aurora, CO_.

Background: Genomic fusions between ROS proto-oncogene 1 receptor tyrosine kinase (ROS1) and more than 20 genes drive many types of cancer. Only a handful of ROS1-positive (ROS1+) cancer models exist to use in research and developing new treatments. Few research facilities encounter ROS1+ patients--the incidence of ROS1+ cancer is only 1% to 2% in non-small cell lung cancer (NSCLC) and ROS1 testing is rarely done in other cancers. To address this, a group of ROS1+ patients and caregivers ("the ROS1ders") collaborated with advocacy organizations, researchers and industry to form the Global ROS1 Initiative.

Methods: The ROS1ders and partners informed patients, caregivers, and clinicians of the initiative through social media, patient blogs, websites, distribution of fliers in several languages at cancer conferences, and contacts with their clinicians. The ROS1der online community shared ROS1+ information about treatment options and side effects, expert clinicians, clinical trials, research and general support. An epidemiologic survey captured data about ROS1 patient histories and treatments. When a member in the ROS1der community shared news of an upcoming surgery, biopsy or thoracentesis, a ROS1der leader told them of an opportunity to donate excess fresh specimens to create cancer models. Tumor samples and resulting cell lines underwent drug testing and sequencing for mechanisms of resistance.

Results: The ROS1ders members include 166 patients (more than twice the largest ROS1 clinical trial cohort to date) from 16 countries representing 6 types of ROS1+ cancer. Four attempted fresh specimen donations resulted in 2 new ROS1+ cells lines, CUTO27 (CD74-ROS1) and CUTO28 (TPM3-ROS1). These cell lines have already been distributed to academic and industry partners to increase the value of these rare resources. The ROS1ders shared study results within their online group. The Initiative is now working to continue the longitudinal epidemiologic survey and create clinical studies that will generate ROS1+ cell lines and/or patient-derived xenograft (PDX) mouse models. We will conduct genomic analysis of the models to determine the presence of resistance mutations as well as ROS1 pairing partner genes. The resultant renewable tissue resources of PDX models and cell lines, when linked to the clinically annotated genomic database, will be broadly shared to help clinical and translational research understand mechanisms of response and resistance to therapies, and assist in drug development.

Conclusions: This project demonstrates that partnerships between patient-caregiver groups, advocacy groups, researchers and industry, combined with social media outreach, can increase the available oncogene-driven patient data, specimens, and cancer models in small, geographically distributed patient populations.

#4767

Regional strategies for expanding the evolving continuum of Physical Sciences-Oncology Network (PS-ON) research advocacy experiences.

Susan Samson,1 Nastaran Zahir,2 Sheila M. Judge,3 Stuart Cornew,4 Bob Riter,5 Jeri Francoeur,6 Anne Meyn,7 Laurie Cynkin,8 Jason J. Northey,9 Valerie M. Weaver,9 Carole Baas10. 1 _University of California Comprehensive Cancer Center, Breast and Physical Sciences and Oncology Programs/Breast Science Advocacy Core, San Francisco, CA;_ 2 _Division of Cancer Biology, National Cancer Institute, National Institutes of Health, Rockville, MD;_ 3 _Chemistry of Life Processes Institute, Northwestern University, Evanston, IL;_ 4 _Chicago Region Physical Sciences-Oncology Center, Chicago, IL;_ 5 _Cornell University Physical Sciences-Oncology Center, Ithaca, NY;_ 6 _Moffitt Physical Sciences-Oncology Center, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL; _7 _Physical Sciences-Oncology Center (PS-OC), Houston Methodist Research Institute, Houston, TX;_ 8 _Office of Advocacy Relations, National Cancer Institute, National Institutes of Health, Rockville, MD;_ 9 _Center for Bioengineering and Tissue Regeneration, University of California, San Francisco, San Francisco, CA;_ 10 _Physical Sciences-Oncology Network and Office of Advocacy Relations, National Cancer Institute, National Institutes of Health, Rockville, MD_.

Background: The National Cancer Institute (NCI) Physical Sciences-Oncology Network (PS-ON), initiated in 2009, is an interdisciplinary hub currently consisting of eighteen regions across the nation to support the emergence of new scientific frontiers, principles, and opportunities within physical sciences and oncology. Based on the belief that the increasing momentum for cross-disciplinary connectivity between biologists, physicists, mathematicians, chemists, biomedical engineers, and oncologists would be enriched and enhanced by vigorous and diverse public and/or advocacy support, the PS-ON leadership, at program inception, incorporated the advocate voice in setting a national research agenda.

Methods: While the regional advocacy programs operate independently and utilize multilevel, multimethod strategies to expand the evolving umbrella of research advocacy experiences, they are connected through an administrative structure that communicates NCI program priorities to enhance capacity in the approaches utilized across the eighteen PS-ON regions.

Impact: As integral team members, advocates bring real-time diverse patient experiences, diverse professional expertise, and concerns into pioneering, innovative research practices. PS-ON regional engagement/communication strategies include: 1) integrating advocate perspectives to shape basic science research agendas, 2) developing conceptual models/roadmaps to holistic engagement focusing on organizational foundations and best practice strategies, 3) applying guiding frameworks and toolkits for setting the terms of principled engagement/shared governance/bidirectional collaboration, 4) implementing education, outreach, and professional development programs for early-stage investigators, students, and patient communities, and 5) translating, communicating and disseminating laboratory innovations into society.

Discussion: To better understand and fully address the complexities of intersecting physical sciences and oncology advocacy engagement, we explore the unique culture and guidelines set by selected participating institutions. Meeting key challenges regarding programmatic scope and policy impact requires a shift to a new, rapidly evolving paradigm. In parallel to incentives and policy measures created through federal and professional organizations, we offer recommendations for strengthening regional programs and encouraging equitable partnerships for advocates at earlier stages of research to help propel convergent science innovation.

#4768

Characteristics of patients diagnosed de novo with AJCC7 stage III or regionally advanced cutaneous melanoma and trends in adjuvant therapy: An analysis of the SEER cancer registry in the United States.

Ahmad Tarhini,1 Sameer Ghate,2 Antonio Nakasato,2 Raluca Ionescu-Ittu,3 Sherry Shi,3 Briana Ndife,2 Rebecca Burne,3 François Laliberté,3 Mei S. Duh4. 1 _Cleveland Clinic Taussig Cancer Institute, Cleveland, OH;_ 2 _Novartis Pharmaceuticals Corporation, East Hanover, NJ;_ 3 _Analysis Group, Inc, Montreal, Quebec, Canada;_ 4 _Analysis Group, Inc, Boston, MA_.

Background: The v1.2018 National Comprehensive Cancer Network guidelines adopted nivolumab and dabrafenib/trametinib as potential adjuvant therapies for selected patients (pts) with AJCC7 stage III/regionally advanced melanoma. There is a need to understand which patient characteristics may affect adjuvant treatment choices and what lessons can be learned from the era of adjuvant interferon-alfa and ipilimumab. This study aimed to describe characteristics and use of adjuvant therapy in pts with stage III/regional melanoma.

Methods: Pts newly diagnosed with invasive melanoma were identified in the US Surveillance, Epidemiology, and End Results (SEER) cancer registry (most recent 5 years: 2010 -2014; n = 106,195). Stage III melanoma was defined based on the AJCC7 classification (n = 7,669); pts classified as AJCC7 stages I/II but with reported lymphatic metastasis were considered regionally advanced and included (n =169). Pt characteristics measured at the time of the diagnosis are reported by treatments (systemic and radiation) initiated as the main course of treatment in the 4 months post-diagnosis.

Results: Of 7,838 pts with stage III/regional disease at diagnosis from 2010-2014, 1,414 (18.0%) received radiation therapy (± systemic), 1,001 (12.8%) received systemic therapy (± radiation), 443 (5.7%) received radiation + systemic therapy, and 5,866 (74.8%) were not treated with either radiation or systemic therapy. Table 1 reports selected patient characteristics by treatment received.

Conclusions: The results of this study indicate that many pts with stage III/regional melanoma, even those not treated, have risk factors known to be associated with worse prognosis. Use of radiation therapy or adjuvant systemic therapy was rare during the study period (2010-2014). It remains to be determined how the new treatment landscape will change the use of adjuvant therapy.

Table 1. | |  | |

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

|

Stage III1/regionally advanced melanoma

|

Radiation (± systemic)

(n =1,414 ) | Systemic (± radiation)

(n = 1,001) | Systemic + radiation

(n = 443) | Neither2

(n =5,866 )

Age at diagnosis, median (Q1-Q3) | 65 (55 - 76) | 58 (48 - 68) | 61 (51 - 71) | 62 (51 - 73)

Males, % | 57.3 | 51.1 | 74.3 | 37.6

LN classification (from TNM), % | |  | |

N1 | 45.3 | 47.5 | 45.4 | 61.9

N2 (includes N2c) | 35.3 | 34.1 | 39.7 | 26.7

N3 | 19.1 | 18.1 | 14.0 | 11.4

Ulceration, % | |  | |

Yes | 20.3 | 25.2 | 9.3 | 39.0

No | 22.8 | 24.3 | 8.8 | 46.3

Unknown | 56.9 | 50.5 | 81.9 | 14.7

Number mitoses per square mm, % | |  | |

None | 1.6 | 2.7 | 0.9 | 4.7

1 - 5 | 14.8 | 18.8 | 4.5 | 39.3

≥ 6 | 15.0 | 18.2 | 7.9 | 24.3

Unknown | 68.6 | 60.3 | 86.7 | 31.7

LN involvement (any), % | 91.4 | 94.0 | 91.6 | 93.9

1 LN | 33.6 | 37.4 | 31.8 | 57.7

2 - 3 LNs | 25.3 | 26.3 | 23.9 | 22.5

> 3 LNs | 19.2 | 16.8 | 18.5 | 7.5

Unknown number of LNs | 13.3 | 13.6 | 17.4 | 6.2

Microscopic nodal metastasis3 (regardless of the number of LNs) | 20.2 | 31.2 | 8.8 | 61.7

Satellite / in-transit without LN involvement (N2c), % | 5.3 | 2.6 | 2.5 | 5.2

LN, lymph node; TNM, tumor/node/metastasis (TNM) classification of malignant tumors. [1] Stage III melanoma was defined based on the American Joint Committee on Cancer (AJCC) 7 edition classification; [2] Radiation therapy and chemotherapy are reported in the SEER with moderate sensitivity (80% and 68%, respectively) and high specificity. This implies the "Neither" category may include some false -negative (i.e., treated) pts.[3] Includes pts identified with clinically occult (microscopic) LN metastases only, pts with isolated tumor cells only in LN, and/or pts who underwent sentinel LN biopsy and were found to have LN metastasis.

#4769

Identification of differentially expressed microRNAs in African American women with quadruple-negative breast cancer.

Anusha Angajala,1 Raymond Hughley,1 Windy DeanColomb,2 Shweta Tripathi,1 Ming Tan,3 Clayton Yates1. 1 _Tuskegee University, Tuskegee, AL;_ 2 _Lafayette General Health, University and Clinics, Lafayette, LA;_ 3 _University of South Alabama, Mobile, AL_.

Purpose: Recently, we have demonstrated that triple-negative breast tumors (TNBC) that lack the expression of androgen receptor are quadruple-negative tumors (QNBC) and have increased aggressiveness. Additionally, we determined that this is due to a unique gene signature in the QNBC subtype. However, whether this gene signature is associated with a unique miRNA expression and differential in African American (AA) women compared to Caucasian (CA) women has not been determined. Therefore, the purpose of this study is to determine the miRNA expression in presence or absence of androgen receptor in both African American and Caucasian QNBC patients.

Methods: miRNA level 3 sequencing files were originally downloaded on June 01 2016 from TCGA for 925 breast cancer patients. The miRNA sequence files (RPKM) and gene expression files (FPKM UQ) were obtained for normal and tumor samples for these patients. Median value of AR FPKM UQ was used as cut-off and divided patient group as AR(+ve/-ve). Log2 fold change values were calculated by comparing AR-negative and AR-positive patients. P-value was calculated using two-tail t-test. GSE19783 (mRNA, miRNA data) was also obtained and was used as validating dataset.

Results: Out of 925 patients, 101 patients have TNBC. Among TNBC patients, 107 are QNBC and 8 patients are AR +ve TNBC. Reads per million for 1046 miRNAs were considered for this study.40 miRNAs showed differential expression in QNBC. (p-value and lt 0.05). All BC patients were organized based on race (CA/AA), stage (I/II/III, IV) and subtype (Luminal, Her2type, TNBC).We have found that hsa-mir-452 and hsa-mir-937 expression was high in AR-negative, hsa-mir-190b expression was low in AR -ve African American women. miRNAs expression was changed in QNBC and correlated to AR (hsa-mir-577, hsa-mir-135b and hsa-mir-18a). hsa-mir-375 expression was low in QNBC.hsa-mir-150, hsa-mir-181a-2, hsa-mir-500a, hsa-let-7d, hsa-mir-92a-1, hsa-mir-92a-2, hsa-mir-20a, hsa-mir-17, hsa-mir-30a, hsa-mir-210, hsa-mir-455, hsa-mir-130a are expression was changed in African American women with QNBC. Hsa-mir-3607 is low in African American women, irrespective of AR expression. These results are in process of validation using independent dataset.

Discussion/Conclusion: Standard treatment of breast cancer relies on reliable assessment by IHC analysis of ER, PR, and HER2. Our results suggest that the heterogeneity of TNBC is at least partially associated with the presence or absence of AR expression, suggesting that QNBC should be considered as a clinically relevant breast cancer subtype. IHC analysis of AR appears to be a practical assay to determine the most aggressive TNBC subtypes and identifies tumors that could benefit from available targeted therapies. miRNA isoform or novel miRNA needs to be discovered to further investigate the gene regulation for QNBC patients.

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

### Canonical Targets 1

#4772

Poziotinib overcomes de novo resistance of HER2 exon 20 mutations in NSCLC and other cancers: Preclinical studies and initial clinical testing.

Jacqulyne P. Robichaux,1 Yasir Y. Elamin,1 Zhi Tan,1 Marelo Vailati Negrao,1 Mark Routbort,1 Brent Roeck,2 Shuai Li,3 Shengwu Liu,3 Ting Chen,3 Jordi Rodon Ahnert,1 Lixia Diao,1 Monique B. Nilsson,1 Shuxing Zhang,1 Zane Yang,2 Jing Wang,1 Funda Meric-Bernstam,1 Kwok-Kin Wong,4 John V. Heymach1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _Spectrum Pharmaceuticals, Irvine, CA;_ 3 _Dana Farber Cancer Institute, Harvard Medicine School, Boston, MA;_ 4 _Laura & Isaac Perlmutter Cancer Center, NYU, New York, NY_.

HER2 is mutated in ~3% of NSCLC cases, with most of these mutations occurring within exon 20. HER2 TKIs including afatinib and dacomitinib have objective response rates <30% in NSCLC. We have shown that EGFR exon 20 insertions stabilize the active confirmation, and restrict the size of the ATP pocket; and the small, flexible TKI, poziotinib, potently inhibits EGFR exon 20 insertions. Therefore, we hypothesized that HER2 exon 20 insertions induce similar changes, resulting in limited activity of EGFR/HER2 TKIs; and exon 20 HER2 mutations can be targeted with small, flexible covalent TKIs. To this end, Ba/F3 cells expressing 8 different HER2 exon 20 mutations were generated and screened against TKIs including erlotinib, lapatinib, afatinib, dacomitinib, neratinib, poziotinib, osimertinib and others. In Ba/F3 cells with HER2 exon 20 mutations, 1st and 3rd generation TKIs failed to inhibit cell (IC50 values >115nM). While 2nd generation TKIs had some activity (average IC50 =11nM), poziotinib significantly inhibited the growth of all HER2 exon 20 mutations tested with an average IC50 value of 1.9nM. 3D modeling revealed that HER2 exon 20 insertions induce conformational changes which cause constitutive activation and steric hindrance of C805 reducing the ability of larger TKIs to covalently bind. Therefore the smaller flexible terminal group of poziotinib can overcome the structural changes induced by exon 20 insertions. To test this in vivo HER2 A775insYVMA GEMMs were treated daily with poziotinib. Treatment reduced tumor burden by 60% at 4 weeks and had a durable response of 322 days. Based on preclinical data, a heavily pre-treated patient with HER2 driven NSCLC (HER2 A771insAYVM) was placed on a compassionate use protocol and received 16mg poziotinib daily. After 4 weeks, the patient experienced a significant radiological response with reduction in FDG avidity in the left 7th rib, right sacrum and a right lower lobe nodule among others. Moreover, HER2 A771insAYVM circulating free DNA dropped from 2.4% to <0.3%. Finally the frequency and sensitivity of HER2 exon 20 mutations in other cancers has not been fully characterized. To this end we have examined HER2 mutations across patients treated at MD Anderson Cancer Center. HER2 exon 20 mutations were found to occur in breast, endometrial, esophageal, small intestine, colorectal, melanoma and other cancers. In vitro testing of many of these mutations confirms their sensitivity to poziotinib. Collectively, these preclinical and clinical data indicate that poziotinib is a potent, clinically active inhibitor of HER2 exon 20 mutant-driven cancers. Based on these findings and the preclinical and clinical activity of poziotinib in EGFR exon 20 mutants, two phase II clinical studies of poziotinib in EGFR and HER2 exon 20 mutant NSCLC are currently ongoing and a basket trial for other HER2 and EGFR exon 20 mutant cancers is in development.

#4773

Knockdown of the p300 histone acetylase stimulates the rapid and selective decay of ERBB2 mRNA as induced by class 1 histone deacetylase inhibitors.

Gary K. Scott, Lei Wei, Mariah Alejo, Jacob Rose, Birgit Schilling, Christopher Benz. _Buck Inst. for Research on Aging, Novato, CA_.

We have recently shown that translation-dependent and 3'UTR-targeted decay of ERBB2 and other oncogenic transcripts can be rapidly induced by either protein knockdown or selective enzymatic inhibition of class-1 histone deacetylases (e.g. HDAC1/2) using an approved therapeutic like Romidepsin (FK228). Nanomolar doses of FK228 sufficient to acetylate endogenous histones but not tubulin in ERBB2 amplified and overexpressing SKBr3 cells, produces significant cell growth inhibition and apoptosis detectable by 72 h and preceded by the rapid (within 4 h) decay of ERBB2 transcripts, similar to that produced by a pan-HDAC inhibitor like trichostatin-A (TSA) but not by any class-2 selective HDAC inhibitor. As polysome profiling indicated that TSA or FK228 induced decay of ERBB2 mRNA was associated with 80S ribosome loss coupled with the appearance of pre-40S acetylated proteins, mass spectrometry (MS) was used to identify these acetylated proteins. Prominent among the MS identified acetylated proteins was the histone acetyl transferase, p300, where 12 acetylated lysines localized largely to p300's HAT domain were detected following 2h of FK228 treatment. Of note, p300 was recently identified as a key component of a HDAC1/2 regulated complex broadly regulating mRNA stability via poly(A) length (Sharma et al., Cell, 2016). Although considered to be a chromatin regulator that is predominantly nuclear in its subcellular distribution, in recent immunohistochemical studies of human breast cancers p300 was found to be overexpressed and predominantly localized in the cytoplasm (+/- nucleus) as compared to its nuclear localization in normal breast tissue samples (Fermento et al., Exp Mol Pathol, 2014). In our SKBr3 cell line model, siRNA knockdown of p300 induced a rapid decay in ERBB2 transcript levels (ERBB2/GAPDH = 0.3 at 48 h) comparable to that observed with FK228 treatment and exceeding that produced by partial knockdown of HDAC1/2 proteins (ERBB2/GAPDH = 0.7 at 48 h). Ongoing studies with specific inhibitors of p300 bromo and HAT domains are in progress along with intracellular assays of p300 catalytic activity. As well, our MS search is now focused on potential p300-associated substrates that may also be mechanistically involved with FK228 induced ERBB2 transcript destabilization. Altogether, these recent findings shed new light on the relatively unrecognized subcellular mechanism by which HDACs regulate translational control and the stability of cell growth and cancer-promoting transcripts like ERBB2. As new therapeutic strategies are increasingly needed to counter clinical resistance to currently available ERBB2 receptor targeted agents, the repurposing of selective HDAC1/2 inhibitors as well as the emergence of new p300 inhibitors may find appropriate clinical utility against treatment refractory ERBB2-positive breast cancers.

#4776

Antitumor activity of pan-HER inhibitors in HER2-positive gastric cancer.

Takahiro Yoshioka, Kazuhiko Shien, Yuta Takahashi, Eisuke Kurihara, Kei Namba, Yusuke Ogoshi, Hidejiro Torigoe, Hiroki Sato, Hiromasa Yamamoto, Junichi Soh, Toshiyoshi Fujiwara, Shinichi Toyooka. _Okayama University Graduate School, Okayama, Japan_.

Purpose: Molecularly targeted therapy has enabled outstanding advances in cancer treatment. Whereas various anti-HER2 drugs have been developed, trastuzumab is still the only anti-HER2 drug presently available for gastric cancer. Among HER2 targeting drug, afatinib and neratinib inhibit the activation of all HER family protein, and are called pan-HER inhibitors. In this study, we examined the effect of these pan-HER inhibitors to gastric cancer cells. Materials and Methods: We determined the molecular profiles of 12 gastric cancer cell lines. Protein level of HER2 and down-signal pathway molecules were analyzed by Western blotting, and copy number assay or gene expression assay were performed using qPCR. To detect HER2 mutation, we also performed direct-sequence of these cell lines. And next, we examined the antitumor effect of the pan-HER inhibitors afatinib and neratinib in those cell lines, in vitro and in vivo. In addition, we analyzed HER2 alteration in 123 primary gastric cancers resected from Japanese patients to clarify possible candidates with the potential to respond to these drugs. Results: HER2 was amplified in 5 out of 12 gastric cancer cell lines. Gene expression or protein level of HER2 were generally correlated with the copy number of HER2. HER2 mutation was found in one cell line, ECC10, at kinase domain (L755S). In the drug sensitivity analysis, both afatinib and neratinib showed an anti-tumor effect in all the HER2 amplified cell lines both in vitro and in vivo except MKN7 cell line. When the molecular profiles of the cells were compared based on the drug sensitivities, we found that cancer cells with lower mRNA expression levels of IGFBP7, a tumor suppressor gene by inhibiting the activation of IGF-1R, were less sensitive to pan-HER inhibitors. A combination therapy consisting of pan-HER inhibitors and an IGF-1R inhibitor, picropodophyllin, demonstrated a notable synergistic effect. Regarding HER2 alteration in 123 clinical samples, we found 19 cases of HER2 amplification and 3 cases of oncogenic mutations. Conclusion: Afatinib and neratinib are promising therapeutic options for the treatment of HER2-amplified gastric cancer. In addition to HER2 amplification, IGFBP7 might be a biomarker of sensitivity to these drugs, and IGF-1R-targeting therapy can overcome drug insensitiveness in HER2-amplified gastric cancer.

#4777

**Antitumor effect of neratinib in lung cancers harboring** HER2 **oncogene alterations.**

Yusuke Ogoshi, shinichi Toyooka, Jyunichi Soh, hiromasa Yamamoto, Kazuhiko Shien, Hidejiro torigoe, Hiroki sato, Takahiro Yoshioka, Kei Namba, Yuta Takahashi, Eisuke Kurihara. _Okayama University Graduate School of Medicine, Okayama, Japan_.

Background The HER2-targeted therapy for non-small cell lung cancer (NSCLC) harboring HER2 oncogene alterations is one of promising strategy to improve the clinical outcome of treatment for NSCLC. In this study, we investigated the antitumor effect of neratinib, an irreversible pan-HER tyrosine kinase inhibitor (TKI) in NSCLC cells harboring HER2 alterations including mutation and amplification.

Materials and Methods We examined the sensitivity of neratinib against normal bronchial epithelial cells BEAS-2B which ectopically overexpressing wild-type or mutant HER2. Furthermore, we examined the antitumor activity of neratinib in addition to afatinib in several NSCLC cell lines harboring HER2 or EGFR alterations in in vitro and in vivo experiments, and investigated the association between their genetic alterations and sensitivity to neratinib treatment.

Results BEAS-2B cells ectopically overexpressing wild-type HER2 or mutants (A775insYVMA, G776VC, G776LC, P780insGSP, V659E, G660D, and S310F) showed constitutive autophosphorylation of HER2 and activation of downstream signaling by Western blotting. These BEAS-2B cells were sensitive to neratinib, but insensitive to erlotinib, a first generation EGFR-TKI. Neratinib also showed antiproliferative effects on both HER2-altered (H2170, Calu-3, and H1781) and EGFR-mutant (HCC827, PC9, and HCC4006) NSCLC cell lines. Neratinib administration showed strong antitumor effect on tumor growth in mouse xenograft model using HER2-altered lung cancer cell lines.

Conclusions Our study strongly suggests that neratinib is a promising therapeutic option for the treatment of HER2-altered NSCLC.

#4778

Assessing efficacy of neratinib in HER2-driven lung cancer.

Shuai Li,1 Shengwu Liu,1 Ting Chen,1 Jiehui Deng,1 Min Wu,1 Mari Kuraguchi,1 Cam Anh Tran,1 Paul T. Kirschmeier,1 Francesca Avogadri-Connors,2 Richard E. Cutler,2 Alshad S. Lalani,2 Kwok-Kin Wong1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Puma Biotechnology Inc., Los Angeles, CA_.

HER2 mutations have been classified as oncogenic drivers that are present in 2-4% of lung adenocarcinomas. However, due to limited preclinical studies and clinical trials, there is still no available standard of care for this group of lung cancer patients. To fulfill the clinical need for targeting HER2 mutations in lung cancer patients, we performed a comprehensive preclinical study to evaluate the efficacy of neratinib, an irreversible small-molecule pan-HER tyrosine kinase inhibitor that potently binds and inhibits EGFR, HER2, and HER4. We used the previously described genetically modified mouse model (GEMM) of lung adenocarcinomas driven by the major mutant form of human HER2 exon 20 insertions (A775_G776insYVMA) for testing neratinib either as a single agent or in combination with other therapies including trastuzumab in vivo. Treatment efficacy was evaluated based on both monitoring tumor volume changes and survival. Magnetic resonance imaging (MRI) was used for quantifying tumor burden. Tolerability and the overall health of animals were evaluated based on body weight change. We found that neratinib treatment alone provided a modest yet significant response in inhibiting HER2 mutant lung tumors in this model. Pharmacodynamic studies showed that neratinib effectively abolished HER2 phosphorylation and inhibited major downstream signaling targets such as pAKT and pERK. Moreover, combination therapy with neratinib and trastuzumab showed a greater treatment effect by reducing tumor burden with significantly longer progression-free survival and overall survival compared to single agents tested. In all neratinib-based regimens, body weight of treated animals remained stable. On this basis, our study provides a strong rationale to test the combination of neratinib with trastuzumab for the treatment of HER2 mutant NSCLC patients.

#4779

Rogaratinib, a small molecule pan-FGFR inhibitor potently inhibits FGFR4-phosphorylation and exerts anti-tumor efficacy in vivo and in vitro.

Oliver Politz,1 Sylvia Gruenewald,1 Alexander Walter,1 Franziska Siegel,1 Arne Scholz,1 Sebastian Bender,1 Christoph Kneip,1 Peter Ellinghaus2. 1 _Bayer AG, Berlin, Germany;_ 2 _Bayer AG, Wuppertal, Germany_.

Fibroblast growth factor signaling plays an essential role in many physiological processes, including cell proliferation, survival, differentiation, migration, and apoptosis Over-activation of the pathway can lead to the development of various cancers. There are four different FGFR subtypes with over 20 ligands providing a very complex network of interactions and signaling.

FGFR4 plays an important role in normal physiology such as myogenesis muscle regeneration and bile acid synthesis Activating mutations in the kinase domain of FGFR4 have been shown to mediate cancer development, for example, breast cancer, hepatocellular carcinoma (HCC) or rhabdomyosarcoma.

Rogaratinib is a potent inhibitor of all FGFR subtypes with low nanomolar binding kinetics towards the four kinase domains. We explored the effect of rogaratinib on cell lines with high expression levels of FGFR4 mRNA, such as breast cancer cell line MDA-MB453 and rhabdomyosarcoma cell line SH30. We could show that the proliferation of these cell lines is potently inhibited by rogaratinib and accompanied with induction of apoptosis. The Mode-of-action analysis of rogaratinib activity in these cell lines showed inhibition of FGFR4-phosphorylation and downstream signaling at clinical relevant doses.

The in vitro efficacy of rogaratinib in FGFR4 dependent cell lines was further observed as in vivo efficacy in xenograft models of rhabdomyosarcoma and HCC.

Elevation of FGFR4 expression has been associated with resistance development in several cancer types such as breast cancer.

In line with the observed anti-tumor efficacy of rogartinib in vitro and in vivo in breast cancer, sarcoma and HCC models, RNA in situ hybridization by RNAscope, revealed high FGFR4 mRNA expression in TMA's from these tumor types..)

The combination of rogaratinib with standard-of-care provides further options for addressing resistance mechanisms mediated by elevated FGFR4 expression. In addition we have profiled a number of patient derived HCC xenograft models revealing the high efficacy of rogaratinib in models with elevated FGFR expression including FGFR4.

#4780

A novel, potent and selective FGFR4 inhibitor, HM81422 in hepatocellular carcinoma with FGFR4-driven pathway activation.

JaeHo Lee, Hyunjeong Kang, Kyounghwa Koo, Youngeun Ha, Sun Young Lim, Joo-Yun Byun, Hyunkyung Yu, Taehun Song, Moonsub Lee, Seung Hyun Jung, Taewoo Kim, Hyojeong Bang, Eunyoung Kim, Jahoon Kang, Ho Jeong Lee, YoungHoon Kim, YoungGil Ahn, KweeHyun Suh, Sun-Jin Kim. _Research Center, Hanmi Pharm. Co., Ltd., Hwaseong-si, Republic of Korea_.

Introduction: Hepatocellular carcinoma (HCC) is the sixth most prevalent cancer and the second most frequent cause of cancer-related death, however, treatment options are very limited. In recent studies, aberrant signaling through FGFR4 and its ligand, FGF19 have been identified as the oncogenic driver in a subset of HCCs and reported to be associated with poor prognosis. About 30% of HCC patients have altered FGF19/FGFR4 pathway signaling, therefore, the treatment with FGFR4 inhibitor may produce benefit. Materials and Method: Using the structure-based design, we have generated a novel, potent and selective FGFR4 inhibitor, HM81422 with irreversible-covalent binding mode, and evaluated its anti-tumor activity in a variety of HCC cell lines, HCC cell line xenografts and orthotopic grafts. Results: Biochemical selectivity assays demonstrated that HM81422 is highly selective towards FGFR4 compared to other FGFR isotypes as well as a panel of several kinases. The treatment of HM81422 to FGF19 amplified and overexpressed HCC cell lines led to suppression of FGF19/FGFR4 signaling pathway and concomitant reduction in cell viability in dose-dependent manner. Oral administration of HM81422 to mice bearing FGF19 altered HCC cells showed dose-dependent pharmacokinetics, pharmacodynamic modulation of FGFR4 signaling and antitumor efficacy in xenograft models. And HM81422 demonstrated inhibition of tumor growth in an orthotopic liver xenograft model of FGF19 altered HCC in nude mice. Conclusion: In conclusion, the treatment of HCC patients with a potent and selective FGFR4 inhibitor, HM81422, can be an attractive approach targeting approximately 30% of HCC patients by inhibiting altered FGF19-mediated signaling cascade. Further preclinical studies with HM81422 will be performed and reported soon.

#4781

Changes in intracellular signaling following chronic FGFR inhibition in urothelial bladder cancer models.

Isabel S. Jerchel,1 Atanas Kamburov,1 Ralf Lesche,1 Sabine Zitzmann-Kolbe,1 Alexander Walter,1 Peter Ellinghaus,2 Dominik Mumberg,1 Oliver Politz,1 Sylvia Gruenewald1. 1 _Bayer AG, Berlin, Germany;_ 2 _Bayer AG, Wuppertal, Germany_.

Preclinical and clinical studies have validated the therapeutic potential of FGFR inhibition in urothelial bladder cancer (UBC) patients with FGFR genetic aberrations. Rogaratinib is a potent small-molecule pan-FGFR inhibitor being studied in phase I trials in UBC. Here, we studied the effects of chronic exposure of bladder cancer cells in vitro to FGFR inhibition by rogaratinib to identify changes in signaling and gene expression patterns that may identify possible drug combinations that may enhance efficacy of rogaratinib and help overcome inherent and/or acquired treatment resistance. Cell proliferation in response to rogaratinib was evaluated using crystal violet staining in a panel of 13 UBC cell lines. Continuous culture of two cell lines--JMSU1 and RT112--with either constant or increasing concentrations of rogaratinib in several independent approaches generated the resistant sublines, JMSU1-R1 to -R4 and RT112-R1 to -R4. In both cellular models rogaratinib resistance (defined as >30-fold [JMSU1] or >100-fold [RT112] difference in absolute IC50 of sublines vs. parental lines) arose reproducibly and with various treatment schedules. Morphologic changes were also observed. Transcriptomic (RNAseq) and proteomic (R&D Systems proteome profiler arrays) analyses of these cell lines or rogaratinib-treated versus untreated parental cells, respectively, revealed changes that co-occur with the development of resistance. Analysis of the phospho-proteome showed increased phosphorylation of several receptor tyrosine kinases compared to the parental cell lines. Phosphorylation levels varied among the 4 resistant sublines that were derived from the same parental line. This resulted in alteration of downstream signaling pathways in rogaratinib-treated sublines compared to parental cell lines. In conclusion, exposure of selected bladder cancer cell lines to rogaratinib resulted in development of resistance and changes in FGFR signaling and gene expression pathways that may identify strategies to optimize treatment with FGFR inhibitors.

#4782

Inhibition of FGFR in breast cancer metastasis.

Hang Lin, Saeed S. Akhand, Michael K. Wendt. _Purdue University, West Lafayette, IN_.

Metastatic breast cancer is the most advanced stage of breast cancer. This type of cancer can spread other organs and cause 90% of cancer-related death. However, little is known about the molecular mechanisms which drive metastasis in breast cancer. Epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) promote tumor invasion and metastasis. Previously, it has been reported that fibroblast growth receptor 1 plays a key role during EMT: MET cycle. Therefore, optimizing FGFR inhibitors is crucial for therapeutic targeting of late-stage breast cancer. Here, we examined the efficacies of three FGFR kinase inhibitors, AZD4547, JNJ-42756493 and FIIN4, in the 4TO7 cell line both in vitro and in vivo. The 4TO7 cell line is a syngeneic model of systemic dormancy. Mammary fat pad engraftment of the 4T07 cells onto immune competent Balb/C mice results in systemic dissemination, but no macroscopic metastasis. In contrast, the 4T07 cells can escape cellular dormancy in 3D culture and form robust pulmonary tumors in mice upon tail vein inoculation. Furthermore, the 4TO7 cell line robustly responses to exogenous stimulation with FGF2. The phosphorylation of Erk1/2 downstream of FGFR was stimulated after a five-minute treatment with FGF2 and was effectively blocked with all the inhibitors tested. Furthermore, we developed a 3D culture approach that combines tumor spheroid formation in a non-adherent round bottom dish followed by placement on a bed of matrix. Our in vitro results demonstrate that both JNJ-42756493 and FIIN4 have more potent anti-proliferative activities in 3D culture and longer residence time as compared to AZD4547. In vivo, inhibition of FGFR is highly effective against 4TO7 tumors in the pulmonary microenvironment. Importantly, FIIN4 showed the least toxicity. This result suggested that covalent inhibition of FGFR is a promising cancer therapy for patients with metastatic breast cancer.

#4783

Selective antitumor activity of NSC311152 toward medullary thyroid cancer by downregulating the transcription of RET oncogene.

Daekyu Sun, Vishnu Muthuraj Kumarasamy, Tariq Alqahtani. _Univ. of Arizona College of Pharmacy, Tucson, AZ_.

The RET (REarranged during Transfection) proto-oncogene encodes a tyrosine-kinase receptor specifically expressed in tissues originated from neuroectodermal tissues. Dominant-activating mutations in this RET proto-oncogene is often associated with Multiple Endocrine Neoplasia type 2A and 2B and Familial Medullary Thyroid Carcinoma syndromes, characterized by the occurrence of medullary thyroid carcinomas. Human medullary thyroid carcinoma cell line TT cell line harbours a MEN2A-type mutation, so this cell line could be a useful cell system to investigate the antitumor activity of new agents interfering with the function of RET oncogene in medually thyroid cancer. Recently, ellipticine and its derivatives were identified as excellent RET G-quadruplex stabilizing agents. Furthermore, these compounds also interfered with the transcriptional mechanism of the RET gene in TT cells, subsequently decreasing the endogenous RET protein expression. In present study, we demonstrated the specificity of NSC311152 by using papillary thyroid carcinoma (PTC) cells, the K1, which carry BRAF and PIK3CA mutations but lacks the dominant-activating mutations in this RET proto-oncogene. NSC311152 selectively suppresses cell proliferation by inhibiting the intracellular Raf/MEK/ERK and PI3K/Akt/mTOR signaling pathways in the TT cells. The systemic administration of NSC311152 in a mouse MTC xenograft model significantly inhibited the tumor growth through RET downregulation, whereas NSC31152 only showed a moderate antitumor activity against K1 xenograft tumors.

#4784

TAS0286/HM05, a novel highly selective RET inhibitor, prominently inhibits various RET defective tumor growth.

Hidenori Fujita, Isao Miyazaki, Masanori Kato, Yukari Yamada, Keiji Ishida, Tomonori Haruma, Haruka Nagasaki, Kenjiro Ito, Akihiro Hashimoto, Yasuo Kodama, Kaoru Funabashi, Emanuela Lovati, Kazutaka Miyadera, Kenichi Matsuo, Yoshikazu Iwasawa. _Taiho pharmaceutical co., ltd, Ibaraki, Japan_.

The rearranged during transfection (RET) gene is a well-known proto-oncogene and encodes a single-pass transmembrane receptor tyrosine kinase. RET fusions and point mutations are oncogenic drivers in NSCLC, medullary thyroid cancer and other solid tumors, and therefore potential targets for cancer therapy. Multikinase inhibitors targeting the RET mutations have been tested in clinical trials with moderate efficacy in terms of tumor shrinkage and PFS.Moreover, multikinase inhibitors are characterized by poor tolerability due to off-target kinase inhibitory activities. To widen the therapeutic index, a selective RET kinase inhibitor is highly desirable. TAS0286/HM05 is a novel highly selective and potent RET kinase inhibitor.RET kinase assay was performed by homogeneous time-resolved fluorescence (HTRF) method. In-vitro proliferation studies and pharmacodynamics analyses were conducted in cancer cell lines with the RET fusions and RET activating mutations. The antitumor efficacy of TAS0286/HM05 was evaluated using mice xenograft models implanted with cancer cells with various RET gene abnormality. TAS0286/HM05 was orally administered for 14 or 28 consecutive days after grouping. In-vitro and in-vivo RET phosphorylation was detected using western blot analysis. The IC50 value for RET kinase of TAS0286/HM05 was below 1 nM. TAS0286/HM05 showed highly selective RET inhibitory activity among 283 kinases. In cellular assay, TAS0286/HM05 strongly suppressed phosphorylation of RET expressed in cells with various RET fusions and activating mutations, and inhibited cell proliferation at around 10 nM. At this concentration, apoptosis was also observed. The potency was higher than other multikinase inhibitors with RET inhibitory activity. Furthermore, in in-vivo efficacy studies, TAS0286/HM05 significantly inhibited the growth of tumors harboring various RET fusions and activating mutations at a range of 20 to 100 mg/kg/day without any body weight loss. The antitumor efficacy of TAS0286/HM05 was more potent than pre-existing multikinase inhibitors at their maximum tolerated dose. In particular, TAS0286/HM05 dramatically induced tumor regression of 40% within 15 days in animals implanted with LC-2/ad cells, a human lung adenocarcinoma cell line with CCDC6-RET fusion gene. TAS0286/HM05 is a novel and highly selective RET inhibitor with prominent tolerability. The potency of TAS0286/HM05 against tumors with RET abnormalities was stronger than currently marketed multikinase inhibitors being tested in NSCLC patients with RET fusions. TAS0286/HM05 is a promising agent for future clinical development in patients with RET gene abnormalities.

#4785

NMS-E668, a highly potent orally available RET inhibitor with selectivity towards VEGFR2 and demonstrated antitumor efficacy in multiple RET-driven cancer models.

Elena Ardini, Patrizia Banfi, Nilla Avanzi, Marina Ciomei, Paolo Polucci, Alessandra Cirla, Matteo D'Anello, Andrea Lombardi Borgia, Ilaria Motto, Cinzia Cristiani, Dario Ballinari, Eduard Felder, Daniele Donati, Arturo Galvani, Antonella Isacchi, Maria Menichincheri. _Nerviano Medical Sciences, Nerviano, Italy_.

RET chromosomal rearrangements initially identified in a subset of papillary thyroid cancers, as well as gain-of-function point mutations present in ca. 70% of medullary thyroid carcinomas, are well established as oncogenic events that induce constitutive RET activation. More recently, recurring activating RET gene rearrangements have also been found to be expressed in 1-2% of lung adenocarcinomas and subsets of other solid tumors, including colorectal and salivary gland carcinomas. RET kinase is therefore a validated actionable therapeutic target in multiple tumor types, and several small-molecule inhibitors targeting RET are being explored in clinical settings. A common feature of most advanced agents is their lack of selectivity and in particular their potent cross-reactivity against VEGFR2, a receptor tyrosine kinase whose inhibition has been described to be associated with dose-limiting cardiovascular toxicity. Indeed, the high homology between the two kinases renders identification of ATP competitive compounds that selectively inhibit RET over VEGFR2 a highly challenging task. Here we describe the preclinical activity of NMS-E668, a potent and selective ATP-competitive RET inhibitor characterized by favorable activity, selectivity and ADME profiles. NMS-E668 has low nM potency on RET and excellent biochemical selectivity when tested against a kinome panel, notably including circa 30-fold selectivity over VEGFR2. Importantly, 30-fold selectivity of NMS-E668 for RET vs. VEGFR2 was confirmed at the cellular level using NIH-3T3 cells engineered to express activated forms of the two receptors. NMS-E668 potently and selectively inhibited the proliferation of RET-dependent tumor cells, including TT medullary carcinoma cells harboring a RET C634W activating point mutation and LC-2/ad lung carcinoma cells bearing the oncogenic fusion protein CCDC6-RET. NMS-E668 also potently inhibited IL3-independent growth of Ba/F3 cells expressing KIF5B-RET, the RET rearrangement most commonly found in lung adenocarcinomas. Importantly, the proliferation of circa 100 non-RET-dependent tumor cell lines was not significantly affected by NMS-E668, confirming again its selectivity. Tested in vivo against the TT xenograft tumor model NMS-E668 displayed an excellent tumor growth inhibition with complete tumor regression achieved in all animals treated at the higher dose and with confirmed ex vivo target modulation. Good activity was also observed in additional RET-dependent models following oral administration of the compound. Thus NMS-E668, a potent and VEGFR2-sparing RET inhibitor, is an innovative and highly promising candidate for clinical development.

#4786

WSD0922: A novel, oral bioavailable, brain penetrable and reversible EGFRm+ inhibitor for the treatment of primary and metastatic brain tumors.

Wei Zhong, Jinqiang Zhang, Zhihua Mu, Claire Sun. _Wayshine Biopharm (Shanghai), Shanghai, China_.

Epidermal growth factor receptor (EGFR) is a cell surface protein that regulates normal cellular growth in tissues of epithelial origin. Des-regulated EGFR signaling (caused by mechanisms of overexpression, mutation, autocrine activation) contributes for oncogenesis by inducing cells proliferation and resisting apoptosis. In patients with NSCLC, activating mutants (EGFRm+) occur within exon 18-21 with increased EGFR activity. About 90% of these mutations (G719x, exon 19 deletions and exon 21 mutation) are sensitizing EGFRm+. EGFR TKIs have shown good performance in clinic for systemic NSCLC anchoring EGFRm+. However, up to 65% of NSCLC patients will develop brain metastasis (BM) ultimately. Current approved EGFR TKIs are not so effective for BM due to limited brain penetration as they are substrates of P-gp or BCRP, two main efflux transporters expressed on human BBB. In patients with GBM, the most common mutation is EGFRvIII (exon2-7 deletion) with an overall frequency of 25–64%. Current EGFR TKIs are ineffective in GBM treatment probably due to resistance caused by EGFRvIII as well as limited brain penetration. One-year survival of GBM patients is approximate 43%. Herein, we report a discovery of brain penetrable EGFR inhibitor WSD0922 for NSCLC BM and GBM with IC50 against EGFRm is <10nM. The ex-vivo antiproliferative GI50 in GBM tissues with EGFRvIII amplification is <100nM. WSD0922 is highly selective over other kinases. In-vitro MDCKII transfected cells and Caco-2 assays showed that WSD0922 is highly permeable and not a substrate of P-gp or BCRP. Preclinical CNS PK studies confirmed good brain penetration of WSD0922 with Kp,uu,brain close to unity. Treatment of mice bearing PC-9 (exon 19 del of NSCLC) in both subcutaneous (SC) and intracranial models with WSD0922 at 1-10mpk BID via PO resulted in dose dependent tumor growth inhibition (TGI) or regression with statistically significant survival benefit. Moreover, significant TGI for mice bearing GBM PDX model was achieved by treatment with

WSD0922. No toxicity was observed in the animals during the treatment. Predicted human PK properties are very promising to offer sufficient target engagement in clinic. Taken together, our data provide a good rationale for WSD0922 to be developed toward clinic for the treatment of patients with BM of NSCLC or GBM harboring EGFRm+.

#4787

SKLB188 inhibits the growth of head and neck cancer cell growth by suppressing EGFR signaling.

Shile Huang,1 Mansoureh Barzegar,1 Shuang Ma,2 Shengyong Yang2. 1 _Louisiana State Univ. Health Sciences Ctr., Shreveport, LA;_ 2 _Sichuan University, Chengdu, China_.

Overexpression of epidermal growth factor receptor (EGFR) occurs in approximately 90% of head and neck squamous cell carcinoma (HNSCC), and is correlated with poor prognosis. Thus, targeting EGFR is a promising strategy for treatment of HNSCC. So far, only Cetuximab, an anti-EGFR monoclonal antibody, has been approved for EGFR-target therapy in HNSCC. Here we show that SKLB188, a newly synthesized small molecule compound, inhibited cell proliferation and induced apoptosis of HNSCC cells in vitro and in vivo, by targeting EGFR signaling. SKLB188 induced cell cycle arrest in G1 phase, by downregulating the expression of Cdc25A, cyclins D1/A and cyclin dependent kinases (CDK2/4), and upregulating the expression of CDK inhibitors (p21Cip1 and p27Kip1), leading to decreased phosphorylation of Rb. SKLB188 also downregulated the expression of Mcl-1 and survivin, inducing caspase-dependent apoptosis. Molecular docking predicted that SKLB188 could bind to the kinase domain of EGFR through hydrogen bonds and hydrophobic interactions. In vitro kinase assay revealed that SKLB188 inhibited the activity of a recombinant human EGFR very potently (IC50 = 5 nM). Western blot analysis showed that SKLB188 inhibited the phosphorylation of EGFR and its downstream targets, extracellular signal-regulated protein kinases 1 and 2 (Erk1/2) and Akt in the cells. In addition, SKLB188 dose-dependently inhibited FaDu xenograft growth in nude mice, and concurrently inhibited the phosphorylation of Erk1/2 and Akt in the tumors. Taken together, our results support that SKLB188 inhibits the growth of HNSCC cells in vitro and in vivo by targeting EGFR signaling.

#4788

Preclinical evaluation of E-191, a highly selective EGFR T790M inhibitor.

Liu Xile,1 Lihong Hu,1 Charles Z Ding,2 Weifeng Mao,2 Liqing Wang,2 Xiaohe Shi,2 Chaofeng Long,3 Xiaoxin Chen,3 Haijun Li,3 Xing Liu,3 Zhuowei Liu3. 1 _wuxiapptec, wuhan, China;_ 2 _wuxiapptec, shanghai, China;_ 3 _Guangdong Zhongsheng Pharmaceutical, Guangdong, China_.

Objective: Patients of non-small cell lung cancer (NSCLC) with activating EGFR mutations initially respond to the first generation reversible EGFR tyrosine kinase inhibitors, such as gefitinib and erlotinib. However, most of the patients acquire resistance within 10~16 months. The most common mechanism of resistance is the EGFR T790M mutation. In addition, selectivity of the EGFR inhibitors is important, as inhibition of EGFR wild type can lead to dose limiting toxicities, including rash and diarrhea. This may be the reason why the second generation irreversible EGFR inhibitors, such as afatinib, which also inhibit EGFRWT, is not widely used. Here, we disclose our clinical candidate compound E-191 as a novel irreversible inhibitor that potently inhibits the resistance mutant forms of EGFR with a good selectivity sparing EGFRWT.

Method: The anti-proliferative activity of E-191 was evaluated in NCI-H1975 (EGFRT790M/L58R), HCC827 (EGFRd746-750), and A431 (EGFRWT) cell lines. Daily oral administration of E-191 at 3 doses was used to evaluate the in vivo anti-tumor activity in three cell-derived tumor xenograft (CDX) models, NCI-H1975, HCC827, and Ba/F3 (EGFRT790M /d746-750). The in vivo selectivity was evaluated in the A431 (EGFRWT) CDX model. Effects on EGFR signaling pathway were assessed by western blot analysis of the downstream effector proteins p-EGFR (Y1068), p-AKT (Ser473) and p-ERK (Thr202/Tyr204).

Result: E-191 displayed potent anti-proliferative activity in the EGFR mutant cell lines NCI-H1975 (IC50 = 22 nM) and HCC827 (IC50=1.9 nM), and a much less potent activity in the EGFRWT A431 cell line (IC50 = 531 nM). E-191 has about 24 fold selectivity for EGFRT790M over EGFRWT. E-191 potently inhibited tumor growth at all doses tested (P<0.001) in the NCI-H1975, HCC827 and Ba/F3 (EGFRT790M /d746-750) models, while significantly less so in the A431 model. Western blot analysis of the tumor samples in the NCI-H1975 model study showed that E-191 inhibited p-EGFR (Y1068), p-AKT (Ser473), and p-ERK (Thr202/Tyr204), indicating that the tumor growth inhibition was through inhibition of the resistant mutant EGFR.

Conclusion: We have demonstrated with in vitro and in vivo preclinical models that E-191 is a selective inhibitor of the resistance mutant forms of EGFR, potently inhibiting EGFRT790M but not EGFRWT. These results make us believe that E-191 has a good potential in the clinic for the treatment of resistant EGFR driven NSCLC.

#4789

Clinical candidate DBPR112: A novel epidermal growth factor receptor inhibitor as a promising treatment for non-small cell lung cancer.

Hsing-Pang Hsieh,1 Jang-Yang Chang,2 Ching-Chuan Kuo,1 John Hsu1. 1 _National Health Research Institutes, Zhunan Town, Taiwan;_ 2 _National Cheng Kung University, Tainan, Taiwan_.

Lung cancer is one of the chief causes of cancer death in the world; in addition, non-small cell lung cancer (NSCLC) accounts for 85% of the lung cancer deaths. The development of tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR) have shown remarkable effects in patients but some acquired resistance after treatment. Therefore, the discovery of efficacious EGFR-TKIs poses an utmost priority. Our team at IBPR has designed and synthesized a series of compounds by high throughput parallel synthesis platform. In the subsequent structure-activity relationship study (SAR) and lead optimization, approximate 150 compounds were synthesized, which concluded that the presence of an N,N-dimethyl functional group on the Michael acceptor and the S-chirality of the phenylaminoethanol side chain were both essential. Consequently, DBPR112 was identified as a potent EGFR-TKI clinical candidate showing excellent inhibitory ability on EGFRL858R/T790M and EGFR exon 20 insertion. DBPR112 was orally effective against the growth of human lung H1975 tumors subcutaneously xenografted in nude mice. A dramatic reduction in tumor size was noted with DBPR112 treatment, while displaying negligible body weight loss in all dosing groups. Furthermore, DBPR112 was more tolerable than afatinib in mice. To date, all pre-clinical studies were completed, and the IND application of DBPR112 was approved by US FDA in 2016 and the Phase I clinical trial has been initiated since July 2017 in Taiwan.

#4790

**YH25448, an irreversible 3** rd **generation EGFR TKI, exhibits superior anticancer effects with potent brain BBB penetration in NSCLC.**

Jiyeon Yun,1 Min Hee Hong,2 Seok-Young Kim,1 Chae Won Park,3 So-Young Kim,3 Mi Ran Yun,3 Han Na Kang,3 Kyoung-Ho Pyo,3 Jong Sung Koh,4 Ho-Juhn Song,4 Young- Sung Lee,5 Se-Woong Oh,5 Soongyu Choi,5 Byoung-Chul Cho1. 1 _Yonsei University College of Medicine, Cancer Research Inst., Seoul, Republic of Korea;_ 2 _Yonsei University College of Medicine, Cancer Research Inst, Seoul, Republic of Korea;_ 3 _Yonsei University College of Medicine. Cancer Research Inst., Seoul, Republic of Korea;_ 4 _Genosco Inc., Cambridge, MA;_ 5 _Yuhan R &D Institute, Yuhan Corporation, Seoul, Republic of Korea_.

EGFR mutated lung cancer shows approximately 10-15% of non-small cell lung cancer (NSCLC). Although the best therapeutic EGFR tyrosine kinase inhibitors (TKIs) targeting mutant EGFR, such as gefitinib and erlotinib, are used in the first line treatment of patients with advanced EGFR mutated NSCLC, the acquired resistance to the drugs usually appears in 10-12 months of therapy by the occurrence of a second EGFR mutation T790M. YH25448, a highly mutant-selective and irreversible 3rd generation EGFR TKI potently penetrating blood-brain barrier (BBB) penetration, targets both activating EGFR mutations Del19, L858R and T790M mutation while sparing wild type. In NSCLC cell lines and primary cancer cells from patients harboring EGFR mutations, YH25448 showed more potent inhibition of cancer cell growth and significantly increased tumor cell apoptosis compared to osimertinibs, which is one of 3rd generation EGFR TKIs. In vivo mouse model implanted with H1975 cells, YH25448 treatment at the once-daily showed a dramatic dose-dependent tumor regression in both subcutaneous and intracranial lesions with no abnormal signs such as skin keratosis shown in osimertinib-treated mice. Plasma half life of YH25448 was 5.9-6.8 hr and tumor to plasma AUC0-last ratio was 3.0-5.1 in tumor bearing mice. YH25448 also showed excellent penetration of the BBB, achieving CSF concentrations exceeding the IC50 value for pEGFR inhibition in the tumor-bearing mice. Taken together, these findings suggest important role for the further development of YH25448 as a novel therapeutic for the treatment of EGFR mutant-positive NSCLC patients with brain metastases.

#4791

OMO-1, a potent, highly selective, orally bioavailable, MET kinase inhibitor with a favorable preclinical toxicity profile, shows both monotherapy activity, against MET pathway-driven tumors, and EGFR TKI combination activity in acquired resistance models.

Marion Libouban,1 Eleonora Jovcehva,2 Desiree De Lange,3 Boudewijn Janssens,3 Tinne Verhulst,3 Souichi Ogata,3 Berthold Wroblowski,3 Laurence Mevellec,4 Tianbao Lu,5 Glen Clack,1 Timothy Perera1. 1 _OCTIMET Oncology NV, Geel, Belgium;_ 2 _F. Hoffmann-La Roche Ltd, Basel, Switzerland;_ 3 _Janssen Pharmaceutica NV, Beerse, Belgium;_ 4 _Janssen Pharmaceutica NV, Rouen, Belgium;_ 5 _Johnson & Johnson PRD, Philadelphia, PA_.

Activation of the MET/HGF pathway has been linked to tumor initiation, metastasis, angiogenesis and resistance to therapeutic agents. Here we present pharmacological characterization of OMO-1 (formerly JNJ-38877618), a potent, highly selective, orally bioavailable MET kinase inhibitor with nM binding affinity (Kd=1.4 nM) and enzyme inhibitory activity against wt and M1268T mutant MET (2 and 3 nM IC50). MET inhibitory effects were assessed in proliferation, colony formation and motility assays. OMO-1 displayed nM potency against MET Ampl/mutant and therapy resistant models. In vivo, OMO-1 induced complete inhibition of tumor growth in 3 models: the SNU5 MET amp gastric, U87-MG HGF autocrine glioblastoma and Hs746T MET exon 14 skipping mutant gastric cancer. OMO-1 induced regression of large MET amplified EBC-1 SqNSCLC where OMO-1 led to dose- and time-dependent inhibition of MET kinase activation, with the duration of target shut down considerably exceeding plasma exposure times. Combination treatments were well tolerated and improved EGFR targeted therapy. Although single agent OMO-1 had no effect on NSCLC HCC827 EGFR, combination with Erlotinib led to delayed onset of tumor recurrence. The acquired EGFR inhibitor resistant model HCC827-ER1 was determined to be MET amplified. OMO-1 and erlotinib both inhibited tumor growth of this model whilst combination induced tumor regression. In an EGFR inhibitor resistant PDX having MET amplification, single agent OMO-1 caused tumour stasis whereas MetMab/erlotinib only led to tumor growth delay. The potent preclinical activity we have observed, supports ongoing clinical development of OMO-1 in patients with MET pathway-driven tumors.

#4792

Preclinical evaluation of GST-HG161, a potent and highly selective c-MET inhibitor.

Xiongbin Xu,1 Gang Li,1 Lihong Hu,1 Junguo Hao,2 Charles Z. Ding,2 Shuhui Chen2. 1 _WuXi AppTec (Wuhan), Wuhan, China;_ 2 _WuXi AppTec (Shanghai), Shanghai, China_.

Objective: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. Studies indicate that aberrant activation of mesenchymal-epithelial transition factor (c-MET) is associated with HCC development. Blocking c-MET signaling shows antitumor efficacy in patients with HCC. Thus, c-MET has been an attractive therapeutic target for HCC. We disclose here for the first time a potent and selective c-MET inhibitor GST-HG161 and show the antitumor efficacy data in preclinical models of HCC.

Method: MHCC97H HCC cell line with c-MET overexpression was used to evaluate the anti-proliferative activity of GST-HG161 in a 120-hour viability assay. Selectivity was evaluated with a panel of cell lines without overexpression of c-MET. Daily oral administration of GST-HG161 doses of 1, 3, 10 and 30mg/kg was used to evaluate its in vivo antitumor efficacy in two animal models of HCC with c-MET overexpression, MHCC97H xenograft (CDX) model and LI-03-0317 xenograft (PDX) model. The c-MET protein levels and gene copy number in LI-03-0317 PDX were assessed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), respectively. High c-MET expression in LI-03-0317 PDX was determined by the presence of tumor cells with membrane staining of 3+ intensity. Effect of GST-HG161 on c-MET signaling inhibition was assessed by western blot analysis of downstream effector proteins including p-MET, p-AKT and p-ERK.

Result: GST-HG161 displayed potent anti-proliferative activity in MHCC97H HCC cell line (IC50 = 5 nM) while showed little activity in the cell lines without c-MET overexpression. It significantly inhibited tumor growth at tested doses (3, 10 and 30mg/kg) (P<0.05) in both MHCC97H CDX model and LI-03-0317 PDX model. In MHCC97H CDX model, western blot analysis showed that GST-HG161 potently inhibited p-MET and downstream effector proteins p-AKT and p-ERK in the tumor tissue, indicating that the tumor growth inhibition in this model was mediated through inhibition of c-MET.

Conclusion: GST-HG161 is a potent and highly selective c-MET inhibitor. Its excellent antitumor efficacy as shown in

the HCC mouse models provide us with confidence that GST-HG161 has high potential in the clinic for treating patients of HCC with high c-MET activity.

#4793

WSD1227: A novel, oral bioavailable, brain penetrable and reversible VEGFR inhibitor for the treatment of primary and metastatic brain tumors.

Jinqiang Zhang, Wei Zhong, Zhihua Mu, claire sun. _Wayshine Biopharm (Shanghai), Shanghai, China_.

Occurrence of brain metastases (BM) has been reported in ~10% cancer patients as a later manifestation of the disease with incidence depending on the type of primary tumors1: about 20~40% in NSCLC and BC2,3 and <5% in CRC4,5 and ovarian cancer (OC)6. The incidence of BM is increasing in part due to the earlier diagnosis by using brain imaging techniques and the improved survival offered by systemic therapies. Patients with BM have a very poor prognosis and the overall survival is only several months after diagnosis. Surgery and radiotherapy either as single treatment or in combination are the most common options for the management of BM since many anticancer drugs cannot cross the blood-brain barrier (BBB). VEGFR2, upregulated in many cancers7,8, plays an important role in tumor angiogenesis. Anti-angiogenesis has been proved to be a good strategy to shrink tumor in many cancer treatments, such as bevacizumab in GBM, NSCLC, mBC, cediranib in OC, etc. However, those clinical validated anti-VEGF agents cannot cross BBB effectively due to either large molecule weight or being substrate of BBB efflux transporters. Herein, using our advanced medicinal chemistry with deep knowledge in CNS area, a BBB penetrable, selective and potent VEGFR2 inhibitor WSD1227 is discovered with biochemical IC50 against VEGFR1/2/3 at 0.69/0.35/0.41nM versus against other targets such as PDGFRα IC50 22.9nM, PDGFRβ IC50 19.4nM, cKit IC50 383nM, FLT3 IC50 555 nM and CSF1R IC50 1062nM. In-vitro MDCKII transfected cell assays demonstrated WSD1227 is not a substrate of P-gp or BCRP, two main efflux transporters on human BBB. Preclinical CNS PK studies confirmed good brain penetration of WSD1227 with Kp,uu,brain close to unity. WSD1227 possessed superior PK profile with sufficient free PK exposure to achieve target engagement in mice. Treatment of mice bearing SW620, a CRC xenograft in both subcutaneous (SC) and intracranial models with WSD1227 after oral dosing resulted in

significant tumor growth inhibition (TGI). Similar TGI was observed in SKOV-3 bearing mice, an OC xenograft in both SC and intracranial models. No toxicity was observed in the animals during the treatment. Moreover, predicted human PK properties are very promising to offer sufficient target engagement at a pretty low dose in clinic. Taken together, our data provide a good rationale for WSD1227 to be developed toward clinic for the treatment of patients with BM whose primary tumors have already been controlled well by systemic therapies.

#4794

Preclinical evaluation of WX-0593, a potent orally active ALK inhibitor.

Liu Xile,1 Charles Z Ding,2 Weifeng Mao,2 Yuxin Qin,2 Shansong Zheng,3 Yingying Yang,3 Qingmei Zheng,3 Long Zhang,3 Shuyong Zhao3. 1 _Wuxiapptec, Wuhan, China;_ 2 _Wuxiapptec, Shanghai, China;_ 3 _Qilu Pharmaceutical, Jinan, China_.

Objective: ALK is a tyrosine kinase belonging to the insulin receptor super-family. The constitutively active ALK fusions are oncogenic and linked with multiple types of human cancer. The EML4-ALK fusion has been proven to be the driver of approximately 5% of NSCLC patients. We disclose here for the first time a novel ALK inhibitor WX-0593 and its preclinical evaluation data in preclinical models of ALK driven NSCLC.

Method: The enzyme activities of WX-0593 for ALKWT, ALKL1196M, ALKC1156Y and EGFRL858R/T790M were measured with corresponding kinase assays. The anti-proliferative activity was evaluated in Karpas-299, Ba/F3, Ba/F3 (EML4-ALK-WT), Ba/F3 (EML4-ALK-L1196M), Ba/F3 (EML4-ALK-C1156Y), and NCI-H1975 cell lines. The specific effect (PD) of WX-0593 on ALK signaling was analyzed in NCI-H3122 cells. Antitumor activity of WX-0593 was evaluated in three patient-derived xenograft (PDX) models of NSCLC (LU-01-0015, LU-01-0319, and crizotinib resistant LU-01-0319R) and one cell-derived xenograft (CDX) model of NSCLC (NCI-H3122). PKPD was assessed by western blot analysis of the treated tumor tissues for p-ALK (Tyr1604), p-STAT3 (Tyr705), p-STAT5 (Tyr694), p-AKT, and p-ERK.

Result: WX-0593 displayed potent enzymatic activities for ALKWT, ALKL1196M, ALKC1156Y and EGFRL858R/T790M with IC50 of 5.38, 9.26, 9.28, and 16.74 nM respectively. It also showed expected anti-proliferative activity in six cell lines, Karpas-299, Ba/F3, Ba/F3 (EML4-ALK-WT), Ba/F3 (EML4- ALK-L1196M), Ba/F3 (EML4-ALK-C1156Y), and NCI-H1975, with IC50 of 4.5, 2208, 4, 9.5, 9, and 508 nM, respectively. Western blot study in NCI-H3122 cells showed that WX-0593 could fully inhibit p-ALK, p-ERK,p-STAT5 at 11.1 nM and p-AKT at 100 nM. WX-0593 significantly inhibited tumor growth at all doses tested (P<0.001) in the LU-01-0015 (HIP1-ALK), LU-01-0319 (EML4-ALK), and LU-01-0319R NSCLC PDX models as well as in the NCI-H3122 CDX model. Western blot analysis of the tumor samples showed that WX-0593 inhibited p-ALK (Tyr1604), p-STAT3 (Tyr705), p-STAT5 (Tyr694), p-AKT, and p-ERK, indicating that the tumor growth inhibition was mediated by inhibition of ALK signaling.

Conclusion: We have identified a potent orally active second generation ALK inhibitor, WX-0593. It can inhibit the activity of both wide type and crizotinib resistant ALK and showed strong antitumor activity in in vitro and in vivo preclinical models. These results are considered highly promising and warrant moving the compound forward to clinical investigation.

#4795

Potency of a new ALK/ROS1 inhibitor TPX-0005 to ALK G1202R mutation and ROS1 G2032R mutation.

Satoshi Yoda,1 Leila Dardaei,1 Kylie Prutisto-Chang,1 Jean Cui,2 Alice T. Shaw,1 Aaron N. Hata1. 1 _Massachusetts General Hospital, Charlestown, MA;_ 2 _TP Therapeutics, Inc., San Diego, CA_.

ALK rearrangements and ROS1 rearrangements are important therapeutic targets in non-small cell lung cancer (NSCLC). ALK-positive NSCLC is currently treated with the first-generation ALK inhibitor crizotinib followed by more potent, second-generation ALK inhibitors, such as ceritinib, alectinib, or brigatinib, and ROS1-positve NSCLC is currently treated with crizotinib. Even though these drugs produce responses in most patients, drug resistance eventually develops. Notable mechanisms of resistance are kinase domain solvent front mutations, such as ALK G1202R and ROS1 G2032R. Genomic analysis of relapsed cases show ALK G1202R in 21%, 29%, or 43% of ceritinib-, alectinib-, or brigarinib-resistant cases, and ROS1 G2032R in 41% of crizotinib-resistant cases.

The novel ALK/ROS1 inhibitor TPX-0005 was designed to overcome clinical resistance mutations, especially solvent front mutations, and is currently being evaluated in a phase 1/2 study. Engineered preclinical models have demonstrated the potency of TPX-0005 against ALK G1202R and ROS1 G2032R. For example, Ba/F3 cells expressing EML4-ALK G1202R or CD74-ROS1 G2032R resistant to second-generation ALK inhibitors or crizotinib, respectively, but were sensitive to TPX-0005. Here we investigate the potency of TPX-0005 to overcome solvent front mutations in resistant patient-derived models. We generated cell lines and patient-derived xenograft mouse models from ALK G1202R and ROS1 G2032R tumors at the time of relapse on second-generation ALK inhibitors or crizotinib, respectively. As expected, these models were resistant to second-generation ALK inhibitors or crizotinib. In contrast, TPX-0005 suppressed ALK or ROS1 phosphorylation, and led to decreased cell viability and tumor regression in vivo. These data demonstrate that TPX-0005 overcomes solvent front mutations in clinically relevant models and provides rationale for the clinical development of TPX-0005 for patients harboring ALK G1202R or ROS1 G2032R resistance mutations.

#4796

Ropotrectinib is a novel polypharmacology kinase inhibitor against WT and mutant ROS1, TRK and ALK.

Wei Deng, Dayong Zhai, John Huang, Evan Rogers, Jeffrey Whitten, John Lim, Yishan Li, Jean Cui. _TP Therapeutics, Inc., San Diego, CA_.

Tyrosine kinase inhibitors (TKI) have achieved great success for cancer patients with aberrant target genes such as ROS1, NTRKs and ALK. However, the inevitable emergence of drug resistance limits their long-term clinical benefits. Ropotrectinib (TPX-0005), a brain-tumor penetrable ROS1/TRK/ALK kinase inhibitor, was designed not only to target abnormal activity of ROS1, TRK and ALK but also to overcome drug resistance caused by previous ROS1, NTRK and ALK inhibitors. The unique structure design of roporectinib granted it the unique ability to overcome acquired solvent front mutations in the targeted kinase domain caused by other TKIs. Moreover, ropotrectinib displays inhibitory activity for SRC/FAK/JAK2, rendering its potential to overcome drug resistance caused by "by-passing" mechanisms such as EMT.

Ropotrectinib is a superb ROS1 inhibitor. The ALK/ROS1/MET inhibitor crizotinib has been approved by FDA to treat NSCLC patients harboring a rearranged ROS1 fusion gene. Acquired secondary mutations in ROS1 kinase domain is a common drug resistance mechanism in 50-60% patients progressed on crizotinib treatment, with solvent front mutations most frequently observed. Compared to other ROS1 inhibitors, such as lorlatinib, ceritinib, brigatinib and entrectinib, ropotrectinb demonstrated most potent activity against WT and mutant ROS1, especially solvent front mutations such as G2032R in cellular assays and potently inhibited xenograft tumors.

Ropotrectinib is the most potent TRK inhibitors in clinic, with IC50s < 1nM in cellular assays. Preclinic in vitro and in vivo studies further demonstrated that ropotrectinib overcomes clinical resistance mutations which occur in patients progressed on TRK inhibitors, such as entrectinib and larotrectinib. In a phosphorylation assay using engineered NIH3T3 cells expressing a TRKAG595R fusion gene, ropotrectinib (IC50 < 0.1nM) exhibited activities at least ten folds more potent than the recently reported activity of LOXO-195 (IC50 = 7nM), a second generation of TRK inhibitor in clinic.

Ropotrectinib is also a ALK inhibitor with CNS activity, inhibiting growth and viability of ALK+ cells, inducing tumor regression in a ALK+ PDX tumor model and significantly improving overall survival of mice bearing CNS ALK\+ H2228 tumors. In particular, compared to previous generations of ALK inhibitors (e.g. crizontinib, ceritinib, alectinib, and brigatinib), it exhibited superb activity in those acquired mutations, especially the G1202R solvent front mutation and double mutations containing L1198F. Roporectinib dose-dependently down-regulated EGFR, CD44 and vimentin expression levels via SRC/FAK inhibition, suggesting a potential to prevent EMT.

In summary, ropotrectinib exerts unique pharmacological properties for clinical applications in patients harboring aberrant ROS1/TRK/ALK. A Phase 1/2 clinical trial of TPX-0005 is actively pursued (NCT03093116).

#4797

**In vitro and in vivo effect of ARQ 531 on Trk family kinases** **.**

Yi Yu, Gabi Schwartz, Pascal Schamber, Ron E. Savage, Sudharshan Eathiraj, Terence Hall, Brian Schwartz. _ArQule, Inc., Burlington, MA_.

The tropomycin kinase (Trk) receptor family contain Trk A, B and C that are encoded by the NTRK1, NTRK2, and NTRK3 genes respectively. Trk family kinases have been shown to play a very important role in the development and physiological functions of the nervous system. Recently, chimeric Trk kinases resulted from gene fusions of NTRK genes are involved in initiation and progression of various cancers such as sarcoma, thyroid, salivary gland cancers, etc. Targeting such alteration of Trk family kinases becomes a new avenue for the therapeutic intervention. ARQ 531 has biochemical IC50 values of 1.3 nM, 1.8 nM and 1.8 nM for Trk A, B and C, respectively with targeting a unique set of other kinases. In a cell based assay, ARQ 531 inhibits kinase activity of Trk A, B and C in UOS2 cells overexpressing Trk family kinases with IC50 values of 50.5 nM, 28.0 nM and 4.1 nM respectively. Pathway analysis shows that ARQ 531 abolished autophosphorylation of TMP3-Trk A in colon cancer KM-12 cells after 2-hour treatment, concomitant with marked inhibition of pAKT, pERK and pPLCγ1. Moreover, in Trk A-overexpressing K562 cells, ARQ 531 suppressed phosphorylation of Trk A and its downstream targets AKT and MAPK. Anti-proliferative activity of ARQ 531 has been demonstrated in Ba/F3 system over-expressing fusion Tel-Trk family kinases (ETV6-NTRK1, 2 or 3), exhibiting IC50 value of 300, 481, and 341 nM for Tel-Trk A, B or C respectively. In TMP3-TrkA(TMP3-NTRK1) fusion-driven KM12 cells driven by ARQ 531 strongly suppressed cell proliferation with an IC50 value of 130 nM. Subsequently, apoptotic response was observed in KM-12 cells after treatment of ARQ 531 for 24 hours, which was demonstrated by the increase in cleaved PARP1 and cleaved caspase 3. Significant anti-tumor activity of ARQ 531 was observed in KM-12 xenograft mouse model. In conclusion, ARQ 531 is a potent Trk family kinase inhibitor and the results from preclinical study provides rationale for testing ARQ 531 in a clinical setting, particularly in cancers driven by Trk fusion kinases.

#4798

Targeting hTERT for treatment of glioblastoma (GBM).

Saumya Reddy Bollam,1 Hyun-Jin Kang,2 Sen Peng,1 Vijay Gokhale,2 Laurence Hurley,2 Michael Berens,1 Harshil Dhruv1. 1 _TGen (Translational Genomics Research Inst.), Phoenix, AZ;_ 2 _University of Arizona, AZ_.

Approximately 86% of GBM tumors exhibit a mutation at -124 or -146 bp upstream from the ATG start site in the transcription activating promoter region of Human telomerase reverse transcriptase (hTERT). Mutations in the hTERT promoter are known to impair repression, leading to overexpression of hTERT and tumor maintenance. Yet, complete understanding of mechanistic implications is still necessary. While overexpressed hTERT is associated with oncogenesis and resistance to apoptosis, we also observe phenotypes unrelated to the reverse transcription function. We characterized long-term glioma cell lines and glioma PDX models by hTERT promoter mutation status, hTERT mRNA expression, and hTERT protein expression in subcellular fractions. The -124 and -146 mutations are located in the major 5-12 G-quadruplex and result in misfolding of the silencer element, thus causing over-expression of hTERT. Using a diverse small molecule library, we identified a small drug-like pharmacological chaperone (pharmacoperone) molecule, TG-4260, which binds to the 26 mer base-pair heteroduplex loop, which is the nucleation site for cooperative folding of the major 5-12 G-quadruplex. The chaperone effect of TG-4260 corrects DNA hTERT G-quadruplex misfolding resulting from the promoter mutations and restores the silencer function of the G-quadruplex. TG-4260 directly decreases the transcription activity of the −124, −124/125, −138/139, and −146 mutants to a similar extent and suppresses the downstream gene BCL2, which activates caspase-3 and produces cell-cycle arrest, leading to cell death. This is the first example of the use of a pharmacoperone molecule to correct the misfolding of a DNA G-quadruplex element resulting from mutations in an early folding intermediate. We screened GBM cell models against this novel small molecule inhibitor that interferes with mutated hTERT promoter and demonstrated that TG-4260 selectively suppresses glioma cell viability without affecting non-transformed normal human astrocytes. Finally, telomere phenotypes from treated cells indicate non-canonical functions of telomerase may also contribute to glioma pathogenesis.

#4799

Nef-M1 peptide inhibits CXCR4 driven Wnt/β-catenin signaling in breast cancer.

Venkat R. Katkoori,1 Upender Manne,2 Harvey L. Bumpers1. 1 _Michigan State Univ., East Lansing, MI;_ 2 _University of Alabama at Birmingham, Birmingham, AL_.

Introduction: The Nef-M1 peptide (Nef-M1) competes effectively with the natural ligand of CXCR4, SDF-1α, and exhibits anti-tumorigenic activity in breast cancer (BC). However, the molecular mechanisms of action of Nef-M1 on BC remain unclear. In this study, we evaluated the Nef-M1 inhibition of CXCR4 driven Wnt/β-catenin pathway in BC. Experimental Design: Cell line derived xenografts (CDX), patient derived explant (PDE) and in vitro cell based models were used to examine the mechanisms of action of Nef-M1. The severe combined immunodeficient mice with tumor were treated intraperitoneally either with Nef-M1 or sNef-M1 (control). Preparation of BC tissue, explant complete media, and cultures and treatment for 48 hours was performed for the PDE study. Sections from tumors were evaluated by immunostaining (IHC) for signaling proteins implicating BC progression. Western blot (WB) analyses were also performed on lysates of both cell lines and tumors to assess the effect of Nef-M1 on the Wnt/β-catenin, stem cell or mitogen-activated protein kinase (MAPK) pathways. Results: The present study revealed that PDE recapitulate multiple biological features of the disease and these were found to be very similar to the corresponding original tumors. IHC analysis for Ub-H2B, a stem cell signaling protein that correlates with advanced disease and metastasis indicated that Nef-M1 treated CDX or PDE tumors had low expression of Ub-H2B. However, sNef-M1 treated tumors had high expression of Ub-H2B. WB analyses indicated that Nef-M1 not only suppressed the expression of Ub-H2B, but also significantly suppressed the activation of MAPK (e.g. p38, ERK) and Akt proteins in BC. Nef-M1 treated BC displayed inhibition of Wnt/β-catenin signaling as demonstrated by an increased expression of p21 and decreased expression of beta-catenin, cyclin D1, Axin 2 and survivin. Cells expressing CXCR4 became susceptible to Nef-M1-induced inhibition of MAPK, Akt, Wnt/beta-catenin and stem cell signaling pathways. Conclusions: These results indicate that Nef-M1 suppresses CXCR4 mediated activation of Wnt/β-catenin and MAPK pathways. Therefore, Nef-M1 inhibition of these signaling pathways may be a promising therapeutic strategy for BC. This work was supported by NIH/NCI Workforce Diversity Grant R21-CA171251.

#4800

Targeting the polyamine pathway as a novel therapeutic treatment against diffuse intrinsic pontine glioma.

Aaminah Khan,1 Maria Tsoli,1 Denise Yu,1 Swapna Joshi,1 Laura Gamble,1 Laura Franshaw,1 Michelle Haber,1 David S. Ziegler2. 1 _Children's Cancer Institute, Sydney, Australia;_ 2 _Sydney Children's Hospital, Sydney, Australia_.

Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive paediatric brainstem tumour, with a peak incidence in middle childhood and a median survival of less than 1 year in the majority of cases. The dismal prognosis associated with DIPG is exacerbated by the repeated failure of over 250 clinical trials to improve survival over standard radiotherapy. Therefore novel and innovative therapeutic approaches are urgently needed to needed to treat this devastating disease. Polyamines are small intracellular polycations that control key aspects of cell biology, such as cell replication/growth, differentiation and survival and can be found up-regulated in many cancers. Difluoromethylornithine (DFMO) is an FDA-approved inhibitor of the enzyme ornithine decarboxylase (ODC1) which is involved in polyamine synthesis. The aim of this study was to investigate the efficacy of polyamine pathway inhibitors such as DFMO as a therapeutic strategy against DIPG. By RT-qPCR and western blotting, we observed high expression levels of key players of the polyamine pathway in a panel of DIPG samples. Through alamar blue cytotoxicity assays and soft-agar clonogenic assays, DFMO was found to be effective against a panel of neurosphere-forming DIPG cells. The activity of DFMO was synergistically enhanced when combined with a polyamine transport inhibitor, AMXT-1501. Increased apoptotic activity following either DFMO or AMXT-1501 treatment, alone or in combination, was observed by western blotting and flow cytometric analysis of Annexin V/7AAD stained cells. In addition, reduced uptake of the radiolabelled polyamine, spermidine, in the presence of AMXT-1501 confirmed polyamine transport inhibition in DIPG cells. Consistent with the in vitro results, the combination of DFMO and AMXT-1501 significantly prolonged the survival of mice bearing DIPG orthografts, and the combination therapy represents the most effective combination treatment we have tested to date in our DIPG animal model. Our results suggest that the polyamine pathway is a potential therapeutic target for this highly aggressive paediatric brain tumour while a number of clinical trials based on this approach are currently underway in a variety of malignancies.

#4801

Potential of mebendazole as an anti-tumor agent for adenoid cystic carcinoma and other rare cancers.

Jamie Dempsey Barber, Mahta Samizadeh, Nannan Jia, William Siders, Johanne Kaplan. _Shepherd Therapeutics, Natick, MA_.

Adenoid Cystic Carcinoma (ACC) is a rare adenocarcinoma arising most commonly in the major and minor salivary glands of the head and neck. Despite a deepening understanding of the molecular events that lead to sustained tumor growth, there are currently no approved therapies for ACC or many other rare cancers. Repurposing of drugs for use in oncology is of increasing interest to promote the rapid development of new therapies and address the significant unmet medical need that remains for rare cancers. The FDA-approved anti-helminthic drug mebendazole (MBZ) has been identified as an agent that can affect several cancer relevant pathways involved in tumor growth and metastasis. Anti-tumor effects of MBZ include G2/M cell cycle arrest leading to apoptosis, inhibition of the IGF1R/AKT pathway, and induction of MYB degradation by the proteasome. Based on this biology, several rare cancer tumor cell lines including neuroblastoma, rhabdomyosarcoma and Ewing sarcoma, were analyzed for sensitivity to MBZ treatment in vitro. Cultured cells were exposed to increasing concentrations of MBZ and viability was measured after 72hours. Treatment with MBZ resulted in a significant decrease in cell viability with IC50s ranging from 0.05-1 μM suggesting that MBZ may represent a potent anti-cancer agent for several rare cancer indications. Identification of therapies for ACC has been hampered by the lack of ACC cell lines and transgenic mouse models. To circumvent these issues, in collaboration with the ACC Research Foundation, the activity of MBZ was tested in several ACC patient-derived xenograft (PDX) models. Mice were implanted subcutaneously with PDX tumors and treatment was initiated when tumors reached a size of 125-300 mm3. Mice were randomized into three treatment groups to receive daily treatment with vehicle or MBZ at either 50 or 200 mg/kg for the duration of the study. Daily oral dosing with MBZ was well tolerated with no overt toxicities. Significant anti-tumor activity was observed in 2 out of 3 ACC PDX models. In the most aggressive ACCX9 PDX model, MBZ showed statistically significant anti-tumor activity at the highest dose of 200 mg/kg resulting in a significant increase in median survival compared to vehicle-treated mice (38 vs 29.5 days respectively, p=0.0013). Inhibition of tumor growth was also observed in the less aggressive ACCX6 PDX model at both the 50 and 200 mg/kg dose levels compared to vehicle and was accompanied by an increase in median survival (95.5 and 64.5 days vs 42 days respectively; p=0.0085 and p=0.083). In the reportedly more refractory ACCX5M1 PDX model, treatment with MBZ did not inhibit tumor growth. Overall, the in vitro and in vivo results suggest that MBZ potentially represents a novel therapeutic option for the treatment of rare cancers including ACC.

### Combination Chemotherapy 1

#4802

Combination therapy in PDAC involving blockade of the APE1/Ref-1 signaling pathway: An investigation into drug synthetic lethality and anti-neuropathy therapeutic approach.

Fenil L. Shah,1 Nadia Atallah,2 Michelle Grimard,1 Chunlu Guo,1 Chi Zhang,1 Jill Fehrenbacher,1 Mark R. Kelley,1 Melissa Fishel1. 1 _Indiana Univ. School of Medicine, Indianapolis, IN;_ 2 _Purdue University Center for Cancer Research, West Lafayette, IN_.

Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related mortality in the US. Most patients present with advanced disease and ~93% die within five years, with most surviving less than six months. Combination therapies including Gemcitabine (GemzarTM) and sustained release, nab-paclitaxel (AbraxaneTM) and FOLFIRINOX (5-FU/leucovorin/irinotecan/oxaliplatin) offer modest improvement in survival, albeit at an increase in side effects including chemotherapy-induced peripheral neuropathy. Data is presented on Apurinic/apyrimidinic endonuclease/redox factor-1 (APE1/Ref-1 or APE1) and redox-specific APE1 inhibitor, APX3330 and its effects on tumor cell growth and sensory neuron function.

APE1 is a multifunctional protein involved in repairing DNA damage via endonuclease activity and in redox regulation of transcription factors such as HIF-1α, NFkB and STAT3. High expression levels of APE1 indicate decreased survival in PDAC as well as other cancers. Because APE1 is essential for cell viability, generation of APE1 knockout cell lines and determining a comprehensive list of genes regulated by APE1 has been difficult.

To circumvent this, we performed single cell RNA-Sequencing on PDAC cells following APE1 knockdown under normoxia and hypoxia to identify differentially expressed genes and further explore APE1's effects on HIF-1α and STAT3 signaling under both conditions. Proteomic analysis on PDAC cells following APE1 knockdown in normoxia and hypoxia revealed changes in signaling downstream of APE1, complementing the transcriptomic data and providing a more complete understanding of pathways affected by APE1.

We used the newly identified APE1 targets and pathways along with drug sensitivity data of cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE) to generate potential combination therapies of FDA approved drugs and the APE1 redox inhibitor, APX3330 and next generation analogs. These combinations were tested using an ex vivo 3D tumor-stroma model system using patient derived cells from the tumor as well as cancer-associated fibroblasts. We identified synergy with agents such as Napabucasin and Entinostat.

We also tested APX3330 in combination with drugs that are part of PDAC standard of care. In vivo studies combining APX3330 with Gemcitabine showed significantly decreased tumor volume. Combining oxaliplatin (part of FOLFIRINOX) with APX3330 caused a significant reduction in oxaliplatin-induced DNA damage in sensory neurons from a KPC orthotopic graft model, without hindering its anti-cancer activity. With the phase I clinical trial for APX3330 underway (IND 125360), the potential for APE1 targeted therapy enhancing tumor efficacy while providing neuroprotective effects in the sensory neurons provides a win-win scenario.

#4803

CXCR6-directed therapeutic approach potentiates efficacy of docetaxel in prostate cancer.

Neeraj Kapur,1 Hina Mir,1 Guru Sonpavde,2 James W. Lillard,1 Shailesh Singh1. 1 _Morehouse School of Medicine, Atlanta, GA;_ 2 _Dana Farber Cancer Institute, Harvard Medical School, Boston, MA_.

Development of chemo-resistance negatively impacts therapeutic outcome of advanced prostate cancer (PCa). Molecular mechanism used by cancer cells to overcome therapeutic effect is still undefined. Here, we demonstrate the influence of CXCR6/CXCL16 mediated molecular signals on the efficacy of docetaxel (DTX). Out data showed poor DTX efficacy on PCa cells (LNCaP, PC3, and DU145) after stimulating CXCR6 with soluble CXCL16, while significant improvement in cytotoxic effect of DTX was observed after CXCR6 blockade using anti-CXCR6 monoclonal antibody. Interestingly, CXCR6, CXCL16, and ADAM-10, a metalloproteinase involved in CXCL16 cleavage from PCa cell membranes, were significantly elevated in response to DTX treatment. This was further confirmed on xenograft tumor by immunohistochemistry. Further, phospho-proteomic profiling and western blot analysis showed activation of pro-survival molecules: GSK-3β, NFκB, ERK/1/2, and survivin following soluble CXCL16 treatment. Activation of CXCR6-CXCL16 axis also inhibited expression of the epithelial marker (E-cadherin) while increased expression of mesenchymal markers (β-catenin, α-SMA, vimentin, snail), which highlights the role of CXCR6 in promoting mesenchymal phenotype that is known to have a significant impact on cancer progression and therapeutic outcome. Thus, our study shows that CXCR6-CXCL16 signaling instigates chemotherapeutic resistance by increasing pro-survival signaling and promoting mesenchymal phenotype. Hence, combining CXCR6 blockade with DTX would be a better treatment option for advanced, drug-resistant PCa.

#4804

Multitargeted kinase inhibition (AEE788) alleviates mTOR inhibitor drug resistance in triple-negative breast cancer.

Jichao He, Ronan P. McLaughlin, Vera van der Noord, Gerard van Westen, Yinghui Zhang, Bob van de Water. _Leiden University, Leiden, Netherlands_.

Triple negative breast cancer (TNBC), although accounting for 15-20% of breast cancer, is disproportionally responsible for female cancer death. Despite therapeutic strategies being advanced, the clinical outcome of mTOR-targeted chemotherapy remains poor in the past decades. Recent data have evidenced the interdependence of kinase signaling networks and their involvement in treatment refractory TNBC settings. Thus, a combinatorial therapy targeting multiple components of signaling cascade is urgently needed. We assessed drug response phenotype across 20 TNBC cell lines by profiling the effect of a kinase inhibitor library consisting of >350 different kinase inhibitors (KIs). A panel of TNBC cell lines clustered with resistance phenotype against rapalogues (rapamycin analogues). Rapamycin efficacy on TNBCs was detected by performing proliferation assays and calculating their IC50, respectively. Given the role of the mTOR pathway in translational control, the relationship between rapamycin responses (IC50) and translational factors expression in TNBC cell lines was studied by Pearson analysis. Notably, translation initiation factor EIF2B2, EIF2S3, EIF3S8 and EIF1AX positively correlated with rapamycin response, whereas EIF4EBP2 and EIF2AK2 displayed negative correlation. Next, a rapamycin combination KI library screen was performed to identify kinase inhibitors synergizing with mTOR inhibition. AEE788, a multiple kinase targeting inhibitor, synergistically enhanced inhibitory effect of rapamycin on TNBC cell proliferation. Moreover, AEE788 dose-dependently synergized with mTOR targeted therapy in several TNBC cell lines. The combination treatment significantly decreased phosphorylation level of mTOR and 4EBP1 as well as ERK, indicating downregulation of PI3K/AKT and MAPK pathways. ChEMBL-based cheminformatics analysis indicated that, besides VEGFR and EGFR, a number of other kinases were predicted as the targets of AEE788, including RPS6K1, AKT2 and CDK7. Altogether, our data reveals the essential role of rapamycin-associated translation factors in TNBC, and provides insight in the resistance of TNBC to rapamycin analogues as well as potential therapeutic approaches to alleviate rapamycin resistance.

#4805

Plinabulin ameliorates chemotherapy-induced neutropenia: Mechanistic insights.

Amlan K. Ghosh,1 Yanfang P. Zhu,1 Jacqueline F. Miller,2 Catherine C. Hedrick,1 Ramon Mohanlal,3 Huang Lan,3 George K. Lloyd,3 Klaus Ley1. 1 _La Jolla Institute of Allergy and Immunology, La Jolla, CA;_ 2 _La Jolla Institute of Allergy and Immunology, LaJolla, CA;_ 3 _BeyondSpring Pharmaceuticals, New York, NY_.

Chemotherapy-induced Neutropenia (CIN) increases the risk of serious infections, sepsis, hospitalizations and mortality, in patients receiving chemotherapy. CIN is currently being treated with G-CSF, which has significant limitations; G-CSF cannot be administered in the first 24 hours after chemotherapy, and its use is associated with bone pain in the majority of patients. There is a need for new therapies with an improved profile. Plinabulin is an inhibitor of tubulin polymerization, which is currently in Phase 3 clinical trials for the treatment of non-small cell lung canacer (NSCLC) in combination with docetaxel. In a previous Phase 2 trial plinabulin significantly ameliorated CIN in patients undergoing docetaxel chemotherapy and this is currently prospectively being evaluated in a series of Phase 2/3 trials. However, the mechanisms by which plinabulin ameliorates neutropenia are unknown. We investigated the role of plinabulin in the regulation of neutrophil homeostasis under chemotherapy-induced neutropenia in mice. We tested the effect of the novel small molecule plinabulin on blood and bone marrow neutrophils and their precursors using complete differential blood counts (CBC) by electrozoning analyzer and flow cytometry of blood and bone marrow. Neutropenia was induced by single injection of the chemotherapeutic agent docetaxel (25 mg/kg, iv.), with and without plinabulin (10 or 20 mg/kg, ip, 12 mice each group) one hour later. Blood was collected on days 3, 4 and 5 post-injection from the retro-orbital plexus and analyzed for CBC. After 5 days of treatment, bone marrow was harvested and analyzed by flow cytometry for the percentage of neutrophils and their progenitors. Blood Neutrophil counts were significantly decreased in mice as a result of docetaxel treatment. Plinabulin ameliorated the docetaxel-induced neutropenia at day 5 (p<0.05) versus the docetaxel-treated mice. Bone marrow neutrophil counts were decreased by docetaxel monotherapy, but this was significantly prevented when plinabulin was given with docetaxel, (p<0.001 for plinabulin 10 mg/kg; p<0.01 for plinabulin 20 mg/kg). The docetaxel-induced accumulation of LSK cells (uncommitted hematopoietic stem cells) was significantly decreased by adding plinabulin to docetaxel-treated mice. The plinabulin effect was neutrophil-specific, because plinabulin had no effect on monocytes and monocyte precursors in the bone marrow. Collectively, these data suggest that plinabulin ameliorates chemotherapy-induced neutropenia by promoting the transition of LSK to common myeloid precursors in mice. This represents a novel mechanism for the prevention of chemotherapy-induced neutropenia.

#4806

Exploring optimal targeted combination therapies with neratinib for HER2 mutated NSCLC.

Mari Kuraguchi,1 Luke J. Taus,1 Shan Zhou,1 Francesca Avogadri-Connors,2 Richard E. Cutler,2 Alshad S. Lalani,2 Paul Kirschmeier,1 Pasi A. Jänne1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Puma Biotechnology, Inc., Los Angeles, CA_.

Genetic alterations which constitutively activate human epidermal growth factor receptor 2 (HER2; ERBB2) without overexpression have been observed in patients with non-small cell lung cancer (NSCLC) yet currently there are no effective targeted therapies for such patients. Neratinib is an orally available, quinazoline-based, irreversible pan-HER tyrosine kinase inhibitor. Prior clinical studies have demonstrated that neratinib alone or in combination with temsirolimus has low response rates in patients with HER2 mutated NSCLC. Since dual HER2-tageting is showing promising activity in breast cancer, we hypothesized that combination of neratinib with other HER2-targeted agents, such as trastuzumab and ado-trastuzumab emtansine (T-DM1), may provide better anti-tumor efficacy compared to neratinib alone. To address this, we have developed, under an IRB approved protocol, two NSCLC patient-derived xenograft (PDX) models bearing HER2 kinase domain mutations, DFCI-315 (ERBB2 P780_Y781insGSP) and DFCI-359 (ERBB2 755_757LREdelinsPR). These PDX lines were characterized by different tumor kinetics and doubling times of approximately 12 and 52 days, respectively. IHC analysis confirmed the expression, but not overexpression, of HER2 with H-scores of 70.0 and 54.2, respectively. Treatment with single agent neratinib (P.O. QD x 28 days, 40 mg/kg) was effective in both HER2 mutant lung models, leading to a significant tumor growth inhibition (TGI) with tumor stasis in DFCI-315 (p<0.0001) and tumor regression (TR) of >20% in DFCI-359 (p<0.05). DFCI-315 was insensitive to monotherapy with trastuzumab (I.P. 2x/week x 4 weeks, 20 mg/kg) but responded to monotherapy with T-DM1 (I.V. QWK x 4 weeks, 3 mg/kg) resulting in about 50% TGI (p<0.005). Both agents in combination with neratinib showed additional anti-tumor activity leading to TR in the DFCI-315 model (p<0.0001 vs vehicle), though difference from neratinib alone was not significant. In contrast, DFCI-359 model responded dramatically to neratinib plus trastuzumab combination starting from Day 5 of dosing, reaching TR of 62% by 3 weeks, compared to 25-30% TR by either of single agents (p<0.0001 vs vehicle, p<0.05 vs neratinib or trastuzumab). Tumor growth stasis following the combination treatment was durable for at least 10 days after the drug withdrawal, while the tumors regrew immediately after the drug withdrawal following neratinib treatment alone. Surprisingly, T-DM1 had no anti-tumor activity in this model and its combination with neratinib was indifferent from neratinib alone. Taken together, these data demonstrate neratinib has efficacy as single agent in preclinical models of HER2 mutant NSCLC and its anti-tumor activity was enhanced when combined with trastuzumab. Neratinib plus trastuzumab is a promising combination treatment for HER2 mutant lung cancer and is currently under clinical investigation (ClinicalTrials.gov NCT01953926).

#4807

Novel theranostic strategy against peritoneal metastasis of scirrhous gastric cancer: Combination with fluorescence oncolytic adenovirus and chemotherapy.

Wataru Ishikawa,1 Satoru Kikuchi,1 Hiroshi Tazawa,1 Toshiaki Ohara,1 Shinji Kuroda,1 Kazuhiro Noma,1 Hiroyuki Kishimoto,1 Masahiko Nishizaki,1 Yasuo Urata,2 Shunsuke Kagawa,1 Toshiyoshi Fujiwara1. 1 _Okayama University, Okayama, Japan;_ 2 _Oncolys BioPharma, Inc, Tokyo, Japan_.

Background: Scirrhous gastric cancer (SGC), which is characterized by poorly differentiated tumor cells that diffusely infiltrate the gastric wall, accounts for 10% of all gastric cancers, and often causes peritoneal metastasis. The prognosis of SGC patients is very poor, because no effective treatment to overcome peritoneal metastasis of SGC has been developed yet. In recent years, intraperitoneal (i.p.) administration of paclitaxel (PTX) has been focused against various cancers with peritoneal metastasis and has been also attempted to SGC, but regrettably its effect is not satisfactory. Therefore, a novel therapeutic strategy is required for the treatment of SGC with peritoneal metastasis.

Methods: We previously developed a telomerase-specific replication-competent adenovirus, OBP-401 (TelomeScan), which can replicate only within the tumor cells selectively and express green fluorescent protein (GFP). OBP-401 infection also induces tumor-specific cell death in monotherapy and combination therapy with chemotherapy. In this study, we assessed the in vitro and in vivo antitumor effects of combination therapy with OBP-401 and chemotherapeutic agent, paclitaxel (PTX), in human SGC cell lines (GCIY and KATO III) and SGC xenograft peritoneal metastasis model, respectively. The molecular mechanism underlying synergistic effect of the combination therapy was evaluated in aspects of cell death and replication efficiency of OBP-401.

Results: SGC cells were visualized as GFP-positive cells selectively by the infection of OBP-401, whereas normal fibroblast cells were not. OBP-401 synergistically suppressed the viability of human SGC cells in combination with PTX. OBP-401 monotherapy dose-dependently induced apoptosis and autophagy, although PTX monotherapy induced only apoptosis. The combination therapy induced autophagy at a lower concentration of OBP-401. In the time-lapse imaging of treatment, the combination therapy enhanced the replication efficiency of OBP-401 in tumor cells. Moreover, the combination therapy with i.p. administration of OBP-401 and PTX also significantly inhibited the tumor growth of peritoneal metastasis, as compared to monotherapy.

Conclusion: These results suggest that OBP-401 has a promising potential to detect peritoneal micrometastasis of SGC intraoperatively and the combination therapy with i.p. induction of OBP-401 and PTX would be a novel theranostic strategy for the treatment of SGC with peritoneal metastasis.

#4808

Combinations of imatinib mesylate with AKT inhibitor (miransertib, ARQ 751) or FGFR inhibitor (derazantinib) show synergy in GIST cell lines and preclinical models.

Marya Kozinova,1 Shalina Joshi,1 Karthik Devarajan,1 Phillip Zook,1 Jimson W. D'Souza,1 Jeffrey M. Farma,1 Nestor Esnaola,1 Reza Foroughi,2 Yi Yu,2 Brian Schwartz,2 Terence Hall,2 Margaret von Mehren,1 Lori A. Rink1. 1 _Fox Chase Cancer Ctr., Philadelphia, PA;_ 2 _ArQule Inc, Burlington, MA_.

The majority of gastrointestinal stromal tumors (GIST) harbor oncogenic mutations in the receptor tyrosine kinase KIT or platelet-derived growth factor receptor alpha (PDGFRA). Small molecule kinase inhibitors such as imatinib mesylate (IM) have significantly improved the clinical management of GIST by targeting these mutant receptors. Despite strong overall response rates to IM, disease progression generally occurs with time. Inhibiting targets other than, or in addition to, KIT/PDGFRA may provide additional therapeutic benefit in GIST patients. Both AKT and FGFR signaling have recently been reported to be resistance mechanisms associated with survival of IM-resistant GIST cell lines and tumors. In this study, we performed in vitro and in vivo experiments to assess the potential benefit of combining IM with the ArQule AKT inhibitors Miransertib (ARQ 092) and ARQ 751 and the FGFR inhibitor, Derazantinib (ARQ 087). To evaluate in vitro drug sensitivity, a panel of IM-sensitive (GIST-T1, GIST882) and resistant GIST cell lines (GIST-T1/829, GIST430) were subjected to drug treatment for 72 hours before measuring viability with the Cell Titer Blue Viability Assay. Synergy between IM and Miransertib, ARQ 751 and Derazantinib was quantified using the Chou-Talalay algorithm to calculate CI values. CI values <1 are considered synergistic. The 3:1 molar ratio of the three tested combinations demonstrated synergistic CI values in all four GIST lines. Immunoblot assays confirmed that drugs inhibited their intended targets in each cell line following six-hour drug treatment. Interestingly, a significant decrease in the activation of a downstream signaling protein, p-S6, was observed in the AKTi+ IM -treated cells compared to cells treated with single agents. In vivo studies evaluating Miransertib, ARQ 751 and Derazantinib as monotherapies and in combination with IM were performed using GIST-T1 and GIST430 xenograft models, as well, as an IM-resistant, KIT exon 9-mutated GIST PDX model. In both IM-resistant GIST models, all three monotherapies (Miransertib, ARQ 751, Derazantinib) significantly inhibited tumor growth as compared to IM alone. In addition, combination therapy, with both AKTi and FGFRi, provided significantly greater efficacy in both IM-sensitive and resistant xenograft models of GIST. Together, these studies demonstrate that IM in combination with the novel ArQule AKT inhibitors (Miransertib and ARQ 751) and FGFR inhibitor (Derazantinib) provide significantly improved efficacy compared to monotherapy in the tested models. These results provide justification for development of translational trials evaluating these combinations in GIST patients.

#4809

Bardoxolone-methyl suppresses both androgen receptor and its splice-variant ARv7 in prostate cancer cells to enhance the anti-cancer efficacy of enzalutamide.

Namrata Khurana, Partha Chandra, Hogyoung Kim, Asim B Abdel-Mageed, Suresh Sikka, Debasis Mondal. _Tulane University, New Orleans, LA_.

Prostate cancer (PCa) is a leading cause of morbidity and mortality in elderly men in the United States. Despite androgen deprivation therapy (ADT), the recurrence of castration resistant prostate cancer (CRPC) remains a significant challenge. Augmented signaling via the full-length androgen receptor (AR) and the constitutively active AR splice variants, especially ARv7, is associated with ADT-resistance. Therefore, a proper understanding of augmented AR signaling and adjunct strategies to suppress both AR and ARv7 levels are critically needed. ADT-induced oxidative stress (Ox-stress) can facilitate CRPC outgrowth. We previously documented that the Ox-stress induced transcription factor, Nrf2 can suppress AR expression and AR-transactivation function in PCa cells. Recently, we also showed that the Nrf2-inducing natural compound, sulforaphane (SFN) can decrease both AR and ARv7 levels in PCa cells, and co-exposure to SFN enhanced the efficacy of anti-androgens. In this study, we examined whether a synthetic triterpenoid drug, Bardoxolone-methyl (CDDO-me), a potent enhancer of Nrf2, can similarly decrease AR and ARv7 expression. Exposure to nanomolar (nM) concentrations of CDDO-me rapidly downregulated AR levels in the androgen-dependent LNCaP cells and androgen-independent C4-2B cells. Exposure to CDDO-me also suppressed both AR and ARv7 levels in the CRPC line, CWR22Rv1. A biphasic effect of CDDO-me on reactive oxygen species (ROS) levels was observed. Pre-exposure to the antioxidant, n-acetyl cysteine (NAC) blocked the AR-suppressive effect of CDDO-me. Molecular mechanistic studies using actinomycin-D (transcription inhibitor), cycloheximide (translation inhibitor), and MG132 (proteasomal inhibitor) suggested that CDDO-me may decrease AR gene expression and increase degradation of AR and ARv7 proteins. Furthermore, exposure to CDDO-me increased proteasomal activity in PCa cells and suppressed the levels of several AR splicing factors, i.e. the heteronuclear ribonucleoproteins A1 and H1 (hnRNP-A1 and hnRNP-H1). Co-exposure to CDDO-me increased the anti-cancer efficacy of enzalutamide (ENZ) as evident by decreased cell-viability (MTT), migration (scratch-wound) and colony forming unit (CFU) assays. Therefore, adjunct treatment with low-dose CDDO-me may be an effective strategy to suppress AR and ARv7 levels and augment the efficacy of anti-androgens. Most importantly, since CDDO-me is in late-stage clinical trials for chronic kidney disease (CKD), its repositioning as a potent AR suppressive agent may be of significant translational value in PCa patients undergoing ADT.

#4810

Targeting androgen receptor in combination with cisplatin: Effective treatment strategy for muscle invasive bladder cancer.

Ashish Tyagi, Balaji Chandersekaran, Samarpit Rai, Houda Alatassi, Ahmed Q. Haddad, Murali K. Ankem, Chendil Damodaran. _University of Louisville, Louisville, KY_.

Background: Bladder cancer is one of the major causes of cancer death in US and worldwide. Cisplatin is a key component of chemotherapeutic regimens employed in the treatment of advanced bladder cancer. The role of androgen and androgen receptor (AR) signaling in bladder cancer remains uncharacterized.

Aim: The aim of the study is to delineate the role of AR in bladder cancer and to determine whether combination of AR inhibitor, enzalutamide (Enz) and cisplatin-based therapies effectively inhibit the growth of muscle invasive bladder cancer (MIBC).

Methods: AR expression was determined in 75 human bladder cancer specimens and in a panel of bladder cancer cell lines. Cells grown in charcoal stripped media supplemented with dihydrotestosterone (DHT) were treated with cisplatin, enzalutamide (AR inhibitor), or a combination of both. Cellular/phenotypic analysis including MTT assay, apoptotic assay, migration as well as invasion assays and molecular analysis including western blotting, real time PCR analysis were performed. Isobologram analysis for the combination was performed and analyzed with CompuSyn. Experiments were repeated in triplicates and analyzed with unpaired Student's t-test and one way ANOVA *p≤0.05, **p≤0.01, ***p≤0.001.

Results: AR expression was seen in around 40% of bladder cancer patients. Inhibition of AR signaling by enzalutamide effectively inhibited the growth of AR+ MIBC cells. Interestingly, enzalutamide in combination with cisplatin (Enz + Cis) synergistically inhibited the proliferation of MIBC cells, TCCSUP (CI: 0.42, 1.25 + 5 μM) and J82 (CI: 0.79, 2.5 + 5 μM) at low concentrations of enzalutamide and cisplatin resp.. The molecular studies revealed the induction of DNA damage markers (pATM, pATR, pChk1, pHis) and enhanced expression of the pro-apoptotic genes (Bax, caspases-3 and PARP) in Enz+Cis treated AR+ MIBC cells. In addition, we demonstrated abrogation of invasive and migratory potential with Enz+Cis treatment, by downregulation of the mesenchymal markers (N-cadherin, slug, β-catenin, and vimentin) in both cell lines. Our studies suggest combination of Enz + Cis may be effective in patients with AR+ MIBC.

Conclusion: Combination of cisplatin and AR inhibition effectively inhibit bladder tumor growth and migration, and hold promise as synergetic therapies for AR+ bladder cancer patients.

#4811

Inhibition of PLK1 abrogates side population and increases radiation-induced DNA damage in human glioblastoma.

Arvind Pandey,1 Junhua Mai,1 Satyendra C. Tripathi,2 Samir M. Hanash,2 Haifa Shen,1 Sankar Mitra1. 1 _Houston Methodist Research Institute, Hoston, TX;_ 2 _MD Anderson Cancer Center, Hoston, TX_.

Glioblastoma multiforme (GBM), a highly malignant form of brain tumor has limited treatment modalities with poor patient survival. Expression of CD133, a marker of cancer stem cells (CSCs), has been linked to GBM recurrence, metastasis and radiation/drug resistance. The GBM CSCs exhibit high level of ABC transporters, DNA repair enzymes and anti-apoptotic proteins. The side population (SP) phenotype of CSCs with high levels of ABC transporters is associated with chemo-resistance. Overexpression of Polo-like kinase-1 (PLK1), a key cell cycle regulator, has been linked to poor prognosis of various cancers. However, the role of PLK1 in radio/chemo-resistance of GBM is poorly understood. In this study, we explored the use of PLK1 inhibitor Volasertib in combination with temozolomide (TMZ) followed by radiation to overcome resistance in GBM, both in vitro and in vivo.

Our results revealed that Volasertib and TMZ act synergistically at a sub-lethal dose to inhibit cell proliferation and induce apoptosis. Both Volasertib and TMZ strongly induce reactive oxygen species (ROS) which may cause cell death by reducing repair capacity. Combined treatment with TMZ and Volasertib followed by radiation showed persistent DNA damage after removal of drugs for 24 hrs. In contrast to earlier reports, significant decrease in the side population was also observed, even after radiation treatment.

We also observed that Volasertib alone or in combination with TMZ activated STAT1 pathways and inhibited MAPK/AKT activation. As the latter pathway promotes apoptosis, our results suggest higher tumor growth inhibitory effect of combinatorial treatment compared to single agents in xenograft mouse model.

In summary, while Volasertib can be used alone or in the combination with radiation, Volasertib combined with TMZ may be useful to inhibit recurrence of GBM after radiation therapy. Furthermore, increased STAT1 expression may modulate the tumor-microenvironment and improve immunotherapy by MHC class 1 upregulation.

#4812

Vorinostat and fenretinide synergize in preclinical models of T-cell lymphoid malignancies via reactive oxygen species.

Monish Ram Makena,1 Thinh H. Nguyen,2 Balakrishna Koneru,2 Ashly Hindle,2 Wan-Hsi Chen,2 Dattesh U. Verlekar,2 Min H. Kang,2 C. Patrick Reynolds2. 1 _Johns Hopkins Medical School, Baltimore, MD;_ 2 _Texas Tech University Health Sciences Center School of Medicine, Lubbock, TX_.

Introduction: T-cell lymphoid malignancies (TCLMs) are in need of novel and more effective therapies. Pan-histone deacetylase (HDAC) inhibitors vorinostat and belinostat, and class I specific HDAC inhibitor romidepsin have achieved FDA registration as 2nd line therapies for peripheral and/or cutaneous T-cell lymphomas. The cytotoxic retinoid fenretinide achieved durable complete responses against T-cell lymphomas in early-phase clinical trials and T-cell lymphoma patients who failed prior HDAC inhibitor treatment responded to fenretinide (Clinical Cancer Res 23:4550-4555, 2017). Fenretinide is currently being evaluated in a Phase IIa clinical trial for relapsed/refractory PTCL patients (NCT02495415). We have previously shown romidepsin and fenretinide synergize in preclinical models of T-cell lymphoid malignancies (Molecular Cancer Therapeutics 16:649-661, 2017). There exist some key differences in the activity of various classes of HDAC's, which have significantly different chemical structures and metabolic profiles. Therefore we determined if the pan-HDAC inhibitor vorinostat synergizes with fenretinide.

Methods and Results: Using the DIMSCAN cytotoxicity assay, we demonstrated cytotoxic synergy between vorinostat and fenretinide in nine TCLM cell lines at clinically-achievable concentrations that lacked cytotoxicity for non-malignant cells (fibroblasts and blood mononuclear cells). In vivo, vorinostat + fenretinide + ketoconazole (enhances fenretinide exposures by inhibiting fenretinide metabolism) showed greater activity in subcutaneous (COG-LL-317m and TX-LY-183x PDX) TCLM xenograft models than single agent vorinostat or fenretinide + ketoconazole. Fenretinide + vorinostat caused a reactive oxygen species (ROS, measured by DCFDA dye)-dependent increase in apoptosis (via TUNEL assay), and histone acetylation (measured by immunoblotting). The synergistic cytotoxicity, apoptosis, and histone acetylation of fenretinide + vorinostat was abrogated by antioxidant vitamin C. Vorinostat + fenretinide activated p38 and JNK via ROS, and shRNA knockdown of p38 and JNK1 significantly decreased the synergistic cytotoxicity and apoptosis. Vorinostat + fenretinide also showed synergistic cytotoxicity for six B-lymphoid malignancy cell lines.

Conclusion: Like romidepsin, vorinostat combined with fenretinide achieved synergistic activity in preclinical models of TCLMs, but not in non-malignant cells, via a novel molecular mechanism. As vorinostat is an oral agent and not a PGP substrate it may have advantages in such combination therapy. These data support conducting a clinical trial of vorinostat combined with fenretinide in relapsed and refractory TCLMs.

#4813

Enhancement of nab-paclitaxel response by inhibition of insulin-like growth factor (IGF) signaling in experimental esophageal adenocarcinoma.

Md Sazzad Hassan,1 Fiona Williams,2 Niranjan Awasthi,1 Margaret A. Schwarz,1 Roderich E. Schwarz,1 Urs von Holzen1. 1 _Indiana University School of Medicine-South Bend, South Bend, IN;_ 2 _University of Notre Dame, South Bend, IN_.

Introduction: Esophageal adenocarcinoma (EAC) is the fastest growing cancer in the western world and the overall 5 year survival rate of EAC is below 20 percent. Most patients with EAC present with locally advanced or widespread metastatic disease, where current treatment is largely ineffective. Therefore, new therapeutic approaches are urgently needed. Epidemiological studies have linked obesity with EAC. IGF signaling is an important mediator in obesity-associated EAC. Paclitaxel (PT) has been used in combination with carboplatin as a standard combination therapy for advanced EAC. PT required emulsification with solvents which has resulted in serious adverse effects in patients. Nanoparticle albumin-bound paclitaxel (nab-paclitaxel) is an albumin-stabilized, cremophor-free and water soluble nanoparticle formulation of PT. Nab-paclitaxel has recently shown greater efficacy over PT in EAC. Here we evaluated the improvement in nab-paclitaxel response by addition of BMS-754807, a small molecule inhibitor of IGF-1R/IR signaling, in experimental EAC.

Methods: We first evaluated the phosphorylation status of IGF-1R/IR protein by western blot in a panel of EAC cell lines. BMS-754807 and nab-paclitaxel, alone or in combination were tested for effects on cell growth detected by WST-1 assay and on cell apoptosis detected by western blot of cleavage of caspase 3 and PARP. We then explored the antitumor efficacy with survival advantage following BMS-754807 and nab-paclitaxel mono and combination therapies in murine subcutaneous xenograft and peritoneal metastatic survival models of human EAC.

Results: BMS-754807 dose dependently inhibited in-vitro cell proliferation of OE19 and FLO-1 EAC cell lines having strong tyrosine phosphorylation of IGF-1R/IR protein and interestingly the addition of IC25 dose of BMS-754807 significantly decreased the nab-paclitaxel IC50 in these EAC cells. In addition, co-administration of BMS-754807 and nab-paclitaxel effectively enhanced cleavage of caspase-3 and PARP in these EAC cells. In subcutaneous xenografts using OE19 cells, average net tumor growth after two weeks in different therapy groups was 558.67 mm3 in control, 208.47 mm3 after BMS-754807 (p=0.043), 104.60 mm3 after nab-paclitaxel (p=0.013), and 14.30 mm3 after BMS-754807 plus nab-paclitaxel (p=0.0005). In OE19 EAC survival model there was a significant increase in median animal survival after two weeks BMS-754807 plus nab-paclitaxel treatment (85 days) compared to control (47 days, p=0.0034), to BMS-754807 therapy (57 days, p=0.0021) or to nab-paclitaxel (68 days, p=0.0339) therapy.

Conclusion: Thus BMS-754807 with nab-paclitaxel treatment resulted in significantly higher antitumor efficacy and survival benefit. These results support the potential of BMS-754807 in combination with nab-paclitaxel as an effective option for EAC therapy.

#4814

Adaptation of uveal melanoma cells to MEK inhibition can be overcome through HDAC inhibition.

Fernanda Faião-Flores,1 Silvya S. Maria-Engler,2 Zeynep Eroglu,1 Keiran S. Smalley1. 1 _Moffitt Cancer Center & Research Institute, Tampa, FL; _2 _University of São Paulo, São Paulo, Brazil_.

Uveal melanoma is the most common adult cancer of the eye. Although uveal melanoma has a low incidence, it has a high capability to develop metastases, mainly to the liver. Mortality rates are high and new therapeutic strategies are urgently needed. 85-90% of all uveal melanomas harbor driver mutations in GNAQ or GNA11 leading to constitutive activation of many signaling pathways, including the MAPK pathway. Although MEK inhibitors have been evaluated clinically for metastatic uveal melanoma, responses are short-lived, and the mechanisms of adaptation are not well delineated. In the current study we performed mass spectrometry-based proteomics to define the adaptive response of uveal melanoma cells to MEK inhibition at a systems level with the hope of identifying the key escape pathways. These analyses identified MEK inhibition to lead to increased histone phosphorylation and histone deacetylase (HDAC) activity, as well as reorganization of the cytoskeleton associated with cortactin phosphorylation. MEK inhibition also increased receptor tyrosine kinase (RTK) signaling (through ROR1 and IGFIR) and enhanced AKT phosphorylation. Use of pan-HDAC inhibitors sensitized the uveal melanoma cells to MEK inhibition leading to enhanced apoptosis and decreased cell invasion. Mechanistic studies showed the MEK+HDAC inhibitor combination inhibited MAPK, PI3K/AKT and the Hippo signaling, an effect associated with increased cell death inhibition of cell invasion. Chronic MEK inhibitor treatment led to acquired resistance that was associated with increased RTK signaling and the adoption of an invasive phenotype. Studies are ongoing to identify the key HDAC isoform required for the adaptation to MEK inhibitor therapy as well as the anti-invasive potential of the cortactin knockdown with the goal of developing novel combination therapies that can be evaluated clinically.

#4815

Efficacy and toxicity of combinatorial therapy with EZH2 and androgen receptor inhibitor for castration-resistant prostate cancer.

Eswar Shankar,1 Daniel Franco,1 Omair Iqbal,1 Stephen Moreton,2 Rajnee Kanwal,1 Sanjay Gupta1. 1 _Case Western Reserve Univ., Cleveland, OH;_ 2 _Louis Stokes VA Medical Center, Cleveland, OH_.

Enhancer of zeste homolog 2 (EZH2), the enzymatic member of the polycomb repressor complex PRC2 has emerged as a key activator of androgen receptor in castration-resistant prostate cancer (CRPC). Androgen deprivation therapy (ADT) is a main therapeutic modality for advance-stage metastatic prostate cancer targeting AR. Recent studies shed light on the fact that overabundance of EZH2 in localized cancers increases aggressiveness and their recurrence. EZH2 activates AR through methylation-mediated gene suppression. Though prostate cancers respond to ADT treatment initially they subsequently re-emerge as CRPC. In recent years, Enzalutamide (ENZU), a second generation AR antagonist exhibits survival advantage in CRPC patients, but show relapse within a year, activating AR in these tumors. Recent genomic studies reveal AR-regulated genes contribute to CRPC emergence. This apparent association between EZH2 and AR in activating target genes by cooperative recruitment may lead to the development of chemoresistance. Therefore we rationalize that combination treatment targeting EZH2 and AR could be efficacious and significantly inhibit proliferation and metastasis of CRPC cells. Human prostate cancer C4-2B and 22Rv1, representative of CRPC tumors, were treated with EZH2 inhibitor GSK126 and AR antagonist (ENZU) individually and in combination, followed by assessment of cell viability, cell cycle analysis, migration, invasion and expression of various target genes by Western blotting. ELISA was employed to assess the change in activity of histone methylation in these cells. Treatment of C4-2B and 22Rv1 cells individually with GSK126 and ENZU (2.5-80 µM) for 24 h exhibited a partial suppressive effect in cell growth. Using GSK126 and ENZU combination at 1:1, 1:5, 1:10 and 1:20 micro-molar ratio exhibited increased cell growth inhibition, where 1:10 ratio showed synergistic effect in inhibiting cell growth in both cell lines. This combination caused marked increase in G0/G1-phase cell cycle arrest followed by cell death, inhibition of migration and invasion in both cell lines. Furthermore, combination treatment led to significant reduction in the protein expression of AR, AR-V7, EZH2, SUZ12, EED, p-Akt (Ser473) in both cell lines, compared to individual treatments. Combined treatment also caused significant decrease in the levels of H3K27 methylation and its activity. Taken together, our results demonstrate that simultaneous inhibition of AR and EZH2 is more efficacious in inhibiting the growth of CRPC cells. This opens new possibilities of uncovering a novel path of treating CRPC by simultaneously targeting EZH2 and AR using a combinatorial approach.

#4817

EP-100 sensitizes BRCA wild-type ovarian cancer cells to PARP inhibitor.

Shaolin Ma, Sunila Pradeep, Sherry Wu, Mark Seungwook Kim, Wei Hu, Selanere Mangala, Robert L. Coleman, Anil K. Sood. _MD Anderson, Houston, TX_.

EP-100 is a synthetic lytic peptide specific for targeting LHRH receptors on cancer cells that is being clinically tested for treatment of ovarian cancer. In this study, we aimed to identify combination approaches with EP-100 in ovarian cancer models. We carried out a series of in vitro (MTT assay, immunoblot analysis, Q-PCR, reverse phase protein array (RPPA), comet assay and immunofluorescence staining) and in vivo (orthotopic mouse model) experiments in ovarian cancer models to determine the biological effects of EP-100 alone and in combination with standard of care drugs. We first examined the cytotoxic effects of EP-100 alone on 8 ovarian cancer cell lines with variable expression level of LHRH receptors and the IC50 values ranged from 0.5 to 2.0 µM. We then tested the effect of combining EP-100 with standard drugs (e.g., cisplatin, paclitaxel, doxorubicin, topotecan, and olaparib) on ovarian cancer cells. Of those drugs, we found that combination of EP-100 and olaparib was highly synergistic in drug sensitive and multi-drug resistant ovarian cancer cell lines (HeyA8, HeyA8-MDR, A2780, and A2780CP20). The combination indices (CI) were < 0.9 at the ED50 level. Furthermore, the nuclear foci of γH2AX were significantly increased in combination group of EP100 and olaparib after 24 h incubation compared with control, EP-100 alone, or olaparib alone groups (p < 0.0001, ANOVA). In addition, there was increased DNA accumulation in tails using comet assay after treating cells with EP-100 and olaparib for 24 h (p < 0.001, ANOVA). RPPA results identified that PI3K/AKT pathway can be blocked significantly by the combination treatment. The study in HeyA8 xenograft mouse models showed that mice treated with EP-100 and olaparib had the lowest tumor weight (0.06 ± 0.05 g) compared with those treated with vehicle (1.19 ± 0.39 g, p < 0.008), EP-100 alone (0.62 ± 0.28 g, p < 0.008), and olaparib alone (0.50 ± 0.22 g, p < 0.01). EP-100 has efficacy in preclinical models of ovarian cancer, especially in combination with olaparib. Our findings suggested that combining EP-100 with olaparib could be a promising therapeutic strategy for ovarian cancer.

#4818

In vivo efficacy of triplet therapy with osimertinib, cetuximab and bevacizumab for lung cancer cells harboring EGFR T790M.

Kazuya Nishii, Kadoaki Ohashi, Go Makimoto, Hisao Higo, Kiichiro Ninomiya, Hiroe Kayatani, Takashi Ninomiya, Toshio Kubo, Kammei Rai, Eiki Ichihara, Katsuyuki Hotta, Masahiro Tabata, Katsuyuki Kiura. _Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan_.

Background

Osimertinib, a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), is the standard treatment for patients with lung cancer harboring EGFR T790M (Mok T et al. N Engl J Med. 2017). However, acquired resistance to osimertinib is inevitable, and alternative strategies are needed. We recently reported that triplet therapy with afatinib, cetuximab and bevacizumab induced deep remission in lung tumors with EGFR T790M in vivo (Kudo K, et al. Mol Oncol. 2017). Therefore, we hypothesized that the combination of osimertinib with cetuximab and/or bevacizumab is a more effective treatment than osimertinib monotherapy for lung tumors harboring EGFR T790M.

Materials and Methods

RPC-9 cells (5 × 106) harboring EGFR 19DEL+T790M were injected subcutaneously into nude mice as xenograft models. The mice were treated with osimertinib (5 mg/kg, 5 times/week), cetuximab (1 mg/mouse, twice/week) and/or bevacizumab (5 mg/kg, twice/week) for 1 month, and observed for an additional month without treatment. The efficacy and toxicity of the triplet therapy (osimertinib, cetuximab, and bevacizumab) were compared with each of the single and double therapies.

Results

The cell proliferation assay confirmed that RPC-9 cells were sensitive to osimertinib in vitro. In the xenograft model, the doublet therapies (osimertinib plus bevacizumab or osimertinib plus cetuximab) had stronger antitumor effects than osimertinib monotherapy 56 days after drug administration (i.e., 28 days after discontinuation of the treatment). However, no significant differences were observed in inhibitory or CR ratios between mice receiving triplet therapy and those receiving doublet therapies

(osimertinib plus bevacizumab or cetuximab). No intolerable toxicity was observed with the doublet or triplet therapies.

Conclusions

Doublet therapy (osimertinib plus bevacizumab or osimertinib plus cetuximab), induced a significant reduction in lung tumors with EGFR T790M in vivo compared with osimertinib monotherapy. We were not able to prove the superiority of the triplet therapy over the doublet therapy. In future studies, we will examine the effects of the doublet and triplet therapies in other models, including an EGFR-driven transgenic lung cancer mice model.

#4819

High-throughput screening of glioma stem-like cells (GSCs) identifies synergistic therapeutic combination of FGFR inhibitor and CDK4/6 inhibitor.

Jianwen Dong,1 Emmanuel Martinez-Ledesma,1 Nghi Nguyen,2 Shaofang Wu,1 Yuji Piao,1 Ningyi Tiao,1 Soon Young Park,1 David Brunell,2 Clifford Stephan,2 Roel Verhaak,3 Erik Sulman,1 Veerakumar Balasubramaniyan,1 John F. de Groot1. 1 _UT MD Anderson Cancer, Houston, TX;_ 2 _Institute of Biosciences and Technology, Texas A &M Health Science Center, Houston, TX; _3 _The Jackson Laboratory for Genomic Medicine, Farmington, CT_.

High-grade gliomas are challenging tumors to treat. The lack of efficacy of single-agent therapy emphasizes the importance of developing combination strategies to overcome acquired resistance. Quantitative high-throughput screening (HTS) of 2 and 3 drug combinations at 5 concentrations (maximal concentration of 1 uM) of each agent were evaluated in a panel of GSCs to identify potentially novel combination therapy. Genomic, proteiomic (reverse protein lysate array, RPPA) and GSC subtype were correlated with drug IC50 to find biomarkers of drug sensitivity. GSCs were classified as sensitive (IC50 ≤ 1µM) or resistant (IC50 >1µM) for single-agent screening. The Bliss independence model and combination index (CI) criteria were used to quantify the synergy of compound combinations. Evaluation of drug combination data for synergistic combination identified FGFR inhibitor LY2874455 and CDK4/6 inhibitor LY2835219 as the most effective combinations for multiple drug sets. Abnormal activation of FGFR and alterations in the CDKN2A-CDK4/6-Retinoblastoma 1 (RB1) pathway have been implicated in glioblastoma pathogenesis. We further investigated the in vitro combination efficacy of FGFR inhibitor and CDK4/6 inhibitor. Compared to single agent, combination of FGFR inhibitor and CDK4/6 inhibitor induced prominent cleaved poly ADP ribose polymerase (c-PARP), an index of apoptosis, which was further confirmed by Annexin V staining. Combination therapy significantly inhibited p-RB, p-STAT3, p-Akt, p-Erk and p-c-Jun expression compared to single agent. Genomic data demonstrated that high PDGFRα expression and low methylation correlated to sensitivity to the FGFR and CDK4/6 inhibitor combination. These findings were validated by demonstrating that this synergistic sensitivity was decreased by knocking out PDGFRα by CRISP/Cas9 system with the combination index (CI) at IC50 increased from 0.964 to 2.247. In addition, Western blotting showed that expression of PDGFRα and Notch1 was significantly inhibited by combination treatment. However, this combination failed to show any survival and tumor inhibition benefits in intracranial xenograft mouse models.

#4821

A targeted differentiation therapy for the treatment of acute myeloid leukemia.

Sudhakiranmayi Kuravi, Myles Taylor, Tara L. Lin, Jensen Roy, Joseph McGuirk, Ramesh Balusu. _Univ. of Kansas Medical Ctr., Kansas City, KS_.

Objectives: Acute myeloid leukemia (AML) is a heterogeneous disease with diverse genetic abnormalities present in all ages, but mainly prevalent in an elderly population with an average age of above 60 years. The treatment of elderly AML remains a formidable challenge as the long-term outcomes of elderly AML patients have not improved in the last three decades, with ≤5% overall survival recorded at 5 years, calling for novel treatment options. Epigenetic therapy has a significant impact on the management of hematologic malignancies. Recent findings show that using very low dosages of decitabine depletes DNA methyl transferase 1 (DNMT1) without cytotoxicity. Decitabine-mediated epigenetic therapy does not induce FLT3 (fms-like tyrosine kinase 3) ligand, which hinders the effectiveness of FLT3 inhibitors. The inhibition of FLT3 and DNMT1 is associated with terminal myeloid differentiation of AML cells. Therefore, combining the inhibitors of FLT3 and DNMT1 may be an effective therapeutic approach for the treatment of poor-risk AML. Ponatinib is a third-generation receptor tyrosine kinase inhibitor. Various in vitro and in vivo preclinical studies demonstrated antileukemic activities of ponatinib against AML cells bearing FLT3-ITD mutations.

Methods: Apoptosis and CD11b were measured by flow cytometry. FLT3 signaling and DNMT1 levels were analyzed by immunoblotting.

Results: FLT3-ITD expressing AML cell lines MV4-11, MOLM-13, and MOLM-14 were used in the study. Co-treatment with decitabine (10-100 nM) and ponatinib (2 nM) in AML cells induced apoptosis in association with PARP cleavage. Increased levels of pro-apoptotic protein BAD and decreased levels of anti-apoptotic protein MCL1 were observed in these treated cells. The combination of decitabine and ponatinib also showed similar effects in primary AML cells expressing FLT3-ITD. The Western blot analysis demonstrated that treatment of decitabine decreased levels of DNMT1 and ponatinib inhibited FLT3 signaling and activation of downstream effectors STAT5, AKT, and ERK1/2 with induction of PU.1 a key regulator of myeloid differentiation. Co-treatment with ponatinib and decitabine using low concentrations in MV4-11 and MOLM14 cells induced myeloid differentiation. The percentage of differentiated cells was measured by increased surface expression of CD11b analyzed by flow cytometry and granulocytic/monocytic morphology examined by Wright-Giemsa staining.

Conclusions: Mechanistically, the hypomethylating agent initiates the differentiation process and FLT3 inhibition augments differentiation leading to apoptosis of AML cells. Altogether, these preclinical findings of downregulation of DNMT1 and induction of PU.1 are a novel differentiation approach to induce apoptosis in AML cells and warrant future therapeutic potential for the treatment of AML patients expressing FLT3 mutations.

#4822

Anti-MMP9 antibody in combination with standard cytotoxic therapy in preclinical models of pancreatic cancer.

Niranjan Awasthi,1 Amanda Mikels-Vigdal,2 Erin Stefanutti,2 Margaret A. Schwarz,1 Sheena Monahan,1 Victoria Smith,2 Roderich E. Schwarz1. 1 _Indiana Univ. School of Medicine, South Bend, IN;_ 2 _Gilead Sciences, Inc., Foster City, CA_.

BACKGROUND: Matrix metalloproteinase 9 (MMP9) is involved in the proteolysis of extracellular proteins and is reported to play a key role in development, invasion and metastasis of many cancers including pancreatic ductal adenocarcinoma (PDAC). Andecaliximab is a potent and highly selective monoclonal antibody inhibitor of MMP9 that has shown antitumor activity in combination with chemotherapy in gastric cancer. The therapeutic potential of targeted MMP9 inhibition in combination with cytotoxic therapy in preclinical models of PDAC was evaluated through the use of an αMMP9 antibody.

METHODS: Animal survival and qualitative analysis were performed in the peritoneal dissemination model in mice using 7.5x105 AsPC-1 cells. Tumor growth study was performed in subcutaneous xenografts in mice using 7.5x105 AsPC-1 cells. The mechanistic evaluation involved RNASeq, Luminex, IHC and Immunoblot analysis in tumor samples.

RESULTS: Median animal survival compared to controls (17 days) was increased after 2-week therapy with NPT (27 days, a 59% increase), Gem (22 days, +29%) and NPT+Gem (30 days, +76%). Addition of αMMP9 antibody increased survival as follows: NPT+MMP9 (30 days, +76%), Gem+MMP9 (25 days, +47%) and NPT+Gem+MMP9 (33 days, +94%). Evaluation of efficacy of maintenance therapy (6-weeks) revealed that median animal survival (controls: 22 days) was increased after NPT+Gem therapy (63 days, +186%) and further improved by addition of αMMP9 antibody (70 days, +218%). Qualitative assessment of mice after 2-week therapy revealed that αMMP9 therapy led to reduction in jaundice, bloody ascites and metastatic burden, both alone and in combination with NPT+Gem. Tumor lysates demonstrated changes in several proteins in chemotherapy groups including IP-10, MDC, PAI-1, GM-CSF, MIP-1b and IL-12b. αMMP9 therapy increased IL-28 (1.5 fold, p = 0.016). IHC analysis revealed decreased tumor microvessel density based on endomucin staining in αMMP9 therapy groups (59.9%, p<0.0001). Immunoblot analysis showed decreased vimentin expression in αMMP9 therapy groups (42.5%, p =0.03). In tumor growth study, NPT+Gem significantly decreased tumor growth (72%, p=0.0005) compared to controls. αMMP9 therapy caused a non-significant decrease in tumor growth (17%, p=0.24). Tumor tissues revealed low, but detectable levels of MMP9 mRNA in all therapy groups but no difference in MMP9 expression. αMMP9 monotherapy resulted in more gene expression changes in the mouse stromal than the human tumor compartment compared to other treatments. However, NPT+Gem+MMP9 combination therapy resulted in greater numbers of changes in gene expression compared to the other treatments in the tumor compartment.

CONCLUSION: These findings suggest that MMP9 inhibition can augment the effects of standard cytotoxic therapy and support the potential of this combination therapeutic strategy for clinical PDAC therapy.

#4823

Alvocidib enhances the efficacy of cytarabine and daunorubicin (7+3) in non-clinical models of acute myeloid leukemia.

Wontak Kim, Clifford J. Whatcott, Adam Siddiqui-Jain, David J. Bearss, Steven L. Warner. _Tolero Pharmaceuticals, Inc., Lehi, UT_.

The '7+3' induction regimen has been the standard of care for frontline acute myeloid leukemia (AML) for more than 30 years. There are still significant unmet needs for frontline patients, particularly those with high-risk features. Alvocidib, a potent CDK9 inhibitor, has shown clinical activity in multiple studies of frontline and relapsed/refractory AML. In some clinical studies, alvocidib has been administered as part of the ACM (alvocidib, cytarabine, and mitoxantrone) regimen. The ACM regimen has demonstrated greater clinical responses compared to 7+3 in frontline intermediate and high-risk AML patients. The ACM regimen is now being evaluated in a multi-institution, Phase II study using a MCL-1 dependency biomarker to select patients likely to be sensitive to the alvocidib-containing regimen. Alvocidib activity is primarily driven by its inhibition of CDK9/RNA-polymerase II and the subsequent suppression of key anti-apoptotic proteins, including MCL-1. Alvocidib induces apoptosis and potentiates the activity of apoptosis-inducing agents, such as cytarabine and mitoxantrone. The synergy of the ACM regimen led us to hypothesize that alvocidib may enhance the anti-leukemia activity of the 7+3 regimen in preclinical models as well. Cell viability and apoptosis measurements were made following treatment using the CellTiter-Glo and Caspase-Glo assays according to manufacturer protocol. MCL-1 mRNA expression and the expression of additional markers was determined using RT-qPCR. Protein expression was determined using standard immunoblotting techniques. Alvocidib and the 7+3 regimen were also tested in vivo in the MV4-11 AML xenograft model. Here, it was observed that the 72-hour viability assays yielded single agent IC50 values of alvocidib, cytarabine, or daunorubicin in cultured AML cells ranging from 2.2 nM to 567 nM. In apoptosis assays, a modest induction was observed with cytarabine, daunorubicin, or alvocidib. However, the three-drug combination more than doubled the induction of apoptosis compared to any of the agents alone. We also observed that MCL-1 was suppressed in the combination treatment. In a xenograft study, 21.1 and 48.5% tumor growth inhibition (TGI) was observed following either single agent treatment of daunorubicin (0.5 mg/kg) or cytarabine (60 mg/kg), respectively. Treatment of alvocidib (1.25 mg/kg) yielded 60.0% TGI. Combination treatment, however, resulted in tumor shrinkage, yielding 116.2% TGI. While we have reported previously that alvocidib synergizes with cytarabine and mitoxantrone preclinically in the ACM regimen, our data here demonstrates that alvocidib potentiates the activity of cytarabine and daunorubicin as well. Our results here provide additional scientific rationale for an investigational Phase Ib clinical study employing alvocidib in combination with 7+3 in frontline AML patients, which is currently ongoing.

#4824

Rac1b overexpression confers resistance to chemotherapy treatment in colorectal cancer.

Pallavi Chaturvedi,1 Erik Goka,1 Adriana De La Garza,1 Dayrelis Mesa Lopez,1 Marc E. Lippman2. 1 _Geneyus LLC, Miami, FL;_ 2 _University of Miami, Miami, FL_.

Acquired resistance to chemotherapy represents a major limitation in the treatment of colorectal cancer. New strategies to circumvent this problem are critical to prolonging patient survival. Previous studies have shown that Rac1b, a constitutively activate isoform of the small GTPase Rac1, is upregulated with disease progression and promotes cell proliferation and inhibits apoptosis by activation of NFκΒ signaling. In the present study, we show that Rac1b overexpression correlates with colorectal cancer stage with the highest levels identified in metastatic cancer. We show that Rac1b expression is associated with increased growth of colon adenocarcinoma cells and that modulation of Rac1b expression in colon adenocarcinoma cells reduces NFκB activity. The use of a Rac inhibitor in colon cancer cells reduces the 2D and 3D colony formation of colon adenocarcinoma cells, reduces the migratory capacity of these cells, and suppresses NFκB signaling. Rac inhibition also reduces tumor growth in a xenograft model of colon adenocarcinoma cells. Surprisingly, Rac1b expression and NFκB activity was upregulated in colon carcinoma cells treated with 5-FU and oxaliplatin (two components of the standard of care FOLFOX chemotherapy regimen), suggesting that Rac1b facilitates chemo-resistance by activation of NFκB signaling. Knock down of Rac1b in colon cancer cells or Rac inhibitor increases the sensitivity of the cells to oxaliplatin. When used in combination, inhibition of Rac in the colon cancer cells prevents the increase in NFκB activity associated with chemotherapy treatment and increases the sensitivity of the cells to oxaliplatin. Moreover, while Rac inhibition or oxaliplatin treatment alone reduces the growth of colorectal cancer in vivo, combination therapy results in significant further reduction of tumor growth than either single agent alone. Thus, we provide the first evidence that Rac1b expression confers resistance to standard of care chemotherapy in colorectal cancer. Additionally, we show that the use of a Rac inhibitor prevents chemoresistance by blocking activation of chemotherapy induced NFκB signaling providing a novel strategy to overcome resistance to chemotherapy in colorectal cancer.

#4825

Targeting PIM and AKT kinases impairs tumor growth and improves overall survival in a murine model of advanced castration-resistant prostate cancer.

Marco A. De Velasco,1 Yurie Kura,1 Naomi Ando Ando,1 Noriko Sato,1 Barry R. Davies,2 Masahiro Nozawa,1 Kazuhiro Yoshimura,1 Kazuhiro Yoshikawa,3 Kazuto Nishio,1 Hirotsugu Uemura1. 1 _Kindai University Faculty of Medicine, Osaka-Sayama, Japan;_ 2 _Strategy, Oncology, IMED Biotech Unit, AstraZeneca, Alderly Park, United Kingdom;_ 3 _Aichi Medical University, Nagakute, Japan_.

Background: Castration-resistant prostate cancer (CRPC) is an incurable disease with few treatment options, thus novel treatment strategies are required. PIM serine/threonine kinases (PIM-1, PIM-2, and PIM-3) are overexpressed in various cancers and are correlated with human prostate cancer progression. We previously evaluated the antitumor activity of the pan-PIM kinase inhibitor AZD1208 and showed that it could effectively suppress tumor growth in early-stage mouse models of castration-naïve and castration-resistant Pten-deficient prostate cancer. To fully determine the therapeutic potential of PIM inhibition we examined the therapeutic efficacy of AZD1208 in a mouse model of advanced prostate using clinically relevant endpoints. Additionally, since AKT and PIM kinases modulate survival processes by the phosphorylation of common substrates, we also examined the treatment combination with the pan-AKT inhibitor, AZD5363.

Methods: PSACre:Ptenflox/flox/Trp53flox/flox double knockout (Pten/P53-DKO) mice bearing palpable tumors with an initial size of 0.5 cm were surgically castrated and randomized to treatments with control vehicle, AZD1208 (60 mg/kg/day), AZD5363 (100 mg/kg/b.i.d) or AZD1208 + AZD5363 upon tumor progression or two weeks after castration, whichever occurred first. Tolerability was assessed by comparing differences in bodyweight and mouse health performance status. Therapeutic efficacy was determined by overall survival and antitumor activity was assessed by comparing tumor growth rates.

Results: Treatments appeared to be well tolerated by the mice and no significant differences were noted in bodyweight changes between treatment groups. Median overall survival times were 21, 28, 28 and 35 days for vehicle, AZD1208, AZD5363 and AZD1208+AZD5363 treated mice , respectively, P=0.027. Median time to tumor doubling (1→2 cm) was longer for AZD1208 and AZD1208+AZD5363 treatment groups compared to vehicle or AZD5363, 28, 21, 14 and 14 days, respectively, P=0.005.

Conclusions: Treatments with AZD1208 suppressed CRPC growth and improved overall survival times in Pten/P53-DKO mice. Furthermore, targeting both PIM and AKT kinases with combination therapy improved overall survival. This study provides evidence to support further development of therapeutic strategies targeting PIM and AKT for the management of human advanced prostate cancer.

#4826

Synergistic effects of foretinib with lapatinib in MET and HER2 co-activated experimental esophageal adenocarcinoma.

Md Sazzad Hassan,1 Fiona Williams,2 Niranjan Awasthi,1 Margaret A. Schwarz,1 Roderich E. Schwarz,1 Urs von Holzen1. 1 _Indiana University School of Medicine-South Bend, South Bend, IN;_ 2 _University of Notre Dame, South Bend, IN_.

Introduction: Recent studies have demonstrated that HER2 and MET receptor tyrosine kinases are co-overexpressed in a subset esophageal adenocarcinoma (EAC). We therefore studied the usefulness of combining HER2 and MET targeting by small-molecule inhibitors foretinib and lapatinib both in-vitro and in-vivo models of experimental EAC.

Methods: In this study we first characterized MET and HER2 activation in a panel of human EAC cell lines and the differential susceptibility of these EAC cell lines to single agents or combinations of foretinib, a multi-kinase MET inhibitor, with HER2 targeted agent lapatinib. We evaluated the levels of phosphorylation status of MET and HER2 proteins using western blot in EAC cell lines. Foretinib and lapatinib, as single agent or in combination were tested for effect on cell growth as detected by WST-1 assay and on cell apoptosis as detected by western blot of cleavage of caspase 3 and poly ADP ribose polymerase (PARP). In addition, we explored the antitumor efficacy with survival advantage following foretinib and lapatinib mono and combination therapies for two weeks in murine subcutaneous xenograft and peritoneal metastatic survival models of human EAC.

Results: The OE33 EAC cell line with phosphorylation of both MET and HER2, demonstrated reduced sensitivity to foretinib and lapatinib when used as a single agent. The co-administration of foretinib and lapatinib effectively inhibited both MET and HER2 phosphorylation, synergistically inhibited cell growth and induced apoptosis, overcoming single agent resistance. In the OE19 EAC cell line with only HER2 phosphorylation and the ESO51 EAC cell line with only MET phosphorylation, profound cell growth inhibition with induction of apoptosis was observed in response to single agent foretinib and lapatinib, respectively, with lack of enhanced growth inhibition when the two drugs were combined. Foretinib in combination with lapatinib treatment resulted in significantly higher antitumor efficacy and survival benefit compared with foretinib or lapatinib treatment alone. In subcutaneous xenografts using OE33 cells, average net tumor growth after two weeks in different therapy groups was 247.83 mm3 in control, 216.71 mm3 after foretinib (p=0.49), 239.68 mm3 after lapatinib (p=0.74), and 108.06 mm3 after foretinib plus lapatinib (p=0.0011). In the OE33 survival model there was a significant increase in median animal survival after two weeks foretinib plus lapatinib treatment (71 days) compared to control (60 days, p=0.0021), to foretinib therapy (63 days, p=0.0019) or to lapatinib (61 days, p=0.0019) therapy.

Conclusion: These data suggest that combination therapy with foretinib and lapatinib should be tested as a treatment option for HER2 positive patients with MET-overexpressing EAC. Therefore, this combination therapy could be a novel treatment strategy for EAC with MET and HER co-activation.

#4827

**Magnetic iron oxide nanoparticles induce apoptosis and autophagic cell death in prostate cancer cells treated with docetaxel** **via ROS generation and NF-ΚB signaling.**

Kanako Kojima,1 Sanai Takahashi,1 Shungo Saito,1 Tadashi Nittami,1 Eri Usugi,2 Kenichiro Ishii,2 Yoshifumi HIrokawa,2 Masatoshi Watanabe2. 1 _Yokohama National University, School of Engineering, Yokohama, Japan;_ 2 _Mie University, School of Medicine, Tsu, Japan_.

Patients with metastatic castration-resistant prostate cancer (mCRPC) have a poor prognosis. Docetaxel (DTX)-based therapy is one of current standard treatments for mCRPC since 2004 although cabazitaxel, abiraterone acetate, and enzalutamide are three agents recently approved for the treatment of mCRPC. Although DTX-based chemotherapy has been shown to improve the quality of life in patients with the advanced disease, this needs to be improved because of limitations by lack of specificity, toxicity, and progression of docetaxel-resistance. Thus, combination of docetaxel with other compounds or drugs is needed to overcome these problems. We have investigated usefulness of magnetic iron oxide nanoparticles (MNPs- Fe3O4) for cancer diagnosis and therapy, showing that MNPs- Fe3O4 may modify the effect of chemotherapy in prostate cancer cells in vitro. The purpose of this study was to analyze the combined effect of docetaxel and MNPs- Fe3O4 on prostate cancer cell lines, DU145 and PC-3 and clarify their mechanisms. Electron microscopy, flow cytometry and Western blotting were used to study apoptosis and autophagic cell death with/without an autophagic inhibitor. Our results showed that MNPs- Fe3O4 produced reactive oxygen species (ROS) and 8-OHdG, and the combination of docetaxel and MNPs- Fe3O4 inhibited cancer cell growth and induced apoptosis and autophagic cell death. Although docetaxel induced nuclear factor-kappaB (NF-ΚB) expression, combination of docetaxel and MNPs inhibited NF-ΚB expression. These results suggest that MNPs- Fe3O4 may enhance effect of docetaxel on prostate cancer cells via ROS generation and inhibition of NF-ΚB signaling, leading to new tumor ablation therapies.<!--EndFragment-->

#4828

Newly identified p53 pathway-restoring compound CB002 and its derivatives sensitize colorectal and multiple myeloma cancer cell lines to front-line cancer therapies.

Liz J. Hernandez Borrero,1 David T. Dicker,2 Wafik S. El-Deiry2. 1 _The Pennsylvania State University, Philadelphia, PA;_ 2 _Fox Chase Cancer Center, Philadelphia, PA_.

Proapoptotic NOXA protein is a p53 transcriptional target that has been shown to be a crucial mediator of cell death in response to chemotherapy agents. Given the fact that NOXA is downregulated in multiple cancers, induction of this protein by small-molecule exposure makes it an attractive therapeutic strategy. Although cancer patients often respond to initial therapy, later on therapy resistance develops, leaving them with limited therapy options. Therefore, new therapeutic regimens that overcome resistance mechanisms are of high importance. The purpose of this study was to identify new small molecules that restore the p53 pathway and, in combination with chemotherapy agents, could result in therapy sensitization. We have discovered a family of small molecules (CB002 and 12 structural analogs) that restore the p53 pathway through NOXA in various cancer cell lines including SW480, DLD-1 and HCT116 colorectal cancer (CRC) cells. In order to test if CB002 and its derivatives sensitize cells to first-line CRC therapies, we treated SW480 and DLD-1 cells in combination with irinotecan, fluorouracil (5-FU), doxorubicin, or oxaliplatin. Our data suggest that treatment combinations of CB002 with first-line CRC therapies result in enhanced cell death and decreased IC50. Particularly, sensitization to doxorubicin and irinotecan was dependent on NOXA expression as stable NOXA knockdown abolished therapy sensitization. NOXA induction has been shown to overcome bortezomib resistance in multiple myeloma (MM) cells. Thus, we investigated if CB002 and more potent derivatives could resensitize bortezomib-resistant cells. We treated MM1S (bortezomib-sensitive) and 33X (bortezomib-resistant) cells with bortezomib along with our CB002 structural analogs. Our data indicate that combination treatment with our structural analogs sensitizes 33X cells to bortezomib treatment. Taken together, our evidence supports that our novel identified structural analogs of CB002 can be further tested in vivo in combination with front-line therapies to improve therapeutic modalities and overcome resistance in CRC and MM. Our experiments will further assess toxicity to normal cells and tissues. Current experiments are focused on using NOXA as a pharmacodynamic biomarker to predict therapeutic efficacy. Our data argue that p53 pathway restoring compounds are worth pursuing further to determine if they can provide a therapeutic advantage to patients who are not responsive to front-line cancer therapies.

#4829

Plumbagin amplifies the pro-apoptotic and anti-tumorigenic activities of vemurafenib in BRAF-mutant melanoma.

Pooja Sharma, Mary Katherine Montes de Oca, Ross L. Pearlman, Farrukh Afaq. _University of Alabama at Birmingham, Birmingham, AL_.

Melanoma, the deadliest form of skin cancer, originates from the pigment-producing cells in the skin known as melanocytes. The incidence of melanoma is increasing at a more alarming rate than any other cancer worldwide. BRAF mutations occur in ~50% of melanomas that drive oncogenic activation of the BRAF-MEK-ERK resulting in cell proliferation and tumorigenesis. BRAF and MEK inhibitors either alone or in combination have improved the overall survival of melanoma patients with BRAF mutations. However, these patients inevitably develop resistance to these treatments. Thus, there is a need for exploring novel agents to enhance the therapeutic efficacy of these inhibitors for the management of melanoma. Phytochemicals offer promising new options for the development of more effective strategies because of their relatively low toxicities on normal cells. Plumbagin, a naturally occurring naphthoquinone found in flowers and roots of the Plumbaginaceae family, exhibits potent anti-proliferative and anti-tumorigenic properties. Recently, we have shown that plumbagin reduces proliferation and induces apoptosis in melanoma cells by downregulating PI3K signaling. Studies have unequivocally demonstrated that BRAF-MEK-ERK and PI3K-AKT-mTOR signaling pathways play major roles in melanoma development and therapy resistance. Therefore, the objective of this study is to determine whether plumbagin in combination with vemurafenib could offer therapeutic benefit over an individual agent for the treatment of the BRAF-mutant melanoma. Treatment of BRAF-mutant human melanoma cells with plumbagin and vemurafenib in combination more effectively reduced cell growth and colony formation than individual agents. Combination treatment (plumbagin + vemurafenib) also resulted in increased apoptosis as revealed by cleaved caspase-3 and PARP. Furthermore, combination treatment resulted in enhanced expression of Bax and Bak, and increased inhibition of Bcl2 and Mcl-1. We also found that combination treatment was more effective in inhibiting the phosphorylation of MEK1/2 ERK1/2, AKT, and mTOR as well as the protein expression of PI3K. In athymic nude mice subcutaneously implanted with A375 melanoma cells, combination treatment resulted in greater inhibition of tumor growth, when compared to individual agents. Combination treatment also resulted in (i) decreased phosphorylation of MEK1/2, ERK1/2, AKT, and mTOR (ii) decreased proliferation, and (iii) increased apoptosis in tumor samples than individual agents.These data encourage the need for further advanced in vivo studies to evaluate the effects of plumbagin as an adjuvant to current therapies for the management of BRAF-mutant melanoma.

#4830

Selective DNA-PK inhibitor, M3814, boosts p53 apoptotic response to DNA double strand breaks and effectively kills acute leukemia cells: Implications for AML therapy.

Eric Haines,1 Astrid Zimmermann,2 Frank Zenke,2 Andree Blaukat,2 Lyubomir T. Vassilev1. 1 _EMD Serono Research and Development Institute, Inc., Billerica, MA;_ 2 _Merck Biopharma, Merck KGaA, Darmstadt, Germany_.

M3814 is a selective inhibitor of DNA-PK in clinical development. It effectively blocks non-homologous end joining repair of DNA double strand breaks (DSB) and strongly potentiates the antitumor effect of ionizing radiation (IR) and topoisomerase II inhibitors. By inhibiting DNA-PK catalytic activity in the presence of DSB damage, M3814 disrupts a previously unrecognized negative regulatory loop between DNA-PK and ATM, leading to increased activation of the ATM pathway, including its downstream targets, CHK2 and p53. As a result, in cancer cells expressing wild-type p53, DNA-PK inhibitor strongly activates the p53 response to DSB DNA damage and its two main functions- cell cycle arrest and apoptosis. While p53 activation in p53 wild-type epithelial cancer cells causes effective cell cycle arrest, the majority of the epithelial tumors lose their ability to undergo p53 dependent apoptosis. In contrast, tumor cells from patients with acute leukemia express wild-type p53 in with preserved p53 apoptotic function, making them amenable to p53 activation approaches. Here, we investigate the therapeutic potential of M3814 in combination with DSB-inducing agents in acute leukemia cells. M3814 strongly potentiated the activity of IR in three p53 wild-type leukemia cell lines (MOLM-13, MOLT-4, MV4-11) but had minimal effect on the p53-null HL-60 and THP-1 lines. This potentiation was due to enhanced activation of the p53 pathway as demonstrated by substantially increased expression of p53 transcriptional targets, p21 and PUMA, and induction of apoptosis only in p53 wild-type cells. M3814 showed synergism in p53-dependent cell killing with the topoisomerase inhibitors, daunorubicin and idarubicin, and 2-3 fold increase in the potency of daunorubicin/cytarabine combination in p53 wild-type acute myeloid leukemia (AML) cells. Daunorubicin/cytarabine/M3814 combinations were better tolerated by CD34+ bone marrow cells harvested from healthy volunteers and tested in vitro. These results support further exploration of M3814 as a novel combination partner in the treatment of p53 wild-type AML.

#4831

DSF-Cu complex sensitizes patient-derived unmethylated MGMT expressing brain tumor cells to temozolomide.

George C. Bobustuc, Amin B. Kassam, Richard A. Rovin, Deborah Donohoe, Dmitry Bosenko, Santhi D. Konduri. _Aurora Health Care, Milwaukee, WI_.

Introduction: O6 methylguanine DNA methyltransferase (MGMT) repairs the DNA damage caused by alkylating agents [temozolomide (TMZ)] leading to chemoresistence. Members of the aldehyde dehydrogenase (ALDH) family of isoenzymes serve as markers of cancer stem cells and contribute to chemotherapy resistance. Disulfiram (DSF) inhibits MGMT through ubiquitin mediated degradation; it is also a specific inhibitor of ALDH. We therefore, hypothesized that DSF through MGMT and ALDH inhibition decreases stemness and sensitizes GBM cells to TMZ.

Methods: Normal astrocytes as well as established MGMT expressing GBM cell lines (LN18, T98G, U138, U118) and MGMT expressing (unmethylated) patient derived glioblastoma cells (ANII 7730 and ANII 7754) were treated with DSF/Cu +/- TMZ in various doses and combinations. We have also evaluated MGMT, ALDH levels and Sox2 expression. Cell viability, apoptotic assay and caspase 3/7 assay were used to evaluate inhibitory effect of various treatment combinations.

Results: DSF and TMZ have a minimal effect on normal astrocyte growth. DSF alone inhibited MGMT in established MGMT expressing glioblastoma cells (5 to 10 µM). DSF alone was unable to inhibit MGMT in patient derived brain tumor cells (up to 20µM). Addition of copper (</=1µM) significantly inhibited MGMT (~ 90%) in patient derived GBM cells which correlated with significant growth inhibition even when DSF was used at low concentrations (</=1µM). This suggests that addition of copper to DSF may reduce any potential, dose dependent DSF related neurotoxicity. Temozolomide alone did not inhibit MGMT in established cell lines nor patient derived brain tumor cell lines (up to 1000µM). DSF+Cu further sensitized patient derived GBM cells to TMZ and significantly inhibited GBM cell growth (~ 95%) and did not inhibit normal astrocyte growth. Further DSF+Cu and DSF+Cu+TMZ combinations caused significant apoptotic cell death and significantly increased caspase 3/7 in patient derived unmethylated GBM cells. Similarly, combination of DSF+Cu and TMZ+DSF+Cu caused significant drop in ALDH activity in patient derived brain tumor cells compared to untreated controls and single agents. DSF induced ALDH and MGMT inhibition correlated with decrease in SOX2. MGMT expression canceled by CRISPR/Cas9 led to significant decrease in SOX2 expression in primary cell cultures.

Conclusions: Our findings suggest that DSF/Cu treatment, a dual MGMT and ALDH inhibitor, suppresses stemness. Furthermore, our results confirm that combination of TMZ and DSF/Cu significantly inhibited glioblastoma cell growth compared to TMZ alone and untreated controls. 

### DNA Damage and Cell Cycle Regulation Experimental Therapeutics

#4833

Pharmacodynamic and tumor biomarker analysis of a PLK1 inhibitor, PCM-075, in a phase 1b/2 trial for acute myeloid leukemia.

Maya Ridinger, Karena Kosco, Latifa Hassaine, Jeffrey N. Miner, Mark Erlander. _Trovagene, Inc., San Diego, CA_.

Acute myeloid leukemia (AML) is an aggressive hematologic disease that is characterized by the accumulation of immature myeloid precursor cells. Treatment options for patients are selected based on factors such as age, comorbidities, and cytogenetics. For patients deemed ineligible for standard intensive induction chemotherapy, treatments include either low-dose cytarabine (LDAC) or hypomethylating agents (e.g., azacitidine and decitabine) but when used as single agents, relapses are inevitable. Polo-like Kinase 1 (PLK1), a serine/threonine kinase that is a master regulator of cell-cycle progression, is overexpressed in a number of cancer types including AML. Depletion of PLK1 preferentially induces cell death in tumor versus normal cells. Inhibition of PLK1 in preclinical AML models results in significant efficacy. PCM-075, a highly selective PLK1 inhibitor, is currently in a phase 1b/2 trial (NCT03303339) in combination with standard-of care (LDAC or decitabine) for AML. Collection and processing of blood and bone marrow samples from patients is being performed pre- and post-treatment to assess 1) the inhibition of PLK1 kinase activity and downstream effects by measurement of pharmacodynamic (PD) and mechanism of action (MOA) biomarkers (blood), and 2) the genomic profile of blast cell population(s) from individual patients (blood and bone marrow). For each patient, blood is collected into CellSave, EDTA, and PAXgene blood RNA tubes at various time points as per the clinical protocol schedule of events. PBMCs isolated from CellSave blood tubes are used to assess the phosphorylation status of PD biomarkers using Western blot and phospho-flow cytometry. DNA extracted from blasts (EDTA tube) is used to detect somatic alterations and RNA extracted from the total cell population (PAXgene) is used to detect fusions and determine gene expression profiles by next-generation sequencing. To identify potential PD biomarkers that would enable assessment of PLK1 inhibition in AML patients treated with PCM-075, nine candidate substrates phosphorylated by PLK1 were screened in the AML cell line MV4-11 by Western blot using selected phospho-antibodies. Of these, phosphorylation of Translational Control Tumor Protein (TCTP) at Ser46 was both time- and dose-dependently inhibited by PCM-075. pTCTP detection by Western blot and phospho-flow cytometry was first optimized in PBMCs isolated from healthy donors and later in blast cells from AML patients resulting in a functional biomarker. Additional downstream MOA biomarkers of PCM-075 activity (DNA content, phospho-histone H3, cleaved caspase 3) and tumor DNA biomarkers that enable categorization of AML molecular subtypes have been validated. We present preliminary patient data from the ongoing phase 1b/2 trial and describe biomarker assays that will be used to evaluate target engagement of PCM-075 in AML patients.

#4834

CT7001: An orally bioavailable CDK7 inhibitor is a potential therapy for breast, small-cell lung and haematological cancers.

Edward K. Ainscow,1 Andrew Leishman,2 Elaine Sullivan,2 Bo Li,3 William Gallagher,3 Adam Peall,4 Kristopher Clark,4 Stuart Thomson,4 Simak Ali,5 Raoul C. Coombes,6 Ash Bahl1. 1 _Carrick Therapeutics, Loughborough, United Kingdom;_ 2 _Carrick Therapeutics, Oxford, United Kingdom;_ 3 _University College Dublin, Dublin, United Kingdom;_ 4 _Sygnature Discovery, Nottingham, United Kingdom;_ 5 _Imperial College, London, London, United Kingdom;_ 6 _Imperial College, London, Oxford, United Kingdom_.

CDK7 inhibition has emerged as a promising strategy in a range of cancer indications. CDK7 acts as a regulator of transcription through its role in phosphorylating and activating RNA Polymerase II (polII) as well as a master regulator of the cell cycle through phosphorylation of members of the CDK family. Inhibition of CDK7 in cancer cells has been shown to be effective in blocking transcriptional addiction to a defined cluster of genes (Wang et al 2015) and leads to decreased cell cycling, a decrease in the levels of proto-oncogenes such as c-Myc and induction of apoptosis. Here we describe the properties of a potent, selective and orally bioavailable CDK7 inhibitor CT7001 (formerly known as ICEC0942). Profiling across a pan-cancer cell line panel showed that CT7001 has anti-proliferative activity in a range of cancer types including acute myeloid leukaemia (AML), small cell lung (SCL) and hormone sensitive and triple negative breast cancers (TNBC).Mechanistic investigations revealed that CT7001s activity was associated with both a decrease in cell proliferation and an induction of apoptosis. Inhibition of cell proliferation was commensurate with a decrease in the levels of c-Myc, Mcl-1 and phospho-Pol II as determined by Western Blot analysis.CT7001 was then evaluated in a range of pre-clinical cancer models. CT7001 was shown to lead to cell death in a panel of patient derived models of small-cell lung cancer (SCLC) in 3D spheroid assays. In an in vivo orthotopic-patient derived xenograft model of TNBC in nu/nu mice, orally administered CT7001 monotherapy produced strong and sustained regression of the tumour that persisted during the dosing schedule, and strong suppression was still maintained upon cessation of treatment. At doses that produced regression CT7001 was well tolerated with little effect on body weight (<10%). Similarly, CT7001 produced a near complete regression in an MV-4-11 xenograft model of AML. Pre-clinical toxicity studies in rat and dog demonstrated that CT7001 was well tolerated at biologically active doses, with no evidence for neutropenia that is associated with inhibitors of other members of the CDK family.Taken together these studies indicate CT7001 is a potent, selective and orally bioavailable inhibitor of CDK7 that shows excellent promise as a potential new treatment in a number of oncology indications. A Phase I study to explore the safety and tolerability of CT7001 was initiated in November 2017. Reference: Wang et al Cell. 2015 163(1):174-86

#4835

UD-017, a novel highly selective and orally active CDK7 inhibitor, shows a significant anticancer activity in patient-derived cancers.

Takashi Matsushita, Sayaka Ogi, Kazuhiro Onuma, Hidetoshi Sunamoto, Ayumi Ogawa, Toru Hasegawa, Yasunori Tokunaga, Yasuhiro Aga, Shigeru Ushiyama. _UBE Industries, LTD., Ube, Japan_.

Background: Cyclin dependent kinase 7 (CDK7) is an attractive target for anticancer drugs due to its dual roles, cell cycle regulation and gene transcription/RNA processing. We synthesized UD-017, a small-molecule, highly selective, orally active CDK7 inhibitor with a novel chemotype. In this study, we characterized anticancer activities of UD-017 in vitro and in vivo using patient-derived (PD) cancer cells.

Methods: We first evaluated an antiproliferative activity of UD-017 broadly in a panel of cancer cell assay including patient-derived 3-dimensional tumor clonogenic assay. To verify the potential of c-Myc expression as a biomarker, we investigated the correlation between c-Myc expression levels and anticancer activity in vitro and in vivo using a colorectal cancer cell. In the main part, anticancer efficacy of UD-017 was investigated in vivo in PD-xenograft (PDX) models using representative PD cancer cells.

Results: In antiproliferative panel assays using over 100 cancer cell lines, UD-017 broadly inhibited a wide range of cancer cells from colon, breast, lung, kidney, blood, pancreas, osteosarcoma, sarcoma and urinary bladder cancers. Especially, UD-017 showed high sensitivity in solid tumor such as non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), gastric, osteosarcoma and sarcoma cells with IC50s of 10-200 nM. UD-017 showed a good correlation of c-Myc expression levels with antiproliferative activity using colorectal cancer cells in vitro, and UD-017 reduced the intratumoral MYC mRNA levels by an administration at 100 mg/kg in a HCT-116 xenograft model in mice. In PDX models, UD-017 showed strong inhibition and even regression of tumor growth, and the tumors were almost disappeared on day 14 without body weight loss in non-small cell lung cancer (LXFL1121). In a pleuramesothelioma (PXF541) PDX model, UD-017 also showed regressive effect, and in gastric cancer (GXA3067) and sarcoma (SXFS117) PDX models, UD-017 completely inhibited the tumor growth.

Conclusions: We propose UD-017 as a novel type of anticancer drug that shows complete antitumor responses in patient-derived xenograft models of diverse cancer types. c-Myc expression in cancer cells may be a biomarker for the antitumor effect of UD-017. These data support the rationale for further advancing towards clinical development.

#4836

Targeting transcriptional regulation in multiple myeloma with a covalent CDK7 inhibitor THZ1.

Liang Zhou, Yu Zhang, Kanika Sharma, Jacquelyn McCarter, William Craun, Steven Grant. _Virginia Commonwealth Univ., Richmond, VA_.

Previous studies have shown that certain tumors such as MYCN-driven neuroblastoma and T-cell ALL may be sensitive to CDK7 inhibition. However, the impact of CDK7 disruption on the proliferation and survival of multiple myeloma (MM) cells, which are regulated, at least in part, by c-Myc, has not as yet been examined. To address this issue, the effects of THZ1, a selective covalent inhibitor of cyclin-dependent kinase CDK7, a component of the general transcription factor TFIIH, was examined in MM cells. Virtually all MM cell lines tested were sensitive to the growth-inhibitory and pro-apoptotic effects of very low (e.g., nM) THZ1 concentrations administered for 24 hr, including H929, OPM2, RPMI8226, and U266 cells, as well as bortezomib- or revlimid- resistant cells. THZ1 induced G2/M arrest and inhibited CDK7-mediated phosphorylation of CDK1/2 at T161 and T160 sites. Notably, THZ1 significantly inhibited general transcription by blocking the phosphorylation of the CTD (carboxy-terminal domain) of RNA Pol II at Ser5, Ser7, and Ser2 sites. Washout studies, in which cells were washed free of THZ1 and resuspended in drug-free medium, demonstrated thatTHZ1 acted irreversibly in blocking CTD phosphorylation. CDK7 inhibition was accompanied by down-regulation of MCL-1, c-Myc (L-myc in U266) and BCL-XL gene expression, all of which exert pro-survival effects. Concordant results were obtained when protein levels were monitored. Moreover, ectopic expression of c-Myc, MCL-1, or BCL-XL in MM cells markedly diminished THZ1 lethality, documenting the functional significance of down-regulation of these proteins in THZ1 activity. CRISPR/Cas9 editing of the CDK7 gene was them utilized to confirm the on-target impact of CDK7 disruption. Cells treated with constructs encoding two independent small guiding RNAs targeting CDK7 (sgCDK7) displayed markedly reduced CDK7 protein levels and sharply diminished H929 and OPM2 cell growth. Furthermore, co-culture with HS-5 cells failed to protect MM cells from THZ1, indicating that THZ1 can circumvent micro-environment forms of drug- resistance. Notably, THZ1 alone showed pronounced antitumor effects (e.g., inhibition of viability), and significantly enhanced proteasome inhibitor (e.g., bortezomib or carfilzomib) lethality in human primary CD138+ MM cells and primitive MM cells (CD138-/19+/20+/27+), but not normal hematopoietic cells (e.g., CD34+ cord blood cells). Finally, administration of THZ1 reduced tumor burden and significantly enhanced survival (P < 0.001) in flank and orthotopic xenograft mouse MM models with minimal toxicity. Collectively, these findings indicate that CDK7 is a potential therapeutic target in MM, and suggest that abrogating CDK7 activity by small molecules like THZ1 warrants further consideration as a therapeutic strategy in MM.

#4837

Evaluation of anticancer activities of UD-017, a novel selective and orally available CDK7 inhibitor, in blood cancers.

Yasuhiro Aga, Sayaka Ogi, Kazuhiro Onuma, Hidetoshi Sunamoto, Takashi Matsushita, Ayumi Ogawa, toru Hasegawa, Shigeyuki Kono, Noriaki Iwase, Shigeru Ushiyama. _UBE Industries, Ltd., Ube, Japan_.

Background: Cyclin dependent kinase 7 (CDK7) is an attractive target for anticancer drugs due to its dual roles, cell cycle regulation and gene transcription/RNA processing. We synthesized UD-017, a small-molecule, highly selective, orally active CDK7 inhibitor with a novel chemotype. In this study, we characterized anticancer effects on blood cancer cells and in vivo anticancer activity in combination with chemo- or immuno-oncology agents. Methods: We first evaluated antiproliferative activities of UD-017 on blood cancer cell lines and investigated the mechanism of activity of UD-017 using NCI-H929 cells (multiple myeloma). Apoptosis was assayed by FACS analysis of Annexin V-positive and PI negative. In vivo anticancer effect was evaluated in the mouse xenograft model with NCI-H929. We further evaluated the anticancer efficacy of UD-017 in combination with chemotherapeutics in an HCT-116 xenograft mouse model and with anti PD-1 antibody in B16F10 mouse allograft model.

Results: UD-017 strongly inhibited the proliferation of blood cancer cell lines (myeloma) with IC50s of 10-100 nM range, more broadly than other representative CDK inhibitors. In the similar assay, UD-017 showed no cytotoxicity to human peripheral blood mononuclear cells up to 10 μM. In the mechanism study, phosphorylation of RNA polymerase II c-terminal domain and expression of c-Myc were both inhibited concomitant with the antiproliferative activity in NCI-H929 cells. Significant apoptosis was induced at around 100 nM. In vivo, UD-017 almost completely inhibited the cancer growth at 50 mg/kg, q.d. for 14-day treatment in a NCI-H929 xenograft model in mice. In the combination assay in vivo, UD-017 showed a clear synergistic effect at 50 mg/kg with 5-fluorouracil (5-FU, 15 mg/kg, i.p.) without further affecting the side effects of 5-FU in HCT-116 (colorectal carcinoma) xenografted mice. Also, UD-017 (100 mg/kg) in combination with anti PD-1 antibody (250 μg/body, i.p.) showed add-on anticancer effect in B16F10-allografted mice. All of the mice in the combination group survived during the dosing period without decreasing the number of blood cells.

Conclusions: We propose UD-017 as a novel type of anticancer drug that shows complete anticancer responses in xenograft models of myeloma cancer cells, and also shows in vivo synergy in combination with chemotherapy and anti PD-1 antibody. These data support the rationale for further advancing towards clinical development.

#4838

R loop-driven genome instability by G-quadruplex binders in BRCA2-silenced human cancer cells.

Alessio De Magis,1 Stefano Giustino Manzo,1 Marco Russo,1 Olivier Sordet,2 Rita Morigi,1 Giovanni Capranico1. 1 _Univ. of Bologna, Bologna, Italy;_ 2 _Cancer Research Center of Toulouse, Toulouse, France_.

G-quadruplexes (G4s) and R-loops are non-B DNA structures that can regulate basic processes such as transcription and replication. Unscheduled formation of R-loops is regarded as highly deleterious to cells, as R loops can induce replicative stress and DNA damage leading to genome instability. G4s are formed by four guanine residues held together by Hoogsteen hydrogen bonds and stabilized by monovalent cations. R loops are triple-stranded structures that contain an RNA-DNA hybrid duplex and a displaced single-stranded DNA. We have recently shown that Topoisomerase I (Top1) can affect genome-wide levels of R loops in human cancer cells consistently with the knowledge that DNA superhelical tension is a main driving force allowing non-B DNA structure formation. As G4 ligands were suggested to synergize with Top1 inhibitors, we asked the question of whether G4 ligands can affect R loops. We have thus investigated by immunofluorescence microscopy the effects of two different G4 binders, Pyridostatin (PDS) and FG (compound 1 in Amato et al J Med Chem 2016), on R-loops in human cancer cells. These G4 binders can increase both G4s and R loops while an inactive derivative of FG cannot induce them. Interestingly, the induction of G4s and R loops well correlate to each other in time and intensity with PDS being more effective than FG. After 24 hours, G4 binders induce a cell cycle arrest at the G2/M phase associated to double-stranded DNA cleavage as detected by γH2AX and 53BP1 foci formation and to activation of checkpoint response as shown by ATM phosphorylation. Interestingly, overexpression of RNaseH1 reduces both R loops and γH2AX foci induced by G4 binders showing that the ligands induce DNA cleavage via an R loop-mediated mechanism. Then, we silenced the BRCA2 gene by RNAi in U2OS cells and the results show that BRCA2 depletion increased γH2AX foci induced by PDS while overexpression of RNaseH1 rescue DNA cleavage induction. We also determined genomic R loop maps by DRIP-seq, and bioinformatic analyses of the specific location of G4-stabilized R loops provided information consistent with a model in which a G4 opposite to a DNA:RNA hybrid can stabilize R loops. Our study establishes for the first time that G4 binders stabilize either G4s and R loops in human cancer cells, and that they induce genome instability with a mechanism dependent on R loop formation. Partially supported by AIRC, Milan.

#4839

Targeting glioma stem cells by pharmacologic stabilization of G-quadruplexes.

Sachiko Okabe,1 Takahiro Nakamura,1 Daiki Hasegawa,1 Reina Kojima,1 Keiji Okamoto,1 Ichiro Nakano,2 Kazuo Shin-ya,3 Kazuo Nagasawa,4 Hiroyuki Seimiya1. 1 _Japanese Foundation for Cancer Research, Tokyo, Japan;_ 2 _University of Alabama at Birmingham, Birmingham, AL;_ 3 _National Institute of Advanced Industrial Science and Technology, Tokyo, Japan;_ 4 _Tokyo University of Agriculture and Technology, Tokyo, Japan_.

Among heterogeneous cell population in a tumor, cancer stem cells (or tumor-propagating cells) are defined as a cell fraction that shows self-renewal, multipotency, high tumorigenicity, and resistance to chemo/radiotherapy. Because cancer stem cells are supposed to drive initiation, metastasis, and recurrence of the disease, identification of their therapeutic targets would have a profound implication for eradication of cancer. G-quadruplex (G4) is an atypical four-stranded nucleic acid structure that can be formed at guanine-rich sequences, such as the telomeric TTAGGG repeats. Accumulating evidence supports that G4s are widely distributed through the genome in living cells and affect various intracellular events, including DNA replication, gene expression and translation, whereas pharmacologic stabilization of G4s has been implicated for cancer intervention. However, precise mechanisms for the efficacy and target cancer types remain elusive. Here we demonstrate that G4 stabilization by chemical compounds, called G4 ligands, preferentially inhibits the growth of glioma stem cells in culture and in vivo. While the established glioma stem cells maintain stemness under the serum-free sphere culture conditions, serum stimulation induces their differentiation into non-stem glioma cells. We found that these glioma stem cells are highly sensitive to G4 ligands, such as a natural compound telomestatin and its newly synthesized derivative, and a bisquinolinium compound Phen-DC3, all of which can stabilize G4s. Upon the ligand treatment, glioma stem cells activated the replication stress pathway more potently than non-stem glioma cells, which could partly explain the selectivity of the deleterious effect. Consistently, these ligands induced DNA damage response in glioma stem cells but not in non-stem glioma cells. In glioma stem cells, about 20% to 30% of DNA damage foci were telomeric (i.e., telomere dysfunction-induced foci: TIFs) whereas the rest were non-telomeric, suggesting that the G4 ligands recognized both telomeric and non-telomeric G4s. Temozolomide, a clinical alkylating agent for brain tumors, induced DNA damage response but did not preferentially attack telomeres. As a potential pharmacodynamic biomarker, the immunofluorescence intensities of the nuclear G4 foci were enhanced by G4 ligands. Furthermore, these ligands directly inhibited in vitro transcription and translation of mRNAs that contained G4-forming sequences, suggesting additional mode of action. While G4 ligands inhibit telomerase activity, classical telomerase inhibitors without G4-stabilizing activity had no preferential impact on glioma stem cells. Together, these observations suggest that G4 is a promising target for pinpointing intractable glioma stem cells.

#4840

APTO-253 is a new addition to the repertoire of drugs that can exploit DNA BRCA1/2 deficiency.

Cheng-Yu Tsai,1 Si Sun,1 Hongying Zhang,2 Andrea Local,2 William Rice,2 Stephen B. Howell1. 1 _USCD Moores Cancer Ctr., La Jolla, CA;_ 2 _Aptose Biosciences, Inc., San Diego, CA_.

APTO-253 is a small molecule with anti-proliferative activity against cell lines derived from a wide range of human malignancies. The goal of this project was to investigate the mechanism of action so as to identify synthetic lethal interactions that can guide combination drug studies. APTO-253 was found to convert intracellularly to a complex containing one molecule of iron and three molecules of APTO-253 [Fe(253)3] using LC/MS/ESI analysis. The intracellular content of Fe(253)3 exceeded that of the native drug by ~18-fold, and Fe(253)3 appears to be the the most active form. The structure of Fe(253)3 is similar to drugs that stabilize quadruplex DNA (G4) structures, and Fe(253)3 was found to stabilize G4s in vitro and to reduce the expression of Myc. Stabilization of G4 structures has been reported to produce DNA strand breaks, which led us to investigate whether APTO-253 caused DNA damage. Indeed, treatment with APTO-253 produced time and concentration-dependent γH2AX foci formation in an array of diverse cell lines. APTO-253 was also found to produce DNA double strand breaks, as detected by the neutral comet assay, which led us to ask if cells deficient in homologous recombination (i.e., loss of BRCA1/2 function) were hypersensitive to this drug in a manner similar to the effects of PARP inhibitors such as olapirib. BRCA1-deficient sublines of 3 isogenic pairs of cell lines (MCF10A, MCF7 and hTERT-IMEC), and BRCA2-deficient sublines of 2 isogenic pairs (PEO1, HCT116/B18 and HCT116/B43) were all hypersensitive to olaparib and APTO-253. Thus, loss of either BRCA1 or BRCA2 function renders malignant cells hypersensitive to APTO-253. When parental HCT116 and the BRCA2-deficient B18 subline were treated with APTO-253 for 24 h, there were significant increases in γH2AX and cleaved PARP only in B18 clone, suggesting BRCA2 is required to repair DNA damage generated by APTO-253. We report here that APTO-253 is converted intracellularly into an Fe complex, and that this Fe(253)3 complex is likely the active form of the drug. APTO-253 stabilizes certain quadruplex DNA structures, causes DNA damage, and exhibits synthetic lethality comparable to olaparib, albeit through a different mechanism, in cells deficient in either BRCA1 or BRCA2 function. Unlike other drugs for which loss of this repair function results in hypersensitivity, APTO-253 does not produce myelosuppression even at the maximum tolerated dose. The observations reported here also identify γH2AX as a potential biomarker of clinical effect and open the window more detailed studies of how APTO-253 promotes DNA damage and how it might be used clinically to treat patients with tumors harboring deficiencies in DNA repair.

#4841

Prostate cancer cell death triggered by a small molecule interacting with the hTERT G-quadruplex.

Jin H. Song, Libia A. Luevano, Hyunjin Kang, Vijay Gokhale, Laurence H. Hurley, Andrew S. Kraft. _University of Arizona, Tucson, AZ_.

Human telomerase reverse transcriptase (hTERT), a catalytic subunit of telomerase, is widely overexpressed in human cancers including prostate cancer. Along with its regulation of the telomere, hTERT was also shown to regulate various signal transduction mechanisms involving DNA damage response, cell cycle checkpoint, and apoptosis. The majority of human solid tumors display telomerase activity, which was found to be well correlated with hTERT expression, and hTERT inhibition led to reduced telomerase activity and telomere length. However, approaches that directly inhibit the action of telomerase have not produced a therapeutically useful response because of long lag times for subsequent cell division to produce telomere shortening. Alternatively, recent studies demonstrated that this delay problem can be solved through downregulation of hTERT transcription. It was shown that telomeric DNA is capable of folding into four-stranded guanine quadruplex (G4) structures, which then lock the hTERT promoter activity that leads to robust telomere shortening. Our new approach has focused on the discovery of small molecules that could stabilize G-quadruplexes by interacting with the telomeric G-rich overhang stem loop, producing a lead candidate compound that enhances the kinetic folding rate of the G4 silencer element. Our in vitro studies demonstrate that this singular mechanism of action permitted reduced telomerase production and telomere shortening. Significantly, hTERT downregulation with the identified molecule caused prostate cancer cell death, which was characterized by activation of the caspase cascade and increased Annexin V- positive cells. In contrast, this compound did not harm normal prostate epithelial cells. Similarly, mouse prostate cancer cells that do not contain a G-quadruplex were not killed, demonstrating that the unique mechanism of action is mediated through the hTERT promoter G-quadruplex element. Our in vivo study demonstrated that administration of this significantly delayed tumor growth on xenografted mice. The rapid onset of cell death by G4 stands in contrast to the delayed action of classic telomerase inhibition. Therefore, our current study provides important data to support developing inhibitors of hTERT as a cancer drug target.

#4842

A comparative preclinical study of PARP inhibitors demonstrates superb properties for IDX-1197.

Myongjae Lee,1 Joon-Tae Park,1 Yoon Suk Lee,1 An-Na Moon,1 Dong-Gu Jeong,1 Jeong-Ah Kim,1 Ji-Hoon Yang,1 Dohee Kim,1 Jeongcheol Shin,1 In-Gyu Je,1 Kyungsun Kim,1 Hong-Sub Lee,1 Nam Seok Baek,2 Sungsook Lee,2 Hun-Kyo Kim,2 YongMan Kim,3 Jung Yong Kim,2 Soobong Park1. 1 _Ildong Pharmaceutical, Hwaseong-si, Republic of Korea;_ 2 _National OncoVenture, Goyang-si, Republic of Korea;_ 3 _Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea_.

Background PARP inhibitors have demonstrated clinically meaningful increase in progression-free survival as a single agent in women with recurrent ovarian cancer following a response to platinum-based chemotherapy. We aimed to develop a novel PARP inhibitor that may have potent antitumor efficacy. IDX-1197 is a novel, potent, selective, and orally bioavailable poly (ADP-ribose) polymerase (PARP)-1/-2 inhibitor in clinical development. This study examined the pharmacokinetics (PK), pharmacodynamics (PD), and efficacy of IDX-1197 as compared to approved PARP inhibitors in several preclinical models.

Material and Methods In vitro assays were conducted to evaluate IDX-1197 as a PARP inhibitor using PARP enzyme assay and PARP-catalytic inhibition assay. In vivo efficacy and PK/PD of IDX-1197 against tumor growth were evaluated using xenograft models. To investigate PARP inhibition in tumors, tumor PAR levels were measured by ELISA in the xenograft models. The concentrations of IDX-1197 in plasma and tumor were determined using LC-MS/MS method.

Results IDX-1197 potently inhibited PARP-1 and PARP-2 enzymes with IC50 of 1.4 and 1.0 nM, respectively, while not sensitive to PARP-5 (Tankyrase-1) which is linked to toxicities including severe gastrointestinal effects. In the cell viability assays in human cancer cell lines, IDX-1197 was obviously superior to the other PARP inhibitors. In the single agent colony forming assays in human cancer cell lines, 15 of 18 (83.3 %) cells were sensitive to IDX-1197, while 10 of 18 (55.6 %) cells responded to Olaparib. In the xenograft model, oral administration of IDX-1197 exhibited significant PAR inhibition in tumor until 24 hr post dose. IDX-1197 also dose-dependently led to potent tumor growth inhibition compared to Olaparib treatment group.

Conclusions IDX-1197 administration induces potent antitumor activities in multiple preclinical models. The potent antitumor activity induced by IDX-1197 is consistent with its high exposure and durable PARP inhibition in tumor. These preclinical data demonstrate the efficacy of IDX-1197, which has the potential for a best-in-class profile. Based on these findings, IDX-1197 is under clinical Phase 1 trials in Republic of Korea. Funded by National OncoVenture.

#4843

**NMS-P293, a PARP-1 selective inhibitor with no trapping activity and high CNS penetration, possesses potent** in vivo **efficacy and represents a novel therapeutic option for brain localized metastases and glioblastoma.**

Alessia Montagnoli, Gianluca Papeo, Sonia Rainoldi, Francesco Caprera, Marina Ciomei, Eduard Felder, Daniele Donati, Antonella Isacchi, Arturo Galvani. _Nerviano Medical Sciences, Nerviano, Italy_.

NMS-P293 is a novel, potent and selective small molecule inhibitor of poly (ADP-ribose) polymerase (PARP-1 Kd = 2 nM).

NMS-P293 inhibits the poly-ADP ribosylation (PAR) induced by DNA damage, leading to the inability of homologous repair deficient (HRD) tumor cells to repair damage and to apoptotic death, while sparing normal cells.

NMS-P293 significantly differs from other PARP inhibitors in several respects. Firstly, while possessing potent in vitro and in vivo efficacy in HRD tumors (e.g. BRCA, PTEN loss), it does not induce "DNA trapping", i.e. the formation of PARP-chromatin complexes. Secondly, NMS-293 is not a substrate of PgP mediated drug efflux, believed to be a mechanism of resistance to current PARP inhibitors. Finally, NMS-P293 extensively crosses the blood brain barrier in rats and mice, with a brain/plasma ratio of 4-10, significantly higher than that of major comparators and opening the potential for treating primary brain tumors and brain metastasis with PARP inhibitor therapy. Extensive preclinical profiling revealed that NMS-P293 possesses favourable drug-like characteristics, which include high in vitro cross-species metabolic stability, lack of cytochrome interaction, and excellent pharmacokinetic profile, with low clearance and nearly complete oral bioavailability in both rodents and non rodents. Preclinical efficacy and PK/PD studies were conducted to assess the mechanism of action of NMS-P293 in an HRD setting, the MDA-MD-436 (BRCA1 mutant) human breast cancer xenograft model. Inhibition of PAR was observed in tumors following single oral administration of 50 mg/kg NMS-P293, with a > 95% decrease of this biomarker persisting for > 24 h. Inverse correlation was observed between intratumoral PAR levels and NMS-P293 concentrations in plasma and in tumor. Significantly, levels of NMS-P293 in tumor were consistently higher (> 3.5-fold) than in plasma, with an intratumoral concentration of > 2 μM at 24 h, in line with the prolonged PAR inhibition. In agreement with these PD effects, NMS-P293, orally administered as single agent, induced potent dose-related growth inhibition of BRCA mutated tumor model, with cured animals. Furthermore, NMS-P293 also possesses potent synergistic efficacy and tolerability in combination with temozolomide (TMZ) in glioblastoma (GBM) tumor models, including TMZ resistant MGMT hypomethylated GBMs.

The unique preclinical features of NMS-P293, which includes a highly permissive profile in GLP drug safety studies, render this compound an appealing candidate for clinical development, both as a single agent and in drug combination therapy regimes.

#4844

Evidence of PARP-1 dependent cytotoxic agents with low target affinity.

Laura Puentes, Sean Reilly. _University of Pennsylvania, Philadelphia, PA_.

Introduction: Inhibition of nuclear protein poly (ADP-ribose) polymerase-1 (PARP1) is synthetically lethal in BRCA mutated cancers. However, the mechanism by which PARPi exert cytotoxic effects is not fully understood. In this study, we compare 16 olaparib analogs and measured their potency in a radioligand binding assay to measure their affinity for PARP1, and in a cytotoxicity assay to measure their ability to serve as a chemotherapeutic.

Methods: Compounds were prepared by replacing the piperazine core on olaparib with a series of diazaspiro ring systems. PARP1 affinity was assessed using radioligand binding competition assays. In vitro cytotoxicity was performed on BRCA1 methylated and PARP1-/- ovarian cancer cell lines to evaluate the relative potency of the analog compounds.

Results: The 16 analogs displayed lower affinity to PARP1 but comparable or higher potency in cell kill assays when compared to olaparib. Of the 16 compounds, those that contained the diazaspiro core with an N-Boc-functional group displayed the highest degree of potency. Cytotoxicity assays were performed for each compound using PARP1 -/- and PARP2 -/- cells to assess off-target effects. Dose response curves for PARP1-/- cells resulted in a rightward shift; no changes in the dose-response curves were detected for PARP2 -/- cells.

Conclusion: Radioligand binding assays demonstrated that these compounds have low PARP1 affinity. Evaluation of the relative potency of these compounds revealed that compounds having a lower PARP1 affinity relative to olaparib still retained a high potency in cell kill assays. In vitro cytotoxicity assays on PARP1 -/- and PARP2 -/- cells confirmed that these compounds are PARP1 but not PARP2 dependent. Future studies will focus on assessing catalytic inhibition of PARP1 and PARP1 trapping in order to better elucidate the antiproliferative effects of these compounds.

#4845

Preclinical evaluation of HWH340, a novel PARP inhibitor.

Wenjie Sun,1 Yang Yue,1 Hailiang Chen,1 Ronghua Tu,1 Jie Shen,1 Gang Li,2 Guoqiang Ma,3 Zhixiang Zhang,3 Charles Z. Ding,3 Shuhui Chen3. 1 _Humanwell Healthcare Group Co.,Ltd, Wuhan, China;_ 2 _WuXi AppTec (Wuhan), Wuhan, China;_ 3 _WuXi AppTec (Shanghai), Shanghai, China_.

Objective: Poly-ADP-ribose polymerase (PARP) participates in DNA repair, replication and transcription, and is important to maintaining genome stability and integrity. PARP inhibitors have proven to be efficacious in the clinic to treat BRCA-deficient cancers. PARP inhibitors may also be used as sensitizing agent in combination with other antitumor agents. The purpose of this study is to investigate in preclinical models the antitumor activity of HWH340, a novel potent and selective PARP inhibitor currently undergoing clinical investigation.

Method: HWH340 was evaluated for its PARP1/2 enzymatic activity in corresponding enzyme assays and its cellular PARylation inhibitory activity in HCC1937 cells. The anti-proliferative activity and the synergistic effect in combination with temozolamide (TMZ) were evaluated in vitro in a BRCA-deficient cell line MDA-MB-436 and BRCA proficient cell line MDA-MB-231. Antitumor activity was evaluated by daily oral administration of in xenograft tumor models. Synergistic effect in combination with TMZ was also determined. The animal models used were PARP highly expressed patient-derived xenograft (PDX) model of small cell lung cancer (SCLC) LU-01-0547 and BRCA1/2-deficient CDX models of breast cancer MDA-MB-436 and pancreatic cancer CAPAN-1.

Result: HWH340 displayed potent PARP1/2 enzymatic inhibitory activity with IC50 1.14 nM and 1.72 nM respectively. It also potently inhibited cellular PARylation in HCC1937 cell with IC50 2.77 nM. In BRCA proficient cell line MDA-MB-231, HWH340 showed no synergistic effect when combined with TMZ and a weak anti-proliferative activity with IC50 5.83 uM and 4.86 uM. In contrast, it displayed potent anti-proliferative activity and a synergistic effect in combination with TMZ with IC50 of 71 nM and 7 nM in the BRCA1/2-deficient MDA-MB-436 cells, respectively. HWH340 showed excellent in vivo antitumor efficacy in PARP overexpressed SCLC LU-01-0547 PDX model (TGI=81.69%@50mpk, QD). In BRCA-deficient breast cancer MDA-MB-436 CDX model, HWH340 showed potent antitumor efficacy as single agent (TGI=122% @12.5mpk, QD). In BRCA-deficient pancreatic cancer CAPAN-1 CDX model, it showed potent antitumor efficacy in combination with TMZ (TGI=79.61% @25mpk+ TMZ 30mpk, QD).

Conclusion: HWH340 is a potent PARP inhibitor. Its antitumor activity in the preclinical tumor models together with its excellent pharmaceutical properties provide confidence that HWH340 may have high potential in the clinic for treatment of BRCA deficient solid cancers.

#4846

**Targeting** BRCA1 **-mutated cancer cells with elesclomol.**

Maria Kammire,1 Emily Fannin,1 James M. Ford,2 Elizabeth Alli1. 1 _Wake Forest School of Medicine, Winston-Salem, NC;_ 2 _Stanford University School of Medicine, Stanford, CA_.

The breast cancer susceptibility gene 1 (BRCA1) encodes a tumor-suppressor protein that maintains genetic stability through DNA damage response pathways. Specifically, BRCA1 has a well-established role in double-strand DNA break repair, and emerging evidence supports a role for BRCA1 in repair of oxidative DNA damage via the base-excision repair (BER) pathway. We and others have shown that cells with mutant/deficient BRCA1 harbor defective BER of oxidative DNA damage. Interestingly, defective BER of oxidative DNA damage in breast cancer cells rendered sensitivity to elesclomol, an experimental therapeutic that induces reactive oxygen species to levels beyond a viable threshold. Given that BRCA1-mutated cancers associate with a poor prognosis and are in need of targeted treatment options, we hypothesized that breast or ovarian cancer cells with mutant BRCA1 would exhibit selective sensitivity to elesclomol or treatment regimens containing elesclomol. First, we determined the effect of BRCA1 on the cellular response to oxidative stress and elesclomol using human ovarian cancer cells isogenic for BRCA1. Compared to mutant BRCA1 cells (UWB1.289), wild-type BRCA1 cells (UWB1.289+BRCA1) produced significantly greater levels of BRCA1 mRNA (p=0.009), expressed markedly higher levels of BRCA1 protein in the nucleus, and showed significantly greater sensitivity to hydrogen peroxide (p=0.002) as evidenced by RTqPCR, Western blot analysis, and MTT assay, respectively. Importantly, mutant BRCA1 cells displayed an approximate 100-fold greater sensitivity to elesclomol compared to wild-type BRCA1 cells as determined by MTT assay. These data suggest that the oxidative stress induced by elesclomol may be used to selectively target cancer cells with mutant BRCA1 due to their defective BER phenotype. We next asked whether elesclomol may be combined with other agents used clinically or experimentally for the treatment of BRCA1-mutated cancers, including standard chemotherapy drugs and PARP inhibitors. Elesclomol exhibited synergistic sensitivity when combined with DNA-damaging agents, including doxorubicin or cisplatin, in breast cancer cells with mutant BRCA1 (SUM149) as determined by combination index and isobologram analysis. On the other hand, a synergistic effect was not observed with the anti-microtubule agent paclitaxel, which is consistent with a phase III study that evaluated this combination for the treatment of advanced melanoma. However, elesclomol in combination with PARP inhibitors, including rucaparib or talazoparib, indeed showed synergism in breast cancer cells with mutant BRCA1. These data suggest that elesclomol may be used in combination with other anticancer agents to further exploit defects in DNA repair due to mutant BRCA1. In conclusion, elesclomol may be explored as a novel component of treatment regimens for targeting BRCA1-mutated breast and ovarian cancers.

#4847

Evaluation of the combination of olaparib and cediranib in small-cell lung cancer cells.

Naoto Morikawa, Monique B. Nilsson, Irene Guijarro, You-Hong Fan, Alissa Poteete, John V. Heymach. _UT MD Anderson Cancer Center, Houston, TX_.

Metastatic Small cell lung cancer (SCLC) is exceptionally lethal, having a median survival of 9-10 months from the time of diagnosis and a 5 year survival rate of less than 2 percent. More effective treatment approaches to SCLC are desperately needed. Poly ADP-ribose polymerase (PARP) inhibitor such as olaparib exploit the concept of synthetic lethality by selectively targeting cancer cells with defective DNA repair pathways. Preclinical data indicates that SCLC cell lines overexpress PARP and are sensitive to PARP inhibition, and PARP inhibitors show promise in the clinical setting of SCLC. The identification of combination regimens that potentiate the efficacy of PARP inhibition would be of great clinical value. Recent findings indicate that olaparib and the vascular endothelial growth factor receptor (VEGFR) inhibitor, cediranib, is an effective therapeutic combination in patients with BRCA wild-type ovarian cancer. Therefore, we evaluated the efficacy of the olaparib and cediranib combinations in SCLC models. Human SCLC cell lines, NCI-H841, H526, COR-L24 and murine SCLC cell line TKOmTmG were sensitive to olaparib or cediranib therapy in vitro as determined by Cell Titer Glo® assay. Moreover, a significantly greater effect on cell viability was observed when olaparib and cediranib were used in combination. Likewise, in H841 xenograft models, single agent treatment of olaparib or cediranib inhibited tumor growth, and combination treatment of olaparib plus cediranib induced tumor regression. Western blot analysis indicated that treatment with cediranib or the combination of olaparib and cediranib induced PD-L1 protein expression both in vivo and in vitro. In TKOmTmG xenograft model in syngeneic mice, programmed death-ligand 1 (PD-L1) inhibitor plus olaparib or olaparib/cediranib combination resulted in superior combined efficacy compared to the respective single agent therapies in terms of tumor growth inhibition. These data indicate that olaparib plus cediranib may be an effective therapeutic strategy for the treatment of SCLC. Further study is warranted to understand the mechanism of action and clinical activity.

#4848

**Preclinical antitumor activity of novel** DNA polymerase 1 (POLA1) **inhibitors.**

Claudio Pisano,1 Lucio Merlini,2 Sergio Penco,3 Raffaella Cincinelli,2 Nadine Darwiche,4 Mario B. Guglielmi,1 Ilaria La Porta,1 Giacomo Signorino,1 Gabriele De Rubis,1 Fabiana Colelli,1 Francesco Cardile,1 Alessandra Fucci,1 Egildo L. D'Andrea,1 Sabrina Dallavalle2. 1 _Biogem, Centro Ricerche, Ariano Irpino (AV), Italy;_ 2 _University of Milan, Milan, Italy;_ 3 _Ronzoni Institute for Chemical and Biochem Research, Milan, Italy;_ 4 _American University of Beirut, Beirut, Lebanon_.

The anti-proliferative and pro-apoptotic effects of Retinoid-Related Molecules (RRMs) are described as independent from Retinoids' receptors-mediated transcriptional activity. Prototypes of this class are CD437 and its more potent analogue ST1926, which have a strong antitumor activity by targeting DNA polymerase 1 alpha (POLA1) (Han et al. Nat Chem Biol. 2016; Abdel-Samad et al. AJCR, in press).

With aim to identify new RRMs with an improved pharmacological profile, we synthetized and screened a library of RRMs for their antitumor properties and inhibitory activity on POLA1. From this screening, four molecules, MIR002, MIR020, MIR072 and MIR075, were selected. All exert a potent anti-proliferative effect, with G1/S arrest and apoptosis, in more than 50 cancer cell lines derived from human hematological and solid tumors. From a mechanistic point of view, these RRMs modulate, at different extent, POLA1 activity and/or expression.

Notably, NSCLC cells harboring POLA1-L764F mutation (H460-R9A), are resistant to both CD437 and ST1926 (IC50>50 higher than the one of wild type H460 cells), while they are sensitive to MIR002, showing for this RRM-derivative an improved/different pharmacological profile with respect to CD437 and ST1926.

Hints on the possibility of these new RRMs to be orally absorbed were obtained using cancer cells overexpressing or not P-glycoprotein (Pgp), known as the major player limiting the oral absorption of several chemotherapeutic agents. Results from these experiments revealed that the Tested compounds are not Pgp substrate thus suggesting the possibility for their absorption via oral route

MIR002 in vivo activity was assessed in tumors from Malignant Mesothelioma derived cells (MM487), and lung cancer cells (H460 and H460-R9A). In all the evaluated models, MIR002 induced a strong Tumor Growth Inhibition either alone or in combination with cisplatin (TGI>61% and TGI>80-100%, respectively).

The treatment of orthotopic models of malignant mesothelioma (MM487) with MIR072 in combination with Cisplatin, resulted in a impressive synergic antitumor activity if compared to Cisplatin monotherapy (TGI 95% vs 55%).

Tests on orthotopic transplants of hepatocellular carcinoma cells (HepG2), showed that MIR020 has significant antitumor effects (TGI 72%).

Finally, the activity of MIR075 was evaluated on glioblastoma luciferase-expressing cells (U-87MG) intracranically injected. Also this compound displayed a significant tumor growth inhibition (TGI 72%), as measured by IVIS imaging system.

Taken together, the results from in vitro and in vivo experiments indicate that this new class of RRMs, including MIR002, MIR072, MIR020, and MIR075, modulate POLA1 functions and activate pro-apoptotic pathways. The large spectrum of antitumor activity, together with the high tolerability observed, opens the possibility for their clinical investigation in different population of cancer patients.

#4849

Targeting the lethal pediatric atypical teratoid/rhabdoid tumors with the DNA minor-groove binding agent quinacrine.

Harpreet Kaur, Huizi Guo, Charles G. Eberhart, Eric H. Raabe. _Johns Hopkins University, School of Medicine, Baltimore, MD_.

Atypical teratoid/rhabdoid tumors (AT/RT) are rare, incurable, and highly proliferative pediatric brain tumors. Discovering new targets and developing novel therapeutics are urgently needed for this devastating tumor. We have previously shown that AT/RT tumors and cell lines express increased amounts of the epigenetic modifier high mobility group AT-hook 2 (HMGA2). HMGA2 is a DNA-binding oncoprotein that regulates transcription during normal embryogenesis and in cancer stem cells. Targeting HMGA2 using short hairpins significantly decreased AT/RT cell growth and increased survival of xenografted mice in our studies. We hypothesized that pharmacological inhibition of HMGA proteins using DNA minor-groove binding drugs will decrease growth of AT/RT cell lines due to displacement of HMGA proteins from the DNA. We used the minor-groove binding agent quinacrine to test our hypothesis in 3 different AT/RT cell lines (BT37, CHLA-06 and CHLA-04). Quinacrine has been used in millions of humans to treat malaria and other parasitic infections and has a well known safety profile. Quinacrine penetrates the brain, and we can achieve micromolar levels of quinacrine in brain after oral administration. Quinacrine causes a dose-dependent reduction in AT/RT cell growth (MTS assay) and proliferation (BrdU incorporation) compared to vehicle-treated cells (P<0.05). Additionally, treatment of AT/RT cells with quinacrine significantly increased apoptotic cell death (increased cleaved caspase-3 and cleaved PARP expression) in a dose-dependent manner compared to vehicle-treated cells (P<0.05). Our results suggest that minor groove binding drugs like quinacrine are a viable potential treatment strategy for AT/RT. Future studies are aimed at testing the in vivo efficacy and validating the mechanism of action of quinacrine in AT/RT.

#4850

The minor groove binding agent quinacrine inhibits growth and increases apoptotic death in diffuse intrinsic pontine glioma tumor cells.

Huizi Guo, Harpreet Kaur, Sepehr Akhtarkhavari, Charles G. Eberhart, Eric H. Raabe. _Johns Hopkins University, Baltimore, MD_.

Diffuse intrinsic pontine glioma (DIPG) is an invasive, incurable and aggressive pediatric brain tumor found in the brainstem. Improved understanding of the biology of DIPG tumors is urgently needed to develop novel treatments. Our previous studies have shown that DIPG tumors and cell lines express high levels of the DNA-binding stem cell factor high mobility group AT-hook 2 (HMGA2).. Targeting HMGA2 using lentiviral shRNA decreased DIPG cell invasion, proliferation and increased apoptosis in our studies. We hypothesized that inhibiting HMGA2 using DNA minor groove binding drugs like quinacrine would decrease DIPG proliferation and increase apoptotic cell death. We used three DIPG cell lines (JHHDIPG1, JHHDIPG16A, and SUDIPG13) to test the effect of quinacrine. Quinacrine has traditionally been used as an anti-malarial drug and is known to strongly bind to DNA. We used BrdU incorporation as a measure of proliferation and expression of cleaved caspase-3 (CC-3) as a measure of apoptosis. Treatment of DIPG cells with quinacrine showed a dose dependent increase in cell death (CC-3 expression) from 3uM to 20uM, with the most effective doses being from 3uM to 5uM. In all three DIPG cell lines, treatment with 1uM to 5uM quinacrine showed a significant reduction in cell proliferation (BrdU) from 3uM to 5uM compared to vehicle-treated cells (P<0.0001). Additionally, treatment of DIPG cell lines with 1uM to 5uM quinacrine showed a significant increase in apoptotic cell death (CC-3 expression) compared to vehicle control cells from 3uM to 5uM (P<0.001). Our data suggests that minor groove binding agents like quinacrine could be effective therapies for this lethal brain tumor. Our future studies are aimed at testing the efficacy of quinacrine in inhibiting DIPG tumorigenicity and elucidating the mechanism of action.

#4851

Potent cell cycle inhibitors suitable for treatment of multidrug-resistant tumors.

Mohan Sivaraja, Sivan Sizikov, Nilantha Sirisoma, Tamari Kirtadze, Madhuri Chattopadhyay, Makena Ewald, Subhadra Dash, Anne Wong, Georg Neckermann, Elaine To, Stephanie Chang, Timothy P. Shiau, David C. Williams, Kevin M. Short, Angels Estiarte, Anirban Datta, David B. Kita. _Verseon, Fremont, CA_.

We have developed a class of small-molecule cytotoxic compounds that are highly potent in an in vitro tubulin polymerization assay. Preclinical studies show that these drug candidates are potent against a range of cancer cell lines and may be well suited for the treatment of hematologic or solid tumors resistant to existing chemotherapy agents. We will present data on representative members of this class that inhibit cancer cell growth at nanomolar concentrations and are potent against a variety of cancer cell lines, including liver, breast, ovarian, and lung. The compounds induce cell cycle arrest in the G2M state within 24 hours with cell death occurring over multiple days. These tubulin inhibitors also behave as potent antiangiogenesis agents and inhibit endothelial tube formation in HUVEC cells at nanomolar concentrations. Functional assays using efflux pump inhibitors show that, compared to many major chemotherapy agents, our class of drug candidates is significantly less affected by the efflux pumps MDR1 and MRP1, which are commonly overexpressed as a mechanism of multidrug resistance. Our compounds show comparable potency against wild-type and cell lines developed to be resistant to other cancer drugs, while the potency of clinically relevant compounds such as paclitaxel or doxorubicin is reduced between 80x and about 3000x in the same assay. This feature suggests that our tubulin inhibitors may be active against tumors that are resistant to common cancer drugs. Overexpression of the β-III isotype of tubulin, which is known to occur in many aggressive and metastatic tumors, is another clinically relevant mechanism of resistance to microtubule-targeting anticancer agents. It has been correlated with significantly lower response to docetaxel-based chemotherapy in a number of cancers and is considered an indicator of resistance to paclitaxel and vinorelbine. We will present preclinical data showing that our compounds have similar activity against cells showing normal levels and those highly overexpressing β-III tubulin. The compounds display good in vitro physicochemical properties as well as favorable in vivo pharmacokinetics. In addition, we will present in vivo tolerability and efficacy data in mice. The ability of this class of tubulin inhibitors to maintain their efficacy across multiple drug-resistant cancer cell lines makes them attractive candidates for development as chemotherapy agents. In particular, a new anticancer agent that is less susceptible to major transporters and retains its potency when β-III tubulin is overexpressed could lead to more effective precision second-line therapy.

#4852

Induction of HRD by targeting G9a and MTDH in DNA repair proficient endometrial cancer cells.

Xiangbing Meng,1 Shujie Yang,1 Jianling Bi,1 Yiyang Li,1 Yuping Zhang,1 Sudartip Areecheewaku,2 Mary Li,1 Zihan Wang,1 Aliasger K. Salem,2 Kimberly K. Leslie1. 1 _Univ. of Iowa College of Medicine, Iowa City, IA;_ 2 _Univ. of Iowa College of Pharmacy, Iowa City, IA_.

The homologous recombination repair (HRR) pathway is required for high-fidelity repair of double-strand DNA breaks. Cancer cells with HRR deficiency due to somatic BRCA1 or BRCA2 mutations are sensitive to platinum agents and targeted therapy with PARP inhibitors. Endometrial cancer is the most common cancer of the female reproductive system. DNA damage agents are commonly used in treatment but tumors often develop resistance. Metadherin (MTDH) has been shown to be amplified in a wide range of solid tumors as well as in leukemia and lymphoma. Overexpression of MTDH is correlated with angiogenesis, metastasis, and chemoresistance. Histone lysine N-methyl-transferase G9a catalyzes the demethylation of histone H3 at lysine residue 9 (H3K9) resulting in a repressive histone modification. The objective of this study is to validate the function of MTDH and G9a in HRR and test the potential to target MTDH or G9a to increase drug sensitivity. p21 expression was induced when G9a was depleted. DNA damage induced MTDH expression at 4 hours after 8Gy γ-irradiation in the well validated endometrial adenocarcinoma cell line Ishikawa H. However, induction of MTDH protein expression by the same radiation dosage was reduced when G9a was depleted. The G9a inhibitor UNC0642 itself can also induce the expression of MTDH after 24 hours treatment. Ishikawa parental cells and two Ishikawa sub-cell lines in which G9a was depleted by CRISPR were tested for the DNA damage biomarker γH2Ax and Rad51 foci formation at 4 hours following 8Gy irradiation. G9a depletion was shown to decrease Rad51 foci formation and to increase the formation of γH2Ax foci. Two natural products, Celastrol and Pristimerim were found to inhibit MTDH, FANCD2 and FANCI protein levels, which may be used to induce BRCA-ness in homologous recombination repair proficient cancer cells. G9a depletion results in the reduction of irradiation-induced Rad51 foci formation and an increase of irradiation-induced γH2Ax foci formation. We suggest that G9a depletion can increase cancer cell sensitivity to DNA damage agents. MTDH is induced by DNA damage and is a G9a inhibitor. MTDH depletion causes reduction of irradiation-induced Rad51 foci. This study is supported by the University of Iowa Department of Obstetrics and Gynecology Research Development Fund, NIH grant R01CA99908 and R01CA184101.

#4853

Regulation of proteolytic repair of Top2 covalent complexes.

Yilun Sun, Karin C. Nitiss, John L. Nitiss. _Univ. of Illinois College of Pharmacy, Rockford, IL_.

DNA topoisomerases (topos) play an essential role in nuclear processes such as replication, transcription, and chromosome segregation. These enzymes regulate DNA topology by introducing transient breaks in DNA with formation of transient protein/DNA covalent complexes. These complexes can be trapped by aberrant DNA structures, and are also the basis for anti-cancer drugs that target topos. Since topos can be trapped on DNA, leading to strand breaks with protein adducts at the site of the breaks, cells require DNA repair pathways to process protein adducts. We have examined the repair of the type II topos (Top2) in yeast and mammalian cells. We developed a sensitive assay to detect ubiquitylation and other post-translational modifications (PTMs) of Top2 trapped on DNA in S. cerevisiae and have shown that the proteasome plays a key role in repair of Top2 damage induced by the anti-cancer drug etoposide. We demonstrated that sumoylation of Top2 is induced by etoposide, and that deletion of the SUMO ligase Siz2 prevents etoposide induced sumoylation. Deletion of either of the genes encoding the ubiquitin ligase Slx5/Slx8 led to decreased ubiquitylation of trapped Top2. The mammalian ortholog of Slx5/Slx8 is Rnf4, and it too plays a role in ubiquitylation and degradation of Top2 trapped by etoposide. A human cell line completely lacking Rnf4 is defective in ubiquitylation of Top2β, one of the two mammalian Top2 isoforms. Interestingly, repair of the Top2α isoform is unaffected in an Rnf4 knockout cell line. Proteolysis of trapped Top2 is also subject to negative regulation. The UBA domain protein UBAP2L, (also termed NICE4) is amplified in various cancers. Knockout of the UBAP2L increases proteolysis of trapped Top2. Levels of trapped Top2α and Top2β are decreased in the absence of UBAP2L, and the level of trapped Top2 returns to the wild type level when MG132 is added, indicating UBAP2L regulates the proteasome mediated degradation of Top2. UBAP2L physically interacts with Top2β as determined by co-immunoprecipitation, and the interaction is not seen in Rnf4 knockout cells. Finally, knockout of UBAP2L confers high level resistance to etoposide. Our results provide insights to the response of cancer cells to Top2 targeting agents.

#4854

Targeting ribonucleotide reductase M2 using didox causes inhibition of estrogen receptor-negative, inflammatory breast cancer cell proliferation and tumor emboli formation in culture.

Pranalee Patel, Ben Mirman, Renzhi Zhan, Gayathri R. Devi. _Duke Univ. Medical Ctr., Durham, NC_.

Ribonucleotide reductase (RR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides during the rate-limiting step of DNA synthesis. Given its significant role in cell cycle regulation, RR and its catalytic subunits are significant molecular targets for inhibiting tumor cell growth and proliferation. Didox (3,4-dihydroxybenzohydroxamic acid) is a known inhibitor of the catalytic RR subunit of RRM2 and overall DNA synthesis. Previous meta-analyses have revealed that RRM2 is highly expressed in estrogen receptor-negative (ER-) breast cancer tumors. As ER negativity is frequent in inflammatory breast cancer (IBC), a distinct and the most aggressive breast cancer subtype, we proposed to evaluate efficacy of didox in IBC cell models. ER- inflammatory breast cancer (IBC) cell lines SUM149 (ER-, PR-, EGFR-activated), lapatinib-resistant SUM149 (rSUM149), and non-IBC BT474M1 metastatic subline of BT474 (ER+, PR+, HER2+) were compared for the cytotoxic and cytostatic effects of didox using cell viability (trypan blue exclusion) and proliferation (MTT) assays. Protein profiling was performed to evaluate changes in molecular expression of key proteins involved in cellular signaling pathways for apoptosis and proliferation. Only ER- cell lines exhibited decreased dose-dependent decrease in viability following didox treatment. In addition, didox inhibited tumor emboli formation in a novel ex vivo tumor emboli in culture assay that mimics the clinicopathologic hallmark of IBC disease in patients. Moreover, didox decreased proliferation in lapatinib-resistant rSUM149, suggesting that the drug may help circumvent lapatinib resistance. Studies are ongoing to evaluate the mechanism of didox in overcoming lapatinib resistance and evaluation in an in vivo IBC invasion model. IBC is rare but accounts for 10% of breast cancer deaths, and despite multimodal therapy the 5-yr survival is only 25-55%. Further, there is a need to identify new therapies that can inhibit IBC tumor emboli formation and migration. Didox has demonstrated minimal toxicity in human phase I/II cancer clinical trials and therefore is an attractive strategy for inflammatory breast cancer therapy.

Support in part from the Development funds (GRD) of the Duke Cancer Institute as part of the P30 Cancer Center Support Grant NIH CA014236 and Department of Defense Breakthrough Level 2 W81XWH-17-1-0297 (GRD).

#4855

Indotecan (LMP400), Imidotecan (LMP776) and LMP744: A new class of non-camptothecin TOP1 inhibitors selective for cancer cells with homologous recombination deficiencies and high SLFN11 expression.

Yves Pommier,1 Laetitia Marzi,1 Zoe Waever Ohler,1 Ludmila Szabova,1 Shyam Sharan,1 Junko Murai,1 Mark Cushman2. 1 _NCI-CCR, Bethesda, MD;_ 2 _NCI-CCR, Purdue University, Lafayette, IN_.

To relax DNA supercoiling, topoisomerase I (TOP1) induces DNA cleavage complexes (TOP1cc). These TOP1cc can be trapped by camptothecin, leading to replication induced DNA double-strand breaks (DSB). Widely used, camptothecin derivatives have known limitations. It is now possible to overcome these limitations with the non-camptothecin indenoisoquinolines currently in clinical trials (LMP400, LMP776 and LMP744). Homologous recombination (HR) and its key components BRCA1, BRCA2 and PALB2 are needed to repair DSB induced by TOP1 inhibitors (Maede, Y et al. 2014). It has been shown that BRCA1 is a determinant of response to camptothecins in addition to Olaparib. Also, Schlafen11 (SLFN11) expression has been demonstrated to be a highly penetrant determinant of response to camptothecin derivatives (Zoppoli, G et al. 2012). To rationally select patients for phase 2 clinical trials based on cancer-specific genomic alterations, we have determined whether HR components deficiency or SLFN11 expression are determinants of LMPs response. Using isogenic cell lines, we assessed the survival after treatment with the LMPs. We found that SLFN11-positives cells are 10-times hypersensitive to LMPs compare to WT cells. Also, BRCA1-, BRCA2- and PALB2-deficient cells are 3- to 5- times hypersensitive to the LMPs. Adding Olaparib led to a greater cell death, especially in HR-deficient (HRD) cells. Our results show that the indenoisoquinolines are active at nanomolar concentrations and that HR deficiency and SLFN11 expression are strong drug response determinants. They also demonstrate that the LMPs synergize with Olaparib. These findings provide a rationale for personalized treatment and further clinical trials with the indenoisoquinolines in HR-deficient cancers.

### Novel Targets and Inhibitors

#4856

Novel, small-molecule PRMT5 inhibitors for treatment of cancer.

Dhanalakshmi Sivanandhan,1 Shivani Garapaty,1 Saravanan Vadivelu,1 Guru Pavan Kumar Seerapu,1 Reshma Das,1 Ronodip Kar,1 Anuj Kumar Singh,1 Venkatesha Ashokkumar Venkateshappa,1 Natarajan Tamizharasan,2 Indu N. Swamy,2 Nagendra Nagaraju,2 Subramaniyam Kanagaraj,1 Sayanti Sarkar,2 Jagadish D. Tibhe,2 Rudresh G,1 Mohd Zainuddin,1 Saravanan Kandan,1 Sridharan Rajagopal,1 Sriram Rajagopal1. 1 _Jubilant Biosys Ltd., Bangalore, India;_ 2 _Jubilant Biosys Ltd., BENGALURU, India_.

Arginine methylation deregulation in cancer has been well studied and PRMT5, a modulator of symmetric dimethylation of arginine (SDMA) has emerged as an attractive therapeutic strategy in various cancer types. PRMT5 is highly expressed in several cancers, including ovarian, lung, lymphoid, glioblastoma, colon, melanoma, gastric, bladder cancers and germ cell tumors. PRMT5 over-expression is thought to be an important factor in its tumorigenicity due to its repressive function on the expression of tumor suppressor genes. Therefore, PRMT5 has emerged as a therapeutic target and inhibitors selectively targeting PRMT5 could be of high clinical value. Rational design and structure based drug design were used to identify novel PRMT5 inhibitors. FlashPlate® methylation assay was used to assess in vitro potency. Cell based activity of these inhibitors was assessed by measuring the symmetrical dimethylation of known cellular protein SmD3. Long term cell proliferation assays were used to assess the functional effect of PRMT5 inhibition. A number of compounds from two different series showed strong in vitro potency against PRMT5, which were comparable to the reported GSK inhibitor. Multiple co-crystal structures have been solved in-house and are extensively used in optimization of these novel scaffolds. Their cell based potency, as measured by proliferation assay in multiple haematological and solid tumor cell lines was comparable to biochemical potency. JBI-778, from one of the series showed an in vitro potency of 0.0.045 μM (0.110 µM for GSK) and 0.005 µM in inhibiting SmD3 dimethylation. JBI-778 exhibited an GI50 of 0.01 to 1 µM in inhibiting proliferation of lymphoma cell lines, whereas the GI50 in other solid tumor cell lines PDAC, SCLC, GBM were in the range of 0.02 to 1.5 µM, again comparable (or better) than the GSK inhibitor. JBI778 showed good in vitro ADME properties in terms of aqueous solubility and metabolic stability and reasonable oral bioavailability in mouse pharmacokinetics. In Z-138 xenograft model, oral administration of JBI-778 at 50 mg/kg resulted in stronger and complete (~90%) tumor growth inhibition and was tolerated well. At comparable dose, this tumor growth inhibition was better than the GSK inhibitor. In addition, JBI-778 showed reasonable brain exposure sufficient to achieve biomarker modulation in brain. SAR has clearly demonstrated that there is further scope to optimize this series for potency, properties and brain exposure. Further studies are underway to better understand the therapeutic potential of these PRMT5 inhibitors in a number of solid cancers including PDAC, SCLC and GBM. Given the therapeutic importance of PRMT5 in Glioblastoma, these molecules will be extremely valuable in treating this cancer either as standalone therapy or in combination with other standard of care agents.

#4857

Discovery of PF-06855800, a SAM competitive PRMT5 inhibitor with potent antitumor activity.

Indrawan J. Mcalpine, John Tatlock, Joseph Billitti, John Braganza, Alexei Brooun, Deng Ya-Li, Brad Hirakawa, Kristen Jensen-Pergakes, Robert Kumpf, Wei Liu, Karen Maegley, Michele McTigue, Ryan Patman, Eugene Rui, Stephanie Scales, Noah Spiegel, Michelle Tran-Dubé, Fen Wang, Zhenxiong Wang, Shinji Yamazaki, Tao Zhang, Martin Wythes. _Pfizer Global R &D, San Diego, CA_.

Protein arginine methyltransferase 5 (PRMT5) is the primary type II arginine methyltransferase responsible for symmetric dimethylation of protein arginine residues. PRMT5 utilizes S-adenosylmethionine (SAM) to methylate a number of cytoplasmic and nuclear substrates that are involved in tumorigenesis. A number of these substrates affect a variety of biologic functions including RNA splicing, transcriptional regulation and translation, all of which are dysregulated in cancer. The discovery of adenosine as an efficient inhibitor of PRMT5 catalyzed a medicinal chemistry effort aimed at nucleoside optimization. Protein structure-based and ligand property-based design were combined to deliver molecules with desirable potency, selectivity and ADME properties. Here we will describe a series of deazapurine nucleoside analogues that culminate in the identification of PF-06855800, a SAM competitive PRMT5 inhibitor with antiproliferative activity in both in vitro and in vivo models.

#4858

Efficacy of the protein arginine methyltransferase PRMT5 inhibitor GSK591 in glioma stem-like cells.

Vikram Shaw, Yuji Piao, Soon Young Park, Jianwen Dong, Emmanuel Martinez-Ledesma, Caroline Carrillo, Verlene Henry, Ravesanker Ezhilarasan, Erik Sulman, Veerakumar Balasubramaniyan, John F. de Groot. _UT MD Anderson Cancer Center, Houston, TX_.

Protein arginine methyltransferase (PRMTs) function as epigenetic regulators of transcription and play a major role in gene regulation. PRMTs are upregulated in gastric, colorectal and lung cancer, and lymphoma and leukemia. PRMT5 is capable of forming symmetric dimethylarginine (SDMA) residues, and has been reported as a therapeutic target in glioblastoma, as its expression has been correlated with tumor grade and progression. In this study, the in vitro therapeutic efficacy of PRMT5 inhibitor GSK591 was investigated in a panel of gliomas stem-like cell (GSC) lines with specific molecular subtypes. Genomic, proteiomic (reverse protein lysate array, RPPA), methylation status and GSC subtype were correlated with drug IC50 to find predictors of drug sensitivity. The efficacy of inhibiting PRMT5 activity was retained at low dose of GSK591 in a small number of GSCs. Western blotting data showed high expression of PRMT5 and multiple bands of SDMAs in most GSCs tested (n=31), indicating PRMT5 enzymatic activity in GSC cell lines. Evidence of PRMT5 inhibition was demonstrated at low doses (< 1.5 uM) in 4 GSC lines (13%) as shown by the total inhibition of SDMA expression in a dose- and time-dependent fashion after GSK591 treatment. The sensitivity of GSK591 correlated with low methylation of multiple genes pretreatment, including MAGI2, EGR2, and DUSP16. In addition, upregulated genes in sensitive GSCs correlated with proliferation signatures using gene set enrichment analyses (GSEA). Together, our study demonstrated that inhibition of PRMT5 activity and its substrate product SDMA correlated with inhibition of tumor growth. These promising in vitro results have led to ongoing experiments to evaluate the efficacy of GSK591 in intracranial xenograft models and the underlying mechanism of sensitivity and resistance to PRMT5 inhibition.

#4859

**JNJ-64619178, a selective and pseudo-irreversible PRMT5 inhibitor with potent** in vitro **and** in vivo **activity, demonstrated in several lung cancer models.**

Tongfei Wu,1 Hillary Millar,1 Dana Gaffney,2 Lijs Beke,1 Geert Mannens,1 Petra Vinken,1 Ivan Sommers,1 Jan-Willem Thuring,1 Weimei Sun,2 Christopher Moy,2 Vineet Pande,1 Junguo Zhou,2 Nahor Haddish-Berhane,2 Mark Salvati,2 Sylvie Laquerre,2 Matthew V. Lorenzi,2 Dirk Brehmer1. 1 _Johnson & Johnson, Beerse, Belgium; _2 _Johnson & Johnson, Spring House, PA_.

PRMT5 is a type II methyltransferase that symmetrically di-methylates arginine residues on proteins involved in signal transduction and cellular transcription. For example, PRMT5 acts as the enzymatic machinery of the methylosome complex, crucial for spliceosome assembly and activity. Although not frequently mutated or amplified in tumors, an elevated PRMT5 protein level that leads to higher methylosome activity and promotes epithelial–mesenchymal transition, has recently been correlated with a poor survival of cancer patients.

The PRMT5 inhibitor JNJ-64619178 has been selected as a clinical candidate based on its high selectivity and potency, paired with favorable oral pharmacokinetics and safety properties.

JNJ-64619178 binds simultaneously to the SAM- and protein substrate- binding pockets of the PRMT5/MEP50 complex with a pseudo-irreversible mode-of-action. Chemical proteomics, methylomics and RNA-sequencing analyses of PRMT5 inhibitor treated cell line samples support the current biological understanding of PRMT5 as a regulator of alternative splicing events.

JNJ-64619178 showed potent and broad inhibition of cellular growth, observed in several cell line panels that represent diverse cancer histologies. Ongoing investigations will explore the potential synthetic lethal correlation between PRMT5 inhibition and cancer driver pathways, including those addicted to altered splicing.

Oral administration of JNJ-64619178 resulted in efficient inhibition of di-methylation of SMD1/3 proteins, components of the splicing machinery and direct substrates of the methylosome, in several human NSCLC and SCLC cancer mouse xenograft models. JNJ-64619178 demonstrated dose-dependent tumor growth inhibition and regression in several human NSCLC and SCLC cancer mouse xenograft models with sustained blockage of tumor re-growth after dosing cessation.

In summary, JNJ-64619178 has a favorable pre-clinical profile supporting clinical testing in patients with lung cancer and other malignancies.

#4860

A novel class of small-molecule inhibitors of galectin-1 for prostate cancer therapy.

Ruiwu Liu,1 Tsung-Chieh Shih,1 Sophie Kiss,1 Xiaocen Li,1 Ting Wang,2 Jonathan S. Huynh,1 Chun-Te Wu,3 Yong Duan,2 Paramita M. Ghosh,1 Kit S. Lam1. 1 _University of California Davis, Sacramento, CA;_ 2 _University of California Davis, Davis, CA;_ 3 _Chang Gung Memorial Hospital, Keelung, Taiwan_.

Galectin-1 (Gal-1) is a multifunctional carbohydrate-binding protein and plays a key role in cancer cell proliferation, apoptosis, cell cycle, tumor angiogenesis and evasion of immune responses. It is also directly involved in the process of angiogenesis and tumor immune escape. An elevated level of Gal-1 has been found in many cancers, including prostate cancer. We show by immunohistochemistry in human prostate tissue that Gal-1 expression level was very low in all non-tumor samples and highly expressed in prostate cancer tissues. In addition, Gal-1 was progressively upregulated from low- , intermediate- to high-grade prostate cancer. Taken together, Gal-1 is an excellent therapeutic target against cancer. Yet, no inhibitors against Gal-1 have been approved by the FDA for cancer treatment due to lack of potent, specific and in vivo effective molecules. We recently discovered a novel small-molecule inhibitor of Gal-1, named LLS2, through a high-throughput one-bead two-compound combinatorial library approach. LLS2 exhibits cytotoxic activity against prostate cancer and ovarian cancer cells. We synthesized a series of LLS2 analogs and performed structure-activity relationship studies, leading to a more potent inhibitor LLS30 that induced apoptosis in castration-resistant prostate cancer cells expressing high levels of Gal-1. Molecular modeling study showed LLS30 bound to the sugar-binding groove of Gal-1. In enzalutamide (ENZ)-resistant 22Rv1 prostate cancer cells expressing high levels of Gal-1 and androgen receptor (AR) splice variants, LLS30 could inhibit expression of AR splice variants and sensitize the cells to ENZ in a dose-dependent manner. Downregulation of Gal-1 by siRNA prevented the expression of alternatively spliced AR variants. More importantly, LLS30 showed potent in vivo antitumor efficacy and synergistic activities with docetaxel and ENZ against PC-3 and 22Rv1 prostate cancer models, respectively. Blood chemistry test from the treated animal showed minimum effect of LLS30 on the liver and kidney functions, implying low toxicity. LLS30 is an excellent drug candidate for preclinical development as a novel prostate cancer therapeutic.

#4861

Initial preclinical evaluation of a novel inhibitor of mitochondrial metabolism against prostate cancer.

Kiran K. Solingapuram Sai,1 Anirudh Sattiraju,2 Zuzana Zachar,3 Michael S. Dahan,3 Robert Shorr,4 Timothy S. Pardee,5 Paul M. Bingham,3 Akiva Mintz2. 1 _Wake Forest School of Medicine, Winston-Salem, NC;_ 2 _Columbia University, New York, NY;_ 3 _Stony Brook University, Stony Brook, NY;_ 4 _Rafael Pharmaceuticals, Newark, NJ;_ 5 _Wake Forest School of Medicine, Winston Salem, NC_.

CPI-613 is a novel inhibitor of mitochondrial metabolism that has recently shown promise in early stage clinical trials for pancreatic cancer, lymphoma, and leukemia. It is a lipoate analogue that selectively inhibits pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (KGDH) complexes in tumor cells (reviewed in Exp.Rev.Clin.Pharma. 7, 837). Since these two enzymes control the vast majority of carbon flow into the tricarboxylic acid (TCA) cycle, CPI-613 significantly inhibits mitochondrial metabolism. Importantly, it has demonstrated a favorable safety profile in clinical trials, presumably due to its documented selectivity to cancer metabolism. Early phase clinical trials with CPI-613 have demonstrated very promising clinical responses in pancreatic cancer, leukemia, and lymphoma when used in combination with standard chemotherapy. For example, in a recently published Phase I clinical trial in 18 patients with Stage 4 pancreatic cancer in combination with a modified FOLFIRINOX regiment, three (17%) had a complete response, eight (44%) had a partial response, three (17%) had stable disease, and four (22%) had progressive disease. Since altered cancer metabolism is a hallmark of cancer in general, we hypothesized that CPI-613 should have efficacy in other cancers, including prostate cancer, which is the second most common malignancy in men that affects over 160,000 men in the U.S annually. To test our hypothesis, we treated prostate cancer cell lines with CPI-613 in vitro and in vivo in a preclinical murine flank model. We found that CPI-613 was highly effective at killing prostate cancer cells in vitro, which was similar to its previously published potency in pancreatic cancer cells. Furthermore, we found in vitro therapeutic synergy with docotaxel, a standard of care treatment of advanced prostate cancer. We therefore tested CPI-613 in a preclinical Pc-3 flank model of androgen-resistant prostate cancer. Groups of mice (n=8) were treated 2-3 times per a week for 5 weeks with 2.5mg/kg of CPI-613, 10mg/kg of CPI-613 and control saline after tumors reached approximately 40mm3. CPI-613 was formulated in the same manner as it was for clinical trials. Both groups of mice treated with CPI-613 demonstrated a significant decrease in tumor growth without any toxicity. There was no observed difference between doses, which may be due to having crossed the threshold of drug uptake and concentration required in the mitochondria for a response. Furthermore, there was a significant increase in survival between treated groups and the control group, again without a dose effect. These results for the first time indicate biological activity of CPI-613 both in vitro and in vivo against prostate cancer and therefore warrants further investigation in representative preclinical prostate cancer models and in combination with standard-of-care therapies.

#4862

Protein arginine methyltransferase 5 as a tumor promoter and therapeutic target in gastrointestinal cancers.

Lakshmi Prabhu,1 Lan Chen,2 Ahmad Safa,1 Murray Korc,1 Zhong-Yin Zhang,2 Tao Lu1. 1 _Indiana University School of Medicine, Indianapolis, IN;_ 2 _Purdue University, West Lafayette, IN_.

Colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) are among the most commonly diagnosed forms of cancer in the United States. Due to their widespread prevalence and high mortality rate, it is vital to develop effective therapeutic drugs to combat these deadly diseases. In both CRC and PDAC, the multifunctional factor nuclear factor kappa B (NF-kB), a central coordinator of immune responses, is activated abnormally, leading to tumorigenesis and cancer progression. Therefore, controlling NF-kB activity is critical in the treatment of these cancers. Previously, we discovered a novel mechanism by which NF-kB is activated through methylation by an epigenetic enzyme known as protein arginine methyltransferase 5 (PRMT5). We showed that overexpression of PRMT5 significantly promoted several characteristics associated with cancer, including increased cell proliferation, migration, and anchorage-independent growth in both CRC and PDAC cells, thus, putting forward PRMT5 as a novel therapeutic target in these cancers. In this study, we successfully adapted AlphaLISA technique into a high-throughput screen platform, and further employed this approach to successfully identify PR5-LL-FDA1 as a potent PRMT5 inhibitor from commercially-available screening libraries. Furthermore, we confirmed that treatment of PDAC and CRC cells with PR5-LL-FDA1 led to decreased NF-kB activation, and reduced cancer associated properties in PDAC and CRC cells. Importantly, we show that PR5-LL-FDA1 is more efficacious than the commercial PRMT5 inhibitor, EPZ015666 in both PDAC and CRC. Our work clearly highlights the significant potential of PRMT5 as a therapeutic target in PDAC and CRC and hold the promise to establish PR5-LL-FDA1 as a promising basis for new drug development in the future.

#4863

Targeting the NFAT1-MDM2-MDMX network for prostate cancer therapy.

Jiang-Jiang Qin, Xin Li, Wei Wang, Ruiwen Zhang. _University of Houston, Houston, TX_.

The MDM2 and MDMX oncogenes are overexpressed in human prostate cancer, promoting tumor growth and metastasis. We recently demonstrate that the transcription factor NFAT1 activates MDM2 oncogene, independent of p53. The present study was designed to examine the effectiveness of targeting the NFAT1-MDM2-MDMX network for prostate cancer therapy. We identified a natural product NFAT1-MDM2 inhibitor, Inulanolide A (InuA) in the present study. The anticancer activity of InuA and its effects on the expression and stability of NFAT1, MDM2, and MDMX were examined in human prostate cancer cell lines with different genetic background, including LNCaP (p53 wild-type, AR positive), PC3 (p53 null, AR negative) and DU145 (p53 mutant, AR negative) cells. Our molecular docking studies predicted the binding ability and binding sites of InuA on NFAT1, MDM2, and MDMX. Its binding affinity and specificity were investigated using recombinant proteins and biotinylated InuA. To further demonstrate the specificity of InuA in targeting NFAT1-MDM2-MDMX network and the importance of NFAT1-MDM2-MDMX network in InuA's anticancer activity, the transient overexpression (OE) and knockdown (KD) experiments of these genes, as well as the pharmacological activators and inhibitors of NFAT1 signaling, were used. Our results indicated that InuA directly bound to the DNA binding domain of NFAT1, leading to a marked inhibition of MDM2 transcription. The compound also bound to the RING domains of MDM2 and MDMX with high affinity, inhibited the MDM2-MDMX binding, and enhanced MDM2 protein degradation. InuA inhibited the proliferation, migration, and invasion of prostate cancer cells, regardless of their p53 status and AR responsiveness. The expression of NFAT1, MDM2, and MDMX were important for InuA's anticancer activity, as demonstrated using the cancer cell lines with transient OE and KD of these genes. In conclusion, InuA represents a novel class of NFAT1-MDM2-MDMX inhibitor, and inhibiting the NFAT1-MDM2-MDMX network may be a promising strategy for prostate cancer therapy. (Supported by NIH R01 CA186662 and R01CA214019 and ACS RSG-15-009-01-CDD.)

#4864

Small-molecule MDM2 antagonists attenuate the senescence-associated secretory phenotype.

Nicholas Schaum,1 Christopher Wiley,2 Fatouma Almirah,2 Jose A. Lopez-Dominguez,2 Gary Scott,2 Christopher Benz,2 Judith Campisi,2 Albert R. Davalos2. 1 _Stanford University, Palo Alto, CA;_ 2 _Buck Institute for Research on Aging, Novato, CA_.

Processes that have been linked to aging and cancer include an inflammatory milieu driven by senescent cells. Senescent cells lose the ability to divide, essentially irreversibly, and secrete numerous cytokines, growth factors and proteases, termed the senescence-associated secretory phenotype (SASP). Senescent cells that lack p53 tumor suppressor function show an exaggerated SASP, suggesting the SASP is negatively controlled by p53. Here, we show that increased p53 activity caused by small molecule inhibitors of MDM2, which promotes p53 degradation, reduces inflammatory cytokine production by senescent cells. Upon treatment with the MDM2 inhibitors nutlin-3a or MI-63, human cells acquired a senescence-like growth arrest, but the arrest was reversible. Importantly, the inhibitors reduced expression of the signature SASP factors IL-6 and IL-1A by cells made senescent by genotoxic stimuli, and suppressed the ability of senescent fibroblasts to stimulate breast cancer cell aggressiveness. Our findings suggest that MDM2 inhibitors could reduce cancer progression in part by reducing the pro-inflammatory environment created by senescent cells.

#4865

BI 907828: A highly potent MDM2 inhibitor with low human dose estimation, designed for high-dose intermittent schedules in the clinic.

Joerg Rinnenthal, Dorothea Rudolph, Sophia Blake, Andreas Gollner, Andreas Wernitznig, Ulrike Weyer-Czernilofsky, Christian Haslinger, Pilar Garin-Chesa, Jürgen Moll, Norbert Kraut, Darryl McConnell, Jens Quant. _Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria_.

MDM2 inhibitors block the interaction between the Tumor Protein p53 (TP53) and MDM2, its key negative regulator, and represent a new therapeutic concept for cancer therapy. MDM2 inhibitors are designed to restore p53 activity in TP53 wild-type tumors. Several MDM2 inhibitors are currently being evaluated in early clinical development with mainly daily dosing regimens. However, recent clinical data suggest myelosuppression as an on-target, dose-limiting toxicity for this class of inhibitors. Particularly thrombocytopenia could limit the clinical utility of MDM2 inhibitors. Hence there is a need to mitigate these side effects and to improve the therapeutic window. One approach is less frequent dosing to allow bone marrow recovery while still maintaining efficacious exposure levels and clinical activity. Here we present pharmacokinetic data on BI 907828, a novel and potent MDM2 inhibitor with optimized drug-like properties. Due to its high permeability, good physiological solubility and low systemic clearance, the compound shows a high bioavailability in mice, rats, dogs and minipigs. BI 907828 is not absorption-limited, even at multiples of the efficacious dose, and shows a reliable, dose-linear PK with low variability in AUC and Cmax across species. Based on a good PK-PD correlation with efficacy in preclinical models and a robust human PK prediction based on in vivo PK data across species, a low human efficacious dose is predicted. The PK properties of BI 907828 enable a variety of different clinical dosing schedules with the potential to improve the therapeutic index with respect to on-target safety. In summary, BI 907828 is a novel, potent, orally available MDM2 inhibitor with excellent PK properties and low estimated human dose suitable for high-dose intermittent dose schedules in the clinic.

#4866

BI 907828: A novel, potent MDM2 inhibitor that induces antitumor immunologic memory and acts synergistically with an anti-PD-1 antibody in syngeneic mouse models of cancer.

Dorothea Rudolph, Markus Reschke, Sophia Blake, Jörg Rinnenthal, Andreas Wernitznig, Ulrike Weyer-Czernilofsky, Andreas Gollner, Christian Haslinger, Pilar Garin-Chesa, Jens Quant, Darryl B. McConnell, Kraut Norbert, Jürgen Moll. _Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria_.

MDM2 inhibitors block the interaction between the Tumor Protein p53 (TP53) and MDM2, its key negative regulator, and represent a new therapeutic concept in cancer therapy. MDM2 inhibitors are designed to restore p53 activity in TP53 wild-type tumors. Several MDM2 inhibitors are currently being evaluated in early clinical development. While the tumor targeting activity of MDM2 inhibitors has been well documented, recent preclinical data with the tool compound Nutlin-3 have shown that p53 activation by local, intra-tumoral injections induces anti-tumor immunity (Cancer Res 2017, 77(9) 2292-2305). Here we present data on BI 907828, a novel and potent MDM2 inhibitor with optimized drug-like properties that is suitable for intermittent dose schedules in syngeneic mouse models of cancer. Our data show that immune modulation contributes to efficacy of BI 907828 after oral administration in a Colon-26 syngeneic mouse model. Responding mice develop anti-tumor immunity that can control the tumor growth of a secondary tumor without any further treatment in a re-challenge study. A T cell depletion study shows a contribution of CD8+ T cells but not of CD4+ T cells to single-agent efficacy with BI 907828. Moreover, preclinical data in a Colon-26 syngeneic mouse model of cancer show synergistic efficacy for the combination of a PD-1 checkpoint inhibitor with BI 907828. In summary, BI 907828 is a novel, potent, orally bioavailable MDM2 inhibitor that shows synergistic efficacy in combination with a PD-1 checkpoint inhibitor in syngeneic mouse models of cancer.

#4867

Treating hepatocellular carcinoma metastasis and overcoming chemoresistance through inhibiting the MDM2 oncogene.

Wei Wang,1 Bo Hu,2 Jiang-Jiang Qin,1 Jianwen Cheng,2 Xin Li,1 Ming Hu,1 John Buolamwini,3 Xin-Rong Yang,2 Jia Fan,2 Ruiwen Zhang1. 1 _University of Houston, Houston, TX;_ 2 _Fudan University, Shanghai, China;_ 3 _Rosalind Franklin University of Medicine and Science, North Chicago, IL_.

Hepatocellular carcinoma (HCC) poses a major health problem worldwide and novel, effective therapeutic approaches are urgently needed. The MDM2 oncogene is amplified and overexpressed in HCC and associated with its initiation, progression, metastasis, and chemoresistence. MDM2 has been demonstrated to be as a promising therapeutic target for the treatment of HCC. However, most of the current MDM2 inhibitors have been designed to block the binding between MDM2 and p53, and have limited efficacy against tumors with mutant or deficient p53. We recently performed a high-throughput virtual and cell-based screening, yielding a novel MDM2 inhibitor, termed SP141. The present study was designed to evaluate the anti-HCC activity of SP141, its potential to chemosensitize HCC cells to other anticancer agents, and to determine the underlying mechanism(s) of action of SP141 in various in vitro and in vivo models with different backgrounds of p53 (wildtype, mutant or null). Our results demonstrated that SP141 inhibited cell growth and prevented cell migration and invasion, independent of p53. Mechanistically, SP141 directly bound to the MDM2 protein and promoted MDM2 degradation. The inhibition of MDM2 by SP141 also increased the sensitivity of HCC cells to sorafenib. In addition, in orthotopic and patient-derived xenograft (PDX) models, SP141 inhibited MDM2 expression and suppressed tumor growth and metastasis, without any host toxicity. Furthermore, mechanistic study demonstrated that the inhibition of MDM2 by SP141 was essential for its anti-HCC activities. In conclusion, MDM2 inhibition by SP141 resulted in the inhibition of HCC tumor growth and metastasis and sensitization of HCC cells to chemotherapy, regardless of their p53 status. These results provide support for the further development of SP141 as a lead candidate for the treatment of HCC. (Supported by NIH R01 CA186662 and R01CA214019 and ACS RSG-15-009-01-CDD.)

#4868

BI 907828: A novel, potent MDM2 inhibitor that is suitable for high-dose intermittent schedules.

Dorothea Rudolph, Andreas Gollner, Sophia Blake, Jörg Rinnenthal, Andreas Wernitznig, Ulrike Weyer-Czernilofsky, Christian Haslinger, Pilar Garin-Chesa, Jens Quant, Darryl B. McConnell, Jürgen Moll, Kraut Norbert. _Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria_.

MDM2 inhibitors block the interaction between the Tumor Protein p53 (TP53) and MDM2, its key negative regulator, and represent a new therapeutic concept for cancer therapy. MDM2 inhibitors are designed to restore p53 activity in TP53 wild-type tumors. Several MDM2 inhibitors are currently being evaluated in early clinical development with mainly daily dosing regimens. However, recent clinical data suggest myelosuppression as an on-target, dose-limiting toxicity for this class of inhibitors. Particularly, thrombocytopenia could limit the clinical utility of MDM2 inhibitors. Hence there is a need to mitigate these side effects and to improve the therapeutic window. One approach is less frequent dosing to allow bone marrow recovery while still maintaining efficacious exposure levels and clinical activity. Here we present data on BI 907828, a novel and potent MDM2 inhibitor with optimized drug-like properties including a reliable, dose-linear PK across species with good bioavailability after oral dosing that allows various dose schedules. Remarkably, a single oral dose of 2 mg/kg led to tumor regressions in a SJSA-1 xenograft model in all treated mice, as did treatment on a daily low dose schedule. Data on PK, PD and efficacy relationships in this xenograft model will be presented including dose-dependent induction of TP53 target genes and markers of apoptosis. Moreover, BI 907828 significantly prolonged the survival by more than 50 days with a daily oral dose of 2.5 mg/kg in a difficult to treat disseminated MOLM-13 AML model, as noted by profiling of a clinical-stage MDM2 inhibitor. In summary, BI 907828 is a novel, potent, orally bioavailable MDM2 inhibitor that shows excellent efficacy with daily low dose but also intermittent high dose schedules in preclinical models of cancer.

#4869

Discovery and characterization of highly potent and selective USP7 inhibitors and benchmarking against clinical MDM2 antagonists.

Gerald Gavory, Colin O'Dowd, Elias Arkoudis, Oliver Barker, Eamon Cassidy, Anthony Dossang, Jakub Flasz, Matt Helm, Caroline Hughes, Keeva McClelland, Hugues Miel, Ewa Odrzywol, Natalie Page, Tim Harrison. _Almac Discovery, Belfast, United Kingdom_.

Given the importance of USP7 in known oncogenic pathways and its emerging role in immuno-oncology, the identification of USP7 inhibitors has attracted considerable interest in the scientific community. However, despite substantial efforts over the past 15 years, the development of genuine deubiquitinase (DUB) inhibitors, which exhibit both drug-like properties and a well-defined mechanism of action, has proven particularly challenging.

In this study, we report the application of UbiPlexTM, our purpose-built DUB drug discovery platform, to USP7. In particular, we detail the identification, optimization and detailed characterization of a new class of non-covalent and highly potent USP7 inhibitors (IC50 < 10 nM). In addition, these inhibitors have shown exquisite selectivity for USP7 over other DUBs and proteases. We also disclose high-resolution co-crystal structures of USP7 in complex with these inhibitors that reveal an allosteric mode of binding.

Further profiling in cells demonstrated potent target engagement with endogenous USP7 (EC50 <20 nM). In line with the known biology of USP7, this effect induced the proteasomal degradation of MDM2, concomitant stabilization of p53 and induction of p21 in multiple cell lines. The identification and parallel profiling of inactive enantiomers further validated the on-target effect of our lead molecules. Finally, we report the identification of cell lines hyper-sensitive (EC50 < 30 nM) to USP7 inhibitors in both haematological and solid cancer cell line settings and demonstrate equal or superior activity when compared to clinically relevant MDM2 antagonists.

In summary, this work exemplifies the tractability and druggability of USP7 as a cancer target and provides new insights and directions for the potential clinical development of USP7 inhibitors.

#4870

Targeted MDM2 degradation as a novel and efficacious cancer therapy.

Jiuling Yang, Yangbing Li, Angelo Aguilar, Donna McEachern, Sally Przybranowski, Ester Fernandez-Salas, Jianfeng Lu. _University of Michigan, Ann Arbor, MI_.

The E3 ubiquitin ligase MDM2 (Murine Double Minute 2) is the most important negative regulator of p53 tumor suppressor primarily by targeting it for ubiquitination and proteasomal degradation. Amplification or overexpression of the MDM2 gene occurs in many human cancers contributing to tumor development, progression and metastasis. Targeting MDM2 to restore p53 function has become an attractive therapeutic strategy for cancers harboring wild-type TP53. Currently, numerous drugs inhibiting the MDM2-p53 interaction, such as RG7112 (Hoffmann-La Roche), MI-773 (Sanofi) and DS-3032b (Daiichi Sankyo), have entered different stages of clinical trials. However, even in p53 wild-type tumors, MDM2 inhibitors can exert limited efficacy as monotherapy in some models, probably due to inadequate p53 induction and side effects. Therefore novel and improved strategies to effectively target the MDM2-p53 pathway are needed. Recently, we have designed a series of MDM2 degraders by conjugating a small molecule MDM2 inhibitor to phthalimide. The phthalimide moiety interacts with its target protein Cereblon (CRBN) and recruits the CUL4-DDB1-CRBN (also known as CRL4CRBN) E3 ubiquitin ligase complex to promote ubiquitination and proteasome degradation of MDM2. Our initial evaluation focused on acute leukemias that are mostly p53 wild-type and express high levels of MDM2. The MDM2 degraders exert significantly improved growth inhibitory activity compared to the inhibitor in human acute leukemia cell lines with IC50s < 5 nM. We performed Western Blots for MDM2 and p53 protein levels and qRT-PCR analysis for mRNA levels of p53 target genes to demonstrate MDM2 protein degradation and activation of p53 downstream pathways. Flow cytometry analysis confirmed the induction of apoptosis by MDM2 degraders in leukemia cells. In agreement with our design, the action of our MDM2 degraders depends on binding to Cereblon, as demonstrated by competition with free phthalimide, and on proteasome function, as demonstrated by proteasome inhibition. siRNA knockdown of p53 significantly abolished the activity of the degraders demonstrating the dependence upon p53 activation. Our studies demonstrate that the MDM2 degraders robustly activate wild-type p53 by inducing rapid degradation of MDM2 and activation of p53 pathways leading to strong apoptosis in leukemia cell lines in a CRBN-binding, proteasome and p53 dependent manner. More importantly, the MDM2 degraders showed great efficacy in inducing complete tumor regression or strong tumor growth inhibition in human leukemia xenograft models and significantly improved survival in disseminated human leukemia models in mice at well-tolerated dose-schedules. Our data provide strong preclinical rationale to further develop MDM2 degraders as a new class of therapy for the treatment of human acute leukemia and potentially other types of human cancer.

#4871

Antitumor activity of novel reversible LSD1 inhibitor, HM97211 in preclinical models of SCLC.

InHwan Bae, JiSook Kim, ChangHee Park, Hyeongki Kim, Namgoong Gwangmog, Joo-Yun Byun, Seokhyun Hong, Teahun Song, JaeHo Lee, Kyuhang Lee, Myoungsil Ko, Cheolkyung Kim, YoungMi Lee, Ho Jeong Lee, YoungHoon Kim, YoungGil Ahn, KweeHyun Suh, Sun-Jin Kim. _Hanmi Research Center, Hwaseong, Republic of Korea_.

Introduction: Global changes in the epigenetic landscape are known as the hallmark in cancer and the histone demethylase Lysine-specific demethylase 1 (LSD1) is one of the novel target for the therapy of various malignant diseases and LSD1 overexpression is associated with poor prognosis in various cancers. LSD1-mediated epigenetic modification is known to play a key role in the regulation of gene expression by removing the methyl groups from methylated lysine 4 and lysine 9 of histone H3. Alterations in histone methylation lead to aberrant silencing of expression of multiple genes involved in tumor survival and in cell cycle. Moreover, epigenetic dysregulation plays a critical role in pathogenesis of various types of cancers such as acute myeloid leukemia (AML) or small cell lung cancer (SCLC). Herein, Hanmi introduces, a safe drug profile of the novel LSD1 inhibitor, H97211on hematopoiesis differentiation by reversible binding on LSD1. Materials and Methods: Novel reversible LSD1 inhibitor, HM97211 was designed and synthesized as the active biologic inhibitory compound against the SCLC cells. The binding mode on reversibility for HM97211 and biochemical activity on LSD1 were determined by LSD1 Fluorescent assay kit (BPS Bioscience; Venkataswamy Sorna et al, 2013). In vitro proliferation assay was performed by using the CellTiter-Glo (Promega) In vivo therapeutic efficacy of HM97211 was evaluated in NCI-H1417 xenograft mice models. 5 x 106 NCI-H1417 cells/mouse were implanted subcutaneously and mice were treated by daily oral administration of (30mg/kg) HM97211. Tumor sizes were measured and tumor samples were analyzed of the mechanisms of action. Results: HM97211 enhanced not only global methylation of H3K4 but also apoptotic signaling in NCI-H1417 cell line. HM97211 altered expression of neuroendocrine factor ASCL1, inducing programmed cell death in SCLC cells. In NCI-H1417 xenograft models, daily oral administration of HM97211 induced tumor regression without remarkable toxicity. Analyses of tumor samples revealed the important role of potent small molecule LSD1 inhibitor in neuroendocrine-associated transcription and cell proliferation of SCLC. Conclusion: Collectively, results of this study suggested that novel reversible LSD1 inhibitor, HM97211, can be a strong therapeutic agent for SCLC patients. Hanmi presents a new insight to epigenetics application in anticancer therapy.

#4872

ONC201 induction of apoptosis in hPheo1 cell line supports use of this oral agent in ongoing phase 2 clinical trial against neuroendocrine tumors (NCT03034200).

Austin M. Proudfit,1 Peter M. Anderson,2 Neetu Gupta1. 1 _Lerner Research Institute, Cleveland, OH;_ 2 _Cleveland Clinic, Cleveland, OH_.

ONC201 is an imipridone that induces TRAIL and causes apoptosis in cancer cell lines, but not normal cells. Recent data shows the dopamine-like DRD2 receptor binds ONC201 and pheochromocytoma-paragangliomas (PC-PG) have >10x more DRD2 than any other human cancer (TCGA data). We present pre-clinical data on dose response and time course of ONC201 against hPheo1, a PC-PG cell line, that support a recently opened phase 2 clinical trial of weekly oral ONC201 in neuroendocrine tumors (NETs) including PC-PG. The human pheochromocytoma cell line, hPheo1, was treated with ONC201 and studied for in vitro anti-cancer activity. Effects on cell viability/proliferation were measured via cell counting with trypan blue exclusion. Post-treatment target-protein levels were assessed via western blotting. Additionally, cells were stained with Annexin V and analyzed using flow cytometry for markers of apoptosis. Upon treatment with ONC201, hPheo1 cells experienced a dose-dependent decrease in cell viability and proliferation with peak effect at 72 h post-treatment (GI50=3.2 μM). Substantial inhibition of pro-survival kinases, Akt and ERK, were confirmed at 72 hours to further support the drug's efficacy and elucidate on a corresponding mechanism of action. The anti-proliferative effects of the drug were gradual over a 96 h time period, rather than an abrupt, intense response. In addition, apoptosis was confirmed at 24 and 96 h, with Annexin V\+ cells detectable by FACS. Pre-clinical data show excellent anti-cancer efficacy of ONC201 against PC-PG cells at concentrations achievable in vivo. These findings suggest both anti-proliferative effects and induction of apoptosis lead to anti-cancer activity. The gradual influence of ONC201 on PC-PG cells, rather than rapid cell destruction argues against a scenario where immediate catecholamine release could result in severe hypertension. These pre-clinical data support safe use of ONC201 against PC-PG in vivo and other NETs in our ongoing Phase 2 clinical trial (NCT03034200; investigator-initiated IND132665).

#4873

Therapeutic targeting of LSD1 in Ewing sarcoma with SP-2509.

Kathleen Pishas,1 Megann Boone,1 Sunil Sharma,2 Stephen Lessnick1. 1 _Nationwide Children's Hospital, Columbus, OH;_ 2 _Huntsman Cancer Institute, Salt Lake City, UT_.

Objective: Multimodal regimes remain the cornerstone treatment for Ewing sarcoma (ES), the second most common solid bone malignancy diagnosed in pediatric and young adolescent populations. We have reached a therapeutic ceiling with conventional cytotoxic agents, highlighting the need to adopt novel approaches specifically targeting the drivers of ES oncogenesis. As LSD1 (Lysine Specific Demethylase 1) is highly expressed in ES tumors, with elevated expression levels associated with worse overall survival, this study examined biomarkers of sensitivity/mechanisms of cytotoxicity to targeted LSD1 inhibition using SP-2509 (reversible LSD1 inhibitor).

Methods: The antiproliferative effects of SP-2509 were determined through Cell Titre Glo assays following 72hrs of treatment in a panel of 17 ES cell lines with varying STAG2/TP53 mutational status and basal LSD1 expression levels. RNA-seq analysis of six ES cell lines -/+ SP-2509 treatment (2μM) was also conducted.

Results: Innate resistance to SP-2509 was not observed in our cell line cohort (72hr IC50:81nM-1593nM). In contrast, resistance to the clinical next-generation LSD1 irreversible inhibitor GSK-LSD1 was observed across multiple cell lines (144hr IC50>300μM). Although TP53/STAG2 mutational status and basal LSD1 mRNA/protein levels did not correlate with SP-2509 response, induction of LSD2 (homologue of LSD1) following SP-2509 treatment was strongly associated with SP-2509 hypersensitivity (R2=0.562). shRNA mediated knockdown of LSD2 significantly reduced the cytotoxic effects of SP-2509 (4.3 fold IC50 increase) only in hypersensitive cell lines. Mechanistically, RNA-seq analysis revealed that SP-2509 imparts apoptosis through engagement of the endoplasmic reticulum stress pathway, with hypersensitive cell lines sharing similar transcriptomic profiles. In addition, ETS1/HIST1H2BM were specifically induced/repressed, respectively, following SP-2509 treatment only in our hypersensitive cell lines. Finally, we demonstrate that the transcriptional profile driven by SP-2509 strongly mirrors LSD1 genetic depletion.

Conclusion: Our findings provide key insights into the mechanisms of SP-2509 cytotoxicity as well as biomarkers that can be used to predict LSD1 inhibitor sensitivity in ES.

#4874

Receptor pharmacology and anti-cancer activity of selective DRD2/3 antagonist imipridone ONC206.

Varun Vijay Prabhu,1 Neel Madhukar,1 Rohinton Tarapore,1 Mathew Garnett,2 Ultan McDermott,2 Cyril Benes,3 Neil Charter,4 Sean Deacon,4 Alexander VanEngelenburg,5 Joseph Rucker,6 Benjamin Doranz,6 Olivier Elemento,7 Wolfgang Oster,1 Martin Stogniew,1 Joshua Allen1. 1 _Oncoceutics, Inc, Philadelphia, PA;_ 2 _Wellcome Trust Sanger Institute, Hinxton, United Kingdom;_ 3 _Massachusetts General Hospital, Boston, MA;_ 4 _DiscoverX Corporation, Fremont, CA;_ 5 _HistoTox Labs, Inc, Boulder, CO;_ 6 _Integral Molecular, Inc, Philadelphia, PA;_ 7 _Weill Cornell Medicine, New York, NY_.

Dopamine receptor D2 (DRD2) is a G protein-coupled receptor (GPCR) overexpressed in many cancers and its antagonism causes anti-tumor effects. ONC201, founding member of the imipridone class of small molecules, is a DRD2 antagonist in Phase II advanced cancer clinical trials. We evaluated the binding target and anti-tumor activity of ONC206, an ONC201 analog. An orphan small molecule target prediction algorithm revealed that ONC206, like ONC201, antagonizes DRD2. Experimental GPCR profiling using the PathHunter® β-Arrestin assay, confirmed that ONC206 selectively antagonizes D2-like (DRD2/3) but not D1-like (DRD 1/5) dopamine receptors. In this assay, ONC206 possesses a ~10-fold increased potency for DRD2 compared to ONC201 with a Ki of ~320nM with selectivity that was superior to approved antipsychotics. Shotgun mutagenesis across 350 amino acids of DRD2 identified 7 residues critical for ONC206-mediated antagonism of DRD2-induced calcium flux. Consistent with competitive inhibition, mutated residues were within the orthosteric binding site. While 6 mutated residues were also critical for ONC201 activity, one of the mutated residues was unique to ONC206, suggesting differentiated receptor pharmacology. TCGA analysis and immunohistochemistry of patient-derived tissue microarrays revealed DRD2 was overexpressed in neuroblastoma, sarcoma and pheochromocytoma specimens relative to normal tissues. In vitro profiling of ONC206 in the Genomic of Drug Sensitivity in Cancer collection of cell lines revealed broad nanomolar efficacy across most tumor types (GI50 <78-889nM) and ~5 to 20-fold improved potency relative to ONC201. Bone cancer and neuroblastoma were identified as the most ONC206-responsive solid tumor types that were comparatively less responsive to ONC201. Within bone cancer, Ewing's sarcoma (n=16) was most sensitive to ONC206 with nanomolar sensitivity (GI50 168-303nM) that was superior to ONC201. ONC206 was highly efficacious in neuroblastoma (n=35, GI50 87-589nM) including cell lines derived from metastatic sites and with MYCN amplification associated with poor prognosis. In the PC12 rat pheochromocytoma cell line ONC206 (GI50 200nM) was superior to ONC201. ONC206 time-course experiments revealed anti-cancer effects occurring at 48-72 post-treatment, similar to ONC201. In support of a wide therapeutic window, ONC206 reduced the viability of normal human fibroblasts at relatively high doses (GI50 > 5µM). Efficacy evaluation in the MHH-ES-1 Ewing's sarcoma xenograft model demonstrated that ONC206 (100 mg/kg PO every 10 days) causes significant tumor growth inhibition that was comparable to methotrexate (400 mg/kg, IP) while being better tolerated. Thus, imipridone ONC206 acts as a selective antagonist of DRD2/3 at nanomolar concentrations and may address tumor types where the properties of ONC201 do not permit for complete therapeutic engagement in vivo.

#4875

Addition of RP10107, a novel and potent glutaminase inhibitor, accentuates 5-FU activity in lung cancer cell lines in vitro.

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

Background: Drug resistance remains a significant limitation to the clinical use of 5-Fluorouracil (5-FU) in lung cancer. With cancer cell metabolism emerging as a critical regulator of tumor progression, combining 5-FU with an inhibitor of the metabolic machinery represents a potential therapeutic strategy to prevent lung cancer progression. Cancer cell metabolism is reprogrammed wherein glutamine utilization is increased via elevation of glutaminase activity thereby generating the necessary substrates required for eventual ATP synthesis and energy production. RP10107 is a novel, potent, and selective glutaminase (GLS-1) inhibitor that demonstrated high potency against mouse (IC50=21.2 nM), rat (IC50=18.2 nM) and human (IC50=26.4 nM) enzymes with selectivity over GLS-2 (>380-fold). The objective of this study was to evaluate the effect of a combination of 5-FU and RP10107 in lung cancer cells.

Methods: Glutamate concentrations in lung cancer cell lines (A549, NCI-H460, NCI-H1975, NCI-H441, and NCI-H2170) following treatment with RP10107 was estimated by LC-MS/MS. Synergism with 5-FU was determined using different concentrations of the compounds in a 5 x 5 grid. Synergism, additivity, or antagonism was calculated based on the BLISS score. Apoptosis was determined by Annexin V/7AAD staining using a MUSE® Annexin V and dead cell assay kit (Millipore) while the effect on cell cycle was estimated using Guava Cell Cycle Reagent (Millipore). Expression of caspase 3/7, phosphor-p53 (S15), and Bcl-2 were determined by Western Blotting.

Results: Treatment of lung cancer cell lines with RP10107 resulted in an increased ratio of glutamine to glutamate with a doubling noticed between 100-300 nM. The change in glutamine utilization was accompanied by a dose-dependent inhibition in cell growth with half-maximal inhibitions ranging from 1.3 to 9.0 μM. Synergism or additivity between RP10107 and 5-FU for anti-proliferative activity was noticed in all the cell lines tested with effect being most pronounced in NCI-H460 and NCI-H441. Additionally, incubation with the combination (1-5 μM RP10107 + 3-5 μM 5-FU) for 48-72 h caused a G2/M or S-phase arrest with a corresponding increase in the percent of apoptotic cells (1-5 to 2.5-fold) compared to the individual agents. Combination of RP10107 and 5-FU increased expression of phospho-p53 expression in all cell lines tested while it led to 40-75% reduction in caspase 3, caspase 7, and Bcl-2 in NCI-H2170 and NCI-H460.

Conclusions: Addition of RP10107, a potent glutaminase inhibitor, potentiated 5-FU activity in lung cancer cell lines. Findings provide a rationale for use of the combination in future clinical trials involving lung cancer patients thereby providing a safer and more effective alternative to currently available therapy.

#4877

Allosteric inhibition of SHP2 variants containing cancer-associated activating mutations.

David Wildes, Naing Aay, Andreas Buckl, Daphne Hsieh, Ashutosh S. Jogalekar, Gert Kiss, Elena S. Koltun, Abby Marquez, Kevin T. Mellem, Jason Romero, Mae Saldajeno-Concar, Christopher J. Schulze, Chris M. Semko, Walter Won, Robert J. Nichols, Carlos Stahlhut, Christos Tzitzilonis, Adrian L. Gill, Jacqueline A. Smith. _Revolution Medicines, Redwood City, CA_.

SHP2 (PTPN11) is a non-receptor protein tyrosine phosphatase and scaffold protein that functions in multiple signal transduction pathways. Genetic and pharmacologic evidence supports a role for SHP2 in driving the proliferation of cancer cells dependent upon a range of activated RTKs, certain RAS and BRAF mutations, and NF1 loss-of-function mutations. In addition, dominant activating mutations in PTPN11 fuel pathogenic RAS/MAPK signaling and underlie certain human RASopathies (40% of Noonan syndrome and 80% of LEOPARD syndrome) and cancers (35% of JMML and up to 5% of many other cancers). These mutants destabilize an autoinhibited conformation of SHP2, which can also be activated by binding to diphosphotyrosine motifs in vivo or to synthetic diphosphopeptides in vitro. Multiple selective, allosteric inhibitors that stabilize this autoinhibited state and potently inhibit wild-type (WT) SHP2 have recently been reported, but the impact of these inhibitors on mutant forms of the protein is less well established. Here we investigate the activity of a diverse set of over fifty allosteric SHP2 inhibitors on multiple cancer-associated activating mutants of SHP2, both in the presence and absence of an activating diphosphopeptide. The rank order of potencies of the inhibitors remains identical for all mutants, although a decrease in potency relative to WT is seen in some mutants, which is magnified further in the presence of activating peptide. These results conform to a simple equilibrium model, where inhibitors show a reduction in potency against activated mutants that is proportional to the energetic magnitude of the activating mutation. Surprisingly, some common activating mutations of SHP2 (e.g., D61G) have a relatively modest energetic effect, and exhibit similar sensitivity to allosteric inhibitors as WT SHP2. We follow up these biochemical results with cellular inhibition studies in HEK293 cells stably transfected with the respective SHP2 mutants. Collectively, these results suggest that potent allosteric SHP2 inhibitors will be effective at inhibiting the growth of cancers driven by a subset of activating mutations in SHP2.

#4878

RMC-4550, an allosteric inhibitor of SHP2: Synthesis, structure, and anti-tumor activity.

Elena S. Koltun, Naing Aay, Andreas Buckl, Ashutosh S. Jogalekar, Gert Kiss, Abby Marquez, Kevin T. Mellem, Kasia Mordec, Mae Saldajeno-Concar, Chris M. Semko, Nidhi Tibrewal, Christos Tzitzilonis, Walter Won, Jacqueline A. Smith, Susan E. Wilson, Robert J. Nichols, Zhengping Wang, David Wilds, Mallika Singh, Adrian L. Gill. _Revolution Medicines, Redwood City, CA_.

Genetic and pharmacologic evidence has shown that SHP2, a non-receptor protein tyrosine phosphatase (PTP) and scaffold protein encoded by the PTPN11 gene, is a convergent signal transduction node that integrates growth factor signals from multiple receptors to promote activation of RAS and its downstream effectors. Guided by structural insights from X-ray data, we describe a strategy aimed at the identification of a highly potent and selective allosteric SHP2 inhibitor series. Our efforts led to the discovery of RMC-4550, a potent and selective SHP2 inhibitor which exhibits a high quality, drug-like preclinical profile. RMC-4550 inhibits purified, activated full length human SHP2 with an IC50 of 1.55 nM, and has cellular IC50 of 39 nM in PC9 cells with a pERK readout. RMC-4550 has no detectable inhibitory activity up to 10 µM against the catalytic domain of SHP2, a panel of 14 additional protein phosphatases, and a panel of 468 protein kinases. RMC-4550 exhibits low to moderate cross species in vitro intrinsic clearance (3.6-24 µL/min/million cells) in hepatocytes, a high passive permeability (458 nm/s) and efflux ratio of 1. The ADME properties translate into favorable pharmacokinetic profiles in preclinical species. RMC-4550 has moderate to high bioavailability and has a half-life amenable for once daily oral administration. In the EGFR-driven KYSE-520 human esophageal cancer xenograft model, we observed a dose dependent efficacy consistent with target modulation, assessed by phospho-ERK inhibition in tumors. RMC-4550 is well tolerated at doses that achieved maximal and sustained efficacy in this model. RMC-4550 was synthesized in 5 linear (6 total) steps from the readily accessible or commercially available intermediates. The chemical structure and synthesis of RMC-4550, along with detailed structure-activity relationships will be presented. In summary, RMC-4550 exemplifies a novel class of potent allosteric inhibitors of SHP2 with an excellent drug like property profile.

#4879

Mutation selectivity of the allosteric SHP2 inhibitor SHP099.

Xiaojun Sun,1 Yuan Ren,2 Steven Gunawan,3 Peng Teng,4 Zhengming Chen,2 Harshani R. Lawrence,2 Jianfeng Cai,4 Nicholas J. Lawrence,2 Jie Wu1. 1 _Stephenson Cancer Center, OUHSC, Oklahoma City, OK;_ 2 _H. Lee Moffitt Cancer Center, Tampa, FL;_ 3 _Stephenson Cancer Center, OUHSC, Tampa, FL;_ 4 _University of South Florida, Tampa, FL_.

SHP2 (PTPN11) mediates oncogenic signaling and gain-of-function (GOF) SHP2 mutations are associated with leukemias and other types of cancer. Recently, SHP099 was identified as a novel allosteric SHP2 inhibitor that displayed high potency and selectivity to the wildtype SHP2 in vitro and inhibited protein tyrosine kinase oncogene-driven cancer cells. However, it was unclear whether SHP099 inhibits SHP2 mutants. Using SHP2 mutant-dependent TF-1 leukemia cells, we determined sensitivities of four common oncogenic SHP2 mutants to SHP099. SHP2D61Y-, SHP2A72V-, and SHP2E76K-expressing TF-1 cells were resistant, whereas SHP2E69K-expressing TF-1 cells were sensitive to SHP099 with a potency similar to that of cytokine-stimulated TF-1 cells expressing the wildtype SHP2. Consistently, SHP099 reduced active ERK1/2 and Bcl-XL and induced apoptosis of SHP2E69K-dependent TF-1 cells, but had no effects on the other SHP2 mutant cell lines. SHP2 knockout cells were unresponsive to SHP099, demonstrating specificity. Like the wildtype SHP2, SHP2E69K PTP was inhibited by SHP099 in vitro with a sub-micromolar IC50, whereas micromolar IC50s of SHP099 were observed against SHP2D61Y, SHP2A72V, and SHP2E76K. These results indicate that SHP2D61Y, SHP2A72V, and SHP2E76K mutants are resistant to SHP099 and that SHP2E69K is a SHP099-sensitive oncogenic mutant.

#4880

Novel nano-molar small-molecule STAT3 inhibitor series with antitumor activities against human breast cancer.

Peibin Yue,1 Francisco Lopez-Tapia,1 Christine Brotherton-Pleiss,1 Casie Kubota,1 Jasmine Chen,1 Marcus Tius,2 James Turkson1. 1 _University of Hawaii Cancer Center, Honolulu, HI;_ 2 _University of Hawaii at Manoa, Honolulu, HI_.

Constitutively-active Signal transducer and activator of transcription (STAT) 3 has been well validated as a significant mechanistic underpinning of many human cancers and a target for anticancer therapy. To date, no small molecule STAT3 inhibitor has been successfully developed for clinical application as a therapeutic agent, despite the discovery of many molecular entities that inhibit the protein. To advance the therapeutic development of small molecule STAT3 inhibitors, we focused on the existing lead compounds, BP-1-102, SH4-54, and SH5-07 (all three showing IC50 of 4-7 μM). All three leads are based on an N-methylglycinamide scaffold, with its two amine groups condensed with three different functionalities. Intensive medicinal chemistry campaign was directed towards understanding and optimizing the molecular determinants critical for enhanced STAT3-inhibitory potency and strong antitumor effects. We report the development of nanomolar potent inhibitors of STAT3 DNA-binding activity. Most notably, the analogs H127, H145, H171, H174, H181, and H182 preferentially inhibit STAT3 activity in DNA-binding assay in vitro, with IC50 of 300 - 800 nM, compared to little or no observed effect on STAT1 and STAT5 DNA-binding activity at concentrations up to 20 μM. Treatment of human breast cancer MDA-MB-231 and MDA-MB-468 cells with H171, H174 or H182 inhibited constitutive STAT3 DNA-binding activity and phosphorylation at Tyr705 in both time- and dose-dependent manner. H171, H174 or H182 further blocked the growth and viability, colony formation, and oncogenic transformation of the human breast cancer cells that harbor persistently active STAT3, with IC50 of 1.0 - 1.9 μM, compared to the IC50 of 3.8 - 8.1 μM against the viability of the immortalized breast epithelial cells that do not harbor persistently active STAT3. These compounds represent some of the first nanomolar potent small molecules observed to directly inhibit STAT3 activity, with potent antitumor cell effects.

#4882

Identification and optimization of chemical inhibitors that directly target KRAS G12D mutant.

Xiaohong Tian, Guoyan Geng, Jian Hui Wu. _McGill Univ. Lady Davis Inst., Montréal, Quebec, Canada_.

KRAS mutations are enriched in three lethal cancers, such as pancreatic ductal adenocarcinoma (PDAC) (> 90% of cases), colorectal cancer (CRC) (~50%) and lung cancer (~25%). Within these cancers, specific KRAS mutations dominate. KRAS G12D is the most common mutation in pancreatic and colorectal cancer. To date, accumulating studies have firmly established that constitutive activation of KRAS mutants is a driver of PDAC and CRC. Inhibitors of the downstream components of KRAS signaling only have limited success. Due to the high affinity of KRAS with GTP and no other obvious binding pocket on the KRAS surface for the design of inhibitors, KRAS has been considered 'undruggable' for a long period of time. As a breakthrough, a novel direct inhibitor of the KRASG12C mutant was discovered in 2013, which forms a covalent bond with the cysteine at residue 12. However, such KRASG12C inhibitor is inactive against the G12D mutant as there is no Cysteine at residue 12. In this project, we have learnt from the excellent work of the G12C inhibitors and identified a novel compound as a direct inhibitor of the KRASG12D mutant. Importantly, this compound is active against the G12D mutant, but inactive against the KRAR wild-type. We demonstrated that our compound at 10 uM significantly inhibits the KRAS signaling in PANC10.05 pancreatic cancer cells and SNU-C2B colorectal cancer cells, both of which express endogenous KRASG12D mutant.

#4883

Suppression of breast cancer by reprogramming glucose metabolism and energy production with HJC0152.

Hyejin Kim,1 Jiabin Dong,1 Jimin Xu,2 Dengfeng Li,1 Zhi Zheng,1 Na Ye,2 Ziwei Zhang,1 Haiying Chen,2 Jia Zhou,2 Qiang Shen1. 1 _MD Anderson Cancer Center, Houton, TX;_ 2 _University of Texas Medical Branch, Galveston, TX_.

Most mammalian cells use glucose as the primary fuel source. Glucose is metabolized via glycolysis to pyruvate, which is further transferred into mitochondria for generation of ATP through the Krebs cycle in normal condition. However, metabolism is characteristically reprogrammed and cancer cells or highly proliferative cells preferably generate ATP through lactate production by lactate dehydrogenase (LDH/LDHA), referred to as the Warburg effect or metabolic reprogramming toward anaerobic glycolysis. Efficient control of energy metabolism is the key to maintaining metabolic homeostasis, and disturbance in energy balance provokes diseases such as obesity, diabetes and cancer. However, the mechanisms underlying efficient energy metabolic homeostasis and breast cancer development are poorly understood. HJC0152, a novel small-molecule glucose metabolism modulator, was developed using structure-/fragment-based drug design strategies and molecular modeling techniques. Aggressively growing and metastatic breast cancer cells of triple-negative subtype (MDA-MB-231) treated with HJC0152 showed inhibited cell growth. Intriguingly, HJC0152 decreased activity and protein level of LDHA, which resulted in a decrease of lactate production in breast cancer cells. In addition, these cells also exhibited a decrease of glucose uptake by reducing Glut1 and HK2 protein level and modulation of the expression of glycolytic enzymes. Furthermore, HJC0152 treatment causes decreased mitochondrial membrane energy potential, resulting in significantly reduced level of intracellular ATP in MDA-MB-231 cells. Our findings suggest that HJC0152 is capable of reprogramming caner metabolism by modulating glucose metabolism and ATP production. These results may provide a rationale to develop HJC0152 as an effective therapeutics for cancer and other metabolic diseases with aberrant glucose metabolism. In addition, HJC0152 can serve as a molecular probing tool for elucidating the key factors responsible for developing breast cancer and other metabolic diseases.

#4884

OXPHOS: A novel target for cancer therapy in oncogene addicted tumor.

Jayshree L. Hirpara,1 Jie Eu Qing,1 Andrea Wong,1 Naoto Ohi,2 Tsunoda Takeshi,1 Boon Cher Goh,1 Shazib Pervaiz1. 1 _National Univ. of Singapore, Singapore, Singapore;_ 2 _Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan_.

Cancer cells are highly dependent on glycolysis over oxidative phosphorylation (OXPHOS). However, there is emerging evidence that (a) dysfunctional OXPHOS may be a major factor driving oncogene addiction and (b) feedback activation of the transcription factor STAT3 is a common surrogate survival pathway in oncogene addicted cancer cells. As such, invasive cancers exhibit enhanced oxidative phosphorylation, mitochondrial biogenesis and mitochondrial oxygen consumption rate. Therefore, inhibiting OXPHOS and STAT3 activity could have potential therapeutic implications for oncogene addicted and OXPHOS-dependent cancers. Here we report that oncogene addicted non-small cell lung carcinoma (NSCLC) cell line HCC827 and its gefitinib resistant clone and malignant melanoma cell line A375 and its vemurafanib resistant clone have significantly higher OXPHOS activity. In addition, these cells express higher level of the mitochondrial electron transport chain complex I protein (NDUFA9) and increased complex I activity as well as higher mitochondrial DNA copy number. Of note, significantly increased activity of STAT3 is detected in oncogene addicted cancer cells, and the increased mitochondrial oxygen consumption and complex I activity could be significantly inhibited by a novel small molecule inhibitor of STAT3, OPB-51602. The latter is shown to be an effect that might be independent of the STAT3 inhibitory activity of the small molecule compound. Most importantly, the novel STAT3 inhibitor showed strong activity in three different murine models of carcinogenesis as well as in patients treated with the STAT3 inhibitor. These data provide evidence that oncogene addicted cancer cells switch to OXPHOS over glycolysis and serve as proof of principle for identifying targeted new OXPHOS inhibitor in recalcitrant cancers. 

### Microenvironmental and Cell Nonautonomous Factors in Mediating Therapeutic Resistance

#4885

**Diverse microenvironmental agonists induce** de novo **resistance to Bcl-2 and BTK targeted combination therapy and cancer cell proliferation in B cell malignancies.**

Kallesh D. Jayappa,1 Craig A. Portell,2 Vicki L. Gordon,1 Timothy P. Bender,1 Michael E. Williams,2 Michael J. Weber3. 1 _Department of Microbiology, Immunology, and Cancer Biology, University of Virginia School of Medicine, Charlottesville, VA; Beirne B. Carter Center for Immunology Research, Charlottesville, VA;_ 2 _Division of Hematology/Oncology, University of Virginia School of Medicine, Charlottesville, VA; Cancer Center, University of Virginia, Charlottesville, VA;_ 3 _Department of Microbiology, Immunology, and Cancer Biology, University of Virginia School of Medicine, Charlottesville, VA; Cancer Center, University of Virginia, Charlottesville, VA; Beirne B. Carter Center for Immunology Research, Charlottesville, VA_.

The Bruton's Tyrosine Kinase (BTK) inhibitor ibrutinib (IBR) is FDA approved for Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL), but de novo resistance and rapid recurrence are common. We showed that venetoclax (VEN), an inhibitor of Bcl-2, induced synergistic cytotoxicity with IBR in cell lines and patient samples ex vivo (Axelrod et al. Leukemia, 2014; Jayappa et al. Blood Adv., 2017) and have initiated a clinical trial to test this combination. However, we noted variable sensitivity to IBR+VEN among CLL/MCL patient samples ex vivo, indicating a high frequency of de novo resistance. Resistance was evident in CLL cells showing an "activation" phenotype (CD5+/CD19+/CD69+) that occurs from interactions with the microenvironment in vivo, suggesting these interactions could induce resistance. This hypothesis was supported when we found that the combination of CpG-ODN, soluble CD40L (sCD40L), and IL10 ("agonist mix") generated resistance to IBR+VEN in most CLL/MCL samples.

In this study, we explored the role of innate immune receptors and other microenvironmental agonists in drug resistance and cancer cell proliferation, singly and in combination. We analyzed induction of apoptosis by IBR+VEN in CLL/MCL patient PBMCs cultured with agonists of various TLRs, NOD1/2, CD40, and IL10R by FACS. Significant resistance to apoptosis by IBR+VEN was noted in most patient samples treated with TLR9 agonist CpG-ODN (10/12) or sCD40L (9/12). IL10 induced modest resistance in a few samples (4/12). TLR1/2, TLR7, and NOD1/2 agonists generated moderate resistance only in MCL samples (N=4). Our preliminary FACS data suggest that CLL cells expressing ZAP-70, a marker of poor disease prognosis, responded with enhanced proliferation to CpG-ODN, sCD40L, and agonist mix as compared to ZAP-70- cells (3/3). In addition, prior exposure to CpG-ODN enhanced the proliferation and drug resistance response to sCD40L and vice versa (4/4), predicting mutually reinforcing microenvironmental interactions in vivo. CpG-ODN or agonist mix induced NF-kB dependent over expression of Mcl-1 and Bcl-xL in CLL/MCL cells, and increased ratio of these proteins to the cognate pro-apoptotic proteins (Puma, BIM, and BAD), indicating enhanced dependence of leukemic B cells for Mcl-1 and Bcl-xL. This presumably caused resistance by lowering cancer cell dependence for VEN target Bcl-2 in presence of agonists.

In conclusion, several microenvironmental agonists, particularly the agonist of TLR9 and CD40, induce resistance to IBR+VEN and cell proliferation in CLL/MCL. Response to these agonists is enhanced in ZAP-70+ cells and when agonist exposure occurs combinatorially. Drug resistance by these diverse agonists was generated by a convergent mechanism of NF-kB dependent over-expression of Mcl-1 and Bcl-xL, which provide additional targets to overcome drug resistance.

#4886

Ligand-independent CXCR7 activation of MAPK signaling leads to prostate cancer enzalutamide resistance.

Shangze Li, Ka-Wing Fong, Galina Gritsina, Ali Zhang, Jonathan C. Zhao, Jung Kim, Jindan Yu. _Northwestern University, CHICAGO, IL_.

Prostate cancer (PC) is the most commonly diagnosed cancer among males in the US. The standard of care for patients with metastatic PC is based on androgen-depletion therapy (ADT), including a first-line chemical castration, and a second-line, treatment with high-affinity antagonists for androgen receptor (AR), e.g. enzalutamide (Enz). Unfortunately, most of the patients eventually relapse with castration-resistant metastatic PC (CRPC). Further, use of Enz is thought to cause a growing concern for prevalence of highly aggressive AR-independent CRPC. Therefore, there is an urgent need for understanding the molecular mechanisms of Enz-resistant (EnzR) CRPC in order to identify novel targeted therapies for deadly PC.

To approach Enz-resistant CRPC we have created EnzR PC cell lines by passaging cells in the presence of sub-lethal doses of Enz for over 6 months. Analyzing the global profiles of expressed genes, we determined CXCR7 (atypical chemokine receptor ACKR3) as one of the top upregulated genes in EnzR cells. Utilizing ChIP-Seq technique we found that AR directly binds to promoter and, at larger scale, enhancer of CXCR7 gene. We identified AR-binding negatively regulates CXCR7 expression; thus, anti-AR therapy unleashes CXCR7 expression in EnzR PC cells. We determined that EnzR PC cells rely on CXCR7-driven signaling in order to survive anti-AR therapy in vitro and in vivo when injected in immunodeficient mice. Moreover, we found that patients with metastatic CRPC increase CXCR7 expression comparing to localized PC tumors. Further, patients who relapsed on second-line ADT demonstrate even higher level of CXCR7 expression.

CXCR7 has a structure of 7-transmembrane (7TM) domains receptor. Activated CXCR7 recruits ARRB2, a cytoplasmic scaffold protein, and internalizes, facilitating activation of MAPK cascade: Raf, MEK, and ERK kinases. CXCR7 can be activated by CXCL12 (SDF1), CXCL11 (ITAC), and MIF. Interestingly, we found that in EnzR PC cells CXCR7 is activated independently from ligand stimulation, causing sustainable ERK phosphorylation (pERK). Thus, we identified that EnzR PC exhibit constitutive accumulation of pro-survival pERK. Accordingly, when we targeted pERK with small molecule inhibitors, we observed decreased EnzR PC survival and growth in vitro and in vivo.

Taken together these data suggest that CXCR7-ERK signaling axis plays a pivotal role in progression to Enz-resistant CRPC, facilitating survival and growth of tumor cells during and after second-line of ADT. Currently, we are focusing on further understanding the molecular mechanisms of CXCR7-ERK tumor-promoting activity. In parallel, we are evaluating the efficacy of CXCR7-ERK targeting for Enz-resistant CRPC and its translatability to the clinics as a novel targeted therapy for patients who relapsed on advanced ADT with deadly CRPC.

#4887

Mitochondrial SOD2 is the mainstay to protect the stemness-featured glioblastoma cells against drug-induced reactive oxygen stress.

Kwang-Yu Chang,1 Chia-Hung Chien,1 Jui-Mei Chu,1 Pei-Hsuan Chung,1 Ming-Sheng Liu,2 Jian-Ying Chuang2. 1 _National Health Research Institutes, Tainan, Taiwan;_ 2 _Taipei Medical University, Taipei, Taiwan_.

Glioblastoma (GBM) is a highly aggressive primary brain tumor with presence of stemness-featured cells that are prone to survive from cancer treatment. We reported Sp1-induced SOD2 was associated with the process to acquire temozolomide (TMZ) resistance that the significance remained to be elucidated. Resistant models were established through long-term coincubation with TMZ in GBM cell lines, which expressed significantly higher stemness properties in extreme limiting dilution analysis (ELDA) and serial transplantation. These cells also showed increased expression of SOD2. Enrichment of stemness-featured cells by spheroid culturing or by CD133-positive cells selection resulted in increased SOD2, suggesting their associations. The impact of the protein to glioma stem cells (GSCs) was then studied through SOD2 RNA interference (RNAi), displaying downregulated stemness features in the knockdown models. Functional studies of the resistant cells showed superior scavenging ability in TMZ- or H2O2-induced ROS that would be dysfunctioned in pretreatment with SOD2 RNAi. In addition, co-treatment with the reagents over the knockdown cells resulted in enhancement of apoptosis as well as inhibition in tumor spheroid culture and cell clonogenic formation, suggesting re-sensitization to the drug. By establishing in vivo model from patient derived xenograft implantation that was obtained from clinical resistant disease, the mouse receiving combination treatment with TMZ and SOD inhibitor had lower tumor proliferation comparing to those receiving TMZ alone. These data indicate that there was an association between SOD2 and the stemness features, suggesting the protein to play important roles in regulation of GSCs behavior. Down-regulation of SOD2 levels may thus be a potential therapeutic strategy for modulating stemness properties of GSCs, making these cells more susceptive to the chemo-therapeutic agent.

#4888

Disrupting MUC1/IFITM1 crosstalk reverses the aggressive phenotype of aromatase inhibitor-resistant breast cancer.

Taylor E. Escher, Eric Geanes, Asona Lui, Joan Lewis-Wambi. _University of Kansas Medical Center, Kansas City, KS_.

Breast cancer is the most commonly diagnosed and second most lethal cancer among women, affecting over 252,000 women in 2017. The ER+/PR+ subtype of breast cancer is the most frequently diagnosed, representing approximately 70% of all cases. Estrogen depletion through aromatase inhibitors (AIs) is the standard of care to cause tumor regression in the ER+ subtype. Our research focuses on targeting the AI-resistant tumors that develop in 30% of women. The human oncoprotein, mucin 1 (MUC1), drives tumorigenesis in breast carcinomas promoting the epithelial-mesenchymal transition, epigenetic reprogramming, and evasion of the immune response. MUC1 interacts with the STAT proteins, via the JAK/STAT signaling pathway, and stimulates transcription of interferon-stimulated genes (ISGs) including IFITM1. Our laboratory has shown that IFITM1 is overexpressed in AI-resistant breast cancer cells and AI-resistant tumors and promotes an aggressive phenotype. In this study, we demonstrate that MUC1 is differentially regulated in endocrine-sensitive (MCF-7 and T-47D) compared to AI-resistant (MCF-7:5C) cells and that it plays a critical role in enhancing IFITM1 expression in the resistant cells. Analysis of a tumor microarray of 94 ER+ human breast tumors indicated that co-expression of both MUC1 and IFITM1 correlated with poor recurrence-free survival, poor overall survival, and AI resistance. To investigate how disruption of the MUC1/IFITM1 crosstalk effects cell survival and proliferation, we used an AI-resistant cell line (MCF-7:5C) that overexpresses MUC1, P-STAT1, and IFITM1. We genetically manipulated breast cancer cells by knocking down the levels of MUC1 with siRNA and observed lowered expression of IFITM1 at the mRNA and protein level and reduced IFITM1 promoter activation. Additionally, knockdown of MUC1 and IFITM1 levels with siRNA induced cell death in AI-resistant cells (MCF-7:5C). We verified this effect with the pharmacologic inhibitors GO-201 (a MUC1 inhibitor) and ruxolitinib (a JAK/STAT inhibitor) to interfere with the MUC1/IFITM1 crosstalk in vitro. Both agents significantly reduced cell proliferation in resistant cells (MCF-7:5C) but not in estrogen-sensitive cell lines (MCF-7 and T-47D). Co-IP confirmed the physical interaction between MUC1 and STAT1/2 and ruxolitinib treatment blocked this interaction by inhibiting STAT phosphorylation in both AI-resistant and -sensitive cells. In vivo studies using ruxolitinib significantly reduced tumor size in NSG mice and decreased expression of MUC1, P-STAT1, and IFITM1. The research discussed here highlights a novel mechanism by which breast cancer cells develop AI resistance and suggests that disrupting MUC1/IFITM1 crosstalk might be therapeutically beneficial to patients with AI-resistant disease.

#4889

**Blockade of FAK and Src activation enhances antitumor activity of sorafenib in non-small cell lung carcinomas harboring G12S** KRAS **mutation.**

Qingyu Stephanie Zhou, Mitchell West. _Univ. of South Florida College of Pharmacy, Tampa, FL_.

Background:

Mutations in the KRAS gene are present in approximately 25% of lung adenocarcinoma cases and are associated with poor prognosis and resistance to anti-EGFR therapy. Although sorafenib has shown considerable promise in the treatment of KRAS mutant NSCLC, resistance to the treatment eventually occurs. We have previously reported that acquired sorafenib resistance in KRAS-mutant A549 xenografts involves the compensatory activation of focal adhesion kinase (FAK) and Src, while inhibition of Src alone is insufficient to overcome the resistance.

Objectives:

Our aim in this study was to demonstrate simultaneous blockade of FAK and Src activation as a potential strategy to enhance the antitumor activity of sorafenib in KRAS mutant NSCLC.

Methods:

The cytotoxicity of sorafenib and dasatinib used as a single agent or in combination was initially determined in human NSCLC cells (A549, H358 and H1915 cells), with either non-targeting control shRNA or FAK shRNA knockdown. Based on the initial findings, the cytotoxicity of sorafenib, dasatinib and FAK inhibitor 14 used as a single agent or in combination was examined in A549 cells. Western blot analysis was conducted to reveal possible mechanisms underlying the improved therapeutic effect.

Results:

Among the three selected human NSCLC cell lines, H358 (with G12C KRAS mutation) was the most sensitive and H1915 (with wild-type KRAS) was the most resistant to treatment with dasatinib and sorafenib alone or in combination. In A549 cells with G12S KRAS mutation, FAK knockdown significantly increased the sensitivity of cells to dasatinib treatment (P < 0.01) but not to sorafenib treatment. As a result, combination therapy with dasatinib and sorafenib significantly improved the synergistic effect between dasatinib and sorafenib in FAK-knockdown A549 cells (P < 0.01). The increase in synergistic effect was not observed in FAK-knockdown H358 and H1915 cells, suggesting simultaneous blockade of FAK and Src activation can greatly enhance the antitumor activity of sorafenib in NSCLC harboring G12S KRAS mutation. Further evaluation of the cytotoxicity of treatment with dasatinb, FAK inhibitor 14 and sorafenib alone or in combination in A549 cells co-expressing green fluorescent protein (GFP) and luciferase showed that the triple combination of dasatinib, FAK inhibitor 14 and sorafenib produced an additive effect. Results of the Western blot analysis revealed that FAK inhibitor 14 failed to effectively inhibit Y397-phosphorylation of FAK, which might in part explain why the triple combination did not show therapeutic synergism in A549 cells.

Conclusions:

Results of our in vitro study warrants further evaluation of the triple combination therapy with sorafenib, dasatinib and an effective FAK inhibitor in the treatment of NSCLC with G12S KRAS mutation.

#4890

Understanding the crosstalk between glycosylation and drug resistance.

Padmamalini Thulasiraman, Kelbie Kerr. _University of South Alabama, Mobile, AL_.

Although great progress has been made in the treatment of breast cancer, resistance to chemotherapeutic drugs is a problem facing current cancer research, promoting the need to understand the molecular mechanisms involved in breast cancer and identify future targeted therapy. One of the mechanisms by which chemoresistance is attained in cancer cells is mediated through the expression of MDR-related glycoproteins (MRPs). These transmembrane proteins efflux drugs across the plasma membrane, conferring resistance of cancer cells to anticancer drugs. Elevated expression of Multidrug Resistance Protein 1 (MRP1) has been associated with poor prognosis and survival rate among breast cancer patients. Acquiring drug resistance has been correlated to the emergence of metastasis, accounting for the progression of the disease. Altered cell-surface glycosylation is a common feature of cancer initiation and progression. During glycosylation, the MRP1 undergoes N-linked glycosylation but it is unclear how glycosylation impacts the drug transport activity. To examine the crosstalk between MRP1 and Neuraminidase 1 (Neu1), the enzyme that catalyzes the removal of sialic acid residues from glycoproteins, we initiated our studies using plant derived natural product honokiol to examine the effect of honokiol on Neu1. In this study, we demonstrated that honokiol suppressed Neu1 in two breast cancer cell lines using Western Blot Analysis. To determine whether there is a link between Neu1 and MRP1 in improving the efficacy of doxorubicin-facilitated growth suppression, we treated the breast cancer cells with Neu1 inhibitor, Oseltamivir Phosphate (OP) and demonstrated that at 600 ug/ml and 800 ug/ml, OP suppressed breast cancer cell growth. OP in combination with doxorubicin reduced growth of breast cancer cells and enhanced the efficacy of doxorubicin-mediated growth inhibition. Future studies are underway to examine the effect of OP and honokiol on the glycosylation of MRP1. We propose that these studies will lead to the understanding on how glycosylation of MRP1 contributes to the transport of chemotherapeutic agents and eventually to the spread of the disease.

#4891

Co-inhibition of AXL and FN14 by pharmacologic inhibitors or genetic inhibition in NSCLC mice models.

Dhananjay Suresh, Soumavo Mukherjee, Ajit Zambre, Shreya Ghoshdastidar, Sairam Yadavilli, Anandhi Upendran, Raghuraman Kannan. _University of Missouri, Columbia, MO_.

Non-small Cell Lung Cancer (NSCLC) patients who express EGFR activating mutations are treated with tyrosine kinase inhibitors (TKI) and have a median overall survival time of 22 months. TKI therapy showed only moderate success with poor (37%) survival rate. On the other hand, TKI therapy is ineffective in patients with active KRAS mutation. Acquired drug resistance is one of the major reasons for the failure of the TKI therapy. Studies have shown that overexpression of AXL is one of the possible reasons for the drug resistance. In our previous studies, we have shown that AXL inhibition in NSCLC drug-resistant cells resulted in FN14 survival activation. Subsequently, we have demonstrated that co-inhibition of both AXL and FN14 resulted in increased therapeutic efficacy. Based on these studies, we recognize that both AXL and FN14 play important role in proliferation, invasion and survival pathways. Therefore, synergistic co-inhibition of AXL-FN14 is a viable therapeutic strategy for treating NSCLC. In the present study, we compared the efficacy of inhibition of AXL and FN14 by siRNA (using nanoparticle delivery vehicles) and small-molecule inhibitors in A549 mouse xenografts. Our data further confirms that co-inhibition is effective and results in tumor volume reduction. The details of this study provide a rationale for enhanced treatment of drug resistant NSCLC.

#4892

Del-1 promotes the proliferation and migration of tamoxifen-resistant breast cancer.

Yee Soo Chae,1 Jong Gwang Kim,1 Jae-Hwan Jeong,1 Soo Jung Lee,1 Jeeyeon Lee,1 Jin Hyang Jung,1 Ho Yong Park,1 Moon-Chang Baek,2 Senten Richard J,3 Ji Young Park1. 1 _Kyungpook National University Medical Center, Daegu, Republic of Korea;_ 2 _Kyungpook National University, Daegu, Republic of Korea;_ 3 _University of Virginia Health System, Charlottesville, VA_.

Background: We previously demonstrated a prognostic role of exosomal Del-1 with breast cancer patients. However, the mechanisms of Del-1 expression are barely understood. Development of resistance to tamoxifen is an important clinical issue in the treatment of breast cancer. Accordingly, we investigated the function of Del-1 in tamoxifen-resistant (TAMR) breast cancer cell line and tumor specimens.

Materials and Methods: Del-1 expression in MCF7 and TAMR MCF7 cells was performed by quantitative RT-PCR, western blot, and ELISA. The effects of Del-1 with RNA interference on proliferation, migration and invasion of TAMR MCF7 cells were observed by MTT, wound healing and Matrigel transwell assay. Meanwhile, the tumoral expression of Del-1 was determined immunohistochemistry (IHC) from 10 tamoxifen resistant-breast cancer patients. Tumor tissue before (tamoxifen sensitive) and after tamoxifen resistance were assessed.

Results: Del-1 was highly expressed in TAMR MCF7 cells compared to MCF7 cells (p<0.001). And the expression of secretory del-1 was also increased in the culture media of TAMR MCF7 cells. Moreover, down-regulation of Del-1 significantly inhibited the proliferation and migration of TAMR MCF7 cells. There was no difference in the invasion of TAMR MCF7 cells. Increased Del-1 expression was preferentially observed in in TAMR tumor tissues when compared to tamoxifen sensitive or normal breast tissue.

Conclusion: Prominent expression of Del-1 in TAMR MCF7 cells was associated with the proliferation and migration of TAMR MCF7 cells. And the tumoral expression of Del-1 was also significantly associated with tamoxifen resistance. These results suggest that the expression of Del-1 promote tamoxifen resistance in breast cancer cells and could be a novel target for anti-breast cancer treatment.

#4893

The use of glycyrrhetinic acid derivatives to inhibit blebbishield emergency program pathogenesis overcoming erlotinib resistance in non-small cell lung cancer.

Ebony Nottingham, Elizabeth Mazzio, Arindam Mondal, Mandip Sachdeva. _Florida A &M Univ. College of Pharmacy, Tallahassee, FL_.

Background: Bleb transformation is an initial induction of apoptosis leading to cellular compartmentalization into vesicles, increased glycolysis/mitochondrial activity combined with dynamin mediated endocytosis allowing the cell to tether vesicles to form stem like spheroids protecting exposed genetic material. Our laboratory has found that not only do both Erlotinib sensitive and resistant cell lines undergo blebbishield transformation in response to treatment, but the addition of Glycyrrhetinic acid derivative (CDODA-Me) overwhelms this pathway leading to cellular apoptosis.

Methods: NSCLC cell lines HCC827 (both erlotinib resistant and sensitive), were treated with CDODA-Me and Erlotinib (combination and single treatment). MicroRNA expression profiling was used to identify and validate reference genes involved with the synergistic effects comparing combination treatment to erlotinib single treatment groups. Spheroids were collected after 24hr treatment using blebbishield extraction media (BEM) consisting of 12µM erlotinib alone and in combination with 2µM CDODA-Me. Western Blot analysis was used to confirm identified pathways as well as to quantify the effects of CDODA-Me on VEGF and TGF-β cellular release levels during treatment as well as MET and EGFR kinase activation.

Results: Genomic studies showed an increase in microRNA has-miR-711 (approximately 4-fold) which is responsible for inhibiting dynamin mediated trafficking, various growth factor expression levels (including VEGF and TGF-β), as well as the expression of receptors associated with these growth factors (EGFR, MET, VEGFR). This inhibition was confirmed through western blot analysis showing a decrease in receptor activation but no effects on receptor expression (6 fold and 8 fold decrease when compared to erlotinib single treatment for EGFR and MET respectively). Sphere reattachment was found to be dependent on the presence of growth factors and other unknown cell signaling molecules through the fact that spheres did not attach in fresh media, but attached in as little as 3hrs in media obtained after 24hr incubation in parental cells. Media obtained after incubation in parental cells provided higher levels of VEGF and TGF-β when compared to media obtained after 24hr treatment with (resistant to non-resistant expression ratios of 3.94 and 4.06 for HCC827 (4µM erlotinib resistant) and HCC827 Cl4 respectively). Actin fluorescent staining 48hrs after reseeding spheroids showed that cells grown in untreated parental media regained normal branch like morphology while those grown in CDODA-Me treated parental media remained rounded and died.

Conclusions: Based on the results of these studies, CDODA-Me has been shown to inhibit NSCLC cell types from overcoming apoptosis thereby inhibiting increased drug resistance as well as tumorgenicity.

#4895

Human ATP-binding cassette proteins ABCB1 and ABCG2 confer resistance to histone deacetylase 6 inhibitor ricolinostat (ACY-1215) in cancer cell lines.

Sung-Han Hsiao,1 Shahrooz Vahedi,2 Suresh V. Ambudkar,2 Chung-Pu Wu1. 1 _Chang Gung Univ., Tao-Yuan, Taiwan;_ 2 _National Institutes of Health, Bethesda, MD_.

Histone deacetylase 6 (HDAC6), localized primarily in the cytoplasm, is responsible for the deacetylation of non-histone substrates such as α-tubulin and heat shock protein 90 (Hsp90). HDAC6 has recently emerged as an important epigenetic therapeutic target for cancer, and much effort has been invested into developing novel inhibitors of HDAC6. Ricolinostat (ACY-1215) is the first safe and well tolerated inhibitor of HDAC6 that has progressed into human clinical trials, both as a single agent and in combination therapy. Ricolinostat is currently under evaluation in phase I and II clinical trials in patients with multiple myeloma, relapsed or refractory lymphoid malignancies, relapsed chronic lymphocytic leukemia, unresectable or metastatic breast cancer and for the treatment of gynecological cancer. The overexpression of ATP-binding cassette (ABC) drug transporters ABCB1 and ABCG2 in cancer cells is often associated with the development of multidrug resistance (MDR) phenotype and poor clinical outcome in patients with metastatic breast cancer, chronic lymphocytic leukemia or relapsed multiple myeloma. In this study, we report that ABCB1 and ABCG2 are capable of actively transporting and conferring resistance to ricolinostat. Moreover, we demonstrated that the efficacy of ricolinostat on the activity of HDAC6 and drug-induced apoptosis was significantly reduced in ABCB1- and ABCG2-overexpressing cancer cells, which can be fully restored by an competitive inhibitor of ABCB1 or ABCG2.Our finding is supported by biochemical data and a docking analysis of ricolinostat in the drug-binding pocket of ABCB1 and ABCG2. Taken together, our study indicate that the overexpression ABCB1 or ABCG2 in cancer cells may potentially play a significant role in the development of resistance to ricolinostat in cancer cells.

#4896

The role of SLC7A5 (LAT1) in endocrine therapy-resistant breast cancer.

Catherine M. Sevigny,1 Surojeet Sengupta,1 Zhexun Luo,1 Lu Jin,1 Dominic Pearce,2 Robert Clarke1. 1 _Georgetown University, Washington, DC;_ 2 _University of Edinburgh, Edinburgh, United Kingdom_.

Breast cancer is the leading cancer diagnosis for women in the United States. Endocrine therapies, such as tamoxifen and aromatase inhibitors, are used to treat the estrogen receptor-positive (ER+) breast cancers that comprise 70% of all new cases. Unfortunately, emergence of resistance to these therapies presents a major clinical challenge. Cancer cells can adapt to dysregulate the bioenergetics of cellular metabolism and evade cell death. Solute carrier family 7 member 5 (SLC7A5 or LAT1) is expressed across the cell membrane and is a transporter of large, neutral amino acids (such as leucine or tyrosine) supporting cell proliferation. Using LCC9 breast cancer cells that are resistant to tamoxifen and fulvestrant, we studied the role of LAT1 in endocrine therapy-resistant cells as compared to sensitive MCF7 breast cancer cells. SLC7A5 expression was upregulated by estrogen in MCF7 cells that was blocked in the presence of fulvestrant treatment. A significant 2.75-fold upregulation of SLC7A5 protein and 71-fold upregulation of SLC7A5 mRNA was found in LCC9 cells (as compared with MCF7 cells) without any estrogen regulation. Fulvestrant treatment did not significantly alter SLC7A5 mRNA or protein expression. These data suggest that higher levels of SLC7A5 in resistant cells may help in transporting key amino acids from their microenvironment to support cell growth. Inhibiting the functions of SLC7A5 using a pharmacologic inhibitor, JPH203, or depleting its expression by using siRNA led to significant growth suppression of LCC9 cells. Cell cycle analysis revealed that SLC7A5 depletion increased G1 phase of cell cycle and reduced S phase cells. In five publicly available data sets, of estrogen receptor-positive and tamoxifen-treated breast cancer patients, high expression of SLC7A5 was associated with worse prognosis. Endocrine therapy resistance is partly driven by autophagy in breast cancer cells. We depleted SLC7A5 expression in LCC9s to better understand the role of this transporter in autophagy. This study uncovers a novel adaptive mechanism in endocrine therapy-resistant breast cancer cells that is facilitated by increased SLC7A5 mediated transport of large neutral amino acids enabling them to supplement their increased metabolic needs and promoting cell growth. Therefore, blocking the functions of SLC7A5, perhaps in conjunction with inhibition of autophagy, may offer an avenue of potential therapeutic intervention in endocrine therapy-resistant breast cancers.

#4897

An acquired vulnerability of drug resistant melanoma with therapeutic potential.

Rodrigo Leite de Oliveira. _NKI, Amsterdam, Netherlands_.

BRAF(V600E) mutant melanomas treated with inhibitors of the BRAF and MEK kinases almost invariably develop resistance, which is frequently caused by reactivation of the Mitogen Activated Protein Kinase (MAPK) pathway. To identify novel treatment options for such patients, we searched for acquired vulnerabilities of MAPK inhibitor-resistant melanomas. We find that resistance to BRAF+MEK inhibitors is associated with increased levels of reactive oxygen species (ROS). Subsequent treatment with the histone deacetylase inhibitor (HDACi) vorinostat represses SLC7A11 that leads to a lethal increase in the already elevated levels of ROS in drug-resistant cells, thereby causing selective apoptotic death of only the drug resistant tumor cells. Consistently, treatment of BRAF inhibitor-resistant melanoma with HDACi in mice results in a dramatic tumor regression. In a study in patients with advanced BRAF+MEK inhibitor resistant melanoma, we find that HDACi can selectively ablate drug-resistant tumor cells, providing clinical proof of concept for the novel therapy identified here.

#4898

Reduction of TGFBR2 in osteoblasts by enzalutamide causes drug resistance in prostate cancer bone metastasis.

Alexandra Vander Ark,1 Erica Woodford,1 Xiangqi Meng,2 Sourik Ganguly,1 Xiaotun Zhang,3 Zachary Madaj,1 Xiaohong Li1. 1 _Van Andel Research Institute, Grand Rapids, MI;_ 2 _The Sixth Affiliated Hospital of Sun Yat-sen University, Guangzhou, China;_ 3 _Mayo Clinic, Rochester, MN_.

In the United States, over 80% of prostate cancer patients die with bone metastases. Second-line hormonal therapies, such as enzalutamide, only improve overall patient survival by a few months in about 50% of patients, and almost all patients develop drug resistance. Thus, there is an urgent need to determine the mechanisms of drug resistance in order to provide better treatments for prostate cancer bone metastasis. Enzalutamide is a small-molecule inhibitor of the androgen receptor. Our study showed that although enzalutamide inhibited the tumor growth of castration-resistant prostate cancer C4-2B cells when xenografted subcutaneously, it had no effect on the growth of C4-2B tumors in the bone or on the development of bone lesions. These data suggested a crucial role of the microenvironment in enzalutamide resistance in prostate cancer bone metastasis. To dissect the role of enzalutamide on the bone microenvironment, we treated bone cells, both osteoblasts and osteoclasts, with enzalutamide. We found that enzalutamide significantly and specifically reduced the TGF-β type II receptor (TGFBR2) protein in osteoblasts. This observation was also confirmed in prostate cancer bone metastatic tissue microarray, in which we performed immunohistochemistry and found a significant decrease of TGFBR2 expression specifically in cancer-associated osteoblasts from patients who had undergone treatment with second-line hormonal therapies, either enzalutamide or abiraterone. Statistical analysis showed that the decrease of TGFBR2 in cancer-associated osteoblasts was a causative effect from the hormonal therapies. To determine the role of TGFBR2 in the osteoblasts during bone metastasis, we used a mouse model (Tgfbr2Col1CreERT KO) with inducible Tgfbr2 knockout specifically in the osteoblasts. We found that loss of TGFBR2 in osteoblasts significantly promoted prostate cancer bone metastasis. Together, our data showed for the first time that reduction of TGFBR2 in osteoblasts by enzalutamide causes resistance to the drug in prostate cancer bone metastasis.

#4899

Establishment and characterization of SN-38 resistant metastatic colorectal cancer cells (SW620/SN100) reveals an ABCG2 (BCRP) transport mediated resistance.

Anna Maria Barbuti, Zhe-Sheng Chen. _St. John's University, Queens, NY_.

Despite advances in anticancer drug research, chemotherapy options and response rates for metastatic colorectal cancer (mCRC) are poor. Irinotecan, a topoisomerase I inhibitor, is a commonly used chemotherapy against mCRC as a single agent, or in combination with other anti-cancer drugs such as fluorouracil and leucovorin. However, acquired resistance to chemotherapy, especially in metastatic patients, is a critical cause of treatment failure.

Multi-drug resistance (MDR) in cancer is a major challenge to chemotherapy success. Mechanisms of MDR involve deliberate alteration in cellular expression, function, and/or responses of the cancer. One of the most notable mechanism of MDR is an increased cellular drug efflux through up-regulation of membrane ATP-binding cassette (ABC) transporters.

To investigate a new and clinically relevant mechanism of MDR in mCRC cells, SN-38 (the potent and active metabolite of irinotecan) was regularly administered to SW620 cells with drug-free intervals to reflect a drug induced resistance to treatment. Through cytotoxicity and western blotting assays on the newly formed SW620/SN100 cells, we discovered ABCG2 (BCRP) to be the main mechanism of SN-38 resistance, with no alterations in the topoisomerase I, ABCB1 (P-gp) or ABCC1 (MRP1). These results confirmed our initial hypothesis, given SN-38 is a known substrate of ABCG2. This discovery of SN-38 induced efflux in mCRC is of a larger importance because this mechanism not only limits the efficacy of the chemotherapy, but also poses a threat to the efficacy of a variety of pharmacologically dissimilar drugs which are substrates of ABCG2.

As treatments for mCRC continue to be investigated it is important to also continue investigating resistant mechanisms of these drugs. More discoveries of MDR mechanisms which are specific to cancer patient populations will offer superior diagnosis, proper treatment regimens and greater prognosis for patient success.

#4900

Reactive oxygen species scavenger extends the efficacy of BRAF inhibitors in BRAF-mutant melanoma.

Long Yuan, Rosalin Mishra, Hima Patel, Samar Alanazi, Joan Garrett. _University of Cincinnati, Cincinnati, OH_.

We aim to identify whether reactive oxygen species (ROS) targeted therapy using a ROS scavenger, A100, will inhibit tumor growth and evade BRAF inhibitor induced resistance in melanoma cells harboring the endogenous BRAFV600E mutation. We also propose to investigate the mechanism by which A100 sensitizes the dabrafenib-mediated resistance. Dabrafenib-resistant (DR) melanoma cells (WM-115DR, SK-MEL-24DR and A375DR) were generated using increasing doses of dabrafenib. We conducted flow cytometry experiments to measure ROS levels before and after acquisition of resistance. DCF-DA assay indicated upregulation of hydrogen peroxide levels in dabrafenib resistant cell lines versus parental cell lines. Furthermore, dabrafenib resistant cell lines demonstrated elevated superoxide levels as analyzed by MitoSOX. Crystal violet and three-dimensional matrigel assays were performed to analyze the effect of A100 and dabrafenib on cell growth of dabrafenib resistant and parental melanoma cell lines. We observed a statistically significant decrease in cell proliferation and colony formation of dabrafenib resistant cells when subjected to the combination of A100 and dabrafenib compared to single agent dabrafenib or A100. We also noted a trend of inhibition of cell proliferation in response to combined treatment of A100 and dabrafenib in parental cell lines versus the single agent. There was significant reduction in colony formation under similar condition in parental cells. Dabrafenib as a single agent had similar effect in reducing phosphorylated ERK1/2 as compared to co-treatment with A100 in A375, SK-MEL-24 and WM-115 parental cells. As expected, there was no alteration of MAPK signaling in resistant WM-115DR, SK-MEL-24DR and A375DR cells upon treatment with dabrafenib. A100 has been reported to cause DNA damage in leukemia cells. We analyzed the effect of A100 in the presence or absence of dabrafenib on markers of DNA damage. The data suggested that DNA damage is induced by A100 as assessed by increased levels of p-ATM, p-ATR and γ-H2AX. Further, A100 in combination with dabrafenib increased levels of p-ATM, p-ATR and γ-H2AX in parental and dabrafenib resistant cell lines. Proteomics analysis on WM-115 versus WM-115DR cell line was performed to determine alterations in mitochondrial protein expression upon resistance to BRAF inhibitors. The data indicated upregulation of proteins with antioxidant functions including SOD2 (superoxide dismutase [Mn]) and Peroxiredoxin-1 (PRDX1) in WM-115DR cells compared to WM-115 parental which was confirmed by immunoblotting analysis. Overall, this study suggested that the combination of ROS quenching agent (A100) with BRAF inhibitor (dabrafenib) could be a potential strategy to treat BRAF-mutant melanoma patients.

#4901

Inhibition of γ-secretase sensitizes drug-resistant neuroblastoma cells to doxorubicin.

Kathryn A. Solka, Fei Chu, Yi-Yong Qiu, Sandra H. Clark, Mary Beth Madonna. _Ann and Robert H. Lurie Children's Hospital of Chicago and Northwestern University Feinberg School of Medicine, Chicago, IL_.

Introduction: Neuroblastoma is the most common extracranial solid tumor in childhood and accounts for ~15% of all pediatric cancer mortality. Approximately 650 new cases are reported annually in the USA, with 90% of cases diagnosed by 5 years of age. Novel therapies are needed as ~50% of cases are high-risk neuroblastoma with a poor (<50%) five-year survival rate due to relapse and treatment resistance. Neuroblastoma cells undergo a mesenchymal change, including the activation of Notch pathway genes, in their transition to a drug-resistant state. The Notch pathway has both oncogenic and tumor-suppressive roles and has been implicated in epithelial-mesenchymal transition (EMT) and chemoresistance. This pathway can be blocked by γ-secretase inhibition, which prevents cleavage of the Notch receptor and therefore Notch signal transduction. Thus, targeting the Notch pathway has been considered a promising anticancer strategy. Underlying mechanisms of the Notch signaling pathway in drug-resistant cancers, including neuroblastoma, are not well understood.

Aim: Determine if inhibition of the Notch signaling pathway sensitizes chemoresistant neuroblastoma cells to doxorubicin.

Methods: Doxorubicin-resistant (DoxR) cells were generated by incubating doxorubicin-sensitive, wild-type (WT) patient-derived cell lines with incremental doxorubicin concentrations (1 x 10-9 to 1 x 10-6 M) over six months. Cells were considered chemoresistant after surviving five consecutive passages in 1 µM doxorubicin (MTT assay). Differentially expressed genes were identified by microarray (Illumina HumanHT-12 v4 Expression BeadChip) and validated by RT-qPCR. Cells treated with doxorubicin and/or DAPT (γ-secretase inhibitor) were evaluated for cell viability (MTT assay), proliferation (PCNA immunofluorescence and Western blot), senescence (SA-β-Gal immunocytochemistry and p21 Western blot) and proteomic profiling (2D-DIGE).

Results: Compared to WT cells, DoxR cells differentially expressed the following Notch pathway genes: downregulation of DLK1 and upregulation of HEY1 and SNAI2, as determined by microarray and RT-qPCR. Compared to WT cells, DoxR cells treated with both doxorubicin and DAPT exhibited significantly decreased cell viability, decreased proliferation and increased senescence. DAPT-treated DoxR cells differentially expressed nine proteins using 2D-DIGE: MCM3, MCM6, CAPN10, ACTN4, TUBA1B, HNRNPK, PDP1, NPM1 and BARD1.

Conclusion: The γ-secretase inhibitor DAPT sensitizes DoxR neuroblastoma cells to doxorubicin. Mechanisms underlying the synergistic effect of doxorubicin and DAPT in DoxR cells warrant further investigation.

#4902

Oxidized low-density lipoprotein is a potentially potent mediator of proteasome inhibitor resistance in multiple myeloma.

Edward A. Medina,1 Javier Esparza,1 Srikanth Polusani,1 Valerie Cortez,1 Huynh N. Nguyen,1 Gopalrao V. Velagaleti,1 Hongxin Fan Fan,1 Marsha C. Kinney,1 Reto Asmis2. 1 _UT Health Science Ctr. at San Antonio, San Antonio, TX;_ 2 _Wake Forest School of Medicine, Winston-Salem, NC_.

Multiple myeloma (MM) is a malignancy of plasma cells that accumulate in the bone marrow. While treatment advances, particularly proteasome inhibitors (PIs) and immunomodulatory drugs, have improved survival, MM is incurable. Obesity and insulin resistance are established risk factors for MM mortality. Oxidized low-density lipoprotein (OxLDL), a key atherogenic factor that is elevated in obesity, has emerged as a risk factor for the development and progression of some solid cancers, and it has been shown to stimulate pro-oncogenic signaling; its role in MM is unexplored. We evaluated the influence of OxLDL on MM cell killing by chemotherapeutics used to treat MM. OxLDL, at concentrations within the range reported for patients with metabolic syndrome, suppressed MM cell killing by the therapeutic PIs bortezomib and ixazomib. Native low-density lipoprotein (nLDL) did not affect the anti-MM effects of these PIs, which suggests that the oxidative modification of lipids or apolipoprotein in OxLDL is the mediator of cytoprotection. OxLDL did not affect MM cell killing by other agents with distinct targets such as the immunomodulatory drug lenolinamide and doxorubicin. OxLDL appeared to restore proteasome activity as evidenced by its suppression of bortezomib-induced accumulation of ubiquinated proteins and pro-apoptotic unfolded protein response signaling. The cytoprotective effects of OxLDL were suppressed when lipid hydroperoxides (LOOHs) associated with the lipoprotein were specifically reduced by pretreatment with the glutathione-dependent selenoperoxidase mimetic ebselen. Finally, immunohistochemical analysis of bone marrow biopsy samples from newly diagnosed MM patients demonstrated the presence of OxLDL in macrophages/histiocytes scattered among MM cells. Our findings suggest that OxLDL may be a potent mediator of chemoresistance to therapeutic PIs in obese/insulin-resistant MM patients. OxLDL appears to counteract the anti-MM effects of PIs through LOOH-mediated restoration of proteasome activity, which is significant since LDL is the major carrier of LOOHs in the plasma. Our findings raise the potential benefit of LDL cholesterol-lowering therapy, or pharmacologic targeting of intracellular pathways used by OxLDL, to increase the efficacy of therapeutic PIs and improve the survival of obese/insulin-resistant MM patients.

#4903

AXL inhibition overcomes erlotinib resistance in AXL-activated non-small cell lung cancer cells.

Feng Wang. _Albert Einstein Cancer Center, Bronx, NY_.

Mutations in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) are present in 10-35% patients with NSCLC. In patients with EGFR-mutated NSCLC, EGFR tyrosine kinase inhibitors, including erlotinib, have demonstrated excellent initial responses. However, despite improved outcomes observed in patients treated with EGFR TKIs, most patients eventually develop drug resistance. We and others recently demonstrated that the activation of oncogene AXL is necessary to confers erlotinib resistance in both cell and mouse models as well as in human tumor tissues. While genetic aberrations affecting AXL appear rare, we recently identified two patients with EGFR-mutated lung cancer who simultaneously harbor AXL A273V or AXL V289M mutation. Both patients were resistant to erlotinib treatment (one case of primary and one case of acquired resistance). In this study, we first evaluated whether these AXL mutations conferred resistance to erlotinib treatment. Using HCC827 and PC-9 NSCLC cell lines stably expressing the AXL A273V or V289M mutation, we confirmed that AXL A273V significantly increased resistance to erlotinib treatment. Subsequently we also conducted a preclinical study to test if a new AXL inhibitor MGCD265 could overcome such erlotinib resistance in NSCLC cells. Using an in vitro MTS assay, we first validated that administration of MGCD265 in combination with erlotinib significantly inhibited the viability of erlotinib-resistant HCC827-ER cells, compared to erlotinib- or MGCD265-alone treatment. Through conducting an array of cell functional studies, we found that combination of MGCD265 and erlotinib significantly decreased cell cycle progression in S phase, enhanced apoptosis, and repressed cell migration relative to single-agent erlotinib or MGCD265 treatment in HCC827-ER cells. We further performed RNA-sequencing to evaluate the gene expression profile and the cellular pathways altered in cells treated with erlotinib alone, MGCD265 alone or a combination of erlotinib and MGCD265. RNA-sequencing analysis identified that several groups of genes involved in cell survival inhibition or apoptosis promotion were upregulated, whereas genes involved in cell cycle process, DNA replication or cancer metabolism were downregulated in cells treated with the combination of erlotinib and MGCD265. Taken together, our results indicated that AXL mutations may confer resistance to EGFR TKI treatment, and effective blockade of the AXL pathway may represent a novel strategy to overcome erlotinib resistance for the treatment of NSCLC patients.

#4904

PD-L1 pathway activation as an escape mechanism of resistance to MEK inhibitor treatment in human colorectal cancer models.

Stefania Napolitano,1 Nunzia Matrone,1 Valentina Belli,1 Vincenzo De Falco,1 Erika Martinelli,1 Scott Kopetz,2 Fortunato Ciardiello,1 Teresa Troiani1. 1 _University of Campania "Luigi Vanvitelli", Napoli, Italy;_ 2 _MD Anderson Cancer Center, Houston, TX_.

Molecular mechanisms driving acquired resistance to anti-EGFR therapies in mCRC is complex and biochemically converge to activate the EGFR-RAS-MAPK pathway. In order to understand the mechanism underlying MEKi resistance, SW48 MEKi resistant cell lines (SW48-MR) have been established by in vivo selection. To expand our results, another CRC cell line sensitive to MEK blockade, such as HCT116, has been generated: HCT116-MR. Microarray analysis showed that several genes involved in the PD-L1 pathway were up regulated in SW48-MR versus SW48. Moreover, genes overexpressed in MEKi-resistant tumor were related to the gene signature of CMS4 subtype of CRC. In particular all the pathways that were up regulated in CMS4 tumors were up regulated in the resistant cell line, as confirmed by Western blot and Immunofluorescence analysis. The regulation of PD-L1 expression is regulated by multiple signaling pathways, including JAK/STAT. STAT3 gene is also up regulated in MEKi-resistant tumors. Inhibition of STAT3 expression by siRNA reduced PD-L1 expression and restored MEKi sensitivity. Moreover, a direct correlation has been demonstrated between EGFR and PD-L1. Exogenous activation of EGFR by TGFα stimulation in MEKi sensitive HCT 116 cell lines induced PD-L1 overexpression and resistance to MEKi. Once demonstrated the EGFR role in modulation of PD-L1 expression and subsequently development of acquired resistance to MEKi, we further evaluated the potential mechanism(s) responsible for EGFR activation. One major mechanism is the specific ligand stimulation. Gene expressions analysis revealed that all EGFR ligands were up regulated in MEKi resistant tumors compared to parental tumors. Moreover, we will perform an ELISA assay to confirm this data. To extend these in vitro findings, we will perform an in vivo study using MC38 and CT26 MEKi resistant syngeneic models that we have previously generated. These results suggest a strategy to potentially improve the efficacy of MEK inhibition by co-treatment with other agents and provide an additional therapeutic strategy via modulation of host immune responses in CMS4 CRC tumors.

### Pharmacokinetics and Pharmacodynamics

#4905

Utility of human FcRn transgenic mice for preclinical screening of immunotherapeutics.

Gregory J. Christianson, Emily Lowell, Cat Lutz. _The Jackson Laboratory, Bar Harbor, ME_.

Preclinical assessment is required for the growing number of immunotherapeutics in development. Clinically relevant pharmacokinetic analysis can be achieved by using transgenic mice that uniquely express the human Fc receptor neonatal (hFcRn). To demonstrate the utility of the human FcRn Tg mouse model platform, three immunotherapeutics (pembrolizumab, ipilimumab, and belatacept) were administered IV to Tg32, Tg276, FcRn null, and B6 wild type mice. The mice were blood sampled (25 µL) at 1, 3, 5, 7, 9, 12, 16, 19, 22, 26, and 30 days. Immunotherapeutic plasma concentations, assessed by human IgG ELISA, were pharmacokinetically analyzed. Tg32 mice yielded half-life values for these immunotherapeutics with ranges that mimicked patient data. Though reduced in scale, Tg276 mice also produced half-life data that correlated with the established human half-life data for pembrolizumab, ipilimumab, and belatacept. These results confirm that the human FcRn Tg model platform can be broadly applied to preclinical pharmacokinetic screening of mAb and Fc-fusion based immunotherapeutics.

#4906

Effect of gender on the rodent pharmacokinetics of ON 123300, a dual inhibitor of ARK5 and CDK4/6, for the treatment of cancer.

Chen Ren,1 Jennypher Mudunuru,2 David Taft,2 Manoj Maniar1. 1 _Onconova Therapeutics, Inc., Newtown, PA;_ 2 _Long Island University, Brooklyn, NY_.

Purpose: To evaluate gender differences in the metabolism of ON 123300 in rats in vitro and in vivo. ON 123300, a novel small molecule dual inhibitor of the c-MYC activated kinases ARK5 and CDK4/6, is being developed by Onconova Therapeutics, Inc. as a novel anti-cancer drug candidate.

Methods: In vitro metabolism experiments were performed in rat liver microsomes from male and female donors. ON 123300 was incubated with microsomes, and samples were withdrawn at specified times over 60 minutes and, immediately quenched and centrifuged. The supernatant was analyzed for ON 123300 and its metabolites by HPLC. An in vivo pharmacokinetic study was performed in male and female SD rats. ON 123300 was administered as a single oral dose (100 mg/kg), and blood samples were collected over 24 hours. ON 123300 concentrations were measured by LC-MS/MS. Pharmacokinetic parameters were estimated by non-compartmental analysis.

Results: The results are provided in Table 1. ON 123300 displayed more rapid microsomal degradation in vitro (increase of Clint) in males compared to females. This translated into a significantly higher exposure of ON 123300 (3 fold increase of AUC) following oral administration to female rats. Based on these results, the higher plasma concentrations observed in females can be primarily attributed to reduced hepatic clearance compared to males.

Conclusions: Gender differences in pharmacokinetics and pharmacodynamics can potentially impact the design of safe and effective dosing regimens for treatments. This investigation demonstrated a significantly lower metabolism of ON 123300 in female rats, which resulted in dramatically high systemic exposure. Additional testing is warranted to assess the potential clinical implications of these findings.

Table 1. ON 123300 In Vitro and In Vivo Pharmacokinetic Parameters | |  | |

|

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

In Vitro Metabolism (n = 2/group) | In Vivo Pharmacokinetics (n = 5/group)

Parameter (mean) | Males | Females | Parameter (mean± SD) | Males | Females

t1/2 (min) | 10.8 | 38.2 | Tmax (hr) | 2.3 ± 1.6 | 0.6 ± 0.2

Clint (ml/min/mg) | 130 | 36.7 | Cmax (ng/ml) | 321 ± 66.0 | 1253 ± 660

Predicted In Vivo Cl(ml/min/kg)a | 239.2 | 67.5 | AUC0-∞

(ng-hr/ml) | 1803 ± 757 | 5520 ± 2163

|  | |

Cl/F (ml/min/kg) | 1116 ± 474 | 390 ± 269

|  | |

t1/2 (hr) | 1.9 ± 0.50 | 3.0 ± 0.5

apredicted from venous equilibration model assuming rat blood flow = 13.8 ml/min, 99% protein binding and blood:plasma ratio = 1

#4907

Cell line panel profiling reveals novel drug response biomarkers for BTK and CDK4/6 inhibitors.

Joost C. Uitdehaag, Jeffrey J. Kooijman, Jeroen A.D.M. de Roos, Martine B.W. Prinsen, Jelle Dylus, Judith de Vetter, Nicole Willemsen-Seegers, Jos de Man, Suzanne J.C. van Gerwen, Rogier C. Buijsman, Guido J.R. Zaman. _Netherland Translational Research Ctr. BV, Oss, Netherlands_.

Profiling of new drug candidates on cancer cell line panels is an important tool to identify candidate drug response biomarkers for clinical studies. We have set up a platform, called Oncolines™, that comprises 102 genetically well-characterized cell lines from diverse tumor tissues. The cell lines are screened in parallel in a high-throughput proliferation assay based on ATP-lite read-out with 9 point dose-response curves in duplicate and manual inspection of curve fitting. We have recently shown that this workflow leads to highly reproducible IC50s which are necessary for genomic biomarker discovery [1]. Here we apply this platform to investigate BTK and CDK4/6 inhibitors. In previous work [1,2] we correlated the IC50 profiles of small molecule inhibitors through Anova to curated databases of somatic mutations and copy numbers. To obtain a more comprehensive view of oncogenic signaling inside a cell, we calculated in this study correlations between the log IC50s and basal gene expression levels for more than 18,000 genes. To reduce the number of false positives, we first considered only genes with a known role in cancer biology or drug resistance. Secondly, correlations were corrected for the average correlations seen in an Oncolines™ database of more than 150 anti-cancer agents [1]. Thirdly, we looked at co-correlation of genes that interact on a protein level (StringDB) or at a pathway level (Gene Set Analysis). Our method, called GeneNominator™, was validated by profiling the microtubule binder vincristine, the EGFR inhibitor gefitinib, and the MDM2 antagonist nutlin, and confirmed known pharmacogenomic relations.In a first study we tested three CDK4/6 inhibitors, ribociclib, palbociclib and abemaciclib. Although their response patterns cluster together, they show significant differences. For instance, palbociclib is more active on cell lines that express the transcription factor GATA3, which is linked to cyclin signalling in neuroblastoma.In a second study we tested the BTK inhibitors ibrutinib and acalabrutinib. Both show very different inhibition profiles across the 102 cell lines. Ibrutinib shows considerable activity in cell lines that overexpress ERBB4, FGFR2 and ERBB2, reflecting its biochemical inhibition of these kinases. Acalabrutinib, which is more selective, is most active in cell lines that highly express IRF4, a known genetic driver in multiple myeloma. and the B-cell receptor subunit CD79B, of which mutations are often found in diffuse large B-cell lymphoma. When mutations and copy number changes are studied, acalabrutinib is particularly active in cell lines with CREBBP or EZH2 mutations, which occur in diffuse large B-cell lymphomas. Our data demonstrate that our in vitro cell panel screening method can uncover new mechanistic information on clinically used anti-cancer agents.

[1] Uitdehaag et al. (2016) Mol. Cancer Ther. 15, 3097-3109.[2] Uitdehaag et al. (2014) PLOS ONE 9: e92146

#4908

Exemestane worsens everolimus-associated stomatitis in cancer patients.

Vladmir C. de Lima, Matheus H. de Lima, Fabio A. Alves, Gláucia N. Hajj. _A. C. Camargo Cancer Center, Sao Paulo, Brazil_.

Everolimus is an antitumor drug usually indicated for the treatment of breast, kidney and neuroendocrine malignancies that inhibits the activation of the mammalian Target of Rapamycin (mTOR). Despite the efficacy and success of the treatment, side effects are not uncommon, and stomatitis is quite prevalent. mTOR inhibitor associated stomatitis (mIAS) is described as ulcerated aphthous-like lesions associated with mTOR inhibitors use. mIAS may cause severe pain, leading to nutritional deficiency and general health decline, besides drug dose reduction or suspension. Clinical observation suggested mIAS to be more common among breast cancer patients. Thus, we hypothesized that exemestane could contribute to increase everolimus toxicity in the oral mucosa. We prospectivelly collected clinical data from patients who used everolimus, associated or not with exemestane, from March 2016 to September 2017, and correlated them with information regarding mIAS (date of onset, number of lesions in the oral cavity, grading). We also performed in vitro experiments with Dysplastic Oral Keratinocytes (DOK). Cells were treated with vehicle, everolimus, exemestane or the combination of everolimus and exemestane. Drug induced citotoxicity was evaluated by MTT assay and migration inhibition by scratch wound healing assay. All assays were done in triplicate. Frequencies were compared by Fisher's exact or McNemar test. Log-rank and Wilcoxon tests were employed to compare time to mIAS development. Multivariate ANOVA was used to compare results from in vitro experiments. SPSS and Image J were used to make all statistical analysis. One hundred and thirty patients were evaluated. Mostly were females, with mean age of 60 years, and had previously treated advanced stage (IV) breast cancer. 30.3% developed mIAS. Breast cancer patients (treated with exemestane plus everolimus) had 2.29 higher chance to develop mIAS when compared to neuroendocrine tumos patients (treated with everolimus alone). In vitro experiments showed that the association of everolimus and exemestane increased cell death and migration (wound healing) inhibition of DOK in comparison with cells treated with everolimus or exemestane alone. The clinical data suggests that exemestane increases the risk of developing mIAS, and the in vitro experiments reinforces the idea that exemestane may enhance oral mucosa toxicity mediated by everolimus.

#4909

Adaptive oncology phase 1 study of first-in-class inhibitor of ataxia telangiectasia mutated protein kinase (ATM), in combination with olaparib.

Yingxue Chen,1 Martin Pass,2 Nuria Bui Bruna,2 Christine Stephens,2 Andrew Pierce,2 Wouter Hanekom,2 Hani Gabra,2 Helen Tomkinson,2 Nidal Al-Huniti1. 1 _AstraZeneca Pharmaceuticals LP, Waltham, MA;_ 2 _AstraZeneca Pharmaceuticals LP, Cambridge, United Kingdom_.

Background: AZD0156 is a highly selective first oral ATM inhibitor in the clinic. Coordinating DNA single and double strand break repair, AZD0156 enhances the preclinical activity of olaparib, leading to its development as an antitumor agent in combination. We report here results from an ongoing phase 1 adaptive dose escalation study of AZD0156 in combination with olaparib.

Methods: This phase I study is to assess the safety, tolerability, pharmacokinetics (PK) and preliminary efficacy of escalating doses of AZD0156 in combination with olaparib in patients with advanced malignancies. AZD0156 is mainly metabolized by CYP3A and FMO, which undergo genetic polymorphism. The flexible language incorporated in the protocol also allow rapid dose escalation beyond subtherapeutic doses.

Results: PK data from the first 2 dose cohorts were lower than predicted and the AZD0156 dose was increased 4 X and dosing changed from QD (daily) to BD (twice daily). The mean terminal half-life of AZD0156 was 9-12 hours and PK was dose proportional. No significant drug-drug interactions (DDI) were observed with olaparib. Minor toxicities included nausea, vomiting and anemia in about 40% of patients in all cohorts. Grade 3 and 4 hematologic toxicities were observed in AZD0156 120mg BD with olaparib 200mg BD, and this dose level was considered intolerable. At doses of AZD0156 of 30 and 60mg BD, active target engagement was demonstrated. PK profile was similar across FMO genotype.

Conclusion: Hematologic toxicities, consistent with the mode of action, emerged as dose-limiting toxicities of AZD0156 in combination with olaparib. Pharmacologically active doses of AZD0156 with olaparib were achieved more quickly in patients by using real-time PK, real-time genotyping, safety, DDI assessment, and PK/PD modelling.

#4910

Development of an antagonist to the TWEAK-Fn14 ligand-receptor interaction with enhanced efficacy/potency in glioblastoma cells.

Bryan Harder,1 Chris Sereduk,1 Harshil Dhruv,2 Nghia Millard,2 Anthony Kim,3 Jeffrey Winkles,3 Graeme Woodworth,3 Holly Yin,4 Nhan L. Tran1. 1 _Mayo Clinic, Scottsdale, AZ;_ 2 _Translational Genomics Research Institute, Phoenix, AZ;_ 3 _University of Maryland, Baltimore, MD;_ 4 _City of Hope National Medical Center, Los Angeles, CA_.

Glioblastoma multiforme (GBM) remains the most common and deadly primary brain tumor with minimal advances in therapeutic development for pharmacological treatment. Tumor resection followed by temozolomide (TMZ) and radiotherapy adds months to survival; however, tumor recurrence usually occurs. The fibroblast growth factor-inducible 14 (Fn14) receptor has been shown to be up-regulated in the GBM cells left behind after surgery and contributes to an invasive and pro-survival phenotype, providing a desirable molecule to target after removal of the primary tumor. Previously, L524-0366 was reported by our lab to be the first antagonist of Fn14 signaling; however, minimal efficacy/potency has limited this drug from progressing from a useful research tool to preclinical studies. Structure activity relationship (SAR) studies using the parent L524-0366 compound gave rise to K78, which was found to bind to Fn14 with enhanced affinity, while showing no binding to the structurally-related TNFα receptor. Furthermore, K78 was able to abate the downstream signaling effects initiated by the Fn14 ligand, tumor necrosis factor-like weak inducer of apoptosis (TWEAK), as well as sensitize GBM cells to the cytotoxic effects of TMZ. TWEAK-induced cellular migration was also mitigated with co-treatment of K78 in GBM cells, compared to a vehicle control. It is anticipated that further development of Fn14 antagonists will be of great importance for the advancement of GBM patient treatment and warrant further investigation as primary and combination therapy, especially for the prevention of recurrent disease.

#4911

Metabolic activation of sunitinib: Implications for sunitinib-induced toxicities.

Klarissa D. Jackson, Gracia M. Amaya, Rebecca Durandis, Kahari J. Wines, Arsany A. Abouda, Samuel A. Starks, R. Nathan Daniels. _Lipscomb University College of Pharmacy, Nashville, TN_.

Purpose: Sunitinib is a multi-targeted tyrosine kinase inhibitor approved for the treatment of renal cell carcinoma, gastrointestinal stromal tumors, and pancreatic neuroendocrine tumors. However, clinical use of sunitinib is associated with potentially fatal idiosyncratic liver injury. The mechanisms of this toxicity are unknown. We hypothesized that sunitinib undergoes metabolic activation to form chemically reactive, potentially toxic metabolites, which may contribute to the development of sunitinib-induced liver injury. The purpose of this study was to define the role of cytochrome P450 (CYP) enzymes in the metabolism and bioactivation of sunitinib to provide insight into the mechanisms of drug toxicity.

Methods: Metabolic incubations were performed using individual recombinant P450 enzymes, human liver microsomal fractions, and P450-selective chemical inhibitors. To assess reactive metabolite formation, glutathione (GSH) and a chemical derivative of GSH, dansyl-GSH, were used as trapping agents to detect reactive metabolite-GSH conjugates. Sunitinib metabolites were analyzed by liquid chromatography-tandem mass spectrometry.

Results: Recombinant CYP3A4 was the major enzyme to form the primary active metabolite N-desethyl-sunitinib. Recombinant CYP1A2 generated the highest levels of reactive metabolites trapped as GSH conjugates, with minor contributions from CYP2D6 and CYP3A4. Detection of reactive metabolite dansyl-GSH conjugates from incubations with dansyl-GSH confirmed the generation of reactive metabolites by CYP1A2. In human liver microsomal incubations, the CYP3A inhibitor ketoconazole reduced formation of N-desethyl-sunitinib by 88%, while the CYP1A2 inhibitor furafylline decreased generation of reactive metabolite-GSH conjugates by 62%, compared to control levels. Quinidine (CYP2D6 inhibitor) and ketoconazole (CYP3A inhibitor) also decreased GSH conjugate formation by 54 and 52%, respectively, compared to control. Kinetic assays with recombinant P450s revealed that the overall enzyme efficiency (kcat/Km) for converting sunitinib to reactive metabolite-GSH conjugates was in the following order: CYP1A2 > CYP3A4 > CYP2D6.

Conclusions: Collectively, these data indicate that CYP1A2 and CYP3A4 are important contributors to metabolic activation of sunitinib to generate reactive, potentially toxic metabolites. Factors that alter CYP1A2 and CYP3A4 activity may affect patient risk for sunitinib toxicity. Future studies will focus on examining the impact of P450 genetic variations and environmental factors on sunitinib metabolism and toxicity.

#4912

Targeting cyclin-dependent kinase 7 in gastric cancer.

Jia-Rong Huang, Wen-Ji Zhang, Qi-Wei Jiang, Yang Yang, Meng-Ning Wei, Zhi Shi. _Jinan Univ., Guangzhou, China_.

CDK7 is a member of the cyclin-dependent kinase family, which forms the CDK activating kinase (CAK) complex with Cyclin H and RING finger protein Mat1 to control cell cycle progression and transcription by phosphorylating other CDKs and RNA polymerase II. Upregulation of CDK7 frequently occurs in human gastric cancer (GC). In this study, we demonstrated that inhibition of CDK7 was able to induce cell growth inhibition, cell cycle arrest at G2/M phase and apoptosis with the increasing intracellular reactive oxidative species (ROS) levels. Pretreatment with ROS scavenger N-acety-L-cysteine partly reversed GC cell apoptosis induced by CDK7 inhibition. In the nude mice, inhibition of CDK7 also suppressed the growth of xenograft tumors of GC. Overall, our data showed that inhibition of CDK7 in GC presented outstanding anticancer effect in vitro and in vivo, suggesting that CDK7 is a potential therapeutic target for GC patients.

#4913

A PK/PD model quantitatively describes inhibition and down-regulation of p90RSK by ERK inhibitor AZD0364.

Francis D. Gibbons,1 Linda Sandin,2 Lyndsey Hanson,2 Rebecca Whiteley,2 Paul Farrington,2 Nicola Lindsay,2 Emma Davies,2 J Elizabeth Pease,2 Vikki Flemington2. 1 _AstraZeneca, Boston, MA;_ 2 _AstraZeneca, Cambridge, United Kingdom_.

ERK1/2 is a key protein in the MAPK pathway, regulating phenotypes such as proliferation and migration. Upstream mutations (e.g., KRAS mutations in non-small-cell lung (NSCLC)) can cause the pathway to become constitutively activated, driving tumor growth. AZD0364 is a potent, selective inhibitor of ERK's kinase activity against its cytosolic substrate p90RSK. It is currently in preclinical development, where it has shown dose-dependent, anti-tumor activity in xenograft models of KRAS-mutant NSCLC, including Calu-6 (where it shows regression) and A549. Treatment with AZD0364 demonstrates rapid and near-complete inhibition of phospho-p90RSK. In addition, prolonged inhibition with AZD0364 causes a gradual downregulation of p90RSK protein over time, without any corresponding change in p90RSK mRNA. Here we present a pharmacokinetic/pharmacodynamic (PK/PD) model that links AZD0364 concentration to inhibition of ERK activity through both a direct inhibition of phospho-p90RSK and an indirect down-regulation of total-p90RSK protein. Anti-proliferative and pro-apoptotic effects on efficacy are linked to changes in p90RSK. The model leads to two key implications (i) repeated dosing will cause apparent potency to improve over time, since the pool of available substrate (i.e., p90RSK) is itself being reduced and (ii) recovery of signaling to baseline will depend not on washout of the inhibitor but on protein synthesis rates. Protein half-lives appear quite different between tumor models of KRAS-mutant NSCLC, with A549 (~20h) significantly slower than Calu-6 (~4h). The model provides a conceptual framework on which to link the timescale of PD changes with those seen in efficacy. Taken together, this means that while a new PD steady-state is achieved in Calu-6 in a few days, it also recovers quickly, necessitating constant cover (daily dosing) to drive regression. On the other hand, while A549 is more robust to inhibition, and slower to reach steady-state inhibition (~2 weeks), it is also slower to recover, so that intermittent schedules can achieve efficacy similar to those achievable with daily dosing.

#4914

IFI16 is increased in response to chemotherapeutic agents in breast cancer cells.

Gayoung Lim, Na-Lee Ka, Mi-Ock Lee. _Seoul National University, Seoul, Republic of Korea_.

Breast cancer is the most common cancer in women worldwide and chemotherapy is one of the standard treatment options for breast cancer. Chemotherapeutic agents induce severe DNA damage in cancer cells, which have mutations in genes related with DNA repair, resulting in cancer cell death. Thus, controlling DNA damage response is an important factor in determining outcome upon chemotherapy. In this study, we identified IFN-γ inducible protein 16 (IFI16) as a regulator of DNA damage response in breast cancer cells. First, we found that the expression of IFI16 was increased in response to chemotherapeutic agents such as 5-fluouracil, doxorubicin and cisplatin. However, changes in mRNA level of IFI16 was not observed, suggesting that IFI16 expression could be regulated by post-translation modification. Second, nuclear fractionation and confocal analysis revealed that expression of nuclear IFI16 was increased and cytoplasmic IFI16 was decreased in response to doxorubicin. Taken together, IFI16 could be a possible candidate for regulating factor of DNA damage response upon chemotherapy in breast cancer cells.

#4915

Preclinical pharmacology and toxicology of intravenous BPM31510, a coenzyme Q10-containing lipid nanodispersion.

David Linsenmayer, Shiva Kazerounian, Khampaseuth Thapa, Carrie Spencer, Aishwarya Sarma, John McCook, Stephane Gesta, Vivek Vishnudas, Niven R. Narain, Rangaprasad Sarangarajan. _BERG, Framingham, MA_.

Coenzyme Q10 (CoQ10) plays an important role in cellular metabolism with redox capabilities beyond the mitochondria. Although, the role of CoQ10 in providing benefit in diseases such as cardiovascular, neurodegeneration, and cancer is well established, it has been limited by the absence of delivery methods to achieve physiologically relevant bio-availability. BPM31510 is a CoQ10 (ubidecarenone) containing lipid nanodispersion that delivers supraphysiological concentrations of CoQ10 to tissues, cells and mitochondria. BPM31510 is currently in clinical trials for the treatment of solid tumors including pancreatic cancer and glioblastoma multiforme. This study describes the preclinical pharmacokinetic (PK) and toxicokinetic assessment of BPM31510 (62.5, 125, and 250 mg/kg) after acute (single) intravenous (IV) administration and chronic (3 times a week for 4 weeks), and after a two week recovery from the last exposure in rats. Here, it was demonstrated that the formulation enables an effective delivery of CoQ10 with a robust increase in plasma level and dose-dependent accumulation in several tissues. PK analysis of mean plasma concentration of CoQ10 after a single and repeat dose administration revealed a dose proportion effect on peak (Cmax) and total exposure (AUC0-t). Tissue analysis of CoQ10 levels demonstrates a dose-dependent increase in liver, lung and pancreas 72 hours post-last dose. A dose dependent elimination was observed in both male and females. No difference in pharmacokinetic of CoQ10 using this formulation was observed between males and females. Notably, chronic exposure to BPM31510 does not lead to toxicities as assessed by hematology and coagulation, clinical chemistry, histopathology and gross necropsy. These results indicate that the effective delivery and bioavailability of supraphysiological levels of CoQ10 after IV administration of BPM31510 is well tolerated.

#4916

OCT and MATE transporters mediate uptake and antiproliferative activity of the anticancer drug metformin in endometrial cancer cell lines.

Aruljothi Muralidharan, Victoria L. Bae-Jump, Dhiren R. Thakker. _University of North Carolina, Chapelhill, NC_.

Purpose Metformin, a first-line drug for treatment of Type 2 diabetes, is effective against multiple type of cancers including endometrial cancer. The anticancer effects of metformin, in part, are mediated via activation of its intracellular target adenosine monophosphate-activated protein kinase (AMPK) and subsequent modulation of the downstream signaling of Mammalian Target of Rapamycin (mTOR) and P70S6K. Since metformin is positively charged (pKa 12.4) and hydrophilic (logD -6.13 at pH 6.0) it requires organic cation transporters (OCT) 1-3, plasma membrane monoamine transporter (PMAT) and multidrug and toxin extrusion transporters (MATE) 1 and 2 to mediate its cellular uptake/egress. The goal of this study is to assess the expression of these transporters in the endometrial cancer (EC) cell lines ECC-1, Ishikawa and SPEC-2. Methods EC cells (ECC-1, Ishikawa and SPEC-2) were treated with varying concentrations (0.1 mM-100 mM) of metformin for 72 hours, cell proliferation was assessed by MTT assay. Cellular uptake (5 min) of [14C] metformin at 50μM concentration was measured in the presence or absence of cation-selective transporter inhibitors, 1-methyl-4-phenylpyridinium (MPP+) (500 μM), desipiramine (200 μM) and pyrimethamine (2.4 μM). Expression of 6 cation-selective transporters (OCT 1-3, MATE1, MATE2 and PMAT) and phosphorylation of AMPK and P70S6K was evaluated by Western blot analysis. Results Metformin exhibited dose-dependent anti-proliferative activity against ECC-1, Ishikawa and SPEC-2 cell lines with an IC50 of 4.3, 2.2 and 8.8mM respectively. Protein expression analysis showed that OCT 1-3 were highly expressed in ECC-1 and Ishikawa cells whereas MATE1 and 2 were the predominant in SPEC-2 cells. [14C] metformin uptake into these 3 cell lines was mediated by cation-selective transporters as evidenced by the observation that >80% uptake was inhibited by the pan cation inhibitors MPP+ (500 μM). In the ECC-1 and Ishikawa cell lines, ≈50% of [14C]metformin uptake was inhibited by OCT1-3 and PMAT inhibitor desipiramine (200 μM) and ≈50% of uptake was inhibited by MATE1 and 2 inhibitor pyrimethamine (2.4 μM). In contrast, pyramithamine inhibited 72% of [14C]metformin uptake in SPEC-2 cells. Western blot analysis revealed that 5 µM pyrimethamine reversed metformin-mediated phosphorylation of AMPK and inhibition of pP70S6K in all the EC cell lines. Discussion Our results demonstrate that the OCT 1-3 and MATE 1-2 transporters play an approximately equal role in the uptake of metformin in ECC-1 and Ishikawa cells, whereas MATE 1-2 play a major role in metformin uptake into the SPEC-2 cells. As expected, metformin promoted phosphorylation of AMPK (activation) and attenuated phosphorylation of P70S6K (inhibition) in all of the EC cell lines. This work highlights the importance of transporters in the anti-proliferative activity of metformin in EC cells.

#4917

Tipifarnib is highly active in HRAS mutant lung squamous carcinoma tumor models.

Linda Kessler, Catherine Scholz, Antonio Gualberto, Yi Liu, Francis Burrows. _Kura Oncology, San Diego, CA_.

Genomic profiling of squamous tumors has revealed important similarities between lung squamous cell carcinoma (LSCC) and head and neck squamous cell carcinoma (HNSCC). For instance, HRAS is the most commonly mutated RAS species in both LSCC and HNSCC, observed in approximately 2% and 5% of cases, respectively (TCGA, Nature 2013). Tipifarnib is a potent and selective inhibitor of farnesyltransferase (FT) that catalyzes the post-translational attachment of farnesyl groups to proteins that require localization to the inner cell membrane. Although all RAS isoforms (KRAS/NRAS/HRAS) are FT substrates, HRAS is exclusively dependent upon farnesylation for membrane localization and signaling activation, making HRAS mutant tumors uniquely susceptible to tipifarnib mediated inhibition of FT. With limited treatment options, LSCC and HNSCC remain a significant unmet medical need. Recent evidence supports the clinical utility of tipifarnib for treatment of patients with HRAS-mutant HNSCC, and we present herein data supporting a potential utility of tipifarnib in the treatment of HRAS-mutant LSCC. We have characterized the antitumor activity of tipifarnib in CDX and PDX models of squamous cell carcinoma with activating HRAS mutations. Tipifarnib displayed robust antitumor activity in the majority of these models, including lung and head and neck tumors. Six of seven HRAS-mutant lung SCC PDX models responded to tipifarnib treatment with the majority either fully growth-inhibited or undergoing partial or complete regression. Importantly, even LSCC PDX tumors resistant to chemotherapy responded to tipifarnib, suggesting tipifarnib has the potential to offer clinical benefit in patients relapsed or refractory to standard therapies. The activity of tipifarnib in LSCC models is quite similar to that seen in HNSCC PDX models, where tipifarnib also induced regressions consistent with those observed in patients enrolled in the ongoing Phase 2 study in HRAS-mutant SCCHN patients (NCT02383927), including patients refractory to chemotherapy, cetuximab and/or immunotherapy. These data demonstrate that HRAS is a targetable mutation in lung SCC as well as in HNSCC and illustrate the potential for tipifarnib in the treatment of additional HRAS-mutant squamous cell carcinomas.

#4918

Pharmacokinetics and toxicity studies of AT1965, a B-cell activating immunotherapy.

Aniruddha Sengupta,1 Mallik Samarla,1 Manoj Pandey,1 Arindam Sarkar,1 Monideepa Roy,1 Shiladitya Sengupta2. 1 _Akamara Therapeutics, Inc., DE;_ 2 _Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA_.

Introduction: Cancer immunotherapy is the emerging paradigm in the search for a cure for cancer. AT1965 is a novel therapeutic that demonstrates remarkable anti-tumor activity across different preclinical tumor models, mediated through the activation of B-cells. In this study, we have evaluated the metabolism, pharmacokinetics (PK) and toxicity of AT1965 in rodent and non-rodent species.

Methods: AT1965 metabolism was studied in rodent, canine and primate liver microsomes. The PK of AT1965 was determined in 4T1 tumor bearing female Balb/c mice; female Sprague Dawley rats and female Beagle dogs at a variety of doses and schedules following single intravenous (i.v) injections. The extent and duration of the exposure was monitored over 120 hours in mice and rats, and till 192 hours in dogs, post dosing. Concentrations of AT1965 in plasma samples were determined either by LC-MS/MS or Atomic Absorption Spectroscopy. The toxicity of AT1965 in male and female Sprague Dawley Rats following single dose i.v. administration of 43.7, 87.4 and 174.8 mg/kg dosage of AT1965 were examined. During toxicity studies, clinical health status was observed daily for 14 days after treatment, followed by necropsy.

Results: AT1965 metabolism evaluated in mouse, rat, dog, monkey and human liver microsomes indicated high stability across all species. PK studies reveal that AT1965 exhibited a high exposure, slow systemic clearance and a proportional increase in dose normalized exposure across species. The elimination half-life (T1/2) varied from 34-40 hours in tumor-bearing mice to 29-44 hours in rats and ~ 66 hours in dogs. The excretion of AT1965 was predominantly through hepatobiliary route, with minimal renal clearance. Single i.v. administered acute dose toxicity studies for AT1965 reveal 87.4 mg/kg and 174.8 mg/kg as MTD and toxic doses respectively in rats.

Conclusion: The excellent pharmacokinetic profile of AT1965 showing high exposure and enhanced elimination half-life tested across species, in addition to striking antitumor activity coupled with low toxicity in multiple rodent tumor models, provide a compelling rationale for the development of AT1965 as a novel immunotherapeutic agent in the clinic.

#4919

Preclinical activity of SDX-7320 in mouse models of obesity and obesity-driven cancer.

Peter Cornelius,1 John S. Petersen,2 Benjamin Mayes,1 David Turnquist,3 Kimberly Sullivan,4 Alfred Anderson-Villaluz,4 Robert Lutz,4 Sara Little,5 Andrew Slee,6 Bradley J. Carver,1 James Shanahan1. 1 _Syndevrx Inc., Cambridge, MA;_ 2 _Petersen Consulting, Acton, MA;_ 3 _Xtuit, Waltham, MA;_ 4 _Development Insights, LLC, Beverly, MA;_ 5 _Neosome Life Sciences, Lexington, MA;_ 6 _Cove Bioconsultants, Shrewsbury, MA_.

Cancer patients who are obese face a greater risk of dying from cancer compared to nonobese patients (Calle, 2003). Obesity is believed to contribute to metastasis and progression of cancer via multiple mechanisms: increased secretion of the adipose tissue hormone leptin, decreased secretion of adiponectin, increased production of estrogen in adipose tissue, and elevated insulin (secondary to peripheral insulin resistance) as well as the local effects of inflammatory cytokines (Gucalp, 2016). Small-molecule inhibitors of methionine aminopeptidase type 2 (MetAP2) have previously demonstrated clinical activity in oncology (Kudelka, 1998) as well as obesity/type 2 diabetes (Hughes, 2013; Kim, 2015). However, development of some small-molecule MetAP2 inhibitors has been hampered by CNS side effects (Bhargava, 1999). SDX-7320 is a copolymer-drug conjugate of a novel fumagillin-derived MetAP2 inhibitor (SDX-7539) attached via a cleavable linker to a hydroxypropylmethacrylamide (HPMA) backbone, intended to limit CNS penetration and therefore reduce CNS toxicity. In vitro MetAP2 binding assays showed that SDX-7320 was unable to bind to MetAP2 while SDX-7539 was a potent binder (apparent IC50 = 0.13 nM), indicating that cleavage of SDX-7320 is required for biologic activity. SDX-7539 inhibited proliferation of human umbilical vein endothelial cells (HUVECs) with an apparent IC50 = 0.2 nM. The preclinical efficacy of SDX-7320 was evaluated in mouse models of obesity/insulin resistance and also in syngeneic tumor models coupled with high fat diet-induced obesity (DIO)/metabolic dysfunction. Obesity and insulin resistance were induced in C57Bl/6 mice by feeding them a high-fat diet (HFD) for at least 12 weeks. SDX-7320 was dosed subcutaneously every 4 days (for 28 days) into DIO mice or control mice fed a low-fat diet. SDX-7320 caused significant reduction in body weight and fat mass in obese mice while also reversing insulin resistance relative to controls. In addition, SDX-7320 caused greater reduction in body weight in obese mice relative to lean mice. Circulating levels of leptin and insulin were reduced while levels of adiponectin were increased in response to SDX-7320. In mouse models of obesity-accelerated tumor growth, SDX-7320 was more efficacious in obese relative to lean mice harboring subcutaneous B16F10 melanoma or EO771 mammary gland tumors. These effects occurred without any signs of neurotoxicity. SDX-7320, currently in phase I (solid tumors; all-comers; NCT02743637), is being developed for the treatment of cancers whose progression is accelerated in the setting of obesity and host metabolic dysfunction, termed "metabo-oncology."

#4920

Intratumoral levels of BAX-BAK heterodimer as a specific on-target pharmacodynamic biomarker of drug action by novel BH3 mimetics.

Apurva K. Srivastava,1 Jeevan P. Govindharajulu,1 Dominic Esposito,1 Melinda G. Hollingshead,2 James H. Doroshow,3 Ralph E. Parchment1. 1 _Frederick National Laboratory for Cancer Research, Frederick, MD;_ 2 _National Cancer Institute, Frederick, MD;_ 3 _National Cancer Institute, Bethesda, MD_.

Background: Promising small-molecule inhibitors of pro-survival Bcl-2 family proteins (BH3 mimetics) are currently under clinical development. Bcl-2, Bcl-xL and Mcl-1 proteins sequester BAK and BAX, which are released by BH3 mimetics, allowing them to activate and form homo- or heterodimers at the mitochondrial membrane. Such oligomerization results in mitochondrial membrane porations, release of cytochrome c and activation of executioner caspases leading to apoptosis. Therefore, measurement of BAK-BAX heterodimers on the mitochondrial membrane is considered an on-target pharmacodynamic biomarker of drug action by BH3 mimetics. We describe development and validation of a quantitative BAX-BAK heterodimer assay as well as fitness-for-purpose for proof of mechanism studies during clinical trials.

Method: The BAX-BAK heterodimer sandwich immunoassay was developed on the Luminex® platform using monoclonal antibodies specific to active BAX and BAK proteins. A recombinant fusion protein containing BAX and BAK domains, connected by a 20-amino acid linker, was used as a calibrator to mimic oligomer formed in situ by the noncovalent association of BAX and BAK proteins. Preparation of the mitochondrial fraction from core needle biopsies followed Standard Procedures described previously for a 15-biomarker apoptosis multiplex (Srivastava et al., Clin Can Res 2016). BH3 mimetics, navitoclax (NSC-759659) and an Mcl-1 inhibitor NSC-798846 (aka S63845 by Kotschy et al., Nature 2016) were used to demonstrate effectiveness of the PD assay for monitoring pharmacodynamic response.

Results: The BAX-BAK heterodimer sandwich assay demonstrated satisfactory precision (CV<19%) and accuracy (mean analytical recoveries of 95%), with a minimum sample load of 4-μg protein/well. Using clinically validated sample collection and processing procedures, the assay revealed 10-45 fold increases in BAX-BAK heterodimers in multiple hematologic cancer cell lines (AMO-1, MV411, THP-1) treated for 4-6 hours with 100-1,000 nM NSC-798846 and NSC-759659. Increased levels of BAX-BAK correlated with 10-12 fold increases in cleaved caspase-3 levels measured with the apoptosis multiplex. The BAX-BAK biomarker assay has been extensively evaluated in murine tumor xenograft models and is ready for clinical use to evaluate core needle tumor biopsies.

Conclusion: We have developed and validated an assay for BAX-BAK heterodimers as a biomarker of target engagement by BH3 mimetics. This assay is suitable for use in preclinical and clinical studies not only to confirm target engagement but also to optimize dose and schedule of BH3 mimetics as single agents or in combination regimens. The BAX-BAK biomarker enables pharmacodynamic assessment of investigational BH3 mimetics for clinical proof-of-mechanism studies and early clinical development.

Funded by NCI Contract No HHSN261200800001E.

#4921

Population pharmacokinetic modeling of ibrutinib administered in patients with relapsed or refractory B cell malignancies eligible for autologous stem cell transplantation.

Keyvan Rezai,1 Olivier Madar,1 Christophe Bonnet,2 Jean Dupuis,3 Hervé Tilly,4 Thierry Lamy de la Chapelle,5 Steven Le Gouill,6 Ombeline Verite,7 Julie Assemat,7 Fanny Bret,1 Sophie Weill,1 François Lokiec,1 Gilles Salles8. 1 _Institut Curie, Saint Cloud, France;_ 2 _CHU de LIEGE, Liege, Belgium;_ 3 _Hôpital Henri MONDOR, Créteil, France;_ 4 _Centre Henri BECQUEREL, Rouen, France;_ 5 _CHU Pontchaillou, Rennes, France;_ 6 _CHU de Nantes, Nantes, France;_ 7 _LYSARC, Lyon, France;_ 8 _CHU Lyon Sud, Lyon, France_.

Background: Ibrutinib is a first-in-class selective, irreversible small molecule inhibitor of Bruton's tyrosine kinase (BTK). The present study's aim is to assess the feasibility and safety and pharmacokinetics (PK) of escalating doses of ibrutinib combined with rituximab (R), dexamethasone (D), ara-C (HA) and one of two platinum compounds (cisplatinum (P) or oxaliplatinum (Ox)) in patients with relapsed or refractory lymphoma. As part of this, Population PK (POPPK) modeling, PK parameters, and inter-individual and inter-occasion variabilities of oral ibrutinib in the presence of R-DHA(P/Ox) were assessed during the dose escalation part of this clinical trial.

Materials and methods: Dataset was obtained from an open label, multicenter, dose escalation, phase Ib study of ibrutinib in combination with R-DHA (P/Ox) in patients with B-cell malignancies. Patients received three doses (280, 420, 560 mg) of oral ibrutinib once a day D5 to D18. Blood samples were collected during cycles 1 and 2 on D5, Just before ibrutinib intake (T0) and at 1, 2 and 4 hours after ibrutinib intake and on D15 at T0 and 1 hour after ibrutinib intake. Ibrutinib plasma concentrations were measured using validated ultra-performance liquid chromatography with tandem mass spectrometry detection with a concentration range 1-400ng/mL. Analyses and POPPK modeling were performed with the nonlinear mixed effect modeling software program Monolix version 4.3.2. The following parameters were calculated Lagtime (Tlag), absorption constant (Ka); apparent distribution volumes (V1/F, V2); apparent clearances (CL/F, Q).

Results: 24 pts (18 male, 6 female), have validated PK data with 184 plasma concentrations. A 2-compartment model with linear elimination and lag time adequately described the total ibrutinib time-concentration curve. The main PK parameters (RSE%) estimated for ibrutinib were Tlag=0.6 (15), Ka=3.36 (36) h-1, CL/F= 699 (12) L/h, Q=182 (1) L/h, V1/F=5,060 (15) L, and V2=32,100 (49) L. The main covariate effect on ibrutinib CL was related to body weight (BW). The inter-individual and variabilities could be well estimated for CL and V1 and inter-occasion variabilities were well estimated for Tlag and V1.

Conclusions: The POPPK modeling satisfactorily described the plasma ibrutinib time-concentration curves in patients. Our data revealed the effect of BW on ibrutinib PK parameters. A large volume of the deep compartment suggest that ibrutinib has large tissue diffusion. The clinical data describes the toxicity and efficacy. This is an analysis of correlation between the PK and the clinical outcomes.

#4922

The combination of MK-2206 and WZB117 exerts a synergistic cytotoxic effect against breast cancer cells.

Yu-Liang Li, Hao-Cheng Weng, Lih-Ching Hsu. _National Taiwan University, Taipei, Taiwan_.

Breast cancer is the most commonly diagnosed cancer and the second leading cause of cancer death in women. Hormone receptor-positive breast cancer is usually subjected to hormone therapy, while triple-negative breast cancer is more formidable and poses a therapeutic challenge. The PI3K/Akt/mTOR signaling pathway plays a crucial role in cancer cell growth, survival, and metabolism. It has been demonstrated that MK-2206, a potent allosteric Akt inhibitor, is cytotoxic against many cancer cell lines and is currently in clinical development. Cancer cells consume more glucose and rely on glycolysis for energy production even in the presence of abundant oxygen, a phenomenon called the Warburg effect. Therefore, glucose transporters responsible for delivering glucose into cells have become targets for the development of anticancer drugs. In search for anticancer agents whose effect could be enhanced by a GLUT1 inhibitor WZB117, we found that MK-2206, when combined with WZB117, showed a synergistic effect on growth inhibition of breast cancer cells, including ER(+) MCF-7 cells and triple-negative MDA-MB-231 cells. The combination index values at 50% growth inhibition were 0.45 and 0.21, respectively. Western blot analysis of proteins involved in the Akt/mTOR pathway revealed that Akt phosphorylation was markedly suppressed in both cell lines by MK-2206 and WZB117 treatment. Phosphorylation of mTOR and its downstream effectors p70S6K and 4E-BP-1 was also dramatically downregulated in MCF-7 but the effect was not as significant in MDA-MB-231 cells, suggesting that other mechanisms might also contribute to cytotoxicity induced by MK-2206 and WZB117. Further investigation revealed that the combination of MK-2206 and WZB117 induced γ-H2AX, a DNA damage marker, in both cell lines. Results from the comet assay confirmed that MK-2206 and WZB117 induced DNA damage. Protein levels of Rad51 and Ku80, participating in homologous recombination and non-homologous end joining repair respectively, were also decreased in MCF-7 cells, suggesting that MK-2206 and WZB117 may not only cause DNA damage, but also impair DNA repair. Furthermore, MK-2206 or WZB117 alone significantly induced ROS, and the combination of both further increased the ROS level within a short time period. In conclusion, this new finding indicates that MK-2206 and WZB117 may exert a synergistic cytotoxic effect in both ER(+) MCF-7 and triple-negative MDA-MB-231 breast cancer cells via ROS induction, which may in turn cause DNA damage. The combination may also compromise DNA damage repair, and ultimately lead to cell death. This finding may have clinical implications.

#4923

Pharmacokinetics of the metastatic cancer inhibitor MBQ-167 in mice.

Maria del Mar Maldonado,1 Linette Castillo-Pichardo,2 Joseph Bloom,3 Jorge Duconge,3 Jose F. Rodriguez-Orengo,1 Eliud Hernandez-O'Farrill,3 Cornelis Vlaar,3 Suranganie Dharmawardhane1. 1 _University of Puerto Rico Medical Sciences Campus, San Juan, PR;_ 2 _Universidad Central del Caribe, Bayamon, PR;_ 3 _School of Pharmacy, University of Puerto Rico Medical Sciences Campus, San Juan, PR_.

Metastasis is the principal cause of death for most cancer patients; however, effective treatment options are limited. The Rho GTPases Rac and Cdc42 are critical regulators of cancer cell migration and invasion; processes required for metastatic progression. Hence, targeting of these pivotal regulators is a rational approach for novel metastasis focused therapy design. We previously characterized the small molecule MBQ-167 as a novel Rac/Cdc42 inhibitor that inhibits mammary tumor growth and metastasis in immunocompromised mice by ~90%, which is ten times more potent than other currently available Rac inhibitors (Humphries-Bickley, et al., 2017). However, further studies are needed to determine the pharmacokinetic (PK) parameters of MBQ-167 in rodents to understand the mechanisms of drug distribution and elimination of this compound. The purpose of this study was to determine the pharmacokinetics and bioavailability of MBQ-167 in a single dose input scheme (10 mg/kg BW) following intraperitoneal (IP) and oral gavage (PO) administration. The drug was administered to BALB/c mice (4 mice/group) and plasma was collected at 0.5, 1, 3, 6, 9, and 12 hours after IP or PO administration. We developed and validated a bioanalytical method using supercritical fluid chromatography (SFC) coupled with electrospray ionization tandem mass spectrometry (MS/MS) for the detection of MBQ-167 in plasma. SFC-MS/MS was selected because it uses supercritical carbon dioxide (SCO2) as the mobile phase and offers advantages such as high sensitivity, increased resolution, and rapid analysis times. Pharmacokinetic parameters were obtained by mono-compartmental and bi-compartmental analysis for IP and PO dosing respectively using WinNolin® software, Version 7.0. Preliminary pharmacokinetic analysis revealed that the area under the curve (AUC0-∞) was 749 ng·hr/mL and 255 ng·hr/mL for IP and PO dosing respectively. The elimination half-life (t1/2) was 2.4 hours for IP dosing and the mean residence time (MRT) was 3.4 hours and 2.3 hours for the IP and PO dosing respectively. The maximum plasma drug concentration (Cmax) was 220 ng/mL after IP administration, compared to 127 ng/mL following PO administration. Furthermore, the time-to-peak for both administration routes was 30 minutes. The relative bioavailability of MBQ-167 after oral gavage administration was 34%. This study presents the first analysis of the pharmacokinetics of the Rac/Cdc42 inhibitor MBQ-167 in mice and it supports the continued development of this drug as a potential anti-cancer therapeutic.

#4924

Evaluation of the efficacy of curcumin and Y15 in platinum resistant ovarian cancer cells.

Arkene S. Levy, Appu Rathinavelu, Nicole Coelho, Amanda Ramnot, Thanigaivelan Kanagasabai, Olena Bracho, Robert Smith. _Nova Southeastern University, Fort Lauderdale, FL_.

Curcumin, a compound derived from turmeric (Curcuma longa), inhibits the proliferation of multiple cancer cell types, including ovarian, pulmonary, brain, and breast through various mechanisms. In advanced ovarian cancer, focal adhesion kinase (FAK), a tyrosine kinase, is overexpressed and contributes to platinum resistance. The compound Y15 (1, 2, 4, 5-benzene tetra amine tetrahydrochloride) inhibits FAK phosphorylation and subsequent activation. Therefore, in the present study we evaluated the combined cytotoxic efficacy of Curcumin and Y15 in platinum resistant OVCAR3 ovarian cancer cells. Cells were initially treated with concentrations of Y15 (10-100 μM), and curcumin (0-20µM) to determine IC10 values. Cells were subsequently treated with Y15 (20 μM) and curcumin (5 µM) alone and in combination. The cytotoxic profile of each treatment was assessed using the MTT assay. DNA fragmentation and poly ADP ribose polymerase (PARP) cleavage assays were performed to evaluate the mechanism of cell death. We further evaluated the expression of total FAK, phosphorylated FAK, p53, p21, and BAX in response to treatments using western blot. Y15 significantly increased the cytotoxic efficacy of curcumin when compared to the curcumin only treatment. Cell death by apoptosis was confirmed by PARP cleavage and the presence of DNA fragments in Y15, curcumin, and curcumin plus Y15 treatment groups. The curcumin plus Y15 combination significantly downregulated the expression of phosphorylated FAK when compared to the individual treatments and control. Reduced FAK phosphorylation also correlated with increased expression of P53, P21 and BAX in curcumin and Y15 treatment groups. These results indicate that Y15 significantly enhances the cytotoxic profile of curcumin in platinum resistant OVCAR-3 cells. This study is the first to report a FAK dependent cytotoxic mechanism of curcumin in ovarian cancer and in further work we will evaluate the signaling pathways involved.

#4925

**Curcumin, a polyphenol from the** Curcuma longa **, prevents mercuric chloride-induced liver damage through reversal of oxidative stress and biochemical changes.**

Anshu Agarwal,1 Prabhu Narayan Saxena2. 1 _Jiwaji University, Gwalior, India;_ 2 _Dr. Bhim Rao Ambedkar University, Agra, India_.

Heavy metals globally exist in the environment, and their prolonged exposure causes various disease including cancer. Mercury is a toxic heavy metal. It has been shown to have multiple adverse effects on human health including the nervous system, renal, cardiovascular, hypertension, and weakening the immune system. It is ranked up among the priority heavy metals those affect human health significantly. In 2013, the United States Environmental Protection Agency reported the higher concentration of mercury in the blood of nearly 1.4 million women of reproductive age, and this increased the risk of learning disabilities associated with in-utero exposure. Due to the major excretory organ in the human body, the liver is the primary target sites for the accumulation of environmental heavy metal such as mercury. Phytochemicals have shown and proved their effectiveness against oxidative stress, inflammation, and cancers of several organs. Curcumin, a bioactive compound of turmeric, has been used for the treatment of several diseases including inflammatory disorders, cancer prevention, and various metal toxicities. Here, we investigate the effect of curcumin treatment on mercuric chloride induced hepatotoxicity using the in-vivo animal model system. For the purpose, experimental rats were administered mercury chloride (0.5mg/kg/body weight/day, orally) with and without curcumin (50 mg/kg body weight/day, orally) treatment in a time-dependent manner (7, 14, and 21 days). At the end of the study, we observed that the accumulation of mercury was higher in the liver of mercuric chloride alone group than the liver of non-curcumin treated rats measured using ICP-AES. A concomitant treatment of curcumin significantly reduces mercury accumulation up to 42-60% (p<0.001). In addition to attenuation of the mercury accumulation, curcumin administration also improves the liver functions as assessed through alteration in the levels of serum biomarkers (AST, ALT, Alkaline phosphatase, and GGT). Next, we evaluated the effect of curcumin on oxidative stress markers lipid peroxidation and glutathione. Numerous studies have been shown that mercury mediated oxidative stress play a significant role in hepatotoxicity. The administration of curcumin significantly reduces lipid peroxidation upto 58% (p<0.001) while restored glutathione level upto 39% (p<0.01) compared with the mercuric chloride alone group. Histopathology observations of the liver also justify the effect of curcumin with biochemical findings. Altogether, our findings indicate that curcumin has potential to inhibit mercury-induced hepatotoxicity and may provide useful insights for the prevention of mercury toxicity.

#4926

Preclinical intratumoral pharmacokinetics (PK) of capecitabine (Cap) given +/- eribulin (Eri).

Maria Jove Casulleras,1 Jade Spencer,2 Paul Loadman,2 Malcolm Clench,3 Steve Shnyder,2 Patricia Cooper,2 Cristina Russo,3 Amanda Race,2 Ramon Salazar,1 Chris Twelves4. 1 _Institut Català d'Oncologia, Barcelona, Spain;_ 2 _University of Bradford, Bradford, United Kingdom;_ 3 _Sheffield Hallam University, Sheffield, United Kingdom;_ 4 _St. James`s Institute of Oncology and University of Leeds, Leeds, United Kingdom_.

Background: Eri & Cap are active as single agents in metastatic breast cancer, but the combination appears particularly active & well tolerated clinically. We explored the hypothesis that normalisation of tumor vasculature by Eri seen in preclinical models may enhance delivery of Cap to the tumor when given in combination.

Methods: Mice bearing MDA-MD-231 xenografts were treated with Cap 540mg/kg p.o. day 1 alone or following Eri 1mg/kg i.v. on day -8 then sacrificed on day 1 at 15min, 30min, 1h, 2h, 4h, 6h or 24h after Cap (n=3/ time point). Concentrations of Cap & its metabolites (5'-deoxy-5-fluorocytidine, 5DFCR; 5-Fluoro-5'-deoxyuridine, 5DFUR; 5-Fluorouracil, 5FU; 5-Fluorouridine, 5FUrd; & 5-Fluoro-2'-deoxyuridine, 5FdUrd) in plasma, tumor, skin & muscle were analysed by LC-MS/MS. Half the tumor was reserved to study Cap distribution using matrix-assisted laser desorption/ionization mass spectrometry imaging with haem imaged as a surrogate marker for tumour vasculature.

Results: We found no differences in the plasma, skin, muscle or tumor PK profiles of Cape & its metabolites with the addition of Eri (Table 1). In both treatment arms intratumoral concentrations of 5DFUR, 5FU, 5FUrd & 5FdUrd appeared higher in tumors while those of Cape & 5DFCR appeared higher in normal healthy tissues. To mitigate the variability in tumor Cape concentrations between animals, we also expressed the results as the ratio of drug concentration in tumour:healthy tissue but again found no apparent effect of Eri on intratumoral drug concentrations. Analyses of drug distribution are on-going & will be presented.

Conclusion: No clear effect of Eri on intratumoral concentrations of Cape or its metabolites was seen. We are exploring potential differences in tumour drug distribution.

#4927

Plasma and CNS pharmacokinetics of the CHK-1 inhibitor prexasertib (LY-2606368) in mice bearing orthotopic group 3 medulloblastoma.

Anil Maharaj, Abigail Davis, Bo Zhong, Martine F. Roussel, Clinton F. Stewart. _St. Jude Children's Research Hospital, Memphis, TN_.

Many children with brain tumors have limited treatment options and poor long-term survival. A key protein kinase involved in activating and maintaining the S and G2/M checkpoints is CHK1, which when inhibited in the absence of p53 leads to loss of DNA damage checkpoints and can enhance the activity of many DNA-damaging agents. Prexasertib (LY-2606368), a potent and selective small molecule inhibitor of CHK1 protein kinase activity in vitro (IC50 <1 nM), is currently under clinical evaluation for treatment of pediatric malignancies. To evaluate prexasertib in preclinical efficacy studies for pediatric brain tumor models, the ratio of free drug concentrations between the central nervous system (CNS) and plasma (Kpu,u) is typically computed as a surrogate to measure CNS penetration. To facilitate the prospective determination of preclinical CNS Kpu,u values, we evaluated the plasma pharmacokinetic (PK) disposition of prexasertib in a murine model of medulloblastoma. A plasma PK study was performed using CD1 nude mice orthotopically implanted with group 3 medulloblastoma to obtain initial prexasertib PK parameters. Tumor growth was assessed by bioluminescence imaging (D-Luciferin) and mice were enrolled in the study once tumors reached 1x10~8-9 photons/second. Prexasertib mesylate monohydrate was reconstituted in 20% Captisol and administered subcutaneously at a dosage of 10 mg/kg (free base). A population based study design was used in which 3 samples per mouse were obtained. Plasma samples were prepared using a liquid-liquid extraction technique, and analyzed for prexasertib using a validated LC-MS/MS method (LLOQ = 0.2 µg/L). A two-compartment PK model was fit to the prexasertib concentration-time data using nonlinear mixed effects modeling (NONMEM 7.3, ICON Development Solutions). Plasma PK parameters from this model were used with the D-optimality criterion as implemented in ADAPT (D'Argenio, 1981) to derive a limited sampling model (LSM) for use in upcoming cerebral microdialysis studies. The results of our plasma PK studies showed a Cmax, terminal half-life, and AUC in CD1 nude mice bearing group 3 medulloblastoma of 1015 µg/L, 4.5 hours, and 1773 µg*hr/L, respectively. The LSM for plasma PK that will be used in the cerebral microdialysis studies include the time points of 0.25, 6.6, and 24 hours. Now that the plasma PK and a LSM are defined, we are poised to begin cerebral microdialysis studies in CD1 nude mice bearing group 3 medulloblastoma.

#4928

Role of hepatic organic anion-transporting polypeptides in methotrexate uptake and clearance.

Hannah H. Lee, Brenda F. Leake, Richard H. Ho. _Vanderbilt University Medical Center, Nashville, TN_.

Methotrexate (MTX) is a folate antimetabolite drug extensively used in patients with malignant and autoimmune diseases. Organic anion-transporting polypeptides 1A/1B (OATP1A/1B) are drug uptake transporters that are primarily expressed in pharmacokinetically relevant tissues and are known for broad substrate specificity, including many drugs in clinical use. While MTX is a known substrate for the efflux transporter MRP2, significantly less is known regarding its interactions with drug transporters that could mediate its cellular uptake and clearance. Previous studies have suggested MTX to be a substrate for OATP transporters. We sought to undertake a comprehensive evaluation of OATP transporters to the in vitro and in vivo disposition of MTX. In vitro, using a recombinant vaccinia-based method, we screened an array of OATPs for MTX transport in HeLa cells, indicating that human OATP1A2, OATP1B1, OATP1B3 and their rat orthologs, Oatp1a4, Oatp1a5 and Oatp1b2, were capable of significant MTX uptake (p < 0.01). In addition, an assessment of a panel of single nucleotide polymorphisms in SLCO1A/1B revealed that two nonsynonymous polymorphisms (NSPs; 516A>C and 404A>T) in SLCO1A2, seven NSPs including 521T>C and 388A>G+521T>C in SLCO1B1, and six NSPs in SLCO1B3 were associated with significantly impaired MTX transport. Transcellular vectorial transport (basal→apical) studies of MTX conducted in MDCKII cells stably expressing hepatic OATP1B transporters revealed both OATP1B1 and OATP1B3 transport MTX. There was significantly increased MTX in the apical compartments of MDCKII-OATP1B/MRP2 cells compared to control cells (p < 0.05) at all time points. Furthermore, MTX translocated into the apical compartments was significantly higher in MDCKII-OATP1B/MRP2 cells than MDCKII-OATP1B cells (p < 0.05) at late time points, reflecting active efflux mediated by MRP2. Compared to control cells, intracellular accumulation of MTX was significantly higher in both MDCKII-OATP1B and MDCKII-OATP1B/MRP2 cells up to 2h post incubation (p < 0.05), reflecting active uptake by OATP1B at early time points. Subsequently, in vivo MTX transport studies showed that plasma MTX AUC was 2.8-fold higher and clearance was reduced by 64% in Slco1a/1b-/- versus wild-type (WT) mice (p < 0.01). The liver-to-plasma ratio of MTX was 9-fold higher in WT mice compared to Slco1a/1b-/- mice (p < 0.01) but no difference between WT and humanized TG mice, collectively suggesting hepatic OATP1B transporters are important for hepatic clearance of MTX. In conclusion, we demonstrate significant roles for OATP1B in transporter-mediated uptake and disposition of MTX. Moreover, significantly impaired MTX transport by OATP1A/1B variants may have important toxicological and therapeutic ramifications. Accordingly, these findings reveal important new insights into the relevance of the hepatic OATP1B transporters to the clinical pharmacology of MTX.

#4929

A PK/PD study of Delta-4 abiraterone metabolite in metastatic castration-resistant prostate cancer patients.

Benoit Blanchet,1 Edith Carton,1 Mohammad Alyamani,2 Lisa Golmard,3 Olivier Huillard,1 Audrey Thomas,1 Michel Vidal,1 Francois Goldwasser,1 Nima Sharifi,2 Jerome Alexandre4. 1 _Université Paris Descartes, Cochin - Port Royal, Paris, France;_ 2 _Lerner Research Institute, Cleveland Clinic, Cleveland, OH;_ 3 _Institut Curie, Paris, France;_ 4 _Université Paris Descartes, Cochin - Port Royal, PARIS, France_.

Background : Abiraterone (ABI), a steroidal CYP17A1 inhibitor, blocks the DHT synthesis from adrenal precursor steroids in castration resistant prostate cancer (mCRPC) patients. ABI is converted by 3β-hydroxysteroid dehydrogenase (HSD3B) into D4-abiraterone (D4-ABI). In a previous preclinical study, D4-ABI was shown to block multiple steroidogenic enzymes and antagonize the androgen receptor. Besides, it appeared more active than ABI itself (Li Z et al. Nature 2015). However, the contribution of D4-ABI to the clinical antitumoral activity of abiraterone acetate in men with mCRPC remains unknown.

Methods : From 12/2012 to 10/2014, 61 consecutive mCRPC patients were treated with ABI (1000 mg, once daily) concomitantly with 10 mg of prednisone (Carton et al Eur J Cancer 2017). The study population represents a subgroup of these patients for whom trough plasma ABI and D4-ABI concentrations were assayed using liquid chromatography with fluorescence detection and LC/MS/MS, respectively. The trough plasma concentration of ABI and D4-ABI was assayed one (M1), two (M2) and three (M3) months after treatment initiation. ABI and D4-ABI Cmin were defined as the mean of trough concentrations measured during the first 3 months of treatment. This prospective analysis was in compliance with the Declaration of Helsinki and approved by the local medical ethical board (N°9166). All patients gave their written informed consent to participate in the study.

Results : Thirty-six mCRPC patients were included. Mean plasma ABI and D4-ABI Cmin were 12.6 ± 6.8 ng/mL (coefficient of variation, CV= 54.0%) and 1.6 ± 1.3 ng/mL (CV= 81.3%), respectively. Intra-individual variability for ABI and D4-ABI Cmin was 23.8% and 60.7%, respectively. The mean metabolic ratio (D4-ABI / ABI) was of 0.18 ± 0.25 (CV=140.4%). In regards with in vitro data previously reported for IC50% of ABI and D4-ABI, we estimated that total plasma Cmin enabled to achieve these IC50% in 30 patients (83.3%) and only 2 patients (5.6%), respectively. The univariate Cox proportional-hazard regression model showed that higher D4-ABI Cmin was associated with shorter OS (Hazard ratio, HR 1.54; CI95% 1.06-2.22; p=0.022) but not with PFS. As previously reported, patients with ABI Cmin >8.3 ng/mL exhibited a longer PFS than patients underexposed (322 vs 237 days, p=0.05). The HR associated with the metabolic D4-ABI / ABI ratio for PFS and OS were 7.80 (CI 95% 1.63-37.38; p = 0.010) and 12.52 (CI 95% 1.95-80.47, p = 0.0078), respectively.

Conclusion : It is unlikely that pharmacologic activity of D4-ABI contributes significantly to abiraterone acetate clinical activity for a daily dosing of 1,000 mg. The poor prognosis of higher D4-ABI Cmin / ABI Cmin ratio could be further in relation with a high activity of HSD3B1 enzyme which drive castration resistance by enhancing the DHT synthesis from non-gonadal precursors.

#4930

Epicatechin alleviates DOX-induced cardiovascular toxicity and improves its cytotoxic profile against breast cancer cells.

Ohoud Y. Alshehri,1 Fahad A. Alabbasi,1 Islam F. Mahmoud,1 Alaa T. Abu-Sharib,1 Hany M. El-Bassossy,1 Hossam A. Abdallah,1 Ahmed M. Al-Abd2. 1 _King Abdulaziz University, Jeddah, Saudi Arabia;_ 2 _National Research Ctr. of Egypt, Cairo, Egypt_.

Epicatechin (EPI) has a well known and robust antioxidant properties which could alleviate drug-induced cardiovascular toxicities. On the other side, Doxorubicin (DOX) is a cornerstone anticancer drug used to treat breast cancer. However, DOX-induced cardiovascular complications might limit its treatment course continuation. Herein, we evaluated cardiovascular protective effects of EPI against DOX in-vivo and ex-vivo using isolated aortic ring preparation; meanwhile we assessed the potential ameliorating effects of EPI against DOX-induced cytotoxicity in breast cancer cells. Animals were given DOX (12 mg/kg, single IV injection) which induced cardiac ischemia appeared as increased QT and QTc intervals (1.8 folds for both). EPI (12 mg/kg, orally for 6 days) completely aborted DOX-induced cardiac ischemia and corrected all ECG abnormalities. Using isolated aortic ring preparation, DOX (10µM) impaired aortic ring vasodilatation and induced excessive vasoconstriction. Co-incubation of EPI (10µM) with DOX (10µM) entirely blocked DOX-induced impaired aortic ring vasodilatation, without affecting DOX-induced elevated vasoconstriction. EPI (10µM) combination with DOX in MDA-MB-231, MCF7 and T47D breast cancer cells significantly decreased the IC50's from 70.0±17.3 to 44.0±14.0 nM, from 554.0±30 to 240.0±10.0 nM and from 700.0±36.0 to 561.0±170 nM, respectively. Under hypoxic condition, EPI (10µM) significantly increased DOX cytotoxicity against MCF-7 cells and decreased its IC50 from 8.9±1.2 to 6.1±1.9 μM. This effect could be partially attributed to the inhibitory effect of EPI to P-glycoprotein (P-gp) and related efflux protein which would enhance DOX intracellular concentration. EPI increased the intracellular accumulation of rhodamine (P-gp probe) within MDA-MB-231 cells by 3-4 folds at concentration range from 3 to 100 μM. Moreover, EPI combination with DOX significantly increased cell apoptosis and necrosis assessed by annexin-V/FITC. However, EPI did not influence DOX effect on cell cycle distribution. In conclusion, EPI possesses potential cardiovascular protective effects against DOX. Meanwhile, EPI does not interfere with DOX-induced cytotoxic profile against breast cancer cells.

#4931

Enhanced oral bioavailability of 3,3'-diindolylmethane administered in a self-microemulsifying drug delivery system (SMEDDS).

William D. Johnson,1 Miguel Muzzio,1 Thomas L. Horn,1 Michael A. Zeligs,2 Elizabeth R. Glaze,3 David L. McCormick1. 1 _IIT Research Institute, Chicago, IL;_ 2 _BioResponse, LLC, Boulder, CO;_ 3 _National Cancer Institute, Bethesda, MD_.

3,3'-diindolylmethane (DIM) is a stable, water-insoluble indole that demonstrates chemopreventive activity in several preclinical models for human cancer. Although clinical data have shown that the safety profile of absorption-enhanced, microencapsulated DIM is consistent with its use in cancer chemoprevention in humans, the in vivo biological activity of DIM is limited by poor solubility and very low oral bioavailability. Self-Microemulsifying Drug Delivery Systems (SMEDDS) provide a novel approach to improving the systemic delivery of lipophilic agents with poor oral bioavailability. Lipid-based SMEDDS formulations form oil-in-water microemulsions in aqueous gastric fluid; the microemulsions increase drug solubility and improve systemic absorption. The present study was performed to determine if the oral bioavailability of DIM and its levels in the prostate are increased by administration in a DIM-specific SMEDDS formulation. Fifteen male CD rats per group received a single oral (gavage) dose of either a microencapsulated DIM formulation (BR-DIM®, BioResponse) or a SMEDDS formulation of DIM (BR-9001, BioResponse). Both groups received a DIM-equivalent dose of 30 mg/kg. Gavage administration of each DIM formulation was immediately followed by a second gavage dose of saline to provide uniform intragastric emulsification of BR-9001 and suspension of BR-DIM. Cohorts of three rats per group were bled at time 0 (pre-dose) and at 0.25, 0.5, 0.75, 1.0, 2.0, 4.0, 8.0, and 24 hours post-dosing; each rat was bled twice. After the second bleed, each rat was euthanized and its prostate was collected. Plasma samples and weighed samples of prostate were extracted and analyzed for DIM content by LC-MS/MS, and plasma pharmacokinetics parameters were calculated. The oral bioavailability of DIM was substantially greater in the SMEDDS BR-9001 formulation than in the BR-DIM formulation: at the Tmax (30 min), mean plasma DIM levels in rats receiving the SMEDDS formulation were >400% of mean plasma DIM levels in rats dosed with microencapsulated BR-DIM. For four hours after dosing, plasma DIM levels in SMEDDS-treated rats remained above plasma DIM levels in rats treated with microencapsulated DIM; the AUC for the SMEDDS formulation of DIM was approximately twice that for the microencapsulated DIM formulation. Similarly, at 2, 4, and 6 hours post-dosing, mean prostate DIM levels in rats receiving the SMEDDS formulation were approximately 200% of mean prostate DIM levels in rats receiving microencapsulated BR-DIM. These data demonstrate that plasma and prostate levels of DIM can be increased by using the BR-9001 SMEDDS formulation of DIM. Because the chemopreventive efficacy of DIM appears to be limited by poor oral bioavailability, the use of SMEDDS formulation technology may increase the efficacy of DIM for cancer chemoprevention. [HHSN261201500024I from the NCI, DHHS.]

#4932

Neuro-endocrine negative SCLC is mostly RB1 WT and may be sensitive to CDK4/6 inhibition, especially if CCND1 is amplified.

Dmitriy Sonkin,1 Anish Thomas,2 Beverly A. Teicher3. 1 _National Cancer Institute, Rockville, MD;_ 2 _National Cancer Institute, Bethesda, MD;_ 3 _National Cancer Institute, MD_.

Background: Small cell lung cancer (SCLC) is an aggressive form of lung cancer with limited therapeutic options, a very high mortality rate and is characterized, in most cases, by neuroendocrine features. A small but important subset of SCLC has intact RB1, however, other characteristics of this subset are not well-defined. Here we aim to characterize underlying genomics of SCLC cell lines with functional RB1.

Methods: To comprehensively assess the underlying genomics of SCLC cell lines with functional RB1, we examined 52 SCLC cell lines from the Cancer Cell Line Encyclopedia (CCLE) collection. We also integrated CCLE SCLC genomics data with drug sensitivity data from NCI SCLC screen of 420 approved and investigational oncology drugs.

Results: Out of 7 neuro-endocrine lineage marker-negative SCLC lines, 6 were RB1 WT. Out of 42 neuro-endocrine lineage marker-positive SCLC cell lines, 3 were RB1 WT. Two RB1 WT SCLC cell lines with CCND1 amplification were sensitive to CDK4/6 inhibitor. Oncogenic alterations in genes such as KRAS, NRAS, SMARCA4, and CDKN2A are rare in SCLC; however, these genes were altered in 7 of 9 RB1 WT SCLC cell lines.

Conclusions: Neuro-endocrine lineage marker-negative SCLC are enriched for RB1 WT and may be sensitive to CDK4/6 inhibition especially in context of CCND1 amplification. Neuro-endocrine lineage marker-negative SCLC cell lines are associated with low DLL3 expression. Therefore, RB1 WT SCLC are less likely to respond to DLL3 targeted drugs. However, this may present the opportunity to identify patients with WT RB1 among patients screened for DLL3 ADC clinical trials and having no or very low DLL3 expression.

#4933

Six-month oral toxicity study of endoxifen in rats.

Thomas L. Horn,1 William D. Johnson,1 Carol J. Detrisac,2 Patrick T. Curry,1 Elizabeth R. Glaze,3 David L. McCormick1. 1 _IIT Research Institute, Chicago, IL;_ 2 _Charles River - Pathology Associates, Chicago, IL;_ 3 _National Cancer Institute, Bethesda, MD_.

The tamoxifen metabolite, endoxifen (N-desmethyl-4-hydroxytamoxifen), is being developed for oral and local topical administration for prevention and therapy of breast cancer. As part of this development, the toxicity of oral endoxifen was evaluated in a chronic (six-month) study in rats. Groups of 25 female CD rats received daily oral (gavage) exposure to endoxifen at doses of 0 (vehicle control), 5, or 50 mg/kg/day for six months. In vivo assessments included survival; clinical and physical signs of toxicity; body weight; food consumption; ophthalmology; and clinical pathology (clinical chemistry, hematology, coagulation). Fifteen rats per group were euthanized and necropsied after six months of endoxifen exposure; remaining rats in each group were euthanized and necropsied after a four-week recovery period. All gross lesions and approximately 45 tissues per animal were evaluated microscopically. In addition, bone marrow smears collected from rats euthanized for the six month necropsy were evaluated for DNA damage (micronucleus assessment). Daily oral administration of endoxifen at 5 or 50 mg/kg/day for six months induced no treatment-related mortality, clinical evidence of toxicity, or effects on ophthalmology. Endoxifen was not genotoxic, as it had no effect on the incidence of bone marrow micronuclei. Both dose levels of endoxifen induced similar suppressions of body weight gain; food consumption was also significantly decreased in both endoxifen-treated groups. Both dose levels of endoxifen induced gross and microscopic changes in hormone-sensitive tissues. Gross pathology was identified at necropsy in the ovary (cysts) and uterus (small) in both endoxifen groups; mean relative ovarian weight was increased and mean relative uterine weight was decreased in endoxifen groups. Microscopic findings were identified in the ovaries (corpora lutea depletion, cysts), uterus (atrophy, endometrial hyperplasia), cervix (mucinous hypertrophy), mammary gland (hyperplasia), and pituitary gland (vacuolation). These changes are consistent with the activity of endoxifen as an endocrine disruptor, and suggest effects on the hypothalamic-pituitary-gonadal axis. After six months of daily oral administration, both dose levels of endoxifen induced significant reductions in body weight gain and food consumption; alterations in the weights of hormone-sensitive organs; and gross and microscopic effects, primarily in hormone-sensitive tissues. Most of the effects of endoxifen observed in this study can be ascribed to its potent antiestrogenic activity, and are interpreted as signs of its pharmacologic action. However, the gross and microscopic changes seen at both dose levels of endoxifen used in this study demonstrate that a No Observed (Adverse) Effect Level (NO[A]EL) for daily oral administration of endoxifen to female rats for six months could not be determined. [Supported by HHSN261201500024I from the NCI, DHHS.]

## CLINICAL RESEARCH:

### Liquid Biopsy: Poster Discussion

#5598

Development and optimization of a comprehensive high-sensitivity NGS cancer assay and bioinformatics pipeline for plasma cfDNA profiling.

Juber Patel,1 Maysun Hasan,1 Fanli Meng,1 Xiaohong Jing,1 Dilmi Perera,1 Jonathan Reichel,1 Erika Gedvilaite,1 Julie Yang,1 Maha Shady,1 Sandeep Raj,1 Preethi Srinivasan,1 Ian Johnson,1 Jiashi Wang,2 Mirna Jarosz,2 Aliaksandra Samoila,1 Agnes Viale,1 Bob Li,1 Pedram Razavi,1 Dana Tsui,1 Michael Berger1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Integrated DNA Technologies, IA_.

The accessibility of tumor-derived cell-free DNA (cfDNA) in blood plasma provides a means to non-invasively profile somatic mutations in solid tumor patients. Clinical applications include longitudinal monitoring of disease burden and acquired drug resistance, identification of clinically relevant alterations and mutation signatures, and detection of minimal residual disease. However, the low fraction of tumor-derived cfDNA in plasma in many patients requires assays and bioinformatics methods that are much more sensitive than have been used for traditional tissue-based analysis.

The design of our cfDNA NGS panel is based on prospectively-collected clinical sequencing data obtained from more than 20,000 patients at Memorial Sloan Kettering Cancer Center using MSK-IMPACT, a custom 468-gene sequencing test authorized by the FDA for somatic mutation profiling. Exons harboring hotspot mutations, clinically actionable mutations, and elevated somatic mutation rates were selected for inclusion in the cfDNA panel. Additional non-coding content was included to enable optimal detection of selected copy number alterations, regions of loss of heterozygosity, rearrangement breakpoints, and microsatellite instability. Altogether the panel contains 208 kilobases of sequence from 129 cancer genes. Ultra-deep sequencing and unique molecular indexing enable PCR-generated replicate sequences to be collapsed into error-free consensus sequences, thereby facilitating the high-confidence detection of mutations present at low allele fractions.

We developed an open source bioinformatics tool, Marianas, for collapsing PCR replicates into consensus sequences and computing associated quality and performance metrics. Marianas incorporates many empirically derived features that lead to significant noise reduction. It efficiently processes a bam file with 20,000X coverage in 20 minutes on a single processor. We benchmarked the performance of Marianas against other available tools for collapsing and consensus base calling. The relative contributions of sources of error such as barcode contamination and sample

cross-talk during PCR and sequencing were also quantified. We found that using unique dual sample indexes in multiplexed sequencing runs was essential to suppress these sources of noise.

Applying these aggregated methods to analyze plasma cfDNA samples obtained from patients across a range of solid tumor types and disease stages, we were able to reliably detect clinically relevant mutations with variant allele fractions below 0.003, including subclonal mutations associated with acquired drug resistance. This approach, when applied prospectively on clinical specimens, has the potential to facilitate diagnosis, prognosis, and treatment selection in an era of precision oncology.

#5599

Noninvasive genomic profiling of cerebral spinal fluid in breast cancer patient with leptomeningeal disease.

Masahiro Oikawa, Naveen Ramesh, Emi Sei, Shanshan Bai, Min Hu, de Groot F. John, Murthy Rshmi, Barbara O'Brien, Nicholas Navin. _UT MD Anderson Cancer Center, Houston, TX_.

Leptomeningeal disease (LMD) involves the dissemination of tumor cells from the primary breast tumors into the membranes surrounding the central nervous system and spinal cords. LMD occurs in about 5% of breast cancer patients and is associated with very poor survival. The genomic evolution of LMD in breast cancer patients has remained difficult to study, in part due to technical challenges in collecting longitudinal biopsy samples from the central nervous system. Liquid biopsies of the cerebral spinal fluid (CSF) may provide a unique opportunity to profile circulating tumor DNA (ctDNA) and has not been compared directly to non-invasive monitoring of ctDNA in blood samples. Furthermore, most non-invasive ctDNA profiling methods are limited to a targeted set of genes and have not allowed unbiased whole-genome profiling. To address these limitations, we developed an unbiased method for ctDNA analysis called PEGASUS (Plasma Exome and Genome Analysis by Size-selection and Unbiased Sequencing) that enables whole-genome sequencing of copy number aberrations and somatic mutations from ctDNA. We applied PEGASUS to sequence matched blood samples and CSF from 10 breast cancer patients with LMD. Quantitation of ctDNA levels showed that ctDNA was detected in CSF samples from 9/10 patients, while blood ctDNA was detected in only 1 LMD patient with extensive metastatic disease. Whole-genome copy number profiling at 220kb resolution and mutational profiling of a 2000 cancer gene panel of the matched CSF and blood samples was performed in 10 LMD patients using PEGASUS. Our data identified aneuploid copy number aberrations and somatic mutations (range: 11-25) in the CSF of 7 patients, including mutations in known driver genes such as BRAF, RB1, TP53 and amplifications of MYC and ERBB2. In contrast the matched blood samples showed only diploid copy number profiles and no detectable somatic mutations in 9/10 LMD patients. In one patient with extensive cranial metastatic disease with matched CSF and blood ctDNA, we found a high concordance in copy number profiles (R= 0.75) and modest concordance of somatic mutations (59.5%), but also additional CNAs and mutations that were specific to the CSF. Collectively, these data show that genomic profiling of ctDNA in CSF is technically feasible for patients with LMD, and provides a major advantage over blood ctDNA which cannot be detected in mostcases.

#5600

Establishment and characterization of a unique circulating tumor cells-derived xenograft (CDX) in prostate cancer.

Vincent Faugeroux,1 Emma Pailler,1 Olivier Deas,2 Virginie Marty,1 Kamélia Alexandrova,1 Kiki Andree,3 Jean-Yves Scoazec,1 Nikolas Stoecklein,4 Nicolo Manaresi,5 Dominique Tramalloni,1 Maud Ngo-Camus,1 Claudio Nicotra,1 Leon Terstappen,3 Valérie Lapierre,1 Karim Fizazi,1 Yohann Loriot,1 Jean-Gabriel Judde,2 Françoise Farace1. 1 _Institut Gustave Roussy, Villejuif, France;_ 2 _XenTech, Evry, France;_ 3 _University of Twente, Enschede, Netherlands;_ 4 _University Hospital of the Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany;_ 5 _Menarini Silicon Biosystems S.p.A., Bologna, Italy_.

Background: The rarity of in vivo and in vitro human prostate cancer (PCa) models has hampered progress in understanding disease pathogenesis, metastatic progression and drug resistance mechanisms. Using CTCs from a leukapheresis product of a patient with advanced PCa, we report the establishment of a CDX and an in vitro cell line derived from this CDX. The phenotypic and molecular characterization of patient tumor-biopsies, CTCs, CDX and CDX-derived cell-line are presented.

Methods: Leukapheresis was performed in seven patients with advanced castration-resistant prostate cancer (CRPC). CTCs from the seven leukapheresis products were enriched by RosetteSep and implanted in Nod/Scid-IL2Rγ-/-mice. The CDX tumor was propagated in successive generations of mice. All samples, including eight tumor-biopsies performed at diagnosis two years prior leukapheresis and CTCs isolated at the single cell level during leukapheresis were characterized by immunofluorescence, immunohistochemistry, and whole-exome sequencing (WES).

Results: Based on CellSearch® counts in leukapheresis products, the estimated median number of engrafted CTCs was 697 (range: 10-19988). A mouse engrafted with 19988 CTCs developed a tumor within 193 days. Immunohistochemistry performed on the CDX and two tumor-biopsies indicated that the CDX and biopsies were positive for EpCAM, CK5/6/8/18, negative for CK7 and vimentin, and weakly positive for synaptophysin. While biopsies expressed PSA and the androgen receptor, the CDX was negative for both indicating tumor evolution. In contrast to tumor biopsies, the CDX strongly expressed Ki67, NSE and chromogranin, evidencing emergence of a neuroendocrine phenotype. The in vitro cell line established by culturing dissociated CDX cells for five months, grew in microspheres and expressed epithelial and ALDH and CD133 cancer stem-cell markers. By WES, a high degree of intra-tumor heterogeneity was observed in the eight tumor biopsies and CTCs as already reported in this tumor type. Only 2.8% (58/2087) and 2.3% (49/2087) of the mutations present in the tumor biopsies were identified in CTCs and the CDX respectively, indicating that a very few number of mutations have the potential to support the dissemination and tumorigenic activity of CTC. Trunk mutations in TP53, NF1 and LRP1B genes were identified in all samples including the CDX while PTEN gene loss was acquired lately and detected only in CTCs and the CDX. Mutational similarity of the CDX and the in vitro cell line was 91%. The analysis of copy number variations is ongoing in all samples and will be presented.

Conclusion: We report the first PCa CDX model, demonstrating the tumorigenicity of CTCs from CRPC. This CDX model represents a unique tool to identify clonal mutations associated with the tumor-initiating capacity of CTCs and explore the genetic and phenotypic basis of metastasis and drug resistance in advanced CRPC.

#5601

Single-cell molecular profiling of circulating tumor cells (CTCs) within the TRACERx study reveals heterogeneous patterns in early non-small cell lung cancer (NSCLC).

Francesca Chemi,1 Sakshi Gulati,1 Dominic G. Rothwell,1 Debbie Burt,1 Daniel Slan-Tan,1 Barbara Mesquita,1 Chris Wirth,1 Gareth Wilson,2 Jackie Pierce,1 Ged Brady,1 Charles Swanton,3 Caroline Dive1. 1 _CRUK Manchester Institute, Manchester, United Kingdom;_ 2 _University College London Cancer Institute, The Francis Crick Institute, London, United Kingdom;_ 3 _University College London Cancer Institute, The Francis Crick Institute, On behalf of TRACERx consortium and Cancer Research UK Manchester/UCL Lung Cancer Centre of Excellence, London, United Kingdom_.

Introduction: For early stage NSCLC, surgery with curative intent is the most common therapeutic option; however, tumour recurrence occurs in approximately 50% of cases and most commonly at distant sites. Analysis of CTCs represents a potential means of tracking cancer cells from the primary tumour and has the potential to provide insights into the metastatic process and mechanisms of resistance to therapy linking cell phenotype and genotype. Within the TRACERx study, we are carrying out whole genome analysis of single CTCs obtained from NSCLC patients undergoing surgery in order to examine the relationship between CTCs, the primary tumour and metastatic relapse.

Methods: Blood from 163 patients was collected at surgery from the tumour draining pulmonary vein and from peripheral vein in longitudinal samples. CTCs were enriched based on their EpCAM expression using CellSearch® and then enumerated according to their positivity for epithelial markers (Cytokeratins) and lack of detectable blood markers (CD45). Samples containing at least five CellSearch® CTCs (CS-CTCs) were processed through DEPArray™ for single cell isolation followed by whole genome amplification (WGA) using the Ampli1™ WGA kit. Genome wide copy number alterations (CNA) were detected through low-depth whole genome sequencing (WGS) and mutational analysis of individual CTCs was carried out using whole exome sequencing (WES). CTC genomic profiles were compared to the corresponding spatially separated tumour sections obtained at surgery.

Results: In a pilot study of 30 patients undergoing tumour resection, the presence of pulmonary vein CS-CTCs has been linked to poor clinical outcome (Crosbie et al, J Thorac Oncol, 2016). Here we extended the evaluation of pulmonary vein CS-CTCs to a further 122 patients and confirmed the initial observation that the presence of pulmonary vein CS-CTCs is a poor prognostic indicator. CNA analysis of 100 single CTCs from the first 12 patients revealed heterogeneous patterns between patients and within individual patients. Three types of pulmonary CS-CTC candidates were observed: Type 1, which share clear copy number changes with both tumour and other CS-CTCs; Type 2, where copy number changes were detected but were not shared with the tumour; Type 3, where no copy number changes were detected. Using WES of both CTCs and matching excised tumour regions, we identified common genetic alterations shared by CTCs and the tumour, as well as private genetic changes detected only in CTCs. We are currently evaluating metastatic biopsies from relapsing patients and comparing their molecular profiles to those seen in CS-CTCs obtained at surgery.

Conclusion: The combined tumour and CTC data show that single CTC analysis provides an additional layer of complexity with a valuable new perspective on tumour heterogeneity and early dissemination in NSCLC.

#5602

Detection of circulating cell-free tumor DNA (ctDNA) in patients with small cell lung cancer (SCLC).

Sumitra Mohan,1 Victoria Foy,1 Hui Sun Leong,1 Pietà G. Schofield,1 Mahmood Ayub,1 Nigel K. Smith,1 Sudhakar Sahoo,1 Chang Sik-Kim,1 Lynsey Priest,1 Mathew Carter,2 Hedley T. Carr,3 Crispin Miller,1 Corinne Finn-Faivre,2 Fiona Blackhall,2 Dominic G. Rothwell,1 Caroline Dive,1 Gerard Brady1. 1 _Cancer Research UK-Manchester Institute, Manchester, United Kingdom;_ 2 _The Christie NHS Foundation Trust, Manchester, United Kingdom;_ 3 _Astrazeneca, Cambridge, United Kingdom_.

Introduction: Tumor genomes can be reconstructed from the molecular information obtained from circulating cell-free DNA (cfDNA) and circulating tumor cells (CTCs) obtained from the peripheral blood of patients with cancer. The analysis of cfDNA and CTCs is a minimally invasive approach and represents a powerful research tool, with potential as a companion diagnostic for both patient stratification and monitoring. Here, we use cfDNA next-generation sequencing (NGS) analysis to assess and compare ctDNA profiles in patients with SCLC.

Methods: Whole genome sequencing (WGS) libraries were prepared from cfDNA isolated from the pre-treatment peripheral blood samples from 69 patients and 32 cancer-free controls. Libraries were subjected to WGS to establish genome wide copy number aberrations (CNA) as well as targeted mutation analysis of 110 SCLC associated genes. Quantitative metrics were calculated from CNA such as Percent Genome Altered (PGA; percentage of genomic regions altered), Z-score (measure of standard deviation) and Moran's I (measure of spatial autocorrelation). In addition CellSearch®, an epitope dependent enrichment platform was used to enumerate CTCs from a parallel blood sample.

Results: An examination of CNA patterns revealed SCLC associated changes such as losses on chromosomes 3p, 5q and 17p and gains on chromosome 3q and 5p as well as amplification of MYC in 21/69 (30%) and SOX2 in 36/69 (52%) as well losses on FHIT in 40/69 (58%), RASSFI in 38/69(55%) and RB1 in 24/69 (35%) patients. A combination of three CNA metrics enabled detection of tumor associated changes in 58/64 (84%) patients, with Moran's I emerging as the most sensitive CNA metric for detecting ctDNA. Targeted NGS detected tumor associated mutations in 60/64 (94%) with TP53 mutations detected in 50 patients (83% of patients with any detectable ctDNA). Somatic mutations and CNA were detected in both limited stage SCLC (LS-SCLC, confined to 1 hemithorax) and extensive stage (ES-SCLC, with distant metastases) with statistically significant differences seen for CNA metrics and variant allele frequencies of mutations consistent with higher levels of ctDNA in ES-SCLC. However, no significant differences between ES and LS were observed in the mutation patterns with respect to DNA damage repair, RAS and PI3K and transcriptional regulation pathways between the two stages of SCLC. There was considerable overlap between the detection of ctDNA and CTC counts, with cfDNA NGS readouts detecting tumor related changes in 96% of patients and CTCs were detected only in 76% of patients.

Conclusion: We have established sensitive methods for detecting ctDNA in cfDNA and combined with CTC enumeration we have an effective liquid biopsy for 98% of patients (96% in LS and 100% in ES) in this cohort. Future work will involve utilising this optimised NGS approach in an independent cohort of patients to correlate cfDNA metrics with clinical outcome.

#5603

Analytical validation of a comprehensive 500-gene ctDNA panel designed for immuno-oncology and DNA damage research.

Elena Helman, Carlo Artieri, James V. Vowles, Jennifer Yen, Tracy Nance, Marcin Sikora, Joshua Gourneau, Mohit Goel, Stefanie Mortimer, Darya Chudova, Justin Odegaard, Richard B. Lanman, AmirAli Talasaz. _Guardant Health, Redwood City, CA_.

Background: Translational research and enrollment in clinical trials are limited by the rarity of individual mutations and lack of sufficient tissue for comprehensive testing. To address these limitations, we developed GuardantOMNI (OMNI), a highly sensitive 500-gene cfDNA sequencing test requiring as little as 2 mL of plasma and designed for broad genomic detection of somatic single-nucleotide variants (SNVs) and small indels in 497 genes, copy number amplifications (CNAs) in 106 genes, and fusions in 21 genes. Additionally, the OMNI panel enables assessment of tumor mutational burden (TMB), and DNA damage and mismatch repair, with coverage of over 30 genes associated with the DDR pathway. Here we present the first analytical validation study of OMNI.

Methods: Analytical performance was assessed as per Nex-StoCT Working Group guidelines using precharacterized cell lines and healthy normal donor-derived samples. Qualitative and quantitative orthogonal confirmation was provided by exome sequencing, microarrays, and data from published compendia.

Results: Seventy-three validation and 150 development plasma samples were processed for this study, using both 5ng and 30ng cfDNA input levels, and all samples passed sequencing QC metrics established prior to testing. Reportable ranges for SNVs were ≥0.04% variant allele fraction (VAF), ≥0.02% for indels, ≥2 supporting molecules for fusions, and ≥2.18 copies for CNAs. Cell line-based dilution studies demonstrated 95% limits of detection (LoD) of 0.24-0.6% VAF for SNVs (depending on known cancer association), 0.4-0.8% for non-homopolymeric indels (depending on clinical relevance), 0.1-0.2% for fusions, and 2.2-2.9 copies for 90% of CNA genes targeted. Comparison of diluted cell line and healthy donor samples to orthogonal sequencing and published genotype data demonstrated accuracies of 98.7% for SNVs, 97.2% for indels, and 100% for CNAs and fusions across the reportable range. The analytical false-positive rate per sample measured across 24 healthy donors was 0.25 for SNVs, 0.04 for indels, and 0 for CNAs and fusions, with positive predictive values (PPVs) of 97.5% for SNVs, 98% for indels, and 100% for CNAs and fusions. Quantitative correlation of allele fraction with confirmatory methods was high (r2 > 0.99).

Conclusions: To our knowledge, OMNI is the largest comprehensive ctDNA cancer gene panel available. It detects alterations in genes under study in over 98% of current clinical trials with sensitivity, specificity, and accuracy similar to currently available targeted ctDNA sequencing tests. OMNI has the potential to accelerate clinical trial enrollment, research and discovery with a single, noninvasive blood sample.

#5604

Novel DNA methylation biomarkers show high sensitivity and specificity for blood-based detection of colorectal cancer - A clinical biomarker discovery and validation study.

Sarah Østrup Jensen,1 Mai-Britt Worm Ørntoft,1 Nadia Øgaard,1 Helle Kristensen,2 Mads Heilskov Rasmussen,1 Jesper Bertram Bramsen,1 Peter Mouritzen,2 Mogens Rørbæk,3 Anne-Sofie Kannerup,4 Søren Laurberg,5 Hans Jørgen Nielsen,6 Claus Lindbjerg Andersen1. 1 _Aarhus Univ. Hospital, Aarhus N, Denmark;_ 2 _Exiqon, Vedbæk, Denmark;_ 3 _Regional Hospital West,, Herning, Denmark;_ 4 _Randers Regional Hospital, Randers, Denmark;_ 5 _Aarhus Univ. Hospital, Aarhus C, Denmark;_ 6 _University of Copenhagen, Hvidovre, Denmark_.

Background: Screening for colorectal cancer (CRC) using fecal occult blood tests (FOBT) reduces CRC mortality, and many CRC screening programs therefore use FOBT. However, FOBT is not the best approach; first, because population acceptance is low due to unpleasantness of fecal sampling; second, because bowel tumors bleed only intermittently, which limits FOBT sensitivity. Development of a novel blood-based screening approach may alleviate these problems.

Objective: This study aims to develop and validate novel blood-based biomarker assays to achieve high patient compliance, sensitivity and specificity.

Methods and materials: CRC-specific DNA methylation biomarker candidates were identified using a genome-wide discovery strategy based on >4,000 Illumina 450K DNA methylation arrays. We designed digital droplet PCR assays to detect top biomarker candidates in circulating cell-free DNA (cfDNA) isolated from plasma. Initially, sensitivity and specificity of biomarkers were evaluated in validation cohort 1 consisting of plasma collected from 114 symptomatic CRC patients and 86 colonoscopy-confirmed healthy controls. To ensure comparable technical sensitivities for all assays in all samples, we used a fixed cfDNA input of 4,500 copies per ddPCR reaction. Next, we tested markers in validation cohort 2, a selected cohort of 8 mL plasma collected from participants in the Danish national screening program. This cohort comprised 131 asymptomatic CRCs and 869 controls enriched for comorbidities like adenomas, other cancers, diabetes, arthritis, hypertension, inflammatory bowel disease and arteriosclerosis.

Results: Our discovery identified 12 DNA methylation biomarkers. Their performance was evaluated in validation cohort 1, and the three best performing markers showed a sensitivity of 89% at a specificity of 99%, which is superior to FOBT. Sensitivity increased with stage, reaching 65%, 85%, 78% and 83% for stage I-IV, respectively. When evaluated in validation cohort 2, the sensitivity was 51% for CRC samples with an input of at least 4,500 copies of cfDNA, and sensitivity was reduced equally for all disease stages compared with results from validation cohort 1. Sensitivity correlated positively with cfDNA input, and it is therefore critical to collect sufficient plasma volumes to achieve high sensitivities in clinical practice. Finally, specificity was 92.4 % even though we enriched for comorbidities in this cohort, and specificity was 95.7% in controls with a clean colon and no comorbidities.

Conclusion: Our systematic biomarker discovery and validation study identified a three-gene DNA methylation panel with superior performance in plasma compared to FOBT.

### Emerging Immunotherapy Targets and Combination Strategies to Overcome Treatment Resistance

#4934

Immunostimulatory and oncolytic properties of rotavirus can overcome resistance to immune checkpoint blockade therapy.

Tala Shekarian, Stéphane Depil, Anne-Catherine Jallas, Christophe Bergeron, Christophe Caux, Aurélien Marabelle, Sandrine Valsesia-Wittmann. _Centre Leon Berard, LYON, France_.

Immune checkpoint targeted therapies against PD-1, PD-L1 and CTLA-4 are currently revolutionizing cancer care. However, only a minority of patients generate objective tumor responses with these treatments. Therefore, new therapeutic interventions are needed to increase the immunogenicity of tumors in order to overcome the resistance to immune checkpoint blockade therapy. Pattern recognition receptors (PRR) such as toll-like receptor agonists have been shown to overcome resistance to immune checkpoint targeted therapy in pre-clinical models. Besides their intrinsic ability to stimulate PRR, the oncolytic properties of common viruses can be exploited also for the priming of anti-tumor immune responses. Several oncolytic viruses are currently in clinical development for cancer immunotherapy. However the routine implementation of these therapies is limited by the ongoing regulations on GMOs. With the aim to analyze if anti-infectious vaccines can be used as a source or PRR agonists and/or oncolytic viruses, we infected different tumors cell lines with commercially available anti-infectious vaccines. We first confirmed that commercially available vaccines do have PRR agonist properties. More interestingly, we discovered that rotavirus vaccines also have oncolytic properties. These attenuated viruses can directly kill cancer cells with features of immunogenic cell death such as upregulation of calreticulin on dying cancer cells but doesn't have cytotoxicity on healthy primary fibroblasts. Moreover, they have pro-inflammatory properties and can activate the NF-ΚB pathway in a toll-like receptor and IRF3 independent manner. These in vitro biological properties translate into in vivo anti-tumor activity. Intra-tumoral (IT) rotavirus therapy has anti-tumor effects which are mainly immune mediated as demonstrated by their weaken activity in NSG mice. Interestingly, in immunocompetent syngeneic murine tumor models of neuroblastoma and lymphoma, IT rotavirus therapy can overcome resistance to immune checkpoint targeted therapy and in particular synergies with anti-CTLA4. This therapeutic effect relied on specific modifications of tumor immune infiltrates and immune activation pathways. IT rotavirus vaccines was associated to an increase of myeloid infiltrating cells expressing up-regulated level of CD86 in the tumor microenvironment, and upregulation of activation markers such as OX40/CD137 on T cells. Rotavirus vaccines are clinical grade products, including for children. Therefore, in situ immunization strategies with IT attenuated rotavirus can be implemented quickly in the clinic including in pediatric cancers. IT priming of the anti-tumor immunity with oncolytic and immunostimulatory rotavirus vaccines could be a feasible strategy to overcome resistance to anti-PD-1/anti-CTLA-4 therapy in patients with cancer.* co-last authors.

#4935

High-throughput immune-oncology screen identifies EGFR inhibitors as potent enhancers of CTL antigen-specific tumor cell killing.

Patrick H. Lizotte, Troy Luster, Megan E. Cavanaugh, Luke J. Taus, Abha Dhaneshwar, Naomi Mayman, Aaron Yang, Mark Bittinger, Paul Kirschmeier, Nathanael S. Gray, David A. Barbie, Pasi A. Janne. _Dana-Farber Cancer Inst., Boston, MA_.

As immune checkpoint blocking antibodies increasing become foundational therapies for the treatment of cancer, there is a pressing need to identify compounds that synergize with checkpoint blockade as the basis of combinatorial treatment regimens. We have developed a screening assay in which a luciferized tumor cell line expressing a model antigen is co-cultured with a transgenic CD8+ T cell specifically recognizing the model antigen in a H-2b-restricted manner. The target tumor cell/T cell assay was screened with a small molecule library to identify compounds that inhibit or enhance T cell-mediated killing of tumor cells in an antigen-dependent manner. The EGFR inhibitor Erlotinib was the top hit that enhanced T cell killing of tumor cells. Subsequent experiments with Erlotinib and additional EGFR inhibitors validated the screen result. EGFR inhibitors increase both basal and IFN-γ-induced antigen processing and presentation of MHC class-I, which enhanced recognition and lysis by CD8+ cytotoxic T lymphocytes. The tumor cell line was also transduced to constitutively express Cas9, and a pooled CRISPR screen utilizing the same target tumor cell/T cell assay identified sgRNAs targeting EGFR as sensitizing tumor cells to T cell-mediated killing. Combination of PD-1 blockade with EGFR inhibition showed significant synergistic efficacy in the MC38 syngeneic colon cancer model that was superior to PD-1 blockade or EGFR inhibition alone, further validating EGFR inhibitors as immunomodulatory agents that enhance PD-1 checkpoint blockade. This novel target tumor cell/T cell assay can be screened in high-throughput with small molecule libraries and genome-wide CRISPR/Cas9 libraries to identify both compounds AND target genes, respectively, that enhance or inhibit T cell recognition and killing of tumor cells.

#4936

Combination CD40 agonist and PD-1 antagonist antibody therapy enhances vaccine induced T cell responses in non-immunogenic cancers.

Hayley S. Ma,1 Evanthia Roussos Torres,1 Bibhav Poudel,1 Tara Robinson,1 Brian Christmas,1 Kayla Cruz,1 Skylar Woolman,1 Christine Rafie,1 Blake Scott,1 Valerie Wall,2 Todd Armstrong,1 Elizabeth Jaffee1. 1 _Johns Hopkins University School of Medicine, Baltimore, MD;_ 2 _Benaroya Research Institute at Virginia Mason, Seattle, WA_.

A hallmark of many non-immunogenic cancers is the lack of tumor infiltrating lymphocytes (TIL) and/or failure to mount a robust anti-tumor T cell response via multiple mechanisms. The presence of T regulatory cells and myeloid derived suppressor cells (MDSCs) serve to dampen the immune response, and furthermore, tumor antigen-specific T cell tolerance limits the efficacy of therapeutic cancer vaccines. CD40 signaling is critical to the decision of whether cytotoxic T lymphocytes become primed or tolerized. Administration of monoclonal CD40 agonistic antibody (Ab) has been shown to promote CD8 activation in vivo, and likely alters the myeloid component of the tumor microenvironment. Our study asks the question of whether combining a T cell inducing vaccine and PD1 inhibition with CD40 agonistic Ab can induce T cell priming and TIL activation in non-immunogenic solid malignancies. We utilized mouse models of pancreatic ductal adenocarcinoma (PDAC) and breast cancer to assess the effects of drug combinations on intratumoral immune responses. Tumor-bearing mice were treated with a GM-CSF secreting vaccine (GVAX) + anti-PD1 Ab alone or in combination with CD40 Ab, or isotype control Ab, and monitored for survival. A separate cohort of mice were analyzed by immunohistochemistry and multi-color flow cytometry to assess T cell infiltration/activation and myeloid maturation. In a hemisplenectomy model of PDAC in which tumor cells were surgically implanted into wild-type recipients, mice treated with isotype control Abs succumbed to disease with extensive liver and peritoneal metastases at 35-70 days. GVAX + anti-PD1 Ab treatment displayed some efficacy, although 70% of mice eventually developed fatal liver metastases. In contrast, CD40 Ab was highly active, with 90% long-term survival afforded by a single administration of Ab, and mice treated with GVAX + anti-PD1 Ab + CD40 Ab had 100% survival. Similar trends in treatment efficacy were observed following subcutaneous tumor implantation of PDAC tumor cells in the lower limb. In an orthotopic model in which HER2/neu-expressing breast tumor cells were implanted into the mammary fat pad of syngeneic neu-N mice, we demonstrated delayed tumor progression and increased median survival in mice treated with GVAX + anti-PD1 Ab + CD40 Ab relative to either therapy alone. Further characterization of immune populations was carried out by high dimensional flow cytometric analysis utilizing PhenoGraph clustering and visualized by t-SNE. Changes were observed in monocytic and dendritic cell infiltration and maturation in the tumors of combination-treated mice. A significant decrease in granulocytic MDSCs was associated with response, as well as an increase in mature antigen presenting cells. In conclusion, GVAX, anti-PD1 and CD40 agonist Ab have potential synergy in modulating anti-tumor immunity in non-immunogenic cancers.

#4937

Targeting epithelial membrane protein 2 on breast tumor cells with a fusion construct containing the serine protease granzyme B.

Khalid Mohamedali,1 Shabnam Mohandessi,2 Lawrence Cheung,1 Michael G. Rosenblum,1 Madhuri Wadehra2. 1 _The University of Texas M.D. Anderson Cancer Center, Houston, TX;_ 2 _UCLA David Geffen School of Medicine, Los Angeles, CA_.

Despite significant advances in biology and medicine, the incidence and mortality due to breast cancer world-wide is still unacceptably high. Conventional anticancer therapeutics often suffer from lack of specificity, resulting in poor therapeutic indexes and substantial toxicities to normal healthy tissues. Immunoconjugates represent an innovative therapeutic approach to deliver a deadly payload to a tumor cell with more specificity and potentially less avenues for resistance than either can exert individually. However, it has been a challenge to find candidates as well as implement conjugation strategies to achieve these goals for a number of cancers. Epithelial membrane protein-2 (EMP2) is over-expressed in a number of gynecological cancers, including in 63% of invasive breast cancers and in 100% of ALDH+ breast cancer stem cells. Clinically, EMP2 is a biomarker for disease severity and progression in these cancers as its expression has been linked to characteristics of advanced disease including increased tissue invasion and accelerated growth. We have recently developed an IgG1 targeting the extracellular domain of EMP2 with single digit nM affinity. Administration of the anti-EMP2 IgG1 was successful at reducing breast cancer tumor load. As the antibody-antigen complex rapidly internalizes, we sought to improve on the efficacy of the naked antibody. Granzyme B (GrB) is a serine protease that plays a critical role in the body's defense against viral infection and tumor development by initiating the apoptotic cascade via both caspase-dependent and -independent mechanisms. Granzyme B-containing fusion proteins can circumvent MDR-1 mediated multi-drug resistance when delivered to targeted cancer cells. Accordingly, we developed novel fusion proteins composed of the anti-EMP2 backbone as the targeting moiety and granzyme B as the cytotoxic payload. The designs comprised (a) the anti-EMP2 IgG1 with GrB fused to the Fc domain through a cleavable linker, and (b) active GrB fused to the IgG heavy chain (Fc) domain containing 2 single-chain antibodies against EMP2, a format that results in a longer plasma retention time. The constructs were expressed by transient transfection in HEK293E cells and purified to homogeneity. Flow cytometry and ELISA demonstrated binding properties of the fusion protein identical to the naked antibody. The enzymatic activity of the fusion construct was similar to commercially-available GrB. The constructs were highly cytotoxic to EMP2-positive cells with superior in vitro efficacy over the naked antibody (10 nM vs 50nM, respectively), compared to EMP2-negative cells. Moreover, these agents show minimum toxicity and high anti-tumor potency. We predict that these designs may improve therapeutic outcomes for EMP2+ tumors. Research conducted, in part, by the Clayton Foundation for Research (K.M.) and by R01 CA163971 (M. W.).

#4938

OX40 agonist antibody-based combination therapy with PI3Kβ selective inhibitor enhances T cell immunity.

Weiyi Peng,1 Chunyu Xu,1 Brenda Melendez,1 Heather Jackson,2 Jodi A. McKenzi,1 Leila J. Williams,1 Yuan Chen,1 Rina M. Mbofung,1 Sara E. Leahey E. Leahey,1 Greg Lizee,1 Michael A. Davies,1 Niranjan Yanamandra,2 Patrick Hwu1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _GlaxoSmithKline, PA_.

OX40 (CD134), a tumor necrosis factor (TNF) receptor family member, plays a critical role in initiating signaling cascades required for full activation of tumor-reactive T cells. Due to its distinct mechanism of action, the use of OX40 agonist-based combinations is emerging as a novel avenue to improve the effectiveness of cancer treatments. Our recently published studies demonstrate that oncogenic activation of the PI3K pathway by loss of the expression of tumor suppressor PTEN, imposes an immunosuppressive microenvironment in favor of tumor immune evasion. These results highlight the therapeutic potential of combinational therapy using OX40 agonists and PI3K inhibitors in cancer patients. In this study, we sought to evaluate the antitumor efficacy of the combination of an OX40 agonist antibody and a PI3Kβ selective inhibitor, GSK2636771, in tumors with PTEN loss. By using a genetically engineered mouse (GEM) model of melanoma, which can spontaneously develop tumors with PTEN loss, we observed that combination therapy using anti-OX40 and GSK2636771 significantly delayed tumor growth and improved survival time of mice bearing PTEN loss tumors. This combinational treatment was also well tolerated in experimental mice. Unlike the combination of GSK2636771 and immune checkpoint blockers, this combinational treatment did not result in an increase in the number of CD8+ tumor-infiltrating T cells, but significantly enhanced the percentage of Ki67+ CD8+ T cells at the tumor site when compared to either treatment alone. These results suggest that GSK2636771 treatment can synergize with OX40 agonists to augment effector functions of tumor-reactive T cells. To further confirm this synergistic effect, we measured serum levels of 45 cytokines/chemokines in tumor-bearing mice receiving anti-OX40 alone or in combination with GSK2636771. Serum levels of CCL4, CXCL10 and IFN-γ, which are mainly produced by memory and/or effector T cells, were significantly increased in mice in the combination cohort in comparison to the monotherapy cohorts. More importantly, using a vaccine mouse model, we demonstrate that GSK2636771 in combination with anti-OX40 did not significantly impair the generation and maintenance of memory T cells. Taken together, our results suggest that the combinational approach of an OX40 agonist antibody and GSK2636771 may induce robust and durable antitumor T cell immunity. Our study also provides a rationale to explore the clinical activity of an OX40 agonist antibody in combination with GSK2636771 in cancer patients with PTEN loss tumors.

#4939

Driving natural killer cell antigen-specific targeting of cancer via next generation trivalent molecules.

Martin Felices, Behiye Kodal, Alexander J. Lenvik, Brianna Ettestad, Laura Bendzick, Caitlin Ryan, Dawn K. Schirm, Ron McElmurry, Craig E. Eckfeldt, Melissa A. Geller, Todd R. Lenvik, Daniel A. Vallera, Jeffrey S. Miller. _University of Minnesota, Minneapolis, MN_.

Natural Killer (NK) cells are components of innate immune response that are involved in tumor surveillance and clearance. Due to their ability to lyse tumor targets without prior priming they have gained some notoriety in the tumor immunotherapy setting. Although NK cells sense malignant transformation of cells through a number of stochastic receptors they posses no clonotypic receptors to drive specific recognition of tumor antigens, somewhat limiting their therapeutic applications. In order to address the antigen specificity issue we generated molecules, termed Tri-Specific Killer Engagers (TriKEs), that contain three arms: one arm contains a single chain variable fragment (scFv) that binds a potent NK cell activating receptor (CD16); one arm contains an scFv that binds a tumor antigen; and one arm contains the cytokine IL-15 to drive NK cell priming, expansion and survival. Initial pre-clinical studies with these first generation molecules showed that we could use them to target hematological malignancies (via a CD33 binding arm) both in vitro and in vivo or solid malignancies (via an EpCAM binding arm) in vitro. However, to improve on this platform we altered the molecule by swapping out the anti-CD16 scFv arm with a humanized camelid single domain antibody against CD16 (termed cam16 henceforth). This second generation cam16 TriKE platform potently enhanced NK cell activation against tumor targets and induced far greater NK cell expansion when compared to the first generation TriKE. Using our bacterial expression system we generated and isolated cam16 TriKEs against a number of different tumor antigens (CD33, EpCAM, Mesothelin and ROR1) to display the ability of the second generation TriKE platform to target a variety of cancers. To test these molecules we used flow cytometry based NK cell activation assays, where NK cells co-cultured with tumor targets +/- cam16 TriKEs are evaluated for NK cell degranulation (by expression of surface CD107a) and NK cell inflammatory cytokine production (by expression of intracellular IFNγ). We also used a real-time imaging platform, the IncuCyte Zoom, to evaluate the ability of NK cells to kill tumor targets in the presence or absence of cam16 TriKEs over a 2-3 day period. Our data indicates that cam16 TriKEs drive NK cell specific degranulation, inflammation, and killing of AML (CD33), ovarian cancer (EpCAM and Mesothelin), lung cancer (Mesothelin and EpCAM) and sarcoma (ROR1) tumor targets in vitro. Furthermore cam16 TriKE in vivo efficacy could be seen in humanized mouse models using both luciferase-labeled cell lines and patient derived xenografts. Taken together, these findings clearly highlight the translational potential of cam16 TriKEs in the tumor immunotherapy setting.

#4940

Nonredundant roles for immune checkpoint blockade and agonistic CD40 in mediating T-cell responses in pancreatic ductal adenocarcinoma.

Alexander H. Morrison,1 Katelyn T. Byrne,2 Robert H. Vonderheide3. 1 _University of Pennsylvania, Philadelphia, PA;_ 2 _Parker Institute for Cancer Immunotherapy, University of Pennsylvania, Philadelphia, PA;_ 3 _Abramson Cancer Center, Parker Institute for Cancer Immunotherapy, University of Pennsylvania, Philadelphia, PA_.

Pancreatic ductal adenocarcinoma (PDA) is a highly aggressive cancer that is resistant to most treatments, with a five-year survival rate of ~10%. Immune checkpoint blockade (ICB) has had dramatic effects in many tumor histologies, but is ineffective in PDA. CD40 lies upstream of the immune checkpoints in the T-cell activation pathway, presenting a unique opportunity to intercede with T-cell activation independently of ICB. We hypothesized that administering agonistic CD40 antibody would prime effector T-cell populations, synergizing with ICB to provide therapeutic benefit in PDA. We administered agonistic CD40 monoclonal antibody (clone FGK45, anti-CD40) combined with anti-PD1 and anti-CTLA4 monoclonal antibodies (ICB) to mice bearing subcutaneous PDA tumors established using syngeneic tumor cell lines derived from the KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) genetically engineered mouse model of PDA. While tumor regressions were rare in mice treated with ICB or anti-CD40 alone (1/7 mice in each group), 100% of mice treated with ICB + anti-CD40 experienced tumor regressions (7/7 mice). Cell depletion experiments showed early tumor regressions depended primarily on CD4 T cells, although both CD4 and CD8 T cells were required for long-term remission. 50% of mice (4/8) treated with ICB + anti-CD40 remained tumor-free (median survival 112 days) and 3/4 mice resisted tumor rechallenge, indicative of a long-lived memory T-cell response against PDA. Treatment with ICB + anti-CD40, but neither alone, increased the total intratumoral CD3+ T-cell population 3.8-fold (p<0.01), with increases in both the CD4+ and CD8+ T-cell compartments (6.7-fold and 3.3-fold, respectively, p <0.016). CD40 stimulation, but not ICB, modulated the CD4 compartment in the TME, increasing the proportion of CD43+CD11a+ antigen-specific CD4 T helpers (41 - 43% v. 10%, +/-ICB v. control, p <0.03) and reducing FoxP3+ CD4+ T regulatory cells (Tregs) 2.7 to 3.7-fold (+/- ICB, p < 0.0001). This resulted in an increased ratio of CD8 T effector cells to Tregs (7.5 to 10.7 CD8/Treg, anti-CD40 +/-ICB, p<0.01) specifically after treatment with anti-CD40, regardless of ICB addition. ICB alone increased the proportion of proliferating CD8 T cells in the tumor draining lymph node that were '"reinvigorated" Eomes+Tbet-Ki67+ (17-19% +/- CD40 v. 8% control, p <0.01), but was unable to increase the same CD8 T-cell population in the tumor. However, the addition of CD40 to ICB reduced the frequency of PD1+ CD8 T effectors in the tumor (25% v. 52% control, p < 0.05), suggesting a tumor-specific benefit of ICB on the exhausted T-cell compartment. These studies reveal the nonredundant roles that agonistic CD40 and ICB play in generating a robust T-cell response against PDA, and highlight the clinical potential of combining CD40 activation with ICB as a novel therapeutic approach in ICB-resistant tumors.

## EPIDEMIOLOGY:

### Endogenous and Exogenous Factors in Cancer Risk and Mortality

#4941

Sexually transmitted infections and risk of epithelial ovarian cancer: Results from the Nurses' Health Studies.

Renee Turzanski Fortner,1 Kathryn L. Terry,2 Noemi Bender,1 Tim Waterboer,1 Shelley S. Tworoger3. 1 _German Cancer Research Ctr.(DKFZ), Heidelberg, Germany;_ 2 _Brigham and Women's Hospital and Harvard Medical School, Boston, MA;_ 3 _Moffitt Cancer Cener, Heidelberg, FL_.

Introduction: Sexually transmitted infections (STIs) including bacterial infections Chlamydia trachomatis and Mycoplasma genitalium and infections with the human papillomavirus (HPV), and the herpes simplex virus type 2 (HSV-2), may induce persistent changes in the female genital tract increasing risk of epithelial ovarian cancer (OC). For example, C. trachomatis and M. genitalium infections are associated with pelvic inflammatory disease (PID), salpingitis, and tubal factor infertility. STI-induced tubal pathologies may be relevant to OC development given mounting evidence showing that a proportion of OCs originate in the fallopian tube. Epidemiologic data on STIs and OC risk are sparse.

Methods: A case-control study was nested in the Nurses' Health Study (NHS) and NHSII including 338 cases individually matched to 338 controls. Antibodies indicating past infection with C. trachomatis (pGP3), M. genitalium (MgPa N-Terminus + rMgPa), HPV (E6+E7 oncoproteins of types 16, 18, and 45), and HSV-2 (mgGunique) were measured using validated serological multiplex assays in pre-diagnosis plasma samples. Multivariable conditional logistic regression was used to estimate relative risks (RR) and 95% confidence intervals [CI] comparing seropositive vs. seronegative for the individual infections for OC overall, as well as invasive OC and invasive serous OC. We further cross-classified the infections, comparing women seropositive for C. trachomatis plus any other STI to women seronegative for all STIs.

Results: Study participants were median age 60 years (range: 34-81) at blood collection. The majority were parous (87%) and ever oral contraceptive users (54%), and 19% reported tubal ligation. Of the 338 OC cases, 257 were diagnosed with invasive disease and 156 were diagnosed with invasive serous disease. Cases were diagnosed median 7 years (range: 0.1-25) after blood collection. Seropositivity was observed in 20% of cases (12% of controls) for C. trachomatis, 5% (3%) for M. genitalium and 12% (9%) for HSV-2. Prevalence of infection with HPV ranged from 4% of cases (4% of controls) for HPV18 E6+E7 to 14% (14%) for HPV16 E6+E7. C. trachomatis infection was associated with increased risk of OC overall (RR: 1.98 [1.21-3.26]), invasive OC (1.81 [1.12-2.93]) and invasive serous OC (2.14 [1.23-3.75]). We observed no association between the other STIs and OC risk. Seropositivity to C. trachomatis plus any other STI was associated with a 3-fold increased risk of OC, relative to seronegativity to all infections (3.19 [1.41-7.21]).

Discussion: We provide the first large-scale epidemiologic study on common STIs and OC risk, and show a significant positive association between C. trachomatis infection and OC, and a potentially synergistic effect of infection with multiple STIs. These results suggest STI prevention efforts, including vaccine development, represent an opportunity to reduce OC risk.

#4942

**Serologic markers of infectious agents and ovarian cancer: Markers of prior** Chlamydia trachomatis **infection associated with increased ovarian cancer risk in two independent populations.**

Britton Trabert,1 Tim Waterboer,2 Sally B. Coburn,1 Louise A. Brinton,1 Mark E. Sherman,3 Jolanta Lissowska,4 Beata Peplonska,5 Patricia Hartge,1 Michael Pawlita,2 Nicolas Wentzensen1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 3 _Mayo Clinic, Jacksonville, FL;_ 4 _Cancer Center and M. Sklodowska-Curie Institute of Oncology, Poland;_ 5 _Nofer Institute of Occupational Medicine, Poland_.

BACKGROUND: Pelvic inflammatory disease (PID) has been associated with ovarian cancer and Chlamydia trachomatis (chlamydia) is the leading cause of PID in the developed world. Primary infection with chlamydia is often asymptomatic and may persist for several months/years, thus ascertainment of PID in epidemiologic studies is challenging due to pervasive misclassification and under-reporting. To improve our understanding of the role of chlamydia and other infectious agents in developing ovarian cancer, we evaluated serologic markers of infection with incident ovarian cancer using a staged approach in two independent populations.

METHODS: Studies included: 1) a case-control study conducted in Poland of 278 ovarian cancer cases diagnosed from 2000 to 2003 and age- and study site-matched controls (n=556) and 2) a nested case-control study from the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial including 160 women who eventually developed ovarian cancer and 159 matched controls. We calculated odds ratios (OR) and 95% confidence intervals (CI) for the association between serologic marker titers and ovarian cancer risk using logistic regression adjusted for matching factors and potential confounders. We evaluated laboratory cutpoints indicative of prior chlamydia infection and tested more stringent marker thresholds, identified in Poland and applied in PLCO, that could be relevant for a PID-like infection.

RESULTS: In the Polish study, positivity for the chlamydia plasmid-encoded Pgp3 protein was associated with increased ovarian cancer risk [OR (95% CI): 1.63 (1.20-2.22)]; when a positive result was redefined at a higher titer, ovarian cancer risk increased [cutpoint 2: 2.00 (1.38-2.89); cutpoint 3 (max OR): 2.19 (1.29-3.73)]. All other chlamydia markers measured were associated with increased risk at the more stringent positivity threshold in Poland. In the prospective PLCO study, Pgp3 was associated with elevated risk at the laboratory cutpoint [1.43 (0.78-2.63)]; using higher thresholds, Pgp3 was associated with increased ovarian cancer risk [cutpoint 2: 2.25 (1.07-4.71); cutpoint 3: 2.53 (0.63-10.08)]. In both studies, markers of other infections (i.e., Mycoplasma genitalium, herpes simplex virus-2, human papillomavirus, herpes simplex virus-1, polyomavirus, hepatitis B, hepatitis C, Epstein Barr virus, cytomegalovirus) measured were not associated with risk.

CONCLUSIONS: In two independent populations, the gold standard serologic marker of prior/current chlamydia infection (Pgp3) was associated with a doubling in ovarian cancer risk, whereas markers of other infectious agents were unrelated. Although these findings need to be replicated, they suggest potential ovarian cancer risk reduction through targeted treatment of chlamydia infections.

#4943

Circulating immunologic markers and risk of multiple myeloma and its precursor disease: A nested case-control study.

Jonathan N. Hofmann,1 Ola Landgren,2 Hormuzd Katki,1 Troy Kemp,3 Loredana Santo,1 Charlene McShane,4 Qing Lan,1 Nathaniel Rothman,1 Ligia Pinto,3 Ruth Pfeiffer,1 Allan Hildesheim,1 Mark Purdue1. 1 _NCI-DCEG, Bethesda, MD;_ 2 _Memorial Sloan Kettering Cancer Center, NY;_ 3 _Leidos Biomedical Research, Inc., MD;_ 4 _Queen's University, Belfast, United Kingdom_.

Objective: Severe immune dysregulation is a strong risk factor for multiple myeloma (MM), and experimental studies have implicated chemokines and pro-angiogenic cytokines in MM pathogenesis. We investigated whether circulating immune markers were prospectively associated with MM and its precursor, monoclonal gammopathy of undetermined significance (MGUS), in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial.

Methods: We characterized levels of 43 immune markers in banked sera from 243 MM cases, 506 subjects with MGUS (including those with light-chain disease) who did not progress to MM, and 258 MGUS-free controls using Luminex-based multiplex assay panels. Odds ratios (ORs) and 95% confidence intervals (CIs) for MM and MGUS were estimated using multivariate-adjusted polytomous logistic regression models.

Results: Relative to MGUS-free controls, we observed statistically significant associations with MM risk for six markers using a false discovery rate of 10%, including several chemokines (MCP-2/CCL8, fourth quartile vs first: OR 2.1; 95% CI 1.2-3.7; Ptrend=0.017 and SDF-1/CXCL12, 2.6; 1.5-4.4; Ptrend=2.9x10-4), angiogenesis markers (ANG-2, 3.0; 1.7-5.3; Ptrend=1.3x10-4 and HGF, 3.0; 1.7-5.1; Ptrend=2.8x10-5), and growth factors (EGF, 3.2; 1.9-5.5; Ptrend=1.9x10-6 and BMP-9, 2.3; 1.4-4.0; Ptrend=3.3x10-4). Associations with these markers remained after restricting to MM cases diagnosed ≥8 years after blood collection although risk estimates were somewhat attenuated (Ptrend≤0.07). MCP-2/CCL8, SDF-1/CXCL12, and HGF were also significantly elevated among those with MGUS compared with MGUS-free controls (Ptrend≤0.04). Four markers were associated with progression from MGUS to MM after adjusting for clinical characteristics such as M-protein concentration, serum free light-chain ratio, and immunoglobulin type (ANG-2, fourth quartile vs first: OR 2.5, 95% CI 1.4-4.6; HGF, 2.6, 1.4-5.0; EGF, 3.3, 1.8-6.0; and BMP-9, 1.9, 1.0-3.4); dose-response trends were statistically significant for all four markers (Ptrend≤0.03).

Conclusions: This is, to our knowledge, the first prospective study to investigate circulating immunologic markers in relation to MM risk and progression from MGUS to MM. Our findings provide insights into potential biological mechanisms associated with MM development and may have clinical utility for improving risk stratification models assessing the likelihood of progression from MGUS to clinically manifest MM.

#4944

Insulin resistance and long-term cancer-specific and all-cause mortality: The Women's Health Initiative (WHI).

Kathy Pan,1 Rebecca Nelson,2 Jean Wactawski-Wende,3 Delphine J. Lee,1 JoAnn E. Manson,4 Joanne E. Mortimer,2 Lawrence S. Phillips,5 Thomas Rohan,6 Gloria Y. Ho,7 Nazmus Saquib,8 Aladdin H. Shadyab,9 Rami Nassir,10 Jinnie J. Rhee,11 Arti Hurria,2 Rowan T. Chlebowski2. 1 _Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA;_ 2 _City of Hope National Medical Center, Duarte, CA;_ 3 _University at Buffalo, SUNY, Buffalo, NY;_ 4 _Harvard Medical School, Cambridge, MA;_ 5 _Atlanta VA Medical Center, Atlanta, GA;_ 6 _Albert Einstein College of Medicine, New York, NY;_ 7 _Feinstein Institute for Medical Research, Northwell Health, Manhasset, NY;_ 8 _College of Medicine, Sulaiman AlRajhi Colleges, Al-Qassim, Saudi Arabia;_ 9 _University of California San Diego School of Medicine, San Diego, CA;_ 10 _Umm Al-Qura University, Mecca, Saudi Arabia;_ 11 _Stanford University School of Medicine, Stanford, CA_.

Background: Elevated insulin resistance has been associated with multiple morbid conditions. To our review, only one cohort study has evaluated insulin resistance and all-cause mortality in a general population (Pyorala 2000).

Objective: To examine associations of insulin resistance with long-term cancer-specific and all-cause mortality in postmenopausal women.

Methods: Included were 22,837 postmenopausal women aged 50-79 from the WHI cohort without prior cancer and with available fasting glucose and insulin levels. Using multivariate Cox proportional hazard models, we compared cancer-specific and all-cause mortality across quartiles of insulin resistance estimated by the homeostasis model assessment-insulin resistance (HOMA-IR) index.

Results: Women in the highest HOMA-IR quartile were less educated, had higher BMI and waist circumference, were more likely to be Black, and were more likely to have hypertension than those in lower HOMA-IR quartiles. Women in the highest quartile were also significantly more likely to have a history of treated diabetes than those in lower quartiles (23.5% in Q4 versus 0.6% in Q1). After a median follow-up of 18.1 years, cancer-specific and all-cause mortality were significantly higher in the highest HOMA-IR quartile compared to the lowest (HR 1.25, 95% CI 1.07-1.46 and HR 1.62, 95% CI 1.50-1.75, respectively) (Table 1). In a sensitivity analysis excluding women with treated diabetes (remaining n=21,104), all-cause mortality findings were similar (HR 1.36, 95% CI 1.25-1.48, p<0.0001). Specific causes of the 7,194 total deaths were cancer (1,786, 25%), cardiovascular disease (2,458, 34%), Alzheimer's/dementia (549, 8%), other (2,041, 28%), and unknown (360, 5%).

Conclusion: Insulin resistance by HOMA-IR index identifies a substantial, previously under-recognized population of postmenopausal women at increased risk for cancer-specific and all-cause mortality who could potentially benefit from early intervention.

Table 1. Adjusted* HRs for cancer-specific and all-cause mortality by HOMA-IR quartiles

---

|  | |  | |

HOMA-IR Quartiles | N | Cancer Specific Mortality

HR (95% CI)  | P value | All Cause Mortality

HR (95% CI)  | P value

Q1: 0.04-1.08 | 5707 | 1.0 (reference) | 0.0431 | 1.0 (reference) | <0.0001

Q2: 1.09-1.76 | 5713 | 1.11 (0.97-1.28) | |

1.13 (1.05-1.21)

|

Q3: 1.77-3.02 | 5716 | 1.17 (1.01-1.36) | |

1.20 (1.11-1.29)

|

Q4: 3.03-403 | 5701 | 1.25 (1.07-1.46) | |

1.62 (1.50-1.75)

|

*Adjusted for education level, BMI, age at menopause, parity, alcohol consumption, and Gail score

#4945

Absolute risk prediction models for pancreatic cancer.

Jihye Kim,1 Chen Yuan,2 Ana Babic,2 Ying Bao,3 Lauren K. Brais,2 Marisa W. Welch,2 Meir Stampfer,1 Edward L. Giovannucci,1 Howard D. Sesso,1 JoAnn E. Manson,1 Charles S. Fuchs,4 Brian M. Wolpin,2 Peter Kraft1. 1 _Harvard T.H Chan School of Public Health, Boston, MA;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _Brigham and Women's Hospital and Harvard Medical School, Boston, MA;_ 4 _Yale Cancer Center, New Haven, CT_.

Introduction: Pancreatic cancer is one of the leading causes of cancer deaths in Western countries and has a 5-year survival rate of less than 10% as a high proportion of patients are diagnosed with advanced disease. Efforts are under way to develop biomarkers and imaging technologies for early detection. Given the low incidence of pancreatic cancer in the general population, a targeted approach that screens only those at high risk may be most effective. Here we build and evaluate prediction models for pancreatic cancer, including established lifestyle, clinical, anthropometric and genetic risk factors as well as emerging risk biomarkers.

Methods: We identified incident cases and age-matched controls from four large U.S. population-based cohort studies (Health Professionals Follow-up Study, Nurses' Health Study, Physicians' Health Study, and Women's Health Initiative Observational Study); cases were diagnosed from 1984 to 2010. Data on lifestyle, clinical, and anthropometric factors were obtained from questionnaires completed around the time of blood sample collection; all cases were diagnosed after blood collection. We used conditional logistic regression models to build a relative risk model including (1) alcohol use, body mass index (BMI), waist-to-hip ratio, height, physical activity, periodontal disease, and diabetes; (2) a weighted genetic risk score incorporating 17 common single-nucleotide polymorphisms associated with pancreatic cancer risk; and (3) circulating biomarkers, such as metabolites, proinsulin, leptin, and others. We calculated five-year absolute risks by combining this relative risk model with gender-specific SEER incidence and U.S. mortality rates. We evaluated model discrimination by calculating the area under the ROC curve (AUROC) using a 4-fold cross-validation. We assessed predictive performance by plotting the distribution of age-specific risk in the general population.

Results: We identified 503 incident cases and 1,174 matched controls. The AUROC for the final model was 0.68, which is an improvement over previously published risk models that only included a subset of the lifestyle, clinical, anthropometric, and genetic risk factors considered here (AUROCs ranging from 0.58 to 0.61 [Klein et al., PLoS One 2013]). The final model identified 1.6% of the general population who had greater than or equal to 3 times the population average risk of pancreatic cancer; this represents a four-fold increase over previously published models.

Conclusions: We derived risk prediction models for pancreatic cancer in the general population, incorporating clinical, genetic, and biomarker information. We found that the models improved discrimination over existing models and could identify a small subset of individuals at notably higher risk of pancreatic cancer. These models may be useful to identify a targeted high-risk subset of the general population who could benefit from screening for pancreatic cancer.

#4947

Aspirin use and risk of lethal prostate cancer in the Atherosclerosis Risk in Communities cohort.

Lauren M. Hurwitz,1 Corinne E. Joshu,1 John R. Barber,1 Anna E. Prizment,2 Mara Z. Vitolins,3 Miranda R. Jones,1 Aaron R. Folsom,2 Misop Han,4 Elizabeth A. Platz1. 1 _Johns Hopkins Bloomberg School of Public Health, Baltimore, MD;_ 2 _University of Minnesota School of Public Health, Minneapolis, MN;_ 3 _Wake Forest School of Medicine, Winston-Salem, NC;_ 4 _Johns Hopkins University School of Medicine, Baltimore, MD_.

Background: Aspirin is commonly used to treat pain and inflammation, and is recommended for some individuals for prevention of cardiovascular disease (CVD) and colorectal cancer. Observational studies suggest that aspirin use may also lower risk of prostate cancer. However, there is limited evidence on whether aspirin may protect against lethal prostate cancer in particular, and on whether benefits are consistent in black and white men. This study sought to determine the association between aspirin use and risk of total and lethal prostate cancer, overall and by race, among men in the Atherosclerosis Risk in Communities (ARIC) cohort.

Methods: The ARIC study enrolled individuals from four U.S. communities in 1987-89. This analysis was restricted to white and black men from this cohort who had non-missing data on aspirin use and were cancer-free at baseline. Aspirin use was assessed at four study visits (V1: 1987-89, V2: 1990-92, V3: 1993-95, V4: 1996-98). Indication for aspirin use was reported at V4. Cancer outcomes were ascertained through 2012. Cox proportional hazards regression was used to estimate cause-specific hazard ratios (HRs) and 95% confidence intervals (CIs) for total incident prostate cancer and lethal prostate cancer, defined as cancer that was advanced at diagnosis or fatal during follow-up. Models were adjusted for race, study center, year of birth, education, and family history of prostate cancer (time-fixed), and smoking status, body mass index, use of statins, diabetes, and coronary heart disease (time-varying). Stratified models and likelihood ratio tests were used to test for effect modification by race.

Results: There were 6,594 men (5,060 white, 1,534 black) included in this analysis. Aspirin use was reported by 29%, 33%, 37% and 44% of men at V1, V2, V3, and V4, respectively. Through the end of 2012, 817 total incident prostate cancers, including 97 lethal prostate cancers, were diagnosed. Aspirin use was not associated with risk of total prostate cancer (HR 1.06, 95% CI 0.91-1.23). However, aspirin use was inversely associated with risk of lethal prostate cancer (HR 0.58, 95% CI 0.35-0.95). This association was consistent among both white men (HR 0.64, 95% CI 0.36-1.13) and black men (HR 0.47, 95% CI 0.16-1.35, p-interaction=0.45). When looked at by indication for use, the inverse association with lethal prostate cancer appeared to be only among men who reported using aspirin regularly for CVD prevention (HR 0.63, 95% CI 0.27-1.50).

Conclusions: Aspirin use was inversely associated with lethal prostate cancer, but not total prostate cancer, in this study population. The association was not modified by race and was possibly restricted to men who used aspirin regularly for CVD prevention. Support: NHLBI, NCI, NPCR 

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

### A Therapeutic "About Face": Reversing Drug Resistance

#4948

MDM2 antagonism overcomes resistance to CDK4/6 inhibition in melanoma.

Anna E. Vilgelm,1 Nabil Saleh,1 Kelsie Riemenschneider,2 Lauren Slesur,2 Sheau-Chian Chen,2 Gregory D. Ayers,2 Rondi M. Kauffman,2 Mark C. Kelley,2 Douglas B. Johnson,2 Ann Richmond3. 1 _Vanderbilt University, Nashville, TN;_ 2 _Vanderbilt University Medical Center, Nashville, TN;_ 3 _Tennessee Valley Healthcare System, Nashville, TN_.

Metastatic melanoma is an aggressive disease characterized by resistance to conventional cancer treatments such as radiation and chemotherapy. While immune checkpoint blockade and BRAF inhibitor therapies are effective in many melanoma cases, a significant proportion of patients are resistant to these standard of care treatments and thus require new therapeutic approaches. We hypothesized that targeting CDK4/6 may offer hope to melanoma patients refractory to immunotherapy and BRAF inhibition, based on CDK4 being a key oncogene activated by mutations or deletions of CDKN2A gene, which are very common in melanoma (>50% of cases). However, in vivo studies using our extensive collection of melanoma patient-derived xenografts (PDX) showed that the majority of melanoma tumors are intrinsically resistant to CDK4/6 inhibition. We identified the mechanism of this resistance which was driven by cooperative activities of other CDKs expressed by tumor cells that compensated for the loss of CDK4/6 function to allow cell cycle progression through G1 and S phases. In order to overcome compensatory activation of other CDKs, we used small molecule antagonists of MDM2-p53 interaction, that stabilize p53 protein, which, in turn, upregulates an endogenous pan-CDK inhibitor, p21. Notably, combined MDM2 and CDK4/6 antagonism resulted in greater inhibition of tumor growth than either inhibitor individually. This synergistic effect was observed in multiple melanoma PDX models and in immunocompetent mice bearing mouse melanoma tumors. In summary, our preclinical data suggest that combining CDK4/6 inhibitors with MDM2 antagonists is a promising strategy for the treatment of metastatic melanoma, which should be further tested by a clinical study.

#4949

The role of symmetric cell division in post-therapy glioma-initiating cell expansion.

Shivani Baisiwala, Robert Hall, Louisa Warnke, Anne Christensen, Jack Shireman, Cheol H. Park, Atique U. Ahmed. _Northwestern University, Chicago, IL_.

The purpose of this study is to elucidate the mechanism by which LNX1 regulates Notch1 to increase symmetric self-renewal of glioma-initiating cells (GICs) and thereby to expand the GIC population in glioblastoma (GBM) during therapy. GBM is an aggressive primary malignancy of the brain with an almost 100% recurrence rate. Our lab and others have shown that GBM contains a subpopulation of GICs that may drive the therapeutic resistance capabilities of recurrent GBM. We have shown that the GIC population expands following administration of temozolomide (TMZ), the most commonly used drug for antiglioma chemotherapy. While it has been suggested that this expansion relies solely on Darwinian selection, we and others have demonstrated that cellular plasticity-driven expansion of therapeutically resistant GICs post-therapy may play a role in disease recurrence. GICs follow the normal stem cell pattern of cell division. They can divide in three ways: (1) asymmetric division to form one GIC and one differentiated cancer cell, (2) symmetric self-renewal to form two daughter GICs, or (3) symmetric differentiation to form two cancer cells. Here, we used ImageStream flow cytometry analysis to show that symmetric self-renewal, where one GIC produces two daughter GICs, occurs at a rate of 24% in GBM cells treated with vehicle as compared to 54% following TMZ therapy (p<0.0001). Gene set expression analysis (GSEA) on GBM patient-derived xenograft (PDX) cells after treatment with vehicle or TMZ showed a statistically significant enrichment of 29 genes involved in polarized cell division after TMZ (FDRq<0.25). LNX1, a Notch1 regulator known to regulate symmetric cell division, was found to be enriched 8.7-fold in multiple GBM PDX lines (p<0.0001). Furthermore, Western blotting of multiple GBM PDX cell lines showed that Notch1 antagonist Numb is significantly downregulated and that Notch1 and Notch1 downstream markers (MAML1, Hes1, and p21) are significantly upregulated after therapy. Knocking down LNX1 reverses these changes, causing a significant downregulation of Notch1 downstream marker Hes-1 (p < .001). Furthermore, knocking down LNX1 reduced the frequency of GICs after TMZ therapy. The stem cell frequency in control cells was 1/128 as compared to 1/54 after therapy (p<0.001). In comparison, LNX1 knockdown cells had a stem cell frequency of 1/84 as compared to 1/92 after TMZ therapy (p=0.84). Finally, based on The Cancer Genome Atlas data, LNX1 mRNA expression is negatively correlated with overall survival in patients with GBM (p=0.015). Taken together, we propose that therapeutic stress-induced LNX1 expression can alter the Notch pathway, which subsequently allows symmetric self-renewal to predominate in GBM and therefore provides a potentially valuable therapeutic target to prevent GBM recurrence.

#4950

MPS1 as a novel target in endocrine- and palbociclib-resistant estrogen receptor positive breast cancer.

Joanna Nikitorowicz-Buniak,1 Sunil Pancholi,1 Nikiana Simigdala,1 Ricardo Ribas,1 Spiros Linardopoulos,1 Mitch Dowsett,2 Stephen Johnston,2 Lesley-Ann Martin1. 1 _Institute of Cancer Research, London, United Kingdom;_ 2 _Royal Marsden Hospital, London, United Kingdom_.

Background: Despite treatment, many estrogen receptor positive (ER+) breast cancer (BC) patients, relapse with de novo or acquired endocrine resistant disease. Using a kinome siRNA library screen, we identified MPS1, which is required for recruitment of the spindle assembly checkpoint complex, as strongly associated with resistance to both endocrine therapy and CDK4/6 inhibitor palbociclib, a contemporary first-line combination for advanced ER+ BC. Until now, the target population for MPS1 inhibitors has been triple negative BC. Here we show for the first time, potential efficacy of MPS1 inhibitors in endocrine resistant BC models.

Methods: A panel of ER+ BC cell lines adapted to estrogen independent growth (LTED) or after additional resistance to palbociclib (palboR), was subjected to a siRNA kinome screen targeting 709 genes. Kinases causative of resistance were identified using Z-scores. Cell viability upon MPS1 inhibition (MPS1i) with BOS172722 was assessed 2D and 3D, and the class effect confirmed with other compounds targeting MPS1. Impact of MPS1i on ER-mediated transcription was assessed using luciferase reporter assays. Effect of MPS1i on chromosomal alignment and time spent in mitosis was established by time lapse and confocal microscopy. Cell cycle and DNA content were analysed by FACS. Apoptosis was assessed using PARP cleavage. Efficacy of BOS172722 in a xenograft model of LTED was evaluated.

Results: Kinome knockdown identified MPS1 as the top common hit in LTED and palboR cell lines. Increased MPS1 was evident in MCF7-LTED at the transcript and protein level. Notably, BOS172722 significantly reduced viability of the majority of LTED and palboR cell lines tested (IC50 between 25-100nM), an effect shown to be class specific using a second MPS1i. Upon MPS1i, cells demonstrated shorter time in mitosis, aberration of cell cycle, increased mitotic errors, culminating in apoptosis. Of note, as little as 48hr exposure to BOS172722 reduced colony formation over 3 weeks suggesting early accumulation of errors is sufficient to provide a long term anti-proliferative effect. To evaluate the clinical relevance of MPS1 in ER+ BC treated with endocrine therapy, we interrogated publicly available datasets from patients treated with neoajuvant AI therapy. In the anastrozole cohort, on-treatment MPS1 expression was significantly (p<0.0001) associated with poor response to anastrozole based on a 2-week residual Ki67 score <10%. In the letrozole cohort, increased on-treatment expression of MPS1 (p=0.0118) was associated with poor response based on tumor shrinkage <50%. Finally, in a xenograft model of AI relapse, BOS172722 caused significant tumor shrinkage, compared to vehicle.

Conclusion: For the first time, we show that MPS1 inhibitors are capable of inducing mitotic aberrations and apoptosis in ER+ BC resistant to endocrine therapy and palbociclib providing a new therapeutic strategy.

#4951

Inhibiting guanylate binding protein 1 (GBP1) impedes ovarian cancer progression.

Dhanir Tailor, Vineet Kumar, Mallesh Pandrala, Angel Resendez, Sanjay V. Malhotra. _Stanford University, Palo Alto, CA_.

Ovarian cancer, the most common gynecologic malignancy, is a leading cause of cancer deaths in women. Seventy percent of patients at diagnosis present with stage III or IV disease, in which the tumor has disseminated beyond the ovaries and pelvic organs to distant sites. Despite resection and and chemotherapy, 80% of patients diagnosed with advanced epithelial ovarian cancer develop recurrent disease and overall prognosis is poor. Resistance to chemotherapeutic agents such as carboplatin and paclitaxel (taxane-based drugs) accounts for the lack of effectiveness of current therapy. Characterization of taxane-resistant tumors has shown that Class III β-tubulin (βTUB3) is significantly overexpressed in ovarian cancer as well as other cancers. Also, molecular characterization of the causes of treatment resistance revealed that βTUB3 plays a prominent role in the incorporation of guanylate binding protein 1, a conditionally expressed GTPase normally involved in the inflammatory response, as one of the critical effectors of taxane and radiotherapy resistance in ovarian cancer. Once in the cytoskeleton, GBP1 binds to pro-survival kinases such as serine/threonine-protein kinase pim-1 (PIM1) and initiates a signaling cascade that induces treatment resistance. We hypothesize that inhibiting the activity of GBP1 will restore sensitivity to taxane therapy. This is now well supported by our preliminary proof-of-concept studies. We have demonstrated that natural product-derived small molecule SU093 stabilizes GBP1 in a nonbinding conformation, resulting in the inhibition of GBP1:PIM1 interaction. The mechanistic studies using confocal microscopy showed that SU093 inhibits GBP1 by blocking its nuclear translocation and disrupts the tubulin dynamics. Cell cycle analysis showed that SU093 leads to G1 phase arrest followed by apoptotic cell death. SU093 treatment also induced pro-apoptotic protein Bax and reduced anti-apoptotic protein Bcl2. Overall, our in vitro and in vivo investigations provide a compelling foundation to develop a novel therapy to treat ovarian cancer by inhibition of GBP1.

#4952

Acquired HER2 mutations in ER+ metastatic breast cancer confer resistance to ER-directed therapies.

Utthara Nayar,1 Ofir Cohen,2 Christian Kapstad,1 Adrienne Waks,1 Seth A. Wander,1 Corrie Painter,2 Samuel Freeman,2 Priyanka Ram,1 Nicole Persky,2 Lori Marini,1 Karla Helvie,1 Nelly Oliver,1 Cynthia X. Ma,3 Eric P. Winer,1 Nancy U. Lin,1 Nikhil Wagle1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Broad Institute, Cambridge, MA;_ 3 _Washington University, St. Louis, MO_.

Despite the reduction in cancer recurrence and mortality provided by therapies that target the estrogen receptor (ER), resistant ER+ breast cancer remains the most common cause of breast cancer death. Beyond mutations in ER in 25-30% of patients treated with aromatase inhibitors, which deplete circulating estrogen, our understanding of clinical mechanisms of resistance to agents that target the ER remains incomplete. We used whole exome sequencing (WES) of metastatic biopsies to identify mechanisms of resistance in patients with ER+ metastatic breast cancer (MBC) who had developed resistance to ER-directed agents, including aromatase inhibitors, tamoxifen, and fulvestrant. We noted mutations in human epidermal growth factor receptor 2 (HER2) in metastatic biopsies from 12 patients out of 168. Of these, eight mutations had previously been described as activating. Examination of the treatment-naïve primary tumors in the five patients with activating mutants where WES from the matched treatment-naïve primary tumor was available revealed no evidence of pre-existing mutations in four of the five patients, suggesting that these four mutations were acquired under the selective pressure of ER-directed therapy. These acquired HER2 mutations were mutually exclusive with ER mutations, suggesting a distinct mechanism of resistance to ER-directed therapies. In vitro analysis through expression in ER+/HER2- cell lines confirmed that these mutations conferred estrogen independence. In addition, and in contrast to ER mutations, these HER2 mutations resulted in resistance to tamoxifen, fulvestrant, and the CDK4/6 inhibitor palbociclib. The mutants result in increased AKT and ERK phosphorylation in ER+ cell lines, while simultaneously repressing ER levels and ER-dependent transcriptional targets. One mutation observed in two patients, S653C, occurring in the transmembrane domain and not previously observed in breast cancer, was shown to likely function through constitutive dimerization, a mechanism of activation not previously described for this mutation. Resistance caused by all four mutations was overcome by combining ER-directed therapy with the irreversible HER2 kinase inhibitor neratinib, suggesting a novel effective treatment strategy in these patients. Thus, we have shown that acquired activating HER2 mutations can confer endocrine resistance in ER+/HER2- breast cancer, and that such resistance can be effectively reversed by combination therapies that include an anti-HER2 inhibitor.

#4953

Metabolic targeting of chemoresistance perturbs clonal complexity in pancreatic cancer.

Sara Loponte, Denise Corti, Sahil Seth, Edoardo Del Poggetto, I-Lin Ho, Chieh-Yuan Li, Shan Jiang, Joseph R. Marszalek, Maria Emilia Di Francesco, Giannicola Genovese, Giulio Draetta, Alessandro Carugo, Andrea Viale. _UT MD Anderson Cancer Ctr., Houston, TX_.

A major barrier to achieving durable remission and definitive cure in oncology patients is the emergence of tumor resistance, a common outcome of different disease types independent from the therapeutic approach undertaken. Patients with pancreatic ductal adenocarcinoma (PDAC) continue to have a poor prognosis despite concerted efforts to advance new drugs to the clinic. One reason for this, in PDAC and other tumors, is that tumors are constantly adapting and evolving in response to external perturbations. To better investigate tumor evolution in response to therapy we developed a new clonal tracking platform that enables the in vivo study of long term self-renewing compartments and the generation of cohorts of patient-derived xenografts in which tumors are virtually identical and maintained by the same clones (clonal replica tumors), representing a unique tool to address fundamental questions about clonal dynamics in response to pharmacological treatment. Using this novel approach we demonstrate that standard of care in pancreatic cancer, despite inducing tumor regression, has minimal effect on the clonal composition of tumors that eventually relapse. Transcriptomic and metabolic characterization of residual tumor cells in patient derived xenograft models as well as in patients after chemoradiation shows that resistant cells that contribute to tumor relapse are metabolically rewired to upregulate mitochondrial respiration (OXPHOS). Combining a novel inhibitor of oxidative phosphorylation (IACS-010759), developed at the MD Anderson Institute for Applied Cancer Science and currently in phase I clinical trial in relapsed/refractory acute myelogenous leukemia and advanced solid tumors, with standard of care drugs drastically reduces tumor clonal complexity, underscoring the promise of inhibiting mitochondrial respiration as a new therapeutic strategy to prolong patient survival by eradicating resistant clones that survive chemoradiation.

#4954

Decoding tumor microenvironment to enhance NSCLC targeted therapy.

Haichuan Hu,1 Lecia Sequist,2 Zosia Piotrowska,2 David Kodack,1 Aaron Hata,1 Matt Niederst,1 Cyril Benes,1 Jeffrey Engelman1. 1 _Massachusetts General Hosp. Cancer Ctr., Charlestown, MA;_ 2 _Massachusetts General Hosp. Cancer Ctr., Boston, MA_.

Background: Tyrosine kinase inhibitors (TKI) have yielded promising responses in non-small-cell lung cancer (NSCLC) with EGFR mutations and ALK translocations. However, these and other targeted therapies are limited by intrinsic and acquired drug resistance. The previous study from our group investigated tumor autonomous resistance mechanisms by developing patient-derived cancer models (PDCs). In this study, we aimed to decipher the non-autonomous resistance mechanisms via tumor microenvironment by developing patient-derived fibroblast (PDF) models. Method: Cancer-associated fibroblast cell lines are established directly from individual EGFR mutant NSCLC biopsies. These cell lines, as representative of each patient's tumor microenvironment, are further subjected to functional analysis. An imaging-based high-throughput platform is developed to screen for non-autonomous resistance by co-culturing PDC and PDF models in vitro. In the parallel, two independent approaches are performed to further identify mechanisms underlying the non-autonomous resistance. These include a drug screen to determine the pathway maintaining the cancer cells' survival, and a secretomic analysis on the PDFs to identify the plausible cytokine(s) responsible for the resistance. Result: By co-culturing screening, non-autonomous resistance can be found in a wide spectrum of models. The subsequent drug screen reveals both a canonical HGF dependent and novel HGF independent mechanisms contributing to EGFR TKI resistance. Both of these can be explained by the PDF's variable cytokine secretion and can be overcome by specific therapeutic combinations. Moreover, the microenvironment-driven EGFR TKI resistance has also been validated in vivo. And the prevalence of the identified cytokine is further tested in clinical specimens. Conclusion: PDFs provide a new avenue to explore non-autonomous resistance for targeted therapy. Applying this approach, we identified both the canonical HGF dependent and novel HGF independent mechanisms that putatively conferring EGFR TKI resistance. Taking EGFR TKI therapy as a paradigm, these findings will be valuable to optimize targeted therapy and to inform the design of personalized pharmaceutical interventions.

### Early Novel Drug Development

#4955

Heterogeneous nuclear ribonucleoprotein C as a novel therapeutic target for acute myeloid leukemia.

Vindhya Vijay, Amy Meacham, Jesse Terrell, Lauren Katzell, Leylah Drusbosky, Christopher Cogle. _University of Florida, Gainesville, FL_.

Acute myeloid leukemia (AML) is a highly refractory cancer despite conventional treatment. Previously, we found that blood vessels are sanctuary sites for chemo-refractory AML cells. To target AML cells sequestered in the vascular niche, we developed an in vitro co-culture assay consisting of AML cells on bone marrow- derived endothelial cells (BMECs), mimicking the chemo-protective effect of the vascular niche in the bone marrow microenvironment. Using this assay, we performed high-throughput chemical-phenotypic screening of 31 million compounds and identified a novel polyamine sulfonamide (2470-51) that selectively killed AML cells, while sparing the underlying BMECs. 2470-51 was also cytotoxic to CD34+CD38-CD123+ AML stem cells; however, the compound was non-toxic to hematopoietic stem/progenitor cells and T lymphocytes from healthy volunteers. In vivo AML patient-derived xenograft modeling further validated the efficacy of 2470-51 as a selective anti-leukemic agent compared to cytarabine (conventional control) and vehicle control. Protein target identification experiments using unbiased label-free shotgun proteomic analysis in combination with targeted selected ion monitoring revealed heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNPC) as the covalent target of 2470-51. These findings were further validated by shRNA mediated knock-down of HNRNPC (HNRNPC-kd) in AML cell lines THP-1 and K562. HNRNPC-kd significantly reduced AML cell proliferation as well as AML cell viability by inducing apoptosis, compared to scramble controls. HNRNPC-kd also significantly reduced the clonogenic potential of both AML cell lines. In contrast, normal mesenchymal/fibroblastic (HS5) and endothelial cells (BMECs) were unaffected by HNRNPC-kd, indicating the selective dependence of AML cells on hnRNPC. RNA-Seq analyses revealed alternative splice events associated with HNRNPC-kd. Functional enrichment analyses followed by pathway analyses indicated the involvement of hnRNPC in key oncogenic and cell survival pathways. Depleting hnRNPC significantly altered the post-transcriptional landscape, leading to induction of apoptosis. Clinical studies using TCGA AML datasets showed significant survival advantage associated with lower HNRNPC expression. Collectively, our data indicate that hnRNPC is a critical factor in AML and that inhibiting this splicing repressor may be a new therapeutic strategy.

#4956

Functional characterization of the ivosidenib (AG-120) and azacitidine combination in a mutant IDH1 AML cell model.

Katharine Yen,1 Vivek S. Chopra,2 Erica Tobin,1 Brian Avanzino,2 Konstantinos Mavrommatis,2 Jorge DiMartino,2 Kyle J. MacBeth2. 1 _Agios Pharmaceuticals, Inc., Cambridge, MA;_ 2 _Celgene Corporation, San Francisco, CA_.

Approximately 6-10% of patients with acute myeloid leukemia (AML) have a mutation in the metabolic enzyme isocitrate dehydrogenase 1 (IDH1), which leads to production of the oncometabolite 2-hydroxyglutarate (2-HG). Accumulation of 2-HG inhibits α-ketoglutarate-dependent enzymes, resulting in epigenetic dysregulation and a block in cell differentiation, promoting AML. Ivosidenib (IVO; AG-120) is a targeted inhibitor of the mutant IDH1 (mIDH1) enzyme in development for the treatment of AML with an IDH1 mutation. Clinical responses to IVO have been observed in mIDH1 AML, with differentiation of myeloblasts seen in responders indicative of the mechanism of action. Azacitidine (AZA) is a hypomethylating agent that has shown clinical activity in AML. We hypothesized that the combination of IVO + AZA would have a synergistic effect on the release of differentiation block in mIDH1 cells. In an in vitro erythropoetin differentiation assay, human TF-1 erythroleukemia cells expressing the IDH1-R132H mutation (TF1-IDH1R132H) were treated with vehicle, IVO alone (0.2 or 1 µM), AZA alone (0.1, 0.3, or 1 µM), or IVO + AZA concurrently. Assessments were hemoglobinization (red color change), expression of CD235a (erythroid differentiation marker) by flow cytometry, and Kruppel-like factor 1 (KLF1) and hemoglobin gene A/B (HBG) RNA levels by qRT-PCR. Apoptosis was assessed using IncuCyte ZOOM real-time imaging. AZA alone had little or no effect on hemoglobinization, but IVO alone showed dose-dependent increases in hemoglobinization. Concurrent IVO + AZA treatment had a greater effect on hemoglobinization than IVO alone. Increases in CD235a expression were seen with IVO and AZA as single agents, and expression was further increased with the combination of IVO + AZA (at AZA doses ≥0.3 µM). Expression of HBG and KLF1 RNA was not increased by single agent AZA, but was increased by single agent IVO. Concurrent IVO + AZA treatment resulted in potentiation of HBG expression (46-fold increase with IVO [0.2 µM] + AZA [1.0 µM] combination versus 25-fold and 1-fold increases for IVO [0.2 µM] and AZA [1.0 µM] alone, respectively); potentiation of KLF1 expression was less marked. IVO alone had no effect on induction of apoptosis, AZA increased apoptosis versus control, and concurrent treatment with IVO (0.1 and 0.3 µM) + AZA (1 µM) showed greater induction of apoptosis than AZA alone, suggesting potentiation. We have demonstrated that combining IVO + AZA in the TF1-IDH1R132H AML cell line enhanced cell differentiation, as measured by increases in hemoglobinization and expression of differentiation markers, and potentiated cell death, compared with either agent alone. Further exploration of the AZA + IVO combination in AML is ongoing in two clinical trials (NCT03173248 and NCT02677922).

#4957

The novel imipridone ONC212 highly synergizes with the BCL-2 inhibitor ABT-199 in AML and activates orphan receptor GPR132.

Takenobu Nii,1 Jo Ishizawa,1 Varun V. Prabhu,2 Vivian Ruvolo,1 Neel Madhukar,3 Ran Zhao,1 Hong Mu,1 Lauren Heese,1 Kensuke Kojima,4 Mathew Garnett,5 Ultan McDermott,5 Cyril Benes,6 Neil Charter,7 Sean Deacon,7 Olivier Elemento,3 Joshua Allen,2 Wolfgang Oster,2 Martin Stogniew,2 Michael Andreeff1. 1 _MD Anderson cancer center, Houston, TX;_ 2 _Oncoceutics, Inc., Philadelphia, PA;_ 3 _Institute for Computational Biomedicine Weill Cornell Medical College, New York, NY;_ 4 _Division of Hematology, Respiratory Medicine and Oncology Saga, Saga, Japan;_ 5 _Wellcome Trust Sanger Institute, Hinxton, United Kingdom;_ 6 _Massachusetts General Hospital Cancer Center, Harvard Medical School,, Boston, MA;_ 7 _DiscoverX Corporation, Fremont, CA_.

Imipridones are first-in-class anti-tumor compounds including ONC201, which has shown promising clinical activity. ONC212 was designed as a second-generation imipridone. We first confirmed the ONC212 effects in a collection of 1,088 human cancer cell lines available from the Genomic of Drug Sensitivity in Cancer Project; leukemia was identified as the most sensitive tumor type. In fact, ONC212 exerted prominent apoptogenic effects in acute myeloid leukemia (AML) cell lines and primary AML, but not normal bone marrow (BM) cells. We investigated the effects of ONC212 in vivo in an aggressive systemic AML xenograft model using OCI-AML3 cells. ONC212 markedly inhibited AML expansion and prolonged median survival (controls: 43 d, ONC212: 49 d; p = 0.0003). For in vivo functional assessment of ONC212's anti-tumor effects against leukemia stem and progenitor cells (LSPCs), we treated patient-derived xenograft (PDX) cells with ONC212 (250 nM, 36 hr) ex vivo, and then injected into recipient NSG mice. After one month, the human leukemic CD45+ cells in the peripheral blood, spleen, and BM were significantly decreased in the ONC212 treated group. The median survival was remarkably prolonged (controls: 36 d, ONC212: 82 d; p < 0.0001). These results indicate that ONC212 has anti-LSPC effects to reduce the engraftment potential. We previously demonstrated that the prototype it compound ONC201 induces apoptosis via an atypical integrated stress response (ISR; Ishizawa et al., Sci Signal, 2016). As expected, ONC212 induced the transcription factor ATF4, a key effector of ISR. Because BCL-2 is generally considered to be protective against ISR-mediated apoptosis, we hypothesized that the BCL-2 inhibitor ABT-199 could further sensitize AML cells to ONC212. Indeed, the in vitro combination of ONC212 plus ABT-199 synergistically induced apoptosis in AML cells. Furthermore, the combination showed highly significant synergistic anti-leukemia effects in vivo. The combinatorial treatment prolonged overall median survival (controls: 20 d, each agent: 21 d, the combination 30 d; p < 0.0001). Since the G-protein-coupled receptor (GPCR) dopamine receptor D2 is the putative target of ONC201, we hypothesized that ONC212 also targets GPCRs. The PathHunter β-arrestin screening discovered that ONC212 specifically activated the orphan GPCR GPR132. Consistently, the GPR132 mRNA expression was correlated with ONC212 sensitivity. On the other hand, GPR132 overexpression induced cell death in AML cells, which is consistent with previous reports implicating GPR132 as a tumor suppressor. Furthermore, ONC212 increased GPR132 mRNA expression. These results suggest that GPR132 could be a potential therapeutic target in AML. Taken together, ONC212 has potential as a novel agent for AML therapy. This study provides the first reported opportunity to therapeutically target GPR132 in oncology.

#4958

**VTP50469 is a novel, orally available menin-MLL1 inhibitor effective against** MLL **-rearranged and NPM1-mutant leukemia.**

Andrei V. Krivtsov,1 Benjamin K. Eschle,1 Matthew Witkin,2 Jayant Y. Gadrey,1 Hannah J. Uckelmann,1 Sayuri Kitajima,1 Gerard M. McGeehan,3 Scott A. Armstrong1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 3 _Syndax Pharmaceuticals, Inc., Waltham, MA_.

Inhibition of the menin(MEN) and MLL1(KMT2A) interaction reverses leukemic gene expression driven by MLL-fusion proteins and is a potential targeted therapeutic strategy in MLL-rearranged AML. Recent data show that inhibition of this interaction could also be efficacious in NPM1-mutant AML. We developed potent and selective menin-MLL1 inhibitors that are highly selective and effective in vitro and in vivo. Structure-based design yielded a potent small-molecule inhibitor of the menin-MLL1 binding interaction, VTP-49477 (Ki=12+5 pM). Human and mouse leukemia cell lines driven by MLL-fusion proteins or NPM1 mutations were selectively sensitive to VTP-49477 (IC50 of ~10 nM). Optimization of the inhibitor structure led to an orally available inhibitor, VTP-50469 (Ki=104+30 pM). Similar to VTP-49477, human cell lines and mouse primary cells carrying MLL-fusions or NPM1c-mutations were highly sensitive to treatment with VTP-50469 (IC50 ~20 nM). Mechanistically, treatment of NPM1-mutant OCI-AML3 cells and MLL-AF9 rearranged MOLM13 cells with VTP-50469 caused >20-fold reduction of menin in high-molecular-weight protein complexes as assessed by glycerol gradient sedimentation. Concordantly, VTP-50469 treatment resulted in >10-fold reduction in menin chromatin occupancy as assessed by ChIP-seq in MLL-AF4 rearranged RS4;11 and MOLM13 cells. Furthermore, treatment with either VTP50469 or VTP4777 reduced expression of common MLL-fusion target genes in RS4;11 and MOLM13 cells and also modulated gene expression in NPM1-mutant leukemia cells. Human PDX models were treated with either VTP-49477 or VTP-50469. Treatment of MLL-rearranged B-ALL and AML PDXs (n=3) with VTP-49477 for 28 days (50mg/kg, bid, IP) resulted in significant reduction of leukemia burden (median: 2-fold in BM, 3-fold in SP, and 6-fold in PB). For oral dosing, VTP-50469 was formulated in mouse chow (0.1%, total daily dose ~175mg/kg/day) and PDX models were treated with this chow for 28 days. Mice engrafted with MLL-rearranged B-ALL, MLL-rearranged AML and NPM1-mutant AML PDXs (n=5) showed dramatic reductions of human leukemia cells in peripheral blood, spleen and bone marrow as compared to control treated mice (median: 225-fold in BM, 14-fold in SP and 129-fold in PB). AML cells showed increased differentiation with elevated expression of CD11b, CD13, CD86 and loss of common MLL-fusion target gene expression. Treatment of MLL1wt and NPM1wt B-ALL and AML PDXs with VTP-50469 did not reduce leukemia burden, confirming VTP-50469 menin-MLL1 target selectivity. Treatment with VTP-50469 did not alter normal peripheral blood counts and no weight loss was observed during treatment. Therefore, at a highly effective dose, VTP-50469 does not have detectable toxicity. Based on these data, inhibition of the menin-MLL1 interaction with VTP-50469 may have beneficial activity in up to 40% of human AML and most infant leukemias.

#4959

CP-506, a next generation hypoxia-activated prodrug, as promising novel anti-cancer therapeutic.

sophie thiolloy,1 Sofie Deschoemaeker,1 Nicolas Ongenae,1 Julie Gilissen,1 Ludwig Dubois,2 Ala Yaromina,2 Amir Ashoorzadeh,3 Jeff Smaill,3 Adam Patterson,3 Philippe Lambin,2 Arne Heyerick1. 1 _Convert Pharmaceuticals, Liège, Belgium;_ 2 _Maastricht University, Maastricht, Netherlands;_ 3 _University of Auckland, Auckland, Australia_.

Hypoxia is a characteristic of many solid tumors and is defined as a low level or absence of oxygen due to an insufficient vascularization of the tumor or a transient blockage of blood vessels. Hypoxia activates a survival response within the tumor cells driving cancer progression and is associated with poor prognosis. Hypoxia-activated prodrugs (HAPs) are anti-neoplastic agents that can solely be activated in hypoxic areas allowing the targeted delivery of cytotoxic compounds in the hypoxic tumor niches. Convert Pharmaceuticals is developing CP-506, a novel generation of HAP presenting a strong bystander effect. A key step in the activation of CP-506 is its conversion by one-electron reductases to an oxygen-sensing intermediate. Under normoxic conditions, the intermediate is rapidly re-oxidized to the pro-drug. However, under severe hypoxic conditions, further reduction steps lead to the generation of the active cytotoxic metabolite, CP-506M. In an in vitro cytotoxicity assay, CP-506 significantly inhibited the viability of several tumor cell lines specifically under anoxic conditions. In spheroid models, CP-506 demonstrates a strong bystander effect i.e. the ability of the active metabolite to locally diffuse beyond the hypoxic core and induce tumor cell death. CP-506 was administered intraperitoneally at a dose of 600 mg/kg once a day for 5 consecutive days and showed a significant inhibition of tumor growth in four tumor xenograft models (triple negative breast cancer, lung and pancreatic cancers). In all tumor models, CP-506 treatment significantly increased the survival of treated mice assessed by the time to reach four times the start volume as a surrogate endpoint for survival. Different dosing regimens for CP-506 were investigated and all demonstrated a significant tumor growth inhibition compared to the vehicle group. The most profound anti-tumor effect was observed for the repeated cycles of daily consecutive administrations. Finally, no cumulative toxicity was observed for any of the repeated dosing schedules as indicated by bodyweight change. The successful development of CP-506 will require combination with treatments targeting well-oxygenated cells and the careful selection of patients predicted to benefit from a HAP treatment. To this end, we are currently identifying key parameters (presence of tumor hypoxia, expression of reductases and DNA repair capacity) in the activation of CP-506 to define predictive gene signatures in liquid biopsies enabling patient selection. Our current pre-clinical data strongly suggest that CP-506 is a potent, highly selective HAP with a favorable pharmacokinetic profile and the addition of predictive gene signatures or imaging biomarkers for patient selection holds great promise for the treatment of cancer.

#4960

First-in-class, highly BDII-selective BET family inhibitor ABBV-744 displays potent anti-tumor activity in androgen receptor positive prostate cancer models and an improved tolerability profile.

Emily J. Faivre, Denise Wilcox, Mai Ha-Bui, Paul Hessler, Vasudha Sehgal, Xin Lu, Tamar Uziel, Gaurav Mehta, Daniel H. Albert, Keith McDaniel, Warren Kati, Yu Shen. _AbbVie Inc., North Chicago, IL_.

First generation BET inhibitors, including ABBV-075, bind with nearly equimolar affinity to two highly conserved bromodomains (BDI and BDII) in the N-terminus of BRD2, BRD3, BRD4, and BRDt. These "pan" inhibitors compete with the natural acetylated lysine substrates of BDI and BDII to displace BET family proteins from chromatin. In so doing, pan-BET inhibitors generally block transcription and induce potent anti-tumor activity across a wide range of pre-clinical models. Consequently, numerous phase I clinical trials have been initiated with pan-BET inhibitors. The initial reports of responses in the leading indication of AML have been encouraging; however, thrombocytopenia and other dose limiting toxicities may prevent realization of a therapeutic dose in most solid tumor indications. We hypothesized that selective inhibition of BDII but not BDI of BET family proteins would improve tolerability while retaining efficacy. Medicinal chemistry efforts produced ABBV-744, a potent inhibitor specific for BDII of BRD2/3/4, with >250x differential binding preference for BDII over BDI and excellent drug-like properties. In striking contrast to the broad range of cell growth inhibition effected by pan BET inhibitor ABBV-075, BDII-selective ABBV-744 anti-proliferative activity was largely but not exclusively restricted to AML and androgen receptor (AR) positive prostate cancer cell line models, including models of Enzalutamide resistance. RNA-Seq and qPCR revealed that ABBV-744 was a potent and selective inhibitor of the AR transcription pathway. ChIP-Seq and ChIP-qPCR revealed a BDII-inhibitor displacement of BRD4 from AR-occupied enhancers and promoters, consistent with a BRD4 BDII-specific dependency of AR to sustain an oncogenic gene expression program. In vivo, doses of ABBV-744 at fractions of its MTD in LNCaP and MDA-PCa-2b xenograft models induced tumor growth inhibition that was comparable to that observed with ABBV-075 when dosed at its MTD. Together, these in vitro and in vivo results provide proof of concept that selective BDII BET family inhibitors may have improved tolerability relative to pan-BET inhibitors while maintaining tumor growth inhibition in AR positive prostate cancer. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.

#4961

A highly potent novel class of SRC-3 inhibitors for the treatment of uveal melanoma.

Salma Kaochar, Cristian Coarfa, Jin Wang, Bert O'Malley, Nicholas Mitsiades. _Baylor College of Medicine, Houston, TX_.

Background: Uveal melanoma (UM), the most common intraocular malignancy in adults, is uniformly refractory to all available systemic chemotherapies, and, as a result, is universally lethal when metastatic, creating an unmet need for novel, effective, targeted therapies for this orphan disease. Somatic activating mutations in G(alpha)q and G(alpha)11, present in a mutually exclusive pattern in ~80% of UMs, activate the PKC pathway and steroid receptor coactivator (SRC)-3. The latter co-localizes on chromatin and co-operates with microphthalmia-associated transcription factor (MITF), a critical transcription factor for melanocytes, to drive oncogenic signaling. While numerous studies have demonstrated the important roles of the p160 SRCs as oncogenes in many cancers, they were historically considered "undruggable", because they lack a natural ligand-binding site or enzymatic activity that can be targeted by small molecule inhibitors (SMIs). This notion was first challenged by observations from our group that natural polyphenols and the cardiac glycoside bufalin can inhibit p160 SRC function.

Methods: We recently identified a novel family of SRC SMIs with improved drug-like properties and strong potential for clinical development (Song X, et al. PNAS 2016;113(18):4970-5). We examined the effects of SI-2, the parent compound of this new class, as well as several derivatives with improved PK properties (longer half-life in the circulation), on cellular proliferation, migration, and gene expression of a panel of UM cell lines. We combined global RNA-Seq and ChIP-Seq data to dissect the critical role of SRC-3 in UM cells and the effects of SI-2 on it.

Results: G(alpha)q-mutant UM cells are exquisitely dependent on SRC-3 for proliferation/survival both in vitro and in vivo. Our novel p160 SRC SMIs exerted potent anti-proliferative activity against UM cell lines (IC50s in the low nM range). Gene expression profiling revealed that they strongly suppress genes associated with UM aggressiveness, cellular proliferation, DNA repair, and overall transcriptional programs associated with stem-like cell state. Our novel p160 SRC SMIs potently suppressed mTOR/MAPK signaling and the transcriptomic footprint of the p160 SRCs, blocked the ability of cancer cells to migrate in vitro and had potent in vivo anticancer activity against xenograft UM models.

Conclusions: We propose a 'first-in-field' approach to target the previously undruggable family of the p160 SRC oncogenes. Our preclinical studies demonstrate that our novel p160 SRC SMIs exert potent activity against UM cell lines in vitro, including against cells that are resistant to conventional therapies, at low nM concentrations that are easily achievable in mouse models. Based on these strong preliminary studies, we are confident that the class of SI-2 derivative compounds are highly promising drug candidates as p160 SRC SMIs for UM treatment.

## IMMUNOLOGY:

### Epigenetic and Metabolic Regulation of Cancer Immunity

#4962

Metabolic rewiring of macrophages by CpG stimulates anti-tumor activity that overrides CD47-resistance in pancreatic cancer.

Mingen Liu, Roddy S. O'Connor, Sophie Trefely, Nathaniel W. Snyder, Gregory L. Beatty. _University of Pennsylvania, Philadelphia, PA_.

Although macrophages commonly support cancer growth, they can be induced with anti-tumor functions - such as the ability to phagocytose and kill tumor cells. This activity is governed by stimulatory as well as inhibitory signals (e.g. CD47). The role of central carbon metabolism in determining macrophage function, however, remains poorly understood. Here, we identify CpG-activated changes in the metabolism of macrophages to be a major determinant of their phagocytic and anti-tumor capabilities, including the ability to overcome inhibitory CD47 expressed by tumor cells. In a fully syngeneic model of murine pancreatic ductal adenocarcinoma (PDAC), systemic delivery of CpG re-directed macrophages to mediate anti-tumor activity against PDAC tumor cells independent of CD47 expressed by tumor cells. The anti-tumor potential stimulated by CpG was not associated with polarization of macrophages toward classical M1 or M2 phenotypes, but rather was dependent on a hybrid state of metabolic features found in M1 and M2 macrophages. These metabolic changes required shunting of tricarboxylic acid (TCA) cycle intermediates for de novo biosynthesis of lipids and elevated fatty acid oxidation (FAO) that was supported by glutamine anaplerosis. Coordination of these processes by carnitine palmitoyltransferase 1A (CPT1A) and ATP citrate lyase (ACLY) supported the ability of macrophages to bypass the CD47 inhibitory pathway and mediate anti-tumor functions. Our findings underscore a novel role for the metabolic state of macrophages in overriding barriers to anti-tumor activity, and reveal potential targets that may enhance CD47-based therapies.

#4963

Targeting glutamine metabolism as a means of enhancing antitumor T-cell responses.

Robert Leone,1 Judson Englert,2 Im-Meng Sun,1 Min-Hee Oh,1 Jesse Alt,3 Im-Hong Sun,1 Ada J. Tam,1 Pavel Majer,4 Rana Rais,3 Barbara Slusher,3 Jonathan Powell1. 1 _Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University School of Medicine, Baltimore, MD;_ 2 _MedImmune, Gaithersburg, MD;_ 3 _Johns Hopkins Drug Discovery Program, Baltimore, MD;_ 4 _Institute of Organic Chemistry and Biochemistry, Czech Republic_.

Tumors possess specialized metabolic programming that facilitates their rapid growth. We have developed a novel glutamine antagonist (JHU-083) that not only demonstrates single-agent efficacy in a variety of mouse tumor models but also synergizes with anti-PD-1, adoptive cellular therapy (ACT) and A2aR antagonism to overcome resistance to immunotherapy and promote durable cures. Interestingly, we have observed that JHU-083 markedly enhances endogenous antitumor T-cell responses even in the absence of additional immunotherapy. Flow cytometry analyses of tumor-infiltrating lymphocyte (TIL) reveal markedly increased infiltration of CD8+ T cells with increased proliferative index (Ki67) and the generation of robust memory phenotypes in JHU-083 treated mice. Interestingly, Gene Set Enrichment Analyses (GSEA) using RNA-sequencing data revealed that CD8+ TIL from vehicle treated mice showed significantly enhanced expression of apoptotic transcriptional programs compared with TIL from mice treated with JHU-083. Consistent with flow cytometry data, GSEA showed CD8+ TILs from mice treated with glutamine antagonism expressed transcriptional programs characteristic of long-lived memory cells. To this end, vaccination experiments in mice treated with JHU-083 demonstrate that many of the phenotypic changes observed in TIL can be generated outside the TME and are due to direct effects of glutamine blockade on effector T cells. Our studies show that these transcriptional changes are associated with profound remodeling of the histone epigenetic code and correlate with decreased intracellular levels of α-ketoglutarate in response to glutamine antagonism. Supplementation with dimethyl α-ketoglutarate, a cell-permeable form of α-ketoglutarate, partially reverses phenotypic and epigenetic changes in CD8 T cells undergoing activation in the setting of glutamine antagonism. These findings are consistent with inhibition of a family of enzymes known as α-ketoglutarate dependent dioxygenases, which are responsible for a broad range of demethylation reactions, including histone and DNA demethylation. Overall, our work demonstrates that glutamine antagonism can directly inhibit tumor growth and survival while also reprogramming maladaptive antitumor T-cell responses to enhance endogenous antitumor immunity. Such observations support the development of our novel glutamine antagonists as both monotherapy and in combination with immunotherapy.

#4964

Metabolically activated macrophages mediate obesity-driven TNBC progression.

Payal Tiwari,1 Ariane Blank,1 Chang Cui,1 Kelly Schoenfelt,1 Seema A. Khan,2 Marsha R. Rosner,1 Lev Becker1. 1 _University of Chicago, Chicago, IL;_ 2 _Northwestern University, Chicago, IL_.

Triple-negative breast cancer (TNBC) patients have the poor prognosis due to their high metastatic potential and lack of targeted therapies. Emerging epidemiological data suggest that obesity is associated with increased incidence of more aggressive triple-negative breast cancer; however, mechanisms are unclear. During obesity, adipocytes within breast tissues recruit macrophages. M2-like macrophages can exacerbate TNBC progression; however, obese breast tissue is reported to be enriched with M1-like adipose tissue macrophages (ATMs), which are anti-tumorigenic. These results raise a paradox: how does obesity promote tumor progression if it activates an anti-tumorigenic macrophage phenotype?

Here we show that obesity promotes a distinct pro-inflammatory phenotype (metabolically activated macrophages; MMe) in breast ATMs, which promotes TNBC progression. We showed that ATMs isolated from obese women express cell surface markers of MMe (CD36, ABCA1) but not M1 (CD38, CD319) macrophages. We further found that pretreating TNBC cells with MMe (but not M1) derived conditioned media increase tumorsphere growth, and frequency of tumor-incidence when injected into mice in limited dilutions. Remarkably, we saw the same effect with obesity on tumor-incidence in both spontaneous and syngeneic TNBC mouse models. Moreover, blocking cytokine expression specifically in MMe using myeloid specific genetic knockout mice diminishes the effect of obesity on tumor-incidence. Interestingly, a subset of these MMe-secreted cytokines shared a common signal transducer, gp130, which led us to investigate the role of gp130 in tumors. Knocking down gp130 in cancer cells inhibits the MMe-induced stat-3 phosphorylation and tumorsphere growth in vitro, and diminishes the MMe-mediated effect of obesity on tumor-incidence in mice. Together these findings suggest that obesity-induced metabolically activated macrophages (MMe) in breast adipose tissue secrete inflammatory cytokines that promotes the TNBC tumor-progression by activating gp130 in cancer cells.

A comprehensive understanding of the signaling mechanism(s) involved in metabolic activation of mammary ATMs would enable development of targeted therapies towards this specific pro-tumorigenic macrophage phenotype, thereby leaving the immune system of cancer patients intact.

#4965

Entinostat transforms the suppressive tumor microenvironment of breast cancer and promotes survival and anti-responses when combined with checkpoint inhibition.

Evanthia T. Roussos Torres, Christine Rafie, Hayley Ma, Brian Christmas, Todd Armstrong, Elizabeth M. Jaffee. _Johns Hopkins University, Baltimore, MD_.

Checkpoint inhibition has revolutionized treatment in cancers that are naturally immunogenic by enabling infiltration of T cells into the tumor microenvironment (TME) and promoting cytotoxic signaling pathways. While this strategy has shown some efficacy in metastatic breast cancer, most breast cancers lack immunogenic potential due to a strictly sustained immunosuppressive microenvironment and a lack of tumor antigen expression and recognition. One strategy to transform the breast TME is to use epigenetic modulation to affect activation and trafficking of myeloid derived suppressor cells (MDSCs), known to alter the immunogenicity of the TME and sensitize tumors to checkpoint modulation. We show that combinations of an epigenetic agent, the histone deacetylase inhibitor entinostat (ENT), checkpoint inhibitors anti -programmed cell death protein (a-PD-1) and anti-cytotoxic T-lymphocyte-associated protein 4 (a-CTLA-4) antibodies, with and without anti-HER2 antibodies significantly improves survival and anti-tumor effects in HER-2/neu transgenic mouse models. Using fluorescence-activated cell sorting, nanostring gene expression profiling, and other functional assays, we have characterized the various types of tumor infiltrating lymphocytes and myeloid cells and their functional capabilities within primary tumors. We show that addition of ENT to checkpoint inhibition leads to significantly decreased suppressive ability of granulocytic-MDSCs into the TME. We also demonstrate an increase in CD8+ T effector cells in mice treated with combination therapy. Flow cytometric evaluation of markers of T cell activation, exhaustion, and MDSC function demonstrate significantly increased T cell activation, exhaustion, and decreased myeloid function. Nanostring gene expression profiling of both MDSCs and lymphocytes infiltrating tumors determined significant changes in immune related pathways that likely lead to our observed outcomes. In summary, addition of ENT to checkpoint inhibition significantly increases infiltration of innate and adaptive immune cells into the highly tolerant neu-N breast tumors and leads to improved survival and decreased tumor burden. Functional assays demonstrate that this drug combination promotes a less suppressive TME and allows a more robust anti-tumor effect of infiltrating cells. It is our hope that these novel findings will provide further rationale for combination therapy and improve the response rate of these immune therapies in patients with breast cancer.

#4966

The SUV39H1-H3K9me3 axis mediates colon carcinoma cell intrinsic apoptosis and immune evasion.

Chunwan Lu,1 Dafeng Yang,1 John D. Klement,1 Aaron H. Colby,2 Mark W. Grinstaff,2 Cedric Pearce,3 Nicholas H. Oberlies,4 Thomas Albers,5 Iryna Lebedyeva,5 Kebin Liu1. 1 _Medical College of Georgia, Augusta, GA;_ 2 _Boston University, MA;_ 3 _Mycosynthetix, Inc., Hillsborough, NC;_ 4 _University of North Carolina at Greensboro, Greensboro, NC;_ 5 _Augusta University, Augusta, GA_.

Human colon cancer, except for the small subset of microsatellite instable (MSI) colon cancer, does not respond to anti-PD-L1/PD-1 immune checkpoint inhibitor (ICI) immunotherapy. A well-established notion is that MSI colon cancer harbors various mutations that serve as neoantigens to generate tumor-specific cytotoxic T lymphocytes (CTLs). Because CTLs suppress tumor through inducing tumor cell apoptosis, we hypothesized that colon cancer cell intrinsic apoptosis resistance is potentially another mechanism underlying microsatellite stable (MSS) colon cancer nonresponse to ICI immunotherapy. If tumor cells are not sensitive to apoptosis induction, then tumor cells cannot be killed by CTLs regardless of how potent the CTLs are. To test this hypothesis, we analyzed MSI and MSS human colon carcinoma specimens and developed a small molecule that targets H3K9me3-mediated cell death pathways as a sensitizer for colon cancer ICI immunotherapy. As expected, we observed high level of CTL infiltration in all eight MSI colon carcinoma specimens examined. However, five of the nine MSS colon carcinoma specimens also exhibited high level of CTL infiltration, suggesting that CTL level is not the sole mechanism underlying MSS colon cancer non-response to ICI immunotherapy. Analysis of the TCGA database revealed that SUV39H1, a histone methyltransferase that catalyzes H3K9me3, is significantly elevated in human colon carcinoma specimens compared to normal human colon tissues. A SUV39H1 structure-based virtual chemical library screening in combination with functional assays identified a small-molecule SUV39H1 inhibitor. Further analysis of the top forty-three hits identified a potent SUV39H1 inhibitor. Structure modifications were then performed and a chemical synthesis procedure was developed to synthesize a novel SUV39H1 inhibitor, termed F5446. F5446 has an EC50 of 0.496 µM against SUV39H1 enzymatic activity and suppresses human colon carcinoma cell growth in a dose-dependent manner in vitro. F5446 suppresses colon carcinoma growth through upregulating key cell cycle regulators to induce cell cycle arrest at the S phase. Furthermore, F5446 also decreased H3K9me3 at the FAS promoter to upregulate Fas expression. Consequently, a sublethal dose of F5446 effectively overcame human colon carcinoma cell resistance to apoptosis induced by FasL, a ligand on activated CTLs in vitro. Treatment of colon carcinoma-bearing mice with F5446 and anti-PD-1 both significantly suppress tumor growth in vivo. Our data determined that F5446 is a novel SUV39H1 inhibitor that has the potential to be further developed as a sensitizer to overcome colon carcinoma resistance to ICI immunotherapy through activating the Fas-FasL pathway.

#4967

HDAC11 function as a transcriptional regulator in immature myeloid cells to myeloid-derived suppressor cells transition.

Jie Chen,1 Fengdong Cheng,1 Eva Sahakian,2 John Powers,2 Zi Wang,1 Alejandro Villagra,1 Javier Pinilla-Ibarz,2 Eduardo M. Sotomayor1. 1 _The George Washington University, Washington, DC;_ 2 _H.Lee Moffitt Cancer Center, Tampa, FL_.

In normal myelopoiesis, immature myeloid cells (IMCs) differentiate into macrophages, neutrophils or dendritic cells, a process that is tightly controlled by transcription factors and epigenetic regulators. However, under tumor burden, IMCs differentiate into myeloid derived suppressor cells (MDSCs) and with subsequent up-regulation of immune suppressive factors and a pro-tumor effect. In prior studies, we found that MDSCs from HDAC11 KO mice displayed an increased T-cell suppressive activity that was associated with a more aggressive tumor growth as compared to MDSCs from wild type control mice. Unlike MDSC's in which absence of HDAC11 is associated with a suppressive phenotype, T-cell lacking HDAC11 are hyper-reactive and endowed with strong antitumor activity. To assess which phenotype will be the dominant one in vivo, we performed adoptive immune cell transfer experiments of MDSC and/or T-cells from HDAC11 KO mice into C57BL/6 tumor-bearing mice. The transfer of HDAC11KO MDSCs was able to eliminate, at least partially, the anti-tumor effect elicited by the adoptive transfer of HDAC11KO T cells.

Mechanistically we have found that MDSCs lacking HDAC11 displayed up-regulation of expression and enzymatic activity of arginase 1 and Nos2, two enzymes that are crucial in regulating MDSCs suppressive function. The aberrant enzymatic activities of Arg1 and Nos2 in HDAC11KO MDSCs correlate with over-expression of the lineage-specific transcription factor C/EBPβ, which has been shown to be essential for the differentiation of functional MDSCs. Furthermore, ChIP analysis confirmed that HDAC11 is recruited to the C/EBPβ gene promoter where exerts a negative regulatory effect upon gene transcription.

Taken together, we have uncovered a previously unknown role for HDAC11 as a transcriptional regulator of MDSCs function. A better understanding of this novel role of HDAC11 in myeloid biology will lead to targeted epigenetic therapies to manipulate the suppressive effect of these immunoregulatory cells.

#4968

Tumor innate immunity primed by specific interferon stimulated endogenous retroviruses.

David A. Barbie. _Dana-Farber Cancer Inst., Boston, MA_.

Introduction: Tumor cell heterogeneity is a key determinant of cancer progression and drug resistance, which is often mediated by mesenchymal cell subpopulations. While these subclones can secrete growth factors, chemokines and cytokines, the immune signaling networks that fuel this pro-tumorigenic state remain incompletely defined. Elucidating what underlies this state would provide insights into tumor biology and inform clinical strategies to improve anti-cancer therapies.

Methods: Because of their well-defined nature, we used the phenotypically distinct H69M and H69AR Small Cell Lung Cancer (SCLC) mesenchymal subclones to uncover a novel mechanism of dysregulated innate immune signaling as compared with parental neuroendocrine H69 cells. Analysis of gene signatures across TCGA and CCLE databases, functional studies in additional cell lines, and ex vivo testing of patient-derived organotypic tumor spheroids (PDOTS) were conducted to determine the broader relevance across human cancers.

Results: We discovered a novel epigenetically regulated subclass of endogenous retroviruses (ERVs) that engages innate immune signaling in mesenchymal cancer subpopulations. Stimulated 3 Prime Antisense Retroviral Coding Sequences (SPARCS) are oriented inversely in 3'UTRs of certain interferon-inducible genes and silenced by EZH2. De-repression of these loci resulted in dsRNA generation following IFNγ exposure due to bi-directional transcription from the STAT1-activated gene promoter and the 5' LTR of the antisense ERV. We found that dsRNA sensing preferentially by MAVS fuels activation of TBK1, IRF3, and STAT1 signaling, sustaining a positive feedback loop. SPARCS induction across specific human tumors and cell lines is tightly associated with downregulation of chromatin modifying enzymes, including EZH2, a mesenchymal AXL positive cell state, and B2M and MHC class 1 antigen expression. SPARCS high tumors were marked by immune infiltration, but also exhibited multiple features of tumor

immune suppression. IFNγ treatment of PDOTS with de-repressed SPARCS markedly enhanced CXCL10 production and sensitized them to PD-1 blockade.

Conclusions: Together, these data unveil a novel subclass of ERVs whose de-repression triggers pathologic innate immune signaling in cancer, with potentially important implications for cancer immunotherapy.

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

### Metabolism: Emerging Concepts and Therapy

#4969

Autophagy modulates lipid metabolism to support Liver Kinase B1 (LKB1) - deficient lung tumor growth.

Vrushank D. Bhatt, Zhixian Hu, Xiaoyang Su, Jessie Yanxiang Guo. _Cancer Institute of New Jersey, Rutgers University, New Brunswick, NJ_.

Autophagy degrades and recycles macromolecules for cells to survive starvation. In genetically engineered mouse models (GEMMs) for human non-small cell lung cancer (NSCLC), autophagy supports Kras-driven lung tumor growth with or without Trp53. Tumor suppressor liver kinase B1 (LKB1) activates 5'-adenosine monophosphate protein kinase (AMPK) to maintain energy homeostasis. LKB1 mutations are detected in 20-30% of NSCLC, causing aggressive tumor growth and resistance to chemotherapy. Identifying novel target to improve LKB1-deficient tumor treatment is urgently needed. Using GEMMs for NSCLC with oncogenic Kras and LKB1 loss (KL), we found that autophagy deficiency increased the survival of the mice bearing Atg7-/- tumors compared to mice bearing wild-type (WT) tumors. To determine the mechanism of autophagy in supporting LKB1-deficient Kras-driven lung tumor growth, tumor derived cell lines (TDCLs) were generated from the lung tumors of these mice. We found that Atg7 null TDCLs were more sensitive to glucose and glutamine deprivation-induced cell death compared to Atg7 WT TDCLs. Atg7 null TDCLs were also more sensitive to starvation-induced cell death than Atg7 WT TDCLs, which can be rescued by glucose, glutamine, pyruvate, lactate and nucleotide supplementation. Thus, autophagy is required for KL TDCLs to tolerate metabolic stress. Furthermore, we observed that palmitate supplementation successfully rescued starvation-induced Atg7 null TDCLs death, indicating that autophagy is required to maintain free fatty acid level for cells to survive starvation. We further performed metabolomics in TDCLs in normal and starvation conditions and found that level of amino acids and intermediates for glycolysis and TCA cycle metabolism were significantly lower in Atg7 null TDCLs compared to Atg7 WT TDCLs during HBSS starvation. Surprisingly, we observed a significant increase in the levels of biotin, a precursor for fatty acid synthesis, in Atg7 null TDCLs than that in WT TDCLs, indicating that accumulation of biotin in autophagy deficient cells might be due to defective fatty acid synthesis. In support of this, we found that the lipid droplets accumulation is significantly lower in Atg7 null KL TDCLs than that in Atg7 WT cells. To further evaluate the functional consequences of autophagy-mediated lipid metabolism in supporting KL tumor growth, we treated Atg7 null and WT cells with etomoxir, an irreversible inhibitor of carnitine palmitoyltransferase-1 (CPT-1) to inhibit fatty acid oxidation, in normal and starvation conditions. We found that Atg7 null TDCLs are much more sensitive to etomoxir treatment than WT cells. Taken together, autophagy plays a critical role in supporting lipid metabolism for cells to survive metabolic stress. Thus, a combination of autophagy inhibition with interruption of lipid metabolism could be a novel therapeutic strategy to treat LKB1-deficient lung tumor.

#4970

Mitochondrial trafficking in the bone marrow microenvironment promotes bioenergetic flexibility in multiple myeloma.

Christopher R. Marlein, Rachel E. Piddock, Charlotte Hellmich, Lyubov Zaitseva, Martin J. Auger, Kristian M. Bowles, Stuart A. Rushworth. _University of East Anglia, Norwich, United Kingdom_.

Multiple myeloma (MM) is an incurable malignancy of terminally differentiated plasma cells which is highly dependent on the bone marrow microenvironment (BMM). MM tumor survival, proliferation and drug resistance is reliant on ATP production however the mechanisms by which the malignant cells generate ATP are poorly defined. In this study, we investigate the contributions of oxidative phosphorylation and aerobic glycolysis to tumor cell ATP generation within BMM. Furthermore, we address whether ATP generation within tumor cells is enabled by the transfer of mitochondria from the non-malignant stromal cells of the BMM to the malignant plasma cell.

Bone marrow was obtained from patients under approval from the UK Health Research Authority. In-vivo experiments were performed with local Animal Welfare and Ethical Review Board approval and under license from the UK Home Office.

Using Seahorse extracellular flux analysis we show that primary MM cells have high baseline rates of mitochondrial respiration compared to MM cell lines. Furthermore, mitochondrial respiration rates increased ex-vivo after co-culture with non-malignant bone marrow stromal cells (BMSC). We engrafted the malignant MM1S cell line into NSG mice and examined the mitochondrial respiration rate from isolated tumor cells compared to MM1S cells cultured in-vitro. We found increased rates of mitochondrial respiration in MM1S cells isolated from mouse BM. To determine whether the increase in tumor oxidative phosphorylation observed in the presence of micro-environment cells was a result of mitochondrial transfer from BMSC to MM, we used three methods. Firstly we stained BMSC with the mitochondrial stain MitoTracker Green and cultured these with primary MM cells. Using a combination of flow cytometry and confocal microscopy we detected MitoTracker fluorescence in the MM cells after co-culture, showing that the stained mitochondria are transferred from BMSC to MM cells in-vitro. Secondly, mitochondrial transfer was directly observed between BMSC and MM cells, visualized by the acquisition of a mCherry labeled mitochondrial protein we transfected into BMSC. Finally, using a human MM cell line engrafted into an NSG mouse xenograft model, we detected murine mitochondrial DNA in sorted human MM1S and U266 tumor cells post-transplant. Through fixed cell confocal microscopy we found that mitochondria move through tunnelling nanotubes (TNTs), and inhibition of TNT formation by treatment with cytochalasin B resulted in reduced mitochondrial transfer and tumor cell oxidative phosphorylation.

Here we show that MM cellular metabolism favors oxidative phosphorylation over glycolysis, in the setting of the BMM. This is due to mitochondrial transfer occurring between non-malignant BMSC and malignant plasma cells. This process is necessary for optimum tumor growth in-vivo and forms part of the malignant phenotype of MM.

#4971

Identification of new modulators of nucleotide metabolism and replication stress in PDAC.

Evan R. Abt, Amanda Dann, Thuc M. Le, Joe R. Capri, Chloe M. Cheng, Juna Yi, Soumya Poddar, Woosuk Kim, Timothy R. Donahue, Caius G. Radu. _UCLA, Los Angeles, CA_.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths in the United States, with an overall survival of less than one year. An improved knowledge of PDAC biology, to uncover vulnerabilities specific to cancer cells, is needed to develop more effective therapeutic options. We are investigating the intersection between three aspects of PDAC biology that can, ultimately, be developed for therapeutics: (i) cytokine signaling, with a particular focus on the metabolic effects of interferons (IFNs), which are present in the highly inflamed and dense PDAC stromal microenvironment; (ii) nucleotide metabolism, a network of tightly regulated biochemical pathways that produce deoxyribonucleotide triphosphates (dNTPs), which are required for DNA replication; and (iii) the replication stress response pathway, an intracellular signaling mechanism that is activated by perturbations in DNA replication, and has been recently shown to govern key aspects of nucleotide metabolism. We hypothesize that IFN signaling reduces the levels of already limited dNTP pools in PDAC cancer cells, which respond by initiating metabolic and signaling mechanisms that coordinately function to increase dNTP recycling and biosynthetic mechanisms while simultaneously reducing their consumption. Our results, which include integrated metabolic, transcriptomic, and proteomic analyses, indicate that IFN signaling in PDAC cells induces a switch in nucleotide metabolism from a biosynthetic to a predominantly dNTP catabolic phenotype. This switch appears to be mediated by dNTP phosphohydrolysis catalyzed by the Sterile Alpha Motif and Histidine/aspartic acid Domain-containing protein (SAMHD1). Furthermore, we have investigated the effects of IFN signaling on dNTP phosphohydrolysis to deoxyribonucleosides across a panel of PDAC cancer cells, resembling the spectrum of the human disease. We also examined the biochemical fates of the deoxyribonucleoside products of SAMHD1 using targeted LC-MS/MS metabolic tracing experiments. We have also demonstrated a role for the replication stress response kinase Ataxia Telangiectasia and Rad3-related protein (ATR) in regulating dNTP levels in PDAC cells exposed to IFN. Furthermore, we have demonstrated that ATR activity is an actionable co-dependency of IFN-exposed cells and that pharmacologic inhibition of ATR eradicates PDAC cells exposed to IFN. Collectively, these studies increase our understanding of the interplay between cell extrinsic (IFN signaling) and intrinsic (replication stress) signal transduction networks, and the regulation of nucleotide metabolism in PDAC, and uncovered critical vulnerabilities to be exploited by new therapeutic approaches against this extremely aggressive and difficult to treat malignancy.

#4972

PDK inhibition sensitizes bladder tumors to cisplatin.

Sambantham Shanmugam,1 Erika Abbott,1 Justin Penticuff,1 Dharamainder Choudhary,2 Benjamin L. Woolbright,1 John A. Taylor1. 1 _University of Kansas Medical Center, Kansas City, KS;_ 2 _University of Connecticut, Farmington, CT_.

Pyruvate Dehydrogenase Kinase-4 (PDK4) is a member of the PDK enzyme family. PDKs phosphorylate and inactivate the pyruvate dehydrogenase (PDH) complex, shunting pyruvate into the cytoplasm for oxidative metabolism, a phenomenon known as aerobic glycolysis or the Warburg Effect. This confers both growth advantages and resistance to chemotherapy. We previously noted that high grade human bladder tumors have substantial upregulation of PDK4, in the absence of other PDKs. We hypothesized PDK4 would be upregulated in bladder tumors, and inhibition of PDK4 would reduce bladder tumor growth and sensitize cells to cisplatin. We explored tested this hypothesis in cell culture and xenograft and carcinogen induced mouse models. Substantial overexpression of PDK4 (20-120 fold) was confirmed by qPCR in multiple bladder cancer cell lines as compared to UROtsa cells, a benign urothelial cell line. Treatment with dichloroacetate (DCA), a pan-specific PDK inhibitor, significantly increased PDH activity and reduced cellular proliferation in HTB-9 and HTB-5 cancer cells. In line with previous studies in other tumors, DCA also sensitized HTB-9 and HTB-5 cells to cisplatin induced cell death. Notably, this was not through enhanced apoptosis, as caspase activity did not increase, but rather co-treatment resulted in significant increases in LDH release, a marker of necrosis, and increased PI/Annexin V positive cells in DCA plus cisplatin treated cells compared to DCA or cisplatin alone. Inhibition of apoptosis with z-VAD-fmk confirmed the increased necrosis, as DCA plus cisplatin treated cells still had significant increases in cell death compared to DCA or cisplatin alone. Treatment of HTB-9 tumor xenografts with DCA or cisplatin did not reduce tumor volumes; however, co-treatment with both compounds resulted in significant reductions in viable tumor weights, along with increased TUNEL staining. As DCA is a pan-specific PDK inhibitor (PDK1-4), we also used the recently generated PS10 compound with equal specificity for PDK2 and PDK4. PS10 also significantly increased PDH activity, sensitized UM-UC3 cells to cisplatin and reduced cellular proliferation at concentrations at, and above 20µM. To further validate PDK inhibition as a therapeutic target, C57Bl/6J WT mice were treated with 0.05% BBN in the drinking water for 14 weeks to initiate tumorigenesis, and then treated with 70mg/kg PS10 daily +/- weekly[B1] ip cisplatin for 3 weeks. PS10 alone had no effect. While cisplatin reduced bladder weights, only PS10 plus cisplatin resulted in significant reduction in bladder tumor weight. Inhibition of PDKs sensitizes bladder cancers to cisplatin both in vitro and in vivo, as well as having direct anti-proliferative effects in vitro. PDK inhibition may be a useful and novel way to enhance cisplatin based therapies in bladder cancer.

#4973

MYCN mediates cysteine addiction and sensitizes to ferroptosis in cancer cells.

Frank Westermann,1 Hamed Alborzinia,2 Sina Gogolin,1 Andrés F. Flórez,1 Lena M. Brückner,1 Moritz Gartlgruber,1 Sabine Hartlieb,1 Daniel Dreidax,1 Michal Nadler-Holly,3 Matthias Ziehm,3 Chunxuan Shao,1 Matthias Selbach,3 Carlo Stresemann,4 Gernot Poschet,2 Barbara Nicke,4 Stefan Wölfl,2 Kai O. Henrich,1 Thomas Höfer1. 1 _German Cancer Research Center, Heidelberg, Germany;_ 2 _University of Heidelberg, Heidelberg, Germany;_ 3 _Max-Delbrück-Center for Molecular Medicine, Berlin, Germany;_ 4 _Bayer AG, Berlin, Germany_.

Aberrant expression of MYC family members predicts poor clinical outcome in many human cancers. Oncogenic MYC profoundly alters metabolism and mediates an antioxidant response to maintain redox balance. The purpose of the study was to analyze the interplay of oncogenic MYCN or c-MYC, referred to here as MYC(N), activity with cysteine metabolism and ferroptosis, an oxidative, non-apoptotic, and iron dependent form of regulated cell death caused by ROS-mediated massive lipid peroxidation (L-ROS), using MYC(N)-driven childhood neuroblastoma as a model.The intracellular amino acid levels at MYC(N)-high and MYC(N)-low cellular states were analyzed by HPLC. Effects on cell viability upon depletion of individual amino acids from the growth medium was tested in various cancer cell lines with regulable MYC(N). An unbiased high-throughput MYCN synthetic lethal siRNA screen was used to identify genes preferentially acting in the 'MYC(N)-high' state and protecting cells from ROS accumulation and ferroptosis. The capacity of cyst(e)ine uptake, intracellular cysteine synthesis via transsulfuration and glutathione biosynthesis was assessed in various neuroblastoma cell lines and tissues using metabolome, RNAseq, ChiP-seq and global proteome analyses. To investigate L-ROS formation at various conditions cells were stained with the lipid peroxidation sensor, C11-BODIPY, and flow cytometrically analyzed. Ferroptosis inducers (FINs) and inhibitors of transsulfuration were used to test their activity in various MYC(N)-dependent neuroblastoma cell lines and in vivo xenografts.We found that intracellular cysteine depletion in a 'MYC(N)-high' context induces cell death by ferroptosis and identified multiple points in glutathione synthesis and metabolism, particularly detoxification of L-ROS, that are vulnerable in the 'MYC(N)-high' state as compared to the 'MYC(N)-low' context. We could show that ferroptosis was dependent on MYC(N) expression and was enhanced by iron. We further demonstrated that both cystine import and intracellular cysteine synthesis via transulfuration achieved the intracellular state supportive of oncogenic MYC(N)-driven growth without endangering the cell to ferroptosis. We demonstrated the MYC(N) drives increased transsulfuration activity, rather than cysteine import, in tumor cells to maintain the cellular cysteine supply for glutathione synthesis. Our findings together with new descriptions of the ferroptotic process establish a novel functional link between oncogenic MYC(N) and ferroptosis, and imply regulation by cysteine-dependent glutathione availability. In MYCN-amplified childhood neuroblastoma, MYCN mediates resistance to ferroptosis by activating transsulfuration of methionine to cysteine. We identified enzymes and antiporter proteins crucial to ferroptotic escape, providing multiple previously unknown sites that may be acted on therapeutically.

#4974

Prospective study of untargeted urinary metabolomics and risk of lung cancer among female never-smokers in Shanghai, China.

Wei Jie Seow,1 Xiao-Ou Shu,2 Jeremy Nicholson,3 Elaine Holmes,3 Wei Hu,4 Qiuyin Cai,2 Yu-Tang Gao,5 Yong-Bing Xiang,5 Steve Moore,4 Bryan A. Bassig,4 Jason Yy Wong,4 Jinming Zhang,4 Bu-Tian Ji,4 Claire Boulange,3 Manuja Kaluarachchi,3 Kyrillos F. Adesina-Georgiadis,3 Anisha Wijeyesekera,3 Wei Zheng,2 Paul Elliot,3 Nathaniel Rothman,4 Qing Lan4. 1 _National University of Singapore, Singapore, Singapore;_ 2 _Vanderbilt University Medical Center, Nashville, TN;_ 3 _Imperial College, London, United Kingdom;_ 4 _National Cancer Institute, Bethesda, MD;_ 5 _Shanghai Cancer Institute, China_.

The lung cancer rate among never-smokers is the highest among Asian women, however its etiology and any relevant non-smoking related biomarkers are still unclear. Pre-diagnostic lung cancer-related metabolic biomarkers may provide novel insights into lung cancer mechanisms, and may contribute to the discovery of etiologic factors for the high lung cancer prevalence among Asian women. We evaluated the role of the urinary metabolome in lung cancer development among female never-smokers in China by conducting a nested case-control study of 275 lung cancer cases and 289 healthy controls from the Shanghai Women's Health Study, a prospective cohort comprised of 73,363 Chinese female never-smokers. Metabolic profiling of urinary chemical features was conducted using ultrahigh-performance liquid chromatography - tandem mass spectrometry (UPLC-MS) (39,409 spectral features) and 600 MHz 1H nuclear magnetic resonance (NMR) spectroscopy (386 features). Unconditional logistic regression models were used to estimate the odds ratios (ORs) and 95% confidence intervals (CIs) for the association between each log-transformed metabolite level and lung cancer risk, adjusting for potential confounders such as age, body mass index, history of respiratory disease and passive smoking. Spearman correlation and linear regression were used to estimate associations between the most significant metabolites and pre-diagnosis dietary factors. Three detected UPLC-MS urinary metabolites were negatively associated with lung cancer risk with a false discovery rate of less than 10%: pos_2.61_127.0382m/z (OR = 0.57, 95% CI = 0.46-0.72, P = 1.98 x 10-6), neg_2.60_369.0408m/z (OR = 0.97, 95% CI = 0.96-0.98, P = 1.36 x 10-6), and pos_2.61_184.0325n (OR = 0.55, 95% CI = 0.43-0.71, P = 4.91 x 10-6). These were strongly correlated with each other (rho > 0.65, p < 0.0001). The significant metabolite (pos_2.61_127.0382m/z) was identified as 5-methyl-2-furoic acid and was moderately correlated with self-reported dietary intake of soy (rho = 0.21, p < 0.001). In conclusion, we identified a metabolite in urine (5-methyl-2-furoic acid) that provides support for the protective association of soy-based foods on lung cancer risk that was previously observed in this population of never-smoking women. Further studies are warranted to replicate these findings.

#4975

Tumor metabolism and cognitive dysfunction in CNS lymphoma.

Lakshmi Subbaraj, Huimin Geng, Jigyasa Sharma, Marisa LaFontaine, James L. Rubenstein. _UCSF Helen Diller Family Comp. Cancer Ctr., San Francisco, CA_.

Background: The etiologic basis for neurocognitive and neuropsychological deficits in cancer patients and cancer survivors is poorly understood. Because of ongoing improvements in therapy and advances in survival, the problem of cancer-associated cognitive dysfunction is increasingly significant. While there is agreement that T2-weighted imaging abnormalities on MRI correlate with neurocognitive deficits, there has been limited insight into the neurochemical abnormalities associated with cognitive dysfunction in brain tumor patients. We are testing the hypothesis that tumor metabolism directly impairs neurotransmitter pathways and cognitive function, independent of anatomic extent of the cancer.

Methods: Our approach has been to focus on a type of brain tumor, CNS lymphoma, in which we are able to simultaneously monitor cognitive function, as assayed by repeat Mini-Mental Status Examinations (MMSE), tumor volume, as assessed by MRI, and dynamic changes in the tumor metabolic microenvironment, as characterized by quantitative measurement of tumor-associated metabolites in correlation with neurotransmitters that we hypothesize to be linked to normal cognitive function. Our initial study was to evaluate 14 subjects with CNS lymphoma that were treated on a phase I trial of the immunomodulatory agent lenalidomide. Volumetric analysis of CNS lymphomas was performed using Smartbrush Software (Brainlab) on pre-and post-therapy MRI's conducted at baseline and at monthly restaging. Metabolomic analysis of cerebrospinal fluid (CSF), using GC/MS, was conducted at baseline and at timepoints within 1 week of corresponding MRI. MMSE tests were conducted in all subjects at baseline and at corresponding monthly restaging examinations.

Results: Of 20 CSF metabolites analyzed, including 12 neurotransmitters, elevated CSF lactate correlated most strongly with impaired neurocognitive function as measured by MMSE score. (P=2.5e-6; rho= -0.66). Patients with high lactate had lower relative CSF concentration of the inhibitory neurotransmitter GABA, and higher concentrations of the excitotoxic glutamate. Notably, we determined that CSF lactate concentration more significantly correlated with lower MMSE score than size of the brain tumor, as quantified by volumetric analysis of tumor T2 hyperintensity and lesional contrast-enhancing volume.

Conclusions: To our knowledge, this is the first data linking cancer metabolism, neurotransmitter dysregulation, and neurocognitive deficits in a brain tumor patient population. We anticipate that elucidation of the mechanistic basis between tumor lactate metabolism, neurotransmitter imbalance, and neurocognitive deficits will provide potential opportunities for pharmacologic intervention to preserve neurologic function and potentially minimize cognitive and neuropsychological deficits in cancer patients.

### Understanding the Genomic Dark Matter

#4976

Small RNA sequencing of preoperative blood plasma identifies microRNA signature enabling to find pancreatic cancer patients who will not benefit from surgical resection.

Natalia Gablo,1 Vladimir Prochazka,2 Tana Machackova,1 Eva Vencovska,1 Zdenek Kala,2 Jiri Sana,1 Parwez Ahmad,1 Jaroslav Juracek,1 Marek Vecera,1 Marek Svoboda,3 Ondrej Slaby1. 1 _CEITEC - Central European Institute of Technology, Masaryk University, Brno, Czech Republic;_ 2 _Department of Surgery, University Hospital Brno, Faculty of Medicine, Masaryk University, Brno, Czech Republic;_ 3 _Masaryk Memorial Cancer Insitute, Brno, Czech Republic_.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the most common and aggressive type of pancreatic cancer, associated with limited treatment options and high mortality rates. Radical resection remains the most effective curative approach for patients in early stage of disease. However, there is a high risk of surgical morbidity/mortality, and therefore development of prognostic assays to identify those patients who will not benefit from surgical resection, present important unmet medical need. miRNAs are small, non-coding RNAs, that post-transcriptionally regulate gene expression. miRNAs are frequently deregulated under pathological conditions including PDAC and therefore present promising candidates for new prognostic biomarkers in PDAC.

Materials and methods: We analyzed preoperative plasma samples obtained from 45 PDAC patients who underwent radical tumor resection. Patients were divided into two prognostic groups: 23 patients with poor prognosis (median overall survival (OS) = 10 months) and 22 patients with good prognosis (median OS = 25 months). cDNA libraries were prepared using CleanTaq Small RNA Library Prep Kit (TriLink). The sequencing analysis were performed by Next 500/550 High Output v2 Kit - 75 cycles using the NextSeq 500 instrument (both Illumina). For miRNA mapping and analysis, an online tool Chimira was used. Obtained data were statistically evaluated using the Bioconductor edgeR and DESeq2 package.

Results: When miRNA expression profiles of the patients from good and poor prognostic group were compared, 41 miRNAs were identified to have significantly different plasma levels between the two groups (P<0.05). Out of these miRNAs, 33 were found to have significantly higher levels in plasma samples of patients with poor prognosis (e.g. mir-376a-3p, mir-6763-5p, mir-885-3p, mir-1258, mir-192-5p, mir-6751-5p, mir-891a-5p, mir-3178, mir-3173-3p, mir-378g, mir-4453, mir-6764-5p, mir-556-5p, mir-6836-5p, mir-6516-5p), and 8 miRNAs have lower levels (mir-885-3p, mir-192-5p, mir-3178, mir-8072, mir-378d, mir-99a-5p, mir-30a-5p, mir-1976) compared to those with good prognosis.

Conclusion: Our findings suggest, that there is a blood plasma miRNAs signature associated with the prognosis of PDAC patients, and after independent validation, miRNAs from this signature may allow for a prognostic stratification of PDAC patients. This work was supported by Ministry of Health of the Czech Republic, grant nr.15-33158A, 15-34553A, 15-31627A, 15-34678A, 16-31314A, 16-31765A and by grant of Czech Grant Agency nr. 16-18257S.

#4977

MIR1307 mediates pancreatic cancer resistance to FOLFIRINOX chemotherapy by affecting response to DNA damage.

Pietro Carotenuto,1 Domenico Zito,1 Maria C. Previdi,1 Maya Raj,1 Matteo Fassan,2 Andrea Lampis,1 Francesco Scalafani,3 Andrea Lanese,3 Ian Said-Huntingford,1 Jens C. Hahne,1 Kate Young,3 Ruwaida Begum,3 Zakaria Ethiar,3 Andrew Wotherspoon,3 Naureen Starling,3 Anguraj Sadanandam,1 David Cunningham,3 Ian Chau,3 Paul Workman,1 Rajesh Chopra,1 Nicola Valeri,1 Chiara Braconi1. 1 _The Institute of Cancer Research, London, United Kingdom;_ 2 _University of Padova, Padova, Italy;_ 3 _The Royal Marsden Hospital NHS Trust, London, United Kingdom_.

ntroduction: FOLFIRINOX-regime is a combination-chemotherapy that provides the best clinical benefit in pancreatic cancer (PC) patients, but is associated with severe toxicity. Aim of this study is to explore the role of miRNAs (MIR) as modulators of chemosensitivity and their potential as biomarkers of sensitivity to FOLFIRINOX-chemotherapy. Methods: High-throughput-screening (HTS) of 997 LNA-MIR-inhibitors was performed in PC cell lines (Capan1, MiaPaCa2) treated with a combination of Fluorouracil (F), Oxaliplatin (O) and Irinotecan (I) that mimics FOLFIRINOX-regime. Cell viability was monitored by CellTiter-Blue assay. Validation experiments were carried out with miRvana probes. MIR expression was assessed by TaqMan-assay. Apoptosis was measured by Flow-cytometry and western-blotting. Knock-out of microRNA was acheived by CRISPR-CAS9 in MiaPaca2 cells (MIR1307KO). Results: 41 and 84 miRNA-inhibitors enhanced FOI activity by >30% (p<0.001) in Capan1 and MiaPaCa2. These included MIR1307-inhibitor that was validated in Capan1, MiaPaCa2, Panc1, AspC1, BxPC3, and Su86.86 cell lines. The proportion of cells killed by FOI in comparison to DMSO was greater in cells transfected with MIR1307 inhibitor when compared to NEG CTRL, making the effect of this MIR specific for chemotherapy. MIR1307 was over-expressed in tumour compared to matched-adjacent tissue in 40/60 human PC cases, confirming clinical relevance. MIR1307KO cells were more sensitive to FOI than WT cells. Chemotherapy-induced apoptosis was higher in MIR1307KO cells (caspase 3/7 activity and annexin-V positivity). We observed significant upregulation of different markers of DNA damage (pH2AX2, 8OHdG, DNA breaks in Comet assay) in MIR1307KO cells treated with FOI in comparison to WT treated cells. Re-expression of MIR1307 in MIR1307KO cells could increase resistance to FOI chemotherapy and protected from FOI-induced DNA damage. Bioinformatics analysis identified MIR1307 binding-sites within a number of genes involved in the DNA-repair pathway (p<0.001, folding energy value greater than -12 Kcal/mol). Conclusions: We identified miR-1307 as a potential modulator of sensitivity to FOI-chemotherapy in PC. We showed that miR-1307 inhibition impairs the ability of PC cells to recover from chemotherapy damage and therefore enhances its activity. The assessemnt of it potential as predictive biomarker of response in PC patients is ongoing.

#4978

On circular RNAs in breast cancer.

Marcel Smid,1 Vanja de Weerd,1 Saskia Wilting,1 Anieta Sieuwerts,1 Serena Nik-Zainal,2 John Martens1. 1 _Erasmus Medical Ctr., Rotterdam, Netherlands;_ 2 _University of Cambridge, on Behalf of the BASIS consortium, Cambridge, United Kingdom_.

Introduction Circular RNA was already discovered many decades ago and circRNAs were long considered idiosyncrasies of the splicing machinery processing precursor mRNA to mature mRNA. A much more recent take on this phenomenon showed an unanticipated abundance of circRNA isoforms in human cells. In breast cancer some work has already been done on publicly available TCGA data, but this analysis has a big limitation since the RNA-seq data were prepared using a poly(A) selection step, thereby missing most circRNAs (which lack a poly(A) tail). Here we describe the identification of circRNAs in a large cohort of 348 primary breast tumors, using RNA-seq data obtained without using a poly(A) selection step in the generation of the sequence libraries. We developed a method that, in contrast to previous identification methods, does not rely on unmapped reads or known splice-junctions.

Methods RNA-seq data were generated, using total RNA and removing ribosomal RNA using Ribo Zero (Illumina, USA). Paired-end (75 bases) sequencing was performed on an Illumina HiSeq 2000. The resulting fastq files were mapped to GRCh38 using STAR (version 2.4.2a) and the resulting bam files were sorted and indexed. CircRNAs were identified directly on the bam file generated by STAR; assuming a circular RNA molecule is present, the sequence read that aligns over the crossing of the non-canonical junction would, on a linear reference, get two different mapped locations. The read-mate aligns somewhere in between these two locations with the head of the read-mate pointing toward the head of the junction read, as would be expected in a properly paired read. Finding additional read-pairs showing this configuration, but always with a breakpoint at the exact same location, strengthens the evidence for circular transcripts.

Results In our cohort of 348 primary breast cancer cases we identified 25,783 circRNAs having a junction over exons of a gene. Of these, 13,853 (54%) were identified in at least 2 samples, and 426 circRNAs were found in at least 150 samples. The top recurrent circRNA was CDR1, a known circRNA that functions as a miR-7 sponge. Other previously described circRNAs were also present in our list. Of the circRNAs with sufficient data, 668 (41%) showed a poor (R<0.2) and 210 (13%) showed a negative correlation with the expression level of the linear gene. We validated several circRNAs by PCR and sequencing, including CNOT2 and CREBBP, the former showing prognostic value, the latter a known breast cancer driver gene. Additional analyses are ongoing and will be presented at the annual meeting.

Conclusion circRNAs are an abundant RNA species in breast cancer, several of which may just be a remnant of the splicing machinery. However, several circRNAs have characteristics of being regulated and thus may have a functional role in breast cancer. Due to their structural stability, candidate circRNAs may serve as good biomarkers for diagnosis and therapy-monitoring in liquid biopsies.

#4979

Novel miRNA regulation in an early progression model of pancreatic ductal adenocarcinoma.

Nina J. Chu,1 Elizabeth M. Jaffee2. 1 _Johns Hopkins School of Medicine, Baltimore, MD;_ 2 _Johns Hopkins School of Medicine, Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD_.

Current success of immunotherapy is dependent on infiltration and function of T cells within the tumor microenvironment (TME). However, for many cancers, inflammatory and stromal cells provide formidable barriers to T cell access. Emerging data suggests that the cellular barriers to T cell access and function are regulated by both genetic and epigenetic factors. Identification of these regulators should provide new targets for enhancing immunotherapy.

The overall goal of this research is to construct a comprehensive profile of miRNA expression within the inflammatory and stromal cells that develop in the earliest pre-malignant pancreatic intraepithelial neoplasias (PanINs) in the KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) mouse model, a spontaneous model of pancreatic ductal adenocarcinomas (PDA) tumorigenesis. Specifically, we aim to investigate the functional roles of key differentially expressed miRNAs in establishing and propagating the earliest PanIN lesions within the TME via modulating the signaling between transformed ductal epithelial cells and the recruited cancer associated fibroblasts (CAFs) that comprise the majority of the desmoplastic stroma that characterizes PDA.

We conducted miRNA microarray analysis to determine the levels of 750 unique miRNAs in the pancreata of KPC mice ranging from 4 to 12 weeks of age (pre-PanIN1 to PDA). miRNA expression was quantified by Taqman miRNA OpenArrays and confirmed by qPCR analysis. Out of the 750 rodent miRNAs, 4 miRNAs (miR-21, miR-16, miR-19b, and miR-224) were significantly upregulated throughout PDA development. miR-21 and miR-224 are of particular interest for their regulation of targets in cancer promoting inflammatory pathways and epithelial-mesenchymal transition.

To investigate the roles that miR-21 and miR-224 play in the developing TME, primary KPC pancreatic ductal epithelial and fibroblast cell lines were established via fluorescence activated cell sorting (FACS) in order to perform in vitro miRNA knock-in and knock-out studies. qPCR of these primary cell lines show that miR-21 is significantly upregulated in KPC tumor cells and miR-224 is overexpressed in CAFs. miRNA fluorescence in situ hybridization (miR-FISH) was performed to examine the endogenous spatial expression of these two miRNAs throughout early TME progression. miR-FISH revealed that miR-21 is expressed at low levels in wildtype pancreata, but is highly expressed particularly in ductal epithelial cells of late stage KPC pancreata. Conversely, miR-224 is expressed by the infiltrating stromal compartment surrounding advanced lesions. Stable lentiviral inhibition of miR-21 in KPC tumor cells reduced cell proliferation and migration, whereas overexpression of miR-224 in normal pancreatic fibroblasts increased their migratory capacity. Additional studies are underway to further determine the functional roles of these miRNAs in PDA development and progression.

#4980

Regulating the regulator: Long noncoding RNAs in the p53 network in colorectal cancer.

Ritu Chaudhary, Xiao Ling Li, Ashish Lal. _NCI/NIH, Bethesda, MD_.

Thousands of long noncoding RNA (lncRNA) genes are transcribed from the human genome, yet the biology of the vast majority remains to be investigated. To identify lncRNAs that could be important, we functionally integrate lncRNAs into tumor suppressor pathways, more specifically the p53 pathway. We identified and functionally characterized two intergenic nuclear-retained lncRNAs that we named PINCR (p53 induced noncoding RNA) and PURPL (p53-upregulated regulator of p53 levels). Our results show that both PINCR (Chaudhary et al, eLife, 2017) and PURPL (Li et al, Cell Reports, 2017), function as pro-survival p53-regulated genes during DNA damage and in mouse xenografts. Mechanistically, PINCR and PURPL mediate the effects of p53 in colorectal cancer (CRC) cells by regulating p53 itself or by modulating the expression of a subset of p53 targets via interactions with unique proteins in the nucleus. Targeted deletion of PINCR in CRC cells significantly impaired G1 arrest and induced apoptosis to chemotherapeutic drugs. PINCR regulates the induction of a subset of p53 targets including BTG2, RRM2B and GPX1. Silencing these PINCR targets phenocopies the effect of loss of PINCR in response to DNA damage suggesting that they could be important downstream effectors of this lncRNA. Using CRISPR-Cas9-mediated knock-in of a single S1-tag at the 3'end of PINCR followed by an RNA pulldown approach that utilized endogenous S1-tagged PINCR, we found that PINCR associates with the enhancer region of these genes by binding to RNA-and DNA-binding protein Matrin 3 that, in turn, associates with p53. We show that PINCR is important for the recruitment of Matrin 3 to the enhancer region of these genes and for their complete p53-dependent induction after DNA damage. In addition, our unpublished data provide further insights on the molecular function of these lncRNAs in CRC. Collectively, our studies reveal critical roles of PINCR and PURPL in the p53 network and uncover unique modes by which lncRNAs can function.

#4981

Circulating mir-320 promotes immunosuppressive macrophages M2 phenotype associated with lung cancer progression.

Orazio Fortunato,1 Cristina Borzi,1 Massimo Milione,1 Giovanni Centonze,1 Davide Conte,1 Mattia Boeri,1 Massimo Moro,1 Federica Facchinetti,1 Luca Roz,1 Veronica Huber,1 Chiara Camisaschi,1 Chiara Castelli,1 Licia Rivoltini,1 Valeria Cancila,2 Claudio Tripodo,2 Ugo Pastorino,1 Gabriella Sozzi1. 1 _Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy;_ 2 _University of Palermo, Palermo, Italy_.

INTRODUCTION miRNAs play a role in the complex network of signaling between cancer cells and tumor microenvironment. We previously reported the identification of diagnostic miRNA signatures (MSC) based on 24-miRNAs in plasma samples of lung cancer patients detected by low dose computed tomography (LDCT) screening.

MATERIAL and METHODS To evaluate the potential origin of the miRNAs of the diagnostic signature, we analyzed their expression by real-time or digital PCR in both cells and conditioned medium (CM) from different cell types of the lung microenvironment as well as in plasma samples of heavy smokers and patients. Lung tissues and cell-blocks were analyzed by miRNAs in situ hybridization. Modulation of miRNAs after in vitro treatments, known to induce changes associated with cancer progression, in different cell types was assessed and correlated to changes observed in circulating miRNAs signatures.

RESULTS and DISCUSSION The analysis of 100 pre and post surgery plasma samples from 31 patients suggested a tumor-related origin of those miRNAs which significantly declined to basal levels after curative tumor resection. Other miRNAs (miR-126, miR-92a, miR-320, miR-28-3p, miR-486, miR-451, miR-16) remained deregulated afer surgery likely indicating a "host-related" origin and the persistence of a risk profile. Specific expression of mir-145 in fibroblasts, mir-126 in endothelial cells, mir-133 in skeletal muscle cells, mir-451 and 142-3p in hematopoietic cells was observed with a good degree of correlation between cellular and secreted miRNAs levels in each cell types (Pearson correlation range: 0.59-0.81). In vitro experiments comparing activated vs. resting neutrophils, showed that 17 out of the 24 miRNAs were concordantly modulated as in lung cancer patients' plasma (Pearson r=0.60, p<0.01), indicating a role of activated neutrophils in determining plasma miRNAs profile associated with lung cancer . Interestingly, we found increased miR-320 levels in cells and CM of neutrophils isolated from MSC positive compared to MSC negative subjects (fold increase: 1.7 and 2.4, respectively, p<0.05). Treatment of cultured macrophages with CM of neutrophils resulted in higher levels of the mature form of miR-320 but not of its pri-miRNA expression levels. Exogenous miR-320 induced M2 polarization, as assessed by the increase of IL-10 expression and of CD163 and CD68 positive cells. These effects of mir-320 were abolished when anti-mir-320 was transfected in recipient cells. Mir-320 over-expressing macrophages were able to stimulate cancer growth both in vitro and in vivo models. Furthermore, we demonstrated that STAT4 was a direct target of miR-320 and responsible of the M2-like switch.

CONCLUSION These findings suggest that circulating miRNAs are non cell-autonomous, may act in paracrine signalling and have a causative role in lung carcinogenesis and immunesuppression.

#4982

**Oncogenic role of** THOR **, a conserved cancer/testis long noncoding RNA.**

Yashar Niknafs,1 Yasuyuki Hosono,1 Matthew K,1 John Prensner,2 Rohit Mehra,1 Sethuramasundaram Pitchiaya,1 Jean Tien,1 Rohit Malik,3 Weibin Zhao,1 Arul Chinnaiyan1. 1 _Univ. of Michigan, Ann Arbor, MI;_ 2 _Harvard, Boston, MA;_ 3 _Bristol Meyer Squibb, NJ_.

Large scale transcriptome sequencing efforts have vastly expanded the catalog of long non-coding RNAs (lncRNAs) with varying evolutionary conservation, lineage expression, and cancer specificity. Through a recent large-scale transcriptomic analysis over 6,500 tumor RNA-seq samples, we discovered over 50,000 lncRNAs in the human genome, many of which exhibited highly evolutionarily conserved sequence patterns. Building upon the discovery of these highly conserved lncRNAs, we functionally characterized a novel ultraconserved lncRNA, THOR, which exhibits expression exclusively in testis and a broad range of human cancers. We establish THOR as the first discovered cancer/testis lncRNA, and further investigated its functional significance. THOR knockdown and overexpression in multiple cell lines and animal models alters cell or tumor growth supporting an oncogenic role. Namely, we generated CRISPR knockout cell line models, showing a definitive role for THOR in cancer progression. Additionally, given the sequence conservation of THOR through the mouse and zebrafish, we generated a zebrafish knockout model, and also a zebrafish overexpression model for THOR. Through RNA-pulldown followed by mass spectrometry, we discovered a conserved interaction of THOR with the RNA binding protein, IGF2BP1, in both human and zebrafish cells. We further show that THOR contributes to the mRNA stabilization activities of IGF2BP1. These findings are corroborated by iCLIP data for IGF2BP1. Notably, transgenic THOR knockout produced fertilization defects in zebrafish and also conferred a resistance to melanoma onset in an NRAS K61-induced zebrafish melanoma model. Likewise, ectopic expression of human THOR in zebrafish accelerated the onset of melanoma. THOR represents a novel class of functionally important cancer/testis lncRNAs whose structure and function have undergone positive evolutionary selection.

## PREVENTION RESEARCH:

### Preclinical Studies of Cancer Prevention

#4983

Intermittent dosing regimens of naproxen and aspirin inhibit azoxymethane-induced rat colon adenoma progression to adenocarcinoma and carcinoma invasion.

Altaf Mohammed,1 Naveena B. Janakiram,2 Venkateshwar Madka,2 Yuting Zhang,2 Anil Singh,2 Laura Biddick,2 Qian Li,2 Stan Lightfoot,2 Vernon E. Steele,1 Ronald Lubet,1 Mark S. Miller,1 Chen S. Suen,1 Shizuko Sei,1 Chinthalapally V. Rao2. 1 _National Cancer Institute, Rockville, MD;_ 2 _Univ. of Oklahoma Health Sciences Ctr., Oklahoma City, OK_.

Colorectal Cancer (CRC) is a major public health issue world-wide with an estimated 700,000 deaths annually. Adults 50 to 69 years of age considered high-risk for CRC can take low-dose Aspirin daily for at least 10 years to reduce their risk for CRC, according to a set of recommendations from the US Preventive Services Task Force. Naproxen is a highly efficacious CRC chemopreventive agent in animal models. Continuous/chronic usage of both drugs is limited by GI toxicity and unwanted side effects. Thus, the rationale to establish intermittent dosing regimens of Naproxen and Aspirin may provide efficacy without GI toxicity. Male F344 rats were used to establish Naproxen and Aspirin pharmacodynamic efficacy and dose-response effects. Rat (36 animals/group) colon cancers were induced by two weekly doses of azoxymethane (AOM). At the adenoma stage, rats were fed diets containing Naproxen (200 and 400 ppm) or Aspirin (700, and 1,400 ppm) either continuously, 1 week on/1 week off, or 3 weeks on/3 weeks off, or Aspirin (2,800 ppm) 3 weeks on/3 weeks off. All rats were euthanized 48 weeks after AOM treatment and assessed for efficacy, dose-response effects, and biomarkers in tumor tissues. Dietary administration of Naproxen and Aspirin did not show any overt-toxicities. Administration of 200 and 400 ppm of Naproxen inhibited colon adenocarcinoma multiplicity by 54.5% and 70.5% (p<0.0001) (continuous treatment); 53.3% and 68.4% (p<0.0001) (1 week on/1 week off); and 22.5% (p<0.03) and 61.5% (p<0.0001) (3 weeks on/3 weeks off), respectively. Importantly, inhibition of invasive colon carcinoma was reduced by 53% (p<0.0009) - >88% (p<0.0001) with different treatment regimens of Naproxen. With regard to colon adenocarcinoma multiplicity, Aspirin showed significant inhibitory effect with different treatment regimens with clear dose-response effects. Total adenocarcinomas (both invasive and non-invasive) multiplicities were suppressed by 41% (P<0.003) - 72% (p<0.0001). Particularly, Aspirin showed suppression of invasive colon adenocarcinomas by >67% (p<0.0001) - >91% (p<0.0001) with different treatment regimens. Based on the biomarkers of proliferation and apoptosis, both agents showed significant modulation of proliferative (PCNA, p21) and apoptotic markers (p53, Casp3) in colonic tumors. Transcriptomic data revealed that proinflammatory cytokines, particularly interleukins and metalloproteases, were significantly reduced in tumors of rats exposed to Aspirin and Naproxen. Overall, our results suggest that intermittent dosing with Naproxen or Aspirin demonstrated significant dose-response efficacy on the progression of adenomas to adenocarcinomas, particularly invasive carcinomas. {This work was supported by NCI-N01-CN-250026}

#4984

**Celastrol inhibits high fat diet-induced obesity and intestinal tumorigenesis in APC** Min/+ **mice by modulating gut microbes and inflammation.**

Naveena B. Janakiram,1 Venkateshwar Madka,1 Yuting Zhang,1 Nicole Stratton,1 Hima Bezawada,1 Beth Griesel,1 Altaf Mohammed,2 Ann L. Olson,1 Chinthalapally V. Rao1. 1 _Univ. of Oklahoma Health Sciences Ctr., Oklahoma City, OK;_ 2 _National Cancer Institute (NCI), Bethesda, MD_.

Obesity and inflammation play a vital role in colorectal cancer (CRC). Antiobesity agents may be beneficial for CRC prevention. Celastrol is a triterpene bioactive compound derived from Tripterygium wilfordii (TW) plant, which possesses antiobesity and anti-inflammatory properties. In the present study, we tested celastrol for its intestinal tumor inhibitory efficacy, modulation of intestinal microbiome, induction of UCP-1 in inguinal fat, and inflammation under obese conditions using APCMin/+ mouse model. For efficacy study, six-week-old male and female C57BL/6J-APCMin/+ mice (10 mice/group/gender) were fed high-fat diets (HFD; 60% Kcal fat) containing 0 and 150 ppm celastrol and a group of mice with a low-fat diet (LFD; 10% Kca fat) for 11 weeks. At termination, intestinal tumors were evaluated histologically and serum was assayed for fasting glucose, uric acid, liver enzymes (ALT, AST), triglycerides and cholesterol levels. Untreated and treated intestinal tumors were assayed for apoptosis and inflammatory markers by real-time PCR method. Results suggest that administration of LFD showed lower intestinal tumor formation by 52% (p<0.02) in males; 74% (p<0.0009) in females compared to HFD fed animals. Importantly, administration of HFD containing celastrol suppressed the intestinal polyp formation by 92% (p<0.0001) in males and 83.6% (p<0.0002) in females compared to control HFD fed mice. Also, significant inhibition of colonic tumor suppression was observed in (34%) male and (100%) female mice fed with celastrol. HFD fed animals showed 55% high-grade adenomas whereas HFD containing celastrol treatment showed 25% high-grade adenomas in colon. HFD containing celastrol treatment resulted in significantly reduced body weight gain, p<0.002, compared to HFD alone in both genders with upregulation of UCP1 protein in inguinal fat indicating increased thermogenesis. HFD celastrol treatment significantly reduced fasting glucose and triglycerides levels with an increase in uric acid with no effect on cholesterol, ALT and AST levels compared to control HFD fed mice. A sequence-based analysis of fecal microbiota of mice fed with HFD celastrol showed significantly decreased number of inflammation-causing microbes belonging to genus Prevotella (84%), Dorea (77%), Allobaculum (74%), and Aneroplasma (100%) and increased number of beneficial microbes belonging to genus Bifidobacterium (100%), Akkermansia (99.9%), Mucispirillum (94%) and Coprococcus (97%) compared to control HFD fecal samples. Celastrol treatment altered mRNA expression of IFN-γ, Ccl6, TGFβ1, and Aimp1. This is first study to report the chemopreventive properties of celastrol against the small intestinal and colonic neoplasia, modulation of gut microbiome and inflammation in APCMin/+ mice.

Stephenson Cancer Center Grant.

#4985

M4OC-Prevent: Clinical evaluation of metformin for oral cancer precision prevention.

J. Silvio Gutkind,1 Frank G. Ondrey,2 Denise Laronde,3 Miriam Rosin,4 Alfredo A. Molinolo,1 Charles Coffey,1 Beverly R. Wuertz,2 Leigha D. Rock,3 Huyen Huynh,1 H-H. Sherry Chow,5 Valerie D. Butler,5 Scott M. Lippman,1 Eva Szabo6. 1 _UCSD, Moores Cancer Center, La Jolla, CA;_ 2 _University of Minnesota, Minneapolis, MN;_ 3 _University of British Columbia, Vancouver, British Columbia, Canada;_ 4 _BC Cancer Research Centre, Vancouver, British Columbia, Canada;_ 5 _University of Arizona Cancer Center, Tucson, AZ;_ 6 _NCI, Bethesda, MD_.

Despite encouraging recent results from novel treatment options, such as immunotherapy, for head and neck squamous cell carcinoma (HNSCC), limited progress has been made in improving outcomes for most patients. Prevention and early detection are key to improving the prognosis of HNSCC. We have previously shown that persistent activation of the PI3K/mTOR signaling circuitry is the most frequent dysregulated signaling mechanism in HNSCC, and that PI3K/mTOR inhibition exerts potent antitumor activity in a large series of genetically-defined and chemically-induced HNSCC models. However, potential immunosuppression and other dose-dependent side effects raise concerns regarding long term use of PI3K/mTOR inhibitors as chemopreventive agents. The repurposed drug metformin, used for frontline treatment of type 2 diabetes, decreases mTOR signaling in HPV- and HPV+ experimental HNSCC models and displays potent chemopreventive activity in mouse oral-premalignancy models. Furthermore, two recent large retrospective case-control cohort studies involving more than 300,000 diabetic patients demonstrated a decreased HNSCC risk in patients using metformin. Therefore, we conducted a single-arm, open label phase IIa clinical trial (NCT02581137) in individuals with oral premalignant lesions (OPL; oral leukoplakia or erythroplakia) to explore the potential of metformin for HNSCC prevention. Subjects were eligible if they had an OPL at any site with dysplasia or hyperplasia not associated with mechanical factors, and were otherwise healthy, without diabetes. They underwent pre- and post-treatment clinical exam of the oral cavity with lesion measurement and biopsy, along with saliva and blood collection. Eligible participants received metformin for 12 weeks (500 mg per day for the first week, 1,000 mg per day for the second week, and then 2,000 mg per day for the remaining treatment period). 26 participants were accrued and 22 were assessable for response. The toxicity profile was consistent with the known side effect profile of metformin. The clinical response rate was 18%, lower than the 30% spontaneous regression rate documented in several published long term oral leukoplakia trials. The clinical progression rate was also 18%. The significance of the clinical response assessment was limited by the short duration of treatment (only 12 weeks). Of interest, however, the histologic response rate was 59%, including 13% complete responses, and the histologic progression rate was 18%. Most of the responses consisted of a one grade improvement in histology. Circulating and tissue biomarker analyses, the latter focused on the impact of metformin on the AMPK-mTOR signaling network, are being analyzed. The goal of our studies is to provide a mechanistic framework for the selection of patients who may benefit from metformin for precision prevention of oral cancer.

#4986

Association of sirtuins and diet in cancer development: Studying the roles of SIRT2/3.

Mohamed A. Ahmed, Carol O'Callaghan, Athanasios Vassilopulos. _Northwestern University, Chicago, IL_.

Lifestyle factors, especially diet, affects health and lifespan. Dietary restriction (DR) has

been shown as a beneficial regimen to increase longevity and protect from cancer. On the other

hand, high fat intake has been found to be associated with greater risk of several cancer types.

In this regard, several studies suggest that the beneficial effect of calorie restriction (CR) is

mediated by the increased activity of sirtuins, a family of NAD-dependent deacetylases that plays

a role in aging and age-related diseases. However, the specific role of individual family members

in the inhibitory role of CR in tumorigenesis has not been elucidated. With the exception of SIRT1,

there is no evidence for the contribution of other sirtuins in the diet-induced effects on

tumorigenesis. In this study, we investigated, for the first time, the role of SIRT2 and SIRT3, which

are found predominantly in the cytoplasm and mitochondria respectively, in this context. To do

this, Sirt2-/- and Sirt3-/- mice were crossed with p53-/- mice to generate: p53+/-, (Sirt2-/-; p53+/-),

(Sirt3-/-; p53+/-), p53-/-, (Sirt2-/-; p53-/-), and (Sirt3-/-; p53-/-) mice. Subsequently, mice with the

different genotypes were subjected to 3 dietary regimes; ad libitum control diet, 30% calorie

restriction diet (CR), and high-fat diet (HFD). As expected, CR reduced tumor incidence and

increased survival of mice wild type for both SIRT2 and SIRT3. In contrast, HFD significantly

increased body weight, tumor incidence and decreased overall survival. Interestingly, the

protective effect of CR was enhanced in (Sirt3-/-; p53-/-) compared to (Sirt3+/+; p53-/-) mice, as

evidenced by delayed tumor incidence (median age 257 days vs 189 days) and increased survival.

On the other hand, Sirt2 deletion in both p53+/- and p53-/- backgrounds abolished the protective

effect of CR. In addition, loss of Sirt2 decreased tumor incidence age (111 days vs 184 days) and

accelerated the death of p53 deficient mice on HFD. Our results highlight a new role of SIRT2 in

mediating the diet-induced effect on tumorigenesis.

#4987

Biofilm-producing sulfate-reducing bacteria suppress tumor burden in a rat model of colon cancer.

Susheel Bhanu Busi, Kara B. De Leon, Judy D. Wall, James M. Amos-Landgraf. _University of Missouri - Columbia, Columbia, MO_.

Human epidemiological and animal model studies have shown that the presence of colon cancer is associated with certain microbiota. Previously, we re-derived genetically identical embryos of the Pirc (F344/NTac-Apc+/Pirc) rat model of familial adenomatous polyposis using three strains of surrogate dams (F344/NHsd, LEW/SsNHsd, and Crl:SD), each harboring distinct gut microbiota (GM) that modulated adenoma susceptibility. Bacterial relative abundances as determined by 16S rDNA sequencing showed several taxa correlating with suppression of both tumor growth and phenotype penetrance as early as 1 month of age. One taxon associated with reduced adenoma burden was a sulfate-reducing bacterium, viz. Desulfovibrio spp. To determine if Desulfovibrio was responsible for differences in tumor burden, Pirc rats harboring complex GM were gavaged at 14 and 15 days of age with two genetic isolates of Desulfovibrio (~10^8 colony forming units, CFU): biofilm-forming DvH-MT and biofilm-deficient DvH-MO. We found that the rats stably colonized with the biofilm-forming DvH-MT strain, with the bacteria present at 4 months of age. Adenoma burden at sacrifice showed that the biofilm-competent strain significantly reduced colonic adenoma size (t-test, p<0.05), compared to the biofilm-deficient, DvH-MO-treated rats.

To understand how the biofilm-forming capacity of the DvH-MT strain modulated adenoma burden, the genomes of the DvH-MT and DvH-MO strains were sequenced and 12 variants were identified between the two strains. In the DvH-MO strain one variant in the DVU1017 type-1 secretion system ABC transporter binding protein gene was shown to be required for biofilm-formation by Desulfovibrio in vitro. This gene was inactivated in the parent DvH-MT strain and the new construct was designated JWT716. To determine the presence and proximity of Desulfovibrio to adenomas in the colon, we modified the parental DvH-MT to express the fluorescent protein, dTomato and designated the fluorescent strain as JWT733. Pirc rats were treated with the biofilm-forming, JWT733 and the biofilm-deficient, JWT716.

3-months post-treatment we confirmed via qRT-PCR the presence of the biofilm-forming, JWT733 in fecal samples. JWT716 was undetectable in the fecal samples from most rats. Using a narrow-band filter to detect dTomato, we confirmed JWT733 colonization in the treated rats by colonoscopy. At 4 months of age, the rats were sacrificed and adenoma burden was estimated. We found that the biofilm-forming Desulfovibrio suppressed tumor burden in the colon compared to rats treated with the biofilm-deficient strain (t-test, p<0.05). This implicates the role of biofilm formation and potentially sulfate-reduction by this bacterium in the suppression of tumor multiplicity. In this model of colon cancer, Desulfovibrio alone or in concert may be residing in a niche in the intestinal mucus layer creating a beneficial or protective biofilm.

#4988

PI3Kγ-deficiency protects against pancreatic tumorigenesis at the expense of diet-Induced hyperlipidemia and hepatotoxicity.

Carolina Torres, Daniel R. Principe, Alex Park, Ronald McKinney, Matthew J. Dorman, Ajay Rana, Jose Cordoba-Chacon, Paul J. Grippo. _University of Illinois, Chicago, IL_.

Pancreatic ductal adenocarcinoma (PDAC) presents a significant challenge clinically, with poor overall survival and widespread resistance to conventional therapies. Several novel molecular targets are being considered in the management of PDAC patients. To this end, as hyperactive PI3K signaling is implicated in both disease incidence and progression, the inhibition of PI3K p110γ isoform is showing early promise in vivo and has been proposed as a reasonable target for therapy. Classically, p110γ is expressed predominantly in macrophages. However, using both primary pancreatic cancer samples and established patient databases, we found that p110γ is commonly upregulated in the cancer epithelium. Further, p110γ is expressed in human cell lines, and the pharmacologic inhibition of p110γ with AS-604850 both delayed mtKRAS/mtTP53-induced acinar-to-ductal metaplasia ex vivo, and sensitized tumor cells to gemcitabine in vitro. Given the apparent importance of epithelial p110γ to the neoplastic phenotype, we next generated mice with constitutive activation of p110γ targeted to the exocrine pancreas (PI3KγCA). When combined with a KRAS activating mutation, PI3KγCA mice developed highly advanced disease forms with robust fibrosis, inflammation, and AKT activation/proliferation. In accordance with these observations, both homo- and heterozygous global deletion of p110γ protected against KRAS-induced tumorigenesis, reducing downstream AKT signaling and proliferation in vivo. However, despite the reduction in tumor burden, mutant KRAS mice with p110γ-deficiency displayed early signs of hepatotoxicity. Since p110γ has several key roles in lipid metabolism, we next assessed p110γ loss in the setting of a high-fat diet (HFD). These mtKRAS/p110γKO mice developed pronounced hyperlipidemia and severe hepatic cell injury/steatosis. Additionally, the previously observed protective effect in the pancreas was markedly diminished, and neoplastic tissues displayed pronounced AKT pathway activation, indicating potential compensatory signaling through non-p110γ isoforms. Indeed, how p110γ loss and lipid accumulation in liver impact development of mtKRAS-induced neoplasia and cancer is a very interesting future aim. Combined, these observations suggest that systemic p110γ inhibition should be carefully considered, particularly given the potential for reduced efficacy, hyperlipidemia, and hepatic injury in obese patients, unless specific targeting to the pancreas can be achieved.

#4989

Efficacy of erlotinib and/or naproxen when administered by intermittent dosing schedules in the prevention of chemically induced urinary bladder cancers.

Altaf Mohammed,1 Mark S. Miller,1 Ronald A. Lubet,1 Chen Suen,1 Shizuko Sei,1 Robert H. Shoemaker,1 Clinton J. Grubbs2. 1 _National Cancer Institute, Bethesda, MD;_ 2 _University of Alabama at Birmingham, Birmingham, AL_.

Inflammation and epidermal growth factor receptor (EGFR) signaling dysregulation plays an important role in urinary bladder cancer development. We have previously shown that single agent regimens with erlotinib (EGFR inhibitor) or naproxen (nonsteroidal anti-inflammatory drug; NSAID) when given continuously were highly effective in the prevention of OH-BBN-induced urinary bladder cancers in rats. Better tolerated chemoprevention regimens can be obtained by reducing doses and frequency of administration. Low dose combinations may also achieve substantial efficacy with minimal toxicity by targeting complementary pathways. Female Fischer-344 rats were obtained at 28 days of age, placed on Teklad (4% fat) diet, and received OH-BBN (150 mg/gavage) 2x/week for 8 weeks beginning at 56 days of age. In the first study, beginning one week after the final OH-BBN treatment the rats (25/group) received either:

Group 1, erlotinib (42 mg/kg BW, 1x/week); Group 2, naproxen (30 mg/kg BW/day, 3 weeks on/ 3 weeks off); Group 3, the combination of erlotinib and naproxen (using the same treatment regimens), and Group 4, vehicle. The rats were palpated for urinary bladder tumors, weighed 1x/week and observed daily for signs of toxicity. At the end of the study (10 months after the initial OH-BBN), the average weights of the urinary bladders (bladder plus tumors combined) were determined. The weights in Groups 1-4 were: 194*, 186*, 136* and 354 mg (*P <0.05, Wilcoxon Rank test). This was accompanied by an increase in tumor latency, a decrease in tumor multiplicity, and a decrease in the number of rats with large palpable tumors (> 200 mg). The second study used the same treatment regimens, but administration of the agents was delayed until three months after the final OH-BBN treatment, at which point microscopic transitional cell carcinomas were present. At the end of the study (11 months after the initial OH-BBN), the weights of the individual bladders in Groups 1-4 were: 584, 234*, 138* and 779 mg (*P< 0.05, Wilcoxon Rank test). Thus, naproxen and particularly the combination were still highly effective. This was accompanied by an increase in tumor latency and a decrease in the number of rats with large palpable tumors (> 200 mg). These studies demonstrate that in protocols meant to reduce the toxicity of agents (weekly erlotinib or intermittent dosing with naproxen), high chemopreventive efficacy was achieved. Of importance, starting erlotinib and/or naproxen intermittent treatments at the time microscopic cancers were present still reduced the size of the urinary bladder cancers without showing observable toxicity. The latter is of particular interest based on the clinical FAP trial showing great efficacy of erlotinib and sulindac. Supported by NCI contract number HHSN261201500036I, Task Order HHSN26100002.

## TUMOR BIOLOGY:

### Stemness and Cancer

#4990

Regenerative origin of colorectal metastasis stem cells.

Karuna Ganesh,1 Harihar Basnet,1 Kevin P. O'Rourke,1 Ashley M. Laughney,1 Lan He,1 Eduard Batlle,2 Scott W. Lowe,1 Dana Pe'er,1 Jinru Shia,1 Joan Massague1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Institute for Research in Biomedicine, Barcelona, Spain_.

Metastatic cancers invariably relapse due to the emergence of resistant tumor clones capable of self-renewal, entry into and exit from quiescence, tumor re-initiation and therapy resistance. The origins of such metastasis propagating cells (MPCs), which ultimately cause cancer death, are not well-understood.

To directly scrutinize MPCs in patient metastases, we established ex vivo organoid cultures from surgically resected, chemoresistant residual colorectal cancer (CRC) liver metastases. We show that the neuronal cell-adhesion molecule L1CAM, which is ectopically expressed in many cancer types and strongly associated with poor prognosis, is a marker of MPCs. L1CAM+ cells are largely quiescent in structured neoplastic glands in tumors, but when dissociated from their epithelial niche, proliferate to regenerate heterogeneous organoids or xenografts containing both L1CAM+ and L1CAM- progeny.

To define the relationship between L1CAM+ MPCs and Lgr5+ intestinal stem cells, we performed single cell mRNA sequencing on ~15,000 CRC organoid-derived cells from four patients. We identified only partial overlap between Lgr5+ and L1CAM+ cells. Lgr5high cells consistent with homeostatic stem cells, have low L1CAM levels, while Lgr5low transit amplifying progenitor-like cells have high L1CAM levels. In addition, we identify a separate population of L1CAMhighLgr5- cells. The data suggest that human CRC metastases are derived from an L1CAM+ population of transit-amplifying, partially differentiated cells.

L1CAM is not expressed in intact human or mouse intestinal crypts during homeostasis. However, when the intestinal epithelium is disrupted by dextran sodium sulfate-mediated colitis, L1CAM is strongly induced in cells in the middle of regenerating crypts. Intestinal epithelium specific deletion of L1CAM causes profound weight loss, poor tissue healing and reduces survival in DSS-treated mice. In turn, L1CAM knockdown/knockout in mouse or human CRC cells inhibits regeneration of organoids in vitro, subcutaneous tumors and orthotopic liver metastases in vivo. Mechanistically, L1CAM RNA expression is normally silenced in non-neuronal cells by the transcriptional repressor REST. We show that disruption of epithelial integrity by organoid dissociation or E-cadherin knockdown reduces REST binding to an L1CAM intronic enhancer, thus inducing L1CAM expression.

Our results suggest that L1CAM is dispensable for epithelial homeostasis, but is required for normal and neoplastic epithelial regeneration when tissue integrity is disrupted. During cancer progression, disseminated tumor cells at the invasion front of primary tumors, in the circulation, or in isolated residual disease following therapy, induce and depend on L1CAM for survival and eventual regrowth. Thus, L1CAM represents a crucial vulnerability of disseminated and residual MPCs that could be exploited therapeutically to treat patients with metastatic cancer.

#4992

Sirt6 loss increases stemness and defense against oxidative stress in tumor-propagating cells, promoting tumor growth and maintenance in squamous cell carcinoma.

Jee-Eun Choi,1 Carlos Sebastian,2 Caroline Lewis,3 Murat Cetinbas,4 Gregory Wojtkiewicz,4 Ruslan Sadreyev,4 Leif W. Ellisen,4 Salvador Aznar-Benitah,5 Raul Mostoslavsky4. 1 _Harvard Medical School, Boston, MA;_ 2 _Candiolo Cancer Institute, Torino, Italy;_ 3 _Whitehead Institute for Biomedical Research, Cambridge, MA;_ 4 _Mass. General Hospital, Boston, MA;_ 5 _Institute for Research In Biomedicine, Barcelona, Spain_.

Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers in the US. As such, a better understanding in HNSCC carcinogenic process and contributing molecular mechanisms may pinpoint to new therapeutic interventions for the patients. Notably, despite extensive genetic studies, little is known on the metabolic features of HNSCC. Metabolic reprogramming to aerobic glycolysis has recently emerged as a key event during carcinogenesis. Our lab has shown that the histone deacetylase Sirt6 inhibits HIF-1α- and c-Myc-dependent transcription, both of which are critical in metabolic reprogramming of cells, thereby acting as a tumor suppressor in the context of colon cancer. Based on these results, we aimed to assess whether Sirt6 loss facilitates squamous cell carcinogenesis, specifically affecting glucose metabolism and stemness of cancer cells. In an in vivo model of skin squamous cell carcinogenesis with Sirt6 deletion (K14-cre+; Sirt6fl/fl), tumor onset was faster in Sirt6fl/fl mice compared to WT animals, and those tumors from Sirt6fl/fl mice were bigger. Sirt6 loss also positively regulates tumor maintenance without continuous proliferative cues, suggesting that Sirt6 deletion could impact on the tumor propagating cells (TPCs). Strikingly, loss of Sirt6 increased the percentage of TPCs (a6high/CD34+), most of which were highly glycolytic. Administration of a glycolytic inhibitor (dichloroacetate) in vivo effectively inhibited tumor growth and maintenance, resulting in much fewer percentages of TPCs. Our results indicate that enhanced glucose metabolism is crucial in maintaining and expanding TPCs. In human HNSCC cell lines, Sirt6 negatively regulates glucose metabolism and cell growth in vitro and ex vivo, nicely recapitulating the in vivo observations. Glucose isotope tracing experiments in vitro suggest that pentose phosphate pathway and generation of antioxidant molecules are main metabolic pathways affected by Sirt6. Transcriptomic analysis of TPCs from in vivo skin tumors revealed critical gene expression signatures, where TPCs exhibit decreased differentiation, increased stem-like, and enhanced antioxidative/redox markers. Sirt6 loss even further up-regulated stemness genes and the genes that generate antioxidant molecules, providing molecular mechanisms to explain a more aggressive tumorigenic phenotype in Sirt6-deleted tumors. Overall, our studies indicate that high glycolytic flux (which can contribute to the pentose phosphate pathway and antioxidant molecules) is crucial in TPCs. Our studies clearly demonstrate that Sirt6 acts as a tumor suppressor by regulating glucose metabolism and stemness of tumor cells. This study has further therapeutic implication that targeting antioxidant defense pathways could effectively eradicate TPCs, critical population maintaining a whole tumor.

#4993

Breast epithelial cell lines from normal breast with luminal and intrinsic subtypes -enriched gene expression document inter-individual differences in differentiation cascade.

Brijesh Kumar, Mayuri Prasad, Manjushree Anjanappa, Poornima Nakshatri, Natascia Marino, Anna Maria Storniolo, Xi Rao, Sheng Liu, Jun Wan, Yunlong Liu, Harikrishna Nakshatri. _Indiana Univ. School of Medicine, Indianapolis, IN_.

Breast cancers are classified into five intrinsic subtypes based on gene expression profile. It is suggested that these intrinsic subtypes originate from specific developmental stage of breast epithelial cell hierarchy, stem-progenitor-mature cell. However, normal breast epithelial cell lines representing these intrinsic subtypes are yet to be created. Using normal breast tissues of ancestry-mapped Caucasian, African American, and Hispanic women and a primary cell culturing system that allows growth of normal epithelial cells of different developmental stages including estrogen receptor-positive mature luminal cells, we created 15 human telomerase-immortalized breast epithelial cell lines. These cells formed acini on a matrigel and ductal structures on 3-dimensional collagen or hydrogel, indicating that these cell lines have retained characteristics of normal breast epithelial cells. RNA sequencing and PAM50 intrinsic subtype clustering algorithms were used to identify the intrinsic subtypes of the immortalized cell lines together with two well characterized "normal" breast epithelial cell lines MCF10A and HMEC as well as luminal breast cancer cell line MCF-7. Unlike MCF10A and HMEC, which are enriched for basal-like gene expression pattern, our cell lines classified into luminal A, basal, and normal-like subtypes. This was also reflected in the immunofluorescence staining with basal marker KRT14 and luminal marker KRT19. Few of these cell lines were dual positive for KRT14 and KRT19, but in varying proportions. Cell lines representing claudin-low subtypes were also created, which are phenotypically (CD201+/EpCAM-) different from the above cell lines. Cell lines showed inter-individual differences in stemness/differentiation capabilities and variable basal activity of signaling molecules such as NF-kB, AP-1 and pERK, which is consistent with, possibly reflecting, recent discoveries of genetic variations in gene regulatory regions among general population that contribute to widespread differences in gene expression/signaling under "normal" state. As majority of breast cancers are believed to originate from luminal progenitor cells, which are well represented our cell lines, these cell lines are ideal to delineate the impact of inter-individual and ethnic differences in normal breast biology on breast cancer initiation and progression as well as to determine whether cell-type-origin instead of genomic aberration drives intrinsic subtype-enriched gene expression patterns in breast tumors.

#4994

p53 and RB regulate Hedgehog responsiveness via autophagy-mediated ciliogenesis.

Jason E. Cain,1 Catherine R. Cochrane,1 Vijesh Vaghjiani,1 Anette Szczepny,1 Andrew McCaw,1 Kirstyn Carey,1 Luciano Martelotto,1 Maya Kansara,2 David Thomas,2 Carl Walkley,3 William H. Matsui,4 David N. Watkins2. 1 _Hudson Institute of Medical Research, Clayton, Australia;_ 2 _Graven Institute of Medical Research, Darlinghurst, Australia;_ 3 _St Vincent's Institute of Medical Research, Fitzroy, Australia;_ 4 _Johns Hopkins, Baltimore, MD_.

Hedgehog (Hh) signaling regulates patterning, cell-fate and self-renewal in development. Hh proteins signal via Smoothened (Smo), a G-protein coupled receptor whose activity is dependent on translocation to the primary cilium, a single immotile tubulin-based structure present on most mammalian cells. Activation of Smo results in the stabilization of GLI activator transcription factors, which in turn induce Hh pathway gene expression. Aberrant activation of the Hh pathway is implicated in initiation and progression of a wide range of cancers, yet very few contain genetic mutations of Hh pathway components that account for increased signaling. Instead, the majority of Hh-driven tumors exhibit ligand-dependent signaling but the mechanisms governing this are unknown. We show that genetic inactivation of Trp53 and or Rb1 in mouse embryonic fibroblasts (MEFs) promotes ciliogenesis and cell responsiveness to Hh ligand. Pampliega et al. (Nature 2013) have previously described a functional interaction between autophagy and ciliogenesis. Interestingly, Trp53KO, Rb1KO and Trp53;Rb1KO MEFs exhibit defective autophagic flux and reduced expression of genes associated with autophagy. siRNA knockdown of Atg5, Atg9b, Ctsd and Pik3cg in C57Bl/6 wild-type MEFs resulted in increased Hh ligand responsiveness. To explore this further in a disease setting we assessed an extensive panel of mouse osteosarcoma (mOS) cell lines. In contrast to radiation-induced mOS cell lines that demonstrated variable sensitivity, all genetically induced mOS cell lines (OsxCre;Trp53fl/fl;Rb1fl/fl) were highly sensitive to Hh ligand. In all cases Hh pathway activation could be inhibited by the small-molecule Smo-inhibitor, LDE225. Hh responsiveness correlated to primary cilia frequency with responsive cell lines demonstrating high cilia frequency while nonresponsive cell lines exhibited few if any cilia, under both normal or serum-deprived conditions. Similarly, autophagic flux was significantly reduced in Hh responsive compared to nonresponsive mOS cell lines. Consistent with these findings, in a panel of human osteosarcoma (hOS) cell lines, those with p53 and/or RB-deficient pathways are associated with reduced autophagy and increased primary cilia frequency. Pathway inhibition by LDE225 in in vivo allograft and xenograft models of highly ciliated mOS and hOS cell lines leads to reduced tumor growth, increased survival and intratumoral bone deposition, but has no effect on xenografts of a cell line lacking primary cilia. These data suggest that p53 and Rb control of genes required for autophagy regulates ciliogenesis and ultimately Hh pathway responsiveness to ligand, implicating p53 and Rb mutation status and primary cilia frequency as biomarkers for Hh-ligand sensitivity and potential responsiveness to Hh-inhibitor therapy.

#4995

Identity fraud: Lineage plasticity as a mechanism of anti-androgen resistance and target for therapy.

Alastair H. Davies,1 Chiara Bostock,1 Musaddeque Ahmed,2 Yen-Yi Lin,1 Fraser Johnson,1 Ka Mun Nip,1 Kirsi Ketola,1 Jennifer Bishop,1 Ladan Fazli,1 Haojie Huang,3 David Goodrich,4 Faraz Hach,1 Hansen He,2 Himisha Beltran,5 Amina Zoubeidi1. 1 _Vancouver Prostate Centre, Vancouver, British Columbia, Canada;_ 2 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada;_ 3 _Mayo Clinic Cancer Center, Rochester, NY;_ 4 _Roswell Park Cancer Institute, Buffalo, NY;_ 5 _Weill Cornell Medicine, New York, NY_.

Background: Potent targeting of the androgen receptor (AR) in castration-resistant prostate cancer has altered the archetypal course of the disease, fueling the emergence of aggressive and incurable neuroendocrine prostate cancer (NEPC). Recent evidence suggests that these tumors can arise from non-neuroendocrine cells in response to AR pathway inhibitors (ARPIs), such as enzalutamide (ENZ), an observation consistent with lineage plasticity. What regulates this plasticity that allows cells to shed their dependence on the AR and re-emerge as "AR-indifferent" NEPC? Sequencing studies have uncovered that the evolution toward a NEPC phenotype is aligned with dynamic epigenetic reprogramming, but the molecular basis underlying this phenomenon remains poorly understood.

Methods: We developed an in vivo model of acquired ENZ resistance to (a) identify reprogramming factors that facilitate lineage plasticity, and (b) determine how to best capitalize on therapeutic strategies aimed at blocking or reversing lineage transformation. Cell lines derived from ENZ-resistant tumors were profiled by RNA-seq and ChIP-seq, and functionally assessed for stem cell-associated properties. Our findings were validated across NEPC cell lines (NCI-H660), genetically engineered mouse models (PBCre4:Ptenf/f:Rb1f/f), and patient tumors and organoids. CRISPR/Cas9-mediated genomic editing allowed us to assess the effect of knocking out reprogramming factors on therapy-induced neuroendocrine transdifferentiation.

Results: AR-indifferent ENZ-resistant tumors were enriched for a Polycomb/EZH2 signature; in particular, we identified EZH2 to be phosphorylated at threonine-350 (pEZH2-T350) by CDK1 in NEPC cell lines, mouse models, and patient tumors. Accordingly, RB1 loss was sufficient to enhance pEZH2-T350, which was required for prostate cancer cells to convert to a metastable stem-like state and, in turn, acquire neuroendocrine features under the pressure of ARPIs both in vitro and in patient-derived xenografts. This therapy-induced NEPC transdifferentation was associated with a marked redistribution of EZH2 and H3K27me3, specifically to a core set of genes governing lineage identity. AR colocalized at the reprogrammed EZH2 binding sites, and was found to be part of the same complex with EZH2. Treating AR-indifferent/NEPC cell lines with clinically relevant EZH2 inhibitors reversed the lineage switch and mitigated ENZ resistance.

Conclusions: This research establishes the centrality of epigenetic reprogramming in driving the insurgence of a neuroendocrine phenotype in response to ARPIs, and posits that drugging the epigenome via EZH2 inhibition may reverse or delay lineage transformation to extend the durability of clinically beneficial ARPIs.

#4996

MLL3 haploinsufficiency preserves self-renewal capacity in HSCs with extensive cumulative division histories.

Theresa Owuor, Shaina Porter, Cluster Andrew, Riddhi Patel, Jeffrey Magee. _Washington University, St. Louis, MO_.

KMT2C/MLL3 is one of the most commonly mutated tumor suppressor genes in human cancer. Haploinsufficiency at the KMT2C/MLL3 locus often occurs in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) as a result of mono-allelic 7q deletions. Mll3 haploinsufficiency has been shown to accelerate AML in mice, but it is not clear how it regulates normal hematopoietic progenitors or why it is required to suppress leukemogenesis.

We used a novel loss-of-function mouse allele to characterize hematopoiesis in Mll3-deficient fetal livers and adult bone marrow. Mll3 deletion caused a modest increase in fetal liver hematopoietic stem cells (HSCs) and a modest decrease in lineage restricted progenitors (HPCs). In contrast, Mll3 heterozygosity caused significant expansion of the HSC population in adult bone marrow without affecting more committed progenitor populations. Mll3+/- and Mll3-/- HSCs were able to repopulate lethally irradiated mice, and repopulating activity was enhanced in secondary and tertiary transplants relative to wild type HSCs. Thus, Mll3 haploinsufficiency enhances HSC self-renewal capacity.

We next tested whether Mll3 regulates the balance between self-renewal and commitment in non-transplanted HSCs. We used H2B-GFP pulse-chase assays to assess the division histories of HSCs over time. After a 6-week chase, wild type HSCs retained high levels of H2B-GFP expression, consistent with prior studies showing that adult HSCs divide infrequently. In contrast, almost all Mll3+/- HSCs had low to moderate H2B-GFP expression levels, indicative of an extensive division history. This difference was not associated with an increase in the percentage of cycling (S/G2/M) HSCs, and it was not associated with a loss of self-renewal capacity that occurs with cumulative divisions in wild type HSCs. Thus, Mll3 haploinsufficiency allows cumulative HSC divisions to accrue without compromising self-renewal.

Mll3 encodes a histone methyltransferase that binds active enhancers and promotes transcription. This raised the question of whether Mll3 deletion results in a loss of commitment-related gene expression, increased self-renewal gene expression or altered epigenetic landscapes. To our surprise, gene expression was indistinguishable between wild type and Mll3+/- HSCs. However, ATAC-seq analysis revealed patterns of nucleosome depletion in Mll3+/- HSCs that closely resembled wild type HPCs. No differences were observed in the ATAC-seq profiles of wild type and Mll3+/- HPCs. These observations suggest that as Mll3+/- HSCs self-renew, they remodel their epigenetic landscapes to resemble committed progenitors, yet they do not activate commitment related genes, and they do exit the stem cell pool. The data suggest that stress conditions that drive HSCs into cycle, such as the chemotherapy regimens that often precede 7q- MDS, may select for Mll3 haploinsufficient HSC clones.

### Molecular Mechanisms Driving Metastasis

#4997

Identifying dynamic EMT states and constructing a proteomic EMT landscape of lung cancer using single cell multidimensional analysis.

Loukia G. Karacosta, Benedict Anchang, Samuel Kimmey, Matt van de Rijn, Joseph B. Shrager, Sean C. Bendall, Sylvia K. Plevritis. _Stanford University, Stanford, CA_.

The role of EMT in cancer has been well reported and has been shown to prime cells for invasion and metastasis. EMT can be adopted or reversed (i.e. mesenchymal to epithelial transition, MET) by cells, revealing plasticity that can also lead to stemness and drug resistance. Although it is appreciated that EMT is not a binary process of two extremes but instead a continuum of intermediate states of partial EMT phenotypes, these are poorly defined. Given that intermediate EMT cancer states are viewed as critical for understanding and clinically targeting EMT processes, our aim was to dynamically capture and characterize intermediate EMT states in TGF beta treated lung cancer cells and clinical specimens. With single cell analysis we identified 4 distinct transition states. These states patterned an EMT axis featuring: (1) an epithelial, (2) a partial EMT and (3) a mesenchymal state that branches off to (4) a subset of phenotypically stem-like cells. Transition was reflected by gradual changes in E Cadherin, Vimentin, CD44 and CD24 levels. "True" mesenchymal state (E Cad- Vim+) was isolated to the stem-like cells (CD44hiCD24lo), which were negative for Twist protein expression. To interrogate the dynamism of EMT and MET processes, we performed TGF beta withdrawal experiments, which showed that most cells were able to reverse their behavior. Simultaneous analysis of 30 parameters with CyTOF confirmed the 4-state transition, offering deep dynamic views of a variety of cell surface, signaling, cell cycle, and transcriptional markers. We then proceeded to computationally identify and define additional states of the EMT/MET spectrum that are visited by transitioning cells in a step-wise manner, in order to construct a lung cancer EMT/MET proteomic landscape. To tackle this, we applied CCAST, an algorithm that employs decision trees to identify and discretize homogeneous cell subpopulations among heterogeneous single cell data. When we assembled the high dimensional data in a timely order, we were able to visualize the emerging states that cells visited during their transitions. This revealed the existence of more than one possible EMT/MET trajectories as well as the existence of a transient subpopulation of cells with a distinct phenotype (Twist+, CD34+, E Cad-, Cytokeratin 7+, pEGFR-). Finally, CyTOF analysis and projection of 2 lung adenocarcinoma specimens on the constructed EMT/MET landscape confirmed the existence of states observed in our cell line studies, including the aforementioned Twist+ transient subpopulation. In summary, we provide a lung cancer cell proteomic map that dissects EMT phenotypic plasticity. Clinically, this type of EMT/MET proteomic mapping could help identify, predict and target EMT mechanisms known to have a role in cancer progression, drug resistance and disease recurrence.

#4998

Targeting DHPS to abrogate TGFβ-induced metastasis in breast cancer.

Robert Güth, Lindsay Kutscher, Yvess Adamian, Cameron Geller, Kishan Bahkta, Kayla Meade, Jonathan A. Kelber. _California State University, Northridge, Northridge, CA_.

Progression of solid tumors to a metastatic stage accounts for over 90% of cancer mortality. Thus, it is critical to identify therapeutic strategies that target both primary and metastatic tumors. Epithelial-mesenchymal transition (EMT) negatively correlates with therapy response, contributing to intratumoral heterogeneity and systemic dissemination in breast cancer. We previously reported that pseudopodium-enriched atypical kinase one (PEAK1) promotes breast cancer cell EMT and metastasis by potentiating fibronectin-transforming growth factor beta (TGFβ) signaling cross-talk. Since eukaryotic initiation factor five A (eIF5A), a unique translation factor that is activated by deoxyhypusine synthase (DHPS)-dependent post-translational hypusination, is required for PEAK1 expression, we hypothesized that TGFβ may directly regulate eIF5A activity to promote EMT, and that targeted inhibition of this pathway may provide a novel means to inhibit or reverse metastatic progression. In this regard, we provide evidence of an active eIF5A-EMT program in undifferentiated breast cancer tissue. Notably, blockade of DHPS activity and eIF5A hypusination reduces PEAK1 translation, cell viability and TGFβ-induced EMT in vitro and metastasis in vivo. Conversely, we demonstrate that TGFβ induces post-translational hypusination of eIF5A in metastatic breast cancer cells. TGFβ is known to activate histone deacetylase six (HDAC6) and HDAC6 was independently reported to promote eIF5A deacetylation and nuclear export to support its translation functions. When delivered in combination, HDAC6 and DHPS inhibitors synergize to sequester eIF5A to the nucleus, suppress eIF5A-dependent translation and potently kill metastatic breast cancer cells. To identify candidate pathways downstream of the eIF5A/PEAK1 axis during EMT, we generated a Cytoscape interactome using eIF5A signaling and PEAK1-induced EMT genes as search terms. All interactome component genes were then analyzed across two breast cancer patient studies available on the Cancer BioPortal. Interestingly, SOX2, PIK3CA and EIF4A2 were the interactome nodes that exhibited copy number amplifications among patients harboring genomic alterations in the initial interactome search genes, and SOX2 amplification significantly and independently associated with decreased patient survival (p = 0.0476). Taken together, our results establish a novel node/axis by which TGFβ signaling stimulates HDAC6 and/or DHPS function to activate cytoplasmic eIF5A and promote EMT and survival of breast cancer cells within the metastatic niche, identifying new targeted therapy strategies that may improve cancer patient survival.

#4999

Identifying intercellular phenotypic stability factors for a hybrid epithelial-mesenchymal phenotype.

Mohit Kumar Jolly,1 Federico Bocci,1 Satyendra C. Tripathi,2 Mitzi Aguilar,2 Samir M. Hanash,2 Herbert Levine,1 Jose N. Onuchic1. 1 _Rice University, Houston, TX;_ 2 _UT MD Anderson Cancer Center, Houston, TX_.

Epithelial-mesenchymal transition (EMT) and its reverse Mesenchymal-epithelial transition (MET) are crucial for embryonic development, wound healing and cancer metastasis. Cells need not undergo full EMT or MET, rather they can maintain stably a hybrid epithelial/mesenchymal (E/M) phenotype and thus exhibit collective cell migration, forming clusters of circulating tumor cells - the primary drivers of metastasis. Mechanisms enabling cells to exhibit a hybrid E/M phenotype stably remain elusive. Here, using an integrated experimental-computational approach, we identify 'phenotypic stability factors' (PSFs) that can stabilize a hybrid E/M phenotype by mediating cell-cell communication through Notch signaling pathway. Our mathematical model predicts that Numb or Numb-like (Numbl) can inhibit a full EMT and stabilize a hybrid E/M phenotype. This prediction is validated by the observation that knockdown of Numb in stably hybrid E/M cells H1975 results in a full EMT, thereby indicating that Numb behaves as a PSF. Generalizing the mathematical model to a multi-cell level, Numb is predicted to alter the balance of hybrid E/M versus mesenchymal cells in clusters, potentially resulting in a higher tumor-initiation ability. Our model also suggests another PSF – Nrf2 (nuclear factor E2-related factor 2) – that couples both to EMT and Notch signaling, and stabilizes a hybrid E/M phenotype too. Intriguingly, Nrf2 levels are maximally observed in a hybrid E/M phenotype as compared to purely epithelial or mesenchymal phenotypes. Finally, high levels of Numb or Nrf2 also correlate with a worse survival in multiple independent cancer datasets, strengthening the association of a hybrid E/M phenotype with increased cancer aggressiveness.

#5000

Biomaterial scaffolds that capture metastatic tumor cells in vivo to detect, treat, and study mechanisms of the premetastatic niche and metastasis.

Grace G. Bushnell, Tejaswini P. Hardas, Rachel M. Hartfield, Yining Zhang, Robert S. Oakes, Adeline Hong, Jacqueline S. Jeruss, Lonnie D. Shea. _University of Michigan, Ann Arbor, MI_.

Objective: For most cancers, the formation of distant metastasis is the point at which clinical treatment shifts from curative intent to palliative care. At present, there is no clinical method to detect metastatic dissemination and colonization until radiologically evident, at which point organ function has already been compromised. The Shea laboratory has developed a biomaterial implant that acts as a synthetic pre-metastatic niche and recruits metastatic cancer cells in xenogeneic human and syngeneic mouse models of breast cancer. Scaffold implantation has facilitated detection of metastasis prior to colonization of organs and has been shown to reduce metastatic burden, resulting in enhanced survival with surgical intervention.

Methods: Triple-negative breast cancer models were used in which tdTomato+ 4T1 or MDA-MB-231-BR cells were orthotopically inoculated into balb/c or NSG mice. Microporous polycaprolactone (PCL) scaffolds were fabricated using gas-foaming and poragen leaching and were subcutaneously implanted one month prior to tumor inoculation. High frequency ultrasound (US) was used to probe the scaffold for early detection of tumor cells prior to metastasis to organs. Human tumor cells were isolated from the scaffold (SCAF), primary tumor (PT), and a bone metastasis via MACS mouse cell depletion (Miltenyi) and grown into stable cell lines in vitro. Scratch, migration, invasion, mammosphere, cancer stem cell markers, RNA-seq, qRT-PCR, and HiC assays were performed to investigate behavioral differences between cell lines in vitro. Additionally, cell lines were inoculated into mice and investigated for metastatic ability.

Results: Initial studies applied US for detection of metastatic cell arrival at the scaffold. The analysis of spectral imaging distinguished scaffolds from tumor bearing relative to tumor free mice. A cell line created from the scaffold (SCAF) was more aggressive in vitro, demonstrating higher levels of migration, invasion, mammosphere formation, and proportion of cancer stem cells compared to PT. SCAF cells were also found to be ~30x more metastatic to the lung in vivo compared to PT cells (SCAF 10,150±7792 PT 354±296 cells per lung). RNA-seq identified 14232 genes with measured expression, 2901 of which were differentially expressed (p<0.05 and log fold change > 0.6) between scaffold and primary tumor cells.

Conclusions: Biomaterial scaffolds capable of recruiting metastatic tumor cells in vivo can serve as a platform for early detection and intervention, and also provide a tool to study metastasis in vivo and the properties of the early metastatic cells.

#5001

Identification of a Six2/Sox2/Nanog stem cell axis that promotes breast cancer metastatic colonization.

Michael U. Oliphant,1 Ahwan Pandey,1 Katherine Johnson,2 Rani Powers,1 Matthew Galbraith,1 James Costello,1 Heide L. Ford1. 1 _University of Colorado Denver Anschutz Medical Campus, Aurora, CO;_ 2 _Middlebury College, Middlebury, VT_.

Although significant progress has been made in understanding the molecular mechanisms that lead to metastatic breast cancer, it remains the overwhelming cause of death for patients. Current studies primarily focus on the prevention of early stages of metastasis, such as migration and invasion. But at the time of diagnosis tumor cells have likely left the primary tumor, suggesting that inhibition of the early stages of metastasis may not be the most effective means of inhibiting metastatic burden. Instead, the identification and targeting of molecules required for establishment and survival of cells at secondary sites is imperative for advancing therapies. We are examining the role of the developmental transcription factor Six2 in promoting metastatic burden. Six2 is a member of the Six family of transcription factors and is responsible for the maintenance of stem/progenitor cells during nephrogenesis. Using knockdown (KD) and overexpression (OE) models in murine mammary cells, we demonstrated that Six2 promotes specifically late-stage metastasis (growth at the secondary site). RNA-Seq analysis on control and Six2 OE cells showed a dramatic enrichment in stem cell transcriptional programs downstream of Six2 in mammary/breast cancer cells. Flow cytometry analyses and tumorsphere assays demonstrate that Six2 OE leads to an increase, whereas Six2 KD leads to a decrease, in the mammary stem cell population and tumorsphere formation, respectively. Using an immune competent mouse model, in vivo orthotopic limiting-dilution experiments reveal that Six2 regulates tumor initiation. In addition, we show that Six2 regulates late-stage metastasis of human breast cancer cells in vivo, as measured by metastatic burden after tail vein injection. Thus, our data demonstrate that Six2 similarly promotes stem phenotypes in breast cancer cells as it does during kidney development, and suggest that this attribute may be critical for its ability to promote metastatic outgrowth. To determine the molecular mechanism by which Six2 mediates stem phenotypes and metastasis in breast cancer, we interrogated our RNA-seq data, which suggested that Six2 may control master regulators of stemness. We demonstrate that both Sox2 and Nanog are regulated by Six2. In human breast cancer gene expression datasets, Six2 significantly positively correlates with both Sox2 and Nanog, and combined OE of Six2 with either Sox2 or Nanog results in poor prognosis. Preliminary data suggest that loss of Sox2 downstream of Six2 inhibits metastatic outgrowth, and that Sox2 is upstream of Nanog in the pathway. Together, our data demonstrate that Six2 regulates cancer stem phenotypes to promote metastatic outgrowth, uncovering a novel Six2/Sox2/Nanog axis that is critical for metastatic colonization.

#5002

KRT13 promotes stemness and drives metastasis in breast cancer through direct interaction with plakoglobin-desmoplakin complexes regulating c-Myc signaling pathway.

Lijuan Yin,1 Gina Chia-Yi Chu,1 Qinlong Li,2 Liyuan Yin,1 Michael Lewis,3 Mourad Tighiouart,1 Leland W. K. Chung,1 Haiyen E. Zhau1. 1 _Cedars-Sinai Medical Center, Los Angeles, CA;_ 2 _Xijing Hospital, Fourth Military Medical University, Xi'an, China;_ 3 _Sepulveda Research Corporation VA Medical Center, Los Angeles, CA_.

Introduction and Objective: Keratins (KRTs) were reported to interact with multiple cellular proteins and initiate signaling cascades that promote cell proliferation, migration and metastasis in cancers. Keratin13 (KRT13), a member of the intermediate filament proteins, plays important role in breast cancer progression and metastasis. The objectives of this study are to determine the role and molecular mechanism of KRT13 in promoting breast cancer growth and metastatic progression.

Methods and Results: We evaluated KRT13 protein expression in 58 primary breast cancer tissues by immunohistochesmistry and found that KRT13 is overexpressed in metastatic breast cancer specimens (12/18) (p<0.05). KRT13 expression in primary breast cancer is associated with decreased patient overall survival based on publicly available Oncomine gene expression datasets. To understand the functional roles of KRT13 in breast cancer progression and metastasis, we performed studies by overexpressing and knocking down KRT13 and investigating alterations of breast cancer cell behaviors in vitro and tumor growth and metastasis in mice. KRT13-overexpressing MCF7 cells promote and KRT13-knocked down HCC1954 and MDA-MB-468 cells inhibit in vitro cell proliferation, invasion and migration and in vivo tumor growth and metastasis to bone and lung. KRT13 overexpression increased the stemness and mammosphere-forming ability of MCF7 cells compared to vehicle control. KRT13 directly interacted with plakoglobin(γ-catenin) to form complexes with desmoplakin as assessed by mass spectrometry and confirmed by Western blot. KRT13 decreased the expression and nuclear translocation of plakoglobin, abrogated plakoglobin-mediated c-Myc gene suppression, promoted cancer stemness and tumor metastasis. Conversely, genetic silencing of KRT13 in HCC1954 and MDA-MB-468 cells increased nuclear translocation of plakoglobin and enhanced c-Myc suppression, and decreased breast cancer cell growth and stemness in vitro and xenograft tumor formation and metastasis in vivo.

Conclusion: KRT13 exerts a novel function in the regulation of breast cancer cell growth, progression and metastasis via alterations of nuclear translocation of plakoglobin that modulates downstream c-Myc-dependent signaling, inducing stem cell-like phenotype and metastasis of breast cancer. Our findings could reveal potential novel targeting of breast cancer progression and metastasis.

Funded by an NCI P01 CA098912 and a Board of Governors Chair of Cancer Research fund from Cedars-Sinai Medical Center to LWK Chung.

#5003

RNase activity of MCPIP1 regulates key elements of mesenchymal phenotype of clear cell renal cell carcinoma cells.

Katarzyna Miekus, Paulina Marona, Judyta Górka, Jolanta Jura. _Jagiellonian University, Krakow, Poland_.

Introduction: Monocyte chemotactic protein 1-induced protein 1 (MCPIP1) regulates the mRNA of proinflammatory cytokines, such as IL-6, IL-1, IL-12 and IL-2 and influences NFκB and AP1 activity. MCPIP1 may also suppress miRNA biosynthesis. Last findings show that MCPIP1 affects viability and proliferation of tumor cells, degrades the mRNA of antiapoptotic gene transcripts in breast cancer cells and reduces tumor growth and metastatic disease of breast cancer. We have already demonstrated that MCPIP1 decreases during clear cell renal cell carcinoma (ccRCC) progression and is responsible for better tumor vascularity and the secretion of proangiogenic factors. Moreover, MCPIP1 downregulation promotes the metastatic spread of ccRCC cells.

Aim: The main objective of our study was to determine the role of RNase activity of MCPIP1 in the regulation of key elements in the EMT signaling axis and the acquisition of mesenchymal phenotype of ccRCC.

Materials and methods: Microarray analysis of tumor tissue was used to evaluate EMT signaling axis during ccRCC progression. For stable overexpression of MCPIP1, doxycycline-dependent TetON system was used: pLIX MCPIP1 for MCPIP1 overexpression, pLIX PURO as a control and mutant form pLIX D141N, with abolished MCPIP1 RNase activity. The level of genes and proteins were studied by real-time PCR, western blot, flow cytometry and immunoprecipitation. Chemotaxis, invasion and motility assays were performed to check migration and invasion. Foxn1nu/Foxn1nu and NOD-SCID mice were used in in vivo experiments.

Results: Microarray analysis revealed that the expression of mesenchymal markers increases during ccRCC progression together with decreased level of MCPIP1 protein in tumor tissue. We demonstrate that endonuclease activity of MCPIP1 regulates the mRNA expression and protein level of key elements in the EMT signaling pathways e.g. Snai1 and Snai2, ß-catenin, vimentin, E-cadherin and N-cadherin. Mutated form of MCPIP1 lacking RNAse activity upregulates β-catenin and vimentin and suppressed E-cadherin. Moreover, our study revealed that MCPIP1 influences the cytosolic or nuclear location of the E-cadherin repressors, transcription factor Snai1 and Snai2 and ß-catenin. Furthermore, motile activity of ccRCC cells is strongly increased in ccRCC with abolished MCPIP1 RNase activity. We also show that initial overexpression of MCPIP1 causes no tumor growth whereas the lack of RNase activity of MCPP1 led to an increase of tumor growth. Conclusions: We showed that endonuclease activity of MCPIP1 directly enhanced the mesenchymal phenotype of ccRCC through regulation of cell motility, expression and position of key elements in the EMT signaling pathways and tumor initiation.

Acknowledgement: The study was supported by research grants from the National Science Centre 2013/09/D/NZ/00249, 2015/19/B/NZ5/01405 and KNOW 35p/3/2015.

### Expanding the Definition of the Tumor Microenvironment

#5004

Profiling the extracellular matrix landscape of tumor microenvironments using proteomics.

Alexandra Naba,1 Karl R. Clauser,2 Steven A. Carr,2 Richard O. Hynes3. 1 _University of Illinois at Chicago, Chicago, IL;_ 2 _Broad Institute, Cambridge, MA;_ 3 _Massachusetts Institute of Technology, Cambridge, MA_.

"Your cancer has spread" remains for most cancer patients synonymous with a death sentence. Indeed, the majority of cancer deaths are due to metastases: the dissemination of tumor cells from a primary tumor to distant vital organs. As of today, we do not have efficient treatments to stabilize or cure most metastatic cancers, and for patients diagnosed early, i.e., before metastases are detectable, we do not have reliable markers to predict whether their tumors will remain indolent or will become deadly. Tumor progression critically depends on a locally permissive microenvironment, including a unique tumor-associated extracellular matrix (ECM). The ECM is a complex meshwork of proteins providing architectural support to cells and conferring biomechanical properties to tissues. ECM-derived biochemical signals transduced by cell-surface receptors, such as the integrins, regulate cellular functions such as proliferation and survival, adhesion, and migration, all essential for metastasis. Importantly, clinicians have observed that more aggressive tumors present a higher ECM content. Thus, to fully understand how tumors progress and disseminate, it is essential to study the tumor ECM. To do so, we have developed a pipeline combining bioinformatics and proteomics to comprehensively characterize the ECM composition, or "matrisome," of tissues and tumors. Using this pipeline, we demonstrated that the composition of the ECM varies i) between normal tissues and tumors, ii) between poorly and highly metastatic primary tumors, and iii) between primary tumors and derived metastases. Here we present for the first time "The Tumor ECM Atlas," a compendium of ECM proteins identified by proteomics in the microenvironment of 10+ types of primary tumors and derived metastases, including breast, colorectal and lung cancers, insulinomas and multiple myelomas, which revealed the presence of a unique subset of cancer-specific and cancer type-specific ECM proteins. We believe that this atlas is a powerful tool to formulate novel hypotheses on the roles of the ECM in tumor progression and metastasis. In addition, we propose that the ECM is an underexplored reservoir of potential diagnostic and prognostic markers and therapeutic targets, and that its exploration using proteomics will pave the way for the development of novel approaches to better care for cancer patients.

#5005

Aging promotes changes to peritoneal and omental collagen structure that contribute to increased ovarian cancer metastatic success.

Elizabeth Harper, Elizabeth Loughran, Annemarie Leonard, Tyvette Hilliard, Marwa Asem, Yueying Liu, Jing Yang, Yuliya Klymenko, Jeff Johnson, Emma Sheedy, Zonggao Shi, Matthew Leevy, Matthew Ravosa, M. Sharon Stack. _University of Notre Dame, Notre Dame, IN_.

Ovarian cancer (OvCa) is the deadliest cancer of the female reproductive system, ranking fifth in overall cancer deaths among women. Unlike the majority of cancers, OvCa metastasizes via diffusion through the peritoneal cavity, resulting in multiple metastatic sites, including the omentum and peritoneum. These metastasizing OvCa cells induce rapid mesothelial cell retraction and readily adhere to the sub-mesothelial collagen of the extracellular matrix. Epidemiologic data identifies age as a significant risk factor in OvCa, as about half of diagnoses are in women over the age of 63. Despite this, age is understudied in the OvCa field. Using a C57Bl/6 mouse model of aging, young (Y) mice ranging from 3-6 months of age, and aged (A) mice ranging from 20-23 months of age, corresponding to women aged 20-30 years (Y) and 60-67 years (A) were used to study the role aging has on metastasis. Fluorescently tagged C57Bl/6 syngeneic ID8 p53-/- mouse OvCa surface epithelial cells were injected intraperitoneally in young and aged mice and disease progression was evaluated for 5.5 weeks. Organ-specific tumor burden was quantified with ImageJ, revealing increased tumor burden in aged mice compared to their young counterparts. These results were reproduced in the FVB mouse model using syngeneic PTENshRNA/KRASG12V modified FVB OvCa oviductal epithelial cells. Second Harmonic Generation Microscopy (SHG) was used to visualize collagen of the peritoneal and omental tissues from young and aged C57Bl/6 mice. Distinct structural differences were shown in omental collagen in the Y vs A cohorts and validated with Scanning Electron Microscopy (SEM). Additionally, Nanoindentation illustrated mechanical differences between young and aged peritoneal samples. In conclusion, aging induces changes in the structure and mechanical strength of peritoneal and omental collagen, which contribute to OvCa metastasis.

#5006

Kit-dependent tissue resident macrophage progenitors drive cancer progression.

Paulina Pathria, Hideyuki Takahashi, Megan Kaneda, Judith A. Varner. _UCSD Moores Cancer Ctr., La Jolla, CA_.

Macrophages play a key role in promoting tumor growth and resistance to therapy. Here we show that tissue-resident as well as bone marrow-derived macrophages play critical roles in promoting tumor growth. Tissue-resident macrophages were recently shown to originate in the yolk sac or fetal liver during embryogenesis; these cells self-maintain in post-natal tissues independent of hematopoietic stem cells. Tissue-resident macrophages and bone marrow-derived macrophages rapidly accumulate in tumors where they play independent roles in promoting tumor growth. Tissue-resident macrophages are CD11b+Gr1-F4/80hiCX3CR1hiCCR2-Ki67+ cells that accumulate in tumors independently of trafficking receptors. In contrast, bone marrow-derived CD11b+Gr1+F4/80loCX3CR1loCCR2+ macrophages accumulate in tumors in an integrin α4β1/αLβ2 and CCR2 or CXCR4-dependent manner. Gene expression studies show that tumor-associated tissue-resident macrophages are highly proliferative, immune-suppressive myeloid cells that are less proangiogenic than bone marrow-derived macrophages. Our studies show that tumor cells induce the expansion of tissue-resident macrophage progenitor cells by secreting stem cell factor and mCSF. Here we identify a Kit/KitL-dependent tissue-resident macrophage progenitor that is abundant in tumors but not in normal tissues. Notably, tumor growth and colony-forming activity are significantly inhibited in mice treated with SCF and Kit inhibitors. Tumors adoptively transferred with tissue-resident macrophage progenitor cells exhibited a significant growth advantage over control tumors. Furthermore, Kit inhibitors synergize with other immune therapy regimens to suppress tumor growth. Our studies show that tissue-resident macrophage progenitors promote aggressive tumor growth that can be targeted by Kit/SCF inhibition.

#5007

**Absence of mammary tissue-resident macrophages is associated with reduced breast cancer susceptibility mediated by the cancer-associated** 8q24 **gene desert.**

Bart M. Smits, Anna I. Rissman, Lauren Shunkwiler, Collin Homer-Bouthiette, Yang Zhao, Benjamine van Peel, Jennifer Schulte, Robert Wilson, Adam C. Soloff. _Medical Univ. of South Carolina, Charleston, SC_.

Non-protein coding regions have frequently been associated with cancer susceptibility, with the human 8q24 gene desert linked to decreased incidence of breast cancer. To examine the impact of 8q24 upon mammary tissue development and breast cancer dynamics, we created a novel megadeletion (MD) mouse model lacking 430kb of the region orthologous to the human 8q24 gene desert. Homozygous MD knockout (MD-/-) mice have lower MYC expression in multiple organs, including whole mammary gland and mammary tumors, 10% reduced body weight, and strongly reduced susceptibility to luminal and to a lesser degree basal breast cancer compared to wild type animals. The latter finding mirrors human genetic data that associates the 8q24 gene desert variant more strongly with Estrogen Receptor-positive (ER+) than ER-negative (ER-) disease. In this study, we found that the MD-/- alters mammary gland development, resulting in a decrease in ductal branch points, fewer terminal end buds, and lower luminal/basal ratio. Using a reciprocal mammary gland transplantation assay, we found a strong donor effect and weaker host effect of the mutation on mammary gland development, indicating that the non-protein coding locus affects mammary cell-autonomous as well as non-mammary cell-autonomous processes. Interestingly, MD-/- resulted in a marked decrease in immune cell infiltrate in mammary tissues during development and throughout spontaneous tumor induction driven via the PyMT oncogene. Cellular profiling demonstrated a significant loss of mammary tissue-resident macrophage populations, but not monocyte-derived macrophages, with concomitant polarization away from homeostatic, developmental phenotypes which may be subverted to support breast cancer tumorigenesis. Using an integrative computational approach combining flow cytometry-based cell phenotyping with RNAseq-derived differential gene expression, we identified gene set and motif enrichment networks from mammary epithelium samples from wild type and knockout mice regulating mammary gland development, cancer processes, and immune function including inflammatory processes in regions affected by the MD locus. Taken together, these findings suggest that 8q24-mediated disruption of homeostatic mammary macrophage populations, either via MYC or yet undetermined mechanisms, may deprive neoplastic cells of crucial support from the stroma, inhibiting cancer initiation and progression. Our findings not only elucidate how the murine ortholog of the 8q24 gene desert, and likely MYC expression regulation, alters mammary gland development, but also provides novel insight into potential links between mammary development, immune infiltration, and breast cancer susceptibility.

#5008

Crosstalk between epithelial-IKKα-deletion and symbiotic bacterial-fungal infection in skin carcinogenesis.

Na-Young Song, Jami Willette-Brown, Feng Zhu, Yinling Hu. _NCI-Frederick, Frederick, MD_.

Bacteria and fungi, two major components of the microbiota, generally share niches and develop both antagonistic and symbiotic relationships, regulating the pathological impacts on the host. The epithelium is where the bacterial-fungal interaction occurs most abundantly, but the relationship between the epithelium, bacteria, and fungi on the pathogenesis of numerous diseases, particularly tumorigenesis, is poorly understood. IKKα is one of the crucial factors regulating the homeostasis of squamous epithelial tissues. Recently, our lab has established a mouse model that develops esophageal squamous cell carcinomas (SCCs) associated with IKKα reduction, inflammation and chronic fungal infection. Cladosporium cladosporioides was a major type of fungi identified in this mouse model. Because IKKα deletion in the keratinocytes causes impaired skin barrier, we hypothesized that loss of epithelial IKKα may control fungal colonization through regulating the barrier integrity and inflammation, resulting in tumor promotion. We thus generated IKKαf/f mice with inducible K15.Cre (IKKαf/f/K15.Cre) which is expressed in skin stem cells, which are important for the epidermal formation and hair follicle development. After ablating IKKα in K15 cells in the oral mucosa and skin, IKKαf/f/K15.Cre mice were orally inoculated with Cladosporium cladosporioides. Loss of epithelial IKKα showed disruption of skin barrier functions and bacterial colonization in the oral mucosa and skin, oral dysplasia and skin SCC development. Interestingly, oral infection with Cladosporium further promoted bacterial colonization as well as skin tumorigenesis in IKKαf/f/K15.Cre mice. This suggests that epithelial IKKα loss can provoke oral bacterial-fungal symbiosis which contributes to tumor promotion. To confirm this hypothesis, we treated Cladosporium-infected IKKαf/f/K15.Cre mice with amoxicillin, an antibiotic, through drinking water. Amoxicillin treatment remarkably reduced skin tumor incidence in fungal-infected IKKαf/f/K15.Cre mice, indicating that oral bacterial-fungal symbiosis indeed promotes skin SCC development driven by epithelial IKKα reduction. We also detected bacterial colonization in human skin SCC tissue array as well as IKKα mutation in 17% of human skin SCC patients from the Cancer Genome Atlas database, supporting that the crosstalk between epithelial IKKα and bacterial-fungal symbiosis in skin SCC promotion is relavent to human diseases. Taken together, our data demonstrate that loss of epithelial IKKα induces the bacterial-fungal symbiosis in oral mucosa, promoting skin tumors. This study will shed light on the importance of the epithelial-bacterial-fungal interaction in the pathogenesis, proposing epithelial IKKα as a novel regulator of the bacterial-fungal interaction.

#5009

Defined factors overcome T-cell exhaustion via abscopal effect.

Danny Khalil,1 Luis Felipe Campesato,2 Sadna Budhu,2 Yanyun Li,2 Caitlin Jones,2 Nathan Suek,2 Cailian Liu,2 Billel Gasmi,2 Rachel Giese,2 Stephane Pourpe,2 Taha Merghoub,1 Jedd D. Wolchok1. 1 _Mem. Sloan Kettering Cancer Ctr; Parker Institute for Cancer Immunotherapy, New York, NY;_ 2 _Mem. Sloan Kettering Cancer Ctr., New York, NY_.

Irreversible exhaustion of tumor-associated T cells is an important factor limiting the efficacy of PD-1 blockade in a number of cancers. Recent data suggest that PD-1 blockade functions largely by augmenting T cell costimulation. Given that activated APCs provide necessary T-cell costimulation, we hypothesized that enforced activation of intratumoral APCs would prime tumor-specific T cells and synergize with PD-1 blockade to overcome T-cell exhaustion. After screening various agents in syngeneic murine models, we found that dual CD40/TLR4 activation within a single tumor triggers a systemic tumor-specific CD8 T-cell response that is dependent on BATF3+ dendritic cells. Remarkably, this approach abolishes exhausted PD-1+ intratumoral T cells in treated as well as distant tumors while sparing more proximal T cells outside the tumors. In addition to treating large established tumors, this approach also confers persistent immunity allowing animals to reject reimplanted tumors 90 days after treatment. Dual CD40/TLR4 activation within a single tumor is thus a promising method for overcoming tumor-associated T-cell exhaustion in a manner that may provide durable systemic control of metastatic human cancers while sparing healthy tissues.

#5010

Advantages in using orthotopic syngeneic tumor models to evaluate immune-based approaches for cancer treatment.

Sylvie Maubant, Philippe Slos, Marc Hillairet de Boisferon, Francis Bichat, Jean-François Mirjolet. _Oncodesign S.A., Dijon Cedex, France_.

Immune checkpoint modulators are now accepted as the fourth pillar of cancer care for both solid tumors and hematological malignancies. In addition to CTLA-4, PD-1 or PD-L1 targeting antibodies, novel immune-oncology targets are currently tested in patients but also new strategies such as bispecific T cell engagers or cell therapies including CAR-T cells. Some of them have been already approved. Prediction of preclinical models is still a long-standing debate, even more when considering modulators of the immune system. Despite significant progresses made in humanized mouse model field during the last decade, having a complete and stable immune system comprising both lymphoid and myeloid subpopulation is not yet possible. Syngeneic tumors thus remain the model of choice to interrogate and better understand the mechanisms of action of new compounds targeting immune cell interactions. In order to improve prediction and relevance of these syngeneic models, orthotopic (OT) models were developed in parallel of standard subcutaneous (SC) models. Here, comparison of regular SC versus OT engraftments are provided. Various models, such as EMT-6 (breast), MBT-2 (bladder) as well as RenCa (kidney) will be described in addition to other OT models like Hepa1-6 (liver) or PAN-02 (pancreas) models. In addition to growth characteristics using imaging for OT models, antitumor efficacy of CTLA-4 and PD-1 targeting antibodies will be detailed for both SC and OT models. One of the most interesting model is MBT-2 (bladder) for which response to CTLA-4 targeting antibody is changing depending engraftment site: significant increase in survival was evidenced for the OT model (treated to control ratio (T/C) of 285%) and only moderate efficacy was observed for the SC model (T/C = 66%). For the PD-1 targeting antibody, OT injection seemed to be the preferential site to observe increased efficacy. Attempting to correlate immune profile and response to treatment, differences in immune infiltrate between OT and SC tumors will also be presented. 

# Wednesday, April 18, 2018

## TUMOR BIOLOGY:

### Applications of 3D Models for Tumor Biology and Therapeutic Studies

#5011

Patient-derived organotypic cancer spheroids (PDOCS) as predictive models for the treatment of cancer in a clinically meaningful time frame.

Cheri A. Pasch,1 Peter F. Favreau,2 Alex E. Yueh,1 Kwang P. Nickel,1 Christopher P. Babiarz,1 Philip B. Emmerich,1 Rosabella T. Pitera,1 Susan N. Payne,1 Demetra P. Korkos,1 Joseph T. Sharick,2 Carley M. Sprackling,1 Linda Clipson,1 Kristina A. Matkowskyj,1 Michael A. Newton,1 Melissa C. Skala,2 Michael F. Bassetti,1 Randall J. Kimple,1 Dustin A. Deming1. 1 _University of Wisconsin-Madison, Madison, WI;_ 2 _Morgridge Institute for Research, Madison, WI_.

Background: Standard two-dimensional cell cultures do not retain the key characteristics of the human cancers from which they are derived and treatment effects are not always able to be replicated in vivo, making the development of alternative culturing systems paramount. Specifically, commercially available cell lines do not fully represent the mutation profiles seen in human cancers. Here we investigate the feasibility of three-dimensional PDOCS to more accurately represent the cancers from which they are derived and to predict treatment sensitivity in a clinically meaningful time frame.

Methods: Surgical resection, core needle biopsies, paracentesis or thoracentesis samples from patients with various types of cancer were obtained under an approved IRB protocol, digested and spheroid cultures grown suspended in Matrigel. PDOCS were grown for up to two weeks and passaged at least once prior to treatment. PDOCS were imaged using brightfield imaging (4X) prior to treatment with vehicle or 5-fluorouracil (5-FU; 1, 10, or 100 µM) and/or radiation (2 or 5 Gy). After 2 days of treatment, the 5-FU was removed and the cultures were allowed to grow for an additional 2 days. PDOCS were re-imaged and the relative change in diameter was calculated using ImageJ software and compared to untreated controls. Optical metabolic imaging (OMI) was performed with a multiphoton microscope to probe the fluorescence lifetime and optical redox ratio of metabolic co-enzymes NAD(P)H and FAD. Single-cell analysis of each image was completed using Cell Profiler software to parse resistant cell populations in each PDOCS sample tested. DNA for mutation profile analysis was isolated and sequenced using a QIAseq targeted panel.

Results: PDOCS were successfully isolated from a variety of cancers including colorectal (CRC), pancreas, lung, neuroendocrine, liver, breast, and ovarian. Key phenotypic characteristics of the tumors were retained in PDOCS cultured including crypt-like structures, mucin production and Ki67 proliferation rates. Cancer hot spot sequencing was performed comparing PDOCS and the tumors from which they were derived. Over 90% of the nonsynonymous mutations were identical, except in the setting of microsatellite instability. All driver mutations were identical (i.e., APC, KRAS, PIK3CA, TP53). Differential sensitivity to chemoradiation was observed among 4 different colorectal PDOCS treated with 5-FU and radiation (Median % PDOCS diameter change vs control: Patient A 17.1, p=0.64; Patient B -3.05, p=0.02; Patient C -19.4, p=2x10-5; Patient D -31.3, p=0.002). Similar response data were determined using OMI; however, single-cell analyses identified potentially resistant cell populations.

Conclusions: PDOCS retain key characteristics of the cancers from which they are derived and can be utilized for treatment sensitivity testing in a clinically meaningful time frame.

#5012

Feasibility of manufacturing-scale bioproduction of novel next-generation 3D organoid cancer models in support of the Human Cancer Models Initiative.

James Clinton, Penney McWilliams-Koeppen, Siddhartha Paul, Allison Ruchinskas, Dezhong Yin, Robert Newman. _ATCC, Gaithersburg, MD_.

To meet the need for improved approaches to study cancer in vitro, there has been a surge in the development of novel research models utilizing advanced culture methods. These methods permit in vitro growth of cancer types previously not possible, and/or models with enhanced in vivo relevancy compared to traditional continuous cell lines. However, availability of these early-stage research models is currently limited and there is a lack of data on the ability to scale up production of these models to support the needs of the global research community. We sought to investigate the protocols, expansion capacity, cryopreservation ability, genetic stability, and feasibility of larger-scale bioproduction of a subset of the models generated by the Human Cancer Models Initiative (HCMI), an international collaborative effort between the National Cancer Institute, the foundation Hubrecht Organoid Technology, Cancer Research UK, and the Wellcome Trust Sanger Institute. The HCMI's initial goal is the development of 1,000 novel human cancer models, paired with bioinformatics and patient clinical data, particularly from rare or underrepresented cancer types. One advanced culture method being utilized, three-dimensional organoid "microtissue" culture, potentially poses challenges for traditional large-scale bioproduction processes. It requires growth embedded within an undefined extracellular matrix and complex media formulations containing multiple small molecules and recombinant proteins with unknown stability and shelf-life. Additionally, organoid growth media typically includes multiple sources of undefined conditioned media containing critical growth factors. We cultured organoid models derived from human colon, pancreas, esophagus, and mammary tissues developed by laboratories contributing to the HCMI. Multiple unique donors were available for all tissues and both cancer and non-cancer models were available for two tissue types. Most models were maintained in culture continuously for at least 60 days (7-27 population doublings, > 10 passages). Tissue and donor variability was evident in model characteristics, including morphology (assessed by microscopy and immunocytochemistry), growth rate, and genetic stability (measured by short tandem repeats analysis). All models were amenable to scale up beyond multiwell plates, and all models could recover from cryopreservation. While organoid culture represents a significant divergence from typical two-dimensional monolayer culture of continuous cell lines, our results show that these next-generation in vitro models are suitable for larger-scale bioproduction. This is vital to ensure the widespread availability of these models within the research community to facilitate applications like pre-clinical drug discovery and basic cancer research.

#5013

Rapid generation of phenomic and functional profiles of patient-derived 3D cell culture models for identification of treatment vulnerabilities of breast cancer: Early results of the EFRE-PoP project.

Verena I. Kiver,1 Guido Gambara,2 Olga Gorea,3 Jens-Uwe Blohmer,1 Philipp Jurmeister,1 Carsten Denkert,1 Alessandra Silvestri,4 Caroline M. Schweiger,1 Maxine Silvestrov,4 Ulrich Keilholz,1 Christian R. Regenbrecht4. 1 _Charité - Universitätsmedizin Berlin, Berlin, Germany;_ 2 _Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany;_ 3 _Charite - Universitätsmedizin Berlin, Berlin, Germany;_ 4 _cpo - cellular phenomics & oncology, Berlin, Germany_.

Targeted treatment for breast cancer subsets currently relies on the occurrence of estrogen, progesterone and Her2/neu receptors. For triple negative breast cancer (TNBC) there is no identified targeted therapy. The mutational landscape for breast cancer subsets has been characterized, but drug development has been limited due to the lack of appropriate preclinical models. Development of patient-derived xenograft models (PDX) has been difficult with take rates of below 30%.To establish a series of patient-derived 3D (PD3D) cell culture models as a versatile resource for ex vivo drug sensitivity screens as well as secondary establishment of PDX. We obtained breast cancer specimens either by biopsy or surgical resection. Upon arrival tumor tissue was minced and enzymatically digested. After subsequent filtering, respective tissue size fractions were seeded as Matrix-droplets into 24-well plates and incubated under standard conditions. Growth of PD3Ds was monitored daily by microscopy. PD3Ds were splitted when their diameter reached appropriate size. Organoids were than fixed and embedded. FFPE sections of donor-tissues and derived PD3D models were than used for IHC and inspected by a pathologist. In parallel, mutational profiling of snap-frozen tumor tissue and cell cultures was performed. All models were subjected to a semi-automated multi-drug response assay in a 384-well format to assess individual compound sensitivities.We established a scalable workflow for culturing and screening of PD3D cell cultures from limited quantities of breast cancer tissue from true-cut and vacuum biopsies as well as surgical specimen. Tissue fragments of ca. 3mm in diameter were sufficient to successfully establish PD3D cell cultures at a high yield. The time needed to expand PD3D cell cultures to obtain a sufficient amount of cells for subsequent molecular analyses varies from 2-6 weeks depending on amount and quality of samples as well as grade and stage of the donor tumor tissue. At the time, we can report a rate of culture establishment rate of 75% (11 models from 15 patient samples) for various clinical relevant subgroups, including TNBC, HR+, Her 2 pos. BC and one DCIS sample. The immunohistology and mutational profiles of these samples are currently being confirmed. The established workflow for culturing PD3D cell cultures offers high yield rates within a potentially clinically relevant time-frame for HTP cytotoxicity screenings. We are encouraged that this method is suitable for various molecular subtypes of breast cancer. Once adequately validated in co-clinical trials, PD3D models would make for an intriguing tool in supporting clinical decision-making for individual patients. Particularly patients with breast cancer refractory to standard of care compounds could benefit from this approach.

#5015

Drug screening and phenotypic analysis in a microwell-based 3D cell culture system.

Michael Hiatt, Marta Mroczek, Eric Jervis, Terry E. Thomas, Allen C. Eaves, Sharon Louis. _Stemcell Technologies Inc, Vancouver, British Columbia, Canada_.

Three-dimensional (3D) cell culture models provide more physiologically relevant drug and toxicity screening platforms than traditional 2D platforms (1). Phenotypic analysis is an informative endpoint for high-throughput screens; however, variability in 3D culture systems can make statistically sound identification of good candidate compounds from a small number of replicates difficult. To minimize this variability we tested AggreWell™, a microwell-based 3D cell culture device that is a prototype system for 3D culture including drug and toxicity screening. The geometry of this system provides precise localization, segmentation and uniformity of spheroids derived from various cell types, and is predicted to minimize experimental variability. To test this directly, estrogen receptor-expressing MCF-7 breast cancer cells were seeded into AggreWell™400 plates in MammoCult™ medium in triplicate, and 24 hours after seeding, the resulting spheroids were treated with a single drug or combination of tamoxifen (TMX), the HER2 and EGFR inhibitor lapatinib (LTB), and the γ-secretase and Notch inhibitor DAPT. Three days later, the spheroids were imaged for the following parameters: size, brightness, shape, and specific morphologic features. Spheroids were also dissociated for viable cell counts. The IC50 values for drug effects for TMX and LTB are 21.6 and 6.3 uM, respectively, whereas DAPT had no effect on the spheroids at doses up to 100 uM. These results are consistent with previous published results and allow for correlation of observed changes in morphologic and cell viability post-treatment. For combinatorial drug treatments, concentrations of 20 uM TMX, 5 uM LTB, and 10 uM DAPT were used. TMX treatment exhibited the greatest reduction of cell reduction (52%). Combination of TMX with DAPT, LTB, or both, resulted in synergistic reductions of viability of 64%, 74% and 83%, respectively. Morphologic features of these cultures analyzed by principal component analysis reveal that parameters including circularity and gray values are associated with untreated cultures, whereas spheroid perimeter and area are associated with treated cultures. Graphing combinations of these parameters, for example, area vs. integrated density, delineates a clear decision plane for classifying treated versus untreated conditions. Analysis of variability in aggregate morphology demonstrates that measurement of multiple spheroids per well in AggreWell™ increased experimental power (e.g., power of 0.8 for 16 spheroids versus 0.3 for 4 spheroids, respectively; 𝛼;; = 0.05) and reduced experimental variance. These results confirm that morphologic analysis of spheroids grown on AggreWell™ plates is highly suited for high-throughput 3D drug screening.Reference: 1. Lee GY et al. Nat. Methods 2007.

#5016

A unique ex vivo drug evaluation model: 3D co-cultured system with tumor, stroma and blood microvessels.

Yuki Takahashi,1 Kei Tsukamoto,1 Shiro Kitano,1 Shinji Irie,1 Michiya Matsusaki,2 Satoshi Nagayama,3 Ryohei Katayama,4 Eiji Shinozaki,3 Naoya Fujita4. 1 _Toppan Printing Co., Ltd., Saitama, Japan;_ 2 _Graduate School of Engineering, Osaka University, Osaka, Japan;_ 3 _Cancer Institute Hospital, Japanese Foundation for Cancer Research, Tokyo, Japan;_ 4 _Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan_.

Background; Two dimensional single-layer culture still remains the preferred platform for most laboratory preclinical studies, although interaction between cancer and stromal cells has been well reported to be important in tumor progression and resistance against therapies. In order to mimic the patient tumor tissues, ex vivo model which recaptures the tumor microenvironment is required.

Methods; Layered 3D stromal tissues were produced by culturing normal human dermal fibroblasts (NHDFs) and human umbilical vein endothelial cells (HUVECs) coated with extra-cellular matrix (ECM) and natural polysaccharide, namely collagen and heparin. The layered 3D stromal tissues and overlaid tumors were morphologically characterized by HE stain, immunohistochemistry (IHC) and immunofluorescence (IF). Furthermore, drug sensitivity assays were conducted using popular colorectal cancer cell lines, and patient-derived cell lines (PDCs) established in the laboratory of Japanese Foundation for Cancer Research. Cancer cell viability was evaluated by fluorescent labeling, enzymatic dissociation and cell counting analysis. IF with cancer specific markers and imaging analyses were also performed.

Results; The 3D stromal tissues including CD31 positive luminal structure were multi-layered (approximately 20 layers), and formation of microvascular network was observed within several days. In comparison with 2D mono-culture or 3D mono/co-cultured spheroid model, decreased drug sensitivities were represented in our 3D co-cultured model. In the simultaneous treatment with cytotoxic anticancer agents and molecular targeted drugs, dose-responses were significantly different between the2D and 3D models.

Conclusion; We developed the layered 3D stromal tissue culture system including blood micro-vessels. Drug evaluation with the co-cultured tumors may reflect the drug sensitivity of cancer cells in vivo. Our unique 3D ex vivo model represents a valuable tool for drug development in a fully human cell and matrix microenvironment, and thus testing patient-derived cells and approved compounds also enable better prediction their efficacy.

#5017

Characterization of novel pancreatic tumor organoid lines generated under hypoxic and pressurized conditions reveals molecularly distinct subclones relevant for target discovery and drug screening applications.

Zhe Wang,1 James I. Lim,2 Yan Kristoff Wang,3 Jie Fu,4 Haifeng Song5. 1 _GS Medical Science and Technology Development Co., Ltd., Beijing, China;_ 2 _Xcell Biosciences, San Francisco, CA;_ 3 _GS medical Science and Technology Development Co, Ltd., Beijing, China;_ 4 _Beijing C &N International Sci-tech Co., Ltd., Beijing, China; _5 _National Center for Protein Sciences Beijing, Beijing, China_.

Cancer is a complex and heterogeneous disease, composed of molecularly distinct tumor cells and various supporting cell types. Pancreatic cancer in particular is associated with poor prognosis and low survival, with majority of patients exhibiting metastasis at diagnosis. Therefore, development of future therapies requires disease-relevant cell models generated from patient biopsies for target discovery and drug screening applications. To investigate the heterogeneity and aggressive nature of pancreatic cancer cells, we collected tumor biopsies from patients with pancreatic ductal adenocarcinoma (PDAC) and created novel organoid lines expanded under discrete oxygen and pressure conditions to mimic the metastatic microenvironments of the liver and bone. Fresh tumor biopsies from four patients with PDAC were dissociated into single cells and cultured using serum-free culture media supplemented with recombinant growth factors. Organoids were passaged and expanded in co-culture with tumor-derived stromal cells at 1% O2 or 5% O2, and 0 PSI or 2PSI. We performed whole-exome and transcriptomic sequencing to characterize organoid subclones propagated under low oxygen and pressurized culture conditions (n = 10). Sequencing analysis revealed mutations commonly associated with PDAC, including KRAS, TP53, CDKN2A and SMAD4. Organoids could be distinctly categorized based on the unique combination of these mutations present in a subclone, along with changes in gene copy number of known oncogenes. As expected, transcriptomic profiling of subclones showed significant differences in gene expression as a function of the oxygen concentration (1% O2 vs. 5% O2) and in the presence of applied atmospheric pressure (0 PSI versus 2 PSI). Drastic changes in gene expression governing metabolic processes were observed under low-oxygen and high-pressure culture conditions. These include genes regulating glucose uptake and glycolysis, such as glucose transporters, lactate dehydrogenases, and hexokinases. Interestingly, genes involved in stem cell maintenance were also upregulated (OCT4, SOX2, and NANOG). These findings indicate that organoids generated under low oxygen and pressure retain both the genetic features and clonal architecture of PDAC. Continued evaluation of these cells through functional and xenograft studies is currently under way.

#5018

Bioprinted (3D) co-cultured spheroids with NSCLC PDX cells and cancer associated fibroblasts (CAFs) using alginate/gelatin hydrogel.

Arindam Mondal,1 Aragaw Gebeyehu,1 Ramakrishnan Subramanian,2 Arun Rishi,3 Mandip Singh1. 1 _Florida A &M University, Tallahassee, FL; _2 _Florida State University, Tallahassee, FL;_ 3 _Wayne State University, Detroit, MI_.

3D bioprinting which is a significant improvement over 2D models offers layer by layer positioning of various cells which can provide superior cell-matrix interactions in the tumor microenvironment in vitro. The objective of the present study was to develop a co-culture model using Non-small cell lung cancer (NSCLC) patient derived xenograft (PDX) cells and lung cancer associated fibroblasts (CAFs) in sodium alginate/ gelatin hydrogel using a 3D bioprinter.

Various concentrations of sodium alginate/gelatin hydrogels (w/w) were prepared and rheological properties were evaluated. PDX cells (EGFR T790M, obtained from Dr. Rishi's Laboratory, Wayne State University) and CAFs (AA0022, obtained from Dr. Noyes Laboratory, Moffit Cancer Center) were mixed with the hydrogel and printed usingINKREDIBLE bioprinter (Cellink, Sweden). A two layered cube was designed in Slic3r software, converted to g-code file and printed through a 22G needle. Printed scaffolds were cross linked by Crosslinking Agent (Cellink, Sweden) for 5 minutes, washed with HBSS and maintained with media at 37°C and 5% CO2. Spheroid formation was observed using an inverted microscope (Olympus IX70). Live/Dead and NucBlue/ActinGreen staining was performed to determine cell viability and spheroid formation. Immuno-fluorescence staining was performed for E-cadherin, α-SMA and vimentin to study cellular crosstalk within the spheroid.

Printable hydrogels were prepared by adding 3.25% sodium alginate to 4% gelatin which was found to be the most optimal mixture. Decrease in storage modulus (G') from 995.29± 30.69 Pa to 12.16± 8.83 Pa and loss modulus (G") from 314.10± 17.54 Pa to 22.22± 12.69 Pa was observed from 18°C to 37°C when using a temperature sweep test. At lowest shear rate (1s-1), the viscosity of the hydrogel was 2.75± 0.37 Pa.s and at 100s-1 the viscosity was 0.50± 0.12 Pa.s. Decrease in hydrogel viscosity with increasing shear rate confirmed shear thinning. The height, width and length of the printed scaffolds were found as 1.45± 0.06 mm, 9.56± 0.15 mm and10.67± 0.13 mm respectively. Formation of spheroids was observed after 4 days and after 15 days, 59.88± 14.81% small (0-400µm2), 15.43± 4.86% medium (401-1000 µm2) and 24.67± 10.14% large size spheroids (1001 µm2-above) were observed which indicated proliferation of individual cells within the scaffolds. LIVE/ DEAD assay showed approximately 82% cells were viable in the printed scaffold. Up-regulation of vimentin and α-SMA and concomitant loss of E-cadherin was observed in the printed co-cultures indicating cross talk between the two cells.

This study demonstrates the preparation and characterization of NSCLC lung PDX and CAF co-culture spheroids in 3D bio-printed sodium alginate/ gelatin scaffold. This model can be further used for high throughput drug screening platform.

#5019

Everolimus in combination with ABT263 in patient-derived organotypic neuroendocrine tumor spheroids.

Christopher P. Babiarz,1 Amani Gillette,2 Mitchell G. Depke,1 Cheri A. Pasch,1 Susan N. Payne,1 Carley M. Sprackling,1 Linda Clipson,1 Kristina A. Matkowskyj,1 Melissa C. Skala,2 Dustin A. Deming1. 1 _Univ. of Wisconsin, Madison, WI;_ 2 _Morgridge Insitute for Research, Madison, WI_.

Background: Neuroendocrine tumors (NETs) are among the most understudied types of human cancer. This is partially a result of their uncommon occurrence, but is mainly related to the lack of models that represent the human disease. Spheroid cultures effectively recapitulate the physiology of these tumors. For the current treatment of patients with NETs everolimus, an mTOR inhibitor, is commonly used, but with limited efficacy. A means by which to enhance the response of NETs to everolimus is a great clinical need. Therefore, we sought to determine the potential of the proapoptotic agent ABT263 (a BCL-2, BCL-xL, and BCL-W inhibitor) to enhance the response to everolimus in NET spheroids.

Methods: NET spheroids derived from patient biopsies or surgical samples (n=6) were digested, plated for spheroid culture in Matrigel, and covered with media supplemented with epidermal growth factor. Cultures were treated with control (normal feeding media), 200 nM everolimus, 250 nM ABT263, or the combination. Brightfield images (4x) of the spheroids were taken pre- and 48 hours post-treatment and changes in spheroid diameter measured. Additionally, optical metabolic imaging was performed, examining the inherent auto-fluorescence of NADH and FAD+, as a marker of the metabolic activity of the cancer cells and an optical redox ratio measured. Spheroid culture whole mounts were stained pre- and post-treatment using immunofluorescence.

Results: A total of 5 different patient-derived NET lines were generated from surgical specimens. In all instances the Ki67 index and synaptophysin/chromogranin A staining demonstrated that these cultures are similar to the tumors from which they are derived. Everolimus and the combination of everolimus and ABT263 resulted in a significant reduction in median spheroid diameter compared to the control and ABT263-only groups in 3 of the 5 NET spheroid lines with a change in median spheroid diameter of -30.16%, -15.94%, and -3.34% (p<0.001). Optical metabolic imaging demonstrated similar efficacy to that seen with the diameter measurements with significant responses especially in the setting of the combination treatment. Reduction of cell proliferation and induction of apoptosis in the combination treatment were both confirmed using immunofluorescence imaging for Ki-67 and cleaved caspase 3, respectively.

Conclusion: This study establishes the utility of NET spheroids as a more representative model of this otherwise difficult to study cancer. The addition of ABT263 to everolimus enhances the treatment response for a subset of NETs. Future investigations will identify the molecular subset most likely to respond and explore this combination regimen clinically.

#5020

A patient-derived organoid model of neuroendocrine prostate cancer transdifferentiation informing the role of the BAF complex component ARID1A.

Mike L. Beshiri, Caitlin M. Tice, Adam G. Sowalsky, Crystal Tran, Fatima Karzai, William Dahut, Kathy Kelly. _NCI, Bethesda, MD_.

Metastatic castration-resistant prostate cancer (mCRPC) is a genetically and phenotypically heterogeneous disease. Resistance to contemporary androgen-deprivation therapy (ADT) is inevitable, and occurs via several different mechanisms. One of these is lineage plasticity leading to transdifferentiation of AR-positive adenocarcinoma (ACPC) to the neuroendocrine (NEPC) subtype. The modern era of second-generation ADT has seen a significant increase in the percentage of NEPC, yet little is known about the genetic and epigenetic drivers of this phenotype. Using modified organoid media conditions that we previously developed, we have established a set of mCRPC organoid lines derived from four biopsies (two samples before and two after carboplatin + etoposide treatment, 1 year apart) of a single patient tumor that capture the genetic and phenotypic heterogeneity of the original metastatic tumor. Genomic and histologic analysis show that the original patient tumor is composed of both adenocarcinoma and neuroendocrine subpopulations that are maintained in the organoids at a ratio of approximately 1:4, respectively. The two subpopulations share a common ancestor and a putative early driver event in the homozygous loss of CDKN1B, but our analysis of clonal evolution indicates that the majority NEPC population did not transdifferentiate directly from the minority ACPC population in the metastasis, but rather in the primary tumor or in a prior metastatic lesion. The ACPC subpopulation contains a high copy gain of an upstream regulatory region of the AR gene and is genetically distinct from the NEPC population that lacks this alteration, but uniquely contains a truncating mutation in the ARID1A gene. ARID1A is frequently mutated and/or deleted in several different cancer types. It has been found to be altered in ~2% of prostate cancer patients, but its role is not understood. It is a key component of the BAF complex, which is a known regulator of neural development. Immunofluourescent staining of our organoid cells shows that mutant ARID1A correlates with the NEPC population. Using single-cell RNA-seq and principle component clustering analysis, we have identified five different transcriptionally defined populations: one population that corresponds to the genetically defined ACPC population and four additional transcriptionally defined populations that represent different states of differentiation of the NEPC component, as determined by expression of lineage markers. A full understanding of this model with respect to differentiation of the NEPC population and the specific role of ARID1A in the process will inform the development of mechanism-based treatments to inhibit NEPC formation.

#5021

Assessment of RAS-RAF-MAPK pathway perturbations and PD-L1 expression in an Isogenic 3D tissue-culture model of drug-resistant melanoma.

Elizabeth Turner,1 Luping Chen,1 Johnathan Haag,1 Alexei Miagkov,1 Lysa-Anne Volpe,1 Metewo Selase Enuameh,2 Robert Newman,2 Fang Tian1. 1 _ATCC, Manassas, VA;_ 2 _ATCC Cell Systems, Gaithersburg, MD_.

Development of the next generation of anti-cancer drugs, biologics, and immunotherapies is currently hampered by extremely poor success rates of seemingly promising experimental therapies in human clinical trials. This poor success rate is partially due to a lack of biologically relevant cancer model systems. The 2D culture system, while relatively inexpensive and easy to use, may not always be adequately representative of the tumor microenvironment. Conversely, the use of animal models is costly and time consuming. The 3D culture system is able to represent biologically relevant complexities such as cell-cell communication, differential proliferation rates, and compound penetration. Combining 3D culture with CRISPR/Cas9 genome editing technologies can generate powerful cell-based model systems with both physiological relevance and well-controlled genetic and drug-susceptibility profiles. These new models will enable the investigation of specific molecular mechanisms, biofunctional outcomes of newly identified genetic alternations, and targeted therapeutic drug responses within a more biologically intricate context. In this study, we use CRISPR/Cas9 to generate isogenic drug-resistant melanoma models that can be used as either 2D or 3D cancer models, as well as for studies of the acquired drug resistance. Two different models were generated starting with the BRAF-inhibitor sensitive A375 melanoma cell line. We introduced either the NRASQ61K or the KRASG13D point mutations, both of which are known to confer BRAF inhibitor resistance and are commonly encountered in BRAF-resistant tumor samples. We then assessed the susceptibility of these new isogenic lines to BRAF inhibitors in both 2D and 3D model systems. We also determined the specific effect of these point mutations on the RAS-RAF-MAPK signaling pathway, a key component of cell-cycle escape and tumor proliferation. Furthermore, we assessed the impact of these mutations on the expression of Programmed Death-Ligand 1 (PD-L1), which recent advances in cancer immunology have directly linked to cancer immune evasion and poor clinical outcomes. Our results show that A375 melanoma isogenic cells carrying KRASG13D have dramatically increased EGFR expression levels, while isogenic cells carrying NRASQ61K have constant activation of the MEK-ERK pathway. We also found a significant increase in PD-L1 expression in KRASG13D, but not in NRASQ61K A375 cells, indicating that expression of PD-L1 is directly linked to KRAS mutation or the downstream effects thereof, rather than to a general increase in RAS-RAF-MAPK signaling. These results have direct implications for cancer immune checkpoint molecule studies, and highlight the utility of these isogenic melanoma models in both 2D and 3D applications in research and development of novel anti-cancer drugs and combination therapies.

#5022

Precision medicine: High-throughput 3D bioprinting of embedded multicellular cancer spheroids.

Lakmali Atapattu,1 Robert Utama,2 Aiden O'Mahony,3 Christopher Fife,1 Jongho Baek,2 Théophile Allard,2 Kieran O'Mahony,4 Julio Ribeiro,3 Katharina Gaus,2 Justin Gooding,2 Maria Kavallaris1. 1 _Children's Cancer Inst. Australia, Randwick, Australia;_ 2 _University of New South Wales, Australia;_ 3 _Inventia Life Sciences, Australia;_ 4 _Omko, Ireland_.

Three-dimensional (3D) cancer cell models, such as 3D multicellular spheroids, are superior models of in vivo systems than the more popular two-dimensional (2D) cell culture. 3D cancer spheroids provide a closer reflection of tumor gene expression and biology than 2D cultures. This is particularly relevant for drug development and precision medicine programs were cell culture conditions that better reflect the 3D tumor environments, including matrix stiffness, would be highly advantageous. A key challenge for the routine application of embedded 3D cancer spheroids in precision and drug programs is the lack of high throughput and uniform production of spheroids in a biocompatible matrix. To address this, we have developed a high-throughput method of producing 3D multicellular cancer spheroids embedded inside a tissue-like matrix using a custom-built drop-on-demand 3D bioprinter. The 3D bioprinter is capable of printing a cell-suspension ink with up to 300 million cells/mL and a cell viability greater than 95%. We have validated the 3D bioprinting using glioblastoma, neuroblastoma and lung cancer cells. The 3D bioprinted spheroids were confirmed to maintain all the biological characteristics typically found in a cancer spheroid. In particular, the 3D bioprinted cancer spheroids were shown to be viable and proliferating, preserved the apoptotic and cancer-stemness characteristics and using super-resolution lattice-light sheet microscopy, we showed the spheroids maintained the same structural integrity as manually formed spheroids. The potential application of the 3D bioprinted spheroids for high-throughput drug screening in 3D environments was demonstrated using doxorubicin as the model drug. 3D-bioprinting of patient-derived cancer cells for precision medicine applications is now underway. High-throughput embedded 3D bioprinting has enormous potential to accelerate precision medicine, drug discovery and cancer biology.

#5023

A 3D perfusable platform for high-throughput screening diverse racial/ethnic prostate cancer specimens.

Lindsey Sablatura,1 Kristin M. Bircsak,2 Mary C. Farach-Carson,3 Nora Navone,4 Peter Shepherd,4 Thomas Zarembinski,5 Pamela E. Constantinou,1 Anthony Saleh,2 Daniel A. Harrington3. 1 _Rice University, Houston, TX;_ 2 _Mimetas, Gaithersburg, MD;_ 3 _The University of Texas Health Science Center Houston School of Dentistry, Houston, TX;_ 4 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 5 _BioTime, Alameda, CA_.

Prostate cancer (PCa) incidence and mortality in African American men is more than 1.5 times greater than men of other races/ethnicities (i.e., Caucasian, Hispanic, American Indian/Alaska Native, Asian/Pacific Islander) in the United States. While elucidating the underlying mechanisms for this cancer health disparity is critical to patient treatment and improved patient outcome, currently, there are no in vitro models available to adequately recapitulate the PCa tumor microenvironment (TME) and aid in this effort. Advanced in vitro culture conditions, such as extended ex vivo culture of patient-derived xenografts (PDXs), 3-dimensional (3D) culture of cells within a defined extracellular matrix (ECM), miniaturization to formats compatible with high-throughput screening (HTS), controlled media perfusion, and co-culture of cancer, stroma, and endothelium, are key elements for improving PCa study, but few systems include two or more of these features. We have optimized culture conditions of a 3D in vitro model system, the OncoPlate, which mimics the PCa TME. In the OncoPlate, cells are seeded in engineered HyStem® hyaluronic acid (HA)-enriched hydrogels, optionally modified with migration-permissive peptides to mimic the tumor ECM. PDX-derived PCa cells are seeded in 3D in MIMETAS' microfluidic OrganoPlate® platform and co-cultured with stromal fibroblasts and endothelial blood vessel mimics under continuous perfusion. We predict that this engineered "tumor-on-a-chip" will better predict patient responses and, by incorporating PCa cells from patients with diverse racial backgrounds, support cancer health disparity research. Here we report culture of several PDX-derived PCa models of diverse racial origin cultured in our OncoPlate format. Live/dead staining followed by automated high content fluorescence imaging was utilized at various time points to characterize viability and growth rate of each model. Immunofluorescent staining for PSA (PCa cells), FSP (cancer associated fibroblasts), and VE-cadherin (endothelial cells) was used to display phenotype and health of each cell type in the culture over a week and formation of 3D structures characteristic of prostate cancer histology. Studies are ongoing to screen each model with a panel of chemotherapeutic drugs for which in vivo mouse PDX response data is available with the goal to improve predictive accuracy by using our perfusable OncoPlate PCa models.

#5024

Drug screening of biopsy-derived multicellular spheroids using microfluidic technology.

Theresa E. Mulholland,1 Milly McAllister,2 Samantha Patek,2 David Flint,1 Mark Underwood,3 Alexander Sim,4 Joanne Edwards,2 Michele Zagnoni1. 1 _University of Strathclyde, Glasgow, United Kingdom;_ 2 _University of Glasgow, Glasgow, United Kingdom;_ 3 _Queen Elizabeth University Hospital, Glasgow, United Kingdom;_ 4 _AMS Biotechnology (Europe) Ltd, Abingdon, United Kingdom_.

Performing drug screening, of physiologically relevant three-dimensional (3D) tumor models, for personalized treatment remains challenging, due to the small amount of tissue available from most biopsies. New microfluidic technologies, enabling greater control over cell positioning and fluid behavior at the micro-scale, allow extensive testing of anticancer agents on human tumor tissue preparations in 3D and offer new solutions for the development of anticancer compounds and personalized medicine. We have developed a microfluidic platform for extensive drug screening of tumor biopsies in a cost-effective manner and validated the system with tumor prostate patient samples. As a typical drug screening assay, up to 22 drug concentration-response curves could be generated from a single biopsy, within a time frame of up to 4 weeks. Biopsy tissue, grown as a heterogeneous co-culture from the primary sample, was prepared as cancer-cell enriched multicellular spheroids, cultured for 3 to 5 days prior to the application of a panel of standard-of-care drugs for prostate cancer. Readouts were obtained via bright-field and epifluorescence microscopy. The microfluidic platform was designed to be operated entirely without the need of external fluid actuation, with the microfluidic network capable of generating long-lasting, stable and repeatable drug concentration gradients across arrays of 240 spheroids. Outcomes were generated as 8-point drug concentration response curves per device, with each drug concentration tested on at least 24 spheroids. In-house developed software was used to analyze bright-field and fluorescent images to provide readouts of spheroid growth and viability, as well as information on drug penetration and drug efficacy over time. Following platform and assay validation using cancer cell lines, proof-of-concept screening was performed on prostate biopsies from 2 different patients. Results showed that biopsy-derived spheroids were more resistant to treatment than LNCaP spheroids, a prostate cancer cell line. For one biopsy, spheroids were sensitive to docetaxel, but resistant to enzalutamide, despite the presence of intact androgen receptors. This preliminary data outlines how this technology could become a useful tool to investigate patient-specific drug resistance and to test novel anticancer agents in a cost-effective manner, based on maximized screening of human tumor tissue in a 3D format.

#5025

From OR to bench: Identification of variables affecting success in generating patient derived organotypic spheroids (pDOTS).

Dalia Larios,1 Amir Aref,2 Elena Ivanova,2 Brandon Piel,2 Andrew Portell,2 David A. Barbie,2 Raphael Bueno,3 Cloud Paweletz2. 1 _Harvard Medical School, Boston, MA;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _Brigham and Women's Hospital, Boston, MA_.

Background: The complex interaction between tumor cells and the immune system has led to increased efforts focused on development of ex vivo systems that recapitulate the tumor microenvironment and model responses to immune checkpoint blockade. Recently, we demonstrated successful ex vivo growth of organotypic tumor spheroids and profiling of PD-1 blockade in syngeneic models and patient tumors1. Optimization of tissue processing from the operating room to the lab for patient-derived materials can further aid these efforts. Here, we identify pre-analytical variables that may impact tumor characterization in ex vivo systems.

Methods: We conducted an evaluation of pre-analytical variables for tumor tissue in a cohort of mesothelioma and non-small cell lung cancer (NSCLC) cases. Pre-analytical variables included neoadjuvant chemotherapy administration, pleurodesis status, type of surgery, presence of fibrosis, and onset of warm and cold ischemia during tissue retrieval and processing. Prior to surgery, medical records were reviewed for pathology, chemotherapy, and pleurodesis status. On surgery day, time of tissue removal or clamping of the pulmonary artery was recorded as onset of warm ischemia. Tissue was transported to pathology and allocations to research were made. Specimens were placed in culture media and ice; placement on ice was recorded as onset of cold ischemia. Arrival of tissue in lab was recorded. Chart and observational data were correlated with tissue analysis at the bench followed by 4-color immunofluorescence to characterize tumor and immune components of spheroids.

Results: A cohort of mesothelioma and NSCLC samples were followed from the operating room to the research lab. The average time between onset of warm and cold ischemia was 60 minutes (range 33-100 min). Mean time for removal of tissue in the OR to arrival in lab was 104 minutes (range 87-137 min). Two patients received neoadjuvant chemotherapy and were among 2 of 3 cases where fibrotic samples were collected. Fibrosis was often associated with more extensive surgery via extrapleural pneumonectomy and lower tumor content per microscopy and immunofluorescence.

Conclusions: Prolonged ischemic times were associated with poor tissue viability. In two cases, pre-therapeutic status was linked to tissue fibrosis, extensive surgery, low tumor yield, and failed spheroid generation. More data can help elucidate variable significance. These initial findings highlight the unique challenges associated with performing studies on live tumor tissue, and suggest that further streamlining of processes for tissue collection in the OR can optimize success of ex vivo cancer models in guiding functional precision therapies.

#5026

Alcohol enriches therapy-resistant cancer cells with high autophagy, CD44 expression and tumor initiating capability.

Koji Tanaka,1 Takashi Kijima,2 Prasanna Modayur Chandramouleeswaran,2 Kelly A Whelan,3 Yuta Kasagi,2 Masataka Shimonosono,4 Haruna Furukawa,1 Takeo Hara,1 Tomoki Makino,1 Makoto Yamasaki,1 Andres J Klein-Szanto,5 Shoji Natsugoe,4 Masaki Mori,1 Yuichiro Doki,1 Hiroshi Nakagawa2. 1 _Osaka University, Suita, Japan;_ 2 _University of Pennsylvania, Philadelphia, PA;_ 3 _Temple University, Philadelphia, PA;_ 4 _Kagoshima University, Kagoshima, Japan;_ 5 _Fox Chase Cancer Center, Philadelphia, PA_.

Introduction: Esophageal squamous cell carcinoma (ESCC) is the deadliest of all human cancers with alcohol (EtOH) as a major risk factor. Malignant properties of ESCC have been attributed to ESCC cells with high CD44 expression (CD44H) and autophagy; however, the pathogenic role of EtOH in ESCC remains elusive

Methods: With a goal of translation in personalized medicine, we analyzed therapy-naïve ESCC tumor biopsies from patients and ESCC cell lines in three-dimensional (3D) organoids for functional characterization of ESCC cells following exposure to 5-fluorouracil (5FU) or EtOH ex vivo. Tumor growth was assessed in EtOH-fed mice carrying xenograft tumors with two independent ESCC cell lines in the presence or absence of 4-methylpyrazole (4MP), an alcohol dehydrogenase inhibitor. 3D organoids and tumors were analyzed by flow cytometry and immunostaining for CD44, Ki67, p53 and autophagic vesicle (AV) content.

Results: ESCC patient biopsies contained CD44H cells with increased AV content. ESCC 3D organoids were grown successfully from 11 out of 16 tumors (68.8%) and passaged with recapitulation of the histopathology, proliferation, p53 and CD44 expression and autophagy present in the original in situ tumors. Successful organoid formation was significantly associated with poor chemoradiation therapy response (progressive and stable diseases, n=10 vs. partial response, n=6). In 3D organoids surviving 5FU treatment, CD44H cells with high autophagic activity was found to be enriched. Pharmacological autophagy flux inhibition by chloroquine augmented 5FU-mediated cytotoxicity in 3D ESCC organoids. 5FU-resistant CD44H cells were more capable of forming 3D organoids compared to bulk populations of ESCC cells. EtOH promoted expansion of CD44H cells with significantly increased proliferation and organoid formation rate upon serial passages, suggesting the increased self-renewal of CD44H cells. In xenograft tumors, alcohol drinking not only promoted tumor growth but increased the intratumoral CD44H cell content which was antagonized by 4MP. Moreover, pharmacological autophagy flux impairment depleted CD44H cells in xenograft tumors.

Conclusions: The novel single cell-based 3D ESCC organoid system may serve as a highly efficient platform to explore the role of alcohol and other environmental factors as well as cancer therapeutics and therapy resistance mechanisms in conjunction with morphological and functional assays with implications for translation in personalized medicine.

#5027

Heterocellular 3D scaffolds as biomimetic to recapitulate the tumor microenvironment of peritoneal metastases in vitro and in vivo.

Olivier De Wever, Emiel Dejaeghere, Elly De Vlieghere. _Ghent Univ. Hospital, Ghent, Belgium_.

Peritoneal metastases cause cancer mortality and preclinical models are urgently needed to boost therapeutic progress. This study reports on a hybrid hydrogel-polylactic acid (PLA) scaffold that mimics the architecture of peritoneal metastases at the qualitative, quantitative and spatial level. Porous PLA scaffolds with controllable pore size, shape, and surface properties are functionalized by type I collagen hydrogel and co-seeding of cancer-associated fibroblast (CAF) increases cancer cell adhesion, recovery, and exponential growth by in situ heterocellular spheroid formation. Scaffold implantation at the peritoneal wall allows long-term follow-up (>14 weeks) and results in a time-dependent increase in vascularization, which correlates with cancer cell colonization in vivo. CAF, endothelial cells, macrophages and cancer cells show spatial and quantitative aspects as similarly observed in patient-derived peritoneal metastases. CAF provide long-term secretion of complementary paracrine factors implicated in spheroid formation in vitro as well as in recruitment and organization of host cells in vivo. In conclusion, the multifaceted heterocellular interactions that occur within peritoneal metastases are reproduced in this tissue-engineered implantable scaffold model.

#5028

Mapping metabolic networks and drug resistance in 3D spheroids using stable isotope-resolved metabolomics (SIRM).

Teresa W. Fan, Salim S. El-Amouri, Jessica K. Macedo, Qiushi Sun, Andrew N. Lane. _Univ. of Kentucky, Lexington, KY_.

Introduction: 2D cells lack cell-cell and cell-extracellular matrix interactions, critical for regulating cell functions. 3D cells in matrigel, hydrogels, hanging drops, and micropatterned plates can overcome these drawbacks but suffer from long spheroid formation times with variable efficiency. Magnetic 3D bioprinting (M3DB) can circumvent these issues by utilizing nanoparticles-magnetized cells for spheroid formation. M3DB spheroids can emulate tissue microenvironments and exhibit higher resistance to toxic agents than their 2D counterparts. It is, however, unclear how M3DB spheroids influence toxic responses in cells via metabolic perturbations. We employed Stable Isotope-Resolved Metabolomics (SIRM) with 13C6-glucose to map metabolic networks in 2D cells and M3DB spheroids formed from lung (A549) and pancreatic (PANC1) adenocarcinoma cells ± anti-cancer Na2SeO3.

Experimental: A549 and PANC1 cells were grown in 2D culture in DMEM to ca. 70% confluence for loading with magnetic nanoparticles (Nanoshuttle, N3D Biosciences), detached, and placed in cell repellent plates for spheroid formation. Spheroids were then treated in [U-13C]-glucose ± Na2SeO3 for 24 h. Metabolites were extracted in cold 70% methanol and analyzed by IC-FTMS and NMR 1,2 to determine the 13C isotopologue and isotopomer distributions for reconstructing metabolic networks.

Findings: We found that spheroids remained metabolically active in 10-day cultures. 13C label incorporation into glycolytic, Krebs cycle, and nucleotide metabolites was similar between spheroids and their 2D counterparts. The exceptions were higher 13C-ribose incorporation into uracil nucleotides in 2D than M3DB cultures of A549 cells and activation of gluconeogenesis in PANC1 M3DB spheroids. Selenite inhibited these pathways less in spheroids than 2D counterparts for both cell lines, consistent with the growth changes. Thus, the increased resistance of cancer spheroids to selenite may be mediated by these metabolic pathways central to growth and survival.

Conclusions: 3D spheroids offer convenient models with more relevant 3-D architectures for preclinical studies on metabolic reprogramming and drug response of human cancer cells and cancer-stromal cell co-cultures. Spheroid formation in the M3DB system is fast, high throughput without matrix interferences for metabolic analyses.

Supported by NCI P01CA163223-01A1, P30 CA177558 and NIDDK 1U24DK097215-01A1

1. Sun RC, Fan TW-M, Deng P, et al. Liquid diet delivery of stable isotopes into PDX mice for deep metabolic pathway tracing. Nat. Commun. 2017;9:1646.

2. Fan TW-M, Warmoes MO, Sun Q, et al. Distinctly perturbed metabolic networks underlie differential tumor tissue damages induced by immune modulator β-glucan in a two-case ex vivo non-small cell lung cancer study. CSH Molec Case Studies 2016;2:a000893.

#5029

Precision cancer medicine based on 3D drug profiling of patient-derived cancer cell spheroid models.

Piia Mikkonen,1 Laura Turunen,1 Lauri Paasonen,2 Swapnil Potdar,1 Lassi Paavolainen,1 Astrid Murumägi,1 Olli Kallioniemi,1 Vilja M. Pietiäinen1. 1 _Institute For Molecular Medicine Finland -FIMM, Helsinki, Finland;_ 2 _UPM-Kymmene, Helsinki, Finland_.

We have set up a precision medicine strategy for solid tumors to i) understand biological heterogeneity and driver signaling pathways in cancer, ii) identify new drug opportunities, iii) develop biomarkers for drug responses, and iv) eventually tailor effective treatments for individual patients. Fresh cancer tissue is obtained directly from clinics and processed to provide patient -derived cells (PDCs). PDCs and original tumor tissues are characterized using genetic profiling and image-based phenotyping, phenomics. Systematic drug sensitivity and resistance testing (DSRT) is carried out on the representative PDC models. The PDCs are plated in 384-well plates and treated with oncological compounds, each in five concentrations in 384-well plates. The cell viability and toxicity are measured as drug responses using plate readers. Alternatively, drug-treated cells are immunostained and subjected to automated high-content imaging and image analysis.

Central to the success of this approach is to grow and test the patient derived cells in ex vivo conditions mimicking the tumor microenvironment. To this aim, we have developed 3D drug profiling for PDC spheroids from ovarian and renal cancers. For the 3D drug profiling, cells were cultivated either in Matrigel or in cellulose-based hydrogel, GrowDex, which has earlier been shown to support 3D growth of cell lines and stem cells. The pipetting robot Biomek FXp (Beckman Coulter) was utilized for transfer of matrices and cells to 384-well plates, and acoustic dispenser Echo 550 (Labcyte) for delivery of tailored drug library of 52 drugs to the spheroids. The drug sensitivities of cancer cell spheroids were scored using cell viability measurement as well as high-content confocal imaging.

Our results with primary cells and cell lines suggest that both Matrigel and cellulose-based hydrogel are applicable in the 384-well plate drug profiling assay, and PDC spheroids are formed in both conditions. When the drug responses of ovarian cancer PDCs grown in different 2D and 3D conditions were systematically compared, we observed significant differences in sensitivity to several drugs. Here, we describe the individual drug effects in all conditions, such as some chemotherapeutics being less effective in 3D. As a conclusion, the comparison of results from 2D and 3D drug profiling increases our understanding of mechanisms of drugs, and may aid to select the most representative drugs for the patient.

#5030

Development and optimization of Matrigel-based multi-spheroid 3D tumor assays using real-time live-cell analysis.

Kalpana Patel,1 Miniver Oliver,1 Nevine Holtz,2 Tim Jackson,2 Nicholas Dana,2 Gillian Lovell,1 Nicola J. Bevan,1 Tim J. Dale,1 Derek J. Trezise1. 1 _Essen BioScience Ltd, Welwyn Garden City, United Kingdom;_ 2 _Essen BioScience Ltd, Ann Arbor, MI_.

The tumor-associated extracellular matrix (ECM) provides critical biochemical micro-environment cues, as well as an essential structural scaffold, for solid tumors to survive and grow (see Pickup et al. 2014 for review). With a view to enabling more translational and turnkey 3D in vitro assays for cancer biology, we have developed and optimized techniques for seeding, growing and automatically quantifying the properties of multiple tumor spheroids in ECMs in 96-well format using real-time live-cell analysis. Matrigel (Corning) was dispensed across a range of volumes (20 - 50 μL) and concentrations (1 - 5 mg mL-1) into flat-bottomed 96-well TC micro-plates to form a solidified base layer. Tumor cells (A549, MCF-7, SKOV-3, MDA-MB-231) were seeded on top (1 - 2K cells per well), and in some experiments a full ECM sandwich was created by addition of a further volume of Matrigel (2 - 25%, 0.2 - 5 mg mL-1). Using a custom autofocusing method, phase contrast, bright-field and fluorescence images (10x) were captured every 6h for 7 days from within the cell incubator (IncuCyte S3 live-cell analysis system). Typically, 20 - 80 spheroids were analyzed in each well. All four cell types formed multiple cell aggregates within the first 3 days, ranging in diameter from 30 - 80 μM. A549, SKOV-3 and MCF-7 multi-spheroids grew as round aggregates while MDA-MB-231 spheroids displayed stellate branching characteristic of an invasive morphology. At Matrigel volumes less than 40 μL or concentrations less than 3 mg mL-1, cells penetrated to the base of the plate and grew as 'flat 2D' cultures. Using a novel bright-field image analysis algorithm, the number, area and average size of the spheroids could be computed over time non-invasively and without the use of fluorescent labels. Once formed, A549, SKOV-3, MCF-7 and MDA-MB-231 multi-spheroids increased 3.0-, 1.6-, 3.8- and 3.3-fold in size over 4 days, respectively. Treatment of A549 multi-spheroids with the DNA enzyme topo-isomerase inhibitor camptothecin (1μM) inhibited growth with comparable spheroid size at day 0 and day 7 post treatment (average brightfield area 1.4 x104 μM2). Using fluorescent protein reporters for apoptosis (Annexin V) and cell viability (IncuCyte CytoTox Green) we could verify camptothecin-induced cell death (fluorescence values 149±16% of control (Annexin V) and 243±51% of control (CytoTox). A concomitant decrease of stably expressed RFP (to 3±1% of control) was observed. The combination of protocol developments, novel image acquisition/analysis algorithms and cell health reporters creates an integrated solution for measuring growth and vitality of multiple small spheroids in a relevant and 3D bio-matrix over time. This approach should be applicable to primary- and patient-derived organoid tumor samples as well as cancer cell lines. Pickup, MW, Muow, JK, Weaver, MW (2014), EMBO Rep. 15(12): 1243-1253

#5031

Organotypic brain explant culture as a drug evaluation system for malignant brain tumors.

Noriaki Minami. _Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan_.

Background: Inhibition of major metabolic pathways is being assessed for several intractable tumors, including glioblastoma. Following screening in 2D-systems, evaluating metabolic inhibitors in assays that include both tumor and normal brain tissue is indispensable for selecting compounds that can affect gliomas within their microenvironment. Here we asked whether organotypic brain slices from adult mice can be used to evaluate the effects of metabolic inhibitors on malignant glioma cells. Methods: Retroviral expression of H-RasV12 and dsRed in Ink4a/Arf-/- neural stem / progenitor cells was adopted to establish glioma initiating cells (GICs). GICs with different extracellular acidification potential were derived by single cell cloning and used after one in vivo passage. GICs or their clones were implanted into the forebrain of 6-weeks-old wild-type mice. Tumor-bearing brain explants were established as 200 µm coronal brain slices, treated with cisplatin, dichloroacetate or phenformin. The effects of the drugs were assessed by fluorescence microscopy of the live explants, as well as immunohistochemical staining at the end of the treatment. Results: The feasibility of assessing drug effects in brain tumor explants from adult mice was confirmed with the use of the DNA-damaging agent cisplatin. Treatment of brain slices carrying tumors formed by GICs with 50 μM cisplatin for 4 days effectively decreased the tumor burden, as shown by a significant reduction of the tumor area on fluorescence imaging and a marked increase in tumor cells positive for cleaved caspase 3 on immunohistochemical analysis. The action of metabolic inhibitors was assessed in explants with tumors formed by GIC-derived populations that predominantly use either glycolysis or mitochondrial respiration. Treatment of the brain slices with dichloroacetate, an inhibitor of pyruvate dehydrogenase kinase, resulted in a marked increase in cleaved caspase 3-positive cells in glycolytic tumors. In contrast, phenformin, which inhibits respiratory complex I, had a pronounced pro-apoptotic effect in tumors formed by cells with a high mitochondrial activity. Conclusion: Inhibitors of both glycolysis and oxidative phosphorylation effectively and selectively induced apoptosis of the targeted tumor cells in cultured brain slices. Our results suggest that brain explants from adult mice can be used to assess the effects of anti-metabolic drugs on the survival of glioma initiating cells within a syngeneic microenvironment. Explants could thus be useful in the evaluation of new metabolic inhibitors.

#5032

Tumor in a Dish (TiD): Novel approach for precision therapy using patient-derived cells.

Prabhu Ramamoorthy,1 Sufi Thomas,1 Sathish Ramalingam,1 Gaurav Fnu,1 Sita G. Sittampalam,2 Roy A. Jensen,1 Shrikant Anant1. 1 _Univ. of Kansas Medical Ctr., Kansas, KS;_ 2 _National Center for Advancing Translational Sciences, Bethesda, MD_.

Metastatic colorectal cancer is a major cause of cancer-related deaths. Most patients with metastatic disease fail to respond to current chemotherapeutic agents. Current, traditional culture model is not appropriate to study the metastatic disease in in vitro condition. Hence, there is an ominous need for developing new therapeutic and preventive agents that can target metastatic cancer. Unfortunately, there are no good in vitro as well as in vivo models available for studying metastatic disease. Hence, in this abstract, we demonstrate the development of a new lung model system, called Primitive Lung in a Dish or PLiD with cancer cells called "Tumor in a Dish" or TiD. The PLiD system shows expression of specific cell type marker like E-cadherin (epithelial cell) vimentin (fibroblast), CD31 (heme-endothelial cells) and LYVE1 (lymph-endothelial cells). Interestingly, we showed expression of lung functional protein like surfactant protein B and D. In the multicell type TiD system, cancer cells were grown in a 3-dimensional (3D) PLiD system containing normal epithelial cells, fibroblasts and endothelial cells. The model resembles in vivo tumor microenvironments, including cell-cell contact, tumor architecture, and the influence of different cell types. We next determined the efficacy of standard colon cancer chemotherapeutic agents. We observed differential activity of 5FU in TiD system when compared to standard 2D and single-cell type 3D cultures. Another surprising result we found was with freshly isolated colon cancer cells from patient samples. Without knowing the genetic characteristics of the cancer tissue, the TiD system was able to identify cells that were resistant to oxaliplatin. Moreover, using this method, we have developed novel drugs that target cancer cells while not affecting the normal tissue. More importantly, the technique has significant utility in precision/personalized medicine, wherein this phenotypic screen can be coupled with current DNA pharmacogenetics to identify the ideal therapeutic agent, thereby increasing the probability of response to treatment while reducing unnecessary side effects.

#5033

Patient-derived xenograft screening in a three-dimensional tumor growth assay incorporating stromal elements to recapitulate the human tumor microenvironment.

Simon Jiang, Jane Wrigley, Sumanjeet Malhi, Jamie Wood, Stephanie Morton, Louise Wainwright, Yinfei Yin, Rajendra Kumari. _Crown Bioscience UK, Loughborough, United Kingdom_.

BACKGROUND: Pre-clinical tumour models lack human stromal cells and are inadequate for the assessment of novel tumour microenvironment targeted therapies. An increase in combination opportunities requires a higher throughput in vitro assay that utilised the patient relevant model. The 3D-tumour growth assay (3D-TGA) enables patient-derived tumour cells to be admixed with a basement membrane extract and bone marrow-derived mesenchymal stem cells (bmMSCs) in a 96-well format for screening. Response to standard of care (SoC) agents such as paclitaxel, targeted agents such as Erlotinib were compared to subcutaneous xenograft responses in vivo. METHODS: Tumour cells or xenograft material which is disaggregated to yield a pure epithelial population, are admixed and suspended in basement membrane extract (Cultrex®, Trevigen) with and without bmMSCs. Cells were maintained for three days before they were treated with a panel of test agents, such as paclitaxel, AZD2014, Erlotinib, carboplatin for lung PDX and final cell viability was determined on day 7 by alamar blue assay. IC50 curves were determined using GraphPad Prism 6.0. Subcutaneous tumour growth was evaluated three times a week by measuring the tumour in two dimensions using electronic callipers for the duration of the study and tumour volumes were estimated using length and width measurements. Treatment was initiated when tumour growth was established. RESULTS: A panel of PDX models which contain various mutation sites (e.g. EGFR, KRAS) were assessed with a range of SoC (e.g. carboplatin, pemetrexed and paclitaxel) or targeted agents (e.g. Erlotinib, AZ2014). A squamous NSCLC model LU6422, which harbours a heterozygous mutation in the EGF-R intracellular kinase domain (c.2573T>G (L858R)) showed exquisite sensitivity to Erlotinib in both 3D TGA and xenograft studies. Similarly, a squamous NSCLC model LU6402 was assessed with AZD2014 and Paclitaxel in 3D-TGA in the presence of bmMSCs which showed an additive effect of AZD2014 in combination of Paclitaxel on tumour cell growth. A similar efficacy was observed in subcutaneous in vivo xenograft; however, a combination finding was not evident when the stromal cells bmMSCs were absent. The data illustrates that the comparable sensitivities to various test agents between 3D-TGA and in vivo xenograft studies was observed. CONCLUSIONS: The 3D tumour growth assay fast and simple approach to restoring the interaction between the tumour and stroma in a 96-well format enabling screening to multiple dug concentrations and combinations. This assays provide unique opportunity for interrogating therapies that target the tumour microenvironment using patient relevant models.

#5034

Time-lapse analysis of drug response and invasive capacity using patient-derived CRC microfluidic 3D cancer models under oxygen-controlled conditions.

Dora Sabino, Isabelle Fixe, Alexandra Foucher, Eric Mennesson, Nadia Normand. _tebu-bio, Le Perray-en-Yvelines, France_.

The development of relevant, reliable, human-derived, and cost-effective cellular models is a present need to bring precision medicine to fruition and to accelerate drug discovery. 3D cultures show robust genomic stability and tissue-specific gene expression with high fidelity to their tissue of origin, making them ideal for preserving healthy and cancer tissue molecular and morphological traits. Our objective is to expand the number of relevant human cancer models available and implement methods that allow cellular response quantification of 3D cultures. We sought to establish and implement 3D cell-based assays suitable for detection of known molecular biomarkers in cancer using spherical cancer models (multicellular tumor spheroids and tissue-derived tumor spheres). We validate our procedures by detecting colorectal cancer (CRC) biomarkers both in patient-derived cultures and cell-lines (HCT116, HT29, LS174T). We expanded the study's scope to the analysis of specific CRC transcriptional and proteomic signatures in 3D. To this end, we employ primary cultures derived from CRC patients, and cell lines. Our methodology takes advantage of multiplex antibody microarrays to detect proteomic levels of 10 metalloproteinases of which MMP-13 and MMP- 9, as well as of surface proteins known to be involved in colorectal cancer invasion. Alterations in oxygen tension are milestones in tumor progression, and low oxygen tension (hypoxia) has been demonstrated in CRC. The Hypoxia Inducible Factor (HIF) transcription factor family is central to the response to hypoxic stress and is correlated with CRC carcinogenesis, invasion, VEGF expression, and poorer diagnosis. To increase the relevance of our model we use low oxygen conditions (3-5% physioxia conditions), and atmospheric oxygen levels (normoxia conditions) finding relevant differences at the proteomic, transcriptional and miRNA levels (also verified by qRT-PCR). We test the spherical cancer models on microfluidics chip and assess their vessel invasion capacity in normoxia and hypoxia, finding a correlation between hypoxia stress response and augmented invasiveness. Additionally, we test cellular responses to standard drugs and vasculature invasive capacity by time-lapse microscopy on-chip.

We design, develop and validate cellular assays for human cancer 3D culture generation, long-term maintenance and analysis. We analyze and quantify cellular responses to cytotoxic drugs, image and quantify invasion in fluidic chambers, determine levels of apoptosis, cell proliferation.

We demonstrate that a combination of controllable assays can successfully be used for identification of biomarker expression and compound screening in preclinical studies. 3D models are, therefore, a cost-effective in vitro model capable of recapitulating reliably physiological responses.

#5035

Identification of novel therapeutics of bladder cancer cells through upregulation of SPARC expression in using 2D cell cultures and 3D bladder organoids.

Chirayu M. Patel,1 Cristina Ivan,2 Anthony Atala,2 Neveen Said1. 1 _Wake Forest Health Sciences, Winston-Salem, NC;_ 2 _Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC_.

We have recently reported the tumor suppressor effect of SPARC in urinary bladder cancer. We have shown that the loss of SPARC in bladder cancer resulted accelerated bladder carcinogenesis and metastasis multiple pathways involving cell cycle deregulation, inflammation and angiogenesis. We have also reported that SPARC protein expression significantly decreased in the cancerous compartment in advanced human bladder cancer as well as in carcinogen-induced murine urothelial cancer. However, the mechanism of the downregulation of SPARC expression in cancer in general and in bladder cancer in particular is still unraveled. The purpose of our study is to identify therapeutic candidates as regulators of SPARC expression in bladder cancer. We developed an in vitro 3-D bladder model system "bladder organoids" that comprises the normal and cancerous urothelial cell, bladder smooth muscle cells, fibroblasts and endothelial cells. We differentially expressed fluorescent SPARC promoter reports in the cancerous and non-cancerous cells to screen for the differential expression of SPARC in each cell type using FDA-approved and natural product drug libraries and to determine the cytotoxicity on cancerous vs non-cancerous cells. Real time monitoring of changes in SPARC expression was done by measuring fluorescence intensity using IncuCyte® ZOOM and InCell Analyzer 1000 Live Imaging Analysis Systems. We identified drugs that consistently exhibited dose-dependent augmentation or down-regulation of SPARC expression in cancer cells. Our screens using the identified novel up-regulators of SPARC expression that differentially reduce the viability of cancer cells and spare non-cancer cells in the organoids. Our efforts produced a platform that mimics bladder cancer microenvironment that can be adapted for throughput screening for targeted personalized therapies.

#5036

Visualization of cancer-derived exosome-mediated angiogenesis in a layered 3D stromal tissue model.

Shiro Kitano,1 Kei Tsukamoto,2 Rii Morimura,2 Shinji Irie,1 Eiji Shinozaki,3 Kensei Yamaguchi3. 1 _Osaka University, Osaka, Japan;_ 2 _Toppan Printing Co., Ltd., Saitama, Japan;_ 3 _Cancer Institute Hospital of Japanese Foundation for Cancer Research, Tokyo, Japan_.

Background: Exosomes are small vesicles of sizes ranging from 30 to 150 nm that originate from the endosomal luminal membrane and are constantly released by fusion with cell membranes. Several studies have revealed that exosomes play an important role in mediating local and systemic cellular communication through the transmission of nucleic acid materials and information in the form of proteins. Here, we demonstrated a unique layered 3D stromal tissue model of visualization of promoting angiogenesis by exosomes in colorectal cancer (CRC) patients.

Method: The CRC specimens of exosome were obtained from 7 patients in stage IV. Isolation of total exosome was performed according to standard procedure by ultracentrifuge. Layered 3D stromal tissue model was constructed with human umbilical vein endothelial cells (HUVEC) and normal human dermal fibroblasts. Cells were aggregated by heparin and collagen to suspension under cationic condition. The cell suspension was seeded to transwell inserts membrane at a density of 2×106. Media were changed after 24h, and serum, exosome or supernatant after ultracentrifuge of serum (SAUS) was added to the 3D stromal tissue after 6 days. The isolated exosomes were diluted with an equal volume of PBS buffer to the serum sample. After cultivation, the tube formation of HUVEC was stained with Immunofluorescence, and the degree of hypervascularity was compared with healthy volunteers.

Result: Except for one serum specimen, hypervascularization was observed in the 3D stromal tissue model in an additive-dependent manner. Especially when exosome was added, hypervascularization was observed in the layered 3D stromal tissue model more efficiently than serum in all specimens (see Table). Interestingly, the frequency of hypervascularization was drastically reduced in the SAUS.

Conclusion: Our data encourage that this layered 3D stromal tissue model may be applicable to cancer diagnosis using exosomes from CRC patients.

Table. Frequency of hypervascularization by exosomes derived from CRC

---

|

5μl | 10μl | 50μl

Exosome | 5/7 (71.4%) | 7/7 (100.0%) | 7/7 (100.0%)

Serum | 4/7 (57.1%) | 6/7 (85.7%) | 6/7 (85.7%)

SAUS | 1/7 (14.3%) | 0/7 (0.0%) | 0/7 (0.0%)

#5038

Ready to use 3D spheroid cultures as a standard tool to screen for compounds targeting cancer.

Angel Garcia Martin, Olatz Leis, Juan Gumuzio, Inés Prieto, Estibaliz Ruiz. _StemTek Therapeutics, Cambridge, MA_.

The cancer stem cell (CSC) concept has important implications not only for our understanding of carcinogenesis, but also for the development of cancer therapeutics. There is a growing body of preclinical evidence showing that cancer stem cells contribute to chemotherapy and radiation resistance in breast cancer. The use of drugs that interfere with stem cell self-renewal represents the strategy of choice for novel effective anti-cancer treatments, but also a great challenge because cancer stem cells and their normal counterparts share many pathways. The biology of cancer stem cells has proven complex and difficult to translate into effective therapeutic strategies. The question arises as: how do we test compounds for anti-cancer stem cell activity? The answer is: phenotypic screening. There are indeed several functional assays well validated in the scientific literature that have been used for years associated to the ability of cancer cells to demonstrate stem cell behavior. The most relevant is the 3D tumor spheroid assay. This assay has been used to uncover and culture stem cells from many tissues as well as from tumors. There are multiple reports now that show that spheroid derived cells are enriched in tumor initiating or cancer stem cells, derived from cell lines and from natural fresh tumors as well. Here we describe the use of 3D spheroid models to profile compound activity against cancer stem cells. Furthermore, a case of compounds preventing hypoxia-inducible transcription factor (HIFs) activity is presented. Recently, HIF transcription factor biology has been linked to pathways that regulate stem cell self-renewal and pluripotency, suggesting a new mechanism whereby HIF proteins may drive tumor growth, through the generation of tumour-initiating cells or cancer stem cells. Therefore, targeting the HIF pathway may provide a novel therapeutic avenue to target cancer stem cells. We demonstrate that interfering with HIF pathway activation prevents mammosphere formation, validated through indenpendent confirmation through Sox2 promoter activation, Aldefluor® assay and in vivo proof-of concept experiments targeting tumor initiation.

#5039

The relationship between interstitial fluid pressure, collective invasion and YAP activation in engineered human breast tumors.

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

The tumor microenvironment exhibits increased interstitial fluid pressure (IFP) due to combined effects of vascular leakage and inefficient drainage. Elevated IFP affects the transition of metastatic cells from the tumor to the healthy tissue. However, the complex architecture of the heterogeneous tumor tissues has made it difficult to correlate IFP with chemical and physical tumor responses that regulate invasion and metastasis. To overcome these limitations, we have engineered 3D breast tumor models for the quantitative characterization of biophysical and biochemical features that delineate its transition from a passive non-invasive to a metastatic state. In brief, we embed MDA-MB-231 human breast cancer cells in a collagen type I cavity molded within polydimethylsiloxane (PDMS) channels. The multicellular aggregates are subject to gradients of hydrostatic pressure through opposing reservoirs of culture media that are located at the base (Pbase) and the tip (Ptip) of the tumor. The pressure differential |Pbase – Ptip| defines the IFP gradient across the tumor.

We found that the morphology of the engineered breast tumor models varies in response to the differential pressure gradients. For Pbase = Ptip and Pbase > Ptip, the engineered tumors displayed limited invadopodia formation. In contrast, for Pbase < Ptip, the engineered tumors displayed enhanced invadopodia formation. Moreover, we found that IFP affected cellular motility and invasion. Tumor cells demonstrated the highest average speed and directionality and were most invasive for Pbase < Ptip. Furthermore, we found that IFP-induced collective invasion coincides with the elevated expression of epithelial-mesenchymal transition (EMT) markers (vimentin, E-cadherin, Snail and keratin-8) in the engineered breast tumor models. In contrast with the previously demonstrated tumor suppressive activity of E-cadherin, engineered tumors that overexpressed E-cadherin developed a more invasive phenotype.

The IFP-induced invasion through EMT in our engineered breast tumors motivated an investigation into the role of Yes-associated protein (YAP). YAP activation is implicated in EMT and regulated by fluid shear stress. Immunofluorescence staining for nuclear YAP suggests that YAP activation modulates the invasion and cell motility responses to IFP. Traction force microscopy will be used to further examine the relationship between IFP, force transduction into the surrounding microenvironment, and YAP activation.

#5040

Perfusion air culture of tissue slices: A new method to cultivate tumor tissue with minimal culture-dependent tissue stress.

Kathrin Boepple,1 Meng Dong,1 Emma Davis,2 Julia Schueler,3 Heike Walles,4 John Hickman,5 Walter E. Aulitzky,6 Heiko van der Kuip1. 1 _Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology and University of Tuebingen, Stuttgart, Germany;_ 2 _Oncology iMed, Bioscience, AstraZeneca, Macclesfield, United Kingdom;_ 3 _Charles River Discovery Research Services Germany GmbH, Freiburg, Germany;_ 4 _University Hospital Wuerzburg, Wuerzburg Branch of the Fraunhofer-Institute Interfacial Engineering and Biotechnology, IGB, Wuerzburg, Germany;_ 5 _Institut de Recherches Servier, Paris, France;_ 6 _Department of Oncology, Robert Bosch Hospital, Stuttgart, Germany_.

Cultivation of tumor tissue slices provides an ex vivo model capturing both tumor heterogeneity and its native microenvironment. Slices are commonly cultured either free-floating in medium or filter-supported. These conditions lead both to culture-dependent stress (free-floating culture condition) and intra-slice gradients regarding proliferation, marker expression and oxygen supply (filter-supported culture condition) (Davies et al Sci Rep (2015) 10.1038/srep17178). To facilitate homogenous supply with nutrients and oxygen, we developed a new method to culture tumor tissue slices. The precision-cut tissue slices (150µm to 300µm thickness) are kept in-between two organotypic supports and fixed in a special chamber allowing continuous perfusion with medium and drugs. The chamber is settled vertically inside of a 50 ml tube with air exchange capacity and connected with a syringe pump via a silicon tube. The whole system is cultured inside the cell culture incubator. Several different types of mouse xenografts (MCF-7, H1437) and primary human tumor (lung and ovarian cancer) tissue slices have been cultured with this new system and compared with the commonly used filter-support culture. Both breast and lung xenograft tissues slices showed a gradient of proliferation, HIF-1α and hormone receptor (ER) expression in the filter-supported culture condition but not with the new perfusion air culture system. The same results were obtained with primary tumor samples. Primary lung tumor and ovarian cancer tissue slices also showed a gradient of HIF-1α expression after cultivation in the filter-supported system but not in the new perfusion air culture system when cotton membranes are used as scaffold. In addition, the choice of the material of the organotypic support allows a variety of biological studies. Scaffolds from de-cellularized porcine intestine provide niches for migrating cells and are suited for studying tumor invasiveness. When used as organotypic support, primary ovarian cancer can be cultured up to 7 days with good tissue morphology and structure and migrating cells into the scaffold can be counted as a measure of invasiveness. When the tissues are sandwiched between polycarbonate membranes (pore size: 12 µm), oxygen gradients can be generated similar to gradients observed around vessels in vivo. Our perfusion air culture system facilitates the cultivation of tumor tissue slices due to its flexibility and adjustability of all culture conditions such as oxygen, scaffolds and flow rate parameters. It allows studying tumor slices under conditions closely resembling the in vivo situation. 

### Carcinogenesis 3

#5041

Repair of azoxymethane-induced nuclear and mitochondrial DNA lesions in mouse liver.

Carlos A. Torres-Ramos,1 Adlin Rodriguez-Munoz,1 Joan Ballista-Hernandez,1 Ceidy Torres-Ortiz,2 Sylvette Ayala-Pena1. 1 _Univ. of Puerto Rico Med. Sciences Campus, San Juan, PR;_ 2 _Pontificia Universidad Catolica de Puerto Rico, Ponce, PR_.

Background & Objectives: Azoxymethane (AOM) is a carcinogen that induces DNA lesions mainly repaired by the base excision repair (BER) pathway. Apurinic/apyrimidinic endonuclease 1 (APE1) is a central component of BER. Mice with homozygous deletion of the Apex1 gene (which encodes APE1) cannot survive development but heterozygous animals (Apex1+/-) have a normal lifespan. Our aim was to study the role of APE1 in the repair of DNA lesions and changes in mitochondrial DNA (mtDNA) abundance induced by AOM in the liver. Methods: We isolated DNA from 6-month-old C57BL/6 WT and Apex1+/- mice that were treated with a single AOM dose (10 mg/kg) and sacrificed 24, 48 and 72 hours after treatment. To quantify nuclear DNA (nDNA) lesions, mtDNA lesions and changes in mtDNA abundance in liver tissue we applied a PCR based assay (QPCR). Results: Our data show that 24h after AOM treatment both WT and Apex1+/- mice exhibit significant levels of nDNA and mtDNA damage. The lesion number decreases 48 and 72h after treatment but remains statistically elevated as compared to their respective controls. A two tailed ANOVA analysis shows no significant differences in DNA lesions between genotypes. However, Apex1+/- mice show a statistically significant 13% decrease in mtDNA abundance 72 hours after treatment. Conclusions: These results indicate that the liver is a major target of AOM and that APE1 plays a role in preventing AOM-induced loss of mtDNA abundance. Acknowledgments: Supported by R25GM061838, 2G12RR003051 and U54CA096297.

#5042

CREB transgenic mice to study alcohol-associated pancreatic carcinogenesis.

Tulasigeri Totiger,1 Supriya Srinivasan,1 Michael VanSaun,1 Chandrashekhar Joshi,1 Chanjuan Shi,2 Xizi Dai,1 Rajinder Dawra,1 Alexander Gaidarski,1 Eric Nestler,3 Nipun Merchant,1 Nagaraj Nagathihalli1. 1 _Department of Surgery, University of Miami Miller School of Medicine, Sylvester Comprehensive Cancer Center, Miami, FL;_ 2 _Department of Pathology, Vanderbilt Medical Center, Vanderbilt-Ingram Cancer Center, Nashville, TN;_ 3 _Icahn School of Medicine at Mount Sinai, New York, NY_.

Background: Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths in the United States. Chronic alcohol (>60 grams/day) consumption is strongly associated with the risk of PDAC development. The metabolites generated from alcohol have been shown to cause significant pancreatic injury. Although the association of alcohol with PDAC progression has been established, the details of the underlying molecular and cellular mechanisms governing this process are unknown. Our study revealed that alcohol-associated pancreatic carcinogenesis correlates with CXCL12-induced activation of cyclic AMP response element binding protein (CREB). In this study, we examined newly developed CREB knockout (CREBfl/fl) mouse in pancreas.

Methods: Human pancreatic epithelial lines (HPNE), HPNE cells with KRAS (HPNE-KRAS), pancreatic stellate cells (PSCs), pancreatic intraepithelial neoplasia (PanIN) mouse cell lines (LSL-KrasG12D/+; Pdx1Cre/+) and cancer-associated fibroblasts (CAFs) cells were exposed to chronic alcohol (50 mM) and analyzed for phospho-kinases in cell lysates and cytokines in conditioned media. Inducible Ptf1aCreERTM;KRASG12D/+ (iPK) mice and CREB knockout (CREBfl/fl) with KRAS activation [Ptf1aCreERTM;KRASG12D/+;CREBfl/fl (iPKCREBKO)] mice were used to investigate the effect of alcohol on CREB activation and the role of CREB in alcohol-associated PDAC pathogenesis. iPK and iPKCREBKO mice were exposed to Lieber-DeCarli alcohol diet for up to 14 weeks with or without caerulein injections. The number of acinar cells (amylase), ducts (cytokeratin 19), PanIN lesions (alcian blue positive), fibrosis (sirius red) and activation of CREB were measured by immunohistochemistry at 6 and 14 weeks of alcohol exposure in vivo. Serum obtained from alcohol-fed iPK and iPKCREBKO mice were analyzed for significant cytokine release upon alcohol exposure in vivo. We then determined the biologic effects of pharmacologic CREB inhibition in iPK mice exposed to alcohol diet.

Results: We found increased pCREB levels in HPNE and HPNE-KRAS cells upon treatment with alcohol, which was further associated with the up-regulation of the CXCL12 protein in the conditioned media. Serum CXCL12 and tissue pCREB levels were high in alcohol fed iPK mice when compared to their corresponding control diet-fed mice. Blocking CXCL12 with Plerixafor, a CXCL12-CXCR4 axis inhibitor, decreased alcohol-induced pCREB levels. Finally, exposure of iPK mice to an alcohol diet coupled with cerulein administration significantly increased the number of PanIN lesions (alcian blue+ cells), and decreased acinar cells when compared to alcohol-fed iPKCREBKO mice.

Conclusion: These findings implicate CREB as a critical oncogenic driver in alcohol addiction-induced pancreatic carcinogenesis. Additionally, CXCL12 represents an important mediator of CREB activation, which can be pharmacologically targeted.

#5043

Glucocorticoids trigger GR-mediated expression of the chemoresistance-associated oncoproteins LEDGF/p75 and Clusterin: Implications for prostate cancer health disparities.

Leanne Woods-Burnham, Christina K. Cajigas-Du Ross, Anamika Basu, Evelyn S. Sanchez, Arthur Love, Carlos A. Casiano. _Loma Linda Univ. School of Medicine, Loma Linda, CA_.

Prostate cancer (PCa) is more aggressive in African American (AA) men compared to European American (EA) men, resulting in increased incidence and mortality disparities. There is a critical need to identify biological determinants contributing to these disparities. Glucocorticoids—a type of stress hormone—have recently been implicated as biological factors driving PCa progression and response to therapy. The underlying mechanism involves endogenous glucocorticoid binding to its receptor (GR), prompting GR to transcriptionally activate genes that are typically androgen-regulated and that promote tumor aggressiveness and therapy resistance. However, a clinical dilemma exists as glucocorticoids, necessary in the palliative care of PCa patients, are now emerging as accelerators of disease progression. In addition, AA men have chronically elevated levels of glucocorticoids linked to stressful life events, and are also hypersensitive to glucocorticoid exposure. We examined the effects of glucocorticoids on the activation of the stress oncoproteins LEDGF/p75 (Lens Epithelium Derived Growth Factor of 75kd) and CLU (Clusterin), linked by our group and others to prostate tumor aggressivenes and taxane resistance. Our goal is to establish the interplay between glucocorticoids and the expression of these proteins in the context of PCa health disparities. We hypothesized that LEDGF/p75 and CLU may be induced by glucocorticoids through GR in PCa cells. Treatment of a racially diverse panel PCa cell lines (MDA-PCa-2b [AA], 22Rv1 [AA/EA], PC3 [EA], and DU145 [EA]) with 10nM cortisol or dexamethasone for up to 48h significantly upregulated both transcript and protein levels of LEDGF/p75 and CLU. This upregulation was more robust in the AA cell lines. Depletion of GR with siRNAs abrogated dexamethasone-induced upregulation of LEDGF/p75 and CLU. Pharmacological inhibition of GR with mifepristone, in the presence of dexamethasone, also abrogated the induction of these proteins in a cell type-dependent manner. We also observed that dexamethasone exposure increased cell migration, a characteristic of tumor aggressiveness. Furthermore, using an ELISA platform we detected increased serum levels of LEDGF/p75 in AA PCa patients, as well as increased levels of CLU in non-PCa AA men, compared to EA patients and controls. These findings show that glucocorticoids induce the expression of oncoproteins associated with PCa aggressiveness and therapy resistance, and this upregulation may occur more robustly in AA men, priming them to develop highly aggressive PCa tumors.

#5044

Molecular analysis of HBV in histologically confirmed hepatocellular carcinoma in a tertiary hospital in Ghana.

Babatunde M. Duduyemi,1 Du-bois Asante,2 Nicholas Titiloye,1 Mary Afihene1. 1 _Kwame Nkrumah University of Science and Technology, Kumasi, Ghana;_ 2 _University of Cape Coast, Cape Coast, Ghana_.

Background: Hepatocellular carcinoma (HCC) is a leading cause of death in Africa. Viral hepatitis B is a leading cause of hepatocellular cancer in Ghana and most African countries except Egypt where hepatitis C virus is more prevalent. This study aims at reviewing the histopathological patterns of HCC and its association with hepatitis B virus in our environment.

Method: Demographics and histological diagnosis were retrieved from the surgical daybook and archival FFPE tissue samples with histopathologically confirmed HCC were used for this study. Sections (10µm) were taken from the tissues and digested to obtain DNA lysates. The DNA lysates were used in polymerase chain reaction (PCR) to determine the prevalence of HBV in the biopsies.

Result: Of the 24 confirmed cases of HCC seen in the 5 year period, there were 17 males and 7 females with M:F ratio of 2.4:1. The mean age of our patients was 39.92 ± 1.98 years with age range 13-85 years. 50% of the cases were moderately differentiated while 25% each were well and poorly differentiated. Out of the 24 archival HCC biopsies screened, HBV DNA PCR amplification was achieved in 11 after the restriction fragment length polymorphism PCR reaction. Out of the 24 archival HCC biopsies screened, HBV DNA PCR amplification was achieved in 11 (45.83%) after the restriction fragment length polymorphism PCR reaction. Eight of the 11 cases were found in the male and 3 in females. Of the 11 (45.83%) samples that were positive for HBV DNA, 3 were above 40 years and 8 were 40 years and below.

Conclusion: The overall prevalence of HBV DNA in our study was 45.83% and a greater proportion seen in less than 40 years. This suggests that most of our patients are infected with HBV early in life in our environment.

#5045

**Inflammation is upregulated in the normal colonic epithelium and stroma of** Apc+/Min-FCCC **mice with colon tumors.**

Alyssa A. Leystra, Lisa Vanderveer, Yan Zhou, Harry S. Cooper, Sergei Grivennikov, Margie L. Clapper. _Fox Chase Cancer Center, Philadelphia, PA_.

The Apc+/Min-FCCC mouse harbors a point mutation in the Apc tumor suppressor gene and is highly susceptible to the development of multiple colon tumors. Despite being maintained for many years as an inbred colony, Apc+/Min-FCCC mice are heterogeneous with respect to the time to colon tumor development and the number of colon tumors that ultimately form. This model therefore serves as a powerful tool with which to better understand the earliest events that lead to tumorigenesis. Identification of early alterations in these animals may provide targets for effective, long-term tumor prevention. The goal of this study was to characterize early changes in gene expression in the nonneoplastic colonic epithelium and stroma that may contribute to tumor formation in Apc +/Min-FCCC mice. Animals were defined as 'tumor-bearing' if they harbored at least one gross colon tumor at necropsy. No lesions were detected at necropsy in the remaining animals ('tumor-free'). Normal tissue was collected for analysis from the distal colon at least 1 cm away from the nearest tumor. Histologically-normal epithelial and stromal cells were microdissected from frozen tissue sections. Gene expression profiling was performed using the Agilent Mouse 4x44K microarray chip. Differentially expressed genes were selected using an FDR of 10% and a ≥ 2-fold change in gene expression. Gene set enrichment analyses confirmed the enrichment of stromal genes in samples of isolated stroma (p< 0.05). Numerous genes were differentially expressed in tumor-free vs. tumor-bearing ApcMin/+ mice, in both the epithelial and stromal compartments. Gene Ontology enrichment analyses revealed the associated biological processes that were dysregulated, including inflammation, immune cell migration, and response to bacterium (FDR<0.1%). These findings indicate that inflammatory processes are altered in the nonneoplastic colon well in advance of tumor formation and represent promising targets for preventive intervention.

#5046

**Bisphenol AF promotes ER** + **breast cancer through amphiregulin-mediated ER-RTK signaling crosstalk.**

Qingxia Zhao,1 Amanda B. Parris,1 Erin W. Howard,1 Ying Xing,2 Xiaohe Yang1. 1 _North Carolina Central University, Kannapolis, NC;_ 2 _Basic Medical College of Zhengzhou University, Henan, China_.

Bisphenol AF (BPAF), a BPA analog, is widely used in the manufacturing of plastics, and has been detected in the environment and in human urine samples. Since BPAF may exhibit more potent endocrine-disrupting activities than BPA, the potential effect of BPAF exposure on breast cancer risk is a health concern. Previous reports highlight the interaction of BPAF with estrogen receptors (ERs), but the molecular pathways of BPAF-mediated cellular activities remain unclear. Therefore, we investigated BPAF-mediated cellular responses in MCF-7 and T47D ER+ breast cancer cells. Results from MTT and clonogenic assays indicated that BPAF (0.01 – 5 µM) induces cell proliferation in a concentration-dependent manner. Cell cycle analysis showed that BPAF (0.5 – 1 µM) significantly increased the percentage of cells in S phase and decreased cells in G0/G1 phase. Data from luciferase reporter assays indicated that BPAF potently activated estrogen response element (ERE)-mediated transcription, which was accompanied by the upregulation and activation/phosphorylation of ERα and ERβ. These data demonstrate that BPAF is a potent endocrine disruptor. Importantly, we also found that BPAF induces ErbB3 phosphorylation, and significantly activates downstream PI3K/Akt and MAPK pathways in both cell lines, as indicated by increased Akt, Erk1/2, and Src phosphorylation. We next examined the mRNA levels of key regulators downstream of ER and receptor tyrosine kinase (RTK) pathways, including AREG, TFF1, MYC, IGF1R/2R, NRG1, EGFR, ERRB3, ESR1/2, and CCND1, in BPAF-treated cells. We found that the most significantly upregulated gene by BPAF was AREG (Amphiregulin), an ER-targeted gene that encodes an RTK ligand protein. Concurrent ER and RTK signaling activation with AREG upregulation suggests that BPAF induces ER-RTK crosstalk through AREG-mediated signaling. To demonstrate the impact of ER signaling on BPAF-induced ER-RTK crosstalk, we blocked estrogen signaling with ICI-182,780 (Fulvestrant) in BPAF-treated cells, and found that estrogen signaling inhibition significantly suppressed BPAF-associated cell growth, ERE-mediated transcriptional activity, and signaling in ER and RTK pathways. We next examined the effect of AREG knockdown on the signaling in both RTK and ER pathways to determine the role of AREG in BPAF-induced ER-RTK crosstalk. Our data indicated that AREG knockdown not only markedly inhibited BPAF-induced proliferation and RTK signaling, but also significantly downregulated ER-mediated transcription. Overall, we demonstrated that ER-RTK crosstalk is essential for BPAF-induced oncogenic cellular responses. Importantly, we identified AREG as a critical mediator of the BPAF-induced signaling interactions. These novel findings highlight the potential clinical implications of BPAF-associated ER+ breast cancer risk and underscore the necessity to further evaluate BPAF as an endocrine disruptor.

#5047

Exosomes derived from HIV-1-infected T cells activate ERK 1/2 through EGFR and TLR3 in cancer cells.

Zhimin Feng, Lechuang Chen, Hong Yue, Bingcheng Wang, Ge Jin. _Case Western Reserve University, Cleveland, OH_.

Exosomes secreted from HIV-1-infected T cells play important roles in coordinating the immune responses and disease processes in HIV-infected people who are treated with antiretroviral therapy. We hypothesize that exosomes from HIV-infected T cells can promote growth and progression of non-AIDS-defining cancer cells. Activation of the MAPK ERK 1/2 is important for cancer cell survival, proliferation and invasion. We investigated the ability of exosomes from HIV-1-infected T cells to activate ERK 1/2 in head neck squamous cell carcinoma (HNSCC) and lung cancer cells for cancer cell proliferation and progression. Exosomes were isolated from Jurkat and J1.1, a latently HIV-1-infected Jurkat cells, using differential ultracentrifugation and quantified by acetylcholinesterase (AchE) activity. HNSCC cell line HSC3 and lung cancer cell lines NCI-H1299 and NCI-H1437 cells were treated with exosomes. To test the involvement of EGFR and TLR3 in ERK 1/2 activation by exosomes, EGFR neutralizing antibody Cetuximab or the TLR3/dsRNA inhibitor complex was added to cancer cells 30 min prior to adding exosomes. To test the entry of exosomes into cells, HEK293 cells were transiently transfected with EGFR-GFP, and seeded into glass bottom 35 mm dishes, time lapse Z-stacking imaging was performed and deconvolution was used to remove out of focus signals. By using Western blotting, we found that exosomes from J1.1 cells (HIV-1-infected), but not those from HIV-negative Jurkat cells, constantly induced ERK 1/2 phosphorylation in HNSCC and lung cancer cells. Cetuximab dramatically decreased ERK activation by exosomes from J1.1 cells, suggesting an EGFR-dependent ERK activation. While EGF induced a whole series of EGFR phosphorylation, exosomes from J1.1 cells failed to do so, suggesting that exosomes from J1.1 cells trigger ERK activation through a non-canonical EGFR pathway. Furthermore, ERK activation was significantly reduced in TLR3/dsRNA inhibitor pretreated cancer cells in response to exosomes from J1.1 cells, indicating exosomes triggered ERK activation is also TLR3 dependent. Time lapse imaging showed that exosomes quickly entered into cells within two minutes; however, Cetuximab greatly delayed exosome entry. To conclude, we found that exosomes from HIV-infected T cells can induce ERK activation in HNSCC and lung cancer cells through interaction with EGFR and TLR3.

#5049

Multiple hepatic lesion sequences in dichloroacetic acid (DCA) stop and promotion studies in the B6C3F1 male mouse.

Julia H. Carter, Harry W. Carter, Anthony B. DeAngelo. _Wood Hudson Cancer Research Lab., Newport, KY_.

Dichloroacetic acid (DCA) is a hepatocellular carcinogen in the male B6C3F1 mouse. Histopathologic analysis of a timed DCA dose response study has demonstrated three hepatic lesion sequences in this model. The purpose of the present study of 465 B6C3F1 male mice was to determine: (1) the persistence of early lesions in a "stop" study and (2) the efficacy of phenobarbital (PB) to promote these early lesions. The persistence of early lesions in mice exposed to drinking water containing 3.5 g/l DCA for 10 week intervals to 50 weeks was determined. One group remained exposed until the study was terminated at 100 weeks. Control animals received distilled water. In the promotion study, groups of animals exposed to drinking water containing 3.5 g/l DCA for 30 weeks were transferred to 0.6% phenobarbital (PB) or water alone and terminated at 60 or 90 weeks. Histopathologic analysis of liver lesions was performed on 1,481 formalin fixed, paraffin embedded tissue sections stained with hematoxylin and eosin. Lesions were identified and classified as altered foci (AF), hyperplastic nodules (HN), adenomas (AD) or carcinomas (CA). 10-50 weeks of DCA exposure doubled the prevalence of atypical (irregular misshapen and hyperchromatic) nuclei (82-95% vs 32% in untreated animals). Lifetime exposure to DCA was not required for induction of neoplastic lesions. Numerous neoplasms arose in animals exposed to 3.5 g/L DCA for 10, 20, 30, 40, or 50 weeks and then placed on water until 100 weeks. The prevalence of AD and CA increased with time to 50 weeks but decreased after 100 weeks, possibly reflecting the dose dependent suppression of proliferation in premalignant hepatic lesions such as hyperplastic nodules and adenomas by DCA. DCA exposure for 10-30 weeks doubled the prevalence of enlarged nuclei which were highly correlated with zonal changes. With increasing DCA exposure there was a shift in frequency of lesion types from hyperplastic nodues (20 weeks) to adenomas (40 weeks) to carcinomas (50 weeks). The major effect of PB promotion was to increase the number of eosinophilic AD at 60 and 90 weeks. These data demonstrate the continued development of preneoplastic hepatocellular lesions following termination of DCA treatment and are in agreement with studies which demonstrated that early-life exposure was as carcinogenic as life-long exposures for 98 weeks. Histopathologic analysis of these (DCA) stop and promotion studies in the B6C3F1 male mouse confirms that carcinomas can arise directly from single initiated cells in liver as well as within hyperplastic nodules and adenomas. Potentially, epigenetic-mediated effects may be involved since earlier reports indicate that RNA and micro-RNA profiles associated with prior DCA treatment were not sustainable. This work as supported by USEPA Cooperative Agreement #CR-814803-01-0.

#5050

Molecular heterogeneity in HPV-dependent and -independent vulvar carcinogenesis: Chromosome 1 gain reflects the cancer risk in vulvar intraepithelial neoplasia.

Maaike CG Bleeker,1 Dorian RA Swarts,1 Quirinus JM Voorham,2 Saskia M. Wilting,3 Marc van Beurden,4 Renske DM Steenbergen1. 1 _VUMC, Amsterdam, Netherlands;_ 2 _PALGA, Houten, Netherlands;_ 3 _EMC, Rotterdam, Netherlands;_ 4 _AVL, Amsterdam, Netherlands_.

Vulvar squamous cell carcinoma (VSCC) and precancerous high-grade vulvar intraepithelial neoplasia (VIN) can develop through human papillomavirus (HPV)-dependent and -independent pathways, indicating a heterogeneous disease. Only a minority of VIN progress, but current clinical and histological classifications are insufficient to predict the cancer risk. Here we analyzed copy number alterations (CNA) to assess the molecular heterogeneity of vulvar lesions in relation to HPV and cancer risk. HPV-status and CNA by means of whole-genome next-generation shallow-sequencing were assessed in 24 VSCC and 41 VIN lesions. The latter included VIN of women with associated VSCC (VINVSCC) and women who did not develop VSCC during >10 year follow-up (VINnoVSCC). HPV-testing resulted in 41 HPV-positive lesions (16 VINVSCC, 14 VINnoVSCC and 11 VSCC) and 24 HPV-negative lesions (11 VINVSCC and 13 VSCC). HPV-positive and -negative VSCC showed a partially overlapping pattern of recurrent CNA, including frequent gains of 3q and 8q. In contrast, amplification of 11q13/cyclinD1 was exclusively found in HPV-negative lesions. HPV-negative VINVSCC had less CNA than VSCC (P = 0.009), mainly 8q gains and 8p losses. In HPV-positive lesions, CNA frequencies were lower in VINnoVSCC than in VINVSCC (P = 0.022) and VSCC. Interestingly, a 1pq gain was detected in 81% of VINVSCC and only 29% of VINnoVSCC. In conclusion, HPV-dependent and -independent vulvar carcinogenesis is characterized by distinct CNA patterns at the VIN stage, while more comparable patterns are present at the cancer stage. Progression risk in VIN is reflected by the extent of CNA, in particularly chromosome 1 gain.

#5051

**STAT3 signaling activates MSK1-mediated histone H3 phosphorylation in** N **-nitrosocompounds induced carcinogenesis.**

Hongyan Qi, Zhiyi Yang, Shuilian Zhang, Xinxin Ke, Chujun Dai, Jimin Shao, Jing Shen. _Zhejiang University, School of Medicine, Hangzhou, China_.

Signal transducer and activator of transcription (STATs) signaling, particularly STAT3, have been demonstrated to be one of the central pathways for cancer initiation and progression. However, the functional role of STAT3 in regulation of epigenetic changes in cancer, such as histone modifications, remains unclear. Our present study performed on N-nitroso compounds (NOC) induced gastric epithelial cell transformation revealed a positive correlation of active STAT3 signaling with increased histone H3 phosphorylation. Further analysis showed that mitogen- and-stress activated protein kinase (MSK1) was upregulated by NOC treatment, thereby promoting H3 Ser 10 phosphorylation (H3S10P). Intriguingly, putative STAT3 binding sites were identified in the MSK1 promoter, and we finally confirmed that MSK1 is a novel target gene of STAT3. It has been reported that STAT3 is a direct substrate for MSK1. Therefore, we analyzed the status of STAT3 activation after MSK1 overexpression and knockdown, and a positive feedback loop was found between them. With specific siRNA or inhibitors, we further demonstrated the aberrant activation of STAT3/MSK1/H3S10P axis in MNU-induced gastric cancer (GC) model in mice, human gastric cancer cells, and GC patients. ChIP-seq analysis was performed to determine the H3S10 phosphorylation distribution pattern in NOC-transformed gastric epithelial cells, and some candidate regulated genes were successfully identified. Together, the results of these studies shed light onto a critical role of STAT3 signaling in epigenetic modification network, mediated at least in part by MSK1. Our results suggest that STAT3/MSK1/H3S10P pathway and downstream key effectors represent interesting therapeutic targets in gastric carcinogenesis.

#5052

ID3 mediated vascular reprogramming of PCB exposed endothelial cells and its potential contribution to lung tumorigenicity.

Mayur Doke, Jayanta Das, Quentin Felty. _Florida International Univ., Miami, FL_.

ID3, a transcription regulator, has been shown to contribute to the aggressive spread of certain cancers by facilitating the generation of tumor blood vessels. Tumor vascular cells characterized as CD133+ endothelial stem/progenitor cells are associated with poor prognosis in lung cancer. Previously, we have shown that ID3 overexpression reprograms adult endothelial cells to acquire vascular stem markers including CD133. However, little is known regarding the molecular contribution of ID3 to tumor vascular reprogramming and how this may impact the aggressive growth of lung cancer. In the current project, we evaluated ID3 mediated vascular reprogramming upon polychlorinated biphenyl (PCB) exposure. This idea is consistent with the literature, which shows that PCBs accumulate in the human lung; PCBs produce pathological vascular remodeling; high levels of PCBs are found in human lung tissue; and epidemiological studies associating lung toxicity with PCBs. Recent studies identify PCB153 as one of the largest contributors for total PCB body burden in humans. Using human lung endothelial and smooth muscle cells, we exposed ID3 overexpressing and vector control cells to PCB153. We observed a significant increase in cell proliferation as determined by the BrdU incorporation assay and FACS analysis. Similarly, a 3D HuBiogel model, which mimics in vivo conditions, showed a significant increase in size and number of vascular spheres upon PCB153 treatment. Pluripotent vascular stem cells showed the loss of VE-cadherin and gain of MMP9, N-cadherin, and vimentin, which are markers of endothelial-mesenchymal transition. RNA-seq analysis showed that PCB153 exposed vascular stem cells had a significant increase in lncRNAs MALAT1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) and HIF1A-AS2. A combination of ChIP-seq and transcriptome analysis identified significantly up-regulated genes (HES1, WEE1, E2F2) and down-regulated genes (SMURF2, CDKN2C, SH2B3). Our findings suggest that ID3 mediates vascular reprogramming through transcriptional regulation of pluripotency factors and epigenetic regulating lncRNAs. ID3 may also serve as a novel diagnostic/prognostic indicator for evaluating chemical-induced vascular reprogramming. Its potential application may be useful in identifying individuals who are susceptible to aggressive lung cancer from exposure to vascular toxicants.

#5053

Mlh1 deficiency increases the risk of hematopoietic malignancies post low- and high-LET radiation exposure.

Rutulkumar Patel,1 Stanton L. Gerson,1 Scott M. Welford2. 1 _Case Western Reserve University, Cleveland, OH;_ 2 _University of Miami, North Miami, FL_.

Introduction/Aim: Natural sources of radiation in space include galactic cosmic rays (GCR), solar energetic particles and trapped energetic particles in a planetary magnetic field. These sources are difficult to shield, thus posing significant health risks to astronauts on long-term inter-planetary missions. During space travel, genomic instability is a major concern where astronauts are exposed to potent sources of ionizing radiation, namely GCR consisting of high energy and charged atomic nuclei. In particular, hematopoietic stem cells (HSCs) are susceptible to internal and external stresses that threaten the integrity of the cell, and accumulation of damage can lead to HSCs dysfunction and oncogenesis. Recent data has demonstrated that humans accumulate microsatellite instability with acquired loss of MLH1 protein in HSCs as a function of age. Therefore, we hypothesized that high-LET (linear energy transfer) radiation characteristic of the GCR that will confront astronauts on space missions will damage HSCs and contribute to induction and progression of hematopoietic malignancies. Methods: To study this hypothesis, we employed a DNA mismatch repair deficient mouse model (Mlh1+/-) to study the effects of low-LET γ-ray vs high-LET 56Fe ion radiation on HSCs of potential astronaut population. In vitro colony forming unit assays and in vivo complete blood count (CBC) plus competitive repopulation assays were carried out to understand harmful impact of radiation and Mlh1 deficiency on HSCs functions. In addition, mice were followed up to 18 months post irradiation to observe HSC malignancies in Mlh1+/+ and Mlh1+/- mice. Results: HSC short- and long-term functional assays showed defects in HSCs/HPCs function caused by irradiation, but not depending on Mlh1 status. CBC 5 and 9 months post irradiation demonstrated no impact of irradiation or Mlh1 status on HSC differentiation. However, 56Fe-ion irradiated Mlh1+/- mice showed a significant higher incidence of lymphomagenesis compared to γ-rays irradiated and sham-irradiated Mlh1+/- mice. In addition, immunohistochemistry analysis of lymphomas displayed significant higher incidence of T-cell rich B-cell lymphomas. Conclusion: Thus, the data show that MMR defects in HSCs leads to sensitization to radiation induced hematopoietic malignancy, and that radiation quality effects exacerbate the sensitivity. The findings could have profound effects on astronaut screening and designing better mitigators for space missions.

#5054

Transcriptional changes in breast cancer initiation.

Natascia Marino,1 Rana German,1 Mariah L. Johnson,2 Xi Rao,1 Xiaoling Xuei,1 Jun Wan,1 Anna Maria V. Storniolo1. 1 _Indiana Univ.-Purdue Univ. Indianapolis, Indianapolis, IN;_ 2 _The Ohio State University, Columbus, OH_.

Background: Identifying the earliest stages of breast cancer carcinogenesis, present well before any clinical signs of disease, is the necessary underpinning of an effective breast cancer prevention strategy. We propose to elucidate transcriptomic changes occurring in the breast tissue during cancer initiation. by analyzing biospecimens donated by women before any clinical sign of sporadic breast tumor (here labeled "susceptible"). In the present work, we report on the transcriptome differences in the microdissected breast compartments (epithelium, stroma and fat) of susceptible versus healthy premenopausal women.

Methods: The specimens were obtained from the Susan G. Komen Tissue Bank at IU Simon Cancer Center. We compared the transcriptome profiles of breast tissues from 7 susceptible and 17 healthy premenopausal women between the age of 34 and 52 years, who were free of breast pathology at the time of donation. Donors in the two experimental groups were matched according to age, racial background and menstrual phase. Differential expression analysis was performed using EdegR. False discovery rate (FDR) was computed from p-values using the Benjamini-Hochberg procedure. Ingenuity Pathway Analysis was used to identify relevant signaling pathways. Because circulating hormones variations during menstrual cycle affect the breast epithelium gene expression, we also examined transcriptome differences independently from the menstrual phase.

Results/Discussion: We found 536 transcripts differentially expressed between the two groups (p<0.05). However, only 412 changed independently from the follicular or luteal status. Among these, 130 transcripts (including 1 linRNA) were downregulated, while 282 transcripts (including 6 lincRNAs and 5 miRNAs) were upregulated in the susceptible versus healthy breasts. Among the upregulated genes, we observed three major affected pathways: 1) lipid metabolism, 2) molecular transport, and 3) energy production. When we set a more stringent cutoff (p<0.05, fold change> 2 and FDR<0.2), only 11 genes were differentially expressed between susceptible and healthy controls; these are involved in cellular metabolism (AKR1C1, AKR1C2, and SDR16C5) and cell adhesion and cytoskeleton organization (CNTNAP2 and XIRP2). The transcription repressor ZFP57 was the only downregulated gene (fold change=-29; p=0.0002; FDR=0.09).The study will be soon corroborated with the transcriptome profiling of the other two breast compartments (stroma and fat).

Conclusion: This study shows that earliest alterations in breast cancer initiation affect metabolic pathway as well as transcriptional regulation. Interestingly, cell adhesion signaling may also be dysregulated at this early stage of cancer development. The findings will contribute to our understanding of the mechanisms of cancer initiation, as well as the identification of new therapeutic targets and thus, improvement of preventive interventions.

#5055

**Expression of Tn and SialylTn tumor antigens in intestinal epithelia in** APC **-mutant mice promotes colorectal carcinogenesis and progression.**

Yuliang Jiang,1 Su-Ryun Kim,2 Shweta Kotian,2 Sean R. Stowell,3 Guangyu An,1 Tongzhong Ju2. 1 _Capital Medical University, Beijing Chao-yang Hospital, Beijing, China;_ 2 _FDA, Silver Spring, MD;_ 3 _Emory University School of Medicine, Atlanta, GA_.

Colorectal cancer (CRC) is a leading cause of cancer deaths in the Western world, and several different animal models for CRC have been established to understand its initiation, progression, and metastasis. Mice with mutations in APC (APCm) is widely accepted in the field because APC is the predominant mutated gene in human CRC. Mice carrying APCm only develop multiple intestinal neoplasia, but not carcinoma. Importantly, emerging evidence shows that tumor associated carbohydrate antigens, Tn and sialylTn (STn) appear at an early stage of colon carcinogenesis, and are associated with progression and metastasis. However, the molecular mechanisms underlying the role of Tn/STn antigens in CRC tumorigenesis and progression are unknown. We identified a novel X-linked gene Cosmc (C1GalT1C1) which encodes a molecular chaperone Cosmc regulating O-glycosylation through assisting correct folding of core 1 β3-galactosyltransferase (T-synthase, C1GalT1). T-synthase is the key enzyme that converts Tn antigen to normal core 1 O-glycans on glycoproteins in all animal cells. Cosmc mutations lead to loss of T-synthase activity and resultant expression of Tn/STn antigens in human carcinoma cells and in other diseases. Moreover, in our study, intestinal epithelial cell (IEC)-specific Cosmc-knockout (IEC-Cosmc-KO) mice spontaneously developed invasive tumors mainly in the rectum from 3-12 months of age through complex pathways. To further investigate the role of Tn/STn antigens in CRC formation, progression and metastasis in a context of APC mutations, we generated IEC-Cosmcnull:APC-/+ (double-knockout, DKO) by gene targeting. Totally, 22 DKO mice (13 male and 9 female) and similar numbers of control mouse groups (APC-KO, Cosmc-KO and WT) were generated. Among them, the DKO male and female mice grew slower companied with stress signs and some mortalities around 3-4 months of age. Importantly, both APC-KO and DKO mice had multiple gross tumors in the small intestine, and all DKO mice had more and larger gross tumors in colorectum while only 15% of APC-KO mice developed tumors with smaller sizes in colon. Consistent with our findings, thickened rectum was observed in Cosmc-KO mice. Furthermore, the enlarged inguinal lymph nodes were also found in the DKO mice. Histology confirmed the Tn/STn expression, as well as adenomas and adenocarcinomas in the DKOs, and adenomas in APC-KOs and Cosmc-KO male mice. The distant metastasis of the CRC was not yet observed in these young DKO-mice, since it may require a longer time to develop. These results clearly indicate that the Cosmc deletion resulted in Tn and STn expression, promotes the colorectal carcinogenesis in APC-mutant mice. We are generating more elder DKO and control mice to continue investigating the role of Tn/STn in CRC progression and metastasis by performing detailed molecular analyses.

#5056

HPV, HIV and male gender as major risk factors for anal neoplastic transformation in African Americans.

Hassan Brim,1 Ali Afsari,1 Nazli Atefi,1 Nicole Retland,1 Muneer Abbas,1 Tammey Naab,1 Babak Shokrani,1 Adeyinka O. Laiyemo,1 Edward L. Lee,1 Seyed M. Nouraie,2 Hassan Ashktorab1. 1 _Howard University, Washington, DC;_ 2 _University of Pittsburgh, Pittsburgh, PA_.

Background: Human Papilloma Virus (HPV) is the most common sexually transmitted infectious agent. The incidence HPV-related anal cancers increased among HIV infected people. Aim: To assess risk factors of anal lesions among African Americans in an inner city hospital. Methods: We reviewed medical records of 370 African-Americans with anal lesions at Howard University Hospital from Jan. 2007 to Dec. 2015. Demographic, clinical and pathological data including; HPV, HIV, HCV (hepatitis C virus), diabetes mellitus (DM), hypertension (HTN) and body mass index (BMI) were collected. Statistical analysis was performed using Chi-square and Student's t-test. Results: 276 (75%) patients were male, with median age of 44 years and BMI of 25.8 kg/m2. The frequency of condyloma, high grade dysplasia, squamous cell carcinoma (SCC) and adenocarcinoma was 191 (52%), 26 (7%), 31 (8%) and 8 (2%), respectively. The frequency of HPV, HIV, and HCV was 231 (68%), 147 (43%) and 42 (12%), respectively. HPV and HIV were risk factors for condyloma and dysplasia (P<0.05). Forty two percent of patients had diabetes (DM) and hypertension (HTN). Conclusion: Our results shows that majority of patients with condyloma are male and young with HPV and HIV infection. A clinical utility of HPV as risk factor may be use for prediction of anal lesions.

#5057

**Exposure to highly energetic charged (hze) particle radiation is associated with higher colitis and colorectal cancer incidence in IL10** -/- **mice.**

Shubhankar Suman, Bo-Hyun Moon, Albert J. Fornace, Kamal Datta. _Lombardi Comprehensive Cancer Center, Georgetown Univ., Washington DC, DC_.

Epidemiological data from atom-bomb survivors and from radiotherapy patients have highlight an underlying risk of colitis and colorectal cancer (CRC) after ionizing radiation-exposure. In contrast, astronauts planning to undertake deep space exploration such as mission to Mars have risk of radiation exposure from highly energetic charged (HZE) particles present in galactic cosmic radiation (GCR) and risk of colitis and CRC after HZE-exposure is yet to be determined. Therefore, animal studies are warranted to address existing uncertainties in colitis and CRC risk prediction after exposure to HZE radiation. Here we report IL10-/- mice as a suitable mouse model for assessment of colitis and CRC risk after exposure to space radiation.

Male mice (n=12 mice/group) were whole-body exposed to sham-radiation, γ-rays and 28Si-ion (300 MeV/n; 70 keV/μm; 1.4 Gy). Mice were sacrificed three months after irradiation, and colon swiss-rolls sections were analyzed for colitis and CRC grade was noted in hematoxylin and eosin stained sections. Immunohistochemistry (β-catenin, Cyclin-D1, phospho-H3, iNOS, phoshoNfKβ-p65, and Cox2) was also performed to assess HZE-induced proliferative and inflammatory signaling. Further, inflammation-associated gene-expression analysis was also conducted in both radiation-induced tumor and adjoining normal tissue area.

Here we report that relative to controls and γ-ray, colitis score, colon tumor incidence, size and grade was significantly higher after space (28Si-ion) radiation exposure. Higher number of phospho-H3 positive cells in 28Si-ion exposed mouse colon demonstrated increased proliferative index and in increased colon tumors of higher grade, could be due to greater activation of β-catenin and its downstream effector cyclin D1. In addition, higher accumulation of iNOS, phoshoNfKβ-p65, and Cox2 in stromal and epithelial cells after space radiation exposure clearly indicates upregulation of both immune and epithelial inflammation as drivers of colitis and CRC development. Inflammation associated protein markers and gene expression signatures were more pronounced after 28Si-ion exposure. Significant up-regulation of GATA4, ICAM1, ITGA2 and EGFR were exclusive to 28Si-ion exposed mouse colon, however PTGES and TGFβ1 were upregulated in both γ and 28Si-ion exposed mice.

Overall, this study suggests that IL10-/- mouse are a suitable model to assess IR-induced colitis and CRC risk, and that space radiation carries higher risk of colitis and CRC incidence, relative to γ-rays at comparable doses. This study has implications for risk prediction in astronauts planning for deep space mission and also for cancer patients planning to undergo particle radiotherapy.

#5058

STRAP-induced Wnt/ß-catenin signaling is important in inactivated Apc-mediated tumorigenicity and stemness of colorectal cancer.

Trung Vu, Arunima Datta, Lin Jin, Pran K. Datta. _University of Alabama at Birmingham, Birmingham, AL_.

Aberrant Inactivation of the Apc gene and activation of Wnt/β-catenin signaling in colorectal cancer (CRC) has been implicated in tumorigenesis and homeostasis of cancer stem-like cells (CSCs), albeit the mechanism remains mostly obscure. We have previously reported the identification of a WD-domain protein, STRAP (Serine-Threonine Kinase Receptor-Associated Protein) that inhibits TGF-ß signaling, interacts with a variety of proteins, and influences a wide range of cellular processes. Upregulation of STRAP in CRC patients had a worse survival with adjuvant therapy. In contrast, patients carrying tumors with normal or low STRAP expression benefited from the treatment. Our preliminary data suggest that homozygous deletion of STRAP causes embryonic lethality at embryonic day E9.5. Therefore, to study the role of STRAP in vivo in CRC, we have generated Strap-conditional knockout mice Villin-Cre;Strapfl/fl(Strap-cko) and used two mouse models of CRC: Apc+/min and AOM/DSS. Conditional deletion of Strap in both mouse models resulted in a dramatic decrease in tumor formation. Our results demonstrate that STRAP is required for tumor formation through activation of Wnt/β-catenin signaling pathway in both models. Immunoblotting and immunohistochemistry analyses of tumors derived from the Strap-cko;Apc+/min mice showed decreased levels of β-catenin and of its nuclear staining. Furthermore, mRNA levels of several Wnt target genes are also decreased in tumors derived from Strap-cko;Apc+/min mice compared to those from Apc+/min mice. Formation and sustained propagation of intestinal enteroids require cells from the stem cell niche. APC mutation accompanied by elevated Wnt signaling promotes spheroid-shaped enteroids as indicated by the absence of crypt buds. We found that enteroids prepared from Strap-cko;Apc+/min mice exhibited more budding compared with the Apc+/min enteroids, indicating the role of STRAP in stemness. Strap deletion in enteroids decrease stem cell markers such as CD133, CD44, and LGR5. By using chromatin immunoprecipitation (ChIP) assay, we have found that STRAP knockdown can affect the recruitment of β-catenin/TCF4 complex on target promoter regions. Interestingly, STRAP protein and mRNA levels were found to be upregulated in Apc+/min induced tumors. STRAP is upregulated by Wnt/β-catenin signaling pathway, as evidenced by downregulation of STRAP in β-catenin/TCF4 inhibitor iCRT3 treated cells. In addition, ChIP assay has identified TCF DNA binding sites in the Strap promoter through which Wnt signaling regulates Strap gene expression. Altogether, our findings reveal that STRAP is a novel target of Wnt/β-catenin signaling and it is required for inactivated Apc- and Wnt/β-catenin-induced stemness and tumorigenesis through a complex feedforward loop.

#5059

Comprehensive molecular profiling of HPV-induced transformation over time.

Iris Babion, Viktorian Miok, Annelieke Jaspers, Wessel N. van Wieringen, Renske D. Steenbergen, Saskia M. Wilting. _VU University Medical Center, Amsterdam, Netherlands_.

Although persistent infection with high-risk human papillomavirus (HPV) is generally acknowledged as necessary cause for cervical cancer, additional molecular changes are required for the development of cancer. Those changes include chromosomal aberrations that result in deregulated expression of coding and non-coding RNAs. In this study we performed a comprehensive and longitudinal molecular characterization of HPV-transformed keratinocyte cell lines, to identify the sequential order and relevance of these molecular alterations for HPV-induced transformation. Genome-wide chromosomal, mRNA and miRNA expression profiles were generated from 4 HPV-transformed keratinocyte cell lines at 8 different passages representing progressive stages of transformation. Unsupervised hierarchical clustering of DNA copy number, mRNA and miRNA profiles revealed that in all 4 cell lines most pronounced changes were associated with the acquisition of anchorage independence, a hallmark of transformation. Approximately one third of differentially expressed mRNAs and miRNAs was directly attributable to DNA copy number alterations. Focal adhesion, TGF-β signalling and mTOR signalling pathways were enriched among these genes. In addition, longitudinal analysis identified more than 600 potential miRNA-mRNA interactions that were negatively correlated during passaging. Four interactions were also predicted by 3 independent prediction algorithms, and have in part already been functionally validated, thereby demonstrating the validity of our approach. In conclusion, integrated longitudinal analysis of our HPV-induced transformation model enabled us to pinpoint relevant interconnected molecular changes and affected signalling pathways. Increased understanding of the interplay between different molecular alterations and affected pathways will be of importance for the clinical management of patients diagnosed with HPV-induced (pre)cancerous lesions.

#5060

Exosomes derived from HIV-infected T cells promote cancer cell growth and progression via the HIV TAR RNA.

Lechuang chen, Zhimin Feng, Hong Yue, Scott Sieg, Bingcheng Wang, Alex Y. Huang, Ge Jin. _Case Western Reserve University, Cleveland, OH_.

Background: Residual and persistent HIV replication plays key roles in the development and progression of non-AIDS-defining cancers (NADCs), including head and neck as well as lung cancers. However, the underlying mechanisms remain elusive. HIV-infected T cells produce immunologically active exosomes to influence intercellular communication and regulate immune response at both local and distant sites, thus potentially contribute to enhanced risk for tumorigenesis. We investigated the role of exosomes derived from HIV-infected T cells in growth and progression of head and neck cancer (HNC) and lung cancer cells. Methods: Exosomes were isolated from conditioned media of HIV-1-infected J1.1 cells and non-infected control Jurkat cells using the ultracentrifugation protocol. Exosomes were validated by western blotting for tetraspanins (CD9, CD63 and CD81) and transmission electron microscope. The acetyl-CoA acetylcholinesterase (AChE) enzyme activity was used to quantify exosomes. HNC cells UM-SCC-104, TR146, HSC3, SCC9 and lung cancer cells NCI-H1299, NCI-H1437 were treated with exosomes for cell growth, migration and gene expression in vitro and in vivo. Exosome HIV RNA was determined by quantitative RT-PCR. Results: Exosomes isolated from HIV-1-infected T cells, but not those from normal control T cells, significantly stimulated HNC and lung cancer cell proliferation, migration and invasion in vitro. Exosomes purified from plasma of HIV-positive individuals also stimulated proliferation of cancer cells. HNC and lung cancer cells co-inoculation with exosomes from HIV-1-infected T cells into nude mice promoted tumor growth compared to those from non-HIV control T cells. Mechanistically, we found that the HIV trans-activation response (TAR) element RNA, which exists in excess of other HIV RNAs in exosomes from HIV-1-infected T cells, enhanced cancer cell proliferation and stimulated EGF- and TLR3-inducible gene expression. The TAR RNA mutant with nucleotide replacements in the loop/bulge region failed to induce gene expression. Moreover, co-transfection of the TAR RNA aptamer blocked induction of gene expression induced by HIV-positive T-cell exosomes. Conclusion: HIV TAR RNA-containing exosomes derived from HIV-1-infected T cells promote growth and progression of NADCs through interaction with EGFR and TLR3.

#5061

ALDH2 restricts esophageal stem/progenitor cell expansion via autophagy under alcohol-induced mitochondrial stress.

Prasanna Modayur Chandramouleeswaran,1 Kelly A. Whelan,2 Noah Engel,3 Jason Correnti,1 Koji Tanaka,1 Satish Srinivasan,1 Manti Guha,1 Rotonya Carr,1 Andres J. Klein-Szanto,4 Balaraman Kalyanaraman,5 Che-hong Chen,6 Daria Mochly-Rosen,6 Narayan G. Avadhani,1 Hiroshi Nakagawa1. 1 _The University of Pennsylvania, Philadelphia, PA;_ 2 _Temple University, Philadelphia, PA;_ 3 _Vanderbilt Univeristy, Nashville, TN;_ 4 _Fox Chase Cancer Center, Philadelphia, PA;_ 5 _Medical College of Wisconsin, Milwaukee, WI;_ 6 _Stanford University, CA_.

Introduction: Esophageal cancer is linked to heavy alcohol (EtOH) drinking and polymorphic aldehyde dehydrogenase 2 (ALDH2) mutation. Mutant ALDH2E487K protein delays mitochondrial clearance of acetaldehyde, the chief metabolite of EtOH and a major human carcinogen. Autophagy maintains mitochondrial redox homeostasis and may act as a tumor suppressor by decreasing oxidative stress. The role of ALDH2 and autophagy in esophageal epithelial homeostasis remains elusive. Methods: We analyzed EtOH-exposed esophageal epithelial cells and EtOH-fed Aldh2E487 or Aldh2WT mice by immunoblotting, immunofluorescence, flow cytometry and respirometry to determine mTORC1 signaling, autophagy, mitochondrial dysfunction and superoxide production. Mitochondrial superoxide was documented via mitochondria-targeted antioxidant mito-CP. Autophagy flux was blocked pharmacologically by chloroquine (CQ) or RNA interference (RNAi) against Beclin, a mediator of autophagy. Mitochondria-targeted autophagy (mitophagy) was further evaluated by RNAi against Parkin. To evaluate esophageal stem/progenitor cell function, we assessed the formation of single cell-derived three-dimensional (3D) organoids from mice with Aldh2E487K or Atg7loxP/loxP (autophagy mediator). We determined organoid formation following EtOH exposure, autophagy inhibition by CQ or adenoviral Cre-mediated Atg7 deletion. Rapamycin was used to inhibit mTORC1 and activate autophagy. Results: EtOH induced mitochondria damage in esophageal epithelial cells in vitro and in vivo with enhanced mitochondrial dysfunction by Aldh2E487K. EtOH induced mitochondrial dysfunction and superoxide production was antagonized by mito-CP. EtOH robustly suppressed mTORC1 signaling with concurrent activation of autophagy flux. Pharmacological or genetic inhibition of autophagy or mitophagy aggravated EtOH-induced mitochondrial dysfunction, superoxide production and cell death. Recapitulating esophageal epithelial proliferation-differentiation gradient ex vivo, Aldh2E487K murine esophageal cells displayed increased 3D organoid formation capability where resulting organoids contained more basaloid (undifferentiated) cells with mitochondrial dysfunction as compared to Aldh2WT cells. Moreover, EtOH stimulated 3D organoid formation by Aldh2E487K cells in a dose-dependent manner. Aldh2 dysfunction was phenocopied by CQ or Atg7 deletion, which increased 3D organoid formation. Interestingly, rapamycin suppressed organoid formation. Conclusions: Our innovative approach reveals that autophagy and ALDH2 protect against EtOH-induced mitochondrial dysfunction to maintain redox homeostasis and prevent abnormal propagation of esophageal stem/progenitor cells, providing novel mechanistic insights into the role of dysfunctional alcohol metabolism and autophagy in esophageal carcinogenesis.

### Carcinoma-Associated Fibroblasts in Tumor Progression

#5062

CAFs in breast cancer circulation: The influence of cancer cell intrinsic mechanisms.

Utsav Sharma,1 Philip Miller,1 Kelsie Medina Saenz,1 Angela Spartz,2 Marc Lippman,1 Dorraya El-Ashry2. 1 _Univ. of Miami Miller School of Medicine, Miami, FL;_ 2 _University of Minnesota, Masonic Cancer Center, Minneapolis, MN_.

Background: Metastatic disease is the primary cause of breast cancer (BC) mortality. Tumor-stromal cell interactions play a pivotal in tumor initiation, progression and metastasis. Cancer-associated fibroblasts (CAFs) are the majority of stroma in BC and are critical to BC tumorigenicity and malignancy. Metastasis occurs due to the transport of circulating tumor cells (CTC) and clusters of CTCs through the vasculature. We recently identified circulating CAFs (cCAFs) as a novel circulating biomarker associated with metastatic BC. cCAFs not only circulate individually, in BC patient blood and in blood from both spontaneous and xenograft murine BC models, but cCAFs are also found in clusters with CTCs. In this study, we examine the egress of CAFs and follow them through to metastatic sites, and evaluate the ability of BC cell subtype/metastatic propensity to influence cCAF and cCAF/CTC cluster egress.

Methods: We used NSG mice with orthotopic xenograft implantation of BC cells, primary CAF cell lines, or co-implantation of BC and CAF cell lines. We used two different BC cell lines, the nonmetastatic BC cell line, MCF-7, and the highly metastatic primary BC cell line, DT28. We also employed the MMTV-PyMT spontaneous model of BC metastasis to evaluate cCAFs and CTCs in a preclinical model. Mice were sacrificed at specific time points, and cardiac blood was collected to ascertain the temporal dynamics of cCAF and CTC presence. Blood was filtered using the faCTChecker microfluidic filtration instrument (Circulogix). Filters were enumerated by IF for cCAFs, CTCs, and cCAF/CTC co-clusters. To explore BC-intrinsic factors that influence cCAF and cCAF/CTC cluster egress, we modeled egress from the primary tumor in vitro using transendothelial cell migration assays, where the chemoattractant for CAF cells was conditioned media from the different BC cells lines.

Results: In both spontaneous and orthotopic xenograft models of BC, cCAFs, CTCs, and cCAF/CTCs appear early in tumor development. cCAF/CTC clusters increase in correlation with tumor burden and metastasis. CAFs injected alone in orthotopic xenografts are able to egress independently of BC cells and cCAFs and cCAF clusters were seen; however, co-inoculation with BC cells resulted in substantially higher numbers of both individual cCAFs and cCAF clusters, and now co-clusters were seen. cCAFs appear about 4 days post-injection, and precede appearance of CTCs and CAF/CTC clusters. CAFs co-injected with BC cells into the MFP appear at metastatic sites. In in vitro transendothelial assays, BC secreted factors that potentiate CAF egress are identified as well as the if cCAF/CTC clusters egress as clusters or cluster in the circulation.

Conclusion: Although CAFs are highly motile and cCAFs precede CTCs into the circulation, BC subtype influences the ability of CAFs to egress. Targeting cCAF egress and/or cCAF/BC cell clusters provides novel avenues to prevent or treat BC metastasis.

#5063

Beta-arrestin-1 function in CAFs is necessary for enhancement of self-renewal of NSCLC stem-like cells.

Mohan Kumar Durai Raj, Namrata Bora-Singhal, Srikumar Chellappan. _Moffitt Cancer Center, Tampa, FL_.

Cigarette smoking is a major risk factor in the genesis of non-small cell lung cancer (NSCLC), which accounts for 85% of all lung cancer. Nicotine, the addictive component of tobacco smoke, has been shown to induce proliferation, invasion and epithelial-mesenchymal transition (EMT) in NSCLC cells in vitro and promote growth and metastasis of NSCLCs in vivo. These nicotine-induced pro-tumorigenic functions are facilitated through activation of nicotinic acetylcholine receptors (nAChRs). The scaffolding protein β-arrestin-1 (ARRB1), which is involved in the desensitization of signals from activated G-protein-coupled receptors (GPCRs), plays a vital role in mediating the proliferative effects of nicotine through nAChR signaling. Nicotine induces the nuclear translocation of ARRB1 and increases the expression of E2F-regulated proliferative and survival genes to promote the growth and progression of NSCLCs. β-arrestin-1 is also necessary for nicotine-mediated induction of EMT; nicotine could induce a variety of mesenchymal genes including fibronectin, vimentin, ZEB1 and ZEB2 in a β-arrestin-1 dependent manner. Further, nicotine promotes stemness of NSCLCs by inducing SCF (Stem cell factor) in a β-arrestin-1 dependent manner. While all these studies from NSCLC derived cell lines and tumor tissues highlight the significance of β-arrestin-1 in cell-autonomous pro-tumorigenic functions, the role of β-arrestin-1 in NSCLC microenvironment is largely unknown. Cancer associated fibroblasts (CAFs) are shown to promote the self-renewal and proliferation of cancer stem cells in vitro and in vivo. In the present study we address whether β-arrestin-1 is necessary for CAFs to promote self-renewal of stem like cells from NSCLCs.

We generated a GFP expressing KRAS mutant NSCLC cell line A549 (A549-GFP). The side population (SP) cells from A549-GFP was isolated and used in 3D co-culture with lung CAFs in a stem cell selective medium. Our results show that CAFs can promote the self-renewal of SP cells, as measured by a sphere formation assay. Interestingly, depletion of β-arrestin-1 in CAFs significantly impaired the ability of CAFs to promote self-renewal and enhance sphere formation. Experiments are under way to assess the downstream mediators of β-arrestin-1 in CAFs that bring about the impairment in self renewal, which includes signaling molecules like TBK1 and a variety of cytokines. These studies are expected to shed new light on the mechanisms by which CAFs promote self-renewal and tumor growth, enabling identification of actionable pathways downstream of β-arrestin-1 that can potentially be targeted for NSCLC therapy.

#5065

**Cancer derived** Escherichia coli **induces tumor-promoting inflammatory cytokine IL-6 in cancer associated fibroblasts (CAFs) in a NF-κB/BRD4 dependent manner.**

Gabriela Uribe, Russel Rourke, Romain Villeger, George Golovko, Kamil Khanipov, Zhiqing Liu, Maria Pimenova, Yuriy Fofanov, Jia Zhou, Allen R. Brasier, Irina V. Pinchuk. _University of Texas Medical Branch, Galveston, TX_.

Introduction: Chronic inflammatory responses within the colonic environment are critical to colorectal cancer (CRC) tumor development. While the exact causes of CRC development are unknown, recent data suggest that dysbiosis in the colonic microbiome results in the overgrowth of bacteria from Enterebacteriaceae, Fusobacteriaceae and Bacteroidaceae taxonomic families, contributing to CRC development. Among bacteria of the Enterebacteriaceae family, adherent invasive Esherichia coli has been shown to promote tumor cell growth via production of colibactin. However, the contribution of E.coli to the tumor promoting inflammatory microenvironment (TME) during sporadic CRC development remains unknown. Cancer associated fibroblasts (CAFs) are a major component of TME and contribute to tumor-promoting inflammatory responses via NF-κB dependent production of IL-6. Herein we investigate the effect of E. coli on CAFs. We hypothesize that stimulation of CAFs by cancer derived E.coli is a key processes in supporting tumor-promoting inflammation in CRC. Methods: Bacterial DNA was extracted from tumor and adjacent normal tissue of 28 CRC patients. High throughput sequencing was done using metabarcoding of 16S rDNA for bacteria and analyzed using CLC Genomics Workbench 8.0.5 Microbial Genomics Module, SILVA v119 database for 16S. E.coli PS092717 strain, isolated from CRC tumor site, was used in co-culture experiments. Real time RT-PCR and multiplex cytokine array was used to analyze fibroblasts gene expression and secretion, respectively. Results: We observed a reduction in microbial diversity in tumor vs adjacent normal tissue, with an increased prevalence of Fusobacteria, and Enterobactereacea, but not Bacteroidacea. We then tested the effect of E.coli PS092717 on colonic primary normal fibroblasts and CAFs. Exposure of N-CMFs to E.coli for 24 h only moderately increased IL-6, while CAF responded with a greater increase in IL-6 production. Analysis of the signaling mechanism demonstrated that this process was NF-κB dependent, and was abrogated in the presence of NF-kB specific inhibitor, triptolide (20 ng/mL). Bromodomain containing protein 4 (Brd4) is an epigenetic regulator that can recruit canonical NF-κB transcription factor RelA. Therefore, we analyzed its involvement in the above processes. NF-κB mediated increase in IL-6 expression and secretion induced by E.coli PS092717 was strongly reduced in CAFs than N-CMFs in the presence of BRD4 specific inhibitor (1 µM ZL0590). A similar observation was made when a major components of the E.coli cell wall, lipopolysaccharide (LPS), was used. Conclusion Taken together our data suggests that, E.coli derived from cancer tissue and its cell wall component LPS can contribute to the tumor promoting inflammation via stimulation of IL-6 production by CAFs and this process is BRD4/NF-κB-dependent.

#5066

Dual-targeting photoimmunotherapy (NIR-PIT) for esophageal cancer cells and cancer-associated fibroblasts (CAFs).

Hiroaki Sato,1 Kazuhiro Noma,1 Toru Narusaka,1 Satoshi Komoto,1 Yuki Katsura,1 Takuya Kato,1 Takayuki Ninomiya,1 Toshiaki Ohara,1 Hiroshi Tazawa,1 Yasuhiro Shirakawa,1 Hisataka Kobayashi,2 Toshiyoshi Fujiwara1. 1 _Okayama UNIV., Okayama, Japan;_ 2 _National Institutes of Health, Bethesda, MD_.

Background: Esophageal cancer has been reported to express HER family, especially EGFR and HER2, of which expressed cases have been reported to show poor prognosis. However, although there are some clinical trials that target those markers, using cetuximab (Cet), panitumumab (Pan), and trasutuzumab (Tra), the result is not sufficient. On the other hand, cancer-associated fibroblasts (CAFs) are one of the major components of tumor microenvironment and suggested to be associated with those chemoprevention, leading to poor prognosis. Thus, to target both cancer cells and CAFs, we focused on near-infrared photoimmunotherapy (NIR-PIT). NIR-PIT is a new molecular targeted therapy, which is based on a near-infrared (NIR) photosensitizer IR700, conjugated to monoclonal antibody (mAb) targeting particular molecules. We developed the CAFs-targeting PIT targeted on fibroblast activated protein (FAP) and have confirmed the efficacy to FAP-expressed CAFs. In this study, we hypothesized that the effect of cancer-targeting PIT is decreased under CAF-rich condition as like other antibody-based therapy. Here we firstly evaluated the effect of cancer-targeting PIT in esophageal cancer cells and furthermore the possibility of additional effect by "dual-targeting PIT" for CAFs and cancer cells.

Materials and Methods: Human esophageal cancer cell lines and human esophageal fibroblasts (FEF3) were used in this study. FEF3 stimulated with conditioned medium (CM) of cancer cells defined as CAFs. We used cetuximab and panitumumab for anti-EGFR antibody and trastuzumab for anti-HER2 antibody. The expression of EGFR, HER2 in esophageal cancer cells was confirmed by Western blot (WB). Cell viability for quantitative evaluation was determined using an XTT Cell Proliferation Kit II.

Result: WB demonstrated that HER2 expression was increased in TE4 and OE19, and EGFR was increased in TE8. FEF3 stimulated with CM was increased FAP expression compared with control. XTT assay demonstrated that the effect of cancer-targeting PIT using Pan-IR700, Tra-IR700 is efficient. Additionally, we confirmed that CAFs-targeting PIT worked in co-culture condition with cancer cells as well as monoculture condition. Furthermore, "dual-targeting PIT" showed rapid cell death of both in co-culture condition, and XTT assay showed the additive effects in vitro, as expected.

Discussion: In this study, dual-targeting therapy has shown cellular specific cell death by targeting to cell-specific antigen and induced both cells death simultaneously. NIR-PIT can be novel effective therapy for esophageal cancer, and also for CAFs in tumor microenvironment. Thus, "dual-targeting PIT," which is combined with both mAb-IR700 for cancer cells and CAFs, can regulate tumor stroma as well as cancer cells simultaneously and can be highly expected to have strong antitumor effect by remodeling tumor microenvironment.

#5067

b1-Integrin promotes inflammatory cytokine-induced vascular leakage.

Elina A. Kiss,1 Laura Hakanpää,1 Guillaume Jacquemet,2 Ilkka Miinalainen,3 Martina Lerche,2 Camilo Guzman,2 Eero Mervaala,1 Lauri Eklund,3 Johanna Ivaska,2 Pipsa Saharinen1. 1 _University of Helsinki, Helsinki, Finland;_ 2 _University of Turku, Turku, Finland;_ 3 _University of Oulu, Oulu, Finland_.

Increased capillary leakage is a potentially life-threatening condition associated with sepsis and cytokine release syndrome (CRS) that develops as a side effect of novel chimeric antigen receptor (CAR) T-cell cancer immunotherapies. CRS and sepsis are characterized by immune activation, resulting in elevated circulating levels of inflammatory cytokines including IL-6 and IL-1β, and thrombin that promote vascular leakage and the development of hypovolemic shock and multiorgan failure. However, molecular mechanisms behind capillary leakage remain unknown, and importantly, targeted approaches for the management of capillary leakage are limited. Here, we found that β1-integrin acts as a critical universal mediator of endothelial permeability induced by various inflammatory agents, including LPS, IL-6, IL-1β and thrombin. In a mouse model of LPS-induced systemic inflammation, resulting in increased inflammatory cytokine production and vascular leakage, function blocking antibodies against β1-integrin prevented vascular leakage and improved integrity of vascular endothelium, leading to protection from LPS-induced cardiac failure. β1-integrin blocking antibodies were effective in reducing vascular leakage even when administered after the onset of systemic inflammation and increase in the serum levels of IL-6, IL-1β and TNF-α. By using an in vitro model, we found that inflammatory agents generated mechanical stress, in a β1-integrin dependent manner, leading to endothelial cell contractility and permeability. Conclusively, our data indicates that β1-integrin promotes vascular leakage in inflammation, and that targeting this mechanism may have therapeutic utility for vascular stabilization in CRS and capillary leak syndromes associated with immune activation and systemic inflammation.

#5068

Distinct pro-inflammatory cytokines in prostate fibroblasts from African-Americans with prostate cancer increase the tumorigenicity of cancer cells.

Marc Gillard,1 Alejandro Morales,2 Susan Crawford,2 Charles Brendler,2 Donald Vander Griend,1 Omar Franco2. 1 _University of Chicago, Chicago, IL;_ 2 _NorthShore University HealthSystem, Evanston, IL_.

Introduction: African-American (AA) men are disproportionately affected by prostate cancer (PCa) compared with Caucasian (CA) men. AA men have higher PCa incidence and, once diagnosed, a higher stage-specific mortality rate (2.4 times higher) than CA men. Although socioeconomic factors partially explain such differences, a growing body of new scientific evidence suggests that biological factors, such as differences at the genetic and molecular level could be more critical than previously thought for the observed racial PCa disparities in incidence and outcome. Recent gene expression studies revealed that differences in the tumor microenvironment (TME) of AA vs. CA prostate tumors could affect the immune-inflammatory milieu. Fibroblasts are important regulators of stromal-epithelial paracrine interactions (SEI) in the TME. We isolated fibroblasts from AA and CA PCa patients and compared their biological effects in vitro and in vivo on PCa cell lines.

Methods: Prostate fibroblasts were isolated from AA (PrF-AA) and CA (PrF-CA) patient tissues. Characterization of PrF included assaying their proliferation and expression of potential markers associated with the activation of carcinoma associated fibroblasts. In vitro effects of AA vs. CA fibroblasts on PCa cell proliferation and motility were studied. In vivo, the pro-tumorigenic properties of fibroblasts were tested using a subrenal xenograft model in SCID mice. RNA-Seq and cytokine array analysis of potential paracrine mediators of tumorigenesis was performed. Immunohistochemical analysis of a panel of putative stromal markers associated with racial differences was assessed in a cohort of PCa tissues from AA and CA.

Results: Prostate fibroblasts from AA vs. CA men exhibit increased proliferation in response to known stromal mitogens. Expression of markers associated with myofibroblast activation (αSMA, vimentin and Tenascin-C) were significantly elevated in AA. Interestingly, expression of the androgen receptor (AR) and activation of AR signaling was significantly increased in PrF-AA compared to PrF-CA. The paracrine effects of PrF-AA on PCa cell proliferation and motility were significantly greater compared to those induced by PrF-CA. In an in vivo model, PRF-AA induced formation of larger and more invasive tumors by the AA PCa cell line E006AA compared to PRF-CA. Analysis of downstream regulatory pathways and potential paracrine mediators identified a panel of pro-inflammatory cytokines notably interleukins (IL6, IL11, IL17, IL18BP), growth factors (VEGF, FGF, BDNF), and other mediators enriched in PRF-AA.

Conclusions: Prostate fibroblasts from African American men show enhanced secretion of pro-inflammatory and pro-proliferative mediators that can potentially increase the tumorigenicity of PCa cells through selective paracrine mechanisms.

#5069

Inflammatory cytokine localization in the prostate tumor microenvironment and their association with prostate cancer racial disparities.

Janielle P. Maynard, Onur Ertunc, Angelo M. De Marzo, Karen S. Sfanos. _Johns Hopkins University, Baltimore, MD_.

Prostate cancer (PCa) is the 3rd leading cause of cancer-related deaths in American men. The major risk factors for PCa development include advanced age, family history and African ancestry. Chronic inflammation has been implicated as a major contributor to PCa and genes involved in inflammatory pathways are reported as more prevalent in tumors from African American (AA) versus Caucasian American (CA) men. Specifically, interleukin-1β (IL1β), IL6, IL8, and IL10 have been identified as over-expressed in the tumor microenvironment of AA men. These interleukins are in involved in the attraction, activation or maintenance of innate immune cells such as neutrophils and macrophages. However, it is not well understood where these interleukins are expressed in the prostate and what cell types produce them. We aim to address these specific questions in this study. The expression pattern and cellular localization of IL1β, IL6, IL8 and IL10 were evaluated using a highly specific RNA in situ hybridization (RISH) assay to analyze formalin-fixed paraffin-embedded tissues of low grade (Gleason ≤ 3+4) and higher grade (Gleason ≥ 4+3) PCa from AA and CA men. IL8 expression was additionally assessed by RISH in two unique tissue microarray (TMA) sets; i) primary prostate tumors obtained by radical prostatectomy from AA and CA men, matched for patient age, tumor grade and stage; ii) distant metastatic tissues obtained by autopsy. Immunohistochemical analysis of neutrophils (CD66ce) and RISH for macrophages (CD68) was used to assess the expression pattern of these immune cells within the same cohorts. Limited IL1β and IL10 expression was observed across all cases. IL6 expression was largely confined to the stromal compartment in endothelial cells and areas of acute inflammation. Modest IL6 expression was observed in atrophic epithelial cells. IL6 was never seen in tumor cells. IL8 was the most abundantly detected cytokine within our cohort and was significantly increased in higher grade cases. In benign regions, IL8 expression was predominantly observed in epithelial cells in regions of prostatic atrophy and in areas surrounding corpora amylacea. We observed marked IL8 expression in urothelial cells in most cases. IL8 expression was also apparent in multiple tumors, both in tumor cells and infiltrating immune cells. Tumor-infiltrating macrophages were markedly increased within a subset of tumors, but did not appear to express IL8. Conversely, a subset of neutrophils expressed IL8. Neutrophils were significantly increased in benign tissues from men with higher grade PCa, particularly in AA men. IL8 expression was also observed in a subset of distant metastases, particularly in liver metastases. There is differential expression of cytokines among men with PCa, and IL8 is the most abundant among the ones we analyzed. Our future studies aim to determine a role for IL8 in prostate carcinogenesis.

#5070

Chemokine expression signatures in infiltrated vs non-infiltrated tumors.

Sarabjot Pabla,1 Jeffrey Conroy,1 Mary Nesline,1 Sean Glenn,1 Blake Burgher,1 Maochun Qin,1 Jonathan Andreas,1 Vincent Giamo,1 Felicia L. Lenzo,1 Angela Omilian,2 Wiam Bshara,2 Antonios Papanicolau-Sengos,1 Yirong Wang,1 Marc Ernstoff,2 Mark Gardner,1 Carl Morrison1. 1 _OmniSeq, Inc., Buffalo, NY;_ 2 _Roswell Park Cancer Institute, Buffalo, NY_.

Background: Previous studies in animal models have shown that expression of specific chemokines determines immune cell infiltration in the tumor microenvironment. More specifically, low expression of CXCL9/10/11, CXCR3, and CCL5, coupled with high expression of CCL2, CCR2, CCR4, CCR5, CCL22, CXCL12, and CXCR4, leads to an exclusion of effector T-cells in the tumor microenvironment while allowing the entry of Treg and MDSC(s).

Methods: 300 formalin-fixed, paraffin-embedded (FFPE) metastatic cutaneous melanoma samples were evaluated by the RNA-seq component of a comprehensive immune profile panel to measure transcript levels of chemokine genes. Resultant data was QC filtered, normalized and ranked based on an assorted reference population of various tumor types. CD8 expression was used to categorize tumors as inflamed (CD8 Rank ≥ 75), borderline ≥ 25 CD8 Rank < 75) and immune deserts (CD8 Rank < 25). T-cell infiltration is defined by CD8 immunohistochemistry with following definitions: Noninfiltrated - Sparse number of CD8+ T-cells that infiltrate nests of neoplastic cells and represent less than 5% of the tumor. Infiltrated - Frequent CD8+ Tcells that infiltrate nests of neoplastic cells in an overlapping fashion at least focally and represent more than 50% of the tumor cells.

Results: CD8 infiltration by immunohistochemistry showed high correlation with CD8 gene expression by RNAseq with infiltrated tumors showing significantly higher expression of CD8 than non-infiltrated tumors (v.test:9.47, p= 2.79E-21; Wilcoxon rank sum test p<0.05). Moreover, 73% of inflamed tumors were categorized as "infiltrated" by IHC while 93% of Immune desert tumors were categorized as "non-infiltrated" by IHC. Chemokine expression significantly correlated with infiltration status by IHC, wherein, CCL5, CCR5, CCL4, CXCR3, CXCL9, CCL2, CCL22, CCL3, and CXCL10 were significantly under expressed in tumors lacking infiltrating effector T-cells (Wilcoxon rank sum test p<0.05, Tukey HSD adjusted p <0.001). Overall ANOVA results showed significant relationship between gene expression of these chemokines and Infiltration status by CD8 IHC (p <0.05).

Conclusion: In 300 metastatic cutaneous melanoma cases, we demonstrated that tumor inflammation status by CD8 expression by RNAseq correlated with CD8 infiltration pattern by IHC. Moreover, expression of Infiltrated and non-infiltrated tumors shows distinct chemokine signatures where higher CD8 T-cell infiltration correlates with higher expression of studied chemokines in the tumor microenvironment. It requires further investigation to better understand the interplay of chemokines and cytokines in the tumor microenvironments that drive the immune cycle in melanoma.

#5071

The functional relevance of Atypical Chemokine Receptor 1 (ACKR1/DARC) genetic isoforms in breast cancer.

Brittany D. Jenkins,1 Rachel N. Martini,1 Inasia Brown,1 Melissa B. Davis2. 1 _Univ. of Georgia, Athens, GA;_ 2 _Henry Ford Health Systems, Detriot, MI_.

Chemokines and their receptors can interact to affect the host's anti-tumor response by influencing the migration of host immune cells indirectly via a chemokine gradient. The regulation and function of these receptors is paramount in ensuring an appropriate immune response to an inflammatory stimulus, especially in the case of cancer. One such receptor, Atypical Chemokine Receptor 1 (ACKR1/DARC), is a genetically diverse transmembrane GPCR that can buffer pro-inflammatory CXC- and CC- chemokine activity in circulation and within tissues, influencing leukocyte migration. While the basic immunological function of this receptor is known, the specific function of its two genetic isoforms, A and B, is not. These isoforms are produced by two alternative promoters and distinct mRNA splicing events. Isoform A is not split by introns, even in the untranslated regions, and offers homology to CXCL8. The alternative splice variant, Isoform B, does contain an intron, and follows the GT-AG splicing rule, including a novel exon coding for an additional 7 amino acids. This localizes to the N-terminus, making up part of the extracellular binding region of the receptor. We aim to determine if the two major ACKR1 isoforms have distinct functional abilities to bind or sequester pro-inflammatory chemokines in epithelial breast cell lines. By creating isoform specific overexpression and knockout constructs, and transiently transfecting the DNA into our subtype specific cell lines, we can use an ELISA assay to determine chemokine concentration in the media, and measure differences based on isoform specificity. Using immunofluorescence, we first show that ACKR1 and our two proinflammatory chemokines, CCL2 and CXCL8, are expressed on the surface of epithelial cells and colocalize. We then indicate that there is an isoform specific difference in chemokine concentration between different breast cancer subtypes, in addition to differences seen in overexpression and knockout cell lines. Determining the functionality of these two isoforms can help us better understand ACKR1's role in the immune system, and its potential role as a prognostic marker in breast cancer.

#5072

Immunohistochemical expression of CC-chemokine receptor-like 2/CCRL2 in a prostate cancer tissue microarray.

Niradiz Reyes,1 Ines Benedetti,1 Jan Geliebter2. 1 _School of Medicine, University of Cartagena, Cartagena, Colombia;_ 2 _New York Medical College, Valhalla, NY_.

Background: CCRL2 is a presumed member of the atypical chemokine receptor family, whose members are increasingly recognized as key components of the regulatory network of inflammation and immunity in cancer and may have a major effect on anti-inflammatory and immunotherapeutic strategies. Recent studies have reported the expression of CCRL2 in different human cancer cell lines and tissues. However, its function and expression in prostate cancer has not been previously addressed. The aim of this work was to determine the protein expression profile of the atypical chemokine receptor CCRL2 in human prostate cancer tissue using a tumor microarray approach.

Methods: A tissue microarray was constructed using 231 cores of matched prostate cancer and benign prostatic tissue from 47 patients with a previous diagnosis of localized prostate cancer, which underwent surgical resection as their primary treatment. CCRL2 expression was evaluated by immunohistochemistry using a monoclonal, anti-human-CCRL2 antibody. An H score was calculated in each spot as the sum of expression intensity (0-3+) by extent (0%-100%). Differences in CCRL2 protein expression were assessed by comparing H-Scores between benign and cancerous tissues using Mann-Whitney test for non-Gaussian distribution.

Results: Immunohistochemical evaluation of CCRL2 at the protein level in a prostate tissue microarray constructed with clinical specimens confirmed its overexpression in malignant prostatic tissue. CCRL2 staining was present predominantly in the cytoplasm of prostate cancer tumor cells. Compared to benign epithelial cells, CCRL2 expression was significantly higher in epithelial tumor cells in cores with prostate cancer compared to cores with benign tissue (p=0.0001).

Conclusions: Immunohistochemistry of prostate cancer tissue specimens arrayed in a tissue microarray revealed that CCRL2 expression was significantly stronger in epithelial cells in cancerous acini compared to epithelial cells in matched adjacent benign acini from the same patient. To this point, the physiological effects of CCRL2 expression in epithelial cancer cells are unclear.

#5073

Involvement of TGFa-EGFR-Akt axis on enhanced proinflammatory chemokines in triple-negative breast cancer cells.

Rosa Mistica Coles Ignacio,1 Carla Gibbs,1 Eunsook Lee,2 Deok-Soo Son1. 1 _Meharry Medical College, Nashville, TN;_ 2 _Florida A &M University, Tallahassee, FL_.

Triple-negative breast cancer (TNBC) is aggressive, leading to poorer outcomes. Chemokines have chemoattractive potential for cancer metastasis. We investigated the signature of chemokine network between TNBC and non-TNBC cells followed by underlining mechanisms on enhanced proinflammatory chemokines in TNBC. Analysis from microarray dataset revealed that basal-like BC subtype representing TNBC expressed dominantly proinflammatory chemokines, such as CXCL1 and 8, compared to non-TNBC. Chemokine PCR array confirmed the dominant proinflammatory chemokines in TNBC cells. As a driving factor for proinflammatory chemokines in TNBC cells, we checked expression profiles for epidermal growth factor receptor (EGFR) family and its downstream signaling. TNBC cells showed higher expression levels of EGFR and phosphorylated Akt compared to non-TNBC cells. In addition, EGF enhanced the proinflammatory chemokines in TNBC cells. Knockdown of Akt reduced the CXCL2 promoter activity, while overexpression of Akt enhanced the activity. MKK2206, an Akt inhibitor, abrogated the CXCL2 promoter activity, but targeting Erk using inhibitor and knockdown did not reduce the activity. As a driving factor for EGFR-mediated Akt activation in TNBC cells, we found transforming growth factor alpha (TGFα) among ligands of EGFR family. MK2206 decreased the TGFα promoter activity, while overexpression of Akt significantly increased the activity in TNBC cells. MK2206 abrogated TGFα protein released by TNBC cells. MK2206 downregulated CXCL2 mRNA, while TGFα upregulated the CXCL2 expression. Taken together, higher expression of proinflammatory chemokines in TNBC involves TGFα-EGFR-Akt axis, probably contributing to the inflammatory burden followed by promoted cancer progression and higher mortality in TNBC.

#5074

Omentum immune microenvironment: Metastatic niche for ovarian cancer.

Venkatesh Krishnan, Paul A. Raju, Koah Vierkoetter, Sonia Patel, Justin Youngyunpipatkul, Supreeti Tallapragada, Bruce Schaar, Ann K. Folkins, Leonore A. Herzenberg, Oliver Dorigo. _Stanford University, Stanford, CA_.

The status of the tumor-immune microenvironment plays a critical role in determining the response to immunotherapy. The omentum, a multifunctional organ that serves as the central regulator of peritoneal homeostasis and plays a central role in immune surveillance, is the most common site of metastasis in early stage ovarian cancer. We analyzed the human omental immune microenvironment that support ovarian cancer metastases.

In experimental models, ovarian cancer cells that colonize the omentum are invariably found in association with immune aggregates known as milky spots. We hypothesize that ovarian cancer cells exploit the unique environment of these omental-immune clusters to promote their own survival and growth. Thus, we formulated a systematic approach to evaluate normal and ovarian cancer-populated omental tissues to characterize the cellular components of human omental milky spots.

We performed immunohistochemistry and flow cytometry to profile immune cell populations present in omental immune clusters. To facilitate the quantitation of immunohistochemical staining, we developed a computational image processing workflow that enables quantitation of multiple immune-biomarkers from serial sections of formalin-fixed paraffin-embedded tissue. In a complementary approach, we used comprehensive multi-color flow cytometry staining panels detecting 28 markers that collectively encompass all innate and adaptive immune cell populations.

We analyzed eight human omental samples (four omenta involved with serous carcinomas and four omenta from benign disease) by immunohistochemistry and found that M2-subtype macrophage (CD163+CD68+) infiltration predominate in high-grade cancer-involved omentum. Conversely, T-regulatory cells (CD25+) predominate cell type in the uninvolved omentum. Hi-D flow cytometry of three cancer-involved omentum samples revealed tumor-associated macrophages (20-40%), PMN myeloid-derived suppressor cells (2-3%), "double positive" CD3+CD4+CD8+ T-cells (3-4%), and "double negative" CD3+CD4-CD8- T-cells (0.9-1%). In addition, we found omental T-cells (Th, Tc, Treg) express immune checkpoint receptors CTLA4 and PD-1, and that omental B-cells express IgD, IgA, and/or IgM, and may be enriched for Ig lambda light chain expression.

As this study is ongoing, we expect to present data for omenta from a larger cohort of patients with either benign or neoplastic disease. However, our findings are already sufficient to show that activated T cells, B cells and macrophages infiltrate omental milky spots in patients with high grade serous ovarian cancers.

#5075

Single-cell RNAseq of human prostate cancer-associated fibroblasts reveals distinct subpopulations that may promote inflammatory cell recruitment to the tumor microenvironment.

Renee E. Vickman,1 Meaghan M. Broman,2 Nadia M. Atallah,2 Ayu Sudyanti,2 Omar E. Franco,1 Timothy L. Ratliff,2 Simon W. Hayward1. 1 _NorthShore University HealthSystem, Evanston, IL;_ 2 _Purdue University, West Lafayette, IN_.

Introduction: Carcinoma-associated fibroblasts (CAF) are a heterogeneous group of cells within the tumor microenvironment that can promote tumorigenesis in the prostate. The full extent of heterogeneity in CAF and its consequences are not understood. A more detailed description of the prostate tumor microenvironment could aid in overcoming major obstacles within the field, including defining indolent versus aggressive disease and uncovering novel therapeutic and disease-stabilizing pathways in a step toward personalized therapy.

Methods: These studies utilized Fluidgm's C1 Single-Cell Auto Prep System and library construction in preparation for single-cell (sc) RNAseq of human prostate CAF. Bioinformatics analysis was used to generate cell clusters. Further examination of the differentially expressed (DE) gene profiles for each cell cluster was performed to elucidate the function and means of communication among clusters and with other cells, particularly those within the immune/inflammatory network. Communication between CAF and immune cells was also analyzed by in vivo testing of tissue recombinants generated with CAF or normal fibroblasts. The resultant tumors were analyzed using FACS to assess the effects of the CAF on inflammatory cell recruitment.

Results: Principal component analysis and unsupervised cell clustering allows for visualization of at least three CAF subpopulations, each with distinct DE gene profiles. Further investigation into the DE genes for each cluster suggests these subpopulations have unique functions within the tumor microenvironment, including a role in immune/inflammatory cell recruitment. For example, one cluster expresses high levels of CCL11 and CXCL1, suggesting a role in the recruitment of neutrophils, myeloid cells, or other inflammatory cells, while a second cluster has decreased expression of chemokines but elevated expression of extracellular matrix-related genes such as COL1A1, MMP16, and FN1, suggesting an alternative, potentially structural, role for this CAF cluster. Previous analysis in vivo demonstrated that CAF drive tumorigenesis in a reporter epithelium. The speculated role of CAF in immune cell recruitment was supported by increased recruitment of myeloid cells, including macrophages and granulocytes, to prostate tissues in the presence of CAF compared to normal fibroblasts.

Conclusions: These studies demonstrate that CAF contain a limited number of distinct subpopulations of cells; the relatively small number of identified CAF clusters allows for feasible biologic study and mathematical modeling. Some CAF highly express extracellular mitotic and chemotactic signaling molecules that may be involved in the recruitment of inflammatory cells as well as direct growth regulation of the tumor by promoting an M2-dominant microenvironment.

#5076

Pancreatic ductal adenocarcinoma associated stellate cells promote a pro-metastatic microenvironment in the liver.

John David Konda,1 Masakazu Hashimoto,1 Jian Zhang,1 Maria Celia Fernandez,1 Ni Wang,1 Stephanie Perrino,1 Laura Montermini,1 Janusz Rak,1 Jean-Sebastien Pelletier,2 Andrew M. Lowy,3 Pnina Brodt1. 1 _McGill University, Research Institute of the McGill University Health Center, Montreal, Quebec, Canada;_ 2 _McGill University, Jewish General Hospital, Montreal, Quebec, Canada;_ 3 _Moores Cancer Center, University of California, San Diego, CA_.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease associated with a 5-year survival rate of ~9%. The most notable clinical features of PDAC are its propensity for aggressive invasion, metastasis (mainly to the liver) and an inherent resistance to conventional therapies. A better understanding of the biology of PDAC metastasis is critical to improving the clinical management of this disease. PDAC is characterized by a dense desmoplastic reaction with up to 50% of the tumor mass consisting of stroma. The major cellular component of the PDAC stroma is the activated pancreatic stellate cell (aPSC). These cells contribute to PDAC progression through extracellular matrix deposition and the secretion of soluble factors. The objective of this study was to determine whether the aPSCs also play a role in PDAC metastasis by contributing to a pro-metastatic microenvironment in the liver.

Methods: We used a syngeneic cell line, LMP-derived from the KPC PDAC mouse model-that recapitulates the clinical course of the disease. When implanted in the pancreas, LMP cells grow rapidly and metastasize aggressively to the liver, and this is associated with PSC activation and expansion. We investigated the ability of PSC and aPSC-derived exosomes to activate hepatic stellate cells (HSC) using a co-culture system in vitro and analyzed the effect of aPSC-derived exosomes on LMP liver metastasis in vivo. Moreover, the protein cargo of PSC-derived exosomes was analyzed by mass spectrometry, in order to identify molecular mediators of PSC-HSC communication that can promote liver colonization by disseminating PDAC cells.

Results: In mice orthotopically implanted with LMP cells, we observed a rapid activation of HSC, an event that preceded tumor cell entry into the liver, as assessed by confocal microscopy and PCR. Similarly, the injection of aPSC-derived exosomes into tumor-naive mice resulted in a liver stromal response, involving HSC and liver-associated fibroblasts. Cultured HSC could be activated by co-culture with aPSCs or by uptake of aPSC-derived exosomes. Moreover, in mice injected with aPSC-derived exosomes, spontaneous liver metastasis was accelerated, resulting in increased metastatic burden. Mass spectometry identified several potential mediators of HSC activation in the aPSC-derived exosomes, including the IGF-2 mRNA binding protein-1 (IMP-1)-an oncofetal, RNA-binding protein involved in the regulation of cytoplasmic mRNA-fate. Finally, IMP-1 silencing in aPSCs reduced HSC activation and the pro-metastatic effect of aPSC-derived exosomes.

Conclusions: Our data identify a novel PSC-HSC crosstalk mechanism that contributes to generating a pro-metastatic microenvironment in the liver and implicate aPSC-derived exosomal IMP-1 in this inter-cellular communication. Our results identify IMP-1 as a potential target for curtailing the metastatic spread of PDAC.

#5077

Cancer-associated fibroblasts stimulate tumor growth and metastatic spread in an orthotopic prostate cancer xenograft model.

Johannes Linxweiler,1 Markus Hammer,1 Christina Körbel,2 Andreas Müller,3 Michael Stöckle,1 Michael D. Menger,2 Kerstin Junker,1 Matthias Saar1. 1 _Saarland University Medical Center, Department of Urology, Homburg/Saar, Germany;_ 2 _Saarland University Medical Center, Institute for clinical and experimental surgery, Homburg/Saar, Germany;_ 3 _Saarland University Medical Center, Department of Radiology, Homburg/Saar, Germany_.

Introduction: In recent years, it has become more and more evident, that the different cell populations comprising the so-called "tumor microenvironment" contribute substantially to the processes of carcinogenesis, local tumor growth and metastatic spread. Especially the tumor-promoting roles of cancer-associated fibroblasts have been the focus of several elaborate in-vitro studies. However, evidence from representative in-vivo models is scarce. In this study, we coinjected prostate cancer (PCa) cells together with different populations of prostate fibroblasts (cancer-associated (CAF), not cancer-associated (NCAF), benign prostate hyperplasia associated (BPHF)) and examined their impact on local tumor growth and the development of metastases.

Material & Methods: We isolated fibroblast primary cultures from tumor-bearing (CAF) and tumor-free (NCAF) tissue samples of a radical prostatectomy specimen as well as from tissue samples of a transvesical prostate adenoma enucleation (BPHF). 5x105 CAF/NCAF/BPHF cells were injected into the prostate of SCID mice together with 5x105 LuCaP136 spheroids or LNCaP cells (n=8 for each group). Tumor growth was monitored by high-resolution ultrasonography (hrUS), micro-CT, 9.4T MRI and serum PSA-measurements. Animals were sacrificed after 10 weeks.

Results: The coinjection of CAFs fostered tumor growth in LuCaP136 but not in LNCaP xenografts. Primary tumor volumes were significantly higher in LuCaP136+CAF mice compared to LuCaP136+NCAF and LuCaP136+BPHF mice (p<0.01 for each time point). Besides that, LuCaP136+CAF mice had significantly higher serum PSA values than their NCAF and BPHF counterparts (p<0.01 for each time point). Significantly more lymph node metastases (LN mets) were seen in LuCaP136+CAF mice (one mouse without, four mice with 2 and three mice with ≥3 LN mets) compared to LuCaP136+NCAF (one mouse without, four mice with 1 and one mouse with 2 LN mets) and LuCaP136+BPHF mice (two mice without, three mice with 1 and two mice with 2 LN mets) (p<0.01). Furthermore, pulmonary metastases were observed more often in LuCaP136 and LNCaP xenografts when PCa cells were coinjected with CAFs (57% for LuCaP136, 71% for LNCaP) as compared to coinjection with NCAFs (0% for LuCaP136, 29% for LNCaP) and BPHFs (14% for LuCaP136, 29% for LNCaP).

Conclusions: Using a representative orthotopic xenograft model and patient-derived fibroblast primary cultures it could be shown that CAFs play a crucial role in local and systemic PCa progression by stimulating primary tumor growth as well as metastatic spread to the lymph nodes and lungs. However, some of the effects were cell-line specific. Future experiments will have to focus on the reproduction of these observations with the use of further sets of fibroblast primary cultures and on the unraveling of the molecular mechanisms underlying the crosstalk between PCa cells and CAFs.

#5078

Stromal MMP3 inhibits oncogenic potential during breast cancer progression.

Maria Cristina Miranda Vergara, Emilia Hartland, Charley Jang, Megan McGarel, Ricardo Romero Moreno, William Kaliney, Laurie E. Littlepage. _University of Notre Dame, South Bend, IN_.

The tumor microenvironment is composed of multiple cell types and extracellular proteins that interact with breast cancer epithelium. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that regulate the microenvironment. MMP3 functions during both breast development and cancer progression. We assessed MMP3 protein localization by immunohistochemistry. MMP3 localized to epithelial ductal cells, adipocytes, and stroma, varying its localization and intensity according to the mouse age and developmental process (pregnancy, lactation, and involution). Roles of stromal and epithelial MMP3 have not been distinguished. To determine the requirement for MMP3 in breast cancer, we transplanted cancer cells (PyMT) into the mammary glands of MMP3 knockout (KO) and heterozygous (Het) syngeneic mice. Tumor burden increased in MMP3 KO recipient mice compared to Het, suggesting an inhibitory role for stromal MMP3. H&Es of primary tumors show similar pathologies between the MMP3 Het and KO cohorts. To identify how stromal MMP3 contributes to tumor progression, we next analyzed proliferation, apoptosis, and immune cell infiltration by immunohistochemistry. The expression of the G2/M cell proliferation marker phospho-histone H3 decreased, while Ki67 did not change in KO recipient tumors. Apoptosis was not significantly different between tumors of Het and KO recipients. Neutrophil recruitment decreased in tumors from KO recipients. In contrast, macrophages and extracellular matrix detected by trichrome staining were not different between tumor cohorts. We also investigated the role of stromal MMP3 during metastasis by analyzing the metastatic tumor burden in recipient mouse lungs. Unlike Het mice, KO recipient mice did not develop visible metastatic tumors in the lung. These data suggest that stromal MMP3 was required for visible lung metastases but does not specify the step in cancer progression where MMP3 was required. MMP3 KO mice have significantly higher levels of trichrome staining in their lungs, which suggests an MMP3-dependent alteration of the extracellular matrix at the metastatic site. To determine if stromal MMP3 plays a similar role after the cancer cells circulate through the vasculature, we injected cancer cells into tail veins of MMP3 Het and KO mice. In vivo imaging of these animals determined that lung metastasis increased in MMP3 KO compared to Het recipient mice, suggesting stromal MMP3 in the lungs inhibits metastatic tumors after tail vein injection. Together, our results suggest that stromal MMP3 primarily has a protective role during breast cancer development by inhibiting primary and metastatic tumor growth. In contrast, epithelial MMP3 promotes initial primary tumor formation. Stromal MMP3 increases early metastasis events between primary tumor invasion and extravasation but inhibits later metastasis during extravasation or colonization steps of the metastatic cascade.

#5079

Role of syntaxin-1A, a synaptic-related protein, in HER2-enriched and luminal B breast cancer progression.

Aleix Noguera-Castells,1 Leire Recalde-Percaz,1 Núria Moragas,2 Patricia Fernández-Nogueira,3 Gemma Fuster,3 Anna López-Plana,3 Patricia Jauregui,2 Pere Gascón,3 Paloma Bragado,3 Mario Mancino2. 1 _Universitat de Barcelona, Barcelona, Spain;_ 2 _Fundació Clínic per a la Recerca Biomèdica (FCRB), Barcelona, Spain;_ 3 _Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain_.

Breast cancer (BC) is the most prevalent cancer in women. It can be classified into 4 subtypes according to their gene expression signature, each one characterized by its aggressiveness and treatment approach. Mortality has declined as a result of earlier diagnoses and the availability of adjuvant therapies. However, a significant number of patients do not respond to these therapies or develop resistance after an initial response. The therapies currently available fail at curing or preventing BC tumor progression and metastasis. Therefore, there is an urgent need to develop novel therapeutic tools to improve BCs patients´ survival rates. The interaction of cancer cells with the stroma has been well established and has led to exciting new therapies. However, the presence and relevance of nerve axons and their secreted neurotransmitters and neuropeptides in the tumor microenvironment has been acknowledged only very recently. We have identified 6 nervous system related genes differentially expressed in BC subtypes. Among them, Syntaxin-1A (STX1A) is up-regulated in luminal B and HER2-enriched tumors, and its expression significantly correlates with a shorter overall survival and metastasis free survival. Therefore, we hypothesized that STX1A regulates luminal B and HER2 enriched tumors progression and metastasis development. STX1A belongs to the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins superfamily that regulate membrane fusion events, including exocytosis and neurotransmission, intracellular trafficking, and cell proliferation. To test its role in breast cancer we have characterized STX1A pathway in a panel of BC cells. Our results showed that STX1A is overexpressed in luminal B and HER2-enriched cell lines at protein and mRNA level, as we have seen in patient's database. Moreover we have analyzed STX1A expression in cell lines that were derived either from lung or brain metastases respectively and we have discovered that the cells that have migrated to the brain or to the lung have increased STX1A expression in comparison to the parentals. Moreover, downregulation of STX1A using siRNA results in inhibition of epithelial to mesenchymal markers, such as Twist-1, indicating a possible role of STX1A in tumor cell migration and metastasis. Furthermore, downregulation of STX1A also represses FGFR2 activation, suggesting that STX1A might regulate tyrosine kinase receptors membrane trafficking. In conclusion we propose STXIA and SNARE proteins as potential biomarkers and targets for novel BC therapy.

#5080

ADAM10 promotes breast cancer via CXCL16 constitutive cleavage and CXCR6 signaling.

Dominique N. Gales, Hina Mir, Neeraj Kapur, James W. Lillard, Shailesh Singh. _Morehouse School of Medicine, Atlanta, GA_.

Despite the survival of breast cancer (BrCa) patients, there is still demand to better understand metastasis as the obvious mark for most aggressive breast cancers. Recent evidence highlighted the possible involvement of chemokines and their cognate receptors in BrCa progression and metastasis. Expression and functional role of CXCR6 and CXCL16 have been investigated in different types of cancer. ADAM10 is also known to regulate the metastatic process by the proteolytic shedding of CXCL16 creating a soluble form of CXCL16. High levels of soluble CXCL16 could support tumor progression by promoting migration and invasion of CXCR6 expressing BrCa cells. Here, we demonstrate the significance of CXCR6-CXCL16 axis in BrCa progression. In addition to increased CXCR6 expression, our tissue microarray analysis (TMA) showed increased expression of CXCL16 and ADAM10 in BrCa tissues. BrCa cell lines (MCF-7 and MBA-231) also showed higher expression of ADAM10 as compared to the normal epithelial cells (MCF-10A). Further, these BrCa cells showed increased migration and invasion towards CXCL16 gradient that is due to high expression of CXCR6 and ADAM10. Hence, our findings suggest the clinical and biological significance of ADAM10 and soluble CXCL16 in BrCa progression. We also highlight the potential of ADAM10 and soluble CXCL16 as prognostic markers and new therapeutic target for BrCa.

#5081

IL-6 trans-signaling pathway promotes early-stage myeloid derived suppressor cells (eMDSCs) via SOCS3 suppression in breast cancer.

Jinpu Yu. _Tianjin Cancer Institute & Hospital, Tianjin, China_.

IL-6 is known to be an important trigger of the expansion and recruitment of myeloid-derived suppressor cells (MDSCs) which are regarded as major coordinators of immunosuppressive microenvironment in cancer. But key molecular events are not fully understood yet. We previously identified a subset of early-stage MDSCs (eMDSCs) with immature myeloid phenotypes in IL-6 over-expressing breast cancer tissues. In this study we elucidated how tumor-derived IL-6 manipulated the development and function of eMDSCs in vitro and vivo. We isolated primary eMDSCs in situ and induced eMDSCs in vitro separately, as well as constructed IL-6 knockdown 4T1 mammary tumor-bearing mice models to explore the molecular events involved. We found that in primary human breast cancer tissues, tumor-derived IL-6 was positively correlated with the accumulation of eMDSCs in situ, which in turn accelerated tumor progression and unfavorable clinical outcomes. IL-6 significantly stimulated the amplification of eMDSCs and promoted their T cell suppressive capacity in vitro. And IL-6 trans-signaling pathway via soluble IL-6 receptor CD126 induced SOCS3 suppression and aberrant hyperphosphorylation of the JAK/STAT signaling pathway in eMDSCs, which promoted the accumulation of eMDSCs in vitro and vivo. Correspondingly, we defined a similar immature subset of CD11b+Gr-1- eMDSCs in IL-6 over-expressing 4T1 murine mammary cancer-bearing mice which displayed more potent suppression on T cell immunity than conventional CD11b+Gr-1+ MDSCs. The proportion of CD11b+Gr-1- eMDSCs in tumor tissues was highly correlated with the tumor size and the numbers of lung metastatic nodules compared to the CD11b+Gr-1+ MDSCs. Tumor-derived IL-6 inducing the inhibition of SOCS3 and the activation of the JAK/STAT signaling pathway was observed in CD11b+Gr-1- MDSCs simultaneously, which further caused the differentiation arrest of myeloid linkage and comparable immunosuppressive capacity of eMDSCs. In order to inhibit IL-6-triggerd activation of the JAK/STAT pathway, specific IL-6R blocking antibody and STAT antagonist JSI-124 were prescribed to 4T1 tumor-bearing mice. We found significant shrinkage of primary tumor and decrease of lung metastatic nodules in vivo, along with recovery of the differentiation of myeloid linkage and attenuation of eMDSC-mediated T cell suppression in vitro. Therefore, we concluded that IL-6-induced dysfunction of the SOCS feedback loop and hyperactivation of the JAK/STAT signaling pathway produced significant differentiation arrest in myeloid linkage, which promoted the accumulation of eMDSCs, the defect in local immune surveillance, and consequential tumor progression in breast cancer. Target therapy against this pathway efficiently decelerated the growth and metastasis of breast cancer in vivo which might be a promising therapeutic strategy for breast cancer treatment.

#5083

Using proteomics profiling to elucidate the interactions of breast cancer-derived exosomes with the blood-brain barrier.

Golnaz Morad,1 Hasan H. Otu,2 Simon T. Dillon,3 Marsha A. Moses1. 1 _Boston Children's Hospital, Harvard Medical School, Boston, MA;_ 2 _University of Nebraska-Lincoln, Lincoln, NE;_ 3 _Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA_.

Breast cancer metastasis to brain is associated with a dismal prognosis commonly attributed to a limited understating of the mechanisms driving this pathological process. Elucidation of the early events leading to brain metastasis is essential to the development of more effective therapeutic and diagnostic approaches. With an interest in the role of breast cancer-derived exosomes in brain metastasis, our group has previously shown that exosomes derived from a brain-seeking variant of the breast cancer cell line MDA-MB-231 (Br-Ex) can facilitate brain metastasis by inducing alterations in the protein expression profile of astrocytes, one of the components of the blood brain barrier (BBB). This observation led us to hypothesize that the interaction(s) between exosomes and astrocytes is more efficient compared to brain endothelial cells (ECs) or pericytes, the two additional major components of the BBB, resulting in more prominent alterations in the protein expression profile of astrocytes. To test this hypothesis, we first quantified and compared the uptake of exosomes by brain ECs, astrocytes, and pericytes in vitro. The uptake of Br-Ex by astrocytes was significantly greater than that of brain ECs (P<4e-3) and pericytes (P<1e-3). In contrast, exosomes derived from parental or bone-seeking MDA-MB-231 cells (P-Ex and Bo-Ex, respectively) did not show a preferential uptake by astrocytes. We have also demonstrated the uptake of Br-Ex by astrocytes in vivo. The uptake of exosomes by different cell types predominantly relies on the interaction of exosomal proteins with different receptors on the recipient cells. To determine the exosomal proteins potentially involved in the preferential uptake of Br-Ex by astrocytes, we performed quantitative mass spectrometry on the P-, Bo-, and Br-Ex via Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) analysis. Database searches were performed against human proteins in the SwissProt database and a total of 126 proteins were detected with over 95% confidence. Pairwise comparisons identified a total of 27 and 21 proteins with statistically significant differential expression (P < 0.05) in the Br-Ex compared to the P- and Bo-Ex, respectively. Functional enrichment analysis of the detected proteins demonstrated that proteins belonging to the cell migration and focal adhesion categories were over-represented in the Br-Ex. Among these proteins, a number of integrins and annexins were highly enriched in the Br-Ex and can potentially be involved in the preferential uptake of these exosomes by astrocytes.

These findings indicate that exosomes derived from brain-seeking breast cancer cells can preferentially interact with astrocytes and these interactions can be driven by exosomal integrins and annexins. (The authors are grateful for the support of the Breast Cancer Research Foundation and the Advanced Medical Research Foundation.)

#5084

Characterizing heterogeneity in the cancer-associated fibroblast population in non-small cell lung cancer: Relating phenotype to function.

Sara Waise, Christopher Hanley, Rachel Parker, Matthew Rose-Zerilli, Christian Ottensmeier, Gareth Thomas. _University of Southampton, Southampton, United Kingdom_.

The aim of this work is to examine the heterogeneity in the cancer-associated fibroblast (CAF) population in non-small cell lung cancer through single-cell RNA sequencing.

Fresh primary lung tissue was obtained directly from surgery, and the disaggregation process optimised to extract the highest number of fibroblasts (using collagenase P for sixty minutes). Single-cell RNA sequencing was performed using a droplet-barcoded sequencing (Drop-seq) platform. Quality control was performed on the raw sequencing data and the resulting digital gene expression matrix. Initial bioinformatic analysis, including cluster identification, was performed using the Seurat package in R. Subsequent cell type identification and gene set enrichment analysis were carried out with the ToppFun tool and GSEA program respectively. Correlations between the presence of fibroblast subtypes and clinical parameters were analysed using CIBERSORT.

Our preliminary analysis of 11 non-small cell lung cancer (NSCLC) tumors and 5 samples of matched non-involved lung revealed the presence of 6 discrete CAF subtypes with differential prognostic impact. Four of the subgroups showed transcriptomic overlap with normal fibroblasts. Of the distinct CAF subtypes, one showed higher expression of genes normally associated with the 'myofibroblastic' CAF phenotype, including multiple collagens, and was enriched for genes associated with the 'extracellular structure organisation' gene ontology (GO) term. The second CAF cluster showed higher expression of genes encoding growth factors and regulators of cell growth, with enrichment of genes in the 'regulation of epithelial cell proliferation' and 'negative regulation of cell death' GO terms. The remaining four subtypes also showed enrichment of genes in keeping with previously-described fibroblast functions: two subtypes were enriched for genes associated with tissue remodelling, including in the 'regulation of proteolysis' and 'regulation of peptidase activity' GO terms. The fifth and sixth clusters showed enrichment for genes in the 'innate immune response' and 'angiogenesis' GO terms, respectively.

Despite their abundance in most solid cancers, CAF remain a poorly characterised cell population. No single molecular marker identifies all CAF, and it is not yet clear whether different CAF phenotypes exist or whether such subgroups have different functions. Our analysis has identified six discrete CAF subtypes in NSCLC. These subtypes have differential gene set enrichment, suggestive of functional differences. In keeping with this, we found that the CAF subgroups differentially impact on patient prognosis. Identification of CAF subgroups associated with aggressive tumor progression may facilitate the development of more specific stromal targeting strategies.

#5085

CXCR2 signaling in pancreatic cancer induces a tumor supporting inflammatory phenotype of the cancer-associated fibroblasts.

Mohammad Awaji, Michelle Varney, Abhilasha Purohit, Surinder Batra, Rakesh Singh. _University of Nebraska Medical Center, Omaha, NE_.

Pancreatic cancer (PC) remains one of the deadliest types of solid malignancies. Making progress in dealing with PC starts by understanding the complex interaction of cellular components in the tumor microenvironment and identifying the crucial checkpoints for tumor progression and metastasis. Desmoplasia and inflammation are two major hallmarks of PC. Cancer-associated fibroblasts (CAFs) are known for inducing fibrosis in several pathologies of the pancreas including PC. Recent reports have described that CAFs can assume the fibrosis-inducing phenotype, when close or have a direct contact with tumor cells, or a tumor-promoting inflammatory phenotype, through distant paracrine signaling. Known for their role in recruiting granulocytes and inducing angiogenesis, CXC chemokines (1-3 and 5-8) contain a conserved motif of Glutamic acid-Leucine-Arginine (ELR) and signal through CXCR1/2 chemokine receptors. In PC, ELR+ chemokines produced by malignant cells and stromal cells contribute to tumor growth, angiogenesis, and tumor-supporting immunosuppression. The deletion of CXCR2 in the genetically engineered PC mouse model (KC) promoted fibrosis and tumor metastasis.

Taken together, we hypothesize that ELR+ chemokines signaling through CXCR2 may trigger the inflammatory phenotype of CAFs in PC that promotes tumor growth and progression. Utilizing unidirectional coculture studies, incubating CAFs in conditioned media of PC cells inhibited their growth, decreased the expression of the fibrotic CAFs-associated markers such as αSMA and collagen I, and increased the expression of immunosuppressive cytokines and tumor-promoting chemokines including IL-4, IL-10, IL-13, and CXCL7. Treating CAFs with exogenous ELR+ chemokines (CXCL1 and CXCL8) exhibited a similar phenotype. Furthermore, treating CAFs with exogenous ELR+ chemokines or PC cells conditioned media in the presence of pharmacological inhibitors of CXCR2 rescued this phenotype. In summary, we believe that ELR+ chemokine signaling through CXCR2 induces the inflammatory phenotype of CAFs that promotes immunosuppression, tumor growth, and tumor progression of PC.

#5086

To identify the critical factors in extracellular vesicles derived from cancer-associated fibroblasts for drug resistance of gastric cancer cells.

Tomoyuki Uchihara, Keisuke Miyake, Eri Oda, Yuki Koga, Taisuke Yagi, Daisuke Kuroda, Tsugio Eto, Yuki Kiyozumi, Kota Arima, Hiroshi Sawayama, Yukiharu Hiyoshi, Masaaki Iwatsuki, Yoshifumi Baba, Yuji Miyamoto, Naoya Yoshida, Hideo Baba, Takatsugu Ishimoto. _Kumamoto university, Kumamoto city, Japan_.

Background: Tumor microenvironment consists of various types of cells. Although cancer associated fibroblasts (CAFs) have been reported to enhance tumor progression and anticancer drug resistance through secretion of soluble factors so far, the crucial mechanism is not fully understood. On the other hand, advanced gastric cancer (GC) is still poor prognosis due to metastasis and recurrence despite the progression of diagnosis and treatment. The aim of this study is to identify the molecular mechanism underlying anticancer drug resistance mediated by CAFs in GC.

Methods: We have isolated CAFs from surgically resected GC tissues and collected conditioned medium (CM) of CAFs. Human GC cell lines with or without CAF-CM were examined the drug sensitivity and analyzed expression of specific genes using quantitative reverse transcription PCR. We isolated extracellular vesicles (EVs) from CAF-CM by ExoQuick-TC ® and ultracentrifugal separation. We observed GC cells with confocal microscopy after the solution of stained EVs treated. We performed EVs from CAF-CM and GC-CM to mass analysis.

Results: GC cells with CAF-CM showed drug resistance on normal coated plates. Moreover, we found that GC cells with CAF-CM on extracellular matrix (ECM)-coated plates showed remarkable morphological change and much higher drug resistance than those without CAF-CM. In contrast, GC cells with GC cell line-CM on ECM- coated plates did not show morphological change. We also found that EVs isolated from CAF-CM caused morphological change and drug resistance of GC cells. Moreover, we identified five proteins included in CAF-EVs as possible candidates involving in GC drug resistance by mass analysis. Gene expression analysis revealed that SNAI2, which is one of the Epithelial Mesenchymal Transition (EMT)-related gene, was significantly up-regulated in GC cells cultured with CAF-CM or EVs from CAF-CM on ECM-coated plates.

Conclusion and future perspective: These findings in current study suggest that EVs from CAFs cause characteristic gene expression change and enhance drug resistance along with morphological change of GC cells. We are trying to identify the critical factor in CAF-EVs for drug resistance of GC cells.

#5087

Human melanoma exosomes induce metabolic reprogramming in human adult dermal fibroblasts.

Shin La Shu,1 Cheryl L. Allen,1 Yunchen Yang,2 Orla Maguire,1 Hans Minderman,1 Arindam Sen,1 Michael J. Ciesielski,1 Katherine A. Collins,1 Peter J. Bush,2 Martin Morgan,1 Yun Wu,2 Richard B. Bankert,2 Marc S. Ernstoff1. 1 _Roswell Park Cancer Institute, Buffalo, NY;_ 2 _University at Buffalo, The State University of New York, Buffalo, NY_.

The "seed and soil" hypothesis of cancer metastasis has led us to examine the role of tumor-derived exosomes on reprogramming stromal cells composing the tumor microenvironment. We used human adult dermal fibroblasts (HADF) in vitro as a model of the premetastatic niche. Specifically, we determined the role of human melanoma-derived exosomes (HMEX) on governing metabolic reprogramming of healthy HADF. HMEX from six melanoma cell lines (3 BRAF V600E mutant cell lines (526-mel, 888-mel and Hs 294T) and 3 BRAF wild-type cell lines (1300-mel, 2183-Her4 and HMCB)) were purified by size-exclusion chromatography. Using the fluorescent tag CellVue (Maroon) we demonstrated via ImageStream that HMEXs are rapidly taken up by HADF. Following 18 hours of exposure to HMEX, HADF increased aerobic glycolysis and reduced oxidative phosphorylation (OXPHOS) in vitro, as measured by Agilent Seahorse XFe96. We have also developed an immuno-biochip that captures exosomes via anti-CD63 antibodies and simultaneously detects exosomal microRNAs by cationic lipoplex nanoparticles (tCLN) containing molecular beacons. With this immuno-biochip, we demonstrated the presence of microRNAs miR-155 and miR-210 in HMEX, both of which work in concert to promote glycolysis and inhibit OXPHOS. The result has been consistent across all HMEXs from the six different melanoma cell lines. Our findings reveal a correlation between metabolic function and the presence of metabolically active microRNAs in HMEX that control aerobic glycolysis and OXPHOS of normal fibroblasts. Melanoma exosomes are potentially key players in promoting tumor progression by modulating normal stromal cell metabolism and creating an environment that is conducive to metastasis.

Oxygen Consumption Rate (OCR) pmol/min

---

|

HADF only | HADF + HMEX 17.5 μg | HADF + HMEX 25 μg | HADF + HMEX 50μg | HADF + HMEX100μg

Basal OCR | 52.67 | 53.58 | 50.96 | 38.20 | 31.56

#5088

Fibroblast-derived factors enhance growth of ovarian cancer cells.

Youngjoo Kwon, Geun Yeong Park. _Ewha Womans Univ., Seoul, Republic of Korea_.

Background: Ovarian cancer is one of major cancers that affect female reproductive organs. Rapid progression of ovarian cancer without symptoms makes this disease difficult to clinically manage and mortal. Tumor microenvironment significantly contributes to tumor malignancy and targeting the tumor microenvironment is being explored as important cancer therapy. MAPK signal transduction pathways mediates cell response to paracrine factors derived from microenvironment. It is important to identify pathways that are specifically activated in response to microenvironment-derived signals and their relevance to pathogenesis of cancer in order to effectively target signaling derived from tumor microenvironment. We studied the effect of fibroblast-derived media on the growth of ovarian cancer cells and the role of three families of MAPK and AKT in fibroblast-induced increase in cancer cell growth. We also evaluated roles of IL-6, IL-8, MCP1, and CXCL1, highly secreted by fibroblasts, in mediating growth increase induced by fibroblasts. Methods: Ovarian cancer cells were cultured in media derived from human ovarian fibroblast (CM1 and CM2), serum free media (SF), and 4% FBS media (REF) and their growth rate and MAPK activation induced by different media were compared. In order to identify, MAPK family that is important in the enhancement of cancer cell growth, cell growth rate was compared in different media in the absence and presence of specific inhibitor. IL-6, IL-8, MCP1, and CXCL1 were treated and their effects on cell growth and MAPK activation were assessed. Results: CM1 and CM2 significantly enhanced cell growth compared to SF at 72 hr in ovarian cancer cells. In OVCAR3 and OVCAR4 cells, phosphorylation of both p38 and ERK were highly induced by both CM1 and CM2 compared to REF and SF. Phosphorylation of JNK and AKT was highly induced by CM1 and CM2 in OVCAR4 cells while that was less apparent in OVCAR3 cells. In contrast, phosphorylation of proteins in MAPK families and AKT was not specifically induced by CMs in SKOV3 cells. In OVCAR3 and OVCAR4 cells, pretreatment with a p38 inhibitor, selectively prevented the cell growth induced by CMs. Treatment of IL-6, IL-8, and MCP1 up to 1 ng/mL did not increase growth of any cancer cells. However, CXCL1 at 1 ng/mL significantly increased cell growth in OVCAR3 and OVCAR4 but not in SKOV3 cells. Treatment of CXCL1 also enhanced phosphorylation of p38, JNK in OVCAR3 and OVCAR4 cells whereas these effects of CXCL1 were not observed in SKOV3 cells. Furthermore, treatment of SB225002 (CXCR2 inhibitor) effectively prevented p38 activation and growth enhancement induced by CM1 and CM2 in OVCAR3 and OVCAR4 cells. Conclusion: Our study demonstrates that the growth of ovarian cancer cells is enhanced by factors derived from ovarian fibroblasts partly by activation of p38. CXCL1 is one of factors that is derived from fibroblast and increases growth of cancer cells.

#5089

A quantitative proteomic survey reveals mechanisms of the bi-directional signaling between lung fibroblasts and lung cancer cells.

MARTIAL BOUTCHUENG DJIDJOU, Jae-Young KIM, Ki-Cheol Han, Gabriela Wright, Anurima Majumder, Bin Fang, John Koomen, Lily.L Remsing Rix, Uwe Rix, Eric Haura. _MOFFITT CANCER RESEARCH CENTER, TAMPA, FL_.

Cancer cells and fibroblasts support each other for increased aggressiveness and drugs resistance. Cancer cells educate fibroblasts to acquire some cancer associated fibroblasts (CAFs) phenotype and CAFs feedback by regulating cancer cells signal rewiring in response to tyrosine kinase inhibitors (TKIs), for their survival and proliferation. The mechanisms underlying this bi-directional interplay at a systems level are poorly understood. To get insights on this complex signaling crosstalk between lung cancer cells (LCs) and lung fibroblasts (LFs), we applied a "Cell Type-specific labelling using Amino acid Precursors" (CTAP) to perform quantitative mass spectrometry data analysis. CTAP allows for simultaneous metabolic labelling of co-cultured cell types leading to subsequent differential proteomic analysis. We generated three sets of proteomic data including: shotgun data for the whole cell proteomes, activity- based protein profiling (ABPP) using a desthiobiotin-ATP probe to target kinase regulation and phospho-tyrosine proteomic data. We selected PC9 lung cancer cells carrying an activating EGFR mutation and the transformed lung fibroblasts (WI-38-VA13). Their mono-cultured cells are used as reference for comparative data analysis. Among the 6319 proteins identified in shotgun data, expression levels of 1305 are changed in LFs by LCs, while 1001 are changed in LCs by LFs. ABPP data consisted of 336 verified ATP-binding sites from 201 proteins including proteins, of the 26s proteasome family associated to cell death regulation, of the ATP-binding cassette family and one hundred kinases. Phosphorylation of one fourth of the (ABPP) kinases matched hundreds of known substrates regulation in phosphoproteomic data as described by PhosphositePlus database. Preliminary analysis from shotgun data using KEGG signaling pathway database, suggest enrichment of proteins of (i) insulin signaling pathways including IGF1R/IRS1/IRS2/AKT axis and (ii) changes of Ephrin receptors associated to cell migration in LFs after co-culture with LCs. Gene set enrichment analysis identified RAS signaling in LFs after co-culture with LCs. Conversely, LFs drive expression changes of AXL receptor in LCs which correlates with phosphorylation of its tyrosine residue (Y702). AXL is associated with TKIs resistance, increased cell survival and metastatic propensity of cancer cells. Its regulation is accompanied by changes of expression (Shotgun data) and activity (ABPP) of non-traditional MAP4K3 proteins as well as regulation of tyrosine phosphorylated protein YAP-1. AXL, MAP4K3 and YAP-1 are linked to hippo signaling pathway and constitute an axis for regulation of cell survival and cell migration in LCs induced by LFs. Taken together, our data nominate new mechanisms of the bi-directional signaling between LCs and LFs, as well as potential targets for therapeutic intervention.

#5090

Exportin-1 enables pro-tumorigenic metabolic reprogramming of lung fibroblasts through relocalization of p53 and p62/SQSTM1.

Antonina Bruccoleri, Francesca Andriani, Federica Facchinetti, Ugo Pastorino, Gabriella Sozzi, Luca Roz. _Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy_.

Bi-directional interactions between cancer cells and the surrounding microenvironment actively contribute to cancer development and progression. Cancer-associated fibroblasts (CAFs) are one of the most prevalent cellular components in the tumor microenvironment and their metabolic reprogramming towards an activated phenotype reminiscent of fibroblasts found at sites of wound healing is involved in the acquisition of pro-tumorigenic properties. Specific determinants of tumor-promoting function of fibroblasts in lung cancer are however not well characterized. Here we used microarray gene expression profiling of fibroblasts isolated from lung cancer patients and functional in vitro and in vivo assays to identify enablers of fibroblasts pro-tumorigenic activity. Gene expression profiles of CAFs and normal fibroblasts (n=60) identified increased levels of the nuclear transporter Exportin 1 (XPO1/CRM1) in patient-derived fibroblasts endowed with an in vivo pro-tumorigenic phenotype in co-injection experiments with lung cancer cells. Elevated levels of XPO1, which controls cellular localization of numerous proteins including oncosuppressors, have been observed in cancer cells but the role of XPO1-related signaling in stromal cells has not been investigated. Pharmacological inhibition of XPO1 in fibroblasts resulted in anti-proliferative effects with a G2/M cell cycle arrest linked to p53 accumulation, increased CDKN1a transcription, induction of senescence (2-4 fold increase of β-galactosidase positive cells) and accumulation of γ-H2AX foci. Interestingly these effects were persistent only in activated fibroblasts compared to control fibroblasts suggesting a central role for XPO1 in mediating and sustaining permanent reprogramming of pro-tumorigenic fibroblasts. Additionally XPO1 inhibition was associated with cellular relocalization of p62/SQSTM1, a master regulator of energy metabolism in tumor stroma. Low levels of nuclear p62 were observed in activated fibroblasts and could be restored to normal levels through XPO1-activity inhibition. p62 nuclear accumulation inhibited DNA-damage repair and reduced extracellular acidification, indicating potential modification of metabolic properties of activated fibroblasts. Consistently, in heterotypic co-cultures experiments, we found that XPO1 inhibition prevented the ability of activated fibroblasts to stimulate migration of lung cancer cell lines and to increase the frequency of CD133+ cancer stem cells thereby reducing their pro-tumorigenic activity in vivo. Overall these evidences suggest that XPO1 is a potent enabler of the tumor promoting functions of fibroblasts and that XPO1-driven metabolic reprogramming in stromal cells could be targeted to affect cancer progression.

#5091

Maintaining long-term stromal depletion therapy for pancreatic cancer.

Sam R. Grimaldo, Mahsa Khatibeghdami. _University of Illinois, Chicago, Chicago, IL_.

A hallmark feature of pancreatic cancer is a dense fibrotic stroma that surrounds the tumor and sequesters blood vessel growth. The lack of blood vessels results in poor perfusion, causing most therapies to fail. Antistromal therapy via targeting the sonic hedgehog (SHH) pathway is a controversial approach to improving drug distribution for pancreatic cancer. While this approach has been shown to be initially effective, over time stroma depletion results in the return of an aggressive cancer. Long-term stromal depletion creates a microenvironment that permits angiogenesis and fuels the return of an aggressive cancer. It is unknown if inhibiting angiogenesis will improve the long-term success of antistromal therapy. We are pursuing two therapeutic strategies that couple an antiangiogenic component with SHH therapy. These therapeutic strategies will be tested in one of the two novel inducible models we have developed, including the standard iKPC model and an SHH-deleted model iKPC-SHH. Preliminary survival analysis shows overall survival of 8 months and 5 months for the iKPC and iKPC-SHH, respectively. Furthermore, increased CD31 blood vessel staining is seen in the iKPC-SHH model, suggesting increased angiogenesis. Using the standard iKPC we will pursue a co-treatment approach with gemcitabine and the SHH inhibitor, itraconazole, to evaluate the effect on median survival. Itraconazole was chosen from a screen of ten SHH inhibitors and found to have a highest efficacy on vascular endothelial cell (IC50 0.02υM). Thereby we expect this molecule to target both the SHH pathway and blood vessels. The second approach we are pursuing is to determine if the antiangiogenic drug, sunitinib, improves median survival in the iKPC-SHH. The iKPC-SHH serves as a surrogate of antistromal therapy and allows for a focused investigation with antiangiogenic compounds. Sunitinib was chosen from the class of antiangiogenic pharmacologics for its direct efficacy on primary pancreatic tumor cells (IC50 2.4υM); thereby it is expected to target blood vessels and tumor cells in vivo. While both treatments approaches are ongoing. current data show that itraconazole co-treatments are living over 7.5 months in the (noninducible) KPC model. Previous reports with an SHH inhibitor in the KPC model showed a survival at approximately 3-4 months. Thereby our initial data show promise that inhibiting angiogenesis can improve the long-term efficacy of antistromal therapy.

### Dissecting Tumorigenesis in Vivo Using Genetic Approaches and Spontaneous Tumor Models

#5092

Establishment of an induction protocol for medulloblastoma during prenatal stages of a floxed-cre inducible model.

Gabriel Gallo-Oller, Ani Azatyan, Ninib Baryawno, Per Kogner, John Inge Johnsen. _Karolinska Institutet, Stockholm, Sweden_.

Advances in mouse genetics in the last decades have improved the site-specific and conditional control of specific genes in vivo. Despite the availability of different recombinases, the most popular and best-characterized model is the Cre-loxP system. To achieve the recombination in specific tissues or cell types, Cre expression is regulated by a cell-specific promoter and it is also possible to use a tamoxifen-inducible form of Cre for temporal control of the recombination process. Different models have been described using this technology. However, the recombination in the central nervous system tissues is still problematic. Here, we describe a pilot study to induce the recombination process in the brain at prenatal stages in a Cre-loxP system.

To test the temporal control of the recombination in the brain, we used a Ptch1fl/fl mouse line in combination with the previously described Math1-creERT2 mouse line that can be induced by tamoxifen administration. All experimental procedures on animals, together with mouse breeding, were performed in accordance with the guidelines of the animal ethics committee of the Karolinska Institutet. The detection of the floxed allele and the cre gene was carried out by PCR at each breeding step.

In order to control the specific time point of the induction, the presence of the males for mating was controlled for two days. After this, females were monitored and the induction of the model was carried out at E14.5 (embryonic days) for two days by oral administration of tamoxifen. Issues due to tamoxifen administrations as problems in delivery and mortality/toxicity in pups were not detected. The litters obtained were genotyped to detect the correct combination, Ptch1fl/fl; Math1creERT2 +/-. Mice with the correct genotype developed neurological symptoms between 7 and 9 weeks old, and once the brain was dissected, an abnormal size of the cerebellum was detected. Cerebellum was dissected and the cells obtained were grown and maintained in different specific culture media. The deleted allele of Ptch1 was detected by PCR in the tumoral tissue and in the cell cultures.

Here, we described a protocol to induce a Cre-loxP system during embryonic stages. The present protocol avoids the problems described for extensive tamoxifen administration. We also described new culture media to grow and expand the tumor cells obtained. Our findings could help to improve the induction of this kind of systems and study mutations during embryonic stages related with the development and progression of medulloblastoma. The culture of cells derived from these systems, can be used to establish therapeutic mouse models and to evaluate novel treatment strategies for this tumor.

#5093

Bcr-Abl leads to mast cell differentiation and promotes degranulation of cells derived from a chronic myeloid leukemia mouse model in a Gab2 dependent manner.

Julia Ellermann,1 Franziska Maria Uhl,1 Martin Köhler,1 Julia Huber,1 Robert Zeiser,1 Steffen Koschmieder,2 Konrad Aumann,1 Tilman Brummer,1 Sebastian Halbach1. 1 _University of Freiburg, Freiburg, Germany;_ 2 _University of Aachen, Germany_.

Chronic myeloid leukemia (CML) is driven by the fusion kinase Bcr-Abl, caused by a chromosomal translocation between chromosome 9 and 22. This tyrosine kinase organizes its own signaling network with various proteins, especially the docking protein Gab2. By being recruited to Bcr-Abl via Grb2, Gab2 plays a critical role in this network. We recently applied a transgenic approach to investigate the role of Gab2 in Bcr-Abl driven disease. Using Gab2 knock-out mice, we analyzed the in vivo role of Gab2 in a chronic-phase CML mouse model in which a tetracycline regulated Bcr-Abl transgene is expressed in hematopoietic stem cells in their native microenvironment. We demonstrated that Gab2 deficiency impairs disease development in a steady-state in vivo setting. In the course of this work, we also identified an abnormal number of mast cells infiltrating the kidneys of Bcr-Abl expressing Gab2 proficient mice, a phenotype associated with an inflammatory urothelium and hydronephrosis. Interestingly, this phenotype was completely absent in Gab2 deficient mice. Therefore, we aimed to analyze the role of mast cells in the CML mouse model in more detail. First, we performed bone marrow transplantations, using Bcr-Abl positive donor mice and C57/BL6N mice as recipients. Also in these mice, we observed high mast cell counts in the bone marrow and developed hydronephrosis in some cases, which demonstrates that these alterations were induced by cell-autonomous properties of the Bcr-Abl positive donor cells. Next, we were interested whether these cells are Bcr-Abl transformed mast cell precursors or reactive mast cells resulting from secondary effects of the leukemic disease. Therefore, we isolated bone marrow cells from Bcr-Abl transgenic mice and analyzed them for Bcr-Abl activity and mast cell properties. Strikingly, Bcr-Abl positive progenitors differentiated into mast cells under cytokine free conditions. Next, we assessed mast cell functionality of these cells by degranulation assays measuring β-hexosaminidase activity and cytokine release. Importantly, Bcr-Abl positive cells were more sensitive towards antigen stimulation and displayed a stronger degranulation und higher levels of secreted cytokines compared to Bcr-Abl negative controls. This suggests that Bcr-Abl positive mast cells could be responsible for the inflammatory urothelium and the hydronephrosis which we observed in our CML mouse model. In line with our previous results, Gab2 deficient cells from Bcr-Abl transgenic mice showed no elevated degranulation.

In summary, we could show that Bcr-Abl can drive the expansion of mast cells. In addition, Bcr-Abl leads to stronger degranulation in a Gab2 dependent manner. This data and our previous work on Gab2 invite for the further evaluation of Gab2 as a biomarker and as a valuable target in the treatment of CML and, possibly, systemic mastocytosis.

#5094

The Jackson Laboratory Repository: Mouse strains relevant to human cancer.

Deborah Boswell, Stephen Rockwood, Cathleen Lutz, The JAX Repository Team. _The Jackson Laboratory, Bar Harbor, ME_.

The Jackson Laboratory Mouse Repository continues to expand its diverse collection of mutant mouse strains, many of which have preclinical applications related to human cancer. Over 11,000 Repository mouse lines are accessible to the scientific community, each described and easily found on the JAX website. Among the hundreds of new strains made available annually by the Repository are mice from NIH-funded mouse projects and programs (SMSR, KOMP, MMRRC). A subset of strains relevant to cancer research is highlighted here. A number of mouse lines have been designed to be models for studying specific types of cancers. Recent additions include a model for small cell lung cancer carrying floxed alleles of Rb1 and Trp53 and a Cre recombinase regulatable Myc<T58A> allele; a conditional strain, in which exon 15 of Apc is floxed, useful for generating models of ovarian and colorectal cancer; and a double floxed Wwtr1 Yap1 line with applications in studies of the role of the Hippo signaling pathway in tumorigenesis and tumor suppression. The set of engraftment models continues to grow. Typically immunodeficient NSG-based (but not exclusively) strains allow xenotransplantation of human tissues, cells or tumors. The set of research tool strains encompassing Cre-lox, Tet-On/-Off and CRISPR cas9 technologies also continues to grow as does the set of reporter strains (fluorescent protein, luciferase, lacZ, etc.), enabling researchers to customize models for particular applications. A transgenic strain expressing Cre recombinase in gastric parietal cells has applications in gastric cancer research. A reporter line that expresses JAG1 and luciferase in Cre-expressing tissues can be used to study prostate cancer. Other resources enabling cancer research at JAX include the JAX patient-derived xenograft (PDX) cancer model resource offering PDX tumor-bearing NSG mice as a preclinical platform for efficacy studies. The Onco-Hu model platform can be used for immunotherapy engraftment studies. The Oncology Therapeutic Area web page is tailored to showcase JAX strains and resources related to cancer research. Repository strains are tested in our comprehensive quality control program to confirm expected mutation identity and genetic background, as well as to screen for the presence of contaminating alleles (GFP, cre, lacZ, etc.). Mice harboring unexpected, often overlooked, genetically engineered alleles can dramatically confound reproducibility between labs. In addition, the JAX Reproductive Sciences group performs cryopreservation to safeguard each strain and can rapidly create cohorts for studies using IVF technology. Researchers wishing to have strains considered for inclusion in the Repository can submit them on the JAX website at www.jax.org/donate-a-mouse. The Jackson Laboratory Repository is supported by the NIH, The Howard Hughes Medical Institute, and several private charitable foundations.

#5095

**Concurrent** Dnmt3a **haploinsufficiency partially abrogates competitive disadvantage in** Smc3 **haploinsufficient myeloid cells.**

Tianjiao Wang, John S. Welch. _Washington University in St. Louis, Saint Louis, MO_.

SMC3 encodes a subunit of cohesin associated with recurrent heterozygous mutations in acute myeloid leukemia (AML) and other myeloid malignancies. About one-third of these mutations are predicted loss-of-function (nonsense and splice-site mutations) and the rest are missense with unclear function. To understand whether these missense mutations might have dominant-negative effects or phenocopy loss-of-function effects, we compared the consequences of Smc3 deficient and haploinsufficient mouse models. We found that both embryonic and somatic deletion of homozygous Smc3 alleles led to complete hematopoietic failure. Furthermore, in competitive transplantation, following engraftment and subsequent tamoxifen treatment, the Smc3fl/fl/ERT2-Cre+/- BM cells were rapidly outcompeted, with earliest cell loss in the Gr1+ myeloid compartment. Hence, Smc3 is indispensable for hematopoiesis and AML-associated SMC3 missense mutations are unlikely to have dominant-negative effects. We examined the consequences of somatic Smc3 haploinsufficiency using Smc3fl/+/ERT2-Cre+/-. We found that Smc3 haploinsufficiency did not lead to increased number of initial methylcellulose colonies, nor did these cells replate beyond two weeks. In competitive transplantations, we again observed a significant competitive disadvantage in the Smc3fl/+/ERT2-Cre+/- BM cells, most pronounced in the Gr1+ myeloid cells. Competitive disadvantage is counter-intuitive for a leukemia-associated mutation. SMC3 mutations frequently co-occur with DNMT3A mutations. We, therefore, asked whether Smc3 haploinsufficiency might lead to a competitive advantage if it occurred in the background of Dnmt3a haploinsufficiency. With the addition of Dnmt3a haploinsufficiency, the severe myeloid competitive disadvantage was partially ameliorated, but the significant competitive disadvantage in other lineages remained intact. In summary, our data demonstrate that homozygous Smc3 deletion was incompatible with embryonic or adult hematopoiesis, with the greatest defects noted within myeloid compartment, indicating that AML-associated SMC3 mutations are unlikely to have dominant-negative effects. Smc3 haploinsufficiency resulted in a myeloid competitive disadvantage, which was partially abrogated in the presence of concurrent Dnmt3a haploinsufficiency, suggesting lineage-specific interactions between the mutations.

#5096

Pre- and early postnatal loss of tsc1/2 induces kidney hybrid oncocytic/chromophobe tumors (HOCT) via c-JUN.

Heng Du,1 Heng-jia Liu,1 Damir Khabibullin,1 Mahsa Zarei,1 John Dreier,1 Chin-Lee Wu,2 Elizabeth Henske,1 David Kwiatkowski1. 1 _Brigham and Women's Hospital, Harvard Medical School, Boston, MA;_ 2 _Massachusetts General Hospital, Harvard Medical School, Boston, MA_.

Renal cell carcinoma (RCC) occurs in up to 4% of tuberous sclerosis complex (TSC) patients, often affecting children and young adults. The mechanisms of TSC-associated RCC are not well understood. We explored the pathogenesis of TSC-associated RCC using three mouse models. First, loss of Tsc2 was induced by i.p. tamoxifen (TM) injection (10mg/kg) at embryonic day 17.5 (E17.5) in Tsc2flox/floxROSA26-CreER+ mice (n=24), examined at age 6 m. Second, loss of Tsc1 was induced by injecting Cre-encoding adenovirus directly into the left kidney of Tsc1flox/flox mice at P1 (n=19), examined at age 1 year. Third, Tsc2 was knock out through 5 consecutive times of i.p. tamoxifen injection (1mg/mouse/day) when the Tsc2flox/floxROSA26-CreER+ mice were 6 weeks old (n=7), examined at age 6 month. We found the following: In the E17.5 TM injection mouse model, each kidney had more than 200 tumor lesions, which almost occupied the whole kidney cortex and medulla. Extensive TSC-associated kidney papillary RCC (PRCC) and hybrid oncocytic/chromophobe tumors (HOCT) were seen in every kidney. The same types of tumor but fewer tumor lesions (~40/kidney) were found in the Cre-encoding adenovirus group. All the females in the P6 week group had the same phenotype as E17.5 TM injection group, but only single cyst was found in the male kidneys. So, the earlier those mice lost TSC1/2, the more severe their tumor burdens. IHC was performed to further validate the diagnosis. All the PRCC showed strong and diffuse expression of phospho-S6-S240/244 (pS6), CK7, and carbonic anhydrase-IX, with negative vimentin staining, while all the HOCT were positive for pS6, S-100 (a diagnostic marker used for human HOCT) and vimentin. The single cysts in the P6 week TM injection male kidneys only showed pS6 and CK7 staining. To further explore the mechanism of tumorigenesis, a phospho-RTK screen was done using lysates from kidney tumors (PRCC and HOCT) and normal kidneys. The pAXL level was 2.8-fold higher in comparison to normal kidney. Immunoblotting revealed high levels of p-AXL and total AXL in kidney tumor lysates compared to normal kidney. AXL mRNA, measured by real-time RT-PCR, was increased 2-fold (p=0.011). c-Jun, a known driver of AXL expression, was also highly expressed in those tumors but negative in normal kidneys as shown by immunoblot. Immunoblot analysis of a cell line established from the kidney tumors showed TSC-dependent c-Jun and AXL expression. These novel mouse models of TSC-related RCC closely resemble those found in TSC patients and elucidate distinct mechanisms of disease development and potential therapeutic targets.

#5097

**Knock-in of a human tumor suppressor (25-kilobase pairs) by traditional and CRISPR/** Cas9 **-stimulated homologous recombination.**

Tiffany Leidy-Davis,1 Kai Cheng,2 Leslie Goodwin,1 Judith Morgan,1 Wen Chun Juan,3 Xavier Roca,4 Sin-Tiong Ong,5 David E. Bergstrom1. 1 _The Jackson Laboratory, Bar Harbor, ME;_ 2 _Charles River Laboratories, Wilmington, MA;_ 3 _Institute of Molecular and Cell Biology, Singapore, Singapore;_ 4 _School of Biological Sciences, Singapore, Singapore;_ 5 _Department of Medical Oncology, Singapore, Singapore_.

Here we describe an expansion of the DNA size limitations associated with CRISPR knock-in technology, more specifically, the physical extent to which mouse genomic DNA can be replaced with donor (in this case, human) DNA at an orthologous locus. Driving our efforts was the desire to create a whole animal model inserting a 25-kbp segment of a human tumor-suppressor gene in place of 17 kbp of its corresponding mouse orthologue. Included within the human fragment is a conditionally removable (by Cre-loxP) segment of the gene's second intron, a cryptic exon immediately 3' of this, and a native exon some 20 kbp downstream. Using two methods, we first carried out the replacement by employing a combination of bacterial artificial chromosome recombineering, classic ES cell targeting, dual selection, and recombinase-driven cassette removal (traditional approach). Using a unique second method, we employed the same vector (devoid of its selectable marker cassettes), microinjecting it along with CRISPR RNA guides and Cas9 into mouse zygotes (CRISPR approach). In both instances, we were able to achieve humanization of the tumor suppressor to the extent designed, remove all selection cassettes, and demonstrate the functionality of the conditionally removable, loxP-flanked, intronic segment.

#5098

Conditional ERK3 overexpression and PTEN deletion induce lung tumors in mice.

Sreeram Vallabhaneni,1 Marion Morel,1 Ming-Jer Tsai,2 Francesco J. Demayo,3 Weiwen Long1. 1 _Wright State University, Dayton, OH;_ 2 _Baylor College of Medicine, Houston, TX;_ 3 _NIEHS, Durham, NC_.

Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen-activated protein kinase (MAPK) involved in cell growth and differentiation. Recent studies have revealed important roles for ERK3 in promoting cancer cell migration and invasion. In addition, ERK3 was shown to be highly upregulated in human lung cancers and to be associated with tumor metastasis. Altogether, these findings suggest an important role for ERK3 in lung tumor formation and progression. However, no in vivo study of ERK3 in lung tumorigenesis has been reported. For this purpose, a transgenic mouse model conditionally overexpressing ERK3 in lung bronchial epithelial cells was generated. First, a transgenic mouse line harboring ERK3 transgene under the control of a ubiquitous promoter and a STOP sequence flanked by two Lox P sites (LSL-ERK3) was generated. The obtained LSL-ERK3 mice were then crossed with a mouse line harboring the Cre recombinase transgene driven by a Clara cell secretory protein gene promoter (CCSP-Cre), resulting in CCSP-Cre/LSL-ERK3 transgenic mice that show ERK3 overexpression in Clara cells, the non-ciliated epithelial cells lining the bronchioles of lung. No clear phenotype, however, was observed in CCSP-Cre/LSL-ERK3 transgenic mice. As lung tumorigenesis usually requires multiple genetic alterations, the CCSP-Cre/LSL-ERK3 transgenic mice were then crossed with a PTENFlox/Flox mouse line in which the exon 5 of the tumor suppressor gene PTEN (Phosphatase and Tensin homolog) is flanked by two Lox P sites. These crossings led to the generation of CCSP-Cre/LSL-ERK3/PTENFlox/Flox mice which display PTEN deletion and ERK3 overexpression in the Clara cells of lung. Thus, four transgenic mouse groups were included in the study and monitored on daily basis: 1) LSL-ERK3; 2) CCSP-Cre/LSL-ERK3; 3) CCSP-Cre/PTENFlox/Flox; 4) CCSP-Cre/LSL-ERK3/PTENFlox/Flox. Tumor growth or other abnormalities in the lungs were analyzed by examining the whole lungs for surface tumors and by histological examination of lung tissue sections. As reported in previous studies, PTEN deletion alone resulted in lung hyperplasia. Interestingly, while ERK3 overexpression alone didn't cause clear phenotype, a combination of ERK3 overexpression with PTEN deletion in CCSP-Cre/LSL-ERK3/ PTENFlox/Flox mice induced lung tumorigenesis as demonstrated by the formation of surface tumors in lungs. Tumor formation in these CCSP-Cre/LSL-ERK3/PTENFlox/Flox mice was confirmed by hematoxylin and eosin (HE) staining of lung sections. Further, immunohistochemical analysis of differential biomarkers suggested that these tumors correspond to lung adenocarcinomas. Taken together, our study demonstrates a promoting role of ERK3 in lung tumor formation in vivo.

#5099

Evaluating Ago2 as an oncogene in Kras-dependent lung carcinoma.

Andrew E. Goodrum, Lisha Wang, Alice Xu, Kristin M. Juckette, Arul M. Chinnaiyan, Jean C. Tien. _University Of Michigan - Ann Arbor, Ann Arbor, MI_.

Background: Lung cancer is the deadliest malignancy in the United States. Non-small cell lung cancer (NSCLC) accounts for 85% of cases, and is frequently driven by activating mutations in the gene encoding the KRAS GTPase (e.g. KRASG12D). Our previous work demonstrated that Argonaute 2 (AGO2)—a component of the RNA induced silencing complex (RISC)—physically interacts with KRAS and stabilizes it at the protein level. In NSCLC cell lines, AGO2 knockdown reduces KRAS protein level and attenuates cell proliferation. We hypothesized that AGO2 acts as an oncogene in KRASG12D-dependent NSCLC in vivo.

Methods: To test the hypothesis, we generated a NSCLC model by intercrossing mice harboring a stop-floxed KRASG12D allele with animals containing a tamoxifen-responsive Cre recombinase gene driven by the (clara cell-specific) CCSP promoter. These animals, which frequently die within 10 weeks of tamoxifen treatment, display pulmonary lesions including hyperplasia, adenoma and frank adenocarcinoma. We evaluated whether concomitant ablation of one or both copies of (floxed) AGO2 influenced the NSCLC phenotype. After tamoxifen administration, we monitored mice daily and harvested tissues when mice were clinically moribund or reached 16 weeks of post-treatment. We harvested lungs for gross/ pathological examination and gene/ protein expression analysis.

Results: While pathologically discernable adenocarcinoma was detectable in AGO2+/+ and AGO2f/+ animals, AGO2f/f mice were free of these lesions. Despite this, deletion of AGO2 had no impact on frequency or extent of hyperplastic or adenomatous lesions. Immunohistochemistry demonstrated absence of AGO2 in tumor tissue of AGO2f/f mice.

Conclusion: AGO2 promotes progression but not initiation of cancerous lesions in a mouse model of KRASG12D-dependent NSCLC.

#5101

Characterization of a mouse model using the Sleeping Beauty transposon method to study diffuse intrinsic pontine glioma (DIPG).

Ramya Ravindran,1 Flor M. Mendez,2 Felipe J. Nunez,2 Carl Koschmann,2 Marta Dzaman,2 Sheeba Pawar,2 Pedro R. Lowenstein,2 Maria G. Castro2. 1 _University of Michigan, Ann Arbor, MI;_ 2 _University of Michigan School of Medicine, Ann Arbor, MI_.

Tumors in the brain-stem are difficult to diagnose and treat primarily owing to the location. Diffuse intrinsic pontine glioma (DIPG) is a fatal paediatric brain-stem tumor located in the ventral pons with 100% fatality. These tumors are often treated on the assumption that DIPGs are molecularly similar to adult high grade gliomas; however, they are molecularly distinct. The ACVR1G328V and the H3.1K27M mutations have been seen to recur in DIPGs and we hypothesis that these mutations play an important role in the disease progression. A mutation in the ACVR1 leads to kinase activation and induces downstream signal transduction by phosphorylation of Smads (1/5). H3.1K27M mutations cause genome wide H3K27me3 hypomethylation. We genetically engineered mouse DIPG models using the Sleeping Beauty Transposon (SBT) method to introduce plasmids containing NRASV12/p53-shRNA/ACVR1G328V or NRASV12/p53-shRNA/H3.1K27M into the 4th ventricle of 1-day old pups adjacent to the brain-stem. This generates DIPGs with specific genetic lesions. NRASV12 is a constitutively active oncogene belonging to the RAS family and p53-shRNA is a short hairpin for TP53 tumor suppressor gene. A control tumor model was engineered using plasmids encoding NRASV12 and p53-shRNA. Tumor growth is monitored periodically over time until animals become symptomatic due to tumor burden. At this time point, animals are euthanized and the tumors extracted. We characterized the model for upregulation of phospho-smad 1/5 and markers of neural precursor cells (nestin, Sox2, olig2, GFAP, PDGFRα). In conclusion, we generated a genetically engineered mouse model of DIPG which represents a novel platform to study the molecular pathways underlying disease pathogenesis. An understanding of the functions of mutations that lead up to the disease can help to develop novel therapies for DIPG.

#5102

Identifying relevant mouse models of human cancer using the mouse tumor biology database (MTB).

Dale A. Begley, Debra M. Krupke, Steven B. Neuhauser, Joel E. Richardson, John P. Sundberg, Carol J. Bult. _The Jackson Laboratory, Bar Harbor, ME_.

Mouse models of human cancer have provided important insights into the genetic and molecular basis of human cancer and have been used to identify promising new treatment options for human patients. Genetically engineered mouse models (GEMMS) have been used to identify and characterize the basis of cancer susceptibility, tumor suppressor and oncogene function, and increasingly co-clinical studies of proposed therapeutic treatments. In recent years patient derived xenograft (PDX) models created by implanting human tumor tissue into immune deficient mouse hosts have become a major in vivo pre-clinical research platform for evaluating novel cancer therapies tailored to genomic properties of a patient's tumor. The diversity and distributed nature of GEMM and PDX mouse models, and the data generated from these models, present a significant challenge to researchers who are searching for mouse models relevant to their research. The Mouse Tumor Biology database (http://tumor.informatics.jax.org) is a comprehensive resource of information on both GEMM and PDX models of human cancer that has been expertly curated from peer-reviewed scientific publications and direct data submissions from individual investigators. MTB provides an easy to use search interface and tools for visualizing associated data from mouse models of human cancer. Standardized annotations using controlled vocabularies and official gene and mouse strain nomenclature ensures that researchers get accurate and comprehensive results to their searches. For GEMMs, MTB contains data from over 24,000 different spontaneous or endogenously induced tumors from genetically defined mice obtained from over 4,400 published manuscripts. Annotations include 88,000 tumor frequency records, over 2,200 pathology reports, and over 6,100 images. MTB also provides access to detailed clinical, pathological, expression and genomics data from over 450 PDX models with over 990 histology images. Information in MTB is cross-reference to cancer models data from other bioinformatics resources including PathBase, the Mouse Phenome Database (MPD), the Gene Expression Omnibus and ArrayExpress. Recent enhancements to MTB include the interactive cancer model summary table linking the most common fatal human cancers to relevant mouse models and interactive plots for dosing studies performed using PDX models. MTB has co-developed the PDX Finder resource in collaboration with EMBL-EBI to provide a comprehensive global catalogue of PDX models available for researchers. MTB is supported by NCI grant CA089713.

#5103

Notch is a master regulator of mesenchymal transformation and therapeutic resistance in glioma.

Matei A. Banu,1 Athanassios Dovas,1 Henar Cuervo,2 Brianna Pereira,1 Aayushi Mahajan,1 Hongxu Ding,1 Mukesh Bansal,3 Sergey Pampou,1 Charles Karan,1 Andrea Califano,1 Peter A. Sims,1 Jan Kitajewski,2 Jeffrey N. Bruce,1 Peter Canoll1. 1 _Columbia University, New York, NY;_ 2 _University of Illinois at Chicago, Chicago, IL;_ 3 _Psychogenics Inc., Paramus, NJ_.

Introduction: Gliomas are highly infiltrative tumors rendering complete resection impossible. As such, prognosis remains dismal. Recent studies have identified both proneural and mesenchymal cell populations. Crucially, recurring gliomas bear a mesenchymal phenotype. Understanding mechanisms driving mesenchymal transformation is important in developing tailored, more efficient therapeutic approaches.

Methods: Using targeted biopsies from a mouse glioma model and human glioma databases, we applied bioinformatics and identified Notch as an additional master regulator (MR) protein driving mesenchymal transformation. To better understand the role of Notch, we overexpressed active forms of Notch1 (N1IC) or Notch3 (N3IC) in primary cells, orthotopic xenotransplants, and transgenic mouse models. We performed mouse survival studies and examined Notch-induced phenotypic alterations in tumor cells and the microenvironment using molecular and immunohistochemical approaches. Finally, we performed high-throughput drug and radiation screening studies to assess responses to treatment.

Results: In silico analyses on RNAseq of biopsies from spatially distinct tumor regions in a mouse glioma model identified Notch as a highly active pathway in the cores of tumors undergoing mesenchymal transformation. Importantly, MR analysis of TCGA samples and single cell transcriptomic data identified the Notch pathway as a driver of proneural to mesenchymal transformation. Immunophenotyping and RNAseq profiling of human GBMs identified Notch receptors on tumor cells and Notch ligands on astrocytes and microglia suggesting potential microenvironment-mediated plasticity since Notch proteins are not frequently mutated in GBM. Overexpression of N1IC or N3IC in proneural cells induced mesenchymal transformation with slow proliferation and a highly invasive phenotype. Orthotransplantation of N3IC-expressing cells in mice led to slower growing tumors, perivascular invasion, increased recruitment of reactive phagocytes and slightly prolonged survival. A novel transgenic mouse model in which N1IC was overexpressed also led to prolonged survival compared to a control population. Importantly, high-throughput screening studies showed that N1IC and N3IC rendered tumor cells significantly more resistant to standard and targeted therapies, including irradiation and etoposide.

Conclusions: Our study identifies Notch signaling as an important, likely microenvironment-driven pathway driving proneural to mesenchymal transformation in glioma. While Notch was shown to slow tumor growth and prolong survival, cells were more invasive and less sensitive to treatment. These findings suggest that Notch plays a role in the mesenchymal transformation and therapeutic resistance seen in post-treatment recurrence. Targeting Notch may therefore be important in improving therapeutic responses against glioma.

#5104

**Prolactin-induced mammary tumors: A preclinical model of luminal B breast cancer which exhibits mutations in the RAS** **pathway.**

Kathleen A. O'Leary,1 Katie M. Campbell,2 Debra E. Rugowski,1 Kilannin Krysiak,2 William A. Mulligan,1 Malachi Griffith,2 Obi L. Griffith,2 Linda A. Schuler1. 1 _University of Wisconsin, Madison, WI;_ 2 _Washington University School of Medicine, St. Louis, MO_.

Metastatic therapy-resistant breast cancers expressing estrogen receptor alpha (ER+) account for the majority of breast cancer deaths. Understanding of the underlying biology of aggressive luminal B cancers would improve preventive/treatment strategies. However, this has been limited by the paucity of preclinical immunocompetent models. Strong epidemiologic data link prolactin (PRL) exposure to development of ER+ metastatic breast cancer. We have capitalized on this relationship to generate a murine model which overexpresses PRL in mammary epithelial cells (NRL-PRL). Transgenic mammary PRL expression leads to the development of spontaneous metastatic ER+ carcinomas with characteristics of luminal B human cancers. In order to understand the mechanisms that underlie the ability of PRL to drive the development of cancers in this model, we used functional genomic analyses to identify genomic alterations and patterns of gene expression with cancer progression. Following development of spontaneous tumors, ER+ tumors and matched adjacent mammary glands and tail samples (N=5) were collected and examined by whole genome sequencing and RNA-seq analysis. A set of mammary cell preparations from the caudal glands of 12-week-old NRL-PRL females (N=5) were also analyzed. Unexpectedly, we found that all of the tumors contained somatic alterations of Kras, including four with canonical hotspot mutations (G12 and Q61) and one with an amplification of the Kras locus. In contrast to the tumors, no Kras mutations were detected in the matched mammary glands and tails, or the cell preparations. Our findings were further validated by targeted sequencing of an extension set consisting of frozen archived spontaneous ER+ tumors from NRL-PRL females (N=15), additional samples from mice with matched tumor, adjacent mammary gland and tails (N=7) and ER+ cell lines generated from PRL-induced spontaneous tumors (N=4). The Kras hotspot mutations were detected in 14 of 22 of the extension tumors and all four cell lines. One additional tumor contained a hotspot activating mutation in Nras. Immunohistochemistry demonstrated that RAS downstream effectors, pAKT and pERK, were upregulated, whereas nuclear STAT5A was decreased, in the tumors compared to the adjacent normal gland. Moreover, RNA-Seq analyses showed that acquisition of RAS mutations by the tumors was associated with alterations in multiple pathways compared to non-tumor glands, including distinct immune cell subpopulations. Although few clinical breast cancers display RAS protein mutations, studies have shown that the RAS pathway is aberrantly activated in more than 50% of all breast lesions. This enhanced RAS activity confers many benefits to these tumors resulting in a poor prognosis. The NRL-PRL model provides a valuable tool to dissect mechanisms that drive metastatic luminal B breast cancer and identify therapeutic opportunities.

#5106

CRISPR/Cas9-based development of RNAi rat models for drug discovery.

Prem K. Premsrirut,1 Chia-Lin Wang,1 Yu-Ting Yang,1 Lukas E. Dow,2 Johannes Zuber,3 Scott W. Lowe4. 1 _Mirimus, Inc., Brooklyn, NY;_ 2 _Weill Cornell Medical College, New York, NY;_ 3 _Research Institute of Molecular Pathology, Vienna, Austria;_ 4 _Memorial Sloan Kettering Cancer Center, New York, NY_.

The cost of drug development has skyrocketed to an estimated $2.6B for every FDA approved drug primarily due to failures from lack of efficacy or safety. This suggests that our current preclinical validation process has been insufficient in predicting therapeutic potential and toxicity in humans. While genetically engineered animal models have been highlighted as the gold standard for dissecting cancer mechanisms and evaluating novel drug targets in vivo, the cost and long lead time to develop them previously prevented their routine use in the oncology drug discovery process. Now, with the advent of CRISPR/Cas9 technology, the speed and precision in which genetically engineered mouse models can be created is unprecedented. When combined with inducible RNA inteference technology, we we can not only generate cancer de novo in a few weeks time, but also mimic drug therapy via RNAi in the same mice, giving us advanced capabilities to perform preclinical studies in vivo. Despite the utility of mouse models, the rat has historically been the major model species in many biomedical fields, notably toxicology and carcinogenicity testing; and for many scientists, the rat still remains the preferred rodent due to their larger size for surgical manipulation, repeat blood sampling, and their cognitive and physiological characteristics that more closely resemble humans than their mouse counterparts. Here, we take advantage of our two-step engineering approach and exploit the efficiency of CRISPR-based targeting to develop RNAi rat models that enable inducible and reversible gene silencing to simulate therapeutic regimes. We demonstrate that our approach allows us to rapidly generate RNAi rat models and mimic the function of the targeted small molecule inhibitors, such as BET inhibitors targeting Brd4. We compare our results to our Brd4 RNAi mice and demonstrate organism variances that provide valuable insight to cross-species differences. These results demonstrate that our high-throughput system currently used to generate RNAi mice is also applicable to the rat system and, by extension, other mammalian models. Inducible RNAi rat models will undoubtedly be powerful tools that can be used to model human cancers, to mimic the action of putative drugs, and to assess the potential of therapeutic targeting strategies in vivo prior to the costly drug development, ultimately guiding the development of safer and more effective drugs.

#5107

A novel MTA1 knock-in mouse model for the mechanistic and therapeutic studies of MTA1-driven prostate cancer.

Avinash Kumar,1 Swati Dhar,2 Nasir A. Butt,2 Pravin R. Phadatare,1 Kshiti Dholakia,1 Jogarao Vedula,3 Virginia Donovan,3 Anait S. Levenson1. 1 _Long Island University, College of Pharmacy and Health Sciences, Brooklyn, NY;_ 2 _University of Mississippi Medical Center, Jackson, MS;_ 3 _NYU Winthrop Hospital, Mineola, NY_.

Metastasis-associated protein 1 is known to play role in prostate cancer progression and metastasis. Studies of MTA1-mediated prostate cancer tumorigenesis, progression and metastasis have been hampered by a lack of suitable experimental system. Here we describe a new conditional mouse model that shows strong MTA1 expression in the prostate. This model was generated through MTA1 transgene knocked into the mouse ROSA26 (R26) locus using CAG-LoxP-Stop-LoxP (LSL)-2HA-MTA1-T2A-GFP-pA construct (Applied StemCell,Inc, Menlo Park, CA). Briefly, after generation of a genetically modified founder mouse line Rosa26-3attP, resulted MTA1 transgene embryos were injected into foster mice to produce MTA1 founders, designated as R26-LSL-MTA1. To monitor tumor formation and development in live animals by using IVIS, we have crossbred R26-LSL-MTA1 with Pb-Cre+;Luc and generated transgenic lines that express MTA1 and Luc upon Cre-mediated removal of the LSL cassette, specifically in the prostate epithelium (Pb-Cre+;R26MTA1;R26Luc) (MTA1-tg). We confirmed prostate-specific expression of the MTA1 transgene on mRNA and protein levels. We evaluated prostate histology for differences in histology of control normal prostates (Pb-Cre-; MTA1-tg) and MTA1-tg mice at as early as 13-weeks of age. MTA1 overexpressing prostate showed signs of mPIN characterized by glandular structures composed of atypical proliferating luminal cells that fill out the lumen. We detected differences in cellular proliferation, as determined by increased PCNA and decreased p27 as well as Ki67 immunofluorescent staining in MTA1-tg prostate. Moreover, expression of several oncogenic MTA1-guided molecules was increased in the prostates of MTA1-tg mice. We have previously reported on the role of MTA1-mediated signaling in prostate tumor progression in the prostate-specific Pten heterozygous (Pb-Cre+; Pten+/f) and Pten null (Pb-Cre+; Ptenf/f ) mice. To determine the role of MTA1 overexpression in the context of PTEN loss, we generated MTA1-tg; Pten+/f and MTA1-tg; Ptenf/f mice to evaluate whether MTA1overexpression in the prostate promotes an earlier onset, greater total incidence, accelerated tumor growth rate and metastasis. We predict that MTA1 overexpression will cause higher activation of downstream pathways to promote cancer cell survival, which will result in increased incidence of invasive adenocarcinoma and probably lymph node metastasis. Mice will be sacrificed at different time points for prostate tissue and lymph nodes isolation and subsequent histopathological, immunochemical and molecular evaluation.

#5108

The role of H2afy in normal and malignant hematopoiesis.

Sanghyun Kim, Monique Chavez, Cara Shirai, Matt Walter. _Washington University in St. Louis, St. Louis, MO_.

Mutations in the spliceosome gene U2AF1 are found in 11% of patients with myelodysplastic syndrome (MDS), a common hematologic malignancy, and at lower frequencies in some solid cancers. We have shown that expression of a common U2AF1 mutant, U2AF1(S34F), causes altered pre-mRNA splicing and abnormal hematopoiesis in mice, including reduced white blood cell counts (WBC) and B-cells, which are phenotypes similar to patients with MDS. We consistently find that expression of mutant U2AF1 results in reduced RNA expression of H2AFY1.1, a splice isoform of H2AFY, in patient samples and mouse hematopoietic cells. H2AFY (also known as macroH2A1) is a histone H2A variant implicated in transcriptional gene regulation, development, and tumorigenesis. This gene encodes two splice isoforms, H2AFY1.1 and H2AFY1.2. Expression of the H2AFY1.1 isoform is also reduced in several solid cancers. We hypothesize that reduced H2AFY1.1 expression contributes to abnormal hematopoiesis induced by mutant U2AF1(S34F). We first characterized hematopoiesis in adult constitutive H2afy knock-out mice (lacking both H2afy1.1 and H2afy1.2 isoforms). We transplanted bone marrow cells from H2afy+/+ or H2afy-/- mice into lethally irradiated congenic recipients to study the hematopoietic cell-intrinsic effects of H2afy loss. At 6 weeks post-transplant, recipients of H2afy-/- bone marrow cells compared to recipients of H2afy+/+ cells exhibited a marked reduction in WBC (5.0 vs. 9.1 K cells/µL, respectively, p<0.001, N=10), peripheral blood B-cells (1.9 vs. 5.3 K cells/µL, respectively, p<0.001, N=10), and bone marrow B-cells (6.33e6 vs. 2.5e6 cells/mouse, p=0.001, N=7). There is also a 2-fold reduction in pre-B methylcellulose colony forming units of H2afy-/- bone marrow cells compared to wild-type controls (p< 0.01, N=20), indicating a role for H2afy in B-cell development. In competitive repopulation assays, we observed a reduction in H2afy-/- donor-derived B-cells in the peripheral blood compared to H2afy+/+ B-cells (14 vs. 58%, respectively, p<0.001, N=14-15) and reductions in bone marrow pro-B-cells through mature B-cells, suggesting potential roles for H2afy in pre-pro- to pro-B-cell development. Viral re-expression of H2AFY1.1 in H2afy-/- bone marrow cells rescued the reduced frequency of peripheral blood B-cells (61 vs. 36%, p<0.001, N=10) and bone marrow B cells (11 vs. 5%, p=0.018, N=5-6) in vivo relative to control virus-transduced H2afy-/- cells, whereas H2AFY1.2 expression did not significantly affect B-cell counts. Collectively, our data indicate that the H2afy1.1 isoform is important for B-cell development and that reductions in the H2afy1.1 isoform may contribute to U2AF1(S34F)-induced abnormal hematopoiesis in mice. Ongoing studies are designed to address how H2afy1.1 affects B-cell development and to determine if the B-cell phenotype in U2AF1(S34F) mice is due to reduced H2afy1.1 isoform expression.

#5109

Somatic CRISPR/Cas9-mediated gene editing enables versatile brain tumor modeling.

Marc Zuckermann,1 Britta Ismer,1 Volker Hovestadt,1 Christiane B. Knobbe-Thomsen,2 Marc Zapatka,1 Paul A. Northcott,3 Martine F. Roussel,3 Guido Reifenberger,2 Peter Lichter,1 Stefan M. Pfister,1 Daisuke Kawauchi,1 Jan Gronych,1 David T. Jones1. 1 _German Cancer Research Center, Heidelberg, Germany;_ 2 _Heinrich Heine University, Düsseldorf, Germany;_ 3 _St Jude Children's Research Hospital, Memphis, TN_.

Despite substantial advances in the molecular classification of pediatric brain tumors over the last decade, many tumors are still incurable, have a poor prognosis or therapy-related detrimental long term effects. To develop innovative and modern therapy approaches, sophisticated mouse models, faithfully reflecting the human disease are urgently needed.

However, the development of transgenic mouse lines is a time consuming process that is confronted with an increasing number of candidate genes resulting from next generation sequencing of tumor specimens. To cope with this discrepancy, more adaptable animal models are needed. Using somatic gene transfer to manipulate cells in situ holds great advantages in terms of speed and flexibility.

We therefore established a system in which we are using the CRISPR/Cas9 system for somatic disruption of candidate tumor suppressor genes (TSGs) in combination with transposon-mediated overexpression of candidate oncogenes to induce murine brain tumors. By deleting Ptch1 in murine cerebella, we induced Sonic Hedgehog (SHH) subgroup medulloblastoma with high penetrance and short latency. We characterized the induced mutations in depth and did not detect recurrent off-targets using unsupervised whole genome sequencing. We further demonstrated that this method is suited to induce other brain tumor entities by targeting multiple TSGs at the same time or by combining TSG targeting with overexpression of certain oncogenes. To date we established five novel mouse models using this system. Two of these resulted from simultaneous deletion of Nf1, Pten and Trp53 or from combining overexpression of a MET fusion with a loss of Trp53, respectively. Both of these combinations led to efficient glioblastoma induction in the murine forebrain. Our results demonstrate a fast and flexible system for validating novel candidate genes and for generating faithful animal models of human cancer.

#5110

Runx knockout in breast luminal stem/progenitor cells induced precancerous lesion via robust expression of ERα.

Junichi Matsuo, Naing Naing Mon, Akihiro Yamamura, Dede L. Heng, Linda SH Chuang, Motomi Osato, Yoshiaki Ito. _National University of Singapore, Singapore, Singapore_.

In breast cancer, 83% of patients were diagnosed as hormone receptor positive, luminal-type, breast cancer (Anderson et al., 2014). Although primary luminal-type breast cancer can be controlled by endocrine therapy, more than 20% of patients have recurrence in 20 years (Pan et al., 2017). Understanding of luminal-type breast cancer would provide benefits to a large number of patients. However, cell of origin and genes responsible for luminal-type breast cancer have not been fully understood. Luminal epithelial cells are considered as origin of breast cancer (Anderson et al., 2014), although not fully proven yet. In addition, extensive genomic analysis of breast cancer patients in TCGA study showed that RUNX genes were downregulated in luminal-type breast cancer: RUNX1 mutation was found in multiple patients, and RUNX3 gene was highly methylated. We previously reported that 270 bp Runx1 enhancer element (eR1) marks hematopoietic stem cell and stomach stem cell (Ng et al., 2010; Matsuo et al., 2017). We also found that this eR1 marks a subset of luminal epithelial cells in mouse mammary gland. Lineage-tracing experiment showed that the eR1+ cells mainly differentiated into luminal cells, but not to basal cells in vivo. These eR1+ cells also contribute to differentiation of alveolar cells in pregnant mice. Interestingly, in vitro lineage tracing by using organoid culture showed that eR1+ cells differentiated into both liminal and basal cells. Runx1 and Runx3 knockout in luminal stem cells showed increase of cell growth, and histology was similar to precancerous lesion such as hyperplasia and ductal carcinoma in situ (DCIS). These lesions showed robust expression of ERα compared with control mice. These results suggested that depletion of Runx in luminal stem cells induces precancerous lesion via robust expression of ERα.

References:

Anderson WF, Rosenberg PS, Prat A, et al. How many etiological subtypes of breast cancer: Two, three, four, or more? J Natl Cancer Inst 2014;106.

Pan H, Gray R, Braybrooke J, et al. 20-year risks of breast-cancer recurrence after stopping endocrine therapy at 5 years. N Engl J Med 2017:1836-46.

Ng CE, Yokomizo T, Yamashita N, et al. A Runx1 intronic enhancer marks hemogenic endothelial cells and hematopoietic stem cells. Stem Cells 2010:1869-81.

Matsuo J, Kimura S, Yamamura A, et al. Identification of stem cells in the epithelium of the stomach corpus and antrum of mice. Gastroenterology 2017:218-31.

#5111

Gene editing in the NSG mouse strain and its genetic derivatives.

David S. Grass, F William Buaas, Michael V. Wiles, Benjamin E. Low, Peter Kutny. _The Jackson Laboratory, Bar Harbor, ME_.

Mouse models continue to be powerful tools in preclinical oncology and immuno-oncology studies. The development of the inbred highly immunocompromised mouse strain NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) has dramatically improved the ability to work with human tumors in an organismal context using xenograft approaches. Furthermore, technical innovations have expanded the utility of this personalized tumor platform via the reconstitution of parts of the human immune system. An example of this is the NSG-SGM3 inbred mouse strain, in which transgenes expressing human IL3, GMCSF and SCF have been stably integrated into the NSG background. This NSG "derivative" strain can effectively support the stable engraftment of human myeloid lineages and the regulatory T cell populations. Direct genetic modification of the NSG genome in mouse embryos was a key technical capability to attain this NSG "derivative" strain rapidly and avoid extensive breeding. Modification of NSG and derivative strains represent an opportunity to optimize the engraftment of other human lineages. The ability to apply modern genome modification technology to different inbred mouse strains is highly desirable but must overcome two major technical hurdles - 1) Reproductive biology constraints that prevent live born progeny after reagent delivery methods such as microinjection or electroporation and 2) Cell intrinsic DNA-repair processes that are a prerequisite enabling gene editing tools. Given the SCID mutation (Prkdcscid) in the NSG background compromises some DNA repair pathways, it was not clear whether nuclease assisted recombination technologies would be a robust tool to modify NSG (and derivatives) genomes at the zygote stage. Here, we demonstrate that CRISPR-Cas9 is an effective tool for introducing genome modifications in NSG and present a specific example using the Hprt locus. Furthermore, we will present general experience with genomic modifications using NSG, and its derivatives, to emphasize direct modification of these genomes is technically feasible and is enabling the rapid sequential genetic tailoring in this high value cancer model strain.

#5112

U2af1 **, a spliceosome gene commonly mutated in MDS, is required for hematopoiesis.**

Brian A. Wadugu, Amanda Heard, Joseph Bradley, Matthew Ndonwi, Matthew J. Walter. _Washington University in St. Louis, St. Louis, MO_.

Somatic mutations in genes encoding factors involved in pre-mRNA splicing (i.e., spliceosome genes) have been identified in more than half of myelodysplastic syndrome (MDS) patients. We are interested in the role of U2AF1, a spliceosome gene mutated in up to 11% of MDS patients. While we reported that mice expressing mutant U2AF1 have altered hematopoiesis and RNA splicing, similar to mutant MDS patients, the role of wild-type (WT) U2AF1 in normal hematopoiesis has not been studied. A complete understanding of the role of WT U2AF1 on hematopoiesis and RNA splicing is critical to our understanding of how mutant U2AF1 contributes to abnormal hematopoiesis and splicing in MDS. In order to understand the role of WT U2af1 in normal hematopoiesis, we created a conditional U2af1 knock-out (KO) mouse. Homozygous embryonic deletion of U2af1 using Vav1-Cre was lethal, suggesting that U2af1 is essential for hematopoiesis during embryonic development. To study the hematopoietic cell-intrinsic effects of U2af1 deletion in adult mice, we performed a non-competitive bone marrow transplant of bone marrow cells from Mx1-Cre/U2af1flox/flox, Mx1-Cre/U2af1flox/wt or Mx1-Cre/U2af1wt/wt into lethally irradiated congenic recipient mice. Following poly I:C-induced U2af1 deletion, homozygous U2af1 KO mice, but not other genotypes, became moribund. Analysis of peripheral blood up to 11 days post poly I:C treatment revealed anemia (decrease >1.7 fold) and multilineage cytopenias in homozygous U2af1 KO mice compared to all other genotypes (p ≤ 0.001, n=5). Deletion of U2af1 also led to rapid bone marrow failure and a reduction in bone marrow neutrophils (p ≤ 0.001), monocytes (p ≤ 0.001), and B-cells (p ≤ 0.05), as well as a depletion of hematopoietic progenitor cells (p ≤ 0.001, n=5). Next, we created mixed bone marrow chimeras (i.e., we mixed equal numbers of homozygous KO and WT congenic bone marrow cells and transplanted them into lethally irradiated congenic recipient mice) to study the effects of U2af1 deletion on hematopoietic stem cells (HSCs). As early as 10 days, and up to 4 months following Mx1-Cre-induction, we observed a significant decrease in white blood cell count chimerism and complete loss of homozygous U2af1 KO HSCs, neutrophils and monocytes, as well as a severe reduction in B-cells and T-cells (p ≤ 0.001, n=3-4 for HSCs. p ≤ 0.001, n=9-10 for all other comparisons). Collectively, the data indicate that normal hematopoiesis is dependent on U2af1 expression. In ongoing studies, we crossed the U2af1 KO mouse to existing transgenic mutant U2AF1-expressing mice to determine if mutant U2AF1 expression is sufficient to rescue cell survival and normal hematopoiesis when endogenous WT U2af1 is deleted. If U2AF1 mutant cells are vulnerable to loss of the WT U2AF1 allele, then selectively targeting the WT U2AF1 allele in heterozygous mutant cells could be a novel therapeutic strategy.

#5113

Spontaneous tumors in aging colonies of C57BL/6J mice.

Andrew Schile,1 Stephanie Dion,2 Denise Imai-Leonard,3 Rosalinda Doty2. 1 _The Jackson Laboratory, Sacramento, CA;_ 2 _The Jackson Laboratory, Bar Harbor, ME;_ 3 _University of California, Davis, Davis, CA_.

The C57BL/6J inbred mouse strain is widely used to study the effects of aging. We have documented 51 spontaneous tumors (39 malignant and 12 benign) as part of a recent effort to establish large colonies of male and female C57BL/6J mice for aging research. Forty six of the tumors were diagnosed in mice submitted for histopathology from typical aging colonies maintained throughout a range of ages (6-18 months) that roughly correspond to 30 - 56 years of age in humans; the remaining cases were from much smaller cohorts aged past 18 months. Sarcomas represented the largest category of malignant tumors (20/51). There were 6 cases (5 male, 1 female) of hemangiosarcoma (4 on the neck, 1 on the liver, and 1 on the ear) and 6 multicentric histocytic sarcomas (4 male, 2 female). Four cases (1 male, 3 female) of rhabdomyosarcoma (2 on the head, 2 on the leg) were also seen. The remaining sarcoma diagnoses included one osteosarcoma (female; leg), one chondrosarcoma (male; ear), and two sarcomas not otherwise specified (both female; one leg, one lateral abdomen). Hematological malignancies constituted the second largest category (9/51), including 7 multicentric lymphomas (3 male, 4 female), a uterine lymphoma, and a multicentric plasma cell tumor (1 male). The four skin malignancies included two melanomas (male: head; female: tail/anus) and two squamous cell carcinomas (male: jaw; female: leg). Three mammary cancers were observed in females, including an adenoacanthoma and two adenocarcinomas, one of which had lung metastases. An ovarian dysgerminoma was the only germ cell tumor documented. The Harderian gland (a lacrimal duct of the eye) was the site of two carcinomas (male) and six adenomas (5 male, 1 female). Other benign tumors included a tail fibroma (male), two pulmonary adenomas (1 male, 1 female), one mammary fibroadenoma (female), a myoepithelioma of the leg (female), and a keratoacanthoma of the paw (female). Tumor incidence was very low overall, representing <0.1% of the mice present on an average week during the typical range of ages (6-18 months) in the aging colonies. Overall, the median age of diagnosis was 239 days for females and 427 days for males. These results should help to define the spectrum of tumors that develop in colonies of C57BL/6J mice that approximately correspond in age to middle-aged humans.

#5114

The role of amphiregulin in mammary gland development and breast cancer.

Serena Mao,1 Minji Park,2 Ramon Cabrera,1 John Christin,1 George Karagiannis,1 Wenjun Guo,1 John Condeelis,1 Jeffrey Segall1. 1 _Albert Einstein College of Medicine, Bronx, NY;_ 2 _Stuyvesant High School, Manhattan, NY_.

Breast cancer is the most prevalent cancer in women worldwide and afflicts more than 3.1 million women in the US alone. The epidermal growth factor receptor (EGFR) is frequently overexpressed in breast cancer and is associated with poor clinical outcome. Several therapies involving anti-EGFR agents have been developed to target breast cancer. However, clinical trials have shown mixed outcomes with these therapies. Amphiregulin (AREG), a ligand of the epidermal growth factor receptor (EGFR), is enriched in invasive breast cancer, particularly ERα-positive breast tumors. In normal physiology, AREG mediates estrogen-induced ductal morphogenesis during mammary gland development. In the absence of AREG, the ductal tree fails to elongate and remains rudimentary throughout adulthood. The structure of a healthy mammary duct consists of luminal cells surrounded by a layer of myoepithelial cells. Studies of mammary gland transplantations suggest that a marker of myoepithelial cells (keratin 14, K14) is only expressed if AREG is present in the mammary epithelium. Multiple studies have provided evidence that myoepithelial cells are natural tumor suppressors. During tumor growth, the disappearance of myoepithelial cells indicates progression to invasive carcinoma. Therefore we hypothesize that AREG plays an important role in maintenance of the maintenance of myoepithelial cells in the mammary epithelium and loss of AREG promotes tumor progression as a result of an early loss of myoepithelial cells. In the mammary glands of AREG WT and AREG KO mice without the PyMT transgene, we have found that mammary ducts in AREG KO mice have significantly fewer myoepithelial cells. Immunofluorescent staining of K14 has revealed a thinner and discontinuous myoepithelial layer in the AREG KO mice. To investigate the role of AREG in breast cancer development and progression, we have utilized a spontaneous mammary tumor mouse model where oncogenesis is driven by expression of the polyoma middle T oncoprotein (PyMT) through the mouse mammary tumor virus (MMTV) promoter. We have crossed AREG knockout mice with the MMTV-PyMT mice to generate AREG KO mice that spontaneously form mammary tumors (AREG KO PyMT). The absence of AREG in both the mammary epithelial and stromal compartments accelerates tumor growth. By 12 weeks of age, tumors of AREG KO PyMT mice are significantly larger than that of AREG WT PyMT mice. AREG KO tumors are histologically less necrotic, apoptotic, and progressed than AREG WT tumors. Furthermore, AREG KO tumors show higher staining of PyMT by immunofluorescence. Together, these results suggest that loss of AREG stimulates tumor initiation but inhibits progression due to fewer myoepithelial cells beginning during development.

#5115

Establishing tumoroid and mouse models for functional validation of progression markers in DCIS.

Stefan J. Hutten, Catrin Lutz, Stefano Annunziato, Ellen Tanger, Jelle Wesseling, Jos Jonkers. _Netherlands Cancer Institute, Amsterdam, Netherlands_.

Ductal Carcinoma in Situ (DCIS) is a starting lesion in the milk duct of the breast, which accounts for 25% of all 'breast cancers' detected since the introduction of breast screening. DCIS is usually treated by surgery combined with radiotherapy, which can have a large impact on the life of patients. However, there is little to no evidence that treatment of low and intermediate grade DCIS reduces mortality, while women diagnosed with DCIS do perceive their risk of dying the same as patients with invasive disease. To reduce the negative perception and the overtreatment of DCIS, but assure proper treatment for high risk DCIS, it is critical to identify which factors can predict whether DCIS stays indolent or becomes invasive.

We have successfully shown that it is possible to propagate tumor cells and organoids in vivo using intraductal injections. Adult female NSG and nude mice were injected intraductally into the mammary gland with suspensions of breast cancer cell and organoid lines as well as DCIS cell lines. Moreover, we were able to instigate a protocol for deriving fresh patient DCIS material to create in vitro primary tumoroid cultures, which we transplanted as suspended cells into NSG and nude mice. Tumor formation was examined via palpation and bioluminescence imaging. The resulting tumors will be compared to the original patient material to show that we are able to create a suitable in vivo model, retaining the morphologic and genomic features of the patient.

In addition to these transplantation models we sought to take advantage of existing non-germline models for establishing a genetic DCIS model. To establish these, genes were selected from literature which are suspected to initiate DCIS formation. These genes were then incorporated in lentiviral vectors and injected in the mammary gland of immunocompetent mice. The mice will be sacrificed at set time points to follow DCIS initiation and progression to Invasive Ductal Carcinoma (IDC).

For characterization, all these models will be subjected to in‐depth histopathologic, transcriptomic, mutational, proteomic, immunologic and methylomic characterization, such as copy number sequencing, RNA sequencing and reverse-phase protein assays. Together these models will aid in finding factors initiating DCIS and finding the key characteristics driving DCIS to IDC switch, and ultimately making it possible to distinguish between high risk and low risk DCIS. On top of this, the in vitro and in vivo human DCIS models will place a unique opportunity for high-throughput drug screening to obtain sensitivity profiles. These will be combined with -omics data to identify (epi)genetic determinants of drug sensitivity.

#5116

Development of murine tumor homograft panels and their genetic fingerprints for their identification to ensure the quality controlled I/O studies using these models.

Jie Cai,1 Wubin Qian,2 Bin Fan,1 Xiaobo Chen,1 Sheng Guo,2 Davy Ouyang,2 Wenqing Yang,2 Annie An,1 Jinglei Bi,1 Dongliang Mo,1 Henry Li1. 1 _Crown Bioscience, Inc., Beijing, China;_ 2 _Crown Bioscience, Inc., Taicang, China_.

Mouse tumors of homografts in the corresponding syngeneic mouse strains have become the key experimental system in immune-oncology (I/O) research and evaluating mouse surrogate I/O therapies. There are several types of mouse tumor homografts, including syngeneic cell line-derived, spontaneous or carcinogen-induced tumor-derived, or tumorigeneic GEMM (genetic engineered mouse model) -derived. We have created an increasingly larger panel of such diverse murine tumor homografts (now > 40 syngeneic cell line-derived and > 60 GEMM primary tumor-derived, or MuPrime®, usually driven by defined activated oncogenes frequently seen in human cancers) to support global I/O researches, including discovery of new drugs or drug targets, discovery/validation of biomarkers of both predictive and pharmacodynamic (PD) natures, as well as mechanism of actions (MOA). We are also systematically characterizing these heterogeneous library of murine tumors, in terms of genomic (Transcriptome-sequencing, or RNAseq), immunological (TILs and cytokine profiles), histopathological (lung, PDAC, breast, prostate, liver, CRC, bladder, kidney carcinoma, lymphoma, melanoma, sarcoma, leukemia, etc), growth and pharmacological (SOC, I/O) profiles. With the broad applications of these models at different laboratories and different times, it is prudent to track and QC the tumor to ensure their absolute correct identification (ID) by a readily routine assay. Unlike human-originated tumors (e.g. xenografts), either cancer cell line-derived or patient tumor-derived (PDX) whose genetic ID can readily be tracked by STR (short tandem repeat) genotyping or HLA tissue typing, murine tumors come from limited strains of inbred experimental mice, thus lack of sufficient genetic and/or tissue diversity to be used, in a readily performed assay, for individual murine model ID. To this end, we have established a novel efficient model quality control and tumor ID method in murine tumor model systems, in which we sought out to identify unique gene features in each individual murine tumor among a cohort of > 100. We have validated many such unique gene features in the original cell lines or primary tumors and/or their subsequent passages. Our results demonstrated that these unique gene features can served as genetic fingerprints for murine model ID.

#5117

A murine hematogenous bone metastatic cancer to model spontaneous metastasis of triple negative breast cancer.

Hye Jin Lee. _Korea University, Seoul, Republic of Korea_.

The development of bone metastasis in breast cancer and its pathologic progression are known to be clinically important problem. It is known that immunologic response in microenvironment is important as soil in the bone metastasis process and progression. Thus, immunologic modulation is expected to play a role in the prevention and treatment of the disease in bone metastasis. However, most studies to date; researches in bone metastasis were carried out using human breast cancer cell lines in xenograft model of immuno-deficient mouse or in vitro co-culture system. These approaches have limitations in regard to interaction among immune cells, cancer cells, and other stroma cells. To overcoming these limitation, we used the C3(1)Tag mouse model to establish a spontaneous bone metastasis model. First, a cell line (M6) obtained from a spontaneously occurring tumor in C3(1)Tag mice was infected with lentiviral-luciferase. M6 cells with lentiviral-luciferase were administered using intracardiac injection and C3(1) Tag mice were monitored until tumors are detected under bioluminescence imaging. In 3 weeks, metastatic bone lesions were apparent in 85.7% of mice. To understand biologic characteristics of in vivo selected bone metastatic subpopulation, metastatic cancer cells were harvested and were analyzed using immunohistochemical staing in comparison with parent cells. Tumor cells metastasized to bone showed differential expression in HIF1A, c-MET, and EMT markers. Unexpectedly, bone metastatic cancers showed better sensitivity to paclitaxel and cisplatin than parent cells in cell viability assay. In addition, immune microenvironment in bone metastatic foci was assessed using multiplex immunohistochemical staining and showed an immune suppressive MDSCs infiltration. Taken together, we established a murine hematogenous bone metastatic cancer and characterized biologic characteristics to model human triple negative breast cancer. The potential targets identified in this study might be a future immunologic target in patients with bone metastasis.

#5118

**Development of new alternative** in vivo **models to study early metastasic dissemination in breast cancer.**

Johanna Elena Sidabra, Carla Sabrina Capobianco, Maria Florencia Gottardo, Daniel Fernando Alonso, Hernan Gabriel Farina. _Quilmes National University, Buenos aires, Argentina_.

Breast cancer is the first cause of death from female cancer. After the treatments against the primary tumor, a few cells can prevail in a state of quiescence for a long period of time, becoming minimal residual disease. There is a clear need for alternative techniques to enable an early detection of the disease for a better understanding of it and the generation of therapies capable of acting with a smaller tumor burden. Although many models have been developed for the detection of macrometastasis, there is a limitation of in vivo models that allow the identification of disseminated cells, micrometastasis, or analyze another steps of the metastasic cascade, as embolization or cell arrest. The aim of this work is the generation of in vivo models to enable the detection of disseminated tumor cells (DTCs) We began our study by evaluating the presence of disseminated tumor cells in three in vivo models and quantification by FACS. To this end, we generated the F3II murine triple negative mammary carcinoma line expressing the fluorescent protein GFP (F3II GFP); the F3II line was used as control. In s.c heterotopic model, we observed the presence of positive cells in tumors and livers of mice inoculated with F3II GFP (Student's t test) 30 days post inoculation. In the s.c orthotopic model, we detected positive cells in tumors of mice inoculated with F3II GFP (Student's t test). In both models, we did not observe significant differences in the presence of lung DTCs. In experimental metastasis models, we detected DTCs 15 days post inoculation in lungs of mice injected i.v with F3II GFP (Mann Whitney test).We carried out the search for cells disseminated at 24 h in order to obtain an in vivo model that evaluates the establishment capacity of these cells, and we observed that the percentage of GFP positive cells was higher in those mice inoculated iv with F3II GFP (Student´s t test). In addition, the presence of DTCs was detected by fluorescence microscopy in blood smears of these mice, suggesting that at 24 h there are fluorescent tumor cells in both lung and blood. In conclusion, although the established models allowed us to detect DTCs, the experimental metastasis model was more efficient in the detection and quantification of disseminated tumor cells. These models allows the detection and quantification of DTCs in the lung and blood circulation within 24 hours and 15 days after inoculation and could be used to study the mechanisms of metastasic dissemination involved in these cells and to test the capacity of various anti-tumor drugs to the early stage of metastasis

#5119

**In vivo** **characterization of the Duffy Antigen Receptor for Chemokines (DARC) in the breast cancer microenvironment.**

Rachel Martini,1 Nancy Manley,1 Melissa Davis2. 1 _University of Georgia, Athens, GA;_ 2 _Henry Ford Health System, Detroit, MI_.

Chemokines are small inflammatory molecules that, in combination with their receptors, recruit lymphocytes to influence immune and inflammatory responses. This work specifically follows an atypical chemokine receptor, known as the Duffy Antigen Receptor for Chemokines (DARC). DARC is a non-signaling receptor able to bind both the CC and CXC chemokine classes, and mainly functions to regulate homeostatic levels of chemokines in circulation, and aid in chemokine transcytosis from sites of inflammation to mediate immune response. DARC serves as a portal of entry for the malaria-causing parasite Plasmodium vivax. In Sub-Saharan Africa, where malaria is endemic, a regulatory variant arose removing DARC expression from red blood cells (RBCs). This mutation, known as the Duffy-Null allele, was highly beneficial in this population, as removal of DARC from RBCs conferred immunity to Duffy-Null individuals. The Duffy-Null allele quickly rose to fixation in this population, and today recent decedents of Sub-Saharan Africans carry this allele. In the context of breast cancer (BCa) we are particularly interested in how Duffy-Null status impacts the tumor microenvironment. In clinic, we observe that pre-menopausal women of African descent have higher incidence rates of BCa, and that regardless of age, these women are disproportionately diagnosed with the most aggressive molecular subtype of BCa, triple-negative BCa. Knowing the important role that DARC plays in immune response, and that the Duffy-Null allele is prevalent in this population of women, we aim to investigate how Duffy-Null status impacts the tumor microenvironment in vivo through establishment of mouse models that depict Duffy-Null status in the BCa environment. To do this, we are using the C3(1)-TAg BCa transgenic mouse, alongside a DARC-KO mouse to characterize cancer progression and aggressiveness in BCa transgenic mice with varying global DARC expression. At 6 and 12 week time points, mice were sacrificed and the following information was obtained: total number of tumors, tumor location, tumor size and weight, and hematoxylin and eosin staining of tumor sections. Removal of DARC could lead to an excess of chemokines in the tumor microenvironment, promoting processes of tumor progression and metastasis, leading to the more aggressing tumor phenotypes we observe in women of African descent in clinic. 

### Metabolism and the Microbiome: Defining the Greater Microenvironment

#5120

Adipocyte-derived lipids mediate melanoma progression via FATP proteins.

Maomao Zhang. _Mem. Sloan Kettering Cancer Ctr., New York, NY_.

During melanoma progression, tumor cells come into direct contact with subcutaneous tissues and these advanced lesions portend a poor prognosis. Despite the fact that subcutaneous tissues are largely composed of adipocytes, little is known about the mechanisms by which adipocytes influence melanoma. Using both in vitro and in vivo models, we find that adipocytes increase proliferation and invasion of adjacent melanoma cells. This is accompanied by direct transfer of fatty acids from adipocytes to melanoma cells, which increases tumor cell metabolism and invasion programs. These adipocyte-derived lipids are transferred to melanoma cells through the FATP/SLC27A family of lipid transporters expressed on the tumor cell surface. Among the six related FATP/SLC27A family members, melanomas significantly overexpress FATP1/SLC27A1. Melanocyte-specific FATP1 expression cooperates with BRAFV600E in transgenic zebrafish to accelerate melanoma development, an effect that is similarly seen in mouse xenograft studies. These data demonstrate that adipocytes in the tumor microenvironment can drive melanoma progression through FATP lipid transporters, and represents a new target aimed at interrupting adipocyte-melanoma crosstalk.

#5121

Obesity and prostate cancer progression: Role of the chemokine CXCL12.

Songyeon Ahn,1 Achinto Saha,1 Mikhail G. Kolonin,2 John DiGiovanni1. 1 _University of Texas at Austin, Austin, TX;_ 2 _The University of Texas Health Sciences Center at Houston, Houston, TX_.

Prostate cancer (PCa) is the third leading cause of cancer in men in the United States. Many epidemiologic studies suggest that obesity increases PCa progression and mortality. However, the mechanistic relationship between PCa progression and obesity is very complex and not fully understood. In previous studies, we have shown that obesity enhances PCa growth and progression in the HiMyc mouse model of PCa by changing the surrounding tumor microenvironment, especially in white adipose tissue (WAT). WAT consists of many cells including mature adipocytes, adipose stromal cells (ASCs) and inflammatory cells, which secrete chemokines, cytokines and proinflammatory molecules. ASCs are an important component of the stromal vascular fraction (SVF) from WAT that secretes such factors. In this study, we evaluated the role of ASCs and ASC-derived CXCL12 in driving PCa progression in obesity. We observed upregulation of mRNA expression of CXCL12 in the SVF of the periprostatic WAT from obese HiMyc mice. In HMVP2 cells, a PCa cell line derived from HiMyc mice expressing high levels of CXCR4 and CXCR7, CXCL12 treatment increased activation of MAPKs (JNK and ERK), AKT, STAT3 and NFkB. In further studies, HMVP2 cells were treated with the CXCR4 antagonist, AMD3100, alone or together with CXCL12 and the CXCL12-induced signaling was abolished in AMD3100-treated cells. Similar results were observed in CXCR4 knockdown cells and CXCR7 knockdown cells. In addition, knockdown of CXCR4 and CXCR7 significantly decreased migration and invasion of the HMVP2 cells. Lastly, a tumor study was performed by injecting HMVP2 cells into the flank of FVB/N mice, which were fed either a control diet or obese diet. AMD3100 significantly inhibited the growth of HMVP2 tumors in both obese and control diet groups. AMD3100 also inhibited proliferation and epithelial-mesenchymal transition (EMT) in both obese and control diet groups. In conclusion, this study provides evidence that the CXCL12/CXCR4/CXCR7 signaling pathway is important for obesity-driven PCa progression in HiMyc mice. Furthermore, this pathway may represent a novel therapeutic target for inhibiting obesity-driven PCa growth and progression.

#5122

Obesity promotes tumor growth, reduces breast tumor latency and correlates with neovascularization.

Roopali Roy*,1 Jiang Yang*,1 Takaya Shimura,1 Lauren Merritt,2 Adelle Dagher,2 Lewis Chodosh,3 Marsha A. Moses1. 1 _Boston Children's Hospital, Harvard Medical School, Boston, MA;_ 2 _Boston Children's Hospital, Boston, MA;_ 3 _Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA_.

Obesity is associated with an increased risk and a poor prognosis for both ER+ and ER- breast cancers (BC). To date, however, few studies have focused on the contribution of obesity to BC. In addition, there is a paucity of in vivo models that reliably recapitulate postmenopausal obesity related to BC development and progression. The goal of this study was to determine whether interactions between adipocytes and breast tumor cells promote BC growth and progression and to identify the underlying mechanism(s) responsible. We found that MDA-MB-436 and MCF-7 BC cells, when treated with the conditioned media of adipocytes from obese individuals (Ad-CM), upregulated a number of angiogenic factors including VEGF-A, Ang-1 and bFGF and suppressed the angiogenic inhibitor Tsp-1. Ad-CM stimulated migration and invasion of MDA-MB-436 and MCF-7 (human) and E-Wnt and M-Wnt (mouse) BC cells. Interestingly, the cellular proliferation rate in response to Ad-CM was stimulated only in the ER+ MCF-7 and E-Wnt cells but not in the ER- BC cells, suggesting that Ad-CM may affect proliferation via an ER-dependent mechanism. After pretreatment with Ad-CM, MDA-MB-436, MCF-7 or M-Wnt BC cells exhibited significantly enhanced EC recruitment as well.

Utilizing two different breast tumor models, an orthotopic model (ER-: luciferase-labeled MDA-436 cells injected into the mammary fat pad of SCID mice) and a transgenic model (ER+: doxycycline-driven conditional MMTV/TWNT/luciferase expression), we investigated whether postmenopausal obesity reduces breast tumor latency, promotes angiogenesis and accelerates tumor growth. To model the effect of obesity on postmenopausal BC, mice were ovariectomized (OVX) before inducing obesity via a high-fat diet (HFD) regimen. In the ER- BC model, obese mice had significantly higher tumor frequency, higher tumor volume, and significantly lower median survival. In the ER+ tumor model, obese mice displayed significantly higher tumor frequency, a significantly shorter tumor latency period and a significantly lower median survival time. Tumors in OVX/HFD animals had significantly higher microvessel density (MVD) and an increased number of SMA+ mature vessels. A majority of tumors in OVX/HFD mice displayed aggressive local invasion into the surrounding fat pad and muscle. Proangiogenic factors such as IL-6 (Interleukin-6) and Lcn2 (lipocalin 2) were ~2-fold higher in the sera of obese tumor-bearing mice as compared to controls. Our data suggest that postmenopausal obesity reduces breast tumor latency and promotes aggressive tumor growth via increased angiogenesis. Ongoing studies in our laboratory are investigating the mechanism(s) by which an obesogenic microenvironment mediates these effects.

*Authors contributed equally.

This work was supported by NIH RO1CA185530.

#5123

Changes of oral microbiome profiles associated with oral squamous cell carcinoma.

Kengo Hashimoto,1 Shuji Nomoto,1 Sho Hirabayashi,1 Sei Ueda,1 Ichiro Oh-iwa,2 Toru Nagao,1 Kazuo Shimozato1. 1 _Aichi-Gakuin University School of Dentistry, Nagoya, Aichi, Japan;_ 2 _Japanese Red Cross, Nagoya Daiichi Hospital, Nagoya, Aichi, Japan_.

[Background] Oral squamous cell carcinoma (OSCC) is the most frequently occurring cancer among the head and neck SCCs worldwide. Despite significant efforts in OSCC research and advanced surgical treatments, radiotherapy, and chemotherapy, the 5-year overall survival rate has remained < 50% for the last few decades. OSCC prognosis remains poor due to late diagnosis. Therefore, clinically effective biomarkers for the early detection of OSCC can greatly improve the survival rate and prognosis. Approximately 15% of oral cancers cannot be explained by major risk factors, such as tobacco and alcohol use. A growing body of evidence supports an association between specific microorganisms, including those in the oral cavity, and various types of cancers. Because the role of bacteria in OSCC development has not been completely described, we investigated the changes in abundance of oral microbiota associated with OSCC. [AIMS] To identify the association between the oral microbiome and OSCC for the early detection and prevention. [Materials and methods] Unstimulated saliva samples were collected from 4 OSCC-free, 6 leukoplakia, and 6 OSCC subjects. Total bacterial genomic DNA from saliva was isolated, and 16S rRNA gene survey was performed by next-generation sequencing of the V4 region. The sequences were analyzed against reference sequences from the Human Oral Microbiome Database, and the relative abundance distribution for phylogenetic groups in 3 different libraries was compared. [Results] In total, 448 bacterial species from combined (OSCC-free, leukoplakia, and OSCC) libraries were detected, which were classified into 133 genera, 69 families, 41 orders, 26 classes, and 12 phyla. The frequency of phylum Bacteroidetes was more notably abundant in OSCC subjects compared with leukoplakia and OSCC-free subjects. In contrast, the abundances of genera Streptococcus and Rothia were significantly lower in OSCC subjects compared with leukoplakia and OSCC-free subjects using the Mann-Whitney U test. [Conclusion] This study revealed some OSCC-associated shifts in oral microbial communities. Salivary screening is suitable for the primary screening procedure because of its noninvasiveness and easy accessibility. Therefore, these changes in the saliva microbiome may have potential application as a useful diagnostic tool for the early detection of OSCC and malignant transformation of pre-cancerous regions.

#5125

**The relationship between microbiome** Fusobacterium nucleatum **and autophagy in esophageal cancer.**

Yang Liu,1 Yoshifumi Baba,2 Shigeki Nakagawa,2 Hiroshi Sawayama,2 Takatsugu Ishimoto,2 Masaaki Iwatsuki,2 Yoichi Yamashita,2 Yuji Miyamoto,2 Naoya Yoshida,2 Hideo Baba2. 1 _Kumamoto University, Kumamoto, Japan;_ 2 _Kumamoto University, Kumotomo, Japan_.

Background: Research into the microbiome is a rapidly advancing field in human cancers. The gut microbiome has recently been shown to play an important role in health, as well as in diseases such as obesity, inflammatory bowel disease, diabetes, non-alcoholic fatty liver disease, and several types of cancers. Fusobacterium nucleatum is a component of the human microbiome that primarily inhabits the oral cavity. It causes periodontal disease and has also been implicated in the development of human cancers. We have recently reported that Fusobacterium nucleatum in esophageal cancer tissues was associated with shorter survival, suggesting a potential role as a prognostic biomarker. In addition, It has been revealed that Fusobacterium nucleatum promotes chemoresistance to colorectal cancer by modulating autophagy. Nonetheless, the relationship between Fusobacterium nucleatum and autophagy in esophageal cancers have been still unknown. We constructed this study to examine the relationship between Fusobacterium nucleatum and autophagy.

Methods: Using 284 esophageal cancers, we detected ATG-7 and ULK -1 expression by IHC staining in tumors.

Results: 21% cases showed ATG-7 positive and 25% cases showed ULK-1 positive. ATG-7 and ULK-1 expression in Fusobacterium nucleatum-positive tumors were significantly higher compared with that of Fusobacterium nucleatum-negative tumors (P=0.026 and P=0.0077). In addition, ATG-7 expression was associated with cancer-specific survival rate in esophageal cancer patients (P=0.0496). There was no relationship of ULK-1 expression and prognosis.

Conclusion: Fusobacterium nucleatum was related with ATG-7 and ULK-1. Our findings may shed lights on understanding the mechanism by which Fusobacterium nucleatum affects chemoresistance in esophageal cancer.

#5126

Modulation of lactate in the tumor microenvironment with lactate transporter inhibitor in a melanoma syngeneic mouse model.

Satish Kumar Reddy Noonepalle,1 Jennifer Kim,1 Melissa Hadley,1 Erica Palmer,1 Debarati Banik,1 Tessa Knox,1 Sophiya Ephrame,1 Prathima Vembu,1 Summer Rudish,1 Sara Moufarrij,1 Jaime Escobedo,2 Vincent Sandanayaka,2 Alejandro Villagra1. 1 _George Washington University, Washington DC, DC;_ 2 _Nirogyone Therapeutics, Natick, MA_.

Solid tumors often have altered metabolism that depend on aerobic glycolysis for energy and macromolecules for cell survival thereby generating lactate as a metabolic byproduct. Lactate is transported across the cell membrane into tumor microenvironment (TME) using monocarboxylate transporters, MCT1 and MCT4. MCTs facilitate proton linked transport of monocarboxylate molecules such as lactate, pyruvate, and ketone bodies across the plasma membrane. Lactate released by glycolytic cells into the TME results in a metabolic symbiosis when other tumor cells utilize lactate as an energy source that undergo oxidative metabolism which is referred to as "lactate shuttle". Moreover, lactate induces inhibitory pathways on immune cells, especially on cytotoxic CD8+ T-cells resulting in local immunosuppression in the TME. Additionally, acidosis of TME through lactate results in local inflammation and angiogenesis by activation of VEGFR signaling. The net result of an increased lactate in TME is that it generates a conducive milieu for tumor growth and metastasis. Although studies show that MCT inhibitors mitigate the effects of lactate and promote immune function, its effect on immune cells in the context of tumor infiltration is yet to be explored. In this study, we used a MCT inhibitor NGY-A to determine the effect of suppressing lactate levels in the TME and restoring the anti-tumor immunity. In our preliminary studies to determine the cytotoxicity, NGY-A showed significant cancer cell killing in multiple cell lines in vitro. To determine the in vivo effect of NGY-A, syngeneic mouse model of SM1 melanoma cells harboring BRAF mutation in C57BL/6 mice was studied. When treated with NGY-A (10 mg/kg, i.p injection) (n=15), a significant decrease in tumor volumes was observed compared to controls (n=15). Gene expression analysis of total RNA isolated from SM1 tumors by quantitative RT-PCR indicated that expression of immunosuppressive molecules such as Cd274 (PD-L1), Cd276 (B7-H3) and Lgals9 (Galactin-9) was down regulated in NGY-A (n=5) treated tumors compared to the control tumors (n=5). An increase in IFN-γ (IFNG) gene expression in NGY-A treated samples indicated an increase in CD8+ T-cell tumor infiltration. Future studies would include analysis of phenotype of tumor infiltrating immune cells and cytokine profiles after treatment with NGY-A in combination with immune checkpoint blockade inhibitors which could potentially be an effective strategy to treat melanoma.

#5127

Eradication of the gut microbiota reduces cancer burden in multiple models by modulating the immune system.

Vrishketan Sethi, Saba Kurtom, Mohd Tarique, Bhuwan Giri, Bharti Garg, Shweta Lavania, Anthony Ferrantella, Zoe Malchiodi, Leonor Hellmund, Harrys Charles Jacob, Rajinder Dawra, Sulagna Banerjee, Sabita Roy, Sundaram Ramakrishnan, Ashok Saluja, Vikas Dudeja. _University of Miami, Miami, FL_.

Introduction: Bacteria residing in the human body outnumber 'human' cells and most of these bacteria exist in the gut. This gut microbiome forms a unique and dynamic metagenome which changes with nutrition and even disease state. A growing body of evidence has implicated the role of the gut microbiome in modulating our immune system. We aimed to investigate this association in murine models of cancer. Methods: C57BL/6 mice were either orally given saline or a gut sterilizing cocktail of poorly absorbable broad-spectrum antibiotics. These mice were used to model multiple cancers, viz. subcutaneous injection of melanoma cells derived from Tyr::CreER; Braf V600E/+;Ptenlox5/lox5 mice, subcutaneous injection of pancreatic cancer cells derived from KPC (Kras LSL.G12D/+; p53 R172H/+ ;Pdx::Cre) mice, intrasplenic injection of KPC pancreatic cancer cells and intrasplenic injection of B16-F10 melanoma cells. The tumors were immunophenotyped through flow cytometry and immunostained for multiple antigens. Mice plasma was used for chemokine analysis. To confirm the role of immunity, subcutaneous experiments were repeated in mice carrying a Rag1tm1Mom mutation (RAG1 knockout), thereby having an immature adaptive immune system. Results: Gut microbiome depletion reduced tumor burden across all four subcutaneous and hepatic metastases models. This effect disappeared when experiments were repeated in immunosuppressed RAG1 knockout mice. Antibiotic-treated tumors had increased TNF-secreting macrophages and NKT cells as well a higher number of iNOS staining cells in the microenvironment. Plasma from antibiotic-treated tumor-bearing mice had decreased levels of CXCL12, a chemokine known to cause tumor immunosuppression. Conclusion: Eradication of the gut microbiota decreases tumor burden by activating the anti-cancer immune system

#5128

Understanding the association of gut microbiota and tumor microenvironment in gastric and esophageal cancer.

Prashant V. Thakkar,1 Chao Zhang,1 Prateek Sharma,2 Sreekar Vennelaganti,2 Doron Betel,1 Manish A. Shah1. 1 _Weill Cornell Medicine, New York, NY;_ 2 _Veterans Affairs Medical Center and University of Kansas School of Medicine, New York, NY_.

Gastric and Esophageal cancers remain one of the leading causes of cancer related deaths. Dysbiosis of gut microbiome is a strong risk factor for gastrointestinal cancer, likely due to altered mucosal immunity and pro-inflammatory immune microenvironment. However, molecular interactions between host human cells and microbiome are poorly understood in context of gastric and esophageal cancer. Our main goal is to understand how altered mucosal microbiome alters tumor microenvironment, thus creating conditions conducive to tumorigenesis. Using a novel computational pipeline combined with low-pass (10-30x) whole genome sequencing (WGS), we examined microbiome composition of 15 gastric cancer (GC) and adjacent normal tissue (n=30). We found a higher overall bacterial content in tumor samples compared to that of matching normal tissues. Furthermore, we observed dysbiosis in H. pylori positive samples, consistent with previous findings. Among other microbes, we found specific enrichment of V. Parvula (12/15) and P. melaninogenica (10/15) in tumor tissues, both of which have been implicated in tumorigenicity. A subset of these samples (10 GC and 12 normal) underwent parallel RNAseq analysis. Using gene expression signature panel of 176 genes, we examined association of various immune cell types with tumor versus normal mucosa. We found an overall higher immune response, and higher association of Th1 and Th2 helper cells and macrophages, with tumors as compared to normal mucosa. We further corroborated these findings using single cell RNAseq approach. Using ELISA, we detected significantly increased expression of pro-inflammatory cytokines such as TNF-α, IL-8, GRO, MCP-1 and IL-1a in tumor samples than normal mucosa. Increased TNF-α and IL-8 expression has been directly correlated to tumor invasion and metastasis, and tumor vascularity respectively. Additionally, MCP-1 is known to recruit macrophages and is secreted by gastric epithelial cells in response to pro-inflammatory cytokines upon H. pylori infection. Using a similar approach, we performed WGS and RNAseq, on 9 normal squamous cell carcinoma (NSCC), 8 non-dysplastic Barrett's esophagus (BE) and 6 esophageal adenocarcinoma samples. WGS analysis revealed higher microbial diversity in NSCCs compared to BE samples with high abundance of V. Parvula, P. melaninogenica and S. parasanguinis. Unsupervised clustering analysis revealed high abundance of F. nucleatum in adenocarcinoma, which has been associated with shorter survival in esophageal cancer. Unsupervised clustering analysis of RNAseq data revealed high enrichment of CD4+ Th1 and Th2 helper cell and CD8+ T cell associated immune response in some adenocarcinoma and BE samples. Together our data suggest that both gastric and esophageal cancers display distinct microbial patterns associated with chronic inflammation and tumor-promoting pro-inflammatory microenvironment.

#5129

Fusobacterium **and co-occurring microbes in primary and metastatic colorectal cancer.**

Susan Bullman,1 Chandra S. Pedamallu,1 Ewa Sicinska,1 Thomas Clancy,2 Shuji Ogino,1 Josep Tabernero,3 Charles Fuchs,4 William C. Hahn,1 Paolo Nuciforo,3 Matthew Meyerson1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Brigham and Women's Hospital, Boston, MA;_ 3 _Vall d'Hebron Institute of Oncology (VHIO), Barcelona, Spain;_ 4 _Yale Cancer Center, New Haven, CT_.

In colorectal cancer, malignant cells are surrounded by a complex microenvironment encompassing a range of non-transformed cells, but also a diverse collection of microorganisms. A growing body of evidence demonstrates the role of particular microorganisms in modulating inflammatory environments and promoting tumor growth and metastasis. Studies by our group, and others, reveal a consistent enrichment of Fusobacterium nucleatum in human colorectal cancer, and F. nucleatum has been shown to accelerate tumorigenesis using both in vitro and in vivo preclinical models.

We recently demonstrated via microbiome analysis and microbial culture that fusobacteria and its co-occurring microbiota, including Bacteroides, Prevotella and Selenomonas species, persist in liver metastasis of Fusobacterium-positive colorectal cancers. Many of the liver metastasis share the same dominant microbiome (>1% relative abundance) as the paired primary colorectal tumor. Additionally, we have cultured fusobacteria from paired primary and metastatic tumors, and following whole genome sequencing analysis reveal the same strains of Fusobacterium are present in the primary tumors and distant site metastasis, despite the tissue being resected months or even years apart. In situ hybridization analysis demonstrate that Fusobacterium is invasive in the primary tumors and distal metastasis, and is associated with cells whose morphology is consistent with malignant cells.

Additionally, we demonstrate via microbiome analysis and microbial culture, that Fusobacterium and its co-occurring microbiome also persist and remain viable in patient derived xenografts of colorectal cancers. Treatment of a patent derived colon cancer xenograft harboring F. nucleatum, with an antibiotic that kills F. nucleatum reduced tumor growth, cancer cell proliferation and tumor fusobacterial load.

We have isolated and sequenced the genomes of over 60 F. nucleatum strains from human colorectal cancer tumors with detailed microbiome and patient metadata. In addition to phenotypic analysis and small molecule inhibitory screens of the F. nucleatum colorectal cancer isolates, we are conducting comparative genomic analysis with F. nucleatum isolates from the oral cavity of non-cancer patients to determine colorectal cancer-specific markers and identify targetable genomic attributes.

In summary, these findings suggest that the tumor microbiota are intrinsic and essential components of the cancer microenvironment and warrant further investigation into the modulation of the tumor microbiota for the treatment of Fusobacterium-associated colorectal cancer in both early and late stage disease.

#5130

Melanoma and breast adenocarcinoma growth inhibition by a 7-ketocholesterol loaded-phosphatidylserine liposome.

Giovani M. Favero,1 Tharcisio C. Tortelli,2 Andreia H. Otake,2 Luciana N. Andrade,2 Daniele P. Faria,3 Camila G. Carneiro,3 Alexandre T. Garcez,3 Fabio L. Marques,3 Roger Chammas2. 1 _State University of Ponta Grossa, Ponta Grossa, Brazil;_ 2 _Centro de Investigação Translacional em Oncologia (LIM24), São Paulo, Brazil;_ 3 _Faculdade de Medicina da USP, São Paulo, Brazil_.

Background: Phosphatidylserine (PS) exposure is one of the first steps of programmed cell death. Phagocytosis on cancer microenvironment is described in tumors and is associated with malignancy and poor prognosis. Tumor Associated Macrophages (TAMs) act suppressing the anticancer immune response. Recently we observed that 7-Ketocholesterol (7KC) induces autophagy on melanoma cell. Aims: Evaluate a 7-ketocholesterol loaded-phosphatidylserine liposome on autophagy and phagocytosis of tumor microenvironment. Methods: Liposomes were constituted by 20 mg PS or PS associated with 5 mg of 7KC extracted, dried, resuspended in phosphate buffer and sonicated. The size and Zeta Pontencial (ZP) were evaluated. A dependent-dose effect of liposomes on J774 macrophages, B16F10 melanoma cells and 4T1 breast cancer cells were assessed by MTT. Cell death evaluations, for the same cells, were performed by flow cytometry. The presence of acid vacuoles related to autophagy was evaluated by acridine orange staining. The effects of the liposomes in vivo were evaluated by B16F10 melanoma bearing C57/bl6 mice and 4T1 breast cancer bearing mice. Endocytosis efficiency of the liposomes was PKH26 fluorescent staining. Liposomes were radiolabeled by adding 1 to 30 mCi of 99mTc radiopharmaceuticals (99mTcO4-, 99mTc-dextran-70, 99mTc-MIBI, 99mTc-DISIDA) and 18FDG. Results: PS liposomes presented 141,9nm + 9,101 size with a -25,2 ZP; PS-7-Ketocholesterol (PS/7KC) liposomes presented 153,9 nm + 10,35 size with a -29,1 ZP.PS/7KC showed intense antiproliferative activity in melanoma cells and breast adenocarcinoma cells. There was 10% more autophagic vacuoles on melanoma cells treated with PS/7KC than the control groups. Both in vivo tumor models had the same antiproliferative effect of the PS/7KC liposomes with daily doses. Daily doses of PS liposomes induced a high size of tumors. 99mTc-MIBI was efficiently and strongly incorporated to liposomes then the other proposed formulations. Conclusion: PS liposomes have effects in vivo and in vitro related to phagocyte and autophagy activities. PS/7KC impairs J774 macrophage, B16F10 melanoma and 4T1 breast adenocarcinoma cells growth. PS/7KC induce the presence of acid vacuoles corresponding to autophagy. The liposomes had a high endocytosis evaluated by PKH 26 labelled particles. PS keep the tumor proliferation and PS/7KC inhibit tumor growth after ten days of daily doses. Supported by CNPq and Fundação Araucária

#5131

Obese conditions induce changes in stromal fibroblast phenotype.

Brittany Harlow,1 Albert Davalos,2 Andrew Brenner,3 Christopher Jolly,1 Stephen Hursting,4 Linda deGraffenried1. 1 _University of Texas, Austin, TX;_ 2 _Buck Institute for Research on Aging, Novato, CA;_ 3 _University of Texas Health Science Center, San Antonio, TX;_ 4 _University of North Carolina, Chapel Hill, NC_.

Background: Over 40% of American women present a BMI ≥30, which is particularly alarming since obesity confers a worse prognosis for both pre- and postmenopausal breast cancer patients. Although the molecular mechanisms behind this remain unclear, obese conditions have been shown to upregulate certain components of the cancer-associated fibroblast (CAF) secretome and senescence associated secretory phenotype (SASP), both directly correlated with tumorigenesis. However, studies have yet to determine whether obesity induces a cohesive SASP or CAF-like phenotype from any one cell type of the breast tumor microenvironment. Because fibroblasts represent the most common breast tumor cell type, it is of particular importance to investigate obesity-induced changes in this cellular compartment and their effects on cancer cell behavior. Methods: Direct effects of obesity were examined by exposing IMR-90 and HCA2 human fibroblasts to media supplemented with 2% sera from obese women and measuring changes in gene and protein expression, while indirect effects were evaluated by exposing the fibroblasts to conditioned media (CM) from obesity-stimulated MCF-7 and T47D cells and assessing changes in expression of proinflammatory cytokines. Breast cancer cells were also exposed to CM from obesity-stimulated fibroblasts and measured for changes in proliferation, survival, motility, and invasion. Results and Conclusions: Obese conditions induced proinflammatory changes in fibroblasts both directly and indirectly and stimulated changes in the paracrine signaling between fibroblasts and breast cancer cells. These data contribute to the growing evidence implicating obesity in the promotion of breast cancer progression. Additionally, these findings provide a new line of research to develop potential therapeutic targets to improve outcome.

#5132

Lactic acidosis-mediated transcriptomic and metabolomic rewiring in pancreatic cancer cells.

Divya Murthy, Jaime Abrego, Ryan James King, Pankaj Kumar Singh. _UNMC, Omaha, NE_.

Tumor microenvironment plays a critical role in regulating tumor cell metabolism and progression. The tumor cells are constantly exposed to hypoxia, nutrient starvation, and extracellular acidification that contribute to tumor malignancy. These malignant cells exhibit Warburg effect by maintaining glycolytic phenotype even in the presence of oxygen and excrete lactate and protons via monocarboxylate transporters, and sodium-hydrogen antiporter 1 (NHE1) causing constant acidification of the tumor microenvironment. The tumor cells must thus adapt to acidosis to thrive in such hostile microenvironment. The purpose of the study is to identify the functional response of pancreatic cancer cells to the acidic tumor microenvironment. We performed integrated transcriptomic and metabolomic analyses to identify the existence of low pH-responsive transcriptional and metabolic regulators. Culturing the cells under low pH resulted in a reduction in cell growth and an increase in oxidative stress within the cells. RNA-seq-based transcriptomic analyses revealed that acidosis drives distinct gene expression patterns in PDAC cells with the expression of 499 and 496 genes up- and downregulated, respectively in PDAC cells. GSEA analyses of significantly modulated genes identified pathways including cell cycle, DNA repair, metabolic pathways, ABC transporters, glutathione metabolism, and genes involved in signaling cascades such as the inositide signaling pathway. As the transcriptomic analyses showed an enrichment of metabolic genes, intracellular metabolomic analyses revealed that chronic acidosis leads to a shift in cellular metabolism towards oxidative metabolism. Our studies demonstrate that while chronic acidosis in the tumor microenvironment leads to reduced proliferation and glycolysis in tumor cells, it also results in increased expression of genes involved in cellular migration, signaling pathways and switching to anaplerotic glutamine metabolism in response to an acidic tumor microenvironment. Overall, our studies demonstrate metabolic reprogramming in PDAC cells in response to low pH microenvironments creating a metabolic vulnerability that could be exploited for therapeutic targeting of pancreatic cancer.

#5133

Lipids and suppressive functions of MDSC in cancer.

Filippo Veglia,1 Vladimir A. Tyurin,2 Valerian E. Kagan,3 Dmitry Gabrilovich1. 1 _Wistar Institute, Philadelphia, PA;_ 2 _University of Pittsburgh, PA;_ 3 _University of Pittsburgh, Pittsburgh, PA_.

Myeloid derived suppressor cells (MDSC) play an important role in the regulation of anti-tumor responses and their presence in cancer patients correlates with a poor prognosis. MDSC contribute to the failure of cancer therapies and favor tumor progression and metastasis. However, despite the recent advances in understanding the biology of MDSC, the mechanisms driving their expansion and suppressive function are not yet well defined. Polymorphonuclear, PMN-MDSC is by far the largest population of MDSC. Here, we found that PMN-MDSC from tumor bearing mice and from cancer patients have higher amount of lipids than their counterpart (neutrophils) in tumor free mice and healthy donors, respectively. The content of lipids in PMN-MDSC was much higher in MDSC infiltrating the tumor, and the accumulation of lipids in PMN-MDSC was caused by the uptake of extracellular lipids. Lipid profile of PMN-MDSC demonstrated accumulation of oxidized fatty acids as part of triglycerides and phospholipids. We demonstrated that fatty acids accumulated by PMN-MDSC are oxidized by myeloperoxidase (MPO) and reactive oxygen species (ROS) generated by Nox2. Genetic ablation of MPO or ROS in MDSC results in a drastic reduction of lipid oxidation and in an impaired ability of MDSC to suppress CD8 T cell responses. In line with these findings, unsaturated fatty acids, highly susceptible to oxidation, favor the expansion of highly suppressive MDSC from hematopoietic progenitors in vitro, contrary to non-oxidizable saturated fatty acids. Together, our data suggest that MDSC have increased ability to pick up lipids from the microenvironment and to oxidize them via their potent oxidative machinery. In turn oxygenated lipids contribute to the suppressive activity of MDSC. In conclusion, the regulation of the oxidation of lipids in MDSC could be an attractive therapeutic strategy in cancer.

#5134

Desacetylvinblastine monohydrazide disrupts tumor vessels by promoting VE-cadherin internalization.

Minfeng Chen, Xueping Lei, Wencai Ye, Dongmei Zhang. _Jinan University, Guangzhou, China_.

Vinca alkaloids, the well-known tubulin-binding agents, are widely used for the clinical treatment of malignant tumors. However, little attention has been paid to their vascular disrupting effects, and the underlying mechanisms remain largely unknown. This study aims to investigate the vascular disrupting effect and the underlying mechanisms of vinca alkaloids.

Methods: The capillary disruption assay and aortic ring assay were performed to evaluate the in vitro vascular disrupting effect of desacetylvinblastine monohydrazide (DAVLBH), a derivate of vinblastine, and the in vivo vascular disrupting effect was assessed on HepG2 xenograft model using magnetic resonance imaging, hematoxylin and eosin staining and immunohistochemistry. Tubulin polymerization, endothelial cell monolayer permeability, western blotting and immunofluorescence assays were performed to explore the underlying mechanisms of DAVLBH-mediated tumor vascular disruption.

Results: DAVLBH has potent vascular disrupting activity both in vitro and in vivo. DAVLBH disrupts tumor vessels in a different manner than classical tubulin-targeting VDAs; it inhibits microtubule polymerization, promotes the internalization of vascular endothelial cadherin (VE-cadherin) and inhibits the recycling of internalized VE-cadherin to the cell membrane, thus increasing endothelial cell permeability and ultimately resulting in vascular disruption. DAVLBH-mediated promotion of VE-cadherin internalization and inhibition of internalized VE-cadherin recycling back to the cell membrane are partly dependent on inhibition of microtubule polymerization, and Src activation is involved in DAVLBH-induced VE-cadherin internalization.

Conclusions: This study sheds light on the tumor vascular disrupting effect and

underlying mechanisms of vinca alkaloids and provides new insight into the molecular mechanism of tubulin-targeting VDAs.

Key words: Vascular disrupting agents; vinca alkaloids; desacetylvinblastine monohydrazide; tubulin polymerization; VE-cadherin internalization

#5135

Retrovirus-transduction promotes cell-to-cell transfer of genetic material in cancer microenvironment mediated by extracellular membrane vesicles.

Berna Uygur, Leonid Chernomordik. _NIH, Rockville, MD_.

Naturally occurring cell-derived extracellular membrane vesicles (EMV) such as exosomes and microvesicles have a significant impact on horizontal gene delivery and communication of cells. According to the Trojan exosome hypothesis, retroviral particles and EMV generated by both infected and not infected cells could have similar characteristics including protein targeting, vesicle biogenesis pathway and moving between cells. In this study we explored the effects of retrovirus-infection on EMV-mediated gene delivery in cancer microenvironment. We focused on genetic material transfer from cancer cells (PC3 cells or U2OS cells) transduced with retroviral vector to express GFP to different receiving cells transduced with lentiviral vector to express mCherry. We observed horizontal GFP transfer (i.e., expression of GFP in mCherry expressing cells) in following conditions (i) co-culturing retrovirus GFP-expressing PC3 cells with lentivirus mCherry expressing PC3 cells, (ii) incubation of mCherry-PC3 cells with conditioned medium from retrovirus GFP-expressing PC3 cells, or (iii) incubation of PC3 cells with EMVs isolated from the medium collected from retrovirus GFP-expressing PC3 cells. EMVs generated by retrovirus-transfected cancer cells carried out horizontal GFP transfer not only to other cancer cells but also to human umbilical vein endothelial cells and human embryonic kidney cells (293T). Interestingly, in contrast to EMVs collected from the retrovirus transfected cells, EMVs collected from lentivirus-transfected cells did not carry out horizontal gene transfer. Therefore, we compared EMVs from retrovirus GFP-expressing cells and lentivirus GFP-expressing PC3 cells by using Western blot and Q-PCR. Exosomes generated by retrovirus-GFP expressing PC3 cells expressed higher human endogenous retroviral proteins syncytin 1 and 2 and contain RNAs encoding these proteins than EMVs from the lentivirus GFP expressing PC3 cells. Blocking either syncytin 1 or syncytin 1 receptor ASCT2 at either EMVs or the receiving cells suppressed the transfer of GFP RNA from EMVs to the receiving cells. These findings suggest that retroviral transduction by yet unclear mechanisms promotes generation of syncytin 1-carrying EMV, which leads genetic material transfer through Syncytin 1 mediated EMV-cell fusion.

#5136

**Development of an RNA** **hybridization technique for the** in situ **visualization of bacteria in cancer tissues.**

Eva Shrestha, Karen S. Sfanos. _Johns Hopkins University School of Medicine, Baltimore, MD_.

There is currently a great interest in understanding direct interactions between human-associated microbial populations and the tumor microenvironment. In this study, we aimed to develop a chromogenic in situ hybridization (CISH) assay for universal detection of bacteria in formalin fixed paraffin embedded (FFPE) cancer tissues. This was accomplished using the RNAscope assay (Advanced Cell Diagnostics) with a CISH probe set designed to target the most highly conserved region in the bacterial 16S ribosomal RNA (rRNA) gene. Optimization of the 16S rRNA CISH assay was conducted using FFPE mouse colon, Mycobacterium tuberculosis infected rabbit lung, and Propionibacterium acnes infected mouse prostate tissues. Positive control (peptidylprolyl isomerase B- PPIB) and negative control (maize –Zm) stains were also conducted on the tissues. We additionally developed immunohistochemistry (IHC) assays with antibodies against lipopolysaccharide (LPS) and lipoteichoic acid (LTA) to visualize gram negative and gram positive bacteria, respectively, as a means to verify the results of the CISH assay. As our laboratory has a specific interest in the interactions between prostate infections and prostate cancer, we used this technique to interrogate a series of 10 radical prostatectomy specimens that contained a very high degree of acute and chronic inflammation, and were suspicious for the presence of an infectious organism. Both CISH and IHC were conducted in FFPE radical prostatectomy samples.

The 16S rRNA CISH assay detected bacteria in all of the control tissues. Interestingly, 3 of the 10 human radical prostatectomy samples that we examined showed bacterial 16S rRNA signal concentrated in areas with acute inflammation where the glandular lumens were filled with neutrophils. Two of the radical prostatectomy samples that showed positive bacterial 16S rRNA CISH signal also showed positive LPS staining indicative of the presence of gram negative bacteria. The remaining positive sample was suspicious for a gram positive intracellular organism. This study shows preliminary data supporting robust detection of bacteria in a subset of human prostate samples. We expect the frequency of bacterial infections of this nature present in radical prostatectomy specimens would be low, and that we were only able to identify these cases because we screened for samples with very high levels of inflammation. Detection of both bacterial RNA and protein in the tissues corroborates the strength and reliability of the 16S rRNA CISH. The use of these two methods concurrently can provide valuable information on bacterial presence in a range of clinical samples. In the future, we plan to use these methods to probe for bacterial presence and in understanding the spatial dynamics of the urinary microbiome. We expect that this method may also be of broader use in the study of many infection-associated cancers.

#5137

Tryptophan catabolism in ovarian cancer.

Lynelle P. Smith, Lucas M. Harrell, Jessica L. Christenson, Benjamin Bitler, Jill Slansky, Jennifer K. Richer. _University of Colorado - Anschutz Medical Campus, Denver, CO_.

Serous epithelial ovarian cancer metastasizes by direct seeding to form disseminated tumors within the peritoneal cavity and has an extremely poor clinical outcome with a 46.5% five-year survival rate. Resistance to anoikis (detachment-induced cell death) is a critical factor in the progression of this disease. We previously demonstrated that triple-negative breast cancer (TNBC) upregulates tryptophan-2,3-dioxygenase (TDO2), a rate limiting enzyme in tryptophan catabolism, under anchorage-independent conditions or by stimulation with an NFκB stimulating cocktail (IL1-β, TNFα). Indoleamine-pyrrole 2,3-dioxygenase (IDO1) is also a rate-limiting enzyme in the tryptophan catabolism pathway, & both TDO2 and ID01 can be expressed by tumors. Kynurenine, a metabolite of tryptophan catabolism acts in an autocrine fashion through the aryl hydrocarbon receptor to provide anti-apoptotic signals that enhance survival under anchorage independent conditions. Through paracrine action, kynurenine can suppress anti-tumor cytotoxic T-cell function. We hypothesize that tryptophan catabolism promotes ovarian cancer progression through these two mechanisms, primarily through TDO2. We mined the publically available Tothill Ovarian Cancer patient cohort (n=293) for correlations between TDO2 or IDO1 & ovarian cancer outcomes. The anoikis resistant serous ovarian cancer cell lines HEY, OV-1847, OVCA-420 & OVCA-433 were used to test levels of enzymes involved in tryptophan catabolism in vitro at baseline, in attached versus suspended conditions & following NFκB stimulation with IL1-β and TNFα. Three enzymes in the pathway; TDO2, IDO1 & Kynureninase (KYNU) were quantified by qRT-PCR & immunohistochemistry. Using a cytokine/chemokine array and follow-up ELISA, we also analyzed factors secreted by ovarian cancer cells surviving in suspension culture as compared to attached. In the ovarian cancer patient cohort, high TDO2 significantly correlated with higher stage disease (p=0.0033), increased recurrence rates (p<0.0001) & lower survival rates (p=0.0034), whereas IDO1 did not show significant correlations. The expression of tryptophan catabolizing enzymes was significantly increased in the suspended condition when compared to attached & following NFκB stimulation. Ovarian cancer cell lines in suspension have increased levels of IL-1α, G-CSF, MIF and IL-6 on arrays, & IL-6 was confirmed by ELISA. Data mining of patient specimens indicates that TDO2 may be the more relevant enzyme responsible for tryptophan metabolism in ovarian cancer since high TDO2 is correlated with higher clinical stage, disease progression & in recurrent disease, suggesting that TDO2 may facilitate disease progression. Tryptophan catabolizing enzymes are elevated in ovarian cancer cells under anchorage independent condition or inflammatory stimuli. Based on these findings, we believe that clinical trials targeting TDO2 in addition to IDO may be warranted for serous ovarian cancer.

#5138

Comparative profiling for bacterial inhabitance in pancreatic ductal adenocarcinoma and matched adjacent normal tissues.

Sheema S. Khan,1 Pratik Banerjee,2 Saini Setua,1 Daleniece Higgins,2 Satish Kedia,2 Yu Jiang,2 Meena Jaggi,1 Subhash Chauhan1. 1 _Univ. of Tennessee Health Science Ctr., Memphis, TN;_ 2 _University of Memphis, Memphis, TN_.

Objective: Composition of resident microbes of the human body is associated with different disease manifestations. The microbiome in bodily fluids and different organs is altered with diverse health outcomes including cancer. Microbiome composition is a potential source for identifying biomarkers in cancers. Studies have shown the association of specific bacterial communities with the occurrence of pancreatic ductal adenocarcinoma (PDAC). However, investigations for the altered microbiota in an individual with the emergence of the disease are not studied. So, this study presents a comparative profiling for bacterial inhabitance in cancer tissues with that of matched adjacent normal tissues. This association will enable discovery of potential microbial biomarker(s) for PDAC.

Methods: Fresh frozen cancer and matched adjacent normal (N= 20 each) tissues were collected from PDAC patients. Genomic DNA was extracted by MO BIO PowerSoil DNA Isolation kit and enriched using NEBNext Microbiome DNA Enrichment kit. Next-generation sequencing (NGS) method, pyrosequencing of 16S rRNA was used to assess the diversity of the tissue-associated microbiota. Operational Taxonomic Units (OTUs) were defined at >97% similarity followed by taxonomic classification using BLASTn against a curated GreenGenes/NCBI/RDP derived database (bTEFAP®). The Bray-Curtis coefficient was used to quantify the compositional dissimilarity between microbiome profiles. Statistical analysis was done using "R" and NCSS 2010. Alpha and beta diversity analyses were conducted to cluster samples by their microbiome profiles. Overall associations between PDAC and microbiome compositions were assessed based on weighted or unweighted UniFrac distances using PERMANOVA.

Results: 16S taxonomic profiling reveal 10 and 11 dominant bacterial genera associated with normal and cancer lesion, respectively. Prominent genera that are only found in cancer lesions include Bacteroides, Enhydrobacter, Lautropia, Mycobacterium, and Phascolarctobacterium, whereas Fusobacterium, Acinetobacter, Propionibacterium, and Pseudomonas are the genera with high relative abundance in both cancer lesion and adjacent normal tissues. The most prevalent bacterial species in all observed samples are Propionibacterium acnes and Pseudomonas pseudoalcaligenes. We found Fusobacterium nucleatum in 4 samples (2 each in tumor and adjacent normal) in our preliminary work (10% prevalence). The PCoA multidimensional plot showed clustering of "tumor" samples, indicating the microbiota of these specimens is distinct from normal adjacent tissues.

Conclusion: This study suggests a plethora of distinct bacterial community structure in PDAC lesions that is significantly different from that of matched adjacent normal tissues. Additionally, it suggests that the tissue-associated microbiota can serve as a useful biomarker for PDAC.

#5139

F. nucleatum **induces invasive phenotypes in oral cancer cells in vitro.**

Amani Harrandah, Sasanka S. Chukkapalli, Ann Progulske-Fox, William Dunn, Kesavalu Lakshmyya, Edward K. Chan. _University of Florida, Gainesville, FL_.

Oral cancer is one of the most common cancers that affect public health with 500,000 new cases diagnosed yearly worldwide. The behavior of oral cancer might be affected by bacterial pathogens present in the oral cavity. The association between periodontitis, a chronic infection that affect the periodontium, and oral cancer have been reported in many studies. However, the effect of interaction between periodontal bacteria and oral cancer cells on cancer progression and aggressiveness is not well studied. Therefore, we aim to investigate the effect of four major periodontal bacteria on cancer cell pathology and treatment response. Three oral cancer cell lines OQ01, BHY and HN were used. Periodontal infection was represented by four periodontal bacteria Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Fusobacterium nucleatum. These bacteria were used to infect the oral cancer cells as polybacterial infection. Further analysis was done on OQ01 with monobacterial infection. The level of different cytokines (IL8 and TGF-β) were quantified in the culture supernatants after 6 and 24 h using ELISA. After RNA isolation from cell lysate, the relative changes in expression of six different oncogenes (MMP1, MMP9, MYC, JAk1, ZEB1, and STAT3) were measured using RT-PCR. To further investigate the role of F. nucleatum in inducing autophagy, three strains of F. nucleatum (Vincinti, Polymorphum and Periodontium) were used and the levels of autophagy markers (ATG7, ATG12 and ULK1) were measured after 6 and 24 hours. The effect of F. nucleatum on cancer cell invasion was was done starting from 4 h after infection. Statistical analysis was done using two-tailed Student's t-test OQ01 alone showed significantly enhance IL-8 secretion after polyinfection (P= 0.0003); however, enhanced TFG-β secretion was detected in all cell lines tested (P= 0.004). Polybacterial infection of oral cancer cell lines also upregulated MMP1 and MMP9 (P= 0.002) (known to enhance cancer cell invasiveness), ZEB1 (known to induce epithelial mesenchymal transition in cancer cells), and MYC, JAK1 and STAT3 (oncogenes involved in cell survival). Further analysis using OQ01 cells in monobacterial infection showed that F. nucleatum alone had the same or greater effect as polybacterial infection (P= 0.001). These results showed that F. nucleatum was the main periodontal bacteria responsible for inducing invasive phenotype in these oral cancer cells. In addition, F. nucleatum was able to enhance cancer cell invasion in vitro. Furthermore, F. nucleatum appear to upregulate autophagy markers in vitro which can lead to enhanced cancer cell survival and chemoresistance. This study demonstrated that the interaction between oral cancer cells and periodontal bacteria might be both cancer cell- and bacteria-specific. Our study can be highly useful in examining how modulating oral cancer environment can improve treatment outcome.

#5140

Lipoxins and their role in Kaposi's sarcoma-associated herpesvirus (KSHV) infection and pathogenesis.

Neelam Sharma-Walia, Jayashree A. Chandrasekharan. _Rosalind Franklin Univ. of Med. & Science, North Chicago, IL_.

Lipoxins are host anti-inflammatory molecules, which play a vital role in restoring tissue homeostasis. The efficacy of lipoxins and their analog epilipoxins in treating inflammation and related diseases have been well documented. Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL) are two well-known inflammation-related diseases caused by Kaposi's sarcoma-associated herpesvirus (KSHV). Controlling inflammation is one of the strategies adopted to treat KS and PEL, a primary motivation for exploring and evaluating the therapeutic potential of using lipoxins. Our study demonstrates how KSHV manipulates and downregulates the secretion of the anti-inflammatory lipoxin A4 in host cells and the viral factors involved in this process using in vitro KS and PEL cells as models. Presence of lipoxin receptor [ALX/Formyl peptidyl receptor (FPR)] in human KS patient tissue sections and in vitro KS and PEL cell models offers a novel possibility for treating KS and PEL with lipoxins. Treating de novo KSHV-infected endothelial cells with lipoxin and epilipoxin creates an anti-inflammatory environment by decreasing levels of NFκB, AKT, ERK1/2, COX-2, and 5-lipoxygenase. Lipoxin treatment on CRISPR/CAS9 technology-mediated ALX/FPR gene-deleted U2OS cells revealed the importance of the lipoxin receptor ALX for effective lipoxin signaling. A viral miRNA cluster was identified as the primary factor contributing to the downregulation of lipoxin A4 secretion in host cells. The KSHV miRNA cluster probably targets enzyme 15-lipoxygenase, which is involved in lipoxin A4 synthesis. Lipoxin treatment in KSHV-infected cells has been shown to reduce cell proliferation and cell survival. Latency factor LANA-1 levels were found to decrease on lipoxin and epilipoxin treatment. Several pro-apoptotic gene levels were upregulated on lipoxin treatment. Several host transcription factors were found to play an important role in regulating KSHV life cycle. This study provides a new insight into the treatment of KS and PEL using nature's own anti-inflammatory molecule, lipoxin.

#5141

Streptococcus gallolyticus **in colorectal cancer development.**

Yi Xu, Ritesh Kumar, John Taylor, Juan Xu. _Texas A &M Health Science Center, Houston, TX_.

Streptococcus gallolyticus subsp. gallolyticus (Sg) has been reported to have a strong clinical correlation with colorectal cancer (CRC) for several decades. Our recent data indicates that Sg is able to actively promote the development of colon tumor and thus is also functionally important to CRC. Further investigations into the Sg-CRC connection revealed that there are variations among Sg strains with respect to their ability to stimulate target host cell proliferation and tumor growth. The ability of Sg to adhere to colon cancer cells and to colonize colonic epithelium in vivo appears to be important. The results suggest that direct interactions between specific Sg factors and colonic epithelial receptors/structures are required to trigger downstream signaling events leading to increased host cell proliferation. Furthermore, we observed that the effect of Sg is also dependent on the host genetic background, suggesting a dynamic and complex interactions between Sg and the host. In addition to the requirement for beta-catenin signaling as we previously reported, we observed that Sg activates yes-associated protein 1 (YAP1), a downstream effector in the Hippo signaling pathway. YAP1 activation is required for Sg to stimulate target host cell proliferation. YAP1 and the Hippo pathway are known to be involved in the development CRC. Bacterial activation of YAP1 has not been described before. Thus, our results suggest a novel mechanism by which bacteria contribute to the development of cancer. Studies are on-going to further investigate the molecular details of this mechanism.

#5142

Elevated glycolysis in pre-malignant ovarian cancer.

Anna E. Lokshin, Mounia Alaoui El Azher, Liudmila Velikokhatnaya, Denise Prosser. _Univ. of Pittsburgh Cancer Inst., Pittsburgh, PA_.

Ovarian cancer is the fifth leading cause of cancer death in women and the most lethal gynecologic malignancy. High-grade serous ovarian carcinoma (HGSC) originates mainly from the fallopian tube epithelium, and is characterized by TP53 mutation identified in all HGSCs as well as the fallopian tubal precursor of serous tubal intraepithelial carcinomas (STICs). The loss of a functional TP53 is an early event in the malignant transformation of fallopian tube epithelium. We have established 4 primary cell cultures of normal fallopian tube epithelial (FTE) cells, and further generated immortalized FTE cells using SV40 large plus small T antigens (FTE-TAg) to disrupt TP53 function. This model mimics the primary lesions of ovarian cancer and is suitable to identify biomarkers and molecular targets of ovarian cancer. Therefore, we compared the secretomes of primary FTE cells with that of their parental isogenic pairs FTE-TAg cells using mass spectrometry analysis. We identified 813 proteins, 228 of which were differentially expressed. Ingenuity Pathway Analysis (IPA) core analysis revealed glycolysis as a major canonical pathway. Overexpression of key enzymes of glycolytic pathway was confirmed by IHC in human STIC lesions. Molecular mechanisms of glycolysis regulation in premalignant lesion of ovarian cancer were determined. Role of glycolysis in the establishment of premalignant ovarian cancer microenvironment (PME) was characterized. Prevention approaches targeting glycolysis were tested in transgenic animal model of ovarian cancer.

#5143

Characterization of the cervicovaginal, endometrial and anorectal microbiota and mycobiota of post-menopausal women with endometrioid and serous endometrial cancers.

Gregory M. Gressel,1 Marina Frimer,1 Christine P. Zolnik,2 Mykhaylo Usyk,2 Devin T. Miller,1 Eirwen M. Miller,1 D. Y.S. Kuo,1 Robert D. Burk2. 1 _Montefiore Medical Center, Bronx, NY;_ 2 _Albert Einstein College of Medicine, Bronx, NY_.

Objectives: Dysbiosis is thought to promote endothelial dysfunction and chronic inflammation, resulting in many chronic human diseases including cancer. This pilot study sought to characterize the cervicovaginal, endometrial and anorectal micro- and mycobiota of post-menopausal women with endometrioid endometrial adenocarcinoma (EAC), uterine serous carcinoma (USC), and controls without cancer.

Methods: Post-menopausal patients undergoing hysterectomy for endometrial cancer or non-cancerous disease were enrolled after informed consent. Women were excluded if they reported history of prior cancer, recent urinary or gynecologic infection, antibiotic or probiotic use. Microbial swabs were obtained from the cervicovaginal, the anorectal, and the endometrial cavity after removal of the uterus. DNA was extracted and PCR amplified using barcoded 16SV4 (bacterial) and ITS1 (fungal) primers. Next generation libraries were prepared and sequenced on an Illumina MiSeq 2x300. Sequence reads were processed using our internally developed bioinformatics pipeline and publically available software.

Results: Next-generation sequencing of bacterial and fungal DNA revealed substantial differences of cervicovaginal, endometrial and anorectal microbiota in patients with USC (n= 8) compared to those with EAC (n= 13) or benign disease (n= 6). Uterine fungal reads were much higher in patients with USC than EAC (average of 3237 reads versus 938 reads respectively, p = 0.05 Wilcox test). Candida species were reduced, but not eliminated in cervicovaginal samples, and there was a dominance of Malassezia restrica in USC and EAC. A non-significant predominance of Porphyromonas species was noted in USC specimens compared to EAC specimens (p = 0.063).

Conclusions: Alpha-diversity of 16S bacterial rRNA and fungal species of the cervicovaginal microbiome was significantly associated with endometrial cancer in our study. USC had a fundamentally different microbiome from control samples and demonstrated a predominance of Porphyromonas species. Further sampling and analysis is needed to confirm these results and to identify rare pathogens. Characterization of microbiota in women with endometrial cancer may help identify risk factors for gynecologic malignancy, and drive investigation regarding preventative and therapeutic intervention.

#5144

Cooperative effects of mushroom polysaccharides and herbal saponins on tumor growth and gut microenvironment in ApcMin/+mice.

Xiao-ang Li, Guoxin Huang Huang, Imran Khan, Wen-rui Xia, Wai kit Leong, W.L. Wendy Hsiao. _State Key Laboratory of Quality Research in Chinese Medicines, Macao, Macao_.

Prebiotics are considered as non-digestible food ingredients that stimulate the growth of beneficial gut bacteria, thus contribute to the health of the host. Recent evidences have suggested that prebiotics treatment may become one of the key therapeutic strategies for many diseases, especially for cancer. In our previous studies, both Gynostemma pentaphyllum saponin (GpS) and Ganoderma lucidum polysaccharides (GlP) demonstrated significant prebiotic effects in mouse models. In addition, GpS alone exerted significant anti-tumor effect by restoring the dysbiosis of the gut microbiota and microenvironment of the ApcMin/+mice. Thus, we hypothesized that supplement with GlP might enhance the anti-cancer effect of GpS through the modulation of the gut microbiota. To test the hypothesis, we treated ApcMin/+mice with four different treatments, i.e. GlP (750mg/kg), GpS (300mg/kg), GlP (750mg/kg) + GpS (300mg/kg) and the drinking water control. The results showed that GlP+GpS was more effective in suppressing the polyp growth compared to the GpS or GlP groups. GlP+GpS also improved the health of gut barriers by inducing Paneth cells and goblet cells, and reinstating the E-cadherin and N-cadherin expressions in a greater extend compared to the other groups. All treatments facilitated the polarization of M1 macrophage to M2 macrophage, and increased the anti-inflammatory cytokines (IL-4 and IL-10). Interestingly, expressions the SCFA receptors, GPR 43, 49 and 119 were markedly elevated in all treatment groups, with the most prominent expression observed in the GlP+GpS group. 16S rRNA sequencing data were analyzed by using alpha diversity analysis and weighted UniFrac distance analysis. Differential alterations of the composition of gut microbiota were observed in all treatment groups. Certain bacteria (Anoerodtipe, Bacteroides, Clostridium, Citribacter, Subdoligranulum, C.

leptum) associated colitis and colorectal carcinoma were significant decreased, while some SCFA-producing bacteria (Blautia sp., Eubacterium saphenum, Clostridium aminobutyricum) were enhanced in the treated groups. We concluded that the anti-tumor effects of GpS and GlP might be in part through the modulation of the gut microbiota and the improvement of epithelial microenvironment, and the stronger effects were produced by the GpS+ GlP combined treatment.

[This study was supported by Macau FDCT 015/2014/A1 and 103/2016/A3]

#5145

PAH-metabolizing microorganism in the oral cavity of smokers.

Guy R. Adami, Lin Tao, Sylvia Pavlova, Stefan J. Green, Ankur Naqib, Benjamin Salameh, Jessica L. Tang, Joel L. Schwartz. _Univ. of Illinois at Chicago, Chicago, IL_.

Certain soil microorganisms have been identified as being not only resistant to polycyclic aromatic hydrocarbons (PAHs) but also capable of metabolizing these compounds. We hypothesized that a subset of oral bacteria may be enriched by the altered oral environment of the tobacco smoker with relatively high levels of PAHs. We identified microorganisms that survived long term in minimal media with PAH as the sole carbon source and showed they were much more likely to be isolated from smokers. To start to determine their contribution to oral health, we measured their level in vivo on two mucosal surfaces. On both gingival and lateral border of the tongue mucosa, a group of bacteria were seen to be at lower relative numbers in those exposed to cigarette smoke, while a minority increased. Three taxons were shown to be enriched on gingiva on the family, Streptococcaceae, or genus level, Staphylococcus and Granulicatella. On tongue, genera Mogibacterium and Dialister were enriched. Of bacteria genera that survived harsh PAH exposure in vitro, one was found at substantial levels in the oral mucosal microbiome. Staphylococcus was enriched approximately 6x in smokers, suggesting that the reason for its increase in smokers is due to its resistance to PAH. We will report on this and other oral microbes that can metabolize PAHs. This study highlights the possibility that the oral microbiome can metabolize PAHs like those in cigarette smoke, resulting in carcinogenic products that may contribute to the cancer process in vivo.

#5146

**Administration of** Parabacteroides distasonis **in the attenuation of colorectal tumorigenesis in a carcinogenic murine model of colorectal cancer.**

Camilo Anthony G. Gacasan. _Texas A &M University, Round Rock, TX_.

Colorectal cancer (CRC) is the second leading cause of related deaths in the United States. Interindividual differences in genetic background result in a complex and dynamic system between the gut microbiome and variable phenotypic response to diet as it relates to CRC risk. Previous research from our laboratory investigated CRC susceptibility in an azoxymethane (AOM) murine model using FVB/NJ (FVB) and C57Bl/6J (B6) mice fed a ketogenic (high fat, no carbohydrate) American (high fat, high carbohydrate) or standard (moderate fat, moderate carbohydrate) mouse chow. Tumor multiplicity was dependent on both strain and diet with a four-fold increase reported in FVB mice fed ketogenic diet relative to those fed standard chow. This response was not observed in FVB mice fed the American diet or in B6 mice on any of the three diets. A subsequent study that investigated the physiological response to diet in multiple genetic backgrounds revealed a decrease in the abundance of the bacterial species Parabacteroides distasonis in FVB mice fed a ketogenic diet. This established an emphasis on the interaction between variable response to diet, gut microbiome and further implications to gut health. To further investigate this relationship in FVB mice we are using an AOM-induced carcinogenic model on FVB mice fed a ketogenic diet supplemented with a biweekly gavage of P. distasonis. Upon necropsy, tumor load of mice receiving the experimental gavage will be compared to the control mice. Droplet digital PCR will be used to quantify the absolute abundance of P. distasonis in fecal samples at several time points before and after the initiation of diet and administration of AOM. Ultimately, we aim to determine the relationship between P. distasonis and CRC tumorigenesis, which will lead to a further understanding of the etiology of colorectal cancer.

#5147

Lactate transporters are a potential therapeutic target for LKB1-deficient lung cancers.

Irene Guijarro, Alissa Poteete, Pan Tong, Jing Wang, John Heymach. _MD Anderson, Houston, TX_.

The serine/threonine kinase LKB1 is mutated and inactivated in 20-30% of lung adenocarcinoma representing the second most commonly altered tumor suppressor in non-small cell lung cancer (NSCLC), and to date there are no effective targeting strategies for tumors bearing this mutation. Therefore, new therapeutic approaches are urgently needed for LKB1-deficient tumors. LKB1 activates AMPK, the master sensor of cellular energy and because of this, many of the best known functions of LKB1 are attributed to its ability to control metabolic alterations. Our laboratory and others have demonstrated that loss of LKB1 can promote enhanced glucose metabolism and a shift from aerobic to anaerobic respiration. A result of enhanced glycolysis is an elevated lactate production even under normoxic conditions. It has been reported that lactate-mediated extracellular acidification is a key factor mediating tumor cell invasion and metastasis. We found that LKB1-deficient NSCLC tumors significantly upregulate lactate transporters and, because these tumors exhibit an immunologically inert phenotype, we propose that inhibition of lactate transporters could represent a rational strategy to inhibit tumor growth and enhance immune responses in LKB1 mutant NSCLC. To characterize lactate transporter expression, a panel of NSCLC cell lines was stably transduced to overexpress LKB1 and with shRNA targeting LKB1. We analyzed gene expression of lactate transporters in TCGA dataset of lung adenocarcinoma (LUAD). We validated these results by qPCR and western blot analysis of expression levels of MCT1, MCT4 and MCT14 in human LKB1-intact and deficient cells and in LKB1 KO murine NSCLC cell lines that were generated using CRISPR/Cas9 in a KRASG12D mutant background. To study lactate transporter expression in tumors in vivo, we generated syngeneic NSCLC mouse models via s.c. injection of LKB1-intact and KO murine cells in immunocompetent mice and analyzed the protein expression levels of MCT4 in these tumor samples. The analysis of TCGA LUAD dataset revealed a significant upregulation of lactate transporter SLC16A14 (MCT14) gene expression levels in LKB1-deficient tumors compared with LKB1 wild-type (p<0.001). In vitro, NSCLC LKB1-deficient cells (A549, H460 and H2030) showed increased RNA and protein expression of MCT4 and MCT14 compared to cells where a copy of LKB1 was introduced. In vivo, KRASG12D mutant LKB1 KO tumors from syngeneic mouse models significantly showed an upregulation of MCT4 protein expression compared with KRASG12D mutant LKB1 wild type tumors (p<0.0001). In conclusion, LKB1-deficient NSCLC showed higher levels of glycolysis and significantly elevated expression of lactate transporters in vitro and in vivo compared to LKB1-wild type NSCLC. Additional studies are ongoing to determine the efficacy of lactate transporter inhibition in LKB1-mutant NSCLC.

#5148

Secreted ccdc80 from hepatic stellate cell promote invasion and epithelial-mesenchymal transition in hepatocellular carcinoma.

Kwang Seock Kim,1 SaeHwan Lee,2 Nayoung Jun,1 Hyog Young Kwon1. 1 _Soonchunhyang Institute of Medi-bio Science, Soonchunhyang University, Cheonan, Republic of Korea;_ 2 _Soonchunhyang University Cheonan Hospital, Cheonan, Republic of Korea_.

Liver cirrhosis is the most important risk factor for hepatocellular carcinoma (HCC) and hepatic stellate cell (HSC) has pivotal role in developing cirrhosis by producing various extracellular matrix (ECM). HSC are essential mediators of tumor microenvironment of HCC through remodeling of ECM and angiogenesis. We found that CCDC80 expression is upregulated in activated HSCs from RNA-sequencing with human fetal stellate cell. However, the role of CCDC80 was not well known in hepatocarcinogenesis. First, we validated mRNA expression of CCDC80 was increased in activated HSC lines and protein expression was also increased in cultured media. Functional analysis of knockdown CCDC80 was investigated in both HCC and HSC cell lines, and HCC cell lines were analyzed for proliferation (MTT assay), migration (using transwell), invasion (using Matrigel-coated transwell), and wound healing assay with condition media of CCDC80 knockdown HSCs. Expression of epithelial-mesenchymal transition (EMT) markers in HCC was evaluated with conditioned media. Knockdown of CCDC80 resulted in reduced cell proliferation and significantly reduced invasion, migration and wound healing behaviors of HCC cell lines. Expression of E-cadherin was increased and snail and vimentin expression were decreased in HCC cell lines cultured with conditioned media of CCDC80 knockdown HSCs. The associations between CCDC80 and EMT markers were validated in CCDC80 transfected HSC cell line compared with control. Taken together, secreted CCDC80 from HSCs affected the aggressiveness and EMT of HCC cell lines. CCDC80 might be a novel biomarker and therapeutic target for patients with HCC.

### Metastasis, Invasion, and Migration 2

#5149

CMTM6 is a potential metastasis regulator of EWS cells.

Yuko Nishiyama,1 Naofumi Asano,2 Yuko Fujiwara,1 Tadashi Kondou,1 Naoto Tsuchiya1. 1 _National Cancer Center Research Institute, Tokyo, Japan;_ 2 _Keio University School of Medicine, Tokyo, Japan_.

Ewing's sarcoma (EWS) is a highly aggressive and metastatic malignant tumor of bone and soft tissues, whose incidence is highest in children and young adulthood. Control of metastasis based on the molecular targeting contributes to improve patient prognosis. To this end, we screened expression of microRNAs in clinical samples to identify target molecules, which regulate malignant properties of EWS cells. Microarray analysis indicated that miR-451a is significantly downregulated in the cases with poor prognosis. Although miR-451a did not affect cell proliferation, it strongly repressed migration of EWS cells. Gene expression analysis revealed that miR-451a targeted CMTM6, a chemokine-like membrane protein with unknown function. Depletion of CMTM6 by siRNAs strongly inhibited migration of EWS cells. Our data suggest that at least the expression of miR-451a and CMTM6 is involved in metastasis onset.

#5150

Loss of CYLD promotes cell invasion via ALK5 stabilization in oral squamous cell carcinoma.

Satoru Shinriki,1 Hirofumi Jono,1 Manabu Maeshiro,1 Takuya Nakamura,1 Jianying Guo,2 Jian-Dong Li,3 Ryoji Yoshida,1 Masanori Shinohara,1 Hideki Nakayama,1 Hirotaka Matsui,1 Yukio Ando1. 1 _Kumamoto University, Kumamoto, Japan;_ 2 _Tianjin First Central Hospital (East Hospital), Tianjin, China;_ 3 _Georgia State University, Atlanta, GA_.

Oral squamous cell carcinoma (OSCC) has a very poor prognosis because of its highly invasive nature, and the 5-year survival rate has not changed appreciably for the past 30 years. Although cylindromatosis (CYLD), a deubiquitinating enzyme, is thought to be a potent tumor suppressor, its biological and clinical significance in OSCC is largely unknown. This study aimed to clarify the roles of CYLD in OSCC progression. Our immunohistochemical analyses revealed significantly reduced CYLD expression in invasive areas in OSCC tissues, whereas CYLD expression was conserved in normal epithelium and carcinoma in situ. Furthermore, downregulation of CYLD by siRNA led to acquisition of mesenchymal features and increased migratory and invasive properties in OSCC cells and HaCaT keratinocytes. It is interesting that CYLD knockdown promoted TGF-β signaling by inducing stabilization of TGF-β receptor I (ALK5) in a cell autonomous fashion. In addition, the response to exogenous TGF-β stimulation was enhanced by CYLD downregulation. The invasive phenotypes induced by CYLD knockdown were completely blocked by an ALK5 inhibitor. Also, lower CYLD expression was significantly associated with the clinical features of deep invasion, poor overall survival, and increased Smad3 phosphorylation, which is an indicator of activation of TGF-β signaling in invasive OSCC. These findings suggest that downregulation of CYLD promotes invasion with mesenchymal transition via ALK5 stabilization in OSCC cells.

#5151

Enhanced chemotactic response of MDA-MB-231 breast cancer cells towards stable gradients of EGF.

Nina Baumann, Verena Kuttenberger, Christine Huemmer, Jan Schwarz, Elias Horn Horn, Roman Zantl. _ibidi, Martinsried, Germany_.

Chemotactic cell migration is a basic mechanism underlying cancer cell invasion and hence metastasis. In this regard, growth factors such as epidermal growth factor (EGF) have been identified as guidance cues. Using ibidi's µ-Slide Chemotaxis we confirm the growth factor EGF as potent guidance cue for directional MDA-MB-231 migration in a physiological 3D environment. Moreover, we find enhanced sensitivity towards gradients of EGF in serum free media supplemented with defined protein composition (UltraCULTURETM). Combining defined, physiological migration conditions and enhanced sensitivity allowed us to quantify EGF dependent MDA-MB-231 chemotaxis in a detailed manor. MDA-MB-231 cells showed increased directional and kinetic response towards temporal and stable spatial gradients of EGF. While EGF guided directional migration was highly concentration dependent with a concentration optimum of 10 ng/mL, we found the chemokinetic effect induced by the presence of EGF being concentration independent. Those results suggest differential response of the migration and signal interpretation machinery of MDA-MB-231 cells. Both, blocking the ligand binding domain of the EGF-Receptor (EGFR) by a blocking antibody and inhibition of the kinase domain of EGFR by a small molecule inhibitor (AG1478) led to a reduction in EGF induced directional sensing and migration velocity. Additionally, low, non-effective single doses of antibody and small molecule inhibitor showed synergistically effective inhibition if combined. Those results support EGF being a potent guidance cue for MDA-MB-231 cell migration and help to understand the mechanisms behind chemotactic migration driven cancer metastasis.

#5153

RKIP-RhoA axis inhibits breast cancer invasion and metastasis by increasing E-cadherin expression.

Gardiyawasam Kalpana, Vu Bach, Clariza Borile, Miranda Yeung, Rafael Garcia-Mata, Kam C. Yeung. _University of Toledo, Toledo, OH_.

Background: Tumor metastasis suppressors are proteins that impede secondary tumor formation by inhibiting one or more steps of the metastasis cascade without stimulating primary tumor growth. Raf-1 kinase inhibitor protein (RKIP) is a metastasis suppressor that inhibits metastasis in breast, prostate, melanoma and several other types of cancers. The molecular mechanism through which RKIP executes its anti-metastasis effects is not yet completely defined. The objective of the current study is to understand how RKIP inhibits breast cancer cell invasion and metastasis in molecular level. Given its primary function in regulating actin cytoskeleton and cell movements, Rho GTPases were studied as possible downstream effectors of RKIP. These small GTPases belong to the Ras superfamily and consist of nearly 22 members with RhoA and Rac1 as the major players affecting cell motility. Rho GTPases oscillate between a GTP-bound active form and a GDP-bound inactive form. The GDP-GTP cycle is regulated by GEFs that facilitate the exchange of bound GDP with GTP, and by GAPs that stimulate the Rho GTPase activity. Aberrantly activated GEFs are reported to drive tumorigenesis, while activated GAPs are assumed to inhibit the cancer formation and metastasis. Hence, Rho GTPases are widely accepted as oncogenes. Contrary to this notion, several recent studies suggested a possible metastasis inhibitory function for wild-type RhoA in triple negative breast cancer and several other types of cancers.

Experimental procedures and results: In this study, several mouse (4T1, 168 FARN) and human (BT20, MDA-MB 231) breast cancer cell lines were used to eliminate possible cell line specific observations. Using in vitro matrigel invasion assay, we showed that RhoA, not Rac1, acts downstream of RKIP and is needed for RKIP-mediated inhibition of breast cancer cell invasion in vitro. GTPase activity pull-down assay results revealed that RKIP specifically increases RhoA activation, thus inhibiting invasiveness of these cells. Similarly, orthotopic mice tumor implantation experiments showed that ectopic expression of dominant negative RhoA in 4T1 cells confers significantly greater proclivity to metastasize in mice. These observations are consistent with RhoA's emerging role as a metastasis suppressor. In BT20 breast cancer cells, RhoA enhanced E-cadherin expression and negatively affected the cell invasiveness. Interestingly, RKIP phenocopied the RhoA effect on E-cadherin and cell invasion, suggesting that RKIP-RhoA axis inhibits breast cancer cell invasiveness by increasing E-cadherin expression.

Conclusions: Our results conclude that RKIP specifically increases RhoA activation in breast cancer cells, and this activated RhoA stabilizes E-cadherin and negatively affects the invasiveness of these cells.

#5154

Semaphorin 6A attenuates the migration capability of lung cancer cells via the NRF2/HMOX1 axis.

Li-Han Chen, Che-Yu Liao, Eric Y. Chuang, Mong-Hsun Tsai. _National Taiwan University, Taipei, Taiwan_.

Cell migration is a fundamental feature of cancer recurrence. Since recurrence is correlated with high mortality in lung cancer, it follows that reducing cell migration would decrease recurrence and increase survival rates. The goal of this study was to determine the role of SEMA6A in cell migration. Semaphorin 6A (SEMA6A), a protein initially known as a regulator of axonal guidance, is down-regulated in lung cancer tissue, and low levels of SEMA6A are associated with cancer recurrence. Thus, we hypothesized that SEMA6A could suppress cancer cell migration. In this study, we found that the migration capability of H1299 lung cancer cells decreased with SEMA6A overexpression, while it increased with SEMA6A silencing. Reduction in migration was observed only with the full length protein and not with the extracellular ectodomain construct, suggesting that a reverse signaling mechanism is used to mediate the effects of SEMA6A on migration. Moreover, silencing of the cellular homeostasis protein HMOX1 and/or the transcription factor NRF2 reversed the migration-suppressing effect of SEMA6A and the SEMA6A-driven alterations in expression of PLAU and IGFBP3, both downstream effectors of HMOX1. Taken together, these results demonstrate that SEMA6A is a potential suppressor of migration that functions through the NRF2/HMOX1 axis. Our results explain why low SEMA6A is linked to high recurrence in the clinical setting and suggest that SEMA6A could be useful as a biomarker or target in lung cancer therapy.

#5155

The modulation of actin dynamics via atypical protein kinase-C activated cofilin regulates migration of colorectal cancer cells.

S M Anisul Islam, Khandker Mohammad Khalid, Raja Reddy Bommareddy, Rekha Patel, Mildred Acevedo-Duncan. _Univ. of South Florida, Tampa, FL_.

Colorectal cancer (CRC) is the second most common cancer in both men and women in the United States. The exact mechanism of CRC cells migration is poorly understood. Actin polymerization is thought to be an initial step in cancer cells motility cycle which drives the formation of cell protrusions that defines the direction of migration and initiate cell crawling. Cofilin is a significant actin regulating molecule that can regulate the migration of cancer cells by the formation of lamellipodia and filopodia. In this study, the effect of atypical Protein Kinase C (aPKC) on cofilin in CRC was studied by using two inhibitors of aPKC: 1) ICA-I (5-amino-1-(2,3-dihydroxy-4-hydroxymethyl) cyclopentyl)-1H-imidazole-4-carboxamide) is a specific inhibitor of PKC-ι and 2) ζ-Stat (8-hydroxy-1, 3, 6-naphthalenetrisulfonic acid) is a specific inhibitor of PKC-ζ. The cell lines tested were CCD18CO normal colon fibroblast and LOVO & RKO metastatic CRC cells. The inhibition of aPKC did not bring any significant toxicity in CCD18CO colon fibroblasts cells. However, our data showed that the inhibition of aPKC blocks the migration of CRC cells migration by increasing the level of cofilin phosphorylation at serine-3. Additionally, the aPKCs inhibition brought a significant reduction of CRC cells proliferation along with the reduction of survival markers. Our findings suggest that the PKC-ι and/or PKC-ζ may be responsible for the migration of CRC cells as well as carcinogenesis. These results suggest the possibility of utilizing aPKCs as the potential therapeutic targets for the CRC cells metastasis.

#5156

RhoC decreases tight junction stabilization in breast cancer cells, revealing a potential therapeutic target.

Hannah Cheriyan, Zhi-Fen Wu, Joel Yates, Sofia D. Merajver. _University of Michigan, Ann Arbor, MI_.

RhoC, a member of the Rho GTPase family, has been shown to have a role specifically in facilitating metastasis of breast cancer cells1, however no conclusive mechanism for how RhoC promotes metastasis currently exists. We demonstrate that CRISPR-Cas9 knockout of RhoC in SUM149 and MDA-MB231 cells results in increased epithelial morphology, cell-cell connections, and tight junction marker (ZO-1, occludin, PATJ/PALS1/CRB3) colocalization compared to wild type cells, as studied via immunofluorescent staining. These results suggest that high expression of RhoC in cancer cells destabilizes tight junctions, which could allow for the increased metastatic potential observed with RhoC-overexpressing cells in prior studies2-4. Ongoing work investigates whether these morphological and molecular changes are observed in other breast cancer cell lines, such as SUM190 and VARI-068, a low-passage PDX-derived triple negative breast cancer cell line. Furthermore, we will determine whether RhoC knockout in these cells lines actively modulates tight junction stability as measured via transepithelial electrical resistance (TEER). Taken together, these data support an important role for RhoC in tight junction modulation, thereby revealing a potential highly specific new therapeutic target in RhoC-driven metastases control.

References

1. Hakem, A. et al. RhoC is dispensable for embryogenesis and tumor initiation but essential for metastasis. Genes Dev. 19, 1974-1979 (2005).

2. van Golen, K. L., Wu, Z. F., Qiao, X. T., Bao, L. W. & Merajver, S. D. RhoC GTPase, a novel transforming oncogene for human mammary epithelial cells that partially recapitulates the inflammatory breast cancer phenotype. Cancer Res. 60, 5832-8 (2000).

3. van Golen, K. L., Wu, Z. F., Qiao, X. T., Bao, L. & Merajver, S. D. RhoC GTPase overexpression modulates induction of angiogenic factors in breast cells. Neoplasia 2, 418-25 (2000).

4. Wu, M., Wu, Z. F., Rosenthal, D. T., Rhee, E. M. & Merajver, S. D. Characterization of the roles of RHOC and RHOA GTPases in invasion, motility, and matrix adhesion in inflammatory and aggressive breast cancers. Cancer 116, 2768-2782 (2010).

#5157

ARF6 controls WNT5A receptor internalization to promote uveal melanoma invasion and metastasis.

Donghan Shin,1 Coulson P. Rich,1 Lehi Acosta,1 Jackson R. Richards,1 Jae Hyuk Yoo,1 Allie H. Grossmann,1 Zongzhong Tong,2 Alan L. Mueller,2 Weiquan Zhu,1 Dean Y. Li,1 Shannon J. Odelberg1. 1 _University of Utah, Salt Lake City, UT;_ 2 _A6 Pharmaceuticals, Salt Lake City, UT_.

Introduction: Uveal melanoma is the most common primary ocular malignancy and there is currently no effective treatment for metastatic uveal melanoma largely because of the lack of understanding of the molecular mechanisms underlying this cancer. Although activating oncogenic mutations in GNAQ and GNA11 are present in 95% of uveal melanoma tumors, virtually nothing is known about the molecular mechanisms that drive oncogenesis in the remaining 5% of uveal melanomas that possess only wild type Gαq alleles and the molecular and cellular mechanisms that promote metastasis have not yet been discovered. This latter deficit in knowledge is extremely vexing, given that metastatic disease causes death in most uveal melanoma patients. We have recently shown that ADP-ribosylation factor 6 (ARF6), a small GTPase, is an immediate downstream effector of oncogenic Gαq and controls the entire major signaling pathways known to drive Gαq-mediated tumor formation and growth in uveal melanoma cells that have an activating mutation in Gαq. Moreover, in cutaneous melanoma, stimulation of the receptor by WNT5A activates ARF6 and controls metastasis via the release of β-catenin from N-cadherin and β-catenin's subsequent nuclear transport. Previous studies have also demonstrated that ARF6 plays a role in the internalization of various classes of membrane receptors including receptor tyrosine kinase (RTK) and G protein coupled receptor (GPCR) to control signal transduction, and WNT5A and its receptor promote tumor invasion and metastasis via receptor internalization in human cancers. Based on these intriguing findings, we postulate that ARF6 might be functioning to control tumor invasion and metastasis in uveal melanomas.

Experimental Procedures: To test our hypothesis, we conducted cell invasion assays on uveal melanoma cell lines while targeting ARF6 expression by siRNA or ARF6 activity by pharmacological inhibition. We then proceeded with cell fractionation to look for any differences in the properties of subcellular components in treated cells compared to untreated cells.

Unpublished findings: Here we show that WNT5A is expressed at high levels in Gαq wild type uveal melanoma cells and that WNT5A activates ARF6 through ROR2 receptor. When we block ARF6 expression by knockdown or ARF6 activity with a small molecule inhibitor, uveal melanoma invasion is significantly reduced. Notably, ARF6 is necessary for WNT5A-mediated ROR2 internalization.

Conclusions: This work indicates that ARF6 functions in the internalization of WNT5A receptor to activate the signaling pathways that drive invasion and metastasis in uveal melanoma. These results would suggest that targeting ARF6 activation might be an effective therapy for the treatment of all uveal melanomas, regardless of their mutational status for Gαq.

#5159

FLOT1 is associated with HGF mediated cell invasion and proliferation by increased MMP9.

Sung Ae Koh, Kyung Hee Lee, Jae Ryong Kim, Sang woon Kim, Byung Ik Jang. _Yeungnam University Hospital, Daegu, Republic of Korea_.

The flotillin protein family, including flotillin-1 (FLOT1 ) and flotillin-2 (FLOT2), have been known as markers of lipid rafts. Recentely, FLOT1 was reported that FLOT1 overexpression was found in several advanced cancer including breast, colorectal, prostate tumors, and renal cell carcinomas and correlate with poor prognosis in advanced tumors. The purpose of this study is to identify the role of HGF upregulated FLOT1 associated with cancer cell proliferation and invasion in gastric cancer. We used cell culture, western blotting, RT-PCR, MTT assays, CHIP assay, zymography and FLOT 1 knock-down with short hairpin RNA (shRNA). First, we confirmed that the expression level of FLOT1 was up-regulated by HGF(hepatocyte growth factor). To determine the role for FLOT1, we used the knock down cell of FLOT1. FLOT1 -sh RNA cells showed a decreased level of MMP9 and NFkB. We also examined to confirm the role of HGF-mediated FLOT1. HGF-mediated cell proliferation and in vitro invasion was decreased in FLOT1 knock down cell. We perforomed the zymography to evaluate the activity of MMP9 . The activity of MMP9 was decreased in FLOT1 knock down cell. The level of AKT phosphorylation was decreased in the knock down cell of FLOT1. Also, We identified the putative binding site of NFkB in MMP9 promotor region and confirmed the function by CHIP assay. Our study showed that upregulation FLOT1 by HGF increased the level of MMP9 through AKT/NFkB pathway and associated with cell proliferation and invasion in gastric cancer.

#5160

Identifying TK1 localization in immortalized placental cell lines and in conditioned placental tissue.

Eliza E. Lawrence, Evita G. Weagel, Juan F. Mejia, Juan Arroyo, Shalee Killpack, Kim L. O'Neill, Richard Robison. _Brigham Young University, Provo, UT_.

Many parallels in growth pattern can be observed between placental and cancer development. In fact, placental trophoblast and cancer cells share similarities in migration and invasion patterns, immune escape strategies, and angiogenesis induction. A common feature of pregnancy complication is aberrant trophoblast behavior. Understanding trophoblast cell behavior can help us identify common patterns in cancer gene and protein regulation. Thymidine kinase 1 (TK1) has been extensively studied as a serum and tissue cancer biomarker. Recently, TK1 has been shown to be upregulated in some cancer tissues and localized on the surface of certain cancer cell lines, and has been suggested as a possible immunotherapeutic target in some cancers. Our goal for this study was to investigate TK1 correlation to placental invasive potential, and therefore establish a function for TK1 localization in these tissues. To do this, we wanted to investigate TK1 placental tissue expression/ localization during normal and obstetric complications and its expression in current placental cell lines. We obtained human placental tissue from various pregnancy conditions including preeclampsia (PE), diabetes (GDM; treated with diet (D) or Insulin (I)) and intrauterine growth restriction (IUGR). For our cell study, immortalized placental cell lines, SW71, JEG-3 and BeWo were used. Immunofluorescence was used for TK1 cell expression in human placental sections. Flow cytometry was performed for TK1 presence in cultured cells. We observed high staining for nuclear and cytosolic expression of TK1 in the control placenta tissues. Compared to controls, there was a marked reduction in TK1 staining in the IUGR and GDM-I placentas which lack nuclear expression of TK1. A decreased for both nuclear and cytosolic TK1 staining, was present in the PE and GDM-D placenta. Flow cytometry showed that SW71 cells had high expression of TK1 on their surface (56%), JEG-3 had low expression of TK1 on their surface (12%), and BeWo cells did not express TK1 on their surface. These findings are interesting because they suggest TK1 surface expression directly correlates with the invasive potential. TK1 protein expression was also measured using standard western blotting techniques to support the data obtained by immunohistochemistry. Furthermore, we established the invasion potential of these placental cell lines using the xCELLigence RTCA DP system between the TK1 positive and negative placenta cell lines. Further confirmation is required; however, these results could assist in understanding the placental/trophoblast invasive mechanism and its correlation in cancer development.

#5161

DARPP-32 and t-DARPP promote non-small cell lung cancer growth through regulation of IKKα-dependent cell migration and Akt/Erk-mediated cell survival.

Sk. Kayum Alam,1 Matteo Astone,1 Ping Liu,2 Stephanie R. Hall,1 Abbygail M. Coyle,1 Erin N. Dankert,1 Dane K. Hoffman,2 Wei Zhang,3 Rui Kuang,3 Anja C. Roden,2 Aaron S. Mansfield,2 Luke H. Hoeppner1. 1 _University of Minnesota, The Hormel Institute, Austin, MN;_ 2 _Mayo Clinic, Rochester, MN;_ 3 _University of Minnesota, Minneapolis, MN_.

Lung cancer is the leading cause of cancer-related death worldwide. In this study, we demonstrate that elevated expression of dopamine and cyclic adenosine monophosphate-regulated phosphoprotein, Mr 32000 (DARPP-32) and its truncated splice variant t-DARPP promotes lung tumor growth, while abrogation of DARPP-32 expression in human non-small cell lung cancer (NSCLC) cells reduces tumor growth in orthotopic mouse models. We observe a novel physical interaction between DARPP-32 and inhibitory kappa B kinase-α (IKKα) that promotes NSCLC cell migration through non-canonical nuclear factor kappa-light-chain-enhancer of activated B cells 2 (NF-κB2) signaling. Bioinformatics analysis of 513 lung adenocarcinoma patients reveals elevated t-DARPP isoform expression is associated with poor overall survival. Histopathological investigation of 62 human lung adenocarcinoma tissues also showed that t-DARPP expression is elevated with increasing tumor (T) stage. Our data suggest that DARPP-32 is a negative prognostic marker associated with increasing stages of NSCLC and may represent a novel therapeutic target.

#5162

Oligodendrocytes might up-regulate the invasiveness of glioblastoma cells via Angiopoietin-2 signaling pathway.

Toshiyuki Kawashima. _Osaka City Univ. Medical School, Osaka, Japan_.

Background & Aims: Glioblastoma (GBM, WHO Grade IV) is considered as the most lethal neoplasm of all solid cancers due to its inherent intensive invasiveness. GBM may arise de novo, or following progression from lower grade gliomas. Recently, solid tumor progression has been recognized as the product of an evolving crosstalk between the cancer cells and its surrounding glial cells. The intensive invasion activity of GBM cells might be regulated by surrounding normal cells such as oligodendrocytes or fibroblasts. In this study, we evaluated the interactive factors between GBM cells and normal glial cells to determine the role of oligodendrocytes in regulating the invasiveness of glioblastoma cells. Methods: Two GBM cell lines, T98G and U251, were used. Two oligodendrocyte cell lines, ODC1 and ODC2, were derived one each from surgical tissues of patients with low-grade glioma (WHO Grade II). Two fibroblasts cell lines, GF1 and GF2, were derived one each from surgical tissues of patients with GBM. Oligodendrocytes and fibroblasts were confirmed by immuno-histochemical staining. The invasive property of GBM cells was analyzed in the presence or absence of conditioned medium from oligodendrocytes or fibroblasts by wound-healing assay and Boyden chamber invasion assay. The proliferation ability of GBM cells was examined by MTT assay. Subsequently Cytokine array was used to examine the cytokines and growth factors in the conditioned medium from oligodendrocytes or fibroblasts. Results: Oligodendrocyte cells, ODC1 and ODC2 cells, significantly (p<0.01) increased the migration and invasion ability of GBM cells, T98G and U251. In contrast, fibroblasts, GF1 and GF2 cells, did not affect the migration and invasion ability of GBM cells. Cytokine array indicated that Angiopoietin-2 was found in the conditioned medium from oligodendrocytes, but not that from fibroblasts. Angiopoietin-2 significantly (p<0.01) increased the invasiveness of GBM, but not their proliferation ability.

Conclusion: Oligodendrocytes might up-regulation of the invasiveness of GBM cells via angiopoietin-2 signaling pathway. Hence, angiopoietin-2 can be considered as a promising target for the treatment of malignant gliomas.

#5163

Rab11 regulates focal adhesion kinase dynamics in sarcoma cell migration.

Ling-Yi Kao, Wan-Chen Wei, Hsiang-Ling Chiu, Yi-Che Wu, Wei-Ting Chao. _Tunghai University, Taichung, Taiwan_.

Focal adhesion (FA) turnover has been demonstrated to play an important role in controlling cell migration and cancer metastasis. FA disassembly was shown to be regulated by endocytosis during cell migration. Rabs are a kind of GTPase that participate in various cellular vesicle transports; however, whether the Rabs play a role in mediating vesicle transport for FA formation is not clear. This study focuses on Rab11, which plays a role in endosome recycling, and examines whether Rab11 has regulatory function in FA formation during cell migration. Wild-type and deficient Rab11 were transfected into human HT1080 fibrosarcoma cells, the cell migration ability was determined by trans-well assay, the localization of Rab11 and focal adhesion molecules were monitored by confocal microscopy and the in vivo study was carried out with subcutaneous xenograft mouse model to evaluate the effect of Rab11 on tumor growth. Results showed Rab11 deficiency inhibited sarcoma cell migration; the Rab11 was also found colocalized with recycled β1 integrin and affected focal adhesion formation. The live image results demonstrated the inactive GDP form Rab11 has no effect on random cell migration; however, the Rab11 knockdown inhibited random cell migration. We further used immunofluorescence and immunoprecipitation and identified the physical interaction of Rab11 and FAK. In the in vivo study, wild-type Rab11 transfected cells increased the tumor volume in xenograft mouse, but not in Rab11-deficient cells. Taken together, the results suggested Rab11 affected cell migration by regulating focal adhesion dynamics through integrin recycling and the effect of Rab11 may be through protein-protein interaction other than GTP activation.

#5164

Mechanosensor MYL9 regulates cancer cell malignancy in gastrointestinal tumors.

Masamitsu Konno, Kiminori Yanagisawa, Katsunori Matsushita, Ayumu Asai, Jun Koseki, Michiya Matsusaki, Shinji Deguchi, Yuichiro Doki, Masaki Mori, Hideshi Ishii. _Osaka University, Suita, Japan_.

Although the stiffness of tissues likely is involved in the malignant behavior of tumors, it remains to be clarified which molecules control the nature, how it is involved in the invasiveness of tumors, or whether any marker is available for the prediction of cancer patient prognosis. In the present study, we studied the role of Myl9, a non-muscle-type, myosin light chain by the experiment in vitro, and assessed the usefulness in the stratification of patients in vivo as the precision medicine. Given that Myl9 is involved in the contraction of cell skeleton, cell hardness, and alterations of cell morphology in various tissues, we first examined whether the expression of the Myl9 is associated with the clinical status of tumors by immunohistochemistry. The results of 45 cases with colon cancer and pancreatic cancer indicated that the increased expression of Myl9 is associated significantly with a reduced provability of overall survival and disease-free survival of those cancers. Moreover, we noted the differential expression of Myl9 in epithelial cancer cells and mesenchymal fibroblasts, i.e., the accumulation of Myl9 staining in cell nuclei of fibroblasts. The experiment of 3-dimensional culture with cancer cells and fibroblasts confirmed the results. Furthermore, we investigated whether Myl9 overexpression is involved in the biologic behavior of gastrointestinal cancer cells. The results showed that the rentiviral-mediated overexpression of Myl9 resulted in an increase of cell proliferation and invasion as well as tumorigenicity in mice. The present study indicates that Myl9 protein can play a fundamental role in the malignant behaviors of gastrointestinal cancer cells.

#5165

Patterning of individual cellular behavior in collective invasion.

Chin-Lin Guo,1 Emilio Sanchez,1 Eileen Fong2. 1 _Academia Sinica, Taipei, Taiwan;_ 2 _NanYang Technology University, Singapore_.

Collective invasion is one of the most important factors accounting for the aggressiveness of solid tumors. Such behavior depends on interactions between tumor cells and between tumor and its surrounding miroenvironment, including stroma cells and extracellular matrix (ECM), which to a certain extent, can be observed in normal cells. Previously we have shown that epithelial cells can use cell-ECM-cell interaction to develop long-range (> 600 microns) invasive patterns. Here, using artificial wounding assay, we show that normal epithelial cells can develop two coexisting, orthogonal migration patterns at the tissue boundary, leading to a periodic patterning of invasion. Such patterning depends on microtubule dynamic instability and results in ROCK activation and YAP1 nuclear translocation, a process to induce epithelial-mesenchymal transition (EMT). Surprisingly, we found that both overexpression and knockdown of an EMT marker, vimentin, can enhance collective invasion, suggesting that the role of vimentin in EMT might be context dependent and needs to be revisited.

#5166

Lysyl oxidase regulates cell surface EGFR, and directionality of cancer cell invasion.

Haoran Tang, Caroline Springer, Richard Marais. _CRUK Manchester Institute, Manchester, United Kingdom_.

Lysyl oxidase (LOX) remodels the tumour microenvironment by cross-linking the extracellular matrix. LOX overexpression is associated with poor cancer outcomes. We find that LOX regulates the epidermal growth factor receptor (EGFR) to drive tumour progression. LOX regulates EGFR by suppressing TGFβ1 signalling through the secreted protease HTRA1. This increases the expression of Matrilin2 (MATN2), an EGF-like domain containing protein that traps EGFR at the cell surface to facilitate its activation by EGF. A novel pharmacological inhibitor of LOX, CCT365623, disrupts EGFR cell surface retention in vitro and delays the growth of primary tumour cells in vivo. In addition to its role in controlling primary tumour growth, LOX expression is also implicated in the processes of cancer cell invasion, and thereby the process of metastasis. Notably, we find that when LOX is depleted or inhibited, cancer cells are no longer able to sence a chemo-attractive gradient, and invade in a directional manner. Importantly, LOX produced by both cancer cells and fibroblasts can mediate this behaviour. Together, we show that LOX regulates EGFR cell surface retention to drive tumour progression. We also find that LOX does not regulate cancer cell invasion per se, but rather that it regulates the directionality of cancer cell migration, and both the cancer and stromal cells appear to contribute to this behaviour.

#5167

Rab27a promotes melanoma cell invasion and metastasis via the regulation of exosome secretion.

DAJIANG GUO,1 Goldie Y. Lui,1 Danae M. Sharp,1 Siew Li Lai,1 Shweta Tikoo,1 Rain Kwan,1 Michelle van Geldermalsen,1 Jeff Holst,1 Nikolas K. Haass,2 Wolfgang Weninger,3 Kimberley A. Beaumont1. 1 _Centenary Institute/University of Sydney, Sydney, Australia;_ 2 _Centenary Institute/University of Queensland Diamantina Institute, Australia;_ 3 _Centenary Institute/University of Sydney/Royal Prince Alfred Hospital, Sydney, Australia_.

Introduction: The Rab GTPase family has been increasingly related to carcinogenesis and cancer biology during the past decade. In melanoma, Rab27a has been identified as a tumor dependency gene. Rab27a is thought to regulate exosome secretion in several cell types, however the exact role of Rab27a in melanoma biology and the underlying mechanisms are not well investigated.

Experimental Procedures: To investigate the influence of Rab27a on melanoma biology and exosome secretion, Rab27a shRNA knockdown (KD) or CRISPR knockout (KO) was performed in human and murine melanoma cell lines. The effect of Rab27a loss on melanoma cell invasion and motility was analyzed by in vitro 3D spheroid invasion and live cell imaging. Rab27a function on melanoma metastasis was investigated using a mouse melanoma spontaneous metastasis model. Exosomes secreted by melanoma cell lines with/without Rab27a KD/KO were purified and characterized by electron microscopy, NanoSight analysis, BCA assay, western blotting and mass spectrometry.

Results: Our results indicate that Rab27a loss in Rab27a high metastatic melanoma cell lines reduced 3D spheroid invasion and cell motility and Rab27a loss also reduced spontaneous melanoma metastasis in vivo. Rab27a KD invasion phenotype can be partially rescued by addition of exosomes from Rab27a replete cell conditioned media, but not exosomes derived from Rab27a KD cells. Loss of Rab27a does not alter the number of exosomes secreted from melanoma cells, but does alter exosome protein composition and morphology. Mass spectrometry analysis has shown that Rab27a replete cell secreted exosomes are more involved in regulating cell invasion and motility than the exosomes from Rab27a KD cells.

Conclusion: In summary, Rab27a promotes the invasion and metastasis of a subset of melanoma cells via the regulation of pro-invasive exosomes, which indicates Rab27a as a potential therapeutic target for preventing melanoma progression.

#5168

Understanding the role of integrins in breast cancer.

Valery Adorno-Cruz,1 Xia Liu,2 Huiping Liu3. 1 _Case Western Reserve Univ., Cleveland Heights, OH;_ 2 _Northwestern University, Chicago, OH;_ 3 _Northwestern University, Chicago, IL_.

Breast cancer is a highly prevalent disease and the second-leading cancer related death in women. Mortality rates have declined a 38% in recent years thanks to early detection and development of treatments. However, the five year survival rate of patients with a distant metastasis is only 26%. Hence, it is important to identify key players that mediate or promote the migration of cells to other organs. Integrins are cell surface receptors that mediate cell adhesion to the extracellular matrix. They are involved in roles such as cell migration and proliferation. In this project we seek to understand the role of integrin α2 in breast cancer metastasis. It has been previously reported that integrin α2 is a metastasis suppressor of a Her2+ model of breast cancer. However, its role in triple negative breast cancer is unknown. We found the integrin α2 gene (ITGA2) a target of has-miR-206 which controls metastasis and stemness of breast cancer. We hypothesize integrin α2 has a metastasis regulating role in triple negative breast cancer. The triple negative breast cancer cell line, MDA-MB-231 has been used to characterize the role of integrin α2 in migration, cell viability and lung colonization of mice injected though tail vein injection. Lung colonization of breast cancer cells in NSG mice is significantly altered by the knockdown expression of integrin α2. Consistent with the phenotypes in migration and invasion. Taken together, these results suggest integrin α2 regulates breast cancer metastasis through modified cell migration and lung colonization.

### Molecular Profiles, Circulating Cancer Cells, and Metastasis

#5169

Novel target molecules for treatment of cancer of unknown primary.

Yoshihiko Fujita,1 Kazuko Sakai,1 Marco De Velasco,1 Takayasu Kurata,2 Hidetoshi Hayashi,1 Kazuhiko Nakagawa,1 Kazuto Nishio1. 1 _Kindai Univ. Faculty of Medicine, Osaka-sayama, Japan;_ 2 _Kansai Medical University, Hirakata, Osaka, Japan_.

We have previously been involved in a multicenter clinical study to predict the primary site of cancer of unknown primary site (CUP) for a site-specific therapy. Based on the microarray analysis of gene expression pattern, we not only predicted the primary site for each CUP patient but also extracted 44 up-regulated genes common to each CUP showing over 2.5-fold change compared to normal tissue (lymph node). To identify genes related to CUP development among these candidate genes, we performed cell-based siRNA screening and estimated to what extend A549 cells reduce the ability of migration, and found 4 genes, GRN, MIF, PRKDC, PSMB4. Konckdown of each of these genes suppressed metastasis of implanted cells from a footpad to a popliteal lymph node. Inhibitors for PRKDC (NU 7441) and PSMB4 (bortezomib) also suppressed the metastasis of parental A549 cells in vivo, suggesting the involvement of these genes in metastasis.

As there is no standard treatment for CUP, drugs targeting immune checkpoints, such as PD-1 or IDO, may reveal promising antitumor activity for CUP. Recent studies suggest that expression levels of PD-L1 (a PD-1's ligand) or IDO may be a biomarker of patient response to anti-PD-1 or anti-IDO therapy, respectively. We analyzed the expression of PD-L1 and IDO for the CUP tissues and found several patients with high expression of either or both proteins who may be most likely respond to these novel immunotherapies.

#5170

The effect of vitamin K on aggressiveness, lipid metabolism and gene expression in triple-negative breast cancer cells.

Leila Kokabee, Sarah Beaudin, JoEllen Welsh. _The State University of New York at Albany, Rensselaer, NY_.

Vitamin K is an essential cofactor in the γ-carboxylation of glutamate (Glu) to γ-carboxyglutamate (Gla), a post-translational modification mediated by the gamma-glutamyl carboxylase enzyme (GGCX) and vitamin K oxidoreductases (VKORC1 or VCORC1L1). All three of these genes are expressed in most breast cancer cell lines and The Cancer Genome Atlas (TCGA) data indicate that upregulation of these genes is associated with reduced survival in breast cancer patients. Since vitamin K is not typically present in cell culture media, γ-carboxylation is limited in vitro despite the presence of GGCX and VKORs. To determine the functional significance of the vitamin K pathway in breast cancer cells, we assessed the effect of vitamin K treatment on cell survival, migration and mammosphere formation. SUM159 and Hs578T cells were maintained in vitamin K1 (5 ug/mL) for at least 3 passages for all assays. Cells passaged with vitamin K produced γ-carboxylated proteins and exhibited increased cell survival after 72h, increased mammosphere formation after 8 days and increased cell migration after 48h compared to cells passaged in ethanol vehicle. Inhibition of VKORC1 with 2 uM warfarin decreased the production of γ-carboxylated proteins and reduced the effects of vitamin K on cell survival and migration but not on mammosphere formation. Microarray profiling and pathway analysis indicated upregulation of several known oncogenic pathways, including PI3K-AKT, VEGF-VEGFR, RANK-RANKL and JAK/STAT in vitamin K-treated SUM159 and Hs578T cells. Thus, γ-carboxylation of proteins driven by vitamin K might indirectly activate these pathways to enhance cell survival, cell migration and mammosphere formation. Additional pathways upregulated in vitamin K-treated cells included adipogenesis, fatty acid biosynthesis and lipid metabolism. We therefore examined lipid droplet formation in cells exposed to vitamin K using Bodipy assays. We detected an increase in lipid droplet formation after 72 hours of vitamin K treatment, which was inhibited by warfarin. Since aggressive breast cancers are often associated with accumulation of cytoplasmic lipid droplets, further studies on the mechanism by which vitamin K increases lipid accumulation are warranted. In conclusion, our data indicate that vitamin K enhances γ-carboxylation, breast cancer aggressiveness and lipid metabolism, suggesting that blocking the vitamin K pathway might be of therapeutic value for patients whose tumors express GGCX and VKORs.

#5171

Tfap2b overexpression contributes to tumor growth & progression of thyroid cancer through AKT&VEGF/PEDF signaling pathway.

Xiaoyan Fu, Zhongyuan Yang, Shuwei Chen. _Sun Yat-sen University Cancer Center, Guangzhou, China_.

TFAP2 is a family of transcription factors implicated in many aspects of development.TFAP2B is a member of the AP2 transcription factor family, which orchestrates a variety of cell processes. However, the roles of TFAP2B in regulating thyroid carcinogenesis remain largely unknown. Here, we investigated the regulatory effects of TFAP2B on thyroid cancer growth & identified the underlying mechanisms of actions in thyroid cancer cells.We examined the expression of TFAP2B in thyroid cancer cell lines & tumor tissues. We also analyzed the TNM staging of TFAP2B in thyroid cancer. Then we investigated the molecular mechanisms by which TFAP2B knockdown or overexpression regulated thyroid cancer cell growth, angiogenesis & apoptosis, & further confirmed the role of TFAP2B in tumor growth in a thyroid cancerxenograft mouse model.TFAP2B was expressed in thyroid cancer lines &tumortissues,especially in anaplastic thyroid cancer. Strong TFAP2B expression showed a positive correlation with the poor differentiation of patients with thyroid cancer . TFAP2B knockdown by siRNA significantly inhibited cell growth & induced apoptosis in thyroid cancer cells in vitro & in a thyroid cancersubcutaneous xenograft model, whereas TFAP2B overexpression promoted cell growth. The observed regulation of cell growth was accompanied by the TFAP2B-mediated modulation of the VEGF/PEDF-dependent signaling pathways in thyroid cancer cells.These results indicate that TFAP2B plays a critical role in regulating thyroid cancer growth & could serve as a promising therapeutic target or diagonistic biomarker for thyroid cancer .

#5172

A functional genomic screen in vivo identifies CEACAM5 as a clinically relevant driver of breast cancer metastasis.

Emily Powell,1 Jiansu Shao,1 Hector Picon,1 Christopher Bristow,1 Zhongqi Ge,1 Caitlin Grzeskowiak,2 Michael Peoples,1 Frederick Robinson,1 Sabrina Jeter-Jones,1 Christopher Schlosberg,3 Fei Yang,1 Yun Wu,1 Ignacio Wistuba,1 Stacy Moulder,1 Fraser Symmans,1 Kenneth Scott,2 John Edwards,3 Han Liang,1 Timothy Heffernan,1 Helen Piwnica-Worms1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Baylor College of Medicine, Houston, TX;_ 3 _Washington University in St. Louis, St. Louis, MO_.

Metastases are responsible for the vast majority of deaths due to breast cancer. Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by high rates of metastasis and poor prognosis. We are employing patient-derived xenograft (PDX) models of TNBC to identify drivers of metastasis. Tumor samples are obtained from the breast tumors of patients with TNBC and engrafted immediately into the humanized mammary fat pads of immune-compromised mice. Lentiviral transduction was employed to express bioluminescent and fluorescent markers in two independent PDX models of TNBC. Using these models, we demonstrated that human breast tumors are capable of completing all stages of the metastatic cascade in mice, and metastatic lesions are observed in organs normally found in patients with metastatic breast cancer including lung, liver, bone, brain, and lymph nodes. Dynamic and reversible shifts in the epithelial-to-mesenchymal transition (EMT) were observed as tumors metastasized to lung and were re-passaged to recipient mouse mammary fat pads (MFPs). Lung metastases were isolated using bioluminescence imaging and lung metastasis gene expression signatures were generated. Metastasis signatures from two independent PDX models were compared to identify genes that were commonly deregulated in lung metastases relative to corresponding mammary tumors. Comprehensive gain-of-function screens were then conducted in vivo to identify functional drivers of TNBC metastasis. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) was identified as a metastatic driver in this screen. CEACAM5 mRNA and protein levels were elevated in lung metastases relative to corresponding MFP tumors in mice. In addition, we demonstrated that CEACAM5 expression was upregulated in the lung metastases of breast cancer patients, and its expression inversely correlated with patient survival. Our data indicate that the metastatic function of CEACAM5 is to promote growth of breast tumors in the lung by inducing MET (mesenchymal-to-epithelial transition).

#5173

Genetic profiling of central nervous system dissemination of B-acute lymphoblastic leukemia reveals clonal selection and therapeutic vulnerability.

Robert J. Vanner,1 Stephanie M. Dobson,2 Ildiko Grandal,3 Olga Gan,2 Jessica McLeod,2 James Kennedy,2 Veroniqu Voisin,1 Abdellatif Daghrach,4 Erwin M. Schoof,5 Cynthia Guidos,3 Jayne Danska,3 Esme Waanders,6 Mark Minden,2 Charles G. Mullighan,7 John E. Dick2. 1 _University of Toronto, Toronto, Ontario, Canada;_ 2 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada;_ 3 _Hospital for Sick Children, Toronto, Ontario, Canada;_ 4 _University Health Network, Toronto, Ontario, Canada;_ 5 _University of Copenhagen, Copenhagen, Denmark;_ 6 _Radboud University, Nijmegen, Netherlands;_ 7 _St Jude Children's Research Hospital, Memphis, TN_.

B-cell acute lymphoblastic leukemia (B-ALL) readily disseminates to the leptomeninges of the central nervous system (CNS). CNS involvement is more frequent in certain poor prognosis subgroups including patients with MLL-AF4 translocations, and late CNS relapse is often lethal. The biology and clonal history of CNS leukemia are poorly defined and consequently therapies exploiting drivers of metastasis are lacking. To characterize leptomeningeal leukemia we performed targeted DNA sequencing, SNP copy number analysis, RNA sequencing, and functional analysis on cells isolated from the bone marrow (BM) and CNS of xenografts generated from a cohort of paired diagnosis and relapse samples from 14 B-ALL patients. The majority of patient samples disseminated to the CNS following intrafemoral injection into irradiated NSG mice, with greater CNS involvement in xenografts derived from relapse patient samples. Secondary transplantation of both BM- and CNS-purified cells demonstrated their capacity to re-engraft BM, CNS, and spleen. Targeted-sequencing results were analyzed using a Bayesian clustering method to determine the clonal composition of matched BM and CNS, demonstrating discordance in subclonal prevalence in nearly half the xenografts tested. Xenografts derived from two patient samples demonstrated recurrent enrichment of a particular subclone in the CNS versus BM. Similarly, copy number analysis identified frequent discordance between BM and CNS tissues within individual mice. All xenografts from one patient exhibited chromosome 6p and 17p hemi-deletions that were exclusive to CNS cells. While these data suggest that individual B-ALLs harbor subclones with CNS tropism, there were no recurrently enriched single nucleotide mutations or copy number alterations across all patients. RNA-sequencing of 45 BM and CNS pairs from primary xenografts demonstrated that CNS-isolated cells were consistently distinct from their matched BM. GSEA analysis of xenografts generated from patients with MLL-AF4 translocations (MLL) (n=2 patients, 26 mice), identified CNS cell enrichment of gene sets related to mRNA translation and nascent peptide elongation compared to BM. MLL-CNS cells exhibited altered rates of protein synthesis compared to BM cells from the same mouse. The clinically-approved translation inhibitor omacetaxine mepesuccinate effectively diminished protein translation rates of CNS isolated cells and reduced CNS engraftment by four fold in xenografts derived from two MLL-AF4 patients. These data demonstrate that the CNS microenvironment selects for the outgrowth of B-ALL cells with genetically and/or biologically distinct properties. Moreover, we demonstrate that in MLL-AF4 patients, altered protein synthesis occurs in CNS dissemination and that targeting this process may clinically benefit patients with CNS disease.

#5174

Lymph node dissemination gene-miRNA signature as a prognosis marker in human thyroid cancer.

Emmanuelle Ml Ruiz, Chih-Hong Wang, Tianhua Niu, Mohamed Hassan, Emad Kandil. _Medecine School of Tulane University, New Orleans, LA_.

Most of the thyroid cancers (TCs) have excellent prognosis if detected early and treated appropriately. 50% of the deaths are due to aggressive variants of metastatic papillary and follicular TCs which have higher risk of recurrence, shortened disease free survival and death. The aim of this study is to identify a gene and/or miRNA signature associated with the lymph node dissemination as a predictive marker for early detection and intervention of TCs and spare patients with non-aggressive TCs from unnecessary surgery and non-surgical treatment.

The bioinformatics analysis suggested a gene/miRNA signature of 25 genes (8 up regulated in N1 samples and 17 down regulated) and 2 miRNAs (1 up and 1 down regulated), based on machine learning regression algorithms classifying the samples between their N0/N1 stage with the highest predictive power estimated by the AUC (Area Under Curve) of a ROC (Receiver Operator Curve) analysis of 0.83 for the gene signature and 0.68 for the miRNA signature. A score, reflecting the expression of the gene/miRNA signatures differentiates the N0 and N1 samples with a p-value inferior to 2x10-16. Mann-Whitney tests were performed to estimate the score's correlation with the clinical parameters. The gene signature seems to be dependent of the cancer type (papillary versus follicular) and independent of the BRAF mutation status and TNM stages.

A combination of a correlation analysis and predictive algorithms have been used to identify potential interactions between the 25 genes of the signature and the 9 differentially expressed miRNAs. Two clusters of genes and two clusters of miRNAs have been identified based on their potential interaction and expression. CLCNKA, MORN5 and AGXT2L1 genes are downregulated in the N1 samples and are potentially targeted by up regulated miR-526b-5p, miR-520a-3p and miR-206. At the opposite, the DSG3, KRT5, FOXR2, PRKAG3 and VGF genes are up regulated in the N1 samples, are potentially targeted by down regulated miR-873-5p, miR-184, miR-483-3p and miR-483-5p.

The gene/miRNA signatures are currently validated. The predictive power of the signature scores has been estimated in other RNAseq datasets. While the q-PCR analysis of thyroid cancer specimen has been designed following a laser capture microdissection. The predictive score will be expected to estimate the probability of disease dissemination to the lymph nodes, computing the expression scores and the probability of the gene/miRNA signature to correctly classify the samples. Moreover, the genes/miRNA clusters interactions will be evaluated by dual luciferase assay and their functions in thyroid cancer dissemination will be assessed.

The preliminary results indicate that the gene/miRNA signature could be a predictive marker for the classification of TCs based on their lymph node dissemination. Targeting this interactome could lead to a new therapeutic strategy for the aggressive thyroid carcinoma.

#5175

Advancing the molecular understanding of stage I colorectal cancer.

Philip Dunne. _Queen's Belfast, Belfast, United Kingdom_.

Background: There are ~1.4 million cases of colorectal cancer (CRC) annually worldwide. Bowel cancer screening (BCS) detects cancers and high-risk adenomas earlier; previously stage I accounted for 12% of CRC, but 42% of screen-detected cancers are now stage I. This study is based on the hypothesis that within the early lesions detected by BCS there are "hopeful monsters"; a term used to describe highly aggressive tumours that are simply being caught earlier while they are still potentially curable. Colorectal tumour evolution models have proposed the "Big Bang" of tumour growth, where a single expansion in adenoma development dictates disease outcome. In line with the "hopeful monsters" theory, this model reason that some tumours are "born-to-be-bad" from the earliest point in CRC tumour evolution.

Aims: This proposal aims to develop a molecular stratifier of lethal vs non-lethal early-invasive disease based on comprehensive molecular pathological profiling, improved biological understanding and multiple tiers of validation to inform the management of CRC disease at the earliest stage. Methods: In contrast to stage II-IV CRC, there are limited stage I molecular studies, reducing opportunities to identify lethal early-disseminating tumours in patients who account for ~50% of screen-detected cancers. This study is undertaking collection and molecular profiling of a cohort of retrospective stage I tissue (n=200), enriched for patients that experienced relapsed, to identify factors associated with early-dissemination.

Results: Pathological characterisation of the stage I cohort indicated that histological factors such as fibroblast content or depth of invasive front are not associated with eventual metastatic relapse. Unsupervised analysis highlighted a detectable shift in transcriptional signalling between recurrent and non-recurrent samples. Further supervised analysis indicates that intrinsic "stem-like" factors may be more prognostic than extrinsic factors in stage I.

Conclusions: In order to find any effective treatment you have to first understand the biology underpinning disease. Given the increasing numbers of early stage patients being diagnosed as a result of BCS, and the paucity of tissue cohorts and focussed molecular studies of stage I CRC, this study aims to increase our understanding of specific factors underpinning prognosis at this early stage.

#5176

Cell free malignant ascites fluid facilitates gastric adenocarcinoma peritoneal metastasis.

Chantelle A. Janeiro,1 Vivian Stavrakos,1 Malak Alzahrani,2 Roni F. Rayes,2 France Bourdeau,2 Betty Giannias,2 Nicholas Bertos,2 Veena Sangwan,2 Jonathan Cools-Lartigue,2 Jonathan D. Spicer,2 Lorenzo E. Ferri2. 1 _McGill University, Montreal, Quebec, Canada;_ 2 _McGill University Health Centre, Montreal, Quebec, Canada_.

Gastric adenocarcinoma is the fastest rising malignancy in North America. It is commonly associated with malignant ascites (MA), the pathological accumulation of fluid containing cancer cells in the peritoneum. Peritoneal metastasis is the most common site of gastric cancer (GC) progression after curative intent surgery and is the leading cause of death. Upon peritoneal dissemination, the malignant process is deemed non-curative as it is rarely amenable to surgical resection and chemotherapeutic regimens are simply palliative. We hypothesize that cell-free MA increases the potential of GC cells to worsen peritoneal metastasis. Gastric adenocarcinoma cell lines (human: MKN-45, SNU-5, KATO III, OKAJIMA; murine NCC-S1, NCC-S1M) were incubated with cell-free MA and their metastatic ability assessed with static in vitro adhesion assays, as well as migration assays. A novel ex vivo peritoneal metastasis model further corroborated the in vitro results, where cancer cell adhesion to stripped human peritoneum was assessed by co-incubation with non- and pre-stimulated cancer cells. Gross liver metastasis was monitored over several weeks after in vivo intra-peritoneal and intra-splenic injections, whereby C57BL/6 mice were inoculated with control media or stimulated NCC-S1M cells. Incubation of human GC cells and/or human peritoneal mesothelial cells (HPMC) with cell-free MA resulted in a significant three to five-fold increase of GC cell adhesion to HPMC compared to non-stimulated condition (P<0.05), as measured by inverted fluorescent microscopy. In addition, the murine GC cells incubated with MA showed a significant eight-fold increase on average (P<0.05) in GC cell adhesion to HPMC compared to non-stimulated condition. Cell-free MA was shown to significantly enhance ex vivo SNU-5 cell adhesion to stripped human peritoneum by a two to four-fold increase (P<0.05) compared to non-stimulated condition. Liver metastases were visible in mice that received in vivo injections of ascites-stimulated NCC-S1M cancer cells by day 25, yet not in mice inoculated with control media. Several factors (ANG-2, HGF, ICAM-1, IL-8, TIMP-2, uPAR, VEGF, NAP-2, MIF) were shown to be upregulated in MA samples compared to a cirrhotic ascites control, using a multiplex ELISA. In particular, VEGF was upregulated 11 to 25-fold, and MIF two to 12-fold. The results demonstrated that MA plays a significant role in facilitating GC cell adhesion to peritoneal mesothelia, an important early step in the peritoneal metastatic cascade. MA must therefore provide an environment that supports tumour growth and spread. A more comprehensive understanding of the molecular network is essential to determine the role of cell free MA fluid in GC progression, allowing for the identification of potential therapeutic targets for this aggressive malignancy.

#5177

Metastasis: Leveraging transcriptomics to identify potential therapeutics.

Matthew Ung, Jason M. Funt, Andrew C. Lysaght, Jenny Zhang, Renan Escalante-Chong, Gregory Koytiger, Sarah Kolitz, Rebecca Kusko, Benjamin Zeskind, Kevin D. Fowler. _Immuneering Corporation, Cambridge, MA_.

Five-year survival rates for patients whose cancer has metastasized are dramatically lower than for those with localized disease. However, few therapeutic development efforts specifically focus on inhibiting the process of metastasis.

Here we perform a systematic pan-cancer analysis of RNA-seq data from The Cancer Genome Atlas to identify targetable gene expression changes in primary tumors that confer metastatic potential. In 4,844 patients comprising 13 different cancer types, we construct a gene expression signature that captures consistent pan-cancer alterations that differ between primary tumors associated with node-negative disease with no recorded distant metastasis (N0 not M1) and primary tumors from patients with node-positive disease (N1, N2, or N3). In order to identify potential therapeutic targets, we query this metastasis signature against drug-induced transcriptomic signatures from CMAP and LINCS. Among the top candidates is Chembl410456, which modulates the TGFβ and Wnt/β-catenin signaling pathways, among others. Genes upregulated in metastatic primary tumors and downregulated by Chembl410456 include SERPINE1 and TWIST1. We observe that low SERPINE1 and TWIST1 expression also associate with improved prognosis in multiple cancers, including colon adenocarcinoma (p=0.007 and p=0.014, respectively). Other top-ranked drugs also target the TGFβ signaling pathway. To summarize, we leverage public RNA-seq data to identify a pan-cancer metastatic signature that can be interrogated to systematically identify potential therapeutics that target metastasis. Building on this, we pinpoint antimetastasis drug candidates including several with mechanisms of action involving modulation of TGFβ and/or Wnt/β-catenin pathways.

#5178

MUC13 is a novel molecular signature, for early detection and metastatic colorectal cancer.

Manish K. Tripathi,1 Chidi Zacheaus,1 Kyle Doxtater,1 Zachary Stiles,1 Fatemeh Keramatnia,1 Nadeem Zafar,2 Mahul Amin,1 Meena Jaggi,1 Subhash Chauhan1. 1 _University of Tennessee Health Science Center, Memphis, TN;_ 2 _VA, Puget Sound, Seattle, WA_.

Objective: Colorectal cancer (CRC) is a leading cause of cancer mortality affecting over a million people every year. Biological markers for early detection and distant metastatic disease in patients with CRC are not well defined. We have identified transmembrane mucin MUC13, which is expressed in normal colon mucosa to be highly expressed in colorectal cancer, but the underlying pathways and the signaling mechanisms involved in CRC pathogenesis are not known. Our studies suggest MUC13 correlation with demographic and clinicopathologic characteristics.

Materials and Methods: Retrospective institutional tumor registry was reviewed to identify patients with resected colon adenocarcinoma. Archived FFPE tissue blocks were reviewed by an experienced pathologist. Selected representative tissue blocks were serially sectioned at 4 µm. IHC staining was performed using an in-house MAb for MUC13. Slides were digitally scanned and analyzed qualitatively as well as quantitatively using a modified H-score based on the intensity of expression and percentage of stained cells. MUC13 was correlated with disease stage, aggressiveness and inflammatory markers, indicating poor prognosis in CRC patients. MUC13 splice variants were probed using novel designed probes. MUC13 SNPs were identified using unbiased approach by analyzing dpGAP database.

Results: 196 tissues, of which 56.1% were female, 52% were white, and the median age at resection was 70. 38 (19.4%) were stage I, 64 (32.7%) stage II, 84 (42.9%) stage III, and 10 (5.1%) stage IV. 100% of colon adenocarcinoma tissues stained positively for MUC13, including definitive tumor epithelial staining, little-to-no background stromal staining, and mild staining of adjacent normal colon mucosa. Typical colon adenocarcinoma cells exhibited strong apical membranous staining with varying degrees of cytoplasmic staining. Advanced stage tumors were noted to more frequently exhibit basolateral and/or circumferential membranous staining compared to early stage tumors which more frequently displayed apical membrane staining alone. Five MUC13 transcript variants were identified by database analysis. Two of the protein coding MUC13 variants (long and short) showed differential expression in aggressive cell lines and patient tissues. Cyclic turnover between the short and long isoforms were observed during Anoikis resistance, cortisol, alcohol and BAP treatments. SNPs have been associated with increased risk of cancer incidence or fatality. Relevant MUC13 SNPs have been identified through systematic analysis of patient databases (dpGAP database). Verification and correlation of identified SNPs with patient disease stage and prognosis will also be presented.

Conclusion: This is the first study to correlate MUC13 expression with disease outcome. Alternative transcripts and single nucleotide polymorphisms gave genetic insight of the role of MUC13 in CRC pathogenesis.

#5179

Uncovering the signaling landscape controlling breast cancer cell migration identifies splicing PRPF4B as a metastasis driver.

Bob van de Water,1 Erik Danen,1 Esmee Koedoot,1 Maria Rogkoti,1 Michiel Fokkelman,1 Sylvia Le Devedec,1 John Martens,2 Peter Stoilov,3 John Foekens2. 1 _Leiden Univ., Leiden, Netherlands;_ 2 _Erasmus Medical Center, Rotterdam, Netherlands;_ 3 _West Virginia University, Morgantown, WV_.

Metastasis is the major cause of death in cancer patients and migration of cancer cells from the primary tumor to distant sites is the prerequisite of metastasis formation. Here we applied an imaging-based RNAi phenotypic cell migration screen uing two highly migratory basal breast cancer cell lines (Hs578T and MDA-MB-231 to provide a repository of candidate metastasis drug targets. We screened ~4,200 individual target genes covering most cell signaling components. We discovered 133 and 113 migratory modulators of Hs578T and MDA-MB-231, respectively, of which 45 genes were common denominators of cell migration. Interaction networks of candidate migratory modulators were in common with networks of different clinical breast cancer prognostic and metastasis classifiers. The splicing factors PRPF4B and BUD31 and the transcription factor BPTF were amplified in human primary breast tumors and the expression was associated with metastasis-free survival. Depletion of PRPF4B, BUD31 and BPTF caused primarily downregulation of genes involved in focal adhesion and ECM-interaction pathways. PRPF4B was essential for TNBC cell migration and critical for breast cancer metastasis formation in vivo, making PRPF4B a candidate for further drug development. Our systematic phenotypic screen provides an important repository for signaling determinants that functionally drive cancer cell migration.

#5180

MicroRNA and triple negative breast cancer.

BIN YI, Ruixia Ma, Yaguang Xi. _LSU Health Sciences Center New Orleans, New Orleans, LA_.

In 2017, a total of 252,710 new cases and 40,610 deaths from breast cancer in the United States were estimated by the American Cancer Society. Breast cancer is the most common malignancy and the second leading cause of death among American women. In this application, we will focus on triple negative breast cancer (TNBC), which is viewed by oncologists as a problematic and unpredictable sub-category of breast cancer because of higher rates of recurrence and poorer prognosis. TNBC accounts for up to 20% of all breast cancers and is highly prevalent in minority and young women. On average, 70% of women with metastatic TNBC die within 5 years, regardless of chemotherapy or other treatments. As such, there is an urgent medical need to develop more effective drugs to manage this deadly disease that raises a health disparity concern, especially in the State of Louisiana. Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used drugs for the treatment of pain, fever, and inflammation. Epidemiological studies have reported that the long term use of NSAIDs can prevent the occurrence multiple types of cancers, including breast cancer. However, their long term use for chemoprevention is not recommended because of toxicities associated with cyclooxygenase (COX) inhibition and the suppression of physiologically important prostaglandins. Our results show that the NSAID, sulindac sulfide (SS) and two non-COX inhibitory derivatives, sulindac sulfide amide (SSA) and sulindac benzylamine (SBA), can significantly inhibit the growth of the major subtypes of TNBC cells (basal-like, mesenchymal, and luminal). In addition, the compounds significantly inhibit tumor cell invasion. While studying the mechanism, we found that four oncogenic miRNAs, miR-10b, miR-17, miR-21, and miR-9 can be downregulated by SS and derivatives, and they were reported to promote tumor metastasis exclusively. Therefore, we conclude that those oncogenic miRNAs are involved in anti-invasive activities of SS and derivatives in TNBC cells.

#5181

KSRP suppresses cell invasion and metastasis through miR-23a-mediated EGR3 mRNA degradation in non-small cell lung cancer.

Ke-Fan Pan,1 Ming-Hsien Chien,2 Wei-Jiunn Lee,3 Michael Hsiao,4 Kuo-Tai Hua1. 1 _Graduate Institution of Toxicology, Taipei City, Taiwan;_ 2 _Graduate Institution of Clinical Medicine, Taipei City, Taiwan;_ 3 _Department of Medical Education and Research, Taipei City, Taiwan;_ 4 _The Genomics Research Center, Taipei City, Taiwan_.

KH-type splicing regulatory protein (KSRP) is a single-strand RNA binding protein which regulates mRNA stability either by binding to AU-rich elements (AREs) of mRNA 3′UTR or by facilitating miRNA biogenesis to target mRNA. Unlike its well-characterized function at the molecular level in maintaining RNA homeostasis, the role of KSRP in cancer progression remains largely unknown. Here we investigate the role of KSRP in non-small cell lung cancer (NSCLC). We first examined KSRP expression by immunohistochemistry in a cohort containing 196 NSCLC patients and observed a strong positive correlation between KSRP expression and survival of NSCLC patients. Multivariate analysis further identified KSRP as an independent prognostic factor. Manipulating KSRP expression significantly affected in vitro cell mobility and in vivo metastatic ability of NSCLC cells. Microarray analysis identified an ARE-containing gene, EGR3, as a downstream effector of KSRP in NSCLC. Interestingly, we found that KSRP decreased EGR3 mRNA stability in an ARE-independent manner. By screening KSRP-regulated miRNAs in NSCLC cells, we further found that miR-23a directly binds to EGR3 3′UTR, reducing EGR3 expression and thereby inhibiting NSCLC cell mobility. Our findings implicate a targetable KSRP/miR-23a/EGR3 signaling axis in advanced tumor phenotypes

#5182

Activity and clinical relevance of autotaxin and lysophosphatidic acid pathways in high-grade serous carcinoma.

Hadil Onallah,1 Claes G Trope,2 Thea E. Hetland Falkenthal,3 Ben Davidson,2 Reuven Reich1. 1 _The Hebrew University of Jerusalem, Jerusalem, Israel;_ 2 _University of Oslo, Oslo, Norway;_ 3 _Oslo University Hospital, Oslo, Norway_.

Lysophosphatidic acid (LPA) is a bioactive phospholipid with mitogenic and growth factor-like activities affecting cell invasion, cancer progression and drug resistance. It is produced mainly by Autotaxin and acts on six G protein-coupled receptors, LPAR1-6. LPA has recently been implicated as a growth factor present in ascites of ovarian cancer patients. However, mitogenic pathways stimulated by LPA via its receptors are still far uncharacterized. Here we show that three LPA receptors are involved in the progression of the malignancy by the activation of both AKT and ERK pathways. We analyzed a set of 155 samples of high grade serous ovarian carcinoma (HGSC) from the three different anatomic sites of the disease. Results show that LPAR2 mRNA was overexpressed in HGSC cells in effusions compared to solid lesions, with opposite findings for LPAR3 and LPAR6 mRNA and ATX protein that was found mainly in exosomes. LPAR3 levels were significantly higher in pre-chemotherapy effusions compared to post-chemotherapy specimens (p=0.025). Higher expression of LPAR1 (p=0.037), LPAR2 (p=0.025) and LPAR5 (p=0.021) was significantly associated with shorter overall survival. Furthermore, we combined 3D (spheroids) and 2D cell culture models with gene editing using the CRISPR/Cas9 method for LPAR2, LPAR3 and LPAR6 and tested the effects of their silencing in vitro using OVCAR3 and ES2 cell lines. The p-ERK/ERK ratio was significantly elevated in LPAR3KO OVCAR3 cells cultured both in 2D and 3D form. Yet, LPAR2KO OVCAR3 cells expressed low levels of p-ERK/ERK in the 3D form, suggesting that LPAR3 inhibits p-ERK activity downstream, while LPAR2 activates it. ES2 LPAR2KO cells expressed elevated levels of p-AKT/AKT ratio in both the 2D and 3D forms. OVCAR3 LPAR6KO had low p-AKT/AKT ratio when cultured in 2D, but high ratio in the 3D form. LPAR KO further inhibited ovarian cancer invasion and motility.Our results demonstrate, for the first time, significant changes in LPARs' mRNA levels and Autotaxin protein levels with changes in anatomic sites of the disease that correlate to clinic-pathological parameters. Our study identifies a specific molecular machinery triggered by LPA and its' receptors that modulate tumor and can serve as therapeutic targets in this malignancy.

#5183

Using transposon elements to elucidate the genetic mechanisms of HCC-associated lung metastases.

Lilian H. Lo,1 Amy P. Chiu,1 Xiao-Xiao Li,1 Barbara R. Tschida,2 Dewi K. Rowlands,3 David A. Largaespada,2 Vincent W. Keng1. 1 _The Hong Kong Polytechnics University, Hong Kong;_ 2 _Masonic Cancer Center, Minneapolis, MN;_ 3 _Laboratory Animal Services Centre, Hong Kong, Hong Kong_.

Metastases account for most cancer-related deaths as a result of primary cancer spreading to distant sites. Amongst human hepatocellular carcinoma (HCC), there is a drastic decrease in the survival rate observed in patients with early stage localized HCC to late stage metastasis-associated HCC. Importantly, the genetic mechanisms associated with metastasis-associated HCC remains elusive. Therefore, a forward genetic screen in mice using Sleeping Beauty (SB) insertional mutagenesis system was developed to identify genes associated with metastasis-associated HCC.

Truncated version of the epidermal growth factor receptor (Egfr) gene was frequently detected in both mouse primary liver tumors and lung metastases, which was confirmed by DNA sequencing and RNA sequencing. This indicates the importance of Egfr mutations for liver tumorigenesis and the metastasis process. More importantly, additional candidate genes involved in the metastasis process were also identified by our forward genetic screen and showed to be relevant in human HCC patients at the M1 cancer metastasis stage. Taken together, our study shows that the SB screen yields a high fraction of relevant events in human liver cancer and can provide valuable information on the evolution of metastasis-associated HCC.

The authors declare that they have no competing interests.

#5184

Expression and role of regulator of G-protein signing 5 in squamous cell carcinoma of the tongue.

Yushi Abe,1 Hirohisa Yano,1 Jun Akiba,2 Reiichirou kondou,1 Sachiko Ogasawara,1 Ken Nakamura,1 Zingo Kusukawa1. 1 _Kurume University School of Medicine, Kurume, Fukuoka, Japan;_ 2 _Kurume university, Kurume, Fukuoka, Japan_.

Objective: Regulator of G-protein signing (RGS) 5 is a member of the RGS family and acts as GTPase-activating proteins for heterotorimeric G protein αsubunits, negatively regulating G-protein signaling. RGS5 was reportedly expressed in the heart, lung, skeletal muscle and small intestine and relates with tumor angiogenesis and gestational hypertension. It was reported that RGS5 was related with the invasion and metastasis of cancers, such as non-small lung cancer and hepatocellular carcinoma. In the present study, we examined RGS5 expression and its relation with invasion in squamous cell carcinoma (SCC) of the tongue. Materials and Methods: For immunohistochemical analysis for RGS5, we used SCC tissues of the tongue obtained from 43 patients who underwent resection at Kurume University Hospital from 2011 to 2015. The staining intensity was graded as 0, negative; 1, weak; 2, moderate; 3, strong. The staining intensity in the normal epithelium was used as an internal control. We examined relationship between RGS5 expression in invasive portions and clinicopathological features (e.g., lymph node metastasis, depth of tumor, and mode of invasion, and so forth). Mode of invasion was classified into 3 levels, i.e., IFNa (expansive), IFNc (invasive), IFNb (between INFa and INFc). Depth of tumor was measured and classified into 2 levels: < 5 mm and ≥ 5mm. Comparison and estimation of cumulative survival rates were performed using the Kaplan-Meier curves and log rank test. Result: Non-cancerous squamous cells expressed RGS5 solely in nucleus. SCC cells in superficial portion of tumor nodules often expressed weak nuclear RGS5 expression. In invasive portions of the tumor nodules, SCC cells expressed significantly lower nuclear and higher cytoplasmic RGS5 expression. A significant correlation was found between the expression score of RGS5 at invasive portions and mode of invasion (P=0.009). When the 43 cases were subdivided into two groups, i.e., expression group (score 1-3) and non-expression group (score 0), significant differences were observed in mode of invasion (P=0.0042), lymph node metastasis (p=0.0314), depth of tumor (p=0.0239) between the two groups. The log-rank test revealed that postoperative early lymph node metastasis was significantly higher in the expression group (p= 0.0415). Conclusion: This study suggests that RGS5 expression is related with tumor invasion in SCC of the tongue and that RGS5 can be a predictor of postoperative early lymph node metastasis.

#5185

Identification of a new candidate therapeutic target for gastric cancer by in silico analysis.

Tomohiro Kohmoto,1 Kiyoshi Masuda,1 Katsutoshi Shoda,2 Yuji Fujita,2 Shoichiro Tange,1 Daisuke Ichikawa,3 Eigo Otsuji,2 Issei Imoto1. 1 _Tokushima University, Tokushima, Japan;_ 2 _Kyoto Prefectural University of Medicine, Kyoto, Japan;_ 3 _Yamanashi University, Yamanashi, Japan_.

Gastric Cancer (GC) is a leading cause of global cancer mortality, with high incidence rates in Asia including Japan. Although recent studies have provided valuable insights into identification of major driver genes, there are still little therapeutic targets and prognostic markers of GC. Here, we report that a novel driver gene, OverExpressed in Gastric Cancer 1 (OEGC1), would induce cell proliferation and migration of GC.Analyzing gene expression profiles by RNA-Seq and relevant clinical data in TCGA dataset, we identified candidate genes whose mRNA expression was higher in tumor tissues than in non-tumorous and associated with poor prognosis. Through further expression analysis and Cox proportional hazards regression analysis of those candidates, we identified OEGC1 as the most prominent candidate for driver gene. OEGC1 mRNA was expressed highly especially in intestinal type GC. The prognosis of the OEGC1 high-expressed intestinal type GC patients was as poor as that of diffuse type GC patients. Immunohistochemical analysis showed that overexpression of OEGC1 was ectopically found in the tumor cell junctions located in the infiltrated region of tumor. By silencing endogenous OEGC1 using siRNAs, cell proliferation was significantly suppressed and G0/G1 arrest was induced in NUGC3, AGS and MKN7 cells with relatively higher OEGC1 expression. Transwell assay showed that the number of migrated cells was significantly lower for siRNA-OEGC1-transfected cells than for control cells. Our findings suggested that aberrant expression of OEGC1 might play an important role in tumorigenesis of GC, and that OEGC1 might be a possible therapeutic target in GC.

#5186

Overexpression of CXCR4 and JUNB in disseminated tumor cells (DTCs) isolated from breast cancer patients before neoadjuvant treatment.

Galatea Kallergi,1 Oliver Hoffmann,2 Nefeli Zacharopoulou,1 Christos Stournaras,1 Vassilis Georgoulias,1 Sabine Kasmir-Bauer2. 1 _Univ. of Crete Medical School, Heraklion, Greece;_ 2 _University Hospital Essen, Germany, Essen, Germany_.

Introduction: The presence of Disseminated (DTCs) and Circulating Tumor Cells (CTCs) in the bone marrow (BM) and the blood of breast cancer (BC) patients are poor prognostic factors. We have recently shown that CTCs derived from metastatic BC patients, overexpressed CXCR4 and JUNB and this expression was related to clinical outcome. In this study we applied the same methodology to BM of non-metastatic BC patients at primary diagnosis, before the onset of therapy.

Methods: Bilateral BM (10 ml) aspirations were assessed for the detection of DTCs by a Ficoll density gradient centrifugation and subsequent centrifugation of the BM cells onto glass slides [1x106 mononuclear cells (MNCs) per slide]. Triple staining experiments with panytokeratin/CXCR4/JUNB antibodies were performed. An expression pattern of the examined proteins was created, using three different BC cell lines (SKBR3, MDA-MB231, and MCF7). Consequently, 10 BM aspirations from BC patients were analyzed. Mean intensity for each examined molecule was calculated automatically with the ARIOL system.

Results: Quantification of CXCR4 among BC cell lines revealed that CXCR4 expression followed the subsequent hierarchy SKBR3>MCF7>MDA-MB231 with higher expression in SKBR3 and lower in MDA-MB 231 cells, respectively. Accordingly, the expression pattern of JUNB in cell lines was SKBR3>MCF7>MDA-MB231. Consequently, we analyzed 10 BM samples from primary BC patients. The (CXCR4+JUNB+CK+) phenotype was observed in 90% (9/10) of the samples. In addition, the (CXCR4-JUNB+CK+) phenotype was detected in 20% (2/10) of the patients. Interestingly, the (CXCR4+JUNB-CK+) phenotype was not observed in any of the patient's samples. Quantification of CXCR4 expression revealed that the mean intensity in DTCs (19.1±2) was statistically higher than in MCF7 (13±0.51; p=0.006) and MDA-MB231 (10.22±0.7; p= 0.0002) cell lines. It was also higher compared to normal donors' PBMCs (10.6±1.01; p=0.0004). In addition, the mean intensity of JUNB was statistically increased in DTCs (17.1±1.66) compared to MCF7 (8.9±0.56; p=0.00001) and MDA-MB231 (6.6±0.15; p= 0.00001) cells, respectively. Furthermore, it was increased compared to normal donors' PBMCs (5.2±0.15; p=0.0002). In two patients, we had available samples after treatment. In one patient, DTCs were eliminated after neoadjuvant therapy, for the other patient, DTCs were reduced from 10 to two DTCs. However, these cells were double positive for CXCR4 and JUNB.

Conclusion: CXCR4 and JUNB were overexpressed in DTCs derived from primary BC patients, in agreement to previous findings in CTCs, implying an upregulation of the CXCR4 pathway in these cells. Quantification of this expression potentially defines a subgroup of patients with high expression of CXCR4 and JUNB that could benefit from targeted therapies.

#5187

Metastatic lung cancer-derived exosomes stimulate premetastatic niche in liver: Beyond seed soil hypothesis.

Kanisha Shah, Dr. Rakesh M. Rawal. _Gujarat University, Ahmedabad, India_.

Background: Tumor-derived exosomes (TDEs) are small membrane bound extracellular vesicles (EVs) that have the ability to alter their immediate microenvironment (ME) through cell-cell interaction and are known to be critical modulators of pre-metastatic niche. Lung cancer is a highly metastatic disease, with metastases often occurring when the tumor is in a clinically early stage. It is known to metastasize to the liver in the Asian population, but the underlying mechanism still remains unclear. Thus, understanding the role of TDEs in development of liver metastasis and its contributions to improvise the prognostic and therapeutic implications is of great importance.

Methodology: Exosomes isolated from serum of lung cancer liver metastasis patients were characterized by nanoparticle tracking analysis, electron microscopy and flow cytometry with antibody directed at CD63. These exosomes were further co-cultured with A549 cells to check for their uptake at various time intervals. Moreover, effect of isolated exosomes was tested on: 1. Cell motility and invasiveness of A549 cells determined by transwell chamber migration assay and scratch assay; 2. Gene expression profiles and cell cycle regulation studies; and 3. Histopathological changes and liver function tests after exosome were intraperitoneally injected in Wistar rats to investigate the early events involved in the metastatic spread.

Results: We found that CD63 - FITC labelled exosomes isolated from serum of liver metastatic patients were actively incorporated by A549 cells. The exosome uptake by A549 cells induced migratory/invasive phenotypic and morphologic changes in a concentration and time- dependent manner. Moreover, on further evaluating the effect of TDEs on different phases of cell cycle it was observed that cellular uptake of exosomes was increased during G2/M phase stimulating the cells to enter these cell cycle phases leading to cell proliferation. Further, we observed that E-cadherin, beta catenin, VEGFA, CDKN2A and TGFBR2 were differentially expressed when A549 cells were treated with exosomes demonstrating an important role of these TDEs in altering the tumor microenvironment. Liver function test demonstrated an increase in serum SGPT and SGOT levels whereas when histopathological examination was done the lungs showed patches of pneumonitis and the liver was highly inflamed.

Conclusion: Collectively this study indicated that metastatic tumor derived exosomes are capable of increasing the migratory and invasive property of the primary lung cancer cells. Furthermore, our results depict an undisputable role of these exosomes as key modulators in the formation of the pre-metastatic niche required for the colonization of circulating tumor cells (CTCs) ultimately leading to distant metastasis. Thus understanding and targeting tumor derived exosomes could lead to better and novel therapeutic strategies.

#5188

MDM2 and MDMX promote p53-independent initiation of circulating tumor cells from triple negative breast cancers.

Chong Gao,1 Gu Xiao,2 Ale Piersiga,3 Jiangtao Gou,2 Olorunseun Ogunwobi,1 Jill Bargonetti1. 1 _Hunter College and Graduate Center of City University of New York, New York, NY;_ 2 _Hunter College of City University of New York, New York, NY;_ 3 _Weill Cornell Medical College, The Rockefeller University, and Memorial Sloan Kettering Cancer Center, New York, NY_.

Many human breast cancers overexpress the E3 ubiquitin ligase MDM2 and its homolog MDMX. The Cancer Genome Atlas (TCGA) shows that high MDM2 and MDMX expression correlates with both estrogen receptor positive (ER+) and triple negative breast cancer (TNBC) subtypes. Overexpression of MDM2 and MDMX can drive breast cancer progression through molecular pathways that do not require wild-type p53 degradation. ER positive breast cancers often express wild-type p53 but 80% of TNBC express mutant p53 (mtp53). It has not been determined how MDM2 and MDMX expression in different sub-types of breast cancer, with mutant p53, influence proliferation and metastasis. We compared MDM2 and MDMX-dependent biological outcomes for mtp53 expressing ER+ T47D (mtp53 L194F) and TNBC cell type MDA-MB-231 (mtp53 R280K). In the ER+ cells no change was detected for real time cell migration or E-cadherin protein levels when MDM2 was knocked down by stable shRNA expression but cell proliferation was reduced. In support of this in vitro data, these MDM2 knockdown cells tested in an orthotopic xenograft model in NSG mice demonstrated no MDM2-mediated change in primary tumor invasiveness but did show a reduced tumor volume. Importantly, in the TNBC MDA-MB-231 cells, knocking down either MDM2 or MDMX did not reduce cell proliferation in vitro but greatly blocked cell migration. In support of this in vitro data, these MDA-MB-231 cells with either MDM2 or MDMX knockdown in NSG mice did not show a reduced primary tumor volume but showed a significant inhibition of the production of circulating tumor cells (CTCs). Our data shows that in different subtypes of breast cancers, MDM2 and MDMX can promote alternative p53-independent biological outcomes. The estrogen-activated MDM2 strongly promotes cell proliferation while MDM2 and MDMX overexpression in TNBC promotes circulating tumor cells and potentially metastasis. Understanding the alternative p53-independent roles of MDM2 and MDMX in ER+ and triple negative breast cancers will provide insight for the development of MDMs-targeted theranostics.

#5189

Evaluation of thymidylate synthase mRNA expression in circulating tumor cells from non-metastatic colon and rectal patients.

Bianca Troncarelli Flores, Emne Ali Abdallah, Virgílio Souza e Silva, Celso Abdon Mello, Samuel Aguiar, Renata Mayumi Takahashi, Vanessa Silva Alves, Bruna Elisa Kupper, Ludmilla T. Chinen. _ACCamargo Cancer Center, São Paulo, Brazil_.

Background: colorectal cancer is the third leading cause of cancer death in the United States. With different behaviors, rectal cancer represents worse prognosis, even in the locally advanced setting, with therapy following three steps, consisting of neoadjuvant chemoradiation, surgery and adjuvant chemotherapy. For non-metastatic colon cancer, the main treatment is surgery and then, patients can or not undergo adjuvant chemotherapy. Studies have shown that circulating tumor cells (CTCs) may be involved in metastatization process. Thymidylate synthase (TYMS) acts in the 5-Fluorouracil metabolism, and when overexpressed, can confer resistance to treatment, which is commonly used for neoadjuvant treatment (rectal cancer) and in combination with oxaliplatin for adjuvant treatment (colon cancer). Excision repair cross complementation Group 1 (ERCC1) is a protein involved in damage DNA repair process that is described to confer resistance to platinum-based chemotherapy. Objective: evaluate the TYMS messenger RNA (mRNA) expression in CTCs isolated from patients with locally advanced colon and rectal cancers; and its predictable value in treatment resistance. For patients with colon cancer, we also evaluated the ERCC1 protein expression in order to verify the adjuvant therapy resistance related to oxaliplatin. Methods: We collected 10 mL of blood before the beginning of treatment and processed it on ISET® (Rarecells) device. RNAscope® Technology is a novel chromogenic in situ hybridization (CISH) assay for detection of target RNA within intact cells. The expression of TYMS target gene was performed using the kit from ACDbio, as described on the protocol, but standardized to cytology. Results: there were included 12 patients of each type of tumor. We analyzed samples from 12 rectal cancer patients, and found 10 with overexpression of TYMS mRNA in CTCs. Curiously, of the two patients who had TYMSneg mRNA expression; one had complete pathological response and the other substantial down-staging. In the positive cases, 50% presented response (partial or complete) and 50% present no response and systemic progression. This result will better understood when we perform the evaluation of the protein TYMS. Among the samples from colon cancer patients, we found 8 out 12 as CTCs TYMSpos mRNA expression. Unfortunately, our recent data does not allow us to do analysis of recurrence. However, one of the positive cases had recurrence. Among the 4 TYMSneg, one had recurrence. This can be explained by the positive immunocytochemical expression of the ERCC1 protein, which confers resistance to oxaliplatin. Our next steps are to expand the CISH experiments in a larger group of patients as well as to evaluate the TYMS protein expression in the same cases. Conclusion: our preliminary results point molecular analysis of CTCs as predictor marker of colon/rectal cancer treatments.

#5190

Development of a multi-parameter immunofluorescence assay for identification of circulating tumor cells with epithelial-mesenchymal phenotype.

Arturo Ramirez,1 Nolan Ericson,1 Daniel Campton,1 Melinda Duplessis,1 Tanisha Mojica,2 Alisa Clein,3 Celestia Higano,3 VK Gadi,3 Daniel E. Sabath,3 Eric Kaldjian1. 1 _RareCyte, Inc., Seattle, WA;_ 2 _Seattle Cancer Care Alliance, Seattle, WA;_ 3 _University of Washington, Seattle, WA_.

Background. The epithelial-mesenchymal transition (EMT) is understood to be an important step in invasion and metastasis of cancer. It is of increasing investigational interest to identify circulating tumor cells (CTCs) that express mesenchymal markers that indicate entrance into EMT. Such cells may not express surface epithelial markers (such as EpCAM) that are often used to capture CTCs. RareCyte has developed a platform for automated visual identification and retrieval of rare cells in blood by immunofluorescence (IF) that does not rely on surface marker capture. We developed a 5-parameter assay to identify epithelial CTCs with or without mesenchymal differentiation. Methods. Buffy coats isolated from blood by AccuCyte® separation were spread onto slides. A 5-parameter IF assay was developed with the following markers: Sytox orange (nuclear dye) / cytokeratin and EpCAM (epithelial) / vimentin (mesenchymal) / CD45, CD11b and CD105 (exclusion) / EGFR (investigational biomarker). Slides were stained with the assay on an auto-stainer, and were scanned and imaged with CyteFinder®. Specificity of the assay was validated on positive and negative control cell lines spiked into normal donor blood. It was applied to a pilot set of blood samples from 3 prostate and 3 breast cancer patients that were collected under an IRB-approved protocol. Nucleated cells that were epithelial marker-positive and exclusion marker-negative were identified as CTCs. CTCs were categorized as epithelial (epiCTC) if they were vimentin-negative and epithelial-mesenchymal (E-M) if they were vimentin-positive. In a sample from a patient with known mutation in TP53 (G244S) from tissue tumor analysis, individual epiCTCs, E-M CTCs and WBC control cells were mechanically retrieved by CytePicker® and whole genome amplification was performed. The resulting DNA was PCR amplified at the TP53 mutation site and sequenced. Results. Between 15 and 193 CTCs were counted in the patient samples. The average percentage of epiCTCs was 74% (range 33 - 100%) and of E-M CTCs was 26% (range 0 - 67%). E-M CTC fraction was higher in breast than in prostate cancer samples. EGFR-positive epiCTCs and E-M CTCs were identified. In the patient with the tumor-associated TP53 G244S mutation, 2 of 2 epiCTC, 6 of 7 E-M CTC and 0 of 6 WBC contained the mutation. Conclusions. We have developed a multi-parameter immunofluorescence assay for identification of circulating tumor cells with epithelial-mesenchymal phenotype and applied it to cancer patient samples. Confirmation of malignancy of epiCTCs and E-M CTCs by single cell mutational analysis was demonstrated. The expression of EGFR on CTCs in prostate and breast cancer confirms literature reports.

#5191

Targeting CX3CR1 Impairs the reseeding and colonization of circulating tumor cells and decelerates metastatic progression.

Chen Qian,1 Asurayya Worrede-Mahdi,1 Ramanpreet Kaur,1 Fei Shen,2 Joseph Salvino,3 Olimpia Meucci,1 Alessandro Fatatis1. 1 _Drexel Univ., Philadelphia, PA;_ 2 _Janssen Pharmaceuticals, Titusville, NJ;_ 3 _The Wistar Institute, Philadelphia, PA_.

This study aims to understand the mechanisms and dynamics by which cancer cells, departing from existing metastases as circulating cancer cells (CTCs), further seed and colonize skeleton and soft tissues, expanding metastatic spreading and precipitating the clinical progression to terminal disease. We have previously shown that the chemokine receptor CX3CR1 is implicated in the metastatic seeding of breast cancer cells and that FX-68, a novel small-molecule antagonist for this receptor, effectively contains both number of lesions and total tumor burden in animal models of metastatic disease. Here we established that FX-68 impairs the reseeding of skeleton and soft tissue and that the CTCs unable to reseed and forced to remain in the blood eventually succumb to apoptotic death. The metastatic potential of CTCs was confirmed by reinoculation experiments as well as by forcefully mobilizing cancer cells from metastases back in the blood and assessing the number of additional lesions generated, in the presence and absence of FX-68. Furthermore, we found that prolonging the permanence of CTCs in the blood by targeting CX3CR1 with FX-68 improved exposure of cancer cells to the chemotherapy drugs doxorubicin or docetaxel significantly decreased tumor progression and extended overall survival. Finally, we found evidence that cancer cells seeding the skeleton despite the inhibition of CX3CR1 failed to develop into tumors, suggesting a role of CX3CR1 expression in the metastasis-initiating properties of breast cancer cells. In summary, our results strongly support developing CX3CR1 antagonists and promoting their clinical use, as this approach will provide novel and effective tools to contain the progression of metastatic disease in patients.

#5192

Detection of disseminated prostate cancer cells in bone marrow using tyramide signal amplification.

Sounak Roy,1 Kenneth C. Valkenburg,2 Kenneth J. Pienta2. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _Johns Hopkins Medical Institutions, Baltimore, MD_.

Approximately 28,000 men die of prostate cancer (PCa) each year in the US, and 90-100% of them are due to bone metastasis. It has been demonstrated that PCa cells detach from the primary tumor, "home" to bone, and develop into these incurable metastases. Existing methods of detecting cells early that can lead to bone metastasis have limited sensitivity and current efforts involve liquid biopsy assays to capture and detect disseminated and circulating tumor cells (DTCs and CTCs respectively). A significant challenge during this detection lies in the optimization of the signal-to-noise ratio during immunofluorescent staining. Noise during immunostaining can originate from endogenous immune cells and other bone marrow matrix components and lead to false staining which reduces sensitivity. We have developed an optimized protocol to reduce background noise from these factors while also enhancing signal using tyramide signal amplification (TSA). Following immuno-staining with PCa cell-specific markers, TSA amplifies beyond primary and secondary antibody binding using horseradish peroxidase (HRP) and fluorophore binding. To further apply this novel signal amplification protocol to the visualization of disseminated tumor cells in bone marrow, we will detect, quantify, and image parental and modified PCa cells injected into murine blood. We have previously shown that C-X-C chemokine receptor 4 causes PCa cells to home to bone. We have stably overexpressed CXCR4 in several PCa cell lines and will be able to determine the role of CXCR4 expression on the development of bone metastases. This robust protocol will be quickly modifiable for automated staining to yield higher throughput analysis.

#5193

A subpopulation of circulating epithelial tumor cells positive for fatty acid receptor CD36 is linked to aggressiveness of breast cancer disease.

Monika Pizon, Dorothea Schott, Ulrich Pachmann, Katharina Pachmann. _Transfusion Center Bayreuth, Bayreuth, Germany_.

Background: Breast cancer is one of the leading causes of cancer death for women worldwide. Major hurdles for a successful treatment are cancer metastases, resistance to therapy and disease recurrence. The presence of circulating epithelial tumor cells (CETCs) is closely related to metastasis formation, but the mechanisms through which CETCs promote recurrence of disease are still unclear. CD36 is found in the cell membrane of tumor cells and responsible for taking up fatty acid. It is known that CD36 is a general marker of metastatic cells and its amplification specifically correlated with metastasis in a large number of human tumors. Therefore, the aim of this study was to determine the metastatic marker CD36 on CETCs and to correlate it's frequency with clinicopathological features in breast cancer patients.

Methods: CETCs were determined from blood of 42 patients suffering from breast cancer. The number of vital CETCs and their expression of CD 36 was investigated using the maintrac® method.

Results: CD36 expressing CETCs were detected in 95 % of breast cancer patients. Breast cancer patients with metastatic disease had significantly more CD36 positive CETCs as compared to patients without metastases (median 72% vs. 48%; p<0.05). The fraction of CD36 positive CETCs was significantly higher in patients with HER2 positive primary tumor (69% vs. 54%; p<0.05). Furthermore, triple negative breast cancer patients had significantly more CD36 positive CETCs as compared to hormone receptor positive tissue (median 62% vs. 52%; p<0.05). Interestingly, frequency of CD36 positive CETCs correlated significantly with a blinded determination of Ki-67 proliferation index in tumor tissue (r=0.75; p<0.01). We observed a significant heterogeneity in CD36 immunostaining intensity across CETCs from the same patients.

Conclusion: We showed for the first time that breast cancer patients have detectable CETCs with a high frequency of CD36 which highly correlated with progression of cancer disease. Our findings suggest that using a CD36 antibody treatment may be an effective strategy to fight cancer.

#5194

In vivo capture of early metastatic cells through a CXCL12 loaded-dermal filler.

Caterina Ieranò,1 Crescenzo D'Alterio,1 Simona Giarra,2 Maria Napolitano,1 Assunta Santagata,1 Antonio Barbieri,1 Virginia Campani,2 Antonio Luciano,1 Claudio Arra,1 Annamaria Anniciello,1 Gerardo Botti,1 Laura Mayol,2 Giuseppe De Rosa,2 Roberto Pacelli,2 Stefania Scala1. 1 _INT 'G. Pascale', Naples, Italy;_ 2 _Federico II University, Naples, Italy_.

The chemokine CXCL12 and its receptor CXCR4 are critical for orienting and seeding cells in metastatic sites. To distract CXCR4 expressing circulating tumor cells (CTCs) a "fake" pre-metastatic niche was built with a hyaluronic acid based dermal filler hydrogel-CXCL12-loaded (CLG). CXCR4 expressing human T leukemia cells, CCRF-CEM, and murine melanoma cells human CXCR4 transduced, B16-hCXCR4-GFP, were evaluated for migration in vitro toward CLG. CLGs and EGs (empty gels) were subcutaneously injected in C57BL/6 mice and five days later B16-hCXCR4-GFP (5x105 cells) were intravenously injected. Twenty two days later mice were euthanized and hydrogels recovered. Gel trapped cells were recovered through ialuronidase digestion of the gels and lungs were analyzed to detect metastases. Circulating tumor cells (CD45neg/hCXCR4 plus/GFP plus) were assessed in the blood at day 10 and 22 from cells injection. In vitro, CEM and B16-hCXCR4-GFP cells more efficiently migrated toward CLG compared with EG. In vivo, a significantly higher number of B16-hCXCR4-GFP tumor cells was detected within the CLGs compared with EGs, indicating that the CXCL12 in hydrogel enabled recruitment of tumor cells. Concomitantly, lung metastases were significantly reduced in mice carrying CLG, but not EG, as assessed by reduced B16-hCXCR4-GFP positive tumor cells in the lung (75% reduction in lung metastases in mice carrying CLG; p<0.05). CLG recovered cells by hydrogel enzymatic digestion recapitulated the features of melanoma cell line B16-hCXCR4-GFP (epithelial, melanin rich, MELAN A/ S100/ c-kit/CXCR4 +; α-SMA -). CTCs, evaluated at 10 and at 22 days post cells injection, significantly increased in CLG carrying mice compared to EG carrying mice. Our study suggests that a hyaluronic acid (HA) hydrogel, commercially used as dermal filler, loaded with CXCL12 can divert CXCR4 positive circulating tumor cells B16-hCXCR4-GFP. The cells trapped in the CLG can be recovered and characterized. As a corollary, a reduction of CXCR4 dependent lung metastasis was detected. It suggests that hydrogel-CXCL12 trap may be a therapeutic and diagnostic tool to recover and analyze the biology of circulating tumor cells.

#5195

Increased circulating tumor cell (CTC) clusters are associated with significantly higher levels of HER2 expression and metastasis in stage III/IV breast cancer.

Qiang Zhang, Youbin Zhang, Lisa Flaum, Lorenzo Gerratana, William Gradishar, Leonidas Platanias, Massimo Cristofanilli. _Northwestern Univ., Chicago, IL_.

Introduction: Molecular and genomic characterization of CTCs may help to understand breast cancer prognosis and predict treatment benefit, especially for metastatic or recurrent disease. Overexpression of HER2 protein has been associated with rapid cell division and the metastasis and prognosis of advanced breast cancer in the clinic. Herein, we report a correlation between the detection of CTC clusters and HER2 overexpression in otherwise biomarker negative disease.

Methods: 146 whole blood samples (7.5ml/each) were collected from 95 patients with stage III/IV breast cancer at baseline, before and after systemic therapy. All samples were transported and stored at 15-30 °C in EDTA tubes. CTC enrichment and enumeration were performed in FDA approved semi-automated fluorescence CELLTRACKS ANALYZERII® System (Janssen Diagnostics) by using CELLSEARCH® CXC Kit (Cell Search) contains ferrofluid-based capture reagent particles with a magnetic core surrounded by a polymeric layer coated with antibodies targeting the Epithelial Cell Adhesion Molecule (EpCAM) antigen for capturing CTCs. Furthermore, it includes immunofluorescent staining reagents including Anti-CK-PE which is specific for the intracellular protein cytokeratin in epithelial cells, DAPI stains the cell nucleus, anti-CD45-APC is specific for leukocytes, and anti-HER-2/neu-FLU is specific for HER-2/neu antigen. The CTCs were classified based on morphology and correct phenotype as CK+, EpCAM+, DAPI+ and CD45-. Database of CTCs was generated and linked with clinical database. Kruskal-Wallis test was used for statistics.

Results: Of the 146 samples analyzed, 80 samples contained CTCs, among which 54 cases did not form clusters (Group 1), 11 samples had 1-5 clusters (Group 2), and 15 had >5 clusters (Group 3). The average ratio of CTCs/ total EpCAM+ was directly correlated with the number of clusters and were reported as 15.36%, 25.32% and 43.65% for the 3 cohorts respectively (P<0.01). The highest expression of HER-2 was found in Group 3 (66.67% samples are positive, and 120 positive cells/each sample). This value was significantly higher than Group 2 (27.27% samples are positive, 24.33 positive cells/each sample) and Group 1 (20.37% samples are positive, 6.18 positive cells/each sample) (P<0.001). The results indicated that high level of CTCs clusters strongly associated with over expression of HER2 in CTCs.

Conclusion: We demonstrate that patients with detectable CTCs clusters in comparison with CTCs with no or low numbers of clusters, high levels of CTCs clusters (> 5 clusters/7.5ml blood) is a strong indicator for more aggressive of CTCs with high level of HER2 expression, which tend to be more aggressive in metastasis and worse prognosis than HER2-negative breast cancers. Along with and cancer stage and HER2 status, CTCs clusters helps determine your treatment options in clinic.

#5196

Feasibility investigation of EML4-ALK rearrangements in mNSCLC CTCs using a new in vivo procedure.

Giulia Gallerani, Angelo Delmonte, Claudia Cocchi, Martine Bocchini, Filippo Piccinini, Marco Angelo Burgio, Carla Casadei, Andrea Rocca, Francesco Fabbri. _I.R.S.T., Meldola, Italy_.

Background - Circulating tumor cells (CTCs) are the liquid phase of a solid tumor. An advanced CTC phenotyping and genotyping procedure could allow a superior understanding of tumor heterogeneity aimed at improving personalized treatment selection. However, these investigations are hampered by the difficulty to catch and analyze CTCs. EML4-ALK chromosomal rearrangement is a fundamental marker of sensitivity in metastatic non-small cell lung cancer (mNSCLC), but conventional tissue-based ALK scrutiny is not always possible. A CTC-based ALK evaluation may be an easy and not invasive method for identifying rearrangements and hence patients eligible for specific treatments.

Methods - Analyses were performed via the Gilupi CellCollector CANCER01 (DC01), a device that allows the in vivo isolation of CTCs from a high volume of peripheral blood. The DC01 is a CE-approved functionalized 16 cm long medical wire that includes a functionalized 2 cm long gold-coated tip covalently coupled with antibodies against EpCAM. The DC01 can be inserted through a standard venous cannula into the cubital vein of cancer patients for 30 minutes where it captures CTCs directly from the bloodstream of cancer patients. CTC presence was investigated in 18 mNSCLC patients and CTC-ALK-chromosomal status was studied by an innovative method, the 3D-immuno-FISH, that combined immuno-fluorescence and FISH approaches on a 3D structure (the DC01). Two DC01 per patients were utilized. After removal, on one wire, CTCs were identified and counted by nuclei, EpCAM and cytokeratins staining. On the second wire, CTCs were recognized by EpCAM staining and analyzed for ALK alterations by FISH. Formalin-fixed paraffin-embedded primary tumor tissues from the same patients were analyzed for comparison. Immortalized cell lines attached to DC01 devices were exploited as positive controls.

Results - Using the DC01 it was feasible to isolate and detect CTCs. Among the 18 enrolled patients, 7 presented tumor-tissue ALK-translocation, whereas 11 were wild type. Overall, 61.1% patients were CTC positive (median CTC number, 3.0 per patient). Amid those that owned ALK-translocation, 28.6% were CTC-positive, whereas 81.8 % patient without ALK-translocation were CTC positive. ALK rearrangements were detectable directly on cells attached to the wires. However, the 3D-immuno-FISH analysis in patients was hampered by the low median number of detected CTCs.

Conclusions - The detection of ALK-chromosomal rearrangements with the 3D-immuno-FISH analysis in cells recovered by DC01 was feasible. This method is valid for tracking positive CTC patients, but the few cells retrieved per patients still limit CTC full exploitation. Although further studies are needed to validate our data and to improve DC01 capture efficiency, our results pave the way to CTC-based chromosomal rearrangement identification and therapy personalization. 

### The Systemic Microenvironment in Tumorigenesis

#5197

Single-cell analysis of the brain metastatic microenvironment in breast cancer.

Katrina Evans, Quy Nguyen, Kerrigan Blake, Devon Lawson. _University of California Irvine, Irvine, CA_.

Breast cancer brain metastasis affects 10-20% of patients, and there are no effective treatments. Activated infiltrating microglia and astrocytes are a major component of metastatic breast tumors in the brain, but it is not known whether these cells are involved in the promotion or rejection of breast cancer cells. To characterize the brain metastatic microenvironment, we used an FACS-based approach to capture pure and viable microglia and astrocytes from normal and metastatic mouse brains for single-cell mRNA sequencing analysis. Using this approach, we have captured more than 10,000 microglia and astrocytes from normal and metastatic brains. Applying our analysis pipelines to this data revealed distinct pathways and markers associated with microglia and astrocytes in metastatic brains. Future work will aim to identify spatial locations and function of specific microglia and astrocyte subpopulations in the metastatic brain.

#5198

Androgen receptor signaling affects macrophage differentiation in the human prostate cancer microenvironment.

Bianca Cioni, Ekatarina Nevedomskaya, Suzan Stelloo, Monique Melis, Johan van Burgsteden, Jeroen de Jong, Henk van der Poel, Jan Paul de Boer, Wilbert Zwart, Andries M. Bergman. _Netherlands Cancer Institute, Amsterdam, Netherlands_.

Increasing evidence from epidemiologic and pathologic studies suggests a role of macrophages in the initiation and progression of prostate cancer (PCa). During PCa development, macrophages (CD68+) are recruited into the human prostate cancer microenvironment, including inflammation-associated M1 and cancer-promoting M2 macrophages. Like other prostate cancer-associated stromal cells, macrophages express the androgen receptor (AR); however, its functionality is not known. CD68+ cells generated in vitro from peripheral white blood cells and PMA activated and IFN and LPS polarized human acute monocytic leukemia THP-1 cells also expressed AR. In these cells AR is an 80KD splice variant, lacking parts of exon 1 and exon 2. AR translocated to the nucleus upon testosterone stimulation, which was counteracted by the AR signaling inhibitor RD162. In vitro generated M1 macrophages expressed the M2 markers CD163 and CD206 upon testosterone stimulation, which was reversed by RD162. AR-ChIP sequencing suggested AR regulated Macrophage Triggering Receptor 1 (TREM1) signaling. Expression of key cytokines (e.g., CCL2, CXCL8, CCL7, IL10, IL1B) involved in TREM1 signaling was upregulated upon testosterone stimulation. Actions of these cytokines is generally associated with a protumor phenotype of macrophages. Conditioned medium from macrophages stimulated with testosterone for 8 hrs resulted in enhanced proliferation and migration of human prostate LNCap and CWRR1 cells. In conclusion, AR signaling might affect differentiation of prostate cancer-associated macrophages into cancer-promoting M2, which affects prostate cancer cell proliferation and migration. Maintaining macrophages in M1 might be a novel mechanism of action of androgen receptor inhibitors.

#5199

Microenvironmental control of breast cancer subtype elicited by paracrine platelet-derived growth factor-CC signaling.

Matteo Bocci, Michael Bartoschek, Pernilla Roswall, Kristian Pietras. _Lund University, Lund, Sweden_.

Aim: Breast tumors of the basal-like, hormone receptor-negative subtype, remain an unmet clinical challenge, as patients exhibit the highest recurrence rate, the shortest time to recurrence and the worst overall survival rate due to a paucity of therapeutic targets. Also, as tumors develop, the surrounding microenvironment co-evolves into an activated state through continuous paracrine communication, creating a dynamic signaling circuitry that promotes malignant progression. Thus, novel treatment approaches that also include the targeting of stromal components are urgently required.

Methods: We used experimental mouse models of cancer (transgenic, syngeneic, human cell lines and patient-derived xenografts) for knockout studies and preclinical trials, immunostaining, in vitro assays, western-blot, gene expression analysis and RNA-sequencing, patient cohorts, data mining and bioinformatics analysis.

Results and discussion: We identified a paracrine crosstalk between cancer cells expressing platelet-derived growth factor (PDGF)-CC and cancer-associated fibroblasts (CAFs) expressing the cognate receptors in human basal-like mammary carcinomas. In translational efforts, we found that PDGF-CC expression in the malignant epithelium of breast cancer was associated with a hormone receptor-negative state. We then generated a mouse model to characterise the functional role of PDGF-CC in tumorigenesis. Moreover, we identified and validated factors secreted by CAFs to maintain a basal-like phenotype, e.g. Stanniocalcin (STC)-1, Hepatocyte Growth Factor (HGF) and Insulin Growth Factor Binding Protein (IGFBP)-3. Genetic or pharmacological targeting of PDGF-CC in experimental mouse models resulted in the transcriptional reprogramming and conversion of basal-like breast tumors into a luminal-like state characterised by Estrogen Receptor (ER)-α positivity, that conferred sensitivity to endocrine therapy in previously intrinsically unresponsive tumors.

Conclusions: Specification of the basal-like subtype of breast cancer is under microenvironmental control and therapeutically actionable in order to achieve sensitivity to endocrine therapy. Our work sheds light on previously unknown mechanisms that define breast cancer subtype and has significant therapeutic implications for patients with basal-like breast tumors.

#5200

Extracellular vesicles in the ascites from gastric cancer patients play critical roles to promote peritoneal dissemination in the intraperitoneal cancer microenvironment.

Atene Ito, shunsuke kagawa, Shuichi Sakamoto, Kazuya Kuwada, Satoru Kikuchi, Shinji Kuroda, Ryuichi Yoshida, Hiroshi Tazawa, Toshiyoshi Fujiwara. _Okayama university, Okayama, Japan_.

Although gastric cancer often develops peritoneal dissemination, the underlying mechanism of peritoneal metastasis is still unclear. Extracellular vesicles (EVs) are known to play roles in cell-cell communication. Here, we investigated the existence of EVs in peritoneal ascites of gastric cancer patients and explored their roles in the intraperitoneal cancer microenvironment. We found that EVs actually existed in peritoneal ascites of advanced gastric cancer patients. On the other hand, immune-fluorescent assay of the ascites by using antibodies against CD14 (Monocytes) and CD45 (Leukocytes) revealed that many macrophages existed along with the cancer cells in the peritoneal cavity. EVs purified from peritoneal ascites with cancer dissemination made peripheral blood mononuclear cells-derived macrophages M2-like phenotype which was confirmed by their morphology and expression of CD163/206 (M2 marker). Next, we purified EVs from the culture medium of gastric cancer cell lines. Gastric cancer-derived EVs induced the M2- differentiation in macrophages and the differentiated macrophages promoted migration ability of gastric cancer cells. Finally, we observed that gastric cancer-derived EVs induced apoptosis of cultured mesothelial cells in vitro. These results suggested that EVs in the ascites of gastric cancer patients are possibly derived from gastric cancer and play critical roles in the intraperitoneal cancer microenvironment by affecting the macrophages phenotype and inducing apoptosis in the mesothelial cells, which would be in part the underlying mechanism of peritoneal metastasis.

#5201

Differences in tumor microenvironments between primary lung tumors and brain metastases in lung cancer patients: Therapeutic implications for immune checkpoint inhibitors.

Ryul Kim,1 Bhumsuk Keam,1 Sehui Kim,1 Miso Kim,1 Se Hyun Kim,2 Jin Wook Kim,1 Yu Jung Kim,2 Tae Min Kim,1 Yoon Kyung Jeon,1 Dong-Wan Kim,1 Doo Hyun Chung,1 Jong Seok Lee,2 Dae Seog Heo1. 1 _Seoul National Univ. Hospital, Seoul, Republic of Korea;_ 2 _Seoul National Univ. Bundang Hospital, Seoul, Republic of Korea_.

Background: We aimed to compare intra- and extracranial responses to immune checkpoint inhibitors (ICIs) in lung cancer with brain metastases (BM), and to explore tumor microenvironments of the brain and lungs focusing on the programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) pathway.

Methods: Two cohorts of lung cancer patients with BM were analyzed. Cohort 1 included 18 patients treated with nivolumab or pembrolizumab, and intra- and extracranial responses were assessed. Cohort 2 comprised 20 patients who underwent both primary lung surgery and brain metastasectomy. Specimens from cohort 2 were subjected to immunohistochemical analysis for the following markers: CD3, CD4, CD8, FOXP3, and PD-1 on tumor infiltrating lymphocytes (TIL) and PD-L1 on tumor cells.

Results: Seven patients (38.9%) in cohort 1 showed progressive disease in both primary and intracranial lesions. Although the other 11 patients exhibited a partial response or stable disease in the primary lesion, eight showed a progression in BM. Interestingly, PD-1+ TILs were significantly decreased in BM (P=0.034). For fifteen patients with adenocarcinoma, more distinctive patterns were observed in CD3+ (P=0.078), CD8+ (P=0.055), FOXP3+ (P=0.016), and PD-1+ (P=0.016) TILs.

Conclusions: There may be discordant responses to an ICI of lung cancer between primary lung lesion and BM based on discrepancies in the tumor microenvironment. The diminished infiltration of PD-1+ TILs in tumor tissue within the brain may be one of the major factors that hinder the response to anti-PD-1 antibody in BM.

#5202

Pigment epithelium-derived factor promotes tumor dissemination of ovarian cancer cells through an interaction with peritoneal immune system.

Sayaka Ueno,1 Eiji Sugihara,1 Tamotsu Sudo,2 Hideyuki Saya1. 1 _Keio University, Tokyo, Japan;_ 2 _Hyogo Cancer Center, Akashi, Japan_.

Ovarian cancer is a gynecological malignancy with a high mortality rate. Patients with ovarian cancer are diagnosed at an advanced stage of the disease with massive peritoneal dissemination. However, the underlying mechanism by which ovarian cancer cells preferentially colonize the omentum is still poorly understood. In this study, we have identified the factors and mechanisms which promote ovarian cancer dissemination by regulating peritoneal immune system. First, we established a mouse ovarian cancer cell line, ID8GO2, with an increased ability to disseminate in the peritoneal cavity by in vivo passage of ID8 cells in B6J mice. To determine the candidate genes that drive the dissemination, we examined the differences in gene expression profiles between two cell lines. Expression of the candidate genes in human ovarian cancer tissues and their correlation with the clinical outcomes of patients were also analyzed using public database. We found that pigment epithelium-derived factor (PEDF) was the most relevant gene associated with poor prognosis. To clarify whether PEDF contributes to peritoneal dissemination, ID8 cells were retrovirally transduced with mouse PEDF (ID8-PEDF) or empty vector (ID8-EV) and transplanted into the syngeneic mouse peritoneal cavity. All mice transplanted with ID8-PEDF cells developed intraperitoneal dissemination more rapidly and the overall survival time was significantly shorter than that in mice transplanted with ID8-EV cells. Moreover, ID8-PEDF cells survived longer in the peritoneal cavity. We further found that ID8 cells originally consisted of clones with varied PEDF expression. Transplantation of the PEDF-high clones resulted in faster development of intraperitoneal dissemination and shorter overall survival than that of the PEDF-low clones. These findings suggest that PEDF contributes to peritoneal dissemination of ovarian cancer cells, and cells with high initial PEDF expression are selected during dissemination. Next, to clarify the mechanism underlying the role of PEDF in peritoneal dissemination, intraperitoneal immune cell subsets in tumor bearing mice were analyzed. Peritoneal macrophages in mice transplanted with ID8-PEDF cells displayed alternative activated macrophage phenotype (F4/80+ CD206+) and high IL10 expression. Interestingly, at later time point, regulatory T and regulatory B cells were induced in the peritoneal cavity. Collectively, these findings suggest that PEDF plays an important role in the peritoneal dissemination of ovarian cancer cells potentially by regulating macrophages and regulatory lymphocytes to evade the peritoneal immune system.

#5203

Bitter melon extract modulates immune signaling in prevention of tobacco associated carcinogen induced oral carcinogenesis.

Subhayan Sur, Robert Steele, Ratna B. Ray. _Saint Louis University, St. Louis, MO_.

Oral squamous cell carcinoma (OSCC) is a complex malignancy and tobacco habit is one of the main etiological factors. Our previous study showed potential chemopreventive effect of bitter melon (Momordica charantia) extract (BME) in different pre-clinical cancer models like head and neck, breast, prostate. The aim of this study is to understand the molecular mechanism of BME in prevention of carcinogen induced in-vivo oral carcinogenesis. For this purpose, we induced oral cancer in immunocompetent mice by using tobacco associated carcinogen 4-nitroquinoline 1-oxide (4-NQO), and treated with BME through their drinking water. We observed that BME feeding significantly reduced the incidence of 4-NQO induced oral cancer. Histological analysis showed moderate dysplasia to invasive squamous cell carcinoma in 4NQO treated mice tongue, whereas no remarkable changes were seen in the BME-fed mice. RNA-seq data showed differential expression of several genes during carcinogenesis and during prevention of the cancer. We previously showed that BME treatment decreases the infiltrating regulatory T (Treg) cells by inhibiting FoxP3+ populations in the tumors and in spleens, suggesting an immunomodulatory role of BME in prevention. Here, we observed elevated expression of pro-inflammatory genes s100a9, IL23a, IL-1β and immune check point gene PDCD1/PD1 of the pathway, during oral cancer development. Interestingly, BME treatment significantly reduced the expression of these molecules. Subsequently, we observed the enhancement of MMP9 during carcinogenesis, which was reduced in BME-fed mouse tongue tissues. Taken together, our observation strongly demonstrated a chemopreventive effect of BME on carcinogen induced oral cancer.

#5204

MYC **expression and smoking as drivers of failure of immune response to melanoma.**

Joanna Pozniak,1 Jeremie Nsengimana,1 Jon Laye,1 Sally O'Shea,1 Sabrina Schindler,2 Mitch Levesque,2 Tim Bishop,1 Julia Newton-Bishop1. 1 _University of Leeds, Leeds, United Kingdom;_ 2 _University of Zurich, Zurich, Switzerland_.

There is good evidence that an immune response to melanoma predicts better survival. We report a bioinformatic study designed to identify immune subgroups of primary melanomas in order to better understand the biology of immune cell/tumor interaction, and to identify candidate predictive biomarkers for immunotherapy.

We applied an approach described by Angelova et al. to the inference of tumor immune cell infiltrates using differential expression of signature genes, in 703 primary melanoma transcriptomes from the Leeds Melanoma Cohort (LMC). Consensus clustering of immune cell scores identified 3 subgroups in LMC and replication was sought in The Cancer Genome Atlas (TCGA) data set, (primarily metastatic samples). We then used the whole transcriptomic data to explore associated biological pathways: a protein-protein interaction network analysis of genes was performed in Cytoscape plugin Reactome FIVIZ. Nodal genes were identified and the cellular origin of the gene expression signature further explored using immunohistochemistry (IHC) and in patient-derived melanoma cell culture transcriptomes. Clinicopathological and environmental factors, and immune cell scores associated with survival within immune subgroups, were then analysed using a multivariate Cox proportional hazard model.

We identified three subgroups with transcriptomic patterns indicative of: low, intermediate and high immune cell infiltration. The groups were concordant with histological evidence of tumor infiltrating lymphocytes. Genes enriched in the low, compared with the high immune group, were in cell cycle, metabolic and Beta-catenin pathways. MYC was the nodal (the most influential) gene of the protein-protein interaction network in this group. In the high immune group, genes were enriched in immune pathways, including antigen processing and presentation, with the nodal gene - NFKB1. Based on the literature and our results, we hypothesised that MYC downregulates HLA-class I, hence we tested MYC correlations in the melanoma cell line transcriptomes (in which immune cells were absent). Indeed, MYC was negatively correlated with many HLA class I genes, with HLA-B having the strongest result: R=-0.57. IHC of primary tumors showed tumor nuclear expression of MYC. Reported smoking independently predicted a worse outcome for patients classified as the high immune group (HR=3.52, P=0.008). The transcriptomic analysis showed that smoking correlated with an increased Th2 cell score (P=0.03) and reduced NK56 bright score (P=0.01) in that group.

We report evidence that MYC plays an important role in immune evasion in primary melanoma. The transcriptomic data support in vitro findings that MYC downregulates HLA class I expression. Furthermore, we present important results that smoking status modifies immune response in melanoma and is an independent risk factor for melanoma death. We hypothesise that smoking may impact on immunotherapy efficacy.

#5205

**Primary melanoma expression of the vitamin D receptor (** VDR **) is protective for melanoma survival and is associated with increased tumor immune response, decreased Wnt/B-catenin signaling and tumor proliferation.**

Sathya Muralidhar,1 Jeremie Nsengimana,1 Joanna Pozniak,1 Sally O'Shea,1 Jonathan Laye,1 David Adams,2 Louise van der Weyden,2 Timothy Bishop,1 Julia Newton-Bishop1. 1 _University of Leeds, Leeds, United Kingdom;_ 2 _Wellcome Trust Sanger Institute, Cambridge, United Kingdom_.

VDR loss has been associated with cutaneous melanoma progression (Brozyna et al, 2011) and VDR polymorphisms have been shown to affect melanoma survival (Mocellin et al, 2008) but the genomic basis remains to be explored. We used microarray data from 700 treatment-naïve melanoma primaries (FFPE) from the Leeds Melanoma Cohort (LMC) to perform agnostic bioinformatic analyses that identified candidate pathways/genes associated with VDR expression. The concordance of these In-silico pathways with histopathological measures was assessed, followed by In-vitro and In-vivo experiments to establish cause-effect. We found that VDR gene expression is protective for melanoma survival in the LMC (Haz Ratio=0.51, P=5.7x10-8) independent of AJCC stage (adjusted Haz Ratio=0.77, P=0.002) and also in the TCGA melanoma data (Haz Ratio=0.83, P=0.001). A genome-wide correlation analysis (FDR<5%) produced 2025 positively correlating genes (β>0.2) and 1383 negative correlating genes (β<-0.2), which were then used for functional enrichment analyses (Reactome FIViz, Wu and Stein 2012). The positive correlating genes were enriched for immune function: ECM organization, TNF signaling, IFNγ signaling, IL12-mediated signaling and NFκB signaling. In line with this, VDR expression was higher in tumors with Tumour Infiltrating Lymphocytes (TILs) compared to those without TILs (P=0.02). VDR also correlated positively with imputed immune cells scores (Angelova et al, 2015) particularly central memory CD4 cells, cytotoxic cells, DCs, NK cells, MDSCs, Neutrophils and T cells (P<10-16). Conversely, the negative correlating genes were enriched for proliferation pathways: Mitotic Prophase, Wnt signaling pathway, Mitochondrial translation, TCA cycle and oxidative phosphorylation. In line with this, VDR expression was lower in tumors with increased mitotic cells (P=0.002). Notable among the negatively correlated pathways was Wnt signaling pathway-VDR has been shown to inhibit Wnt/β-catenin signalling in colon carcinoma cells (Larriba et al, 2011) and β-catenin signalling inhibits melanoma immune infiltration (Spranger et al, 2015). Thus we hypothesized that VDR inhibits β-catenin signalling and hence increases tumor immune infiltration. To test this, stable-transfected B16-BL6:VDR mouse melanoma cells were used in an In-vivo metastatic colonisation assay (Speak et al, 2017, ongoing work). Collectively, we conclude that VDR is of prognostic significance in a cohort of 700 primary melanomas and has a pro-immune and anti-proliferative role, based on highly concordant In-silico and histopathological evidence. Taken together, our results provide a novel insight into the effect of VDR in melanoma survival and evidence for the use of VDR as a potentially significant prognostic marker.

#5206

The biological significance of CCL 16 regulated by thyroid hormone in liver cancer cells.

Cheng-Heng Wu, Kwang-Huei Lin. _Chang Gung University, Taoyuan City, Taiwan_.

Thyroid hormones(THs) are multi-function mediators that modulate various physiological processes. Thyroid hormone receptors(TRs) are type II nuclear receptors, TRs binds to thyroid hormone response elements (TREs) of target promoter regions modulates target gene expression at the transcriptional level upon THs binding. Previous literatures illustrate an association between aberrant TR regulation and human hepatocellular carcinoma (HCC). We employed cDNA microarrays and SILAC secretome analysis to identify the T3-regulated secreted proteins after T3 treatment in TR-expressing hepatoma cell lines, Chemokine (C-C motif) ligand 16 (CCL16) expression is positively regulated by T3 treatment. The patients with higher expression of CCL16 have poor recurrence survival rate. Ectopic expression of CCL16 in HCC cell lines increased tumor growth rate in vivo but not in vitro. CCL16 induced chemotaxis of monocytes and macrophages via interacting with its receptors CCR1 and CCR5. Expression of proangiogenic genes in macrophages were increased while co-cultured with HCC cell lines. The motility of HUVECs were increased after treated with conditional medium of macrophages co-cultured with HCC cell lines. Our study revealed that CCL16 recruited macrophage and promoted tumor progression.

#5207

HIF-1α regulates the Tie2 receptor on Tie2-expressing monocytes in PyMT breast tumors and augments angiogenic function and metastatic potential.

Kayla J. Steinberger,1 Mary Forget,2 Xiaokui Mo,2 Randall Evans,2 Amy Gross,2 Leni Moldovan,2 Andrey Bobko,1 Valery Khramstov,1 Clay B. Marsh,1 Timothy D. Eubank1. 1 _West Virginia University School of Medicine, Morgantown, WV;_ 2 _The Ohio State University, Columbus, OH_.

Tissue oxygenation in the tumor microenvironment serves as a significant parameter in tumor pathophysiology. In fact, normalization of tumor oxygen is associated with inhibition of tumor growth and metastatic capacity. Irregular tumor vessels produced during tumor angiogenesis have low blood perfusion; thus, oxygen transport is decreased in tumors. Tie2-expressing monocytes (TEMs) are a distinct subset of proangiogenic monocytes selectively recruited to tumors in breast cancer patients. Due to the hypoxic nature of the tumor microenvironment, we investigated if oxygen regulates the trafficking of these cells into tumors or if oxygen regulates monocyte differentiation into TEMs once inside the tumor proper. To understand the differentiation of F4/80+/Tie2- cells to Tie2-positivity, we orthotopically implanted PyMT breast tumor cells containing particulate lithium octa-n-butoxy-naphthalocyanine (LiNcBuO) into the mammary fat pads of LysM-Cre control and HIF-1αfl/fl/LysM-Cre mice and evaluated tissue oxygenation by electron paramagnetic resonance (EPR), TEM infiltration, tumor angiogenesis, and metastatic potential. Longitudinal monitoring of physiologic oxygen in tumors using EPR demonstrates the significant role of oxygen-driven TEM regulation in tumor angiogenesis and progression.

#5208

Monocyte-produced Chitinase-3-like 1 is a driver of metastatic behavior in prostate cancer patients.

Karen A. Cavassani, Rebecca J. Meza, David M. Habiel, Jie-Fu Chen, Alexander Montes, Manisha Tripathi, Gislaine A. Martins, Timothy R. Crother, Sungyong You, Cory M. Hogaboam, Neil Bhowmick, Edwin M. Posadas. _Cedars-Sinai Medical Center, Los Angeles, CA_.

Purpose: The identification, functional status, distribution, and interactions of immune cells in prostate cancer (PCa) patients have yet to be fully characterized. Understanding these details could provide key insights as to how promising current and developing immunotherapies might be directed toward PCa. Recruited myeloid cells are known to promote cancer initiation, malignant progression, metastasis, and resistance to therapy in the tumor niche. In the present study, we tested the hypothesis that circulating blood monocytes from advanced PCa patients exhibit a protumor phenotype and directly influence the tumor microenvironment in response to tumor-derived signals.

Experimental Design: Monocytes from metastatic (PCa-M) and nonmetastatic (PCa-N) patient blood samples were isolated, cultured, and conditioned media (CM) was obtained. To evaluate the role of monocytes in metastatic behaviors of PCa cells, in vitro invasion (via Matrigel) and motility assays (Incucyte® Live Cell Analysis System) were performed using monocyte-CM. To identify the candidate PCa-M monocyte-secreted protein(s) that might be implicated in tumor progression, we analyzed the CM using Proteome Profiler Analysis (R&D). Data were validated using ELISA and qPCR.

Results: Herein, we report that CM from circulating monocytes in patients with PCa/mCRPC (metastatic castration-resistant PCa) increased motility and invasion of epithelial PCa cells in vitro. Proteome Profiler Analysis revealed that monocyte-derived CM from metastatic PCa/mCRPC patients presented high levels of Chitinase-3-like 1 (CHI3L1, YKL-40) when compared with PCa-N patients and healthy-control individuals (HC). The only described receptor for CHI3L1, interleukin-13 receptor α2 (IL-13Rα2), was significantly upregulated in the human metastatic prostate cancer cell line, ARCaPM. Accordingly, we observed that the activation of IL-13Rα2 from PCa-M CM increased the invasiveness of ARCaPM cells, and siRNA directed against this receptor significantly reduced invasiveness of these cells in the presence of CM from PCa-M patients.

Conclusions: Thus, we show that circulating monocytes from metastatic PCa/mCRPC patients exert a tumor-promoting role via the secretion of CHI3L1, and CHI3L1 demands further exploration as a possible therapeutic target in advanced PCa.

#5209

Overexpression of TGF-β in Smad4 deficient head and neck squamous cell carcinomas promotes myeloid cell infiltration associated with metastatic progression.

Jingjing Yu, Ariel Hernandez, David Raben, Xiao-Jing Wang. _University of Colorado School of Medicine, Aurora, CO_.

Purpose of study: Transforming growth factor-β (TGF-β) has been shown to promote tumor progression when overexpressed and could be an important biomarker for head and neck squamous cell carcinomas (HNSCC) prognosis and treatment. Overexpression of TGF-β in tumor epithelia and stroma is observed in Smad4 deficient HNSCCs and is known to upregulate inflammation and angiogenesis. Our preliminary studies in Smad4 deficient HNSCC mice have revealed that inhibition of the TGF-β receptor (TGF-βR), specifically of the intracellular kinase domain, did not affect tumor growth in an immune compromised background but significantly decreased the number of metastatic lesions. We aim to elucidate the specific mechanisms by which TGF-β signaling promotes this metastatic phenotype in Smad4 deficient HNSCCs. We hypothesized that Smad4 deficient SCCs overexpress TGF-β to create an inflammatory microenvironment and promote angiogenesis to drive tumor progression in vivo.

Methods: Nude mice were injected subcutaneously in the flank with SCC cells derived from keratinocyte-specific Smad4 knockout mice and treated with either a TGF-βR inhibitor (n=5) or a vehicle control (n=6) once primary tumors reached 1mm3. Using immunofluorescence (IF) or immunohistochemistry (IHC), sections of the primary tumor were probed for leukocyte marker CD45, macrophage markers F4/80, iNOS and Arg1, myeloid cell markers CD11b and Ly6G and endothelial cell marker CD31.

Results: Smad4 deficient SCCs treated with the TGF-βR inhibitor showed a significant decrease in CD45+ leukocytes, with a specific decrease in CD11b+Ly6G\+ cells compared to the vehicle control group. However, there was no difference in the total number of F4/80\+ macrophages between TGF-βR inhibitor treated and vehicle control groups. There was also no significant difference in angiogenesis between these two groups as measured by the number of blood vessels/mm2 and the percentage area of the tumor covered by blood vessels.

Conclusions: Smad4 deficient SCC tumors treated with the TGF-βR inhibitor showed a significant decrease in myeloid cell populations, suggesting that overexpressed TGF-β plays a role in mediating inflammation in the tumor stromal environment. Treatment of SCC tumors with the TGF-βR inhibitor, however, did not show an affect on angiogenesis compared to the vehicle control.

#5210

Liver prometastatic genes in patients with and without colorectal cancer.

Fernando Vidal-Vanaclocha. _Valencia Institute of Pathology-UCV, Valencia, Spain_.

Metastasis-related hepatic genes were investigated in the tumor-unaffected hepatic tissue from patients with hepatic colorectal cancer (CRC) metastases. In addition, the contribution of CRC and patient's clinical parameters and comorbidities to hepatic expression of CRC prometastatic genes was also studied in new hepatic biopsies from metastasis-free CRC patients and from CRC-free patients used as controls. The expression level of 58% metastasis-associated hepatic genes significantly changed between patients with and without CRC. Principal component and Linear discriminant statistical analyses, and a hierarchical cluster analysis based on Pearson's correlation indices uncovered a subset of 37 CRC-dependent liver prometastatic genes able to segregate patients with and without CRC. Functionally, they belonged to immune regulation, inflammation, metabolic bioprotection, hepatokines and regeneration factors. More importantly, a multivariate canonical correlation analysis showed the additional contribution of patient's pathophysiology and comorbidities to hepatic gene expression and metastasis risk. The liver prometastatic gene expression pattern represented a patient-specific and heterogeneous hepatic reaction to CRC-specific factors, which developed prior to metastatic recurrence and varied according to location of such CRC in the left and right-sided colonic area and rectum.

#5211

Dual E-selectin and CXCR4 inhibition reduces tumor growth and metastatic progression in an orthotopic model of osteosarcoma.

Wei Ju,1 Choh L. Yeung,1 Arnulfo Mendoza,1 Meera Murgai,1 Sabina Kaczanowska,1 Jennifer Zhu,1 Shil Patel,1 David A. Stewart,2 William E. Fogler,2 John L. Magnani,2 Rosandra N. Kaplan1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _GlycoMimetics, Inc., Rockville, MD_.

Osteosarcoma is the most common bone cancer in children and young adults and has a strong propensity to develop lung metastases. E-selectin is known to be involved in the focal adhesion of tumor cells to cytokine exposed endothelial cells and we postulated may play a central role in osteosarcoma progression. Previously we identified that SDF-1, the main ligand for CXCR4, was upregulated in the pre-metastatic niche (Kaplan et al Nature 2005). Many tumor cells express CXCR4 and may use this signaling pathway to direct disseminated tumor cells to pre- and early metastatic sites in the lung. Based on these findings we examined human osteosarcoma cell lines and primary patient derived xenografts (PDXs) for the expression of CXCR4 and E-selectin ligands by flow cytometry. We found robust expression of these ligands in the majority of both the human osteosarcoma cell lines and PDXs examined. We therefore, investigated the impact of targeting these two axes on metastatic progression of orthotopic osteosarcoma using a small molecule, glycomimetic compound with dual inhibitory activity against E-selectin and CXCR4, GMI-1359. Five days post paratibial injection the HOS cell line, female NMRI-nu mice (n=12/group) were treated with saline; GMI-1359 alone (40 mg/kg IP BID x 25 days); doxorubicin (DOX) alone (5 mg/kg IV days 5, 15 and 25), or the combination of GMI-1359 and DOX. All treatments were well tolerated. The % tumor volume in treatment/control on day 27 of mice treated with GMI-1359, DOX or the combination was 35.5, 36.7 and 32.5, respectively. At study conclusion the incidence of lung metastases was approximately 60% and 50% in mice treated with saline or DOX and 15% in mice treated with GMI-1369 alone or in combination with DOX. Moreover, the extent of ectopic bone formation and/or osteolytic lesions was lower in mice treated with GMI-1359 compared to saline and DOX. These results indicate that the E-selectin and CXCR4 axes are important for the progression of osteosarcoma, and further, that inhibition of these two pro-tumor growth components by GMI-1359 has a therapeutic advantage over chemotherapy alone. Furthermore, studies in the adjuvant setting can provide proof of concept of utility of targeting CXCR4 and E- selectin ligands in the metastatic niche as a therapeutic strategy to limit metastatic progression in high risk patients.

#5212

The inhibitory effect of iron control on breast cancer cells in tumor microenvironment.

Wataru Goto, Shinichiro Kashiwagi, Koji Takada, Yuka Asano, Tsutomu Takashima, Satoru Noda, Naoyoshi Onoda, Kosei Hirakawa, Masaichi Ohira. _Osaka City Univ. Grad. School of Medicine, Abenoku, Osaka, Japan_.

Background: Iron is an essential element for both normal and cancer cells, and it is well known that iron depletion leads to G1-S arrest and apoptosis. Iron depletion is also known to reduce serum hemoglobin and oxygen supply to the tissue, which may induce angiogenesis. On the other hand, it is reported that hypoxia mediates epithelial-mesenchymal transition (EMT) and cancer cell metastasis with angiogenesis. In addition, it is thought that antitumor autoimmune response relates to suppression of EMT, and we investigated previously it with clinical samples. In this study, we examined the mechanism of iron control therapy in breast cancer based on hypoxia, EMT and immune microenvironment.

Material and Methods: Human breast cancer cell lines MDA-MB-231 (triple-negative breast cancer), MCF-7 (luminal type) and OCUB-1 (HER2-enriched type), and iron chelator Deferoxamine (DFO) were used in the present study. The effect of proliferation and migration of breast cancer cells by DFO was determined using MTT assay and wound healing assay. We also examined the influence of cell cycle by DFO using flow cytometry. Furthermore, we investigated the relationship between iron depletion of breast cancer cells and hypoxia, EMT and immune microenvironment using RT-PCR. We produced subcutaneous xenografts of mice, and examined the change in tumor sizes by administration of DFO.

Results: The iron-depleted condition suppressed breast cancer cell proliferation and migration. In cell cycle analysis, DFO increased the population of the G0-G1 phase. In addition, iron depletion induced hypoxia and angiogenesis. Also, N-cadherin and PD-L1 expression of breast cancer cells were increased by iron-depleted condition. Subcutaneous tumors in the DFO administration group were suppressed tumor growth in vivo.

Conclusion: Iron control by DFO showed the effect of tumor suppression, and suggested the possibility of novel treatment for patients with breast cancer. In addition, this mechanism related to hypoxia, EMT and immune microenvironment.

#5213

HRH1 inhibition enhances antitumor therapeutic responses through ERK activation in breast cancer.

Patricia Fernandez-Nogueira,1 Aleix Noguera Castells,1 Gemma Fuster Orellana,1 Leire Recalde Percaz,1 Nuria Moragas Garcia,1 Anna Lopez Plana,1 Patricia Jauregui Jimenez,1 Maria Neus Carbó Carbó,2 Pedro Gascon Villaplana,1 Paloma Bragado Domingo,1 Mario Mancino1. 1 _IDIBAPS, Barcelona, Spain;_ 2 _Universitat de Barcelona, Barcelona, Spain_.

Histamine receptor 1 (HRH1) belongs to the rhodopsin-like G-protein-coupled receptor family. Its activation by histamine, a ubiquitous messenger molecule released mostly from mast cells, has been demonstrated to be involved in cell proliferation, embryonic development, and tumor growth. In particular, high levels of HRH1 have been associated with dual behavior in the regulation of several tumors. Most of the physiological and pathological actions of HRH1 have been shown to depend strictly on its level of expression and the tumor cell type under study. We recently established that HRH1 is upregulated in patients with basal and HER2-enriched breast tumors and that its expression correlates with a worse prognosis. Despite this, the functional role of HRH1 basal and HER2 targeted therapies for resistant breast cancer (BC) progression has not so far been addressed. We assessed HRH1 expression in a wide panel of breast cancer cell lines and found HRH1 overexpressed in basal and HER2-targeted therapy resistant to BC cell lines at the protein and mRNA levels, corroborating data previously published using patient databases. Using terfenadine, a selective chemical inhibitor of HRH1, we showed that inhibition of HRH1 activity in basal BC cells leads to sub-G0 accumulation, suppresses proliferation, promotes cell motility and triggers the activation of ERK signaling, initiating the mitochondrial pathway of caspase cascade activation, inducing apoptosis. Moreover, in vivo experiments showed that terfenadine therapy reduced basal and HER2-targeted therapies for resistant BC cell tumor growth but does not affect luminal and parental tumor growth. In conclusion, our results suggest that targeting HRH1 might be an effective therapeutic treatment in patients with basal and HER2-targeted resistant BC tumors.

#5214

Tumor cell-derived complement components C1r and C1s promote growth of cutaneous squamous cell carcinoma.

Pilvi Riihilä,1 Liisa Nissinen,1 Mehdi Farshchian,1 Markku Kallajoki,1 Atte Kivisaari,1 Seppo Meri,2 Reidar Grénman,1 Sirkku Peltonen,1 Juha Peltonen,1 Veli-Matti Kähäri1. 1 _Univ. of Turku, Turku, Finland;_ 2 _Univ. of Helsinki, Helsinki, Finland_.

The incidence of cutaneous squamous cell carcinoma (cSCC) is increasing worldwide. We have analyzed the role of complement components C1r and C1s in the progression of cSCC. Analysis of cSCC cell lines (n=8) and normal human epidermal keratinocytes (NHEK)(n=11) with oligonucleotide arrays, RNA-seq and quantitative RT-PCR revealed significantly elevated C1r and C1s mRNA levels in cSCC cell lines. Western blot analysis showed increased production of C1r and C1s in cSCC cells compared to NHEKs. The mRNA levels for C1r and C1s were markedly elevated in cSCC tumors (n=6) compared to normal skin (n=11) in vivo. Immunohistochemical analysis for C1r and C1s revealed strong tumor cell specific expression of C1r and C1s in invasive sporadic cSCCs (n=164) and recessive dystrophic epidermolysis bullosa-associated cSCCs (n=16), whereas the expression of both was clearly lower in cSCC in situ (n=63), in premalignant epidermal lesions (actinic keratoses, n=61), and in normal skin (n=9). Knockdown of C1r and C1s expression with two specific siRNAs inhibited proliferation of cSCC cells and growth of human cSCC xenograft tumors in vivo. These results provide evidence for the role of tumor cell-derived C1r and C1s in the progression of cSCC independently of C1q and identify these serine proteinases as putative therapeutic targets in unresectable and metastatic cSCC.

#5215

Vitamin D receptor regulates intestinal barriers in colon cancer.

Yong-guo Zhang, Rong Lu, Danika Bakke, Yinglin Xia, Jun Sun. _University of Illinois at Chicago, Chicago, IL_.

Background Vitamin D and Vitamin D receptor (VDR) play important roles in the development of colon cancer. Tight junction structures are essential in intestinal barrier integrity, inflammation, and cancer. The disruption of tight junctions is a common manifestation of various diseases including cancers. Claudin-5 is a tight junction protein that is expressed in epithelia and endothelia and form paracellular barriers and pores that determine permeability. It is downregulated in colon cancer. Although VDR and Claudin-5 are all involved in colon cancer, it remains unclear whether they are closely related or function independently. Methods In the current study, we investigate the novel role of VDR in regulating Claudin-5, using whole body VDR knockout mice (VDR-/-) and intestinal epithelial VDR knockout mice (VDRΔIEC). In vitro, VDR-/- embryonic fibroblasts (MEF) cells, cultured human intestinal epithelial cells, organoids, and human colon cancer samples were used to determine the molecular mechanism. Results In intestinal samples of colon cancer patients, VDR expression is low, whereas the expression of Claudin-5 is also decreased. In the colon of VDR-/- mice and VDRΔIEC mice, deletion of VDR led to lower claudin-5 at the protein and mRNA levels. This observation was also confirmed by immunostaining of Claudin-5. In VDR-/- MEF cells, both Western blot and real time PCR revealed that VDR deletion led to lower protein and mRNA levels of claudin-5 in cells without any treatment. Vitamin D3 is known to increase VDR expression and activate VDR signaling. Protein and mRNA levels of Claudin-5 were significantly elevated in mice, mouse organoids, and human intestinal epithelial SKCO15 cells treated with 1, 25 vitamin D3. In contrast, knockdown of Claudin-5 using siRNA did not change the expression level of VDR protein in human colonic epithelial cells. Our data suggest that the Claudin-5 gene is a downstream target of the VDR signaling pathway. Conclusion Taken together, we show that VDR is important for the maintenance of physiological level of Claudin-5. This study also reveals a complex role for vitamin D/VDR regulation of intestinal barrier functions in colon cancer.

#5216

Exosome as a vehicle for delivery of membrane protein therapeutics, PH20 for enhanced tumor penetration and anti-tumor efficacy.

yoosoo yang, Yeonsun Hong, Gi Beom Kim, Gihoon Nam, In-san Kim. _Korea Institute of Science and Technology, Seoul, Republic of Korea_.

As biochemical and functional studies of membrane protein remain a challenge, there is growing interest in the application of nanotechnology to solve the difficulties of developing membrane protein therapeutics. Exosome, composed of lipid bilayer enclosed nano-sized extracellular vesicles, is a successful platform for providing a native membrane composition. Here, we report an enzymatic exosome, which harbors native PH20 hyaluronidase (Exo-PH20), was able to penetrate deeply into tumor foci via hyaluronan degradation, allowing tumor growth inhibition and increased T cell infiltration into the tumor. This exosome-based strategy was developed to overcome the immunosuppressive and anti-cancer therapy-resistant tumor microenvironment, which is characterized by an overly accumulated extracellular matrix. Notably, our engineered exosome with the native GPI-anchored form of hyaluronidase had a higher enzymatic activity than a truncated form of the recombinant protein. In addition, the exosome-mediated co-delivery of PH20 hyaluronidase and a chemotherapeutic (doxorubicin) efficiently inhibited tumor growth. This exosome is designed to degrade hyaluronan along the path of its movement, thereby augmenting nanoparticle penetration and drug diffusion. Our results thus show that this is a promising exosome-based platform that harbors not only a membrane-associated enzyme with high activity but also therapeutic payloads.

#5217

Platelet miRNAs shape tumor cell gene expression and inhibit growth of primary pancreatic ductal adenocarcinoma tumors and tumor-induced pro-coagulant state, via platelet microparticle-mediated RNA transfer.

Sophia Lazar,1 Jeremy G. Wurtzel,1 A. Koneti Rao,1 Edna Cukierman,2 Igor Astsaturov,2 Lawrence E. Goldfinger1. 1 _Lewis Katz School of Medicine at Temple University, Philadelphia, PA;_ 2 _Fox Chase Cancer Center, Philadelphia, PA_.

The goal of this study is to determine roles of platelet-derived microRNAs (miRNAs) and platelet microparticles (PMPs) in primary tumor growth and hypercoagulant state in pancreatic cancer. Pancreatic ductal adenocarcinoma (PDAC) has long been associated with greatly increased risk and incidence of venous thromboembolism (VTE), due to transition to a hypercoagulable state, associated with increased platelet activity. Platelet activation and release of PMPs are upregulated in PDAC. Platelet and MP surfaces act as docking sites for essential coagulation factors including Tissue Factor (TF). TF upregulation in carcinoma cells is associated with VTE risk, due to membrane-bound TF on tumor cell-derived MPs and other surfaces such as PMPs. Oncogenic KRAS and MYC and TP53 loss enhance TF expression, and KRAS activation coupled with TP53 loss causes invasive PDAC in patients and mice. We now find that wild type mice bearing ectopic PDAC tumors derived from spontaneous tumors in Kras mutant/Tp53-null/Pdx-Cre mice (KPC tumor cells) have reduced circulating platelet counts, indicating ongoing platelet consumption likely representing disseminated intravascular coagulation. However, plasma MPs were increased in tumor-bearing mice, including PMPs and non-platelet-derived MPs which may be tumor-derived, and both MP populations showed increased surface TF compared to non-tumor-bearing mice. Mice with KPC tumors also had platelets with surface TF as well as increased plasma thrombin-antithrombin complex. We recently demonstrated that PMPs selectively infiltrate solid tumors, but not adjacent unaffected tissue, and transfer platelet-derived miRNAs, suppressing tumor cell gene expression and inhibiting tumor growth. To investigate a specific role for platelet miRNAs in primary PDAC tumor growth, we implanted KPC tumors ectopically in mice with platelet-specific depletion of miRNAs via Pf4-Cre-mediated knockout of floxed Dicer1 endoribonuclease required for miRNA maturation. KPC tumor growth was accelerated in these mice compared to controls, demonstrating that platelet miRNAs per se are tumor suppressive in this model. RNAseq analysis indicated broad suppression of gene expression, including Kras and F3 (TF mRNA), in tumor cells as a result of PMP exposure in vitro. A major miRNA species in platelets and PMPs is the let-7 family, and one isoform, let-7g, is known to target KRAS and MYC. Let-7g over-expression suppressed KPC growth in vitro, reduced TF by qRT-PCR, and substantially reduced TF surface exposure. Thus, platelet miRNAs are tumor suppressive in PDAC and may also reduce hyper-coagulant activities in part through downregulation of genes including TF. Together these results demonstrate an active interplay between platelet miRNAs, tumor growth, and pro-coagulant states in cancer.

#5218

Boosting doxorubicin-induced immunogenic cell death by using tumor microenvironment modulating nanoparticles.

Mohammad Ali Amini,1 Azhar Abbasi,1 Ping Cai,1 Hoyin Lip,1 Jason Li,1 Claudia Gordijo,1 Li Zhang,2 Michael Rauth,2 Xiao Yu Wu1. 1 _Univeristy of Toronto, Toronto, Ontario, Canada;_ 2 _Toronto General Research Institute, the University Health Network, Toronto, Ontario, Canada_.

Background: Immunogenic cell death (ICD) is a cell death modality which triggers an immune response against dead-cell antigens, particularly when they derive from cancer cells. ICD has been reported to be highly induced by anthracycline drugs, such as doxorubicin (DOX), via facilitating tumor antigen presentation to and activation of T cells. However, these immunogenic effects may be compromised due to the immunosuppressive tumor microenvironment (TME), such as excess ROS, particularly H2O2, produced by tumor cells and dysfunctional macrophages in the tumor. We hypothesize that modulating immunosuppressive factors in the TME can enhance tumor response to chemotherapy and boost immunity. Specifically, polymer-lipid encapsulated manganese dioxide nanoparticles (PLMD NPs) are used to reduce tumor hypoxia and ROS by scavenging H2O2 and generating oxygen. Antitumor immunity against the cancer cells can be enhanced by the combination therapy with PLMD and DOX. Methods: PLMD NPs were prepared by dispersing MnO2 precursor particles in melted lipid and polymer in a surfactant-containing aqueous medium. The efficacy of the combination treatment was evaluated in immunocompetent mice-bearing orthotopic murine EMT6 breast tumor. The mice were treated by intravenous (IV) injection of free DOX alone, or 4 h after PLMD NP IV injection. A re-challenge study was performed by re-inoculating EMT6 cells in cured mice 120 days after the initial treatments. to validate the presence of adaptive anti-tumor immunity in the animals treated with PLMD+DOX combination, splenocyte transfer study was carried out. Ex-vivo imaging techniques were used to measure the number of infiltrated CD8+ T cells into the tumor and polarization of the M2 to M1 macrophages. Results: The PLMD+DOX combination treatment resulted in the median survival time by ~ 5.6-fold as compared to DOX alone, and led to 60% cure rate (9 out of 15 mice). This combination therapy also generated anti-tumor immunity against tumor re-inoculation in 88% of surviving mice and provided anti-tumor immunity in naïve mice that received splenocyte transfer with 57% of mice without tumor growth after inoculation. Ex-vivo studies resulted a 400% increase in the number of intratumoral CD8\+ T cells, 5 days after PLMD+DOX treatment, while DOX alone treatment showed only 150% increase when compared to saline treatment. Moreover, M1 macrophages-related CD86 positive cells were found up-regulated by 51% while a significant loss of M2 marker CD163 (-47%) was observed in PLMD+DOX-treated tumors compared to DOX alone-treated ones. Conclusions: The present work demonstrated that clinically suitable PLMD NPs can effectively downregulate TME-associated immunosuppression factors and boost DOX efficacy. The PLMD NPs and DOX combination therapy presents a multimodal and a translational treatment approach to enhancing DOX-induced ICD and boosting anti-tumor immunity.

#5219

Effect of extracellular vesicles from gastric cancer cells on the morphologic and genetic changes of peritoneal mesothelial cells.

Tomohisa Okuno, Masakazu Yashiro, Shingo Togano, Kenji Kuroda, Yuichiro Miki, Hiroaki Kasashima, Kosei Hirakawa, Masaichi Ohira. _Osaka City Univ. Grad. School of Medicine, Osaka city, Japan_.

Background: Gastric cancer is a major cause of cancer death, particularly on the basis of peritoneal metastasis. Human scirrhous gastric carcinoma (SGC), diffusely infiltrating carcinoma, or Borrman type 4 also known as linitis plastica-type carcinoma is characterized by rapid cancer cell infiltration and proliferation accompanied by extensive stromal fibrosis. SGC account for about 10% of all gastric carcinomas, and carry an extremely worse prognosis than other types of gastric carcinoma, reflecting their frequent peritoneal metastasis. We previously reported that SGC cells ("seed") might change the peritoneum to favorable environment, so-called pre-metastatic niche ("soil"), (Cancer 77:1668-75, 1996). Recently, it has been clarified that extracellular vesicles (EVs) play an important role for cell-cell interactions. In this study, we investigated the effect of EVs from SGC cells on the pre-metastatic niche formation of the peritoneum.

Methods: A SGC cell line, OCUM-2MD3, OCUM-12, KATO-III, gastric cancer cell line, MKN74, MKN45, and peritoneal mesothelial cells (PM cells) were used. PKH26-labeled EVs from OCUM-2MD3 cells were intravenously injected into nude mice, and the distribution of EVs was examined by a fluorescence microscope. The morphology of PM cells was investigated in the addiction of EVs from various gastric cancer cell lines using a phase-contrast microscope. And gene expression was examined by RT-PCR in the presence or absence of EVs from OCUM-2MD3. Clinical significance of EVs markers CD9 and CD63 was immunohistochemically evaluated using 63 human gastric cancer specimens.

Results: PKH26-labeled EVs were frequently found in the peritoneum, stomach and liver, but rare in lung and brain of mice. Peritoneal mesothelial cells uptake EVs of gastric cancer cells and change morphology from cobblestone shape to spindle shape in vitro. mRNA expression level of MMPs and integrins in PM cells was increased following the addiction of EVs, in compared with the control. The high expression of CD9 and CD63 was significantly correlated with distant metastasis.

Conclusion: EVs from gastric cancer cells might induce a pre-metastatic niche at peritoneum by the morphologic and genetic changes of mesothelial cells.

#5220

The role of brain-derived neurotrophic factor in ovarian cancer initiation and progression.

Min Kang, Kay Y. Chong, Tobias M. Hartwich, Oluwagbemisola O. Madarikan, Jonah Nucci, Sarah L. Cady, Yang Yang-Hartwich. _Yale School of Medicine, New Haven, CT_.

Ovarian cancer is one of the most devastating cancers in women. A fundamental step toward improving detection and treatment of this lethal disease is to understand the mechanism of its initiation and progression. In this study we demonstrated the role of brain-derived neurotrophic factor (BDNF) in ovarian cancer initiation and progression, particularly, the high-grade serous ovarian carcinoma (HGSOC). BDNF was discovered in the brain as a growth factor and chemoattractant inducing the migration, survival, and differentiation of neurons. It is also secreted by the ovary into the follicular fluid and by the adipocytes in the omentum and peritoneum. BDNF suppresses anoikis, the apoptosis induced by lack of proper cell to extra-cellular matrix (ECM) attachment. BDNF has been associated with tumor progression in colon, breast, lung, and gastric cancers. In ovarian tumors both BDNF and its receptor tropomyosin receptor kinase B (TrkB) are expressed. Their overexpression is associated with poor survival. Fallopian tube epithelial cells (FTEs) are hypothesized to be the cell of origin of HGSOC. We used 3 immortalized human normal FTE cell lines to demonstrate the effects of BDNF on the survival, mobility, and adhesion of FTEs in vitro. CellTiter luminescent cell viability assays were conducted to evaluate the effects of BDNF on the short-term and long-term survival of FTEs in 3D culture conditions. Caspase-3 activity assays were used to quantify the extent of which BDNF inhibited anoikis in FTEs. Trans-well and 3D bioprinting migration assays were used to determine whether BDNF could promote the mobility of FTEs. The ECM-coated beads were co-cultured with FTEs in a 3D model and their attachment to ECM was quantified in order to evaluate the effects of BDNF on the cell adhesion. The BDNF-activated intracellular pathways were identified using Western blots, RNA microarray, RT-QPCR analyses. Our data demonstrated that TrkB was expressed by the FTEs in human and mouse fallopian tubes. BDNF significantly enhanced the survival of FTE cell lines in the serum-free 3D culture condition. Their enhanced survival was also evidenced by the decreased caspase-3 activity indicating the inhibition of anoikis. BDNF increased the ability of FTEs to migrate in the migrations assays. The BDNF-treated FTEs attached to the ECM-coated beads faster than the untreated cells. Western blots showed that BDNF activated the phosphorylation of TrkB, AKT, ERK, PLC-gamma1, and CREB. RNA microarray and QPCR data suggest that epithelial-mesenchymal transition (EMT), anti-oxidative stress, and ECM-related pathways were activated by BDNF leading to the enhanced survival, migration, and cell adhesion. These results revealed the potential role of BDNF to promote ovarian cancer initiation. Understanding this molecular pathway will lead to the development of more specific markers for early detection and better prevention and treatment strategies.

#5221

MAP3K11 regulates hedgehog signaling and suppresses tumor microenvironment in genetic mouse models of pancreatic cancer.

Navin Viswakarma, Gautam Sondarva, Lucas Ibrahimi, Daniel R. Principe, Basabi Rana, Ajay Rana. _University of Illinois at Chicago, Chicago, IL_.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-associated mortality worldwide with an overall five-year survival rate less than 5%. Despite extensive efforts, cytotoxic and targeted therapies have provided only limited efficacy for PDA patients to date. One contributing factor to the failure of systemic therapies may be the abundant tumor stromal content that is characteristic of PDA. Therefore, it is extremely important to identify alternative pathways which can be targeted in PDA. Here we report a MLK-mediated novel pathway that promotes oncogenic potential of Gli1, an effector of hedgehog pathway (Hh), can be inhibited in PDA. Earlier, we reported that MLK3 phosphorylates Pin1 on Ser138 site and increases its catalytic activity and nuclear translocation. In order to identify targets of MLK3 and nuclear p-Pin1, we performed cancer specific gene expression profiling and found 13-fold up-regulation of Gli1 transcript, suggesting MLK3-Pin1-Gli1 axis could activate Hh pathway. Most importantly, MLK3, pMLK3, Pin1 and Gli1 were overexpressed in human pancreatic cancer tissues, compared to normal adjacent tissues. Interestingly, MLK3 was able to directly phosphorylate Gli1 on specific residues and the transcriptional activity of Gli1 was synergistically regulated by Pin1 and MLK3 together, which was partially blocked by pan-MLK inhibitor, CEP-1347. Another key finding was that CEP-1347 reduced the expression of Kras-G12D in the pancreatic cell lines derived from KPC mice. Therefore, we expanded our studies to demonstrate the therapeutic effects of MLK inhibition by CEP-1347 in genetically engineered mouse models of pancreatic cancer. LSL-KrasG12D/+; Pdx1-Cre (KC) mouse model was used to investigate this effect in pancreatic tumorigenesis suppression; LSL-KrasG12D/+; Trp53fl/+; Pdx1-Cre (KPC) mouse model was used to evaluate this in PDAC. Chronic pancreatitis induced a stroma-rich and duct-like structure and increased the formation of ADM and PanIN lesions in KC and KPC mice with an increased cytokeratin 19 (CK19)-stained area. CEP treatment diminished chronic pancreatitis-mediated ADM and PanIN formation. In addition, it alleviated the percent area of Masson's trichrome staining, α-SMA and vimentin expression. In KPC mice, CEP-1347 inhibited tumor growth and the incidence of abdominal invasion. More importantly, it prolonged the overall survival. In conclusion, our findings suggest that pathological activation of MLK3-Pin1 axis could promote survival of pancreatic cancer cells via Hh pathway activation. Collectively our results suggest that targeting MLKs could be an alternative approach to overcome Desmoplasia and PDAC.

#5223

IL-6 regulates the OCT4 expression by epigenetic modulation in hepatocellular carcinoma.

Ssu-Chuan Lai,1 Te-Sheng Chang,2 Yu-Ting Su,1 Yu-Chih Wu,3 Yung-Che Kuo,3 Pei-Chi Lan,3 Yen-Hua Huang4. 1 _Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Tiapei, Taiwan;_ 2 _Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Chang Gung Memorial Hospital, Tiapei, Taiwan;_ 3 _Department of Biochemistry and Molecular Cell Biology, College of Medicine, Tiapei, Taiwan;_ 4 _International PhD Program for Cell Therapy and Regeneration Medicine, College of Medicine, Taipei Medical University, Taipei Medical University Hospital, Tiapei, Taiwan_.

Purpose: Chronic hepatic inflammatory cytokine interleukin 6 (IL-6) appears important role in pluripotent transcription factor OCT4 expression particularly in HBV-related hepatocellular carcinomas (HBV-HCC). However, the underlying mechanism still remains largely unknown. Experimental Design: Serum levels of IL-6 were detected using ELISA assays (n = 120). The effects of IL-6 on DNMT/OCT4 expression in HCC were examined using real-time QPCR analysis, RNA interference, and xenograft animal models. The DMNT/OCT4 protein and phospho-STAT (p-STAT) were detected by western blotting. OCT4, and DNMT expression levels in tissues (n = 144) were analyzed by real-time Q-PCR and was correlated with early tumor recurrence using Kaplan-Meier survival analysis. Results: A high positive correlation between the expression levels of OCT4 and epigenetic DNMT family in human HCC tissues was observed. High serum levels of IL-6 were significantly correlated with high OCT4 and DNMT expression. IL-6 stimulated the DNMT expression STAT3-dependently, which stimulated the OCT4 expression in both the cell lines and the xenografted mouse tumors. The inhibition STAT phosphorylation/DNMT expression by dominant negative STAT-Y705F plasmid transfection or DNMT RNA interference significantly suppressed the IL-6-induced OCT4 expression in vitro. Conclusions: The expression of pluripotency-related OCT4 is associated with early tumor recurrence and is regulated by IL-6-induced epigenetic DNMTs in HCC.

#5224

hMENA isoforms impact NSCLC patient outcome through fibronectin/β1 integrin axis.

Francesca Di Modugno,1 Sheila Spada,1 Belinda Palermo,1 Paolo Visca,1 Pierluigi Iapicca,1 Anna Di Carlo,1 Barbara Antoniani,1 Isabella Sperduti,1 Anna Di Benedetto,1 Irene Terrenato,1 Marcella Mottolese,1 Francesco Gandolfi,2 Francesco Facciolo,1 Emily Chen,3 Martin A. Schwartz,4 Angela Santoni,2 Mina J. Bissell,5 Paola Nisticò1. 1 _Regina Elena National Cancer Inst., Rome, Italy;_ 2 _University Sapienza, Rome, Italy;_ 3 _Columbia University, New York, NY;_ 4 _Yale School of Medicine, New Haven, CT;_ 5 _Lawrence Berkeley National Laboratory, Berkeley, CA_.

The splicing of the actin regulator hMENA generates different isoforms and we have demonstrated that the two alternatively expressed isoforms, hMENA11a and hMENAΔv6, have opposite functions in cell invasiveness. This general mechanism is of great clinical relevance in early NSCLC patients, where the pattern of hMENA isoform expression is a powerful prognostic factor. However the mechanism of action of the two isoforms have remained unclear. Herein, we evaluated whether hMENA and its isoforms influence β1 integrin expression and signaling considering the role of this integrin in cancer cell invasiveness and tumor progression. We performed hMENA silencing by siRNA and shRNA, to evaluate by QRT-PCR and biochemical approaches the expression of β1 integrin; by immunofluorescence the MRTF1 localization, by in vivo assay G-Actin/F-Actin ratio and by luciferase reporter assay the SRF activity. β1 integrin activation and signaling was evaluated by flow cytometry using an antibody specific for the β1 active conformation and by biochemical analysis of the phosphorylation of FAK, SRC and Paxillin. The secretoma of hMENA11a transfected cancer cell lines was analyzed by LC-MS/MS. Immunohistochemical analysis was performed using pan-hMENA, hMENA11a, and fibronectin antibodies in primary cancer tissues from node negative NSCLC patients. The Chi-Square or Fisher Exact tests were used to estimate associations among categorical variables and disease-free survival was calculated by the Kaplan-Meier product limit method. We show that the depletion of all hMENA isoforms inhibits the Serum Response Factor (SRF) activity, and the expression of its target gene β1 Integrin, by affecting G-Actin/F-Actin ratio, critical for the nuclear localization of the SRF co-factor myocardin related transcription factor 1 (MRTF1). Furthermore, we provide new insights into the mechanisms involved in the opposite functions of hMENA11a and hMENAΔv6 in cell invasiveness and we identify a new role of these isoforms in the β1 integrin-ECM signalling axis. Indeed, hMENAΔv6-drives cancer cell invasion by increasing β1 integrin activation and signalling, which is reduced by the anti-invasive hMENA11a isoform. Moreover, exogenous expression of hMENA11a in hMENAΔv6 positive cancer cells dramatically reduces secretion of extracellular matrix (ECM) components, including β1 integrin ligands and metalloproteinases. On the other hand overexpression of the pro-invasive hMENAΔv6 increases fibronectin production. In primary tumors high hMENA11a correlates with low stromal fibronectin and favorable clinical outcome of early node-negative non-small cell lung cancer patients. This newly discovered signature, which pays attention to the alternative splicing of hMENA and ECM components such as fibronectin in the stroma, might help fill in the gap in the still controversial clinical management of early node-negative NSCLC patients.

#5225

PAR1 signaling on tumor cells limits tumor growth by maintaining a mesenchymal phenotype in pancreatic cancer.

Cansu Tekin, Kun Shi, Arnold Spek, Maarten F. Bijlsma. _Academic Medical Center/ University of Amsterdam, Amsterdam, Netherlands_.

Background: Pancreatic cancer is a dismal disease with limited treatment options. Importantly, in recent years an oncogenic role of protease-activated receptor 1 (PAR1) emerged in various cancer types, and PAR1 is generally accepted to promote tumor progression. PAR1 depletion in stroma limits cancer growth in an orthotopic pancreatic cancer model, suggesting that PAR1 would be a prognostic marker for PDAC.

Aim: To determine PAR1 expression in tumor compartment of PDAC microenvironment and its contribution to tumor progression and invasion.

Methods: Kaplan-Meier overall survival analysis for PAR1 expression on PDAC tumor expression sets was performed. In an orthotopic tumor model, short hairpin RNA mediated PAR1 (shPAR1) knockdown cells derived from p48-CRE/LSL-KRAS/p53flox/flox mice (KP cells) and Panc02 cells were used. Additionally, gene set enrichment analysis was done for EMT signatures on PDAC tumor and pancreatic cancer cell lines. Furthermore, in vitro migratory capacity and mRNA levels for emerging mesenchymal signatures were analyzed in PAR1 knockdown PANC-1 cell line.

Results: PAR1 expression does not associate with the overall survival of PDAC patients. However, tumor cell-specific PAR1 silencing potentiates pancreatic tumor growth but decreases metastatic potential in subsequent murine orthotopic pancreatic cancer model. Further analysis of PAR1 expression in PDAC tumors and pancreatic cancer cell lines show positive correlation with mesenchymal signatures and epithelial to mesenchymal transition genes. Indeed, PAR1 knockdown results in well-differentiated pancreatic tumors in vivo, with enhanced epithelial characteristics both in vitro and in vivo. Furthermore, decreased PAR1 activity decreases migratory capacity and in vitro metastatic potential in vivo.

Conclusion: Our results suggest that growth promoting effects of PAR1 in stromal cells are counterbalanced by the growth inhibitory contributions of PAR1 in tumor cells. Overall, tumor cell PAR1 is linked to the maintenance of a mesenchymal-like state and silencing PAR1 in this setup results in an increased epithelial phenotype of cancer cells with an increased proliferative index but a decreased metastatic potential.

#5226

Tiam1 is a key modulator of chemoresistance in colorectal cancer.

Daisuke Izumi. _Baylor Scott &White Research Institute, Dallas, TX_.

Purpose: Emerging evidence indicates that a small subpopulation of cancer cells with stem cell-like properties are responsible for acquisition of drug resistance in colorectal cancer (CRC). Intriguingly, the acquisition of such a stem-like state and subsequent drug resistance has been shown to be induced by the tumor microenvironment. In particular, cancer associated fibroblasts (CAFs) have been identified as a key component of tumor microenvironment which promote cancer stemness. Wnt signaling is a well-recognized pathway that drives self-renewal, and recently one of the Wnt-responsive genes, Tiam1, a guanine nucleotide exchange factor specific for Rac1, was shown to be overexpressed in CRC and associated with an aggressive cancer phenotype. Herein, we have investigated Tiam1 as a key modulator of chemoresistance in CRC.

Methods: Tiam1 expression was assessed in resected CRC tissues from 300 patients who did or did not respond to chemotherapy. We used siRNA and CRISPR/CAS9 approach in three cell lines and animal models to examine whether inhibition of Tiam1 affects chemosensitivity in CRC. In addition, we established CRC patient derived-CAFs and examined whether the drug sensitivity of CRC cells was altered by supplementation of CAF-derived conditioned medium.

Results: High Tiam1 expression significantly correlated with poor prognosis in CRC patients (P=0.032), and emerged as an independent prognostic factor (HR 5.06, 95% CI 1.10-23.2, P=0.038). In addition, Tiam1 overexpression is associated with patients who did not respond to chemotherapy (P=0.0005). We demonstrated that siRNA-mediated inhibition of Tiam1 enhanced sensitivity to three chemotherapeutic drugs (5-fluorouracil, oxaliplatin and irinotecan), and reduced tumor invasiveness in HCT116, SW480 and SW620 cell lines. Silencing of Tiam1 resulted in downregulation of stemness-related genes and spheroid formation ability. Furthermore, we validated that Tiam1-knockdown enhanced drug sensitivity and decreased the expression of stemness-related genes in xenograft tumors. Finally, we illustrated that the conditioned medium (CM) derived from CAFs led to increased stemness and chemoresistance in CRC cells through Tiam1 overexpression, and silencing of Tiam1 in CAFs resulted in reversal of stemness and chemoresistance properties. From a mechanistic standpoint, the target genes of Wnt signaling were also upregulated during co-culture with CM in CRC cells.

Conclusion: We have firstly demonstrated that Tiam1 is overexpressed in CRC patients who did not respond to chemotherapeutic drugs; and furthermore, high Tiam1 expression emerged as an independent prognostic factor in CRC. Mechanistically, CAFs enhanced CRC chemoresistance through Tiam1 overexpression. Collectively, these results suggest that Tiam1 suppression in stroma may sensitize CRC cells to chemotherapeutic agents, suggesting that Tiam1 may be an attractive therapeutic target in colorectal cancer.

## ENDOCRINOLOGY:

### Clinical Endocrinology

#5227

Suppressive role of elevated estrogen on endometrial carcinogenesis.

Ryoichi Asaka, Tsutomu Miyamoto, Koichi Ida, Hodaka Takeuchi, Hirofumi Ando, Yasushi Yamada, David Hamisi Mvunta, Hisanori Kobara, Hiroyasu Kashima, Tanri Shiozawa. _Shinshu Univ. School of Medicine, Matsumoto, Japan_.

Background: Although estrogen has been regarded as a risk factor for endometrial carcinoma, its concentration-dependent carcinogenetic effects remain unclear, because most endometrial carcinomas occur in post-menopausal women, whose serum estrogen levels are relatively low. Because impaired mismatch repair (MMR) was reportedly crucial in the early stages of endometrial carcinogenesis, we focused on the relationship between various estrogen concentration and MMR activity in normal and malignant endometrial glandular cells. In those cells, we reported that the protein and mRNA expression of both hMLH1 and hMSH2, the key factors of MMR, were up-regulated by estrogen stimulation. In addition, we demonstrated that the estrogen stimulation enhanced the MMR activity in those cells using in vitro MMR assay with the M13mp2 phage heteroduplex DNAs and that the proliferating cells without hMLH1/hMSH2 expressions implying "high risk" cells were more frequently observed under relative-low estrogen concentrations. In this study, to further confirm the role of E2 in vivo, we employed a mouse endometrial carcinoma model induced by intra-uterine injection of a carcinogen, N-methyl-N-nitrosourea (MNU) under various E2 concentrations.

Methods: MNU was injected into the uterine cavity of 29 CD-1 mice at 6 weeks of age, and E2 was administered by pellets or orally. Uteri were removed for histological examinations 24 weeks later, and serum E2 levels were measured. The immunohistochemical expression of MMR proteins in uterine epithelia was investigated.

Results: Of 29 mice, 8, 8, 8, and 5 showed atrophic, normal, hyperplastic, and carcinomatous endometria, respectively. The mean E2 levels of each group were 0.2pg/ml, 3.8pg/ml, 190.0pg/ml, and 6.7pg/ml, with significant differences. The expression of the MMR proteins was stronger in mice with elevated E2.

Conclusions: Elevated E2 levels preferentially induced endometrial hyperplasia rather than carcinoma, and this may be mediated by MMR proteins. These results indicate that modest E2 is needed, whereas elevated E2 levels are not necessarily advantageous for carcinogenesis, suggesting the importance of low-chronic (un-opposed) estrogen in human endometrial carcinogenesis.

#5228

Mammary gland tumors in progesterone receptor transgenic mice.

Katelin A. Gibson, Merit L. Goodman, Christy R. Hagan. _University of Kansas Medical Center, Kansas City, KS_.

The role of progesterone and its receptor, the progesterone receptor, in breast cancer remains controversial. Although years of clinical trial data have shown that synthetic progesterone (progestins) increases breast cancer risk, the role for native progesterone in breast cancer development is the source of much debate. PR is post-translationally modified, primarily through phosphorylation, by many mitogenic kinases, including ck2, cdk2 and MAPK. These phosphorylation events on PR dictate many aspects of PR biology, including promoter specificity and transcriptional co-factor interactions. Ck2, a mitogenic kinase shown to be upregulated in breast cancer, has been shown to phosphorylate PR and regulate transcription of a certain subset of PR target genes. In an effort to determine how this translates to murine mammary gland tumor formation, we created a bi-transgenic mouse overexpressing both PR and ck2 in the murine mammary gland. We studied long-term tumor formation in the PR/ck2 bi-transgenic mice, as compared to the single transgene controls (PR only or ck2 only), in both virgin and multiparous mice. Our preliminary data suggest a number of interesting results. First, tumor development is the highest in the PR transgenic mice, as compared to ck2 alone or PR/ck2 bi-transgenic mice. This is of particular interest as previous studies in PR transgenic mice failed to show mammary gland tumor formation, despite the presence of dysplastic mammary gland tissue. Moreover, the rate of tumor formation appears to be unaffected by parity: virgin and multiparous mice develop tumors at the same rate. This is in contrast to ck2 single-transgenic mice, where multiparty increases the rate of tumor formation (as observed by us and others). Interestingly, and most significantly to our studies, the combination of ck2 and PR appears to slow tumor formation rates as compared to either single transgenic mouse control. This phenotype is potentiated by pregnancy, as all multiparous PR/ck2 bi-transgenic animals still remain tumor free. These data, while still preliminary, suggest that ck2-dependent modification of PR may prevent mammary gland tumor formation, suggesting that PR regulation of a subset of genes (those genes regulated by ck2-phopshorylation) may drive anti-proliferative, tumor-preventing gene programs.

#5229

Expression and regulation of chondroitin sulfate in prostate cancer.

Nader Al Nakouzi,1 Chris Kedong Wang,1 Irina Nelepcu,1 Coralie Crouzit,1 Noushin Nabavi,1 Amal Almami,1 Htoo Zarni Oo,1 Thomas Mandel Clausen,2 Tobias Gustavsson,2 Ali Salanti,2 Mads Daugaard1. 1 _Vancouver Prostate Centre, University of British Columbia, Vancouver, British Columbia, Canada;_ 2 _Centre for Medical Parasitology, Copenhagen University Hospital, Copenhagen, Denmark_.

Increased levels of chondroitin sulfate (CS) glycosaminoglycans (GAGs) in prostate cancer (PC) have been observed for more than three decades. In 1984, De Klerk et al. noted that hyperplastic and cancerous prostate contained elevated levels of CS while other GAGs, like heparin sulfate (HS), were decreased. Chondroitin sulfate-modified proteoglycans (CSPGs), such as versican or tomoregulin (TENB2), have been promoted as PC progression markers. They have been associated with cell attachment to the matrix, the metastatic phenotype, disease progression, and androgen independence. Functionally, pericellular enrichment of CSPGs in the PC microenvironment promotes cell motility. Our team has discovered that human tumors display high levels of a specific highly sulfate type of CS normally restricted to placental and fetal tissue compartment. This "oncofetal" CS (ofCS) GAG can be conveniently detected and targeted using recombinant CS-binding VAR2CSA (rVAR2) lectins, derived from the malaria parasite Plasmodium falciparum. Recently, we identified chondroitin sulfate biosynthesis as being controlled by androgens in PC, modulating expression of the CHST11 and CHST13 carbon-4 GalNAc sulfotransferases. We established that these enzymes are under direct control of the androgen receptor (AR), regulating synthesis of the cancer-associated ofCS-modification on CSPGs. Glycosylation has a key role in many important biologic processes in cancer including cell differentiation. We identified CHST11 to be highly increased in high-risk neuroendocrine prostate cancer (NEPC) both in vitro, in vivo, and in situ. Moreover, cells that expressed neuroendocrine markers showed higher level of sulfation and ofCS. Our work reveals that alterations in GAG signatures regulated by AR might be responsible for progression neuroendocrine differentiation in prostate cancer. The prostate is an abundant secretor of PGs, and tumor-specific alterations in GAG signatures such as ofCS therefore constitute an untapped reservoir of potential biomarkers to be exploited as therapeutic targets.

#5230

Development of a multi-parameter immunofluorescence assay for simultaneous detection of androgen receptor and androgen receptor variant 7 in prostate cancer circulating tumor cells.

Yao Sun,1 Daniel Campton,1 Arturo Ramirez,1 Tanisha Mojica,2 Alisa Clein,3 Celestia Higano,3 Daniel Sabath,3 Stephen Plymate,3 Eric Kaldjian1. 1 _RareCyte, Inc., Seattle, WA;_ 2 _Seattle Cancer Care Alliance, Seattle, WA;_ 3 _University of Washington, Seattle, WA_.

Background. Androgen receptor (AR) signaling is the primary driver of prostate cancer. Expression of the AR splice variant AR-V7, is predictive of resistance to anti-androgen therapies enzalutimide and abiratirone. There is great interest to non-invasively identify circulating tumor cells (CTCs) and investigate their expression of AR and AR-V7 and monitor them over time. RareCyte has developed a platform for automated visual identification of CTCs by immunofluorescence (IF) that allows up to 6-parameter assessment. We have developed a set of prototype assays using tyramide amplification to detect AR and AR-V7 individually and in combination with prostate membrane-specific antigen (PSMA). Using a novel method, we also developed an assay to identify AR and AR-V7 simultaneously. Methods. Normal human whole blood samples were spiked with prostate cancer lines serving as model CTCs (mCTCs). Prostate cancer blood from a patient with advanced and known high CTC count were collected under an IRB-approved protocol. Blood was processed onto microscope slides and stained on an automated stainer with CTC detection assays incorporating 4 core parameters (nuclear dye, CD45, pan-cytokeratin, EpCAM) plus the following markers: (1) AR; (2) AR-V7; (3) AR and PSMA; (4) AR-V7 and PSMA; (5) AR and AR-V7. Each assay was applied to the cancer cell lines PC3, LNCaP, and 22RV1 and to the patient sample. An antibody denaturing process was used to sequentially amplify two rabbit monoclonals for the assay containing both AR and AR-V7. No-primary (diluent only) staining controls were run to confirm success of the denaturing step. Results. Staining of spike-in CTC models confirmed the reported AR and AR-V7 phenotype of the cell lines, supporting the specificity of the assays. When applied to an equivalent volume of the patient sample, the assays identified a mean (SD) of 36 (11) CTCs. In the assays staining AR, the mean percent of AR+ CTCs was 70% (range 61 - 86) and for the assays staining AR-V7, the percent of AR-V7+ CTCs was 30% (range 26 - 39). In the assay staining both AR and AR-V7, 10 of 38 CTCs were AR-V7+ (26%); all of these were AR+; 15 CTCs were only AR+ (39%). Conclusions. We have developed multi-parameter IF assays for detection of AR and AR-V7 in prostate CTCs, including the first assay we are aware of to simultaneously visualize AR and AR-V7. It has been validated in prostate cancer CTC models. The percentages of AR and AR-V7-positive CTCs identified in a patient sample with the assay are consistent with the percentages observed when AR and AR-V7 are detected in independent assays, supporting the accuracy of the simultaneous detection assay. All AR-V7-positive CTCs were also AR-positive, supporting specificity of AR-V7 staining.

#5231

**Small-molecule inhibitors of SCF-Skp2-Cks1 ubiquitin E3 ligase stabilize nuclear p27** kip1 **as a novel therapeutic approach to endometrial cancer.**

Chinaza Egbuta,1 Julien Dubrulle,2 Ana Tellechea,1 Miguel A. Manzanares,1 Yunfeng Li,3 Shanshan Duan,1 John K. Dickson,4 Mirco Meniconi,5 Kuo-sen Huang,6 Xinyan Huang,1 Nadim Shohdy,1 Michelle Pagano,1 Bing Hao,3 Michael A. Mancini,2 Leslie I. Gold1. 1 _New York Univ. School of Medicine, New York, NY;_ 2 _Baylor College of Medicine, Houston, TX;_ 3 _University of Connecticul Health Center, Farmington, CT;_ 4 _AptaChem Consulting, Apex, NC;_ 5 _Evotec (UK) Ltd, Oxfordshire, United Kingdom;_ 6 _Cepter Biopartners, Hoboken, NJ_.

We have previously shown that ubiquitin-mediated degradation of the cyclin-dependent kinase inhibitor, p27kip1 (p27), critical for G1 arrest, is involved in the pathogenesis of Type I endometrial cancer (ECA; 80% of ECAs), which is estrogen (E2) induced and usually preceded by hyperplasia. In vitro, E2 induces MAPK phosphorylation of p27 at Thr187, which is required for ubiquitylation of p27 by the SCF-Skp1-Skp2-Cks1E3 ligase to signal its degradation by proteasomes. We have shown that the absence of nuclear p27 in human endometrial hyperplasia and ECA is due to high SCF-Skp2-Cks1 E3 ligase activity, exemplified in vitro by a decrease in nuclear p27 and proliferation of ECA primary cells and cell lines. The 3D structure of Skp2 and Cks1 harboring p27 was previously solved. Two approaches were used to identify small-molecule inhibitors of the SCF-Skp2-Cks1 E3 ligase (Skp2E3LIs) that block p27 ubiquitylation: (i) structure-based virtual screen targeting the protein interaction surfaces formed by Skp2-Cks1 (the p27 binding pocket) and separately, the Skp1-Skp2 interface; and (ii) ligand-based virtual screen of previously published compounds, which target the p27 pocket within Skp2-Cks1 (blocked E2-induced endometrial hyperplasia in mice with an increase in nuclear p27) or the Skp1-Skp2 interface (blocked tumor growth in mice). The virtual screening hits (207 compounds) from both approaches were subjected to time-resolved-FRET (Skp2-Cks1 pocket interface) and ELISA (Skp1-Skp2 interface) for evaluation of their physical binding to Skp1-Skp2-Cks1 (SSC) complex. 35 hits that elicited IC50s ≤120 µM in both assays were further evaluated by high-content image analysis (HCA) for toxicity, uridine (EdU) uptake, and number of p27 fluorescent nuclei. We confirmed 3 Skp2E3LIs that show IC50s less than 60 µM (E12, E17, E27) are nontoxic by trypan blue, have EC50s for growth inhibition ≤15 µM, increase nuclear p27, and block retinoblastoma protein (pRb) phosphorylation, preventing S phase progression. Importantly, Skp2E3LIs block both E2-induced degradation of nuclear p27 and proliferation and induce an increase in nuclear p27 in 3D spheroids composed of ECA cells and ECA cancer-associated fibroblasts (CAFs). The lead compounds will be co-crystallized with the SSC complex and together with SAR studies will be optimized (i.e., biologic activities and druggability parameters) for therapeutic development. SKp2E3LIs represent a new class of specific E3 ligase inhibitors that prevent degradation of cell cycle proteins involved in growth control. Therefore, lead compounds have the potential to be a major therapeutic advancement over current general proteasome inhibitors that indiscriminately block protein degradation including oncogenes. Skp2E3LIs are promising new therapeutics for ECA and other cancers characterized by high Skp2 levels and loss of nuclear p27.

#5232

**1,25-Dihydroxyvitamin D** 3 **mitigates lung cancer cell mediated mitochondrial dysfunction in human skeletal muscle .**

Zachary C. Ryan, Theodore A. Craig, Xuewei Wang, Philippe Delmotte, Jeffrey L. Salisbury, Ian R. Lanza, Gary C. Sieck, Rajiv Kumar. _Mayo Clinic, Rochester, MN_.

To assess cellular mechanisms that could contribute to muscle weakness in the context of cancer cachexia, we evaluated the effect of conditioned medium (CM) from highly tumorigenic Lewis lung carcinoma (LLC1) cells, CM from less tumorigenic MLE12 cells and non-CM on human skeletal muscle mitochondrial morphology and function. We assessed mitochondrial oxygen consumption, morphology, enzyme activity and gene expression in human skeletal muscle cells in the presence of tumor conditioned medium or control medium. We examined the secretome of LLC1 cells for mediators of skeletal muscle changes. We demonstrate that LLC1 CM inhibits cellular O2 consumption rate (OCR) and increases the proton production rate in human skeletal muscle cells. Mitochondrial fragmentation and the expression of the fission mediator, FIS1, are increased, whereas the expression of fusion mediators, OPA1 and MFN2 are reduced by LLC1 CM. LLC1 CM reduces pyruvate dehydrogenase (PDH) activity thereby limiting the production of acetyl CoA. Reduced PDH activity is associated with reduced PDHA1 sub-unit expression. The amount inactive phospho-PDH is increased in association with reduced PDP2 expression and increased PDK4 and SIRT4 expression in human skeletal muscle cells treated with LLC1 CM. The expression of several signaling pathways is altered by LLC1 CM in human skeletal muscle cells. When compared to MLE12 cells, LLC1 cells show increased expression of mRNAs encoding several potential mediators of altered muscle function such as Gdf15, TGFβ-induced protein, IL11, Il27, Il33, Il34, and PTH-like protein. We show that the active vitamin D metabolite, 1α,25(OH)2D3, that we previously demonstrated increases myoblast mitochondrial oxygen consumption and PDH activity, mitigates LLC1 CM effects on hSkMC OCR, mitochondrial fragmentation and mediators thereof. Mitochondrial oxygen consumption, morphology and enzyme activity in human skeletal muscle cells are inhibited or altered by tumor cell conditioned medium, and are reversed by 1α,25(OH)2D3, the active metabolite of vitamin D.

#5233

Associations between somatic mutations and clinical manifestations in South American Hispanic patients with papillary thyroid cancer.

Ana P. Estrada-Florez,1 Mabel E. Bohorquez,1 Carlos S. Duque,2 Jorge Donado,2 Gilbert Mateus,3 Fernando Bolaños,4 Alejandro Velez,2 Erika Estrada,5 Guadalupe Polanco-Echeverry,5 Ruta Sahasrabudhe,5 Elisha Garcia,5 Magdalena Echeverry,1 Luis G. Carvajal-Carmona5. 1 _Universidad del Tolima, Ibague, Colombia;_ 2 _Hospital Pablo Tobon Uribe, Medellin, Colombia;_ 3 _Hospital Federico Lleras Acosta, Ibague, Colombia;_ 4 _Hospital Hernando Moncaleano Perdomo, Neiva, Colombia;_ 5 _University of California Davis, Davis, CA_.

Papillary thyroid cancer (PTC) is the second most commonly diagnosed tumor in U.S. Hispanic women, representing ~9% of all incident female cancer cases in this minority. In Colombia, a country with a predominantly Hispanic population, PTC is the fifth most commonly diagnosed cancer in women. Several studies, predominantly in non-Hispanic whites (NHW), have associated the RAS-RAF gene and TERT promoter mutations with severe PTC clinical manifestations. We investigated associations between mutations in these genes and clinicopathologic data (age, gender, histologic type, tumor features and AJCC stage) in 143 Colombian cases (76 with classical variant PTC, CVPTC and 65 with follicular variant PTC, FVPTC). DNA from archival samples was isolated using standard methods and mutation status at hotspots in BRAF (V600E), NRAS (Q61R, Q61K), HRAS (Q61R, Q61K), and TERT (C228T, C250T) were assessed using Sanger sequencing. Clinicopathologic data between mutation carriers and noncarriers and with data from NHWs in the TCGA were carried out with t-tests and odds ratios (OR). BRAF V600E was detected in 59% of all cases, a rate that is similar to NHW (64%; P=0.29). When we stratified the data by subtype, we found a similar V600E frequency in CVPTC in Colombian cases than in NHWs (67% vs 68%; P=0.87) but a borderline significantly higher frequency of the mutation in FVPTC (49% vs 29%; P=0.06). TERT promoter mutations were found in 17% of all cases, a rate that is significantly higher than in NHW (10%, p= 0.03). 5% of cases had mutations in HRAS/NRAS, which is lower than in NHW (11%; P=0.04). Given the low RAS gene mutation frequency, comparisons with clinical data were carried out only with BRAF and TERT data. BRAF/TERT mutation-positive samples (BRAF+/TERT+, n=18) were compared to mutation-negative cases for either or both genes (n=123). BRAF+/TERT+ tumors were more common in CVPTC than in FVPTC (17% vs. 7%; OR=4.1; P=0.02), and in cases with tumors >2cm (20% vs. 6%; OR=4.0; P=0.03), with capsular invasion (18% vs. 5%; OR=5.6; P=0.02), with lymph node metastasis (19% vs. 6%; OR=7.1; P=0.006), with extrathyroidal extension (23% vs. 6%; OR=4.8; P=0.03), and advanced AJCC stage (29% vs. 4%; OR= 8.7; P=5.00 x 10-4). PTC is considered a relatively benign malignancy with good prognosis and survival in the U.S., but we found a high fraction of Colombian cases in advanced AJCC stage (32%) and with distant metastasis (6%), suggesting that most cases were not the result of incidental findings, a common phenomenon in developed countries where most PTCs are likely the result of overdiagnosis. In summary, we present the first report of BRAF/RAS/TERT mutations in Hispanics with PTC from Latin America, demonstrate the utility of these mutations to identify cases with severe disease, and generate a body of data that will be important for the establishment of PTC precision medicine in the region.

#5234

Transcriptome profiling of single prostate cancer cells following androgen deprivation.

Ashlee K. Clark, Mitchell Lawrence, Hieu Nim, Natalie Lister, Mark Frydengerg, Gail Risbridger, Renea Taylor. _Monash University, Melbourne, Australia_.

Androgen withdrawal is the standard of care for men with metastatic prostate cancer. While all patients initially respond, resistance is inevitable and lethal castration-resistant prostate cancer ensues. Using patient-derived xenografts (PDX) of localized tumors, we have identified a subpopulation of castration-tolerant prostate cancer cells that survive following castration. Identifying the unique biologic characteristics of these cells is essential in determining their role in tumor progression and potential to be targeted by therapeutic agents. To study the genomic features of castrate-tolerant cells, we enriched for prostate cancer cells from PDXs using FACS and subjected them to single-cell isolation and RNA seq. We efficiently captured and sequenced >50 cells from pre- and post-castration PDXs using the Fluidigm C1 platform. Sequencing of isolated single cells was performed using the Illumina HiSeq in rapid mode with 50 bp fragment sequencing chemistry (3 million reads/cell). Multidimensional scaling showed that the response to castration is not uniform in all human cells. A unique gene set was identified in intact versus castrate-tolerant cells; we identified distinct changes in energy metabolism, including suppression of ATP production to aid cell survival. We also detected consistent upregulation of the retinoic acid signaling pathway, involving upregulation of CRABP2 and RARRES3 expression in castrate-tolerant cells. This is the first study to report on gene expression in single human prostate cells and revealed novel endocrine-related changes prior to and following androgen deprivation. Our data suggest that further and/or alternative hormone suppression may be effective in targeting castration-tolerant prostate cancer cells.

#5235

An investigation of the potential effects of the atypical PKC-ι inhibitor ICA-1 on ovarian cancer proliferation and survival, in estrogen dependent and independent systems.

Tracess Smalley, Mildred Acevedo-Duncan. _Univ. of South Florida, Tampa, FL_.

Ovarian cancer is the most lethal gynecological cancer diagnosis. There is no efficient screening process for ovarian cancers and the average stage of diagnosis is stage III. Atypical PKCs (PKC-ζ and PKC-ι/λ) have been shown to be overexpressed in various malignant cells lines and to be linked to pathways for cellular proliferation and survival. Many investigations suggest that the hormone estrogen regulates cellular proliferation in an ERα dependent manner. Little research shows the effects of estrogen on ERα and β negative cells and its role in cellular proliferation. In this investigation, ovarian cell lines (HEY, COV644, T80) were treated with the atypical PKC-ι inhibitor ICA-1 nucleoside variant in two different systems, with and without estrogen, and assayed to determine the effects on proliferation and cellular survival. These assays included protein quantification, cell proliferation, and wound healing. Our preliminary data suggests that PKC-ι is a novel target in carcinogenesis and inhibition of this protein decreases the rate of proliferation. Our results also suggest that estrogen has a significant effect on cell proliferation, even in estrogen receptor negative cells.

#5236

ARV-110: An androgen receptor PROTAC degrader for prostate cancer.

Taavi Neklesa,1 Lawrence B. Snyder,1 Ryan R. Willard,1 Nicholas Vitale,1 Kanak Raina,1 Jennifer Pizzano,1 Deborah Gordon,1 Mark Bookbinder,1 Jennifer Macaluso,1 Hanqing Dong,1 Zheng Liu,1 Caterina Ferraro,1 Gan Wang,1 Jing Wang,1 Craig M. Crews,2 John Houston,1 Andrew P. Crew,1 Ian Taylor1. 1 _Arvinas, New Haven, CT;_ 2 _Yale University, New Haven, CT_.

The Androgen Receptor (AR) remains the principal driver of castration-resistant prostate cancer during the transition from a localized to metastatic disease. Most patients initially respond to inhibitors of the AR pathway, but the response is often short-lived. The majority of patients progressing on enzalutamide or abiraterone exhibit genetic alterations in the AR locus, either in the form of amplifications or point mutations in the AR gene. Given these mechanisms of resistance, our goal is to eliminate the AR protein using the PROteolysis TArgeting Chimera (PROTAC) technology. Here we report an orally bioavailable small molecule ARV-110 that leads to ubiquitination and degradation of AR. ARV-110 completely degrades AR in all cell lines tested, with an observed 50% degradation concentration (DC50) < 1 nM. PROTAC-mediated AR degradation suppresses the expression of the AR-target genes PSA and FKBP5, inhibits AR-dependent cell proliferation, and induces potent apoptosis in VCaP cells. ARV-110 degrades clinically relevant mutant AR proteins and retains activity in a high androgen environment. In mouse xenograft studies, greater than 90% AR degradation is observed at a 1 mg/kg PO QD dose. Significant inhibition of tumor growth and AR signaling can be achieved in both an intact and castrate setting. Further, ARV-110 demonstrates in vivo efficacy and reduction of oncogenic Erg protein in a long term, castrate, enzalutamide-resistant VCaP tumor model. DMPK and exploratory toxicology studies show robust oral, dose proportional drug exposure in rodent and non-rodent species. In summary, we report preclinical data on ARV-110, an orally bioavailable androgen receptor PROTAC degrader that demonstrates efficacy in enzalutamide-resistant prostate cancer.

#5237

Sex-dependent association of circulating sex steroids, pituitary hormones and treatment-free survival in patients with chronic lymphocytic leukemia.

Eric P. Allain,1 Karin Venzl,2 Patrick Caron,1 Véronique Turcotte,1 David Simonyan,3 Michaela Gruber,2 Trang Le,2 Éric Lévesque,1 Chantal Guillemette,1 Katrina Vanura2. 1 _Pharmacogenomics laboratory, Centre Hospitalier Universitaire de Québec (CHU) Research Center, Université Laval., Québec, Quebec, Canada;_ 2 _Division of Hematology and Hemostaseology, Department of Medecine I, Medical University of Vienna., Vienna, Austria;_ 3 _Statistial and Clinical Research Platform, CHU de Québec Research Center., Québec, Quebec, Canada_.

Chronic lymphocytic leukemia (CLL) is the most frequent adult leukemia in the western world, and is not considered a hormone-regulated cancer but sex is a known risk factor with a significant male/female ratio (2:1) in its incidence with male patients more frequently developing progressive disease. We hypothesized that variable hormonal exposure may have a sexually dimorphic effect on CLL progression. However, a detailed evaluation of sex steroids and pituitary hormones in CLL patients is still lacking. In 156 CLL patients, we quantitatively profiled 15 circulating sex steroids (androgens, estrogens and progesterone) by sensitive and specific mass spectrometry and two pituitary hormones (luteinizing hormone (LH) and follicular secreting hormone (FSH)) by immunoassay. Data were analyzed separately by sex and in relation to treatment-free survival (TFS). Univariate and multivariate analyses of TFS were performed using Cox's proportional hazard model with hormone levels as continuous variables. Median age of CLL patients was 59.8 and 62.9 years for men and postmenopausal women, respectively. Common CLL prognostic markers had very similar frequencies between male and female cases. Median TFS was shorter for male patients than for women (80.7 vs. 135.0 months, P=0.033). Circulating profiles of hormones in CLL patients were significantly different from those of healthy donors whereas male cases had higher steroid levels than female patients. In male CLL cases, sex steroid levels were not significantly associated with TFS; however, higher LH levels were associated with shorter TFS in multivariate analyses with an adjusted hazard ratio (HRadj) of 2.11 (P = 0.004). In female CLL cases, high levels of potent androgens testosterone, dihydrotestosterone (DHT) and the sum of methoxy estrogens were significantly associated with improved TFS with HRadj values of 0.24 (P =0.007), 0.54 (P=0.023) and 0.31 (P =0.034), respectively. This study is the first to establish a link between outcome of CLL patients and circulating sex steroid and pituitary hormones, revealing a sex-specific hormonal imbalance associated with disease progression. This work was supported by a Canadian Institutes of Health Research grant to CG. EPA holds scholarships from Université Laval foundation - Leadership and sustainable development award and CHU de Québec foundation - Fernand Labrie excellence award.

#5238

Characterization of the enzyme generating the cholesterol metabolite and tumor suppressor dendrogenin A in the breast and its deregulations in breast cancer.

Marc Poirot,1 Emmanuel Noguer,2 Florence Dalenc,3 Regis Soules,1 Lisa Barrett,1 Arnaud Rives,2 Hye-Young Kim,4 Brigitta Sjödin,5 Camille Franchet,3 Pilippe Rochaix,3 Raphaelle Duprez-Paumier,3 Magali Lacroix-Triki,3 Thomas Filleron,3 Leonor Chaltiel,3 Louise Jones,6 Emanuala Gadaleta,6 Claude Chalala,6 Sergio Roman-Roman,7 Thierry Dubois,7 Ned A. Porter,4 Bengt Mannervik,5 Michel Record,1 Sandrine Silvente-Poirot1. 1 _Cancer Research Center of Toulouse, Toulouse, France;_ 2 _Affichem, Toulouse, France;_ 3 _Institut Claudius Regaud, IUCT-Oncopole, Toulouse, France;_ 4 _Vanderbilt University, Nashville, TN;_ 5 _Stockholm University, Stockholm, Sweden;_ 6 _Barts Cancer Institute, London, United Kingdom;_ 7 _Institut Curie, Paris, France_.

Dendrogenin A (DDA) is a tumor suppressor metabolite identified in human tissues that arises from the conjugation of 5,6α-epoxycholesterol (5,6α-EC) with histamine (HA) by a yet unidentified enzyme. DDA is present in the normal breast but its levels were found drastically decreased in breast tumors, showing that a deregulation of DDA metabolism occurred during breast carcinogenesis. It was shown that DDA displayed chemopreventive and anticancer properties (de Medina et al, Nat Commun, 2013; Voisin et al, PNAS, 2017; Segala et al, Nat Commun, in press). In addition, DDA blocks the biosynthesis of a newly identified cholesterol tumor promoter named 6-oxo-cholestan-3β,5α-diol (OCDO) (Voisin et al, PNAS, 2017). DDA and OCDO arise from 5,6-EC. We showed the existence of a metabolic balance between these two 5,6-EC derivatives in normal breast and BC that controls or stimulates BC progression (Silvente-Poirot & Poirot, Science, 2014, Voisin et al, PNAS, 2017). We addressed here the question of the identification and characterization of the DDA synthase (DDAS) and we determined whether its expression could reflect DDA levels in patient breast tumor and normal tissue. We report that the recombinant human glutathione transferase A1-1 (GST A1-1) produced DDA from 5,6α-EC and histamine (HA). The chemical characterization of the DDA product was performed by chromatography and mass spectrometry fragmentation. DDAS activity was found to be a new and important activity of GST A1-1 in addition to known glutathione transferase and steroid isomerase activities. The measured Michaelis constants of GST A1-1 for its new substrates were: Km5,6α-EC=0.27±0.05 µM and KmHA=0.35±0.3 µM, and the maximum velocity for the transformation of each substrates Vm5,6α-EC=0.81±0.2 µmol.min-1.mg and VmHA=0.66±0.2 µmol.min-1.mg. Interestingly, we showed that OCDO and other ring-B oxysterols, as well as several natural substrates and product of the GST A1-1, were potent inhibitors of DDAS activity while xenobiotics substrates of GST, and side chain oxysterols were not. Patient BC samples (n=50) showed significant decreased DDA levels and lower GST A1-1 protein expression compared to normal matched tissues, indicating that the decreased production of DDA in tumors is due to decreased expression of its enzyme. The analyses of two human BC mRNA databases from the Barts Cancer Institute (London, UK) and the Curie Institute (Paris, France) showed that the expression of GST A1-1 was lost in ER(+) BC tumors compared to normal breast tissue. Interestingly, DDAS was selectively expressed in the cytoplasm of epithelial cells from lactating ducts and lobular terminal units. Since these cells are the origin of most BC, the loss of DDAS expression and DDA biosynthesis combine to OCDO production, which controls DDAS activity, may constitute a major oncogenic process leading to BC development in human.

#5239

Gli is a primary driver of prostate cancer cell growth.

Na Li, Jackson Moore, Sarah Troung, Mannan Nouri, Jane Foo, Ralph Buttyan. _Vancouver Prostate Ctr., Vancouver, British Columbia, Canada_.

Canonical Hedgehog signaling is a Smoothened- (Smo-) driven process that activates transcription from Gli proteins. However, we have shown that Hedgehog is activated in prostate cancer (PCa) cells by a non-canonical, non-Smo-dependent process mediated by the binding of transcriptionally active androgen receptors (AR) to the protein processing domain on Gli2/3. This binding competes with β-TrCP, and protects Gli3 (the predominant Gli isoform in PCa cells) from cleavage to its truncated repressor form (Gli3R). Our observations are consistent with the idea that AR transcriptional activity (mediated by liganded full-length AR or by truncated ARs) is effectively coupled to Gli activity in PCa. Here we present our evidence that Gli is the primary driver of PCa cell growth and that suppression of Gli transcriptional activity or interference with AR binding to Gli (using a decoy peptide) is a strong growth suppressor of androgen-dependent (AD) and -independent (AI) PCa cell lines. Gli transcription was inhibited by GANT61 or HPI-1 or by exogenous overexpression of a Gli3R cDNA. Each of these conditions significantly suppressed the growth of LNCaP parental (AD) and LNCaP-AI cells. Knockdown of Gli3 with siRNA decreased growth of a wide variety of PCa cell lines up to 75%. Finally, a decoy peptide (270aa) containing the Gli2 AR binding site displaced Gli3 from AR complexes, increased the levels of Gli3R and significantly suppressed growth of AI cell lines. Collectively, our data supports the idea that androgen-driven growth of PCa is a result of non-canonical activation of Gli and that interference with Gli binding to DNA or with the Gli-AR interaction is a means for suppressing PCa cell growth.

#5240

**Functional and therapeutic significance of** ESR1 **gene fusions in breast cancer.**

Jonathan T. Lei,1 Jieya Shao,2 Jin Zhang,2 Michael Iglesia,2 Doug W. Chan,1 Jin Cao,1 Meenakshi Anurag,1 Purba Singh,1 Xiaping He,3 Yoshimasa Kosaka,4 Ryoichi Matsunuma,1 Robert Crowder,2 Jeremy Hoog,2 Chanpheng Phommaly,2 Rodrigo Goncalves,5 Susana Romalho,5 Raquel M. Peres,6 Nindo Punturi,1 Cheryl Schmidt,1 Alex Bartram,7 Eric Jou,7 W V. Lai,8 Oliver Hampton,1 Anna Rogers,1 Ethan Tobias,9 Poojan Parikh,1 Sherri R. Davies,2 Shunqiang Li,2 Cynthia X. Ma,2 Vera Suman,10 Kelly K. Hunt,11 Mark A. Watson,2 Katherine A. Hoadley,3 E A. Thompson,12 Xi Chen,1 Shyam M. Kavuri,1 Chad J. Creighton,1 Christopher A. Maher,2 Charles M. Perou,3 Svasti Haricharan,1 Matthew J. Ellis1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Washington University in St. Louis, St. Louis, MO;_ 3 _University of North Carolina, Chapel Hill, NC;_ 4 _Kitasato University School of Medicine, Kanagawa, Japan;_ 5 _University of São Paulo School of Medicine, Sao Paulo, Brazil;_ 6 _State University of Campinas, Sao Paulo, Brazil;_ 7 _University of Cambridge, Cambridge, United Kingdom;_ 8 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 9 _University of Texas Southwestern Medical Center, Dallas, TX;_ 10 _Mayo Clinic, Rochester, MN;_ 11 _MD Anderson Cancer Center, Houston, TX;_ 12 _Mayo Clinic, Jacksonville, FL_.

RNA sequencing detects estrogen receptor alpha gene (ESR1) fusion transcripts in estrogen receptor positive (ER+) breast cancer but their role in disease pathogenesis remains unclear. Herein we examined multiple in-frame and out-of-frame ESR1 fusions and found only two, both identified in advanced endocrine treatment resistant disease, encoded stable and functional in-frame fusion proteins. In both examples, ESR1-e6>YAP1 and ESR1-e6>PCDH11X, the N-terminal, DNA binding and dimerization motifs encoded by exons 2-6 were fused to C-terminal sequences from the partner gene. Functional properties included estrogen-independent growth, constitutive expression of ER target genes, anti-estrogen resistance, induction of cellular motility in vitro and the development of lung metastasis in vivo. Chromatin immunoprecipitation and RNA sequencing experiments showed both fusions uniquely activated a metastasis-associated transcriptional program. ESR1-e6>YAP1 and ESR1-e6>PCDH11X-induced growth remained sensitive to a CDK4/6 inhibitor, palbociclib, and a patient-derived xenograft (PDX) expressing the ESR1-e6>YAP1 fusion was also responsive. Transcriptionally active ESR1 fusions therefore trigger both endocrine therapy resistance and metastatic progression explaining the association with fatal disease progression, although CDK4/6 inhibitor treatment is predicted to be effective.

#5242

NFĸB pathway activation is a key determinant of tamoxifen tolerance and recurrence in breast cancer.

Irida Kastrati,1 Svitlana D. Brovkovych,1 Joshua D. Stender,2 Elaine T. Alarid,3 Jonna Frasor1. 1 _University of Illinois at Chicago, Chicago, IL;_ 2 _University of California, San Diego, CA;_ 3 _University of Wisconsin-Madison, Madison, WI_.

Nearly 75% of breast tumors express estrogen receptor (ER) and will be treated with endocrine therapies, such as tamoxifen or aromatase inhibitors. Despite their proven success, it is estimated that up to 50% of tumors fail on endocrine therapy and recur as aggressive therapy-resistant tumors. Therefore, preventing recurrence remains a major clinical problem. One hypothesis is that recurrences are driven by cells that survive therapeutic intervention. This poorly understood population is referred to as minimal residual disease that consists of drug-tolerant cells capable of surviving by entering a state of negligible growth. In trying to model tamoxifen tolerance and minimal residual disease in vitro, we have utilized the clonogenic assay where ER+ breast cancer cells' clonal growth and survival in the presence of tamoxifen is examined. Tamoxifen-tolerant cells show activation of the pro-inflammatory NFĸB pathway measured by an NFĸB-reporter, elevated expression of bona-fide target genes, and enhanced nuclear localization of p65 and p50, two major transcription factors of the NFĸB pathway. Tamoxifen does not directly activate the NFĸB pathway, but rather cells with high NFĸB activity proliferate independently of tamoxifen despite retaining ER expression. Mechanistically, NFĸB-dependent ER phosphorylation at S305 residue may contribute to failure to respond to tamoxifen. More importantly blocking NFĸB with multiple NFĸB inhibitors is sufficient to eradicate this population of tamoxifen-tolerant cells, suggesting NFĸB is required and can be exploited for therapy. We find that dimethyl fumarate, an FDA-approved anti-inflammatory and immune-modulatory drug, which we have established as an effective NFĸB inhibitor in breast cancer cells, prevents recurrence modeled both in vitro and in vivo. Altogether this work shows a novel role for the NFĸB pathway in promoting a population of cells that are tamoxifen tolerant and capable of seeding recurrent breast cancer disease. Furthermore, therapeutic targeting by an NFĸB inhibitor such as dimethyl fumarate can be used to eradicate residual disease and prevent breast cancer recurrence.

#5243

Prognostic value of nuclear and non-nuclear estrogen receptor expression by immunohistochemistry with phosphor-integrated dots in hormone receptor-positive early breast cancer.

Zhaorong Guo, Hiroshi Tada, Narufumi Kitamura, Yoh Hamada, Shin-ichi Hayashi, Noriaki Ohuchi, Kosuke Gonda, Takanori Ishida. _Graduate School of Medicine, Tohoku University, Sendai, Japan_.

Background: Estrogen receptor α (ERα) is associated to cell proliferation and survival through genomic (nuclear) and non-genomic (non-nuclear) signaling pathway. Endocrine therapy is the most effective therapies for patients with hormone receptor(HR) positive breast cancer. Recent progress shows that both nuclear and non-nuclear crosstalk was considered to contribute to endocrine resistance. Clinically, ERα is predominantly observed using immunohistochemistry(IHC) as a nuclear protein while unable to detect nuclear and non-nuclear ERα separately. Our group recently created new fluorescent nanoparticles called phosphor-integrated dots (PIDs). PIDs has extremely brightness so that it could be double stained with hematoxylin and quantitated without influence. In addition, the localization of biomarker protein can be visualized and analyzed on cell as PIDs score by this method. Methods: 65 HR+/HER2- breast cancer patients from 2001 to 2003 who treated with postoperative endocrine therapy were selected in this study. Expression levels of total ERα, nuclear-ERα, non-nuclear-ERα, progesterone receptor (PR) was examined by IHC with PIDs. Nuclear-ERα was recognized by double staining using hematoxylin. IHC with DAB was carried out to analyze the expression levels of ER and PR and H score was automatically calculated. In addition, PIDs score of non-nuclear-ERα can be calculated through PIDs score of total ERα and nuclear-ERα by PID analyzer. Optimal cutoff value of PIDs score of each receptor to predict survival were derived and independently tested. The correlation among every clinical characteristic and pathological feature was examined. Result: PIDs score of total ERα(χ2=5.17, p=0.023) and nuclear-ERα is negative correlated with relapse (χ2=6.46, p=0.011), ratio of non-nuclear-ERα to total ERα is positive correlated with relapse (χ2=5.82, p=0.016).The cutoff value as 37.46 of nuclear-ERα PIDs score was statistically proved significant for predicting disease-free survival(DFS) (log-rank p=0.020).We also found that cutoff value as 0.32 which represents ratio of non-nuclear-ERα PIDs score to total ERα PIDs score was statistically proved significant for predicting DFS (log-rank p=0.022). H score of ER, PR and PIDs score of non-nuclear-ERα, PR has no predictive value for recurrence. Conclusion: Our result demonstrates that nuclear-ERα PIDs score obtain a higher prognostic value than that of total-ERα PIDs score and H score detected by DAB-IHC. Ratio of non-nuclear-ERα to total ERα may play a role of prognostic factor in HR positive breast cancer.

#5244

Neratinib induced HER2 dependent and independent intracellular signaling events in human breast cancer cells and the clinical implications.

W. G. Jiang,1 Tracey A. Martin,1 Sioned Owen,1 Ling Xin,2 Yinhua Liu,2 Fiona Ruge,1 Francesca Avogadri-Connors,3 Alshad S. Lalani,3 Richard P. Bryce,3 Eleri Davies4. 1 _Cardiff-Peking Cancer Institute, Cardiff University, Cardiff, United Kingdom;_ 2 _Peking University First Hospital, Beijing, China;_ 3 _Puma Biotechnology, Los Angeles, CA;_ 4 _University Llandough Hospital, Cardiff, United Kingdom_.

Background. Neratinib is an orally available irreversible pan-HER receptor tyrosine kinase inhibitor that has clinical benefit in patients with HER2-positive and HER2-mutated breast cancer. Neratinib also targets the other two members of the HER family, namely EGFR and HER4. However, tentative evidence suggests that neratinib may affect kinases of non-HER family. In the present study, we aimed to explore the pattern of kinases targeted by neratinib, in the context of HER2 non-amplified cancer cells and explored the potential clinical implications. Method. A panel of HER2-low/medium-expressing (MCF-7 and ZR-75-1) and HER2- negative (MDA-MB-231 and BT549) human breast cancer cells were used. The response of a panel of 304 kinases to neratinib was screened using the kinase antibody array. Cellular toxicity was quantified using a colorimetric method. Candidate neratinib-responsive kinases were subject to analyses against the survival of a breast cancer cohort as well as a public database for additional validation. The protein expression level of the kinase targets identified was also validated on tissue arrays from breast cancer by immunohistochemistry. Result. Of all the kinases tested, thirty-six were found to be influenced by neratinib, either via activation or suppression of their phosphorylation. Based on the differential responses to neratinib in HER2 negative and positive breast cancer cells, a small panel of the kinases were classified as HER2 independent. Using clinical cohorts, these kinases collectively had predictive value for the overall survival of the patients (p<0.0001). We further analysed one of the candidate kinases, namely salt inducible kinase-1 (SIK1) and the SIK family in relation with HER2. HER2 expression was found significantly correlated with both SIK1 (r=0.46, p<0.001) and also other two SIK family members. SIK1 protein staining in the tissue array was found to be aberrant in breast tumours. Conclusion. Neratinib has an effect on a subset of HER2 independent kinases in breast cancer cells. These kinases had a prognostic value on patient's survival and correlated with HER2 expression, as in the case of the SIK family.This finding may have important implication in understanding the biology of HER2 inhibitors such as neratinib.

#5245

Identification and characterization of the IDO1 inhibitor LY3381916.

Frank C. Dorsey,1 Karim A. Benhadji,1 Lillian L. Sams,1 Debra A. Young,1 John F. Schindler,1 Karen L. Huss,1 Alexander Nikolayev,1 Carmine Carpenito,2 David Clawson,1 Bonita Jones,1 Andrew L. Faber,1 James E. Thomas,1 Steven A. Haney,1 Gaiying Zhao,1 William T. McMillen,1 Tod Smeal,1 Daniel J. Sall,1 Michael D. Kalos,2 Sandaruwan Geeganage,1 James R. Henry1. 1 _Eli Lilly and Company, Indianapolis, IN;_ 2 _Eli Lilly and Company, New York, NY_.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-dependent enzyme that catalyzes the initial and rate-limiting step of tryptophan catabolism resulting in the local depletion of tryptophan and the concomitant production of kynurenine, both of which are immunosuppressive. Targeting IDO1 in combination with PD-1/PD-L1-targeted antibodies has shown promise in early phase clinical trials in several cancers and strongly suggests that, in some patients, IDO1 expression restrains PD-1/PD-L1-targeted checkpoint therapies.

While some cancers extrinsically express IDO1 in response to IFN-γ produced from an ongoing, yet ineffective immune response, others select for the intrinsic expression of IDO1, independent of an immune response. We identified several cancer cell lines that intrinsically expressed either IDO1 or the related isozyme TDO2. Using these cell lines, we discovered LY3381916, a potent and selective inhibitor of cell-based IDO1 activity (IDO1 7 nM; TDO2 >20 µM). Using a variety of techniques, we demonstrated that LY3381916 binds to newly synthesized apo-IDO1 lacking heme, but does not inhibit mature heme-bound IDO1. Protein x-ray crystallography confirmed that LY3381916 binds to apo-IDO1 where it occupies the heme-binding pocket of IDO1. As a result of this novel mechanism of action, substantial inhibition of IDO1 in tumors requires the turn-over of mature heme-bound IDO1. Modeling of the pre-clinical PK/PD relationship suggests QD dosing of LY3381916 will maintain greater than 90% inhibition over 24 hours. In addition, due to the favorable properties of the drug, significant central nervous system (CNS) penetration has been measured for LY3381916 (rodent kp,uu 0.26).

Kynurenine-mediated agonism of the aryl hydrocarbon receptor (AHR) is immunosuppressive in the tumor microenvironment. Inhibition of IDO1 and the subsequent reduction of kynurenine can relieve this immunosuppression. However, several heme-binding IDO1 inhibitors have been shown to replace kynurenine as an AHR agonist potentially limiting their ability to relieve this IDO1-dependent immunosuppressive mechanism. LY3381916 shows no confounding agonism of AHR up to 100 µM. Additionally, we characterized LY3381916 in pre-clinical tumor models and demonstrated that it was able to enhance LY3300054, anti-PD-L1 antibody (LY3300054) activity, which was associated with an enhanced T cell response. Based

on these characteristics, LY3381916 is currently being investigated in a Phase I clinical trial. These data suggest further development of LY3381916 may be warranted. 

## EPIDEMIOLOGY:

### Diet, Alcohol, Tobacco, and Other Lifestyle Risk Factors

#5246

Lifetime trajectories of cigarette smoking and cancer mortality among older adults in a large cohort in the United States.

Maki Inoue-Choi,1 Scott P. Kelly,1 Kelvin Choi,2 Neal D. Freedman1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _National Institute on Minority Health and Health Disparities, Bethesda, MD_.

Background: Despite declining prevalence, cigarette smoking causes about 30% of all cancer deaths in the United States. More than a quarter of daily cigarette smokers in the United States smoke 10 or less cigarettes per day (CPD), encompassing a heterogeneous group of lifelong low-intensity smokers and formerly heavier smokers who have cut down their CPD. The impact of prior smoking history on the disease risks of low-intensity cigarette smokers is poorly understood.

Methods: In the National Institutes of Health-AARP Diet and Health Study, a prospective cohort study of older adults in the United States, a questionnaire administered in 2004-2005 assessed CPD during nine age-periods over the lifetime (<15 to ≥70). This analysis included 121,025 men and women aged 60-82 who reported never smoking cigarettes (n=110,444) or currently smoking <1 or 1-10 CPD (low intensity smokers, n=10,581). We used fixed-effects latent class trajectory modeling to define lifetime trajectories of prior CPD smoking history among low-intensity cigarette smokers. Then, we calculated sex-specific multivariable-adjusted hazard ratios (HR) and 95% confidence intervals (CI) for each trajectory relative to never smokers using Cox proportional hazards regression for overall and lung cancer mortality.

Results: We identified nine trajectory patterns in both men and women. Mortality risks varied substantially by trajectory: the HRs for overall cancer mortality ranged from 1.17 to 4.03 in men and 0.83 to 3.42 in women. Similarly, the HRs for lung cancer mortality ranged from 5.20 to 23.88 in men and 8.00 to 23.08 in women. Mortality risks were substantially higher among participants who had cut down their CPD than among participants who reported smoking at low-intensity for all age-periods that they smoked. For example, in men, the HR for lung cancer mortality was 23.88 (95% CI=12.10-47.13) among low-intensity smokers who previously smoked 31-40 CPD, whereas it was 9.36 (95% CI=5.92-14.79) among lifelong 1-10 CPD smokers. Similarly, in women, the HR for lung cancer mortality was 19.76 (95% CI=8.71-44.85) among women who previously smoked 21-30 CPD, whereas it was 9.26 (95% CI=2.98-14.34) among lifelong 1-10 CPD smokers.

Conclusions: In a large prospective cohort study of older adults in the United States, risks among low-intensity smokers for overall and lung-cancer mortality varied substantially by prior smoking history. These results suggest that it is important for risk prediction models, such as for lung cancer-screening, to incorporate lifetime smoking history into their algorithms.

#5247

Aspirin use and estrogen receptor-negative breast cancer risk in African American women.

Kimberly A. Bertrand,1 Traci N. Bethea,1 Hanna Gerlovin,1 Patricia Coogan,1 Lucile L. Adams-Campbell,2 Lynn Rosenberg,1 Julie R. Palmer1. 1 _Slone Epidemiology Center at Boston University, Boston, MA;_ 2 _Georgetown Lombardi Comprehensive Cancer Center, Washington, DC_.

Introduction: Use of aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) has been hypothesized to be associated with reduced risk of breast cancer; however, results of epidemiological studies have been mixed. Further, few previous studies investigated these associations among African American women and no studies that included African Americans evaluated whether associations differ by estrogen receptor (ER) status.

Methods: To assess the relation of aspirin use to risk of ER+ and ER- breast cancer in African American women, we conducted a prospective analysis within the Black Women's Health Study, an ongoing nationwide cohort of 59,000 black women that began in 1995. On baseline and biennial follow-up questionnaires, women reported regular aspirin use (defined as use at least three days per week) and years of use. Acetaminophen use was queried similarly. Among women with available information on aspirin use, 1701 invasive breast cancers occurred during follow-up through 2015, including 962 ER+ and 492 ER- tumors. All cancers were confirmed through pathology reports or state cancer registry data. We used age-stratified Cox proportional hazards regression models to estimate multivariable-adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) for associations of aspirin use with risk of ER+ and ER- breast cancer. All models adjusted for age at menarche, oral contraceptive use, parity, age at first birth, estrogen plus progestin use, body mass index, alcohol consumption, and use of other NSAIDs.

Results: Compared to never use, current regular use of aspirin was associated with lower risk of breast cancer overall (HR 0.87; 95% CI 0.76, 1.00). The association was driven by a statistically significant inverse association for ER- breast cancer (HR 0.76; 95% CI 0.58, 0.99); for ER+ breast cancer, the corresponding HR was 0.92 (95% CI 0.77, 1.09). In contrast, we found no associations with acetaminophen use.

Conclusions: Our observation of reduced risk of ER- breast cancer in relation to current regular use of aspirin is consistent with anti-inflammatory effects of aspirin, rather than hormone-dependent pathways. Aspirin may represent a potential opportunity for chemoprevention of ER- breast cancer; however, these findings require corroboration in additional studies.

#5248

Adherence to the WCRF/AICR cancer prevention guidelines and colorectal cancer incidence and mortality in the biracial cohort--the Atherosclerosis Risk in Communities (ARIC) study.

Anna K. Lintelmann,1 Guillaume Onyeaghala,1 Corinne E. Joshu,2 Pamela L. Lutsey,1 Aaron R. Folsom,1 Kimberly Robien,3 Elizabeth A. Platz,2 Anna E. Prizment1. 1 _Univ. of Minnesota School of Public Health, Minneapolis, MN;_ 2 _Johns Hopkins Bloomberg School of Public Health, Baltimore, MD;_ 3 _GW Univ. Milken Institute School of Public Health, Washington, DC_.

Introduction: Greater adherence to the World Cancer Research Fund (WCRF) and the American Institute for Cancer Research (AICR) cancer prevention recommendations has been linked to decreased risk of colorectal cancer (CRC). However, it is unknown whether this association differs by race. We compared the adherence to the WCRF/AICR guidelines between African-Americans and Whites and assessed the association between adherence and CRC incidence and mortality in the Atherosclerosis Risk in Communities (ARIC) prospective analytic cohort (1987-2012).

Methods: Baseline information from 13,822 participants (54.6% women, 26.5% African-American) was used to develop an 8-component adherence score based on the WCRF/AICR guidelines. The adherence score included body mass index (BMI), physical activity, and dietary intake recommendations, including intake of alcohol, fruits and vegetables, dietary fiber, sugar-sweetened beverages, sodium, and red and processed meats. Each recommendation contributed 0, 0.5, or 1 points, with higher scores corresponding to greater adherence. Adherence scores were categorized as tertiles (0.5-4.0, 4.5-5.0, and 5.5-8.0). Cox proportional hazards regression was used to calculate hazard ratios and 95% confidence intervals (HR, 95% CI) for the CRC incidence and mortality associated with the adherence score for the total cohort and stratified by race.

Results: Mean adherence score was 4.31 (range 0.5-8.0) in African-Americans (N=3,668; 101 CRC cases; 31 CRC deaths) and 4.85 (range 1.0-8.0) in Whites (N=10,154; 268 CRC cases; 87 CRC deaths). African-Americans were less likely to follow most of the cancer prevention guidelines than Whites (except alcohol and sodium intake). After adjustment for age, gender, race, center, smoking, education, intake of aspirin, calcium, total calories, and, in women, hormone-replacement therapy, greater adherence was associated with a 28% lower CRC risk (0.55-0.96) for the highest versus lowest adherence score tertile (ptrend=0.03), and were similar in men (HR=0.76, 95% CI: 0.51-1.12) and women (HR=0.69, 95% CI: 0.46-1.03; pinteraction with gender=0.66). When stratified by race, HRs were 0.74 (0.41-1.34) in African-Americans and 0.74 (0.53-1.02) in Whites (pinteraction with race=0.72) for the highest versus lowest tertile, but power was limited in African-Americans. Associations between adherence score and fully-adjusted mortality from CRC showed a similar inverse trend, but were not significant.

Conclusions: Greater adherence to the WCRF/AICR cancer prevention recommendations was associated with decreased CRC risk in the ARIC cohort. The associations between adherence score and CRC risk were similar in African-Americans and Whites.

#5249

Crosstalk between visceral adipose and tumor tissue in colorectal cancer patients: Molecular signals driving host-tumor interaction.

Andreana N. Holowatyj,1 Mariam Haffa,2 Tengda Lin,1 Biljana Gigic,3 Jennifer Ose,1 Christy Warby,1 Caroline Himbert,1 Clare Abbenhardt,2 Juergen Boehm,1 Magnus von Knebel Doeberitz,3 Nina Habermann,4 Esther Herpel,3 Hans-Ulrich Kauczor,3 Matthias Kloor,3 Johanna Nattenmüller,3 Peter Schirmacher,3 Martin Schneider,3 Petra Schrotz-King,3 Thomas Simon,3 Alexis Ulrich,3 Laura Bowers,5 Stephen D. Hursting,5 Cornelia M. Ulrich1. 1 _Huntsman Cancer Institute, University of Utah, Salt Lake City, UT;_ 2 _National Center for Tumor Diseases and German Cancer Research Center, Heidelberg, Germany;_ 3 _University of Heidelberg, Heidelberg, Germany;_ 4 _European Molecular Biology Laboratory, Heidelberg, Germany;_ 5 _University of North Carolina, Chapel Hill, Lineberger Comprehensive Cancer Center, Chapel Hill, NC_.

Adipose tissue has recently emerged as a driver of tumorigenesis, as adipose-associated factors enhance tumor growth and development. Here we investigated for the first time direct crosstalk between visceral adipose tissue (VAT) and tumor tissues in colorectal cancer (CRC) patients. We analyzed paired transcriptomic profiles of tissues and systemic inflammatory biomarkers among CRC patients to elucidate potential mechanisms and molecular signals driving the host-tumor interaction. Paired presurgery blood and tumor adjacent VAT, colorectal tumor and mucosa tissue samples collected during surgery were obtained from 47 Caucasian patients diagnosed with microsatellite-stable primary CRC enrolled in the ColoCare Study. Clinical/demographic information, and lifestyle-related risk factors, were reported. VAT was quantified by computed tomography. RNA was extracted from VAT, colorectal tumor and mucosa tissues, analyzed using HumanHT-12 Expression BeadChips, processed, and validated. Serum-based assays were conducted in 46 of 47 patients for systemic inflammatory markers with the Mesoscale Discovery Platform. High PTGS2 tumor expression was associated with 131 significantly altered VAT genes, of which 85.5% were upregulated. A significant upstream regulator of dysregulated VAT genes was SAA1. Investigation of circulating inflammatory biomarkers corroborated these transcriptomic findings, as patients with high PTGS2 tumor expression were also significantly more likely to have higher serum-based SAA levels. The differential expression of PPAR-γ in VAT identified 284 significantly altered tumor genes. 71.8% of tumor genes were upregulated in association with higher PPAR-γ VAT expression. Differentially expressed tumor genes were enriched in functional cellular movement. Inflammatory biomarkers in patient sera showed marginally significant differences for soluble ICAM-1 in association with differential PPAR-γ VAT expression. Overlap of the 131 altered VAT genes associated with differential PTGS2 tumor expression with the 284 dysregulated tumor genes associated with differential PPAR-γ VAT expression identified 15 common genes. Gene set enrichment analysis demonstrated that phenotypes associated with these genes in humans included cancer invasiveness signatures from tumor microenvironment interactions. Our multi-omics profiling of paired tissue transcriptomics and systemic inflammatory biomarkers from CRC patients established a bidirectional link between VAT and colorectal tumor tissue. We have demonstrated that this direct crosstalk activates signaling cascades to promote proinflammatory interactions and metabolic reprogramming. Future efforts to develop mechanism-based interventions that disrupt these molecular signals at the nexus of VAT-tumor crosstalk may improve obesity-driven CRC prevention and control.

#5250

Overall and central obesity and risk of lung cancer: A pooled analysis of 12 cohort studies.

Danxia Yu,1 Wei Zheng,1 Mattias Johansson,2 Qing Lan,3 Yikyung Park,4 Emily White,5 Charles Matthews,3 Norie Sawada,6 Yu-Tang Gao,7 Kim Robien,8 Rashmi Sinha,3 Arnulf Langhammer,9 Rudolf Kaaks,10 Edward Giovannucci,11 Linda Liao,3 Yong-Bing Xiang,7 DeAnn Lazovich,12 Ulrike Peters,5 Xuehong Zhang,13 Bas Bueno-de-Mesquita,14 Walter Willett,11 Shoichiro Tsugane,6 Yumie Takata,15 Stephanie Smith-Warner,11 William Blot,1 Xiao-Ou Shu1. 1 _Vanderbilt University Medical Center, Nashville, TN;_ 2 _International Agency for Research on Cancer, Lyon, France;_ 3 _National Cancer Institute, Bethesda, MD;_ 4 _Washington University School of Medicine, St. Louis, MO;_ 5 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 6 _National Cancer Center, Tokyo, Japan;_ 7 _Shanghai Jiaotong University School of Medicine, Shanghai, China;_ 8 _George Washington University, Washington, DC;_ 9 _Norwegian University of Science and Technology, Trondheim, Norway;_ 10 _German Cancer Research Center, Heidelberg, Germany;_ 11 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 12 _University of Minnesota, Minneapolis, MN;_ 13 _Brigham and Women's Hospital and Harvard Medical School, Boston, MA;_ 14 _National Institute for Public Health and the Environment, Bilthoven, Netherlands;_ 15 _Oregon State University, Corvallis, OR_.

Background: The obesity-lung cancer association remains controversial. Concerns over confounding by smoking and reverse causation persist. Influence of obesity type and effect modifications by race/ethnicity and tumor histology are largely unexplored.

Methods: We examined associations of body mass index (BMI), waist circumference (WC), and waist-hip ratio (WHR) with lung cancer risk among 1.6 million Americans, Europeans, and Asians.

Results: During an average 12-year follow-up, 23,732 incident lung cancer cases were identified. While BMI was generally associated with a decreased risk, WC and WHR were associated with increased risk after controlling for BMI. These associations were seen 10 years before diagnosis, in smokers and never-smokers, were strongest among Blacks, and varied by histological type. After excluding the first 5 years of follow-up, HRs (95% CI) per 5 kg/m2 increase in BMI were 0.95 (0.90-1.00), 0.92 (0.89-0.95), and 0.89 (0.86-0.91) in never, former, and current smokers, and 0.86 (0.84-0.89), 0.94 (0.90-0.99), and 1.09 (1.03-1.15) for adenocarcinoma, squamous cell, and small cell carcinoma. HRs (95% CI) per 10 cm increase in WC were 1.09 (1.00-1.18), 1.12 (1.07-1.17), and 1.11 (1.07-1.16) in never, former, and current smokers, and 1.06 (1.01-1.12), 1.20 (1.12-1.29), and 1.13 (1.04-1.23) for adenocarcinoma, squamous cell, and small cell carcinoma. Participants with BMI < 25kg/m2 but high WC had a 40% higher risk than those with BMI ≥ 25 kg/m2 but normal/moderate WC (95% CI, 26%-56%).

Conclusions: The inverse BMI-lung cancer association is not entirely due to smoking and reverse causation. Central obesity, particularly concurrent with low BMI, may help identify high-risk populations for lung cancer.

#5251

Prospective investigation of general and visceral obesity, body fat distribution and risk of common cancers using data from the UK Biobank cohort study.

Ying Liu, Wei Zheng. _Vanderbilt University, Nashville, TN_.

Background: General obesity, as measured using body mass index (BMI), has been linked to the risk of multiple cancers, but limited studies have been conducted to evaluate associations of visceral obesity, fat mass and body fat percentage with cancer risk. The aim of this study was to analyze different measurements and aspect of obesity as potential risk factors for common cancers.

Methods: The UK Biobank cohort was designed to recruit middle and old aged participants in UK between 2006 and 2010, and follow them up via linkage to health records in National Health Service. Cox proportional hazard models were fitted to prospectively investigate associations between common site-specific cancers with BMI, waist-to-hip ratio (WHR), body fat percentage (BF%), whole body fat mass (WBFM), and trunk fat percentage (TF%), adjusting for potential confounders. Detailed measurements were obtained at baseline survey conducted in UK Biobank cohort, among approximately 492,000 participants. BF%, WBFM and TF% were ascertained using bio-impedance measures.

Results: A total of 20,628 incident cancers (except for non-melanoma skin cancer) occurred during 5.6 years of follow-up. Each SD increase in BMI was linearly associated with increased cancer risks of all-cancers combined (HR = 1.05, 95%CI = 1.04-1.07, p < 0.001), esophageal adenocarcinoma (1.43, 1.25-1.63, p < 0.001), colon (1.10, 1.04-1.16, p < 0.001), liver (1.22, 1.06-1.41, p = 0.007), gallbladder (1.36, 1.14-1.64, p = 0.001), pancreas (1.18, 1.07-1.31, p = 0.001), kidney (1.38, 1.27-1.50, p < 0.001), postmenopausal breast (1.10, 1.06-1.14, p < 0.001), and endometrium (1.65, 1.56-1.76, p < 0.001), and with decreased risk of prostate cancer (0.90, 0.87-0.94, p < 0.001). Positive associations for these cancers were also observed with WHR, BF%, WBFM and TF%. The association with WHR (per SD increment) remained statistically significant after further adjustment of BMI using residual method for risks of all cancers combined, esophageal adenocarcinoma, colon, liver, bladder, and kidney. For postmenopausal breast cancer, the positive association remained statistically significant for BF%, WBFM, TF%, but not for WHR after adjusting for BMI; for epithelial ovarian cancer, the positive associations retained for WBFM and TFM, but not for WHR and BF% after adjusting for BMI. For endometrial cancer, prostate cancer, non-Hodgkin lymphoma, multiple myeloma, and leukemia, the negative association remained statistically significant for BF% and TF%, even after adjusting for BMI.

Conclusions: Visceral obesity and specific fat mass measurements, including WHR, BF%, WBFM, and TF% confer additional risk to multiple cancers beyond the level contributed by general obesity measured using BMI. Cancer prevention programs should be designed not only to reduce body weight but also visceral fat and body fat mass.

#5252

Prospective cohort study of physical activity and lung cancer risk in the Black Women's Health Study.

Marsha Samson,1 Sarah Nomura,1 Chiranjeev Dash,1 Traci Bethea,2 Lynn Rosenberg,2 Lucile Adams-Campbell1. 1 _Georgetown University, Washington, DC;_ 2 _Boston University, Boston, MA_.

Background: Lung cancer is the second most common site of cancer among Black women. Black women have higher incidence rates of lung cancer despite having a lower smoking prevalence than their White counterparts. These differences are not well understood but may be due to differences in smoking behavior and other potential lung cancer risk factors (e.g., physical activity). Lower reported physical activity levels among Black women compared to White women could contribute to observed lung cancer disparities. Previous studies have shown a 20-50% lower risk of lung cancer among those who are physically active but overall, findings are equivocal. To our knowledge, no previous studies have examined the relationship between physical activity and lung cancer risk in Black women and few studies have examined the associations of physical activity with lung cancer risk stratified by smoking status.

Objective: The objective of this study is to evaluate the association between physical activity over time and lung cancer incidence in the Black Women's Health Study, and determine if tobacco use modifies this association. Methods: In this ongoing prospective cohort of Black women (analytic cohort n=46217), 291 cases of lung cancer were diagnosed between 1995 (baseline) and 2013. Questionnaire data, collected at baseline and updated every other year thereafter, were used to calculate recreational physical activity (walking for exercise, vigorous physical activity) and smoking history. The Anderson-Gill method was used as a time-varying analysis tool to continuously update exposures and covariates over time. Cox proportional hazards models were used to estimate hazard ratios and 95% confidence intervals (CIs). Results: At baseline, 65.5% of women were never smokers and 44% reported <1 hr of vigorous physical activity per week. Women who reported any vigorous activity (≥1 hr/wk) had a lower, albeit not statistically significant, risk of lung cancer compared to women with no vigorous activity. In stratified analyses by smoking status, vigorous activity was inversely associated with lung cancer risk among never smokers (HR: 0.37; 95% CI: 0.15, 0.91), but was not associated with risk in former (HR: 1.39; 95% CI: (0.88, 2.21) or current smokers (HR: 1.06; 95% CI: 0.63, 1.77). Conclusion: Our findings suggest that intensity of physical activity may play a role in reducing lung cancer risk among Black women who had never smoked, but the number of lung cancer cases in that category was relatively small. No studies have examined the relationship between physical activity and lung cancer risk in black women and this relationship should be further explored to help reduce lung cancer disparities in the nation.

#5253

Dietary fiber intake and lung cancer risk: A pooled analysis of 1.44 million individuals in 10 cohorts.

Jae Jeong Yang,1 Danxia Yu,1 Yong-Bing Xiang,2 William Blot,1 Kim Robien,3 Rashmi Sinha,4 Yikyung Park,5 Emily White,6 Yumie Takata,1 Mattias Johansson,7 Wei Zheng,1 Xiao-Ou Shu1. 1 _Vanderbilt University Medical Center, Nashville, TN;_ 2 _Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China;_ 3 _Milken Institute School of Public Health, George Washington University, Washington, DC;_ 4 _National Cancer Institute, Bethesda, MD;_ 5 _Washington University School of Medicine, St. Louis, MO;_ 6 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 7 _International Agency for Research on Cancer, Lyons, France_.

Background: Dietary fiber has been shown to have various health benefits, including anti-inflammation. However, the role of dietary fiber on lung cancer risk has not been well characterized. In this pooled analysis of 10 prospective cohort studies from the US, Europe, and Asia, we evaluated the association between dietary fiber intake and risk of lung cancer and further, potential effect modifications by tobacco smoking, alcohol drinking, and demographic factors (e.g., sex and race/ethnicity).

Methods: Hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated within each cohort using Cox proportional hazard models with adjustment for major lung cancer risk factors. Risk estimates from each cohort were pooled using random-effects meta-analysis. To reduce potential reverse causation due to preclinical dietary changes, all analyses were conducted excluding the first two years of observation.

Results: A total of 18,822 incident lung cancer cases were identified among 1,445,850 participants during mean follow-ups of 9.4 years. After adjustment for lung cancer risk factors (i.e., age, total energy, smoking status, smoking pack-years, gender, race/ethnicity, education, obesity status, diabetes, family history of lung cancer, alcohol consumption, physical activity level, menopausal status in women, and intake of saturated and polyunsaturated fat), dietary fiber intake was inversely associated with lung cancer risk (HR [95% CI] = 0.90 [0.83-0.99] for highest vs. lowest quintile and 0.93 [0.89-0.97] per 10g/day increase). Stratified analyses showed a significant inverse association in men (HRs [95% CIs] per 10g/day increase = 0.92 [0.85-1.00]), women (0.94 [0.90-0.99]), whites (0.92 [0.88-0.95]), smokers (0.92 [0.88-0.97]), and alcohol drinkers (0.89 [0.86-0.94]). However, no significant association was found in Blacks (1.05 [0.92-1.19]), Asians (0.99 [0.85-1.16]), never smokers (0.97 [0.88-1.07]), and never drinkers (0.99 [0.94-1.05]) (P interaction = 0.06 for sex, 0.004 for race, 0.17 for tobacco smoking, and <0.001 for alcohol drinking).

Conclusion: Our findings suggest that a high-fiber diet was associated with a lower risk of lung cancer among whites, smokers, and alcohol drinkers. Further research is needed to determine if the association between dietary fiber intake and lung cancer risk reflects a causal relation.

#5254

The associations of habitual physical inactivity with cancer outcomes: Evidence from the Roswell Park Cancer Institute Data Bank and BioRepository.

Rikki A. Cannioto, Shruti Dighe, Karen Hulme, Alexandra Drake, Susan E. McCann, Christine B. Ambrosone. _Roswell Park Cancer Institute, Buffalo, NY_.

Despite recent position statements asserting that a physically active lifestyle reduces cancer mortality, inverse associations of physical activity (PA) with cancer outcomes are only well established for breast, colorectal, and prostate cancers. Further, the independent associations of physical inactivity with cancer outcomes are rarely reported. Given the prevalence of physical inactivity at the population level and among cancer survivors, we examined the associations of habitual physical inactivity with clinical outcomes in a large patient cohort at Roswell Park Cancer Institute (RPCI). We utilized data from 7,827 patients enrolled in the Data Bank and BioRepository (DBBR) at RPCI between 2003 and 2016. Pre-diagnostic PA, spanning the decade prior to diagnosis, and post-diagnostic PA, representing PA at study enrollment, were assessed by self-administered questionnaire. Patients reporting no regular PA in the decade prior to diagnosis (i.e., walking for exercise, moderate or vigorous PA, or weight training) and no PA after diagnosis were categorized as habitually inactive, our primary analytic exposure of interest. Multivariable Cox proportional hazards models adjusted for age, sex, stage, and tumor site were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) representing associations of habitual inactivity with all-cause mortality, cancer mortality, and disease recurrence. Additional variables (e.g., race, education, smoking, BMI, diabetes, tumor grade, treatment, occupational PA, etc.) were assessed for confounding utilizing the 10% change-in-estimate method. During the follow-up period, median time to follow-up was 54 months and 2,504 (32%) patients died. Collectively, 1,372 patients (17.5%) self-reported habitual inactivity. In comparison to patients who engaged in regular PA before and after diagnosis, habitually inactive patients experienced increased hazards of all-cause mortality (HR=1.41, 95% CI: 1.27-1.56, p<0.001), cancer mortality (HR=1.34, 95% CI: 1.19-1.52, p<0.001) and cancer recurrence (HR=1.15, 95% CI: 1.05-1.26, p=0.004). Associations remained robust and consistent in subgroup analyses by sex, stage and BMI. In sensitivity analyses, associations were strengthened when excluding patients with <3 years of follow-up and remained significant when excluding breast, colorectal and prostate cancer patients for all-cause mortality (HR=1.33, 95% CI: 1.18-1.50, p<0.001), cancer-specific mortality (HR=1.32, 95% CI: 1.14-1.52, p<0.001) and disease recurrence (HR =1.17, 95% CI: 1.04-1.30, p=0.007). We observed consistent evidence that habitual inactivity associates with increased hazards of cancer recurrence and mortality across multiple tumor types. Well-designed survivorship studies are needed to confirm how much mortality can be reduced among regularly active cancer patients.

#5255

A pooled analysis of dietary fiber intake and risk of prostate cancer.

Elkhansa Sidahmed,1 Stephen J. Freedland,2 Molin Wang,1 Kana Wu,1 Jeanine M. Genkinger,3 Stephanie A. Smith-Warner1. 1 _Harvard T.H Chan School of Public Health, Boston, MA;_ 2 _Samuel Oschin Comprehensive Cancer Institute, Los Angeles, CA;_ 3 _Columbia University Mailman School of Public Health and Herbert Irving Comprehensive Cancer Center, New York, NY_.

High dietary fiber intake has been associated with a lower risk of many cancers but evidence from epidemiological studies on dietary fiber intake and risk of advanced prostate cancer and prostate cancer mortality are limited. We examined associations between dietary fiber intakes overall and from grains, fruits, and vegetables and risk of prostate cancer in 15 cohorts in the Pooling Project of Prospective Studies of Diet and Cancer. Among 842,149 men, during maximum follow-up ranging from 9.2-21.9 years across studies, 52,683 incident prostate cancer cases were identified. Prostate cancer outcomes examined were: 1) all cases; 2) advanced cases, which included men with advanced stage prostate cancer at diagnosis (T4, N1, and/or M1) and men who died of prostate cancer during follow-up (n=4,924); 3) advanced (restricted) cases, which included only men with advanced stage prostate cancer at diagnosis (n=3,110) (men diagnosed with localized prostate cancer who died of prostate cancer were excluded) and 4) prostate cancer mortality, death due to prostate cancer (n=3,199). We adjusted dietary fiber intake for energy intake using the residual method. Cox proportional hazards models were used to calculate study-specific multivariable relative risks (MVRR); the study-specific results were pooled using random effects models. Models were adjusted for age, race/ethnicity, education, marital status, body mass index, height, smoking status, family history of prostate cancer, physical activity, history of diabetes, multivitamin use, and energy and alcohol intake. Median dietary fiber intake varied from 6-28 g/d across studies. For total dietary fiber intake, dietary fiber intake from fruits, and dietary fiber intake from vegetables, no statistically significant associations were observed for any outcome; the pooled MVRRs comparing the highest with the lowest quintile ranged from 0.95-1.02. The pooled MVRR comparing the highest with the lowest study-specific quintile of dietary fiber intake from grains was 0.84 (95% confidence interval [CI] 0.76-0.93) for advanced stage and 0.85 (0.74-0.97) for advanced (restricted) stage prostate cancer. The pooled MVRR for the same comparison was 0.78 (95% CI 0.69-0.89) for prostate cancer mortality. Similar results were observed when dietary fiber intake was categorized using common absolute intake cutpoints across studies (e.g., the pooled MVRR for prostate cancer mortality was 0.86, 95% CI 0.75-0.98 comparing ≥8 with <2 g/d of dietary fiber intake from grains). Further adjustment for dietary folate, calcium, and lycopene intake and red meat consumption did not substantially change the results for dietary fiber intake from grains. In this large, international consortium, we found that higher dietary fiber intake from grains, but not intake of dietary fiber overall or from fruits and vegetables, was associated with lower risk of advanced prostate cancer and prostate cancer mortality.

#5256

A prospective study of inflammatory diet potential and risk of hepatocellular carcinoma (HCC).

Yanan Ma,1 Tracey G. Simon,2 Fred K Tabung,3 Lindsay Y. King,4 Dawn Q. Chong,5 Long Nguyen,2 Trang VoPham,3 Charles S. Fuchs,6 Jeffrey A. Meyerhardt,7 Kathleen E. Corey,2 Hamed Khalili,2 Stephanie Smith-Warner,3 Raymond T. Chung,2 Edward L. Giovannucci,3 Andrew T. Chan,2 Xuehong Zhang1. 1 _Brigham and Women's Hospital and Harvard Medical School, Boston, MA;_ 2 _Massachusetts General Hospital, Boston, MA;_ 3 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 4 _Duke University, Durham, NC;_ 5 _National Cancer Centre Singapore, Singapore, Singapore;_ 6 _Yale University Cancer Center, New Haven, CT;_ 7 _Dana-Farber Cancer Center, Boston, MA_.

Worldwide, hepatocellular carcinoma (HCC) is the fifth most common cancer and the second-leading cause of cancer-related death worldwide. In the U.S., the incidence rate of HCC has tripled since 1975. Although preliminary evidence suggesting a biological impact of diet on multiple inflammatory pathways implicated in hepatocarcinogenesis, the most comprehensive review by the WCRF/AICR concluded that the relationship between diet and HCC remains largely unknown. HCC is an inflammation-related cancer but few studies have investigated diet quality based on its inflammatory potential in relation to HCC risk. The empirical dietary inflammatory pattern scores (EDIP) score were calculated from validated food frequency questionnaires. It characterized dietary inflammatory potential based on circulating levels of inflammatory bio-markers. We prospectively followed 49,674 men in the Health Professionals Follow-up Study (1986-2012), and 74,139 women in the Nurses' Health Study (1984-2012). Cox regression analyses were used to calculate hazards ratios (HR) for HCC risk adjusting for body mass index, smoking, history of diabetes, race, physical activity, and regular aspirin use. We documented a total of 89 incident HCC cases (44 in women and 45 in men) over 28 years, encompassing 1,522,972 person-years of follow-up. Compared to the lowest tertile, the highest tertile of the EDIP score was associated with a multivariable HR for HCC of 2.86 (95%CI, 1.20-6.81) among women (P-trend=0.01) and of 0.68 (95%CI, 0.31-1.50) among men (P-trend=0.35). Future study is warranted to confirm this finding and elucidate the potential biological basis.

Key words: inflammation, dietary patterns, HCC

A. T. C. and X. Z contributed equally to this work.

#5257

Prevalence and trends of obesity and metabolic syndrome among U.S. adults diagnosed with cancer, NHANES 1999-2014.

Wambui Grace Gathirua-Mwangi. _Indiana University, Fishers, IN_.

Background: The prevalence of obesity and Metabolic syndrome (MetS) in the general population has been documented extensively. While obesity and MetS are associated with cancer risk and may persist after diagnosis, the prevalence and the secular trends of obesity and MetS among cancer survivors has not been examined in a nationally representative cohort.

Objective: To compare adults diagnosed with cancer and those without cancer on the prevalence of obesity and MetS in 2013-2014, and on the time trends over the past years from 1999 through 2014. Methods: A sample of 39,608 adults, aged 20+ who participated in National Health and Nutrition Examination Survey (NHANES) surveys from 1999-2014 were included. Of those surveyed, 2,811 subjects had been diagnosed with non-skin cancers, and 1,770 with obesity-linked cancers (colorectal, liver, kidney, esophageal, stomach, pancreatic, gallbladder, breast, endometrial, ovarian and prostate). MetS was defined as having at least three of the following five factors: elevated levels of (1) triglyceride, (2) systolic or diastolic blood pressure, or use of anti-hypertensive medication, (3) blood glucose or self-reported diabetes or use of insulin, (4) enlarged waist circumference and (5) lower levels of high-density lipoprotein (HDL) cholesterol or use of cholesterol lowering medication.

Results: In 2013-2014, the prevalence of obesity was 41.1%, 40.5% and 37.8% among adults diagnosed with obesity-linked cancers, total-cancer, and no cancer diagnosis, respectively. The prevalence of obesity have significantly increased over the last 16 years for all three groups. Relative to adults without a cancer diagnosis, the 16-year increase in obesity prevalence was significantly higher for those with obesity-linked cancers (p=0.019), but not with total cancer (p=0.35). In relation to MetS, the 2013-2014 prevalence was significantly higher among adults with obesity-linked cancers (52.7%) and total cancer (50.3%) compared to adults without cancer (29.2%). Over the 16-year period from 1999-2014, the prevalence of MetS has increased among adults with obesity-linked cancers (p=0.008), total cancer (p=<.0001), and those without a cancer diagnosis (p=0.005), respectively. Compared to the adults without a cancer diagnosis, the prevalence of MetS over the years has significantly increased among adults with obesity-linked cancers (p=0.004) and total cancer (p=0.05) were significantly higher.

Conclusion: U.S. adults diagnosed with obesity-linked cancers had a higher prevalence of obesity and MetS, and the prevalence have significantly increased over the last 16 years, compared to those without a cancer diagnosis. The findings of this study underscore the urgent need for clinicians and public health workers to recognize and address cardiometabolic health problems among cancer survivors.

#5258

Determinants of impaired lung function among never-smokers in the UK Biobank Cohort.

Matthew T. Warkentin,1 Rayjean J. Hung2. 1 _University of Toronto, Toronto, Ontario, Canada;_ 2 _Sinai Health System, Toronto, Ontario, Canada_.

Introduction: The role of impaired lung function in lung cancer etiology remains contentious, especially in never-smokers who do not have primary smoking as a major risk factor, and represent an increasing proportion of lung cancers at 10-25%. Lung cancer in never-smokers (LCINS) would be the 7th most incident cancer worldwide if considered separately from smoking-related lung cancer. There has been a dearth of studies to evaluate lung function in a large cohort exclusively of never-smokers. The objective of this study was to address this gap.

Methods: Based on the UK Biobank cohort, which recruited more than 500,000 individuals aged 40-69 between 2006-2010, we analyzed the association between lung function (measured as forced expiratory volume in 1-second, FEV1 and forced vital capacity, FVC) and early life factors, air pollution, and lifestyle exposures. Never-smokers were defined as those who smoked < 100 cigarettes, and impaired lung function was defined by FEV1/FVC based on GOLD criteria, FEV1 percent predicted (pp), or both (based on NICE criteria).

To construct the percent predicted, reference FEV1 values were estimated using linear models in healthy never-smokers, with the predictors height, height2, age, sex, age by sex interaction, and ethnicity. Logistic regression was used to evaluate risk of impaired lung function, adjusted for all other predictors. We constructed risk-prediction models for LCINS incorporating lung function based on area under the curve (AUC) and corrected for optimism using bootstrap validation methods.

Results: After excluding participants with prevalent respiratory cancers, 249,052 never-smokers were included in the analysis. The proportions of never-smokers with lung impairment based on the three criteria were: 11%, 12%, and 5%, for FEV1pp, GOLD, and NICE, respectively. Low birth weight (OR=1.32, 95% CI: 1.24-1.41) and any second-hand smoke exposure (OR=1.18, 95% CI: 1.13-1.23) increased risk of impaired FEV1 among never-smokers, as did air pollution, with PM2.5 (OR=1.83, 95% CI: 1.52-2.21) conferring the greatest increase in risk, and these associations were similar for impaired lung function based on NICE and GOLD criteria. High body mass index (OR=1.70, 95% CI: 1.62-1.77) was associated with increased risk of FEV1 impairment, but the opposite was observed with impairment defined by FEV1/FVC ratio. Risk-prediction models for LCINS incorporating lung function will also be presented.

Conclusions: The association of obesity with FEV1/FVC remains paradoxical. Severe lung impairment can lead to both restrictive (FVC) and obstructive (FEV1) effects, and non-proportional impairment of these measures may help explain some of the paradoxical associations observed in this study and in the literature. Findings from this study suggest that evaluating multiple aspects of lung function may reveal a more complete picture of impairment and may inform approaches toward risk prediction.

#5259

Cancer incidence in relation to body fatness in China: Evidence from a large population-based cohort study.

Lu Wang,1 Guangfu Jin,1 Canqing Yu,2 Jun Lv,2 Yu Guo,3 Zheng Bian,3 Ling Yang,4 Yiping Chen,4 Zhibin Hu,1 Feng Chen,1 Zhengming Chen,4 Liming Li,2 Hongbing Shen1. 1 _Nanjing Medical University, Nanjing, China;_ 2 _Peking University Health Science Center, Beijing, China;_ 3 _Chinese Academy of Medical Sciences, Nanjing, China;_ 4 _University of Oxford, Oxford, United Kingdom_.

Body fatness has been associated with several cancers in developed countries, but little is known in China. The purpose of this study was to examine the association between body fatness and cancer incidence, with different anthropometric measures, and compare their predicting values. Between June 2004 and July 2008, the China Kadoorie Biobank (CKB) study recruited 0.5 million adults aged 30-79 years from 10 geographically diverse areas across China, and followed up until December, 2015. Body-mass index (BMI), body fat percentage (BFP), waist circumference (WC), and waist-to-hip ratio (WHR) were measured at baseline. Cox regression yielded adjusted hazard ratios (HRs) associating incidence of all cancers and 15 site-specific cancers with different anthropometric measures, adjusting for education level, marital status, annual household income, alcohol consumption, smoking status and physical activity, and stratifying by age at risk, sex, and region. We compared the ability of cancer risk prediction between different anthropometric measures through a receiver operating characteristic (ROC) analysis. Among 508,362 participants, the overall mean (SD) BMI was 23.66 (3.38) kg/m2, 31.11% of men and 34.24% of women were overweight or obesity (BMI≥25.0kg/m2), and 4.41% of men and 4.26% of women were underweight (BMI<18.5kg/m2). 21,474 incident cancers were identified over an average of 8.94 years of follow-up. Each 5 kg/m2 increase in BMI was associated with an increased risk of endometrial (HR, 2.01; 95% CI, 1.72 to 2.35), postmenopausal breast (HR, 1.29; 95% CI, 1.18 to 1.40), colorectal (HR, 1.17; 95% CI, 1.10 to 1.25) and cervical (HR, 1.15; 95% CI, 1.03 to1.29) cancer, whereas it was associated with a reduced risk of oesophageal (HR, 0.73; 95% CI, 0.67 to 0.79), lung (HR, 0.78; 95% CI, 0.74 to 0.82), liver (HR, 0.85; 95% CI, 0.79 to 0.92), and stomach (HR, 0.88; 95% CI, 0.82 to 0.94) cancer. BMI was the best risk prediction measure for postmenopausal breast and endometrial cancer, whereas BFP were the best for oesophageal, lung, liver and stomach cancer, and WC and WHR did for colorectal cancer. BMI is related to the incidence of several cancers in China. Obesity and overweight, as well as underweight, contribute to the cancer incidence in China. Other anthropometric measures may add information for cancer-specific risk prediction.

#5260

Alcohol consumption and risk of gastric cardia adenocarcinoma and gastric non-cardia adenocarcinoma: A prospective analysis from the NIH-AARP Diet and Health cohort.

ShaoMing Wang, Neal D. Freedman, Erikka Loftfield, Xing Hua, Christian C. Abnet. _National Cancer Institute, Rockville, MD_.

Objective: The 2016 WCRF Continuous Update Project reported that consumption of alcoholic drinks probably causes gastric cancer, but the magnitude of the association was small, there was moderate heterogeneity among the studies included in the meta-analysis, and most studies were from Asian populations, with only 401 cases from North American studies. Therefore, we re-evaluated the association between alcohol intake and gastric cancer in a large US cohort that has accrued more than 1300 gastric cancer cases.

Methods: We prospectively investigated the associations of alcohol consumption with gastric cardia adenocarcinoma (GCA) and gastric non-cardia adenocarcinoma (GNCA) in 490,605 members aged 50-71 years at baseline in the NIH-AARP Diet and Health Study cohort. Alcohol consumption was assessed during the baseline questionnaire in 1995/1996 and evaluated as grams of ethanol intake and as a categorical variable: never, up to or including one drink/day, one to three drinks/day, and greater than three drinks/day. We used Cox proportional hazard regression to calculate the hazard ratios and 95% confidence intervals for associations between alcohol intake and risk of gastric adenocarcinomas with adjustment for several potential confounders.

Results: Through 2011, 662 incident cases of GCA and 713 of GNCA occurred in the cohort. We found no association between higher alcohol consumption and GCA or GNCA, regardless of whether analyzed as total alcoholic beverage intake or the individual beverage types of beer, wine and liquor and no evidence for a dose-response. Furthermore, we observed no association by stratum of sex, ethnic group, educational level, or smoking status. Although there were no overall associations, we found some evidence for lower risk of GNCA among people who drank up to one drink per day (HR=0.81, 95%CIs:0.67-0.97) compared to nondrinkers.

Conclusion: Alcohol consumption was not associated with increased risk of gastric cardia adenocarcinoma or gastric non-cardia adenocarcinoma in this large US prospective cohort.

#5261

Indices of diet quality and risk of lung cancer incidence and mortality in the Women's Health Initiative Observational Study.

Ajay A. Myneni,1 Gary A. Giovino,1 Amy E. Millen,1 Michael J. LaMonte,1 Jean Wactawski-Wende,1 Marian L. Neuhouser,2 Jiwei Zhao,1 James M. Shikany,3 Lina Mu1. 1 _University at Buffalo, State University of New York, Buffalo, NY;_ 2 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 3 _University of Alabama at Birmingham School of Medicine, Birmingham, AL_.

Background: Prospective evidence on the association between diet patterns and lung cancer risk is limited, particularly in older women. We investigated whether commonly used diet quality indices - Healthy Eating Index 2010 (HEI), Alternate Healthy Eating Index 2010 (AHEI), alternate Mediterranean Diet (aMED) and Dietary Approaches to Stop Hypertension (DASH) - were associated with lung cancer incidence and mortality in the Women's Health Initiative Observational Study (WHI OS).

Methods: Self-reported diet intake (food frequency questionnaires) and relevant covariate information was obtained from 86,090 race-ethnically diverse postmenopausal women aged 50-79 years at baseline (1993-1998). During a median follow-up of approximately 17 years, 1,491 lung cancer cases and 1,393 lung cancer deaths were documented. We used cox proportional hazards regression models to estimate hazards risks and 95% confidence intervals [HR (95% CI)] after controlling for age, race/ethnicity, education, body mass index, smoking, physical activity and energy intake.

Results and conclusions: Diet quality indices were not associated with lung cancer incidence overall but all four dietary indices showed a protective association (highest versus lowest quintile) against squamous cell carcinoma cases (12.8% of total lung cancer cases) [HEI: 0.56 (0.33-0.96), AHEI: 0.42 (0.24-0.76), aMED: 0.65 (0.39-1.08), DASH: 0.56 (0.32-0.97)]. Diet quality indices were not associated with lung cancer mortality overall or when stratified by histological type, except for HEI with lung cancer mortality overall [fourth versus lowest quintile: 0.82 (0.69-0.99)]. Smoking status did not modify the association between diet quality indices and lung cancer incidence or mortality. Further studies in other populations may help elucidate the relationship between diet patterns and lung cancer, especially by histological type.

#5262

Associations between intake of calcium, magnesium, and phosphorus and risk of pancreatic cancer in a population-based case-control study in Minnesota.

Hao Fan,1 Andrew Marley,1 Margaret Hoyt,1 Haocheng Nan,1 Kristin Anderson,2 Jianjun Zhang1. 1 _Indiana University, Indianapolis, IN;_ 2 _University of Minnesota, Minneapolis, MN_.

Pancreatic cancer is the fourth leading cause of cancer death among both men and women in the U.S. and has a dismal prognosis among all cancers due to its aggressive nature and lack of effective screening tests. Pancreatic cancer etiology remains elusive, with few well-established risk factors aside from cigarette smoking; therefore, it is critical to identify other modifiable risk factors for primary prevention. Calcium, magnesium, and phosphorus are essential minerals for bone health and other important metabolic processes. Emerging evidence indicates that these minerals are involved in the carcinogenesis of the colon and other organs through their influence on cellular proliferation, systematic inflammation, immune functions, and/or genomic stability. In the present study, we aimed to investigate the associations between intake of calcium, magnesium, and phosphorus and risk of pancreatic cancer in a case-control study conducted during 1994-1998 in Minnesota. Cases (n=150), aged 20 years or older, were ascertained from all hospitals in the metropolitan area of the Twin Cities and the Mayo Clinic; from the latter, only cases residing in the Upper Midwest of the U.S. were recruited. Controls (n=459) were randomly selected from the general population and frequency matched to cases by age (within 5 years) and sex. Dietary and supplemental intake of three minerals of interest and other nutrients was estimated from a validated food frequency questionnaire. Odds ratios (OR) and 95% confidence intervals (95% CI) were estimated using unconditional logistic regression. After adjustment for age, sex, race, education, physical activity, cigarette smoking, alcohol use, and intake of energy, total fat, fiber, fruits, and vegetables, risk of pancreatic cancer decreased with an increasing dietary intake of calcium [OR (95% CI) for quartiles 2, 3, and 4 vs. quartile 1: 0.96 (0.54-1.67), 0.65 (0.35-1.17), and 0.58 (0.23-1.40), p-trend = 0.09. A similar inverse but not significant association was observed for total calcium intake. A significantly reduced risk of pancreatic cancer was found among subjects in the third quartile of total magnesium intake compared with those in the first (lowest) quartile [OR (95% CI): 0.35 (0.17-0.69)], although it may be a chance finding due to multiple comparisons. No apparent associations existed between dietary and total intake of phosphorus and risk of pancreatic cancer. Our study shows that there are suggestive, but inconsistent beneficial effects of dietary and/or supplemental intake of calcium and magnesium on the occurrence of pancreatic cancer. This observation warrants confirmation in other epidemiologic studies.

#5263

Association between urinary tea catechins and risk of gastric cancer: A nested case-control study within the Shanghai Cohort Study.

Hamed Samavat,1 Renwei Wang,1 Chung S. Yang,2 Yu-Tang Gao,3 Jian-Min Yuan1. 1 _Univ. of Pittsburgh, Pittsburgh, PA;_ 2 _Rutgers University, Piscataway, NJ;_ 3 _Shanghai Cancer Institute, Shanghai, China_.

Background: Findings from in vitro and animal studies suggest a protective role for green tea consumption against gastric cancer risk; however, data from observational studies are inconsistent and mostly based on self-reported dietary intake. To date, very few studies have investigated the association between tea catechins and gastric cancer risk using a biomarker-based approach.

Methods: We analyzed data from 300 incident gastric cancer cases and 905 controls nested within a prospective population-based cohort of 18,244 middle-aged and older men in Shanghai, China. Control subjects were matched to case patients by age (± 2 years), date of sample collection (± 1 month), and neighborhood of residence at recruitment. Tea catechins including epicatechin (EC); epigallocatechin (EGC); methylated-EGC (Me-EGC); 5-(3′,4′,5′-trihydroxy-phenyl)-γ-valerolactone (M4); and 5-(3′,4′-dihydroxy-phenyl)-γ-valerolactone (M6) were measured in pre-diagnostic urine specimens collected between 1986 and 1989 using a validated high-performance liquid chromatography method. Odds ratios (ORs) and 95% confidence intervals (CIs) of gastric cancer by quintiles of tea catechins levels were estimated by unconditional logistic regression models, adjusting for potential confounders.

Results: Compared to control subjects, cancer patients were less educated with higher prevalences of smoking, alcohol drinking and seropositivity of Helicobactor pylori. Although not statistically significant, cases showed higher levels of urinary tea catechins relative to controls. Among control subjects, levels of urinary catechins were significantly positively associated with consumption of tea. After adjusting for matching factors and selected known risk factors, men with the highest quintiles of urinary EC, Me-EGC, and M6 had significantly higher risk of gastric cancer compared to those in the lowest quintiles (OR = 1.59, 95% CI = 0.98 to 2.58, P trend = 0.04 for EC; OR = 1.47, 95% CI = 0.86 to 2.49, P trend = 0.02 for Me-EGC; OR = 1.64, 95% CI = 1.06 to 2.55, P trend = 0.01 for M6). Corresponding risks were slightly stronger among cases with Helicobactor pylori seropositivity status. When the analysis was limited to ever smokers or drinkers, the OR (95% CI) comparing top versus bottom quintiles of Me-EGC were 2.05 (1.06 to 3.97; P trend = 0.01) and 2.17 (0.98 to 4.79; P trend = 0.04), respectively; however, we did not observe significant interaction between Me-EGC and smoking or alcohol drinking status on risk of gastric cancer.

Conclusion: In contrast to some previous findings, we found that tea catechins are associated with increased risk of gastric cancer. The impact of tea catechins on the risk of gastric cancer may differ by smoking or alcohol drinking status.

Funding source: NIH/NCI (R01 CA144034 and UM1 CA182876).

#5264

A prospective study of dietary inflammatory index scores and colorectal cancer risk: The Singapore Chinese Health Study.

Hung N. Luu,1 Renwei Wang,2 Nittin Shivappa,3 James R. Hébert,3 Woon-Puay Koh,4 Jian-Min Yuan2. 1 _Univ. of South Florida, Tampa, FL;_ 2 _University of Pittsburgh Cancer Institute, Pittsburgh, PA;_ 3 _University of South Carolina, Columbia, SC;_ 4 _National University of Singapore, Singapore, Duke-NUS Medical School Singapore, Singapore, Singapore_.

Background. Colorectal cancer (CRC) has increased dramatically in Singapore during the past three decades and becomes the most common cancer when both sexes combined. Extensive evidence shown the specific component of diet may modulate inflammation and thus influence to disease endpoints, including CRC. We determine the association between diet inflammatory index (DII)TM, a literature-derived, population-based score, developed to assess the inflammatory potential of an individual's diet, and CRC risk in Singaporean Chinese, using data from the Singaporean Chinese Health Study (SCHS).

Methods. A total of 61,321 SCHS study participants was included in the current analysis. The DII scores were calculated using validated food frequency questionnaires (FFQs). Cox proportional hazards model was performed to assess the association between DII scores and CRC risk.

Results. There was a positive association between DII score and CRC risk in females (HRs and respective 95% CIs: 1.30 (1.02-1.64), 1.32 (1.03-1.67), and 1.20 (0.93-1.55) for Q2, Q3, Q4 vs. Q1). DII score was associated with increased risk of colon cancer (HRQ4vsQ1=1.35, 95% CI: 1.08-1.68; Ptrend=0.04) and this association appeared stronger in females. Among never-smokers, the highest quartile of DII was associated with statically significant 43% increased risk of developing CRC in women compared to those in the lowest quartile (HRQ4vsQ1=1.43, 95% CI: 1.01-2.02; Ptrend=0.03). There was also a dose-response association between DII scores with colon cancer in women among those with BMI less than 25 (HRQ4vsQ1=1.66, 95% CI: 1.03-2.68; Ptrend=0.04).

Conclusions. Consumption of anti-inflammatory foods will reduce the risk of colorectal cancer, particularly among females and non-smokers. This will have substantial public health impact as prevention program should focus on promoting healthy and anti-inflammatory foods to reduce the risk of colorectal cancer.

#5265

Association of dietary patterns with serum testosterone levels in men.

David S. Lopez,1 Lydia Liu,2 Stephanie A. Smith-Warner,2 Eric B. Rimm,2 Konstantinos K. Tsilidis,3 Steven Canfield,4 Edward Giovannucci2. 1 _UT School of Public Health, Houston, TX;_ 2 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 3 _University of Ioannina School of Medicine, Ioannina, Greece;_ 4 _UTHealth McGovern Medical School, Houston, TX_.

Background: Testosterone (T) plays an important role in men's health and its deficiency has been linked with prostate cancer (PCa).1-3 In the U.S., there are approximately 2.4 million men (40-69 years old) with T deficiency4-7 and this number is projected to increase to 6.5 million men by 2025.8 Although this may be due, in part, to the increasing elderly population and body size, dietary factors may also play a role in T levels.9,16 Dietary patterns have been linked with PCa, and it has been hypothesized that T levels may be involved in the biological pathway.10-15 Therefore, we investigated the associations of prudent, western and alternative healthy eating index (AHEI) dietary pattern scores with serum T levels and whether these associations varied by obesity status (body mass index ≥ 30 kg/m2). Methods: We performed a cross-sectional analysis for the associations of the dietary pattern scores with T levels in 1,376 male participants of the Health Professionals Follow-up Study (HPFS). Total testosterone was measured by chemiluminescent immunoassay in blood samples collected in three assay batches: blood-draw to 1996, 1996-1998, 1998-2000. T deficiency was defined as total testosterone ≤ 350 ng/dL.17 We assessed diet every 4 years using a validated food frequency questionnaire.18 Western (high in red meat, refined grains, and high-fat dairy) and prudent (high in fruits, vegetables, and whole grains) dietary patterns were identified using principal component analysis. AHEI-2010 score is a modified version of the Healthy Eating Index 2005. Multivariable linear and logistic regression analyses were conducted to investigate these associations.Results: After adjusting for risk factors of T levels, we did not identify significant associations between quintiles (lowest to highest) of prudent, western, and AHEI dietary pattern scores with T levels, Ptrend ≥ 0.05 for all. However, there seemed to be a weak association with AHEI, Ptrend ≥ 0.07. Furthermore, a potential nonlinear relationship with the prudent diet was observed (Pnonlinearity = 0.0005). The associations of the three dietary patterns with T deficiency did not reach statistical significance, Ptrend ≥ 0.48 for all. In stratified analysis, we identified a weak linear relationship between prudent diet and T levels in obese men (Ptrend = 0.05).Conclusion: With the exception of a weak association with AHEI, we did not find a significant linear relationship between prudent and western dietary patterns and T levels, but this doesn't exclude potential nonlinear associations. Although we only identified a weak relationship between prudent diet and T levels in obese men, it seemed that obesity overwhelmed any impact of the three dietary patterns on T levels because in general the T levels across the quintiles of the three dietary patterns scores were lower among obese than non-obese men irrespective of the diet.

#5266

Current smoking habit is a risk factor for poor surgical outcome of non-B, non-C hepatocellular carcinoma.

Keita Kai, Hiroki Koga, Shinichi Aishima, Atsushi Kawaguchi, Koutaro Yamaji, Takao Ide, Junji Ueda, Hirokazu Noshiro. _Saga Univ., Saga, Japan_.

Background: Many factors are recognized as risks for the development of hepatocellular carcinoma (HCC) in non-B non-C (NBNC) patients. Although cigarette smoking has been recognized as one of the risk factors for HCC, the surgical outcomes and clinicopathological characteristics according to smoking habits of NBNC-HCC patients are unclear.

Methods: Consecutive cases of 83 NBNC-HCC patients who underwent curative surgical treatment for the primary lesion at Saga University Hospital were enrolled the study. The information about possibly carcinogenic factors such as alcohol abuse, diabetes mellitus, obesity and smoking habit were collected from medical records. Smoking habits were subcategorized as never, ex- and current smoker at the time of surgery. The diagnosis of non-alcoholic steatohepatitis (NASH) was based on both clinical information and pathological confirmation.

Results: Alcohol abuse, diabetes mellitus, obesity and NASH had no significant effect on the surgical outcomes. Current smoking status was strongly correlated with both overall survival (p=0.0058) and disease-specific survival (p=0.0105) by multivariate analyses. Subset analyses revealed that current smokers were significantly younger at the time of surgery (p=0.0002) and more likely to abuse alcohol (p=0.0188) and to have multiple tumors (p=0.023).

Conclusion: Current smoking habit at the time of surgery is a risk factor for poor long-term survival in NBNC-HCC patients. Current smokers tend to have multiple HCCs at a younger age than other patients.

#5268

Interactions between genetic predictors of gene expression and dietary factors associated with risk of colorectal cancer.

Paneen S. Petersen,1 Yu-Ru Su,1 Sonja I. Berndt,2 Stephanie A. Bien,1 Hermann Brenner,3 Graham Casey,4 Andrew T. Chan,5 Jenny Chang-Claude,3 Jane C. Figueiredo,6 Steven J. Gallinger,7 Robert W. Haile,8 Tabitha A. Harrison,1 Michael Hoffmeister,3 Mark A. Jenkins,9 Amit D. Joshi,10 Sébastien Küry,11 Loic Le Marchand,12 Yi Lin,1 Noralane M. Lindor,13 Polly A. Newcomb,1 John D. Potter,1 Robert Schoen,14 Martha L. Slattery,15 Stephen N. Thibodeau,16 Emily White,1 Li Hsu,1 Ulrike Peters,1 CCFR, GECCO. 1 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 2 _National Cancer Institute, National Institutes of Health, Rockville, MD;_ 3 _German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 4 _University of Virginia, Charlottesville, VA;_ 5 _Massachusetts General Hospital, Boston, MA;_ 6 _Keck School of Medicine of University of Southern California, Los Angeles, CA;_ 7 _Mount Sinai Hospital, Toronto, Ontario, Canada;_ 8 _Cedars-Sinai Medical Center, Los Angeles, CA;_ 9 _University of Melbourne, Parkville, VIC, Australia;_ 10 _Harvard School of Public Health, Boston, MA;_ 11 _Centre Hospitalier Universitaire Nantes, Nantes, France;_ 12 _University of Hawaii Cancer Center, Honolulu, HI;_ 13 _Mayo Clinic, Scottsdale, AZ;_ 14 _University of Pittsburgh, Pittsburgh, PA;_ 15 _University of Utah Health Sciences Center, Salt Lake City, UT;_ 16 _Mayo Clinic, Rochester, MN_.

Genome-wide association studies of colorectal cancer (CRC) have identified over 50 susceptibility loci. These variants represent only a small fraction of total heritability for CRC. Gene-environment (GxE) interaction studies may help identify novel loci and biological interactions that give insight to the pathogenesis of CRC. Previous genome-wide GxE studies with dietary factors have identified interactions between loci and processed meat consumption and alcohol; however, limited statistical power remains a primary concern. Set-based SNP testing has the potential to increase statistical power to detect GxE interactions by aggregating functionally relevant SNPs. In this large pooled analysis using 14 case-control and nested case-control studies, we incorporated functional information from the transcriptome prediction tool, PrediXcan, into a novel set-based approach for testing GxE interactions. We used variant weights from the PrediXcan models of tissue-specific gene expression in the transverse colon as a priori variant information for a set-based GxE approach. We restricted our analysis to variants in the PrediXcan transverse colon gene models (n = 4,842). This discovery phase included 10,360 CRC and advanced adenoma cases, and 11,183 controls of European ancestry from the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO) and the Colon Cancer Family Registry. All 14 studies were analyzed together in a pooled data set using the Mixed Effects Score Tests for interactions. We tested for gene interactions with sex- and study- specific quartiles of dietary intake of red meat (servings/day), processed meat (servings/day), vegetables (servings/day), fruits (servings/day), and fiber (g/day). We detected two genes with suggestive interactions (false discovery rate (FDR) < 0.2) with intake of red meat and risk of CRC: Superoxide Dismutase 2 (SOD2) and Ubiquitin Conjugating Enzyme E2 H (UBE2H). No interactions at FDR < 0.2 were observed for processed meat, vegetables, fruits, or fiber. The SOD2 gene, which encodes an enzyme important in apoptotic signaling and clearing of reactive oxygen species, may regulate response to colonic exposure to heme iron or increased bile acid from high-fat content in red meat. UBE2H is part of the ubiquitin-proteasome system that has a role in Wnt signaling, which can be mediated by heme iron in red meat and is commonly found dysregulated in cancer. These findings highlight the efficacy of integrating functional information and set-based testing for novel discovery of genes interacting with known dietary risk factors of CRC. We plan to replicate these findings in additional studies.

#5269

Body mass index may not have causal association with thyroid cancer risk: A Mendelian randomization study.

Jeongseon Kim,1 Soo Ji Lee,2 Quy N. Nguyen,1 Jeonghee Lee,1 Eun Kyung Lee,1 Yul Hwangbo,1 Joohon Sung2. 1 _National Cancer Ctr. Korea, Goyang-si, Republic of Korea;_ 2 _Seoul National University, Seoul, Republic of Korea_.

Background: Thyroid cancer (TC) is one of the endocrine tumors of which incidence rates have shown an upward trend worldwide. Obesity, another endemic health issue, has been recently reported in the association with increased TC risk. We attempted to examine if there is a causal association between body mass index (BMI) and TC using a Mendelian randomization approach.

Methods: Total 17,935 healthy individuals from the Korean Genome Epidemiology Study and 1,214 TC cases from National Cancer Center, Korea, and Seoul National University Hospital were selected with age, sex, BMI, and genetic information. Based on the thirteen previously reported single nucleotide polymorphisms associated with BMI, we constructed a genetic risk score (GRS) as a genetic instrumental variable (IV) representing BMI independent of possible confounding. IV analysis was performed using the ratio (or, Wald) method to test causal association.

Results: The odds of TC were 2.6 times higher in females than males. The cases were younger than the controls. The F-statistic from the regression of BMI on BMI GRS (IV) was 157.2 (usually, F larger than 10 indicates a strong and valid IV), and the IV explained 1.10% of the phenotypic variance for BMI. From IV analysis, genetically instrumented BMI was not associated with TC risk (causal odds ratio per 1kg/m2 increase in BMI 0.98 [0.79 - 1.20 95% confidence interval]).

Conclusion: Our analysis suggests that positive association between obesity and TC risk repeatedly reported from several meta-analyses may be null. Further investigation is needed as the incidence rates differ in different ethnicity.

#5270

**Associations of circulating 25-hydroxyvitamin D** 3 **concentrations with incident, sporadic colorectal adenoma risk according to common vitamin D-binding protein isoforms.**

David C. Gibbs. _Emory University, Atlanta, GA_.

25-hydroxyvitamin D3 (25[OH]D3), the main circulating form of vitamin D, is inversely associated with incident, sporadic colorectal adenoma. We investigated whether this association differs by two functional variants in the vitamin D binding protein gene (GC) that encode for common protein isoforms (Gc1s, Gc1f, Gc2) linked to differences in vitamin D metabolism. We pooled data from three US colonoscopy-based case-control studies (418 adenoma cases, 524 polyp-free controls), and estimated 25(OH)D3-adenoma associations, by GC isoforms, using multivariable logistic regression. Consistent with previous studies, mean-adjusted 25(OH)D3 concentrations were lowest among Gc2 homozygotes (20.7 ng/mL, 95% CI: 26.2, 28.0) and highest among Gc1 (combined Gc1s and Gc1f) homozygotes (27.1 ng/mL, 95% CI: 18.6, 22.7). Higher 25(OH)D3 concentrations were inversely associated with colorectal adenoma among those with the Gc2 isoform (per 10 ng/mL increase in 25(OH)D3 odds ratio = 0.71; 95% confidence interval: 0.56, 0.90), but not among those with only Gc1 isoforms (odds ratio = 1.07; 95% confidence interval: 0.87, 1.32) (Pinteraction = 0.03). These novel findings suggest that the vitamin D-incident, sporadic colorectal adenoma association may differ by common GC isoforms, and that those with the Gc2 isoform may particularly benefit from maintaining higher circulating 25(OH)D3 concentrations for adenoma prevention.

#5271

Exercise intervention alleviates inflammation-related biomarkers in breast cancer survivors.

Aditi Vyas, Yuan Chun Ding, Sarah Flores, Megan Johnson, Gabrielle Riazi, Joanne Mortimer, Susan Neuhausen, Jessica Clague Dehart. _City of Hope Beckman Research Institute, Duarte, CA_.

Purpose: In this pilot study we assessed the feasibility of breast cancer survivors exercising at Curves facilities, and examined stress-related biomarkers to explain the association between exercise and reduction in cancer associated co-morbidities.

Methods: 50 sedentary, estrogen-positive, postmenopausal breast cancer survivors were recruited from the City of Hope, of whom 50% were randomized into a 16-week Curves exercise intervention. Adherence to the exercise/control regimen was followed through the 16 weeks. Body measurements and blood samples were taken at each time point. Samples were analyzed using genome-wide RNA sequencing and inflammation measured using a 13 Plex Immunology Multiplex Assay and a C-reactive protein assay.

Results: Although insignificant variation in weight loss was observed between the two groups after 16 weeks, the exercise group did show a reduction in 12 of the 14 measurable inflammation markers compared to the control group. The average fold change after 16 weeks showed that women in the exercise group had a 40% and 25% reduction in the proinflammatory markers CRP and IL-6, respectively, when compared to the control group. Additionally, a greater number of women in the exercise group showed an overall decrease in proinflammatory markers including IL-6, CRP, TNF-α and IL-8 when compared to the control group; 80% of the women in the exercise group showed reduced levels of IL-8 at 16 weeks compared to control group where only 36.4% of women had any reduction in IL-8 levels. Interestingly, irrespective of the exercise intervention, analyses based on their exposure to radiation therapy (RT), showed that women who underwent RT exhibited increased levels of all 14 inflammation markers at 16-weeks while women who had not been given RT showed a slight decrease in 13 of the 14 inflammation markers. The gene expression profiling results for the exercise group showed 435 differentially expressed (DE) genes post-intervention, when compared to the gene expression at baseline (p=0.05). Several of these DE genes are involved with inflammatory response, interleukin and interferon signaling pathways. Interestingly, the difference at baseline between the breast cancer survivors from this study and a group of women without a history of cancer from a parallel exercise study by our team showed a drastic variation, with a total of 2,543 DE genes (p=0.05), with most of the DE genes associated with catabolic cellular processes.

Conclusion: Our results indicate that moderate levels of exercise, even without weight loss, could potentially be useful in alleviating inflammation and stress-related biomarkers in breast cancer survivors. The expression-level variation at baseline between women with and without a history of breast cancer could be attributed to the oncogenic and chemotherapy-related stress in the breast cancer survivors. Further gene set enrichment analysis is needed for all groups.

## PREVENTION RESEARCH:

### Population and Behavioral Studies in Cancer

#5272

Racial disparities and predictors of receipt of late surgery among patients diagnosed with breast cancer in South Carolina.

Oluwole Adeyemi Babatunde, Swann Adams, Jan Eberth, Robert Moran, Erica Sercey, Samantha Truman, James Hebert. _Univ. of South Carolina, Columbia, SC_.

Background: Diagnosis-to-surgery wait times among breast cancer (BrCa) patients in the United States have increased over the past two decades and have been associated with poorer survival outcomes. Racial disparities have been reported in these diagnosis-to-surgery wait times, with significant odds of delayed treatment found among Blacks. The objective of this study was to identify predictors of late surgery and to assess interactions with race in predicting late surgery among patients diagnosed with BrCa in South Carolina (SC).

Methods: We derived data on all female BrCa cases in 2002-2009 from the SC Central Cancer Registry linked with administrative medical and pharmacy claims data from the Public Employee Benefits Plan (private insurance) and Medicaid. The main outcome variable was diagnosis-to-surgery wait time. The exposures explored were patient race (White vs Black), age at diagnosis, marital status, county of residence (rural versus urban), year of diagnosis, estrogen and progesterone hormone receptor status (positive versus negative), tumor stage, tumor grade and enrollment in a government-funded breast and cervical cancer screening program: Best Chance Network (BCN). Chi-square tests and logistic regression analyses were conducted to compare patients who received early (<30 days after diagnosis) versus late surgery (>30 days after diagnosis) to identify significant predictors of late surgery. Two-way interactions were assessed between covariates hypothesized to modify the effect of race.

Results: Of 2137 total BrCa cases, 1633 received early surgery (1213 White, 77.9%; 422 Black, 70.6%). The crude odds of late receipt of surgery were 1.47 (95% CI: 1.19-1.82), and the adjusted odds were 1.32 (95% CI: 1.05-1.66) among Black compared to White patients. The adjusted odds of late receipt of surgery were 2.25 (95% CI: 1.52-3.31) among those who were BCN enrollees versus non-enrollees. Stratified analysis showed that among patients who lived in rural areas, the odds of late surgery were 2.02 (95% CI: 1.34-3.03) among Blacks compared to Whites, whereas among those who lived in urban areas, the odds of late surgery were 1.31 (95% CI: 1.01-1.69) for Blacks compared to Whites. Among patients who were diagnosed between 2002 and 2004, the odds of late surgery were 2.17 (95% CI: 1.40-3.36) among Blacks compared to Whites, whereas among those who were diagnosed between 2005 and 2007, the odds of late surgery were 1.72 (95% CI: 1.17-2.52) for Blacks compared to Whites.

Conclusions: Groups that are at higher risk of having late surgery are Blacks who live in rural areas and those on BCN. Despite the trend toward shorter intervals in Blacks vs Whites in the more recent data, all of these disparate groups should be targeted for more intensive navigation efforts.

#5273

Outcomes from a religiously tailored intervention to enhance mammography uptake among American Muslims.

Aasim I. Padela,1 Sana Malik,2 Shaheen Nageeb,1 Stephen Hall,1 Fatema Mirza,3 Monica Peek,1 Michael Quinn1. 1 _University of Chicago, Chicago, IL;_ 2 _Stony Brook University, Stony Brook, NY;_ 3 _Worry Free Community, Glen Ellyn, IL_.

Objective: Describe outcomes of a religiously-tailored peer-led group educational intervention addressing mammography-related barrier beliefs of American Muslims.

Methods: We used focus groups and interviews with a diverse group of Muslim women aged 40 and older to identify salient behavioral, normative and control beliefs regarding mammography. We used these data to design the curriculum and messaging for a mosque-based intervention involving peer-led group education. A novel model for religious tailoring was developed to address barrier beliefs by reframing, reprioritizing, and/or reforming belief structures. Peer educators were recruited/trained from mosques to match the ethnic composition of target population. The classes involved facilitated discussions and guest-led didactics covering religion and health, and mammography guidelines. Survey data was collected pre-, post, 6 months, and one-year post-intervention which captured changes in three primary outcome variables (1) perceived mammography intention, likelihood, confidence, (2) breast cancer knowledge and (3) receipt of mammograms. Resonance with barrier and facilitator beliefs was also measured.

Results: Fifty-eight Muslim women who had not had a mammogram in the past two years participated. Mean age was 50 years, with 29 being South Asian and 18 Arab. With respect to changed intention/likelihood/confidence pre-post there was a significant increase in mean perception of likelihood to obtain a mammogram (0.29, P = 0.01). Mammography knowledge also significantly increased following the intervention (p = 0.0002). Importantly, 22 participants received a mammogram at one-year follow-up. With respect to belief structures, a marginally significant increase was observed in level of agreement with mammography facilitator beliefs (p = 0.08) post-intervention. Although overall level of agreement with the outcome variable of mammography barrier beliefs did not change following the intervention (p = 0.94), there was a significant decrease in agreement with the specific belief in fatalism (p = 0.03). Multivariable models demonstrated that higher levels of agreement with fatalistic beliefs at baseline was significantly associated with a lower odds of increased mammography knowledge following the intervention (OR = 0.73), while higher religiosity at baseline was significantly associated with higher odds of increased mammography knowledge following the intervention (OR = 1.90).

Conclusion: Our mosque-based intervention involving religiously-tailored messages and peer-led classes demonstrated efficacy in behavioral change by improving Muslim women's self-reported likelihood of obtaining mammograms and having 38% of participants obtained a mammogram within a year.

#5274

Role of obesity and dietary chemopreventive nutrients on risk for breast cancer among ethnically diverse women.

Padma P. Tadi Uppala,1 Sozina Katuli,1 Alfredo Mejia,1 Sherine Brown-Fraser,1 Brian Yuen Yau Wong,1 Ryan Hayes,1 Maheswari Senthil,2 Carlos Garberoglio2. 1 _Andrews University, Berrien Springs, MI;_ 2 _Loma Linda University, Loma Linda, CA_.

Dietary factors have been inconsistently associated with breast cancer risk among various ethnicities. Diet however, may influence breast cancer through mechanisms of obesity. The purpose of this study was to compare intake of dietary chemopreventive nutrients and obesity across three racial/ethnic groups: Latina, African American and Asian women. Methods: Eighty subjects from Inland Empire, California were enrolled into the study from five different sites. Dietary intake was assessed using a validated food frequency questionnaire. The questionnaire includes 19 adjustment questions used to modify food-item responses in regard to fat, fiber, and sodium intake followed by 122 specific food item-lines that capture frequency and serving sizes for select foods. Women also completed a demographic and breast cancer survey. Chi-square tests were used for categorical variables, ANOVA, and Kruskal-Wallis one-way analysis of variance was used for nutrient variables. Results: The age of the study population ranged from 24 to 79. Subjects included 30 Latina, 35 African American and 15 Asian women. The mean body mass index for Latina, African American and Asian women was 26.8±4.2, 30.8±7.24, 24.41±3.85 respectively. Mean fat percent for the three races was 34.21±5.80 for Latina, 41.57±6.33 for African American and 35.5±8.74 for Asian. African American women were significantly (p=0.001) more obese compared to Latina and Asian women. Total breast cancer cases for Latina is 3, African American 6 and Asian 2. Family history of reported breast cancer among Latina, African American and Asian was 0, 11, 0 respectively. There was a significant difference among racially ethic groups in alcohol consumption (p=0.001), alpha carotene (p=0.005), beta-carotene (p=0.031), beta-Cryptoxanthin (p=0.022), Lycopene (p=0.000) MUFA (p=0.017), PUFA (p=0.000) animal protein (0.49) and total Vitamin A (0.044). Conclusion: Significant differences were found in obesity and dietary intake of chemopreventive nutrients among the three racial groups. African Americans had higher rates of obesity and lower consumption of total vegetable intake. This data is consistent with the general US population data in terms of obesity and risk for breast cancer. Further studies will include larger sample size to identify specific nutrients that may protect against breast cancer risk among diverse populations.

#5275

Comparing self-reported cognitive function among Caucasian and African American breast cancer survivors.

Kayla A. Lewis, Jacqueline B. Vo, Kendra J. Royston, Silvia Gisiger-Camata, Karen Meneses. _University of Alabama at Birmingham, Birmingham, AL_.

Approximately 35% of breast cancer survivors (BCS) report changes in cognitive function after cancer treatment. The purpose of this abstract is to compare self-reported cognitive changes between Caucasian (CAU) and African American (AA) BCS who participated in the Think Well program.

Think Well is an educational program to increase awareness of cognitive function and promote healthy living. Sixteen Think Well seminars were delivered in-person to urban and rural counties throughout North Central Alabama. Following each seminar, participants completed an evaluation of demographic information and cognitive changes. Descriptive statistics and chi-squared tests were used. Data were analyzed using SPSS v23.

Among BCS who completed the evaluation, 82 (54%) were CAU, and 62 (41%) were AA. Many BCS were employed (32%) or disabled/retired (51%). CAU were more likely to be married than AA BCS (p<0.01). More CAU self-reported "poor/below average" in domains of memory, ability to think, speed of problem solving, and ability to pay attention than AA BCS (p<0.01). CAU were more likely than AA BCS to report "moderately" or "very much/extremely" in changes in ability to think (p<0.01), speed of problem solving (p=0.02), and ability to pay attention (p=0.01) since completion of breast cancer treatment.

CAU BCS self-reported poorer cognitive function and changes since cancer treatment. Differences in self-reported changes between AA and CAU may be contributed to geographic location, racial differences in perception of cognitive function, or marital status. Race-related stigmas may affect self-reporting of cognitive function. Future research is warranted to examine factors that contribute to racial differences in cognitive function and interventions aimed at improving cognitive function among both AA and CAU BCS.

Acknowledgement: Think Well is supported by a grant from the North Central Alabama Affiliate of Susan G. Komen. Authors are also supported by funding: Susan G. Komen Graduate Traineeship in Disparities Research, Robert Wood Johnson Foundation Future of Nursing Scholarship, and American Cancer Society Doctoral Degree Scholarship in Cancer Nursing (DSCN-17-076-01).

#5277

Gender differences in awareness of lung cancer screening in the Health Information National Trends Survey, 2013-2017.

Erica Warner,1 Christopher S. Lathan2. 1 _Harvard Medical School, Boston, MA;_ 2 _Dana-Farber Cancer Institute, Boston, MA_.

Background: Lung cancer is the leading cause of cancer death in U.S. men and women with just a 17.7% five-year survival rate. Based largely on the results of the National Lung Screening Trial, the U.S. Preventive Services Task Force recommends, and Medicare now covers, low-dose computed tomography for current and former smokers age 55 or older with at least 30 pack-year smoking history. While lung cancer incidence mortality rates are higher in men than women, this is in large part due differences in smoking status. Previous research shows that women worry about breast cancer to a greater extent than lung cancer, and there is a suggestion of greater lung cancer susceptibility in female smokers. We investigated whether there were gender differences in discussions with a doctor about lung cancer screening and awareness of a screening test for lung cancer.

Methods: Data are from the three cycles [4.2 (2013); 4.4(2015) and 5.1 (2017)] of the nationally representative Health Information National Trends Survey (HINTS). We included 4,658 respondents age 55 to 80 who responded to the following question: "In the past year, have you talked with your doctor about having a test to check for lung cancer?" and had available data on smoking status and gender. We assessed responses across survey cycles to see if the proportion responding "yes" to this question has increased over time, and whether this differed by gender. We used logistic regression accounting for complex sample weighting to generate adjusted odds ratios (AOR) and 95% confidence intervals (CI) for gender's association with discussion of lung cancer screening with a doctor in HINTS cycles 4.2, 4.4 and 5.1, and with awareness of lung cancer screening tests in cycle 4.4, accounting for potential confounders.

Results: Reported discussions with a doctor about lung cancer screening have not increased over time. In the most recent cycle, just 15.7% of current smokers (19.9% of males; 15.7% of females) and 9.9% of former smokers (12.4% of males; 6.9% of females) said they'd discussed screening. Compared to males, females were 32% less likely to report talking to their doctor about lung cancer screening (AOR: 0.68, 95% CI: 0.50-0.93). Estimates were similar among never (AOR: 0.72, 95% CI: 0.43-1.20), current (AOR: 0.73, 95% CI: 0.39-1.36), and former (AOR: 0.66, 95% CI: 0.40-1.10) smokers. Females were also 32% less likely to report that they'd heard of tests for early detection of lung cancer (OR: 0.68, 95% CI: 0.47-0.99) compared to males.

Conclusions: Though we are unable to assess pack-years of smoking, we found that irrespective of smoking status (never, current or former), women were less likely to have talked with their doctor about lung cancer screening and were less likely to be aware of lung cancer screening tests. Educational efforts may be needed to ensure that women receive the benefits of this important test.

#5278

Tobacco and areca nut use among Chuukese youth in Guam.

Lilnabeth P. Somera,1 Brayan Simsiman,1 Grazyna Badowski,1 Hye-ryeon Lee,2 Kevin Cassel2. 1 _University of Guam, Mangilao, GU;_ 2 _University of Hawaii, Honolulu, HI_.

Background: Little is known regarding the tobacco use of Micronesian population in Guam. According to the Youth Risk Behavior Survey (YRBS) 2013, Micronesian youth have a higher prevalence rate of current smokers (27.2% vs. 15.7% for U.S.) and current smokeless tobacco users (39.7% vs. 8.8% for US) compared to the U.S. They also have the highest rate among all ethnic groups in Guam. This study aims to investigate tobacco use and areca nut use, which may explain high lung and bronchus cancer incidence and mortality rates among Micronesians. The survey in this study focuses on Chuukese youth, the largest group among Micronesians in Guam.

Methods: Respondent-driven sampling method (RDS) was used to recruit 364 Chuukese youth (ages 13-19) to complete the survey. YRBS question items that measured cigarette, cigar, and smokeless tobacco use were included in the survey. Measures for areca nut use were also included. Chi-square tests were performed by bivariate analysis. Only high school-level youth are included in the analysis.

Results: A total of 237 Chuukese high school students completed the survey. Fourteen percent of the participants currently smoke cigarettes, 6.4% currently smoke cigars, and 23.7% currently use smokeless tobacco. In addition, smokeless tobacco remains highest regarding daily use (11%) within the past 30 days prior the survey. Although there is high use of smokeless tobacco among participants, there are significant associations between gender and cigarette and gender and cigar use only. Boys smoke cigarettes and cigar at higher rates than girls. Among the recruited participants, 32% are areca nut users with tobacco product as an ingredient. There is significantly high association between smokeless tobacco users and areca nut with tobacco users. The majority of cigarette smokers (82.4%) have tried to quit smoking. Fifty-six percent of the smokeless tobacco users have tried to quit as well. However, only 17% of the participants are aware of the Guam Youth Quitline.

Conclusion: Results suggest the need to raise awareness among Chuukese youth on tobacco cessation programs on Guam, particularly the smoking cessation quitline. Tobacco cessation efforts should not focus on cigarette use only, but must also be expanded to include the use of smokeless tobacco products and chewing areca nut. Future studies should investigate the roles and implications of tobacco as an ingredient of areca nut chewing among Micronesian youth.

#5279

Prevalence and correlates of perceived harmfulness and addictiveness to traditional and alternative tobacco products among US adults.

Kahee A. Mohammed, Martin W. Schoen, Nosayaba Osazuwa-Peters, Ahmad M. Al-Taee, Gebran W. Khneizer, Lauren D. Arnold, Leslie Hinyard, Thomas E. Burroughs. _Saint Louis University, St. Louis, MO_.

Purpose: Beliefs about addictiveness and harmfulness of tobacco products are associated with intentions to use them. This study aimed to (1) evaluate perceived harmfulness and addictiveness patterns towards multiple tobacco products and (2) examine sociodemographic variables that influence those perceptions.

Methods: Weighted multivariate ordinal logistic regression analyses were performed on 5,474 US adults aged ≥18 years who participated in the 2015 and 2017 cycles of the Health Information National Trends Survey-FDA. The two primary outcomes were perceived harmfulness and addictiveness of traditional (cigarette, cigar, and pipe filled with tobacco) vs alternative tobacco (e-cigarette, hookah, and "roll your own" cigarettes). All models were adjusted for age, gender, race/ethnicity, education, household income, census region, and smoking status.

Results: Overall, 7.9% of adults in the U.S. perceived alternative tobacco as "not harmful at all" compared to 2.4% for the traditional products. Similarly, 16.2% of adults perceived alternative tobacco as "not addictive at all" compared to 13.6% for the traditional products. Beliefs about the harmfulness and addictiveness of traditional and alternative tobacco products differed by sociodemographic characteristics and smoking status: Those who believed traditional and alternative tobacco products are more harmful were more likely to be female, older (compared with those aged 18-34), Black (compared with White), former or never smokers (compared with current smokers), and adults living in West (compared with Northeast). Similarly, those who believed traditional and alternative tobacco products are more addictive were more likely to be female, older, Black, and adults living in West. However, no association between smoking status and perceived addictiveness was noted.

Conclusion: Adults in U.S. perceive tobacco harmfulness and addictiveness differently based on their sociodemographic characteristics and smoking status. These findings are potential areas for targeted behavioral interventions to increase individuals' perceived risk of harm and addiction to all forms of tobacco products in the United States.

#5280

Predicting initiation and sustenance to smoking cessation among smokers using multi-theory model (MTM) approach.

Falguni Patel,1 Richard W. Kim,1 Ghazal Ghafari,1 Bernhard Dietz,1 Vinayak K. Nahar,1 Manoj Sharma2. 1 _Lincoln Memorial University, Harrogate, TN;_ 2 _Jackson State University, Jackson, MS_.

Background and Hypothesis. The purpose of this study was to determine whether the initiation and sustenance of smoking cessation among smokers in the Appalachian region can be predicted using a newly proposed health behavior theory called the multi-theory model (MTM) of health behavior change. MTM may provide an innovative treatment plan for patients trying to quit smoking in order to improve cancer outcomes and decrease disparities in cancer outcomes in the Appalachian region. Smoking rates are 1.8 and 1.6 times greater in women and men, respectively, in Appalachian Kentucky than in the rest of the nation. MTM predicts health behavior change across cultures by proposing that health behavior change occurs in two stages: initiation of the behavior change and sustenance of the behavior change. This model has been validated for several other health behavior changes. The primary research question this study seeks to answer is: to what extent can MTM be used to predict quitting smoking and long-term sustenance of smoking cessation in current smokers? We hypothesized that MTM significantly predicts quitting smoking and long-term sustenance of smoking cessation in smokers. Research Methods. The study utilized a cross sectional design. IRB permission from the university was obtained to conduct the study. Investigators approached mall-goers at the Middlesboro Mall in Middlesboro, KY and asked current tobacco smokers to voluntarily participate in the study. In order to maintain anonymity, no identifiers or signatures were collected from the participants. Exclusion criteria included participants that were illiterate or who had poor vision. The survey instrument was a face and content valid 38 question assessment about the smokers' background and questions pertaining to the constructs of MTM. Data Analyses: Descriptive statistics were computed to summarize the data. In order to assess internal consistency reliability of the scales, Cronbach's alpha were computed. Confirmatory factor analyses were conducted on subscales to establish construct validity. Stepwise multiple regression analyses were performed to determine best possible predictors of initiation and sustenance of smoking cessation among people who were currently smoking. A p-value less than 0.05 was considered significant. Results: Data collection is complete and results are being complied and will be presented at the conference. Conclusion: This study aims to determine to what extent MTM can be utilized to explain quitting smoking and long-term sustenance of smoking cessation in smokers. In the interim, MTM appears to be a useful model for predicting health behavior change, and for designing interventions promoting smoking cessation in smoking patients.

#5281

An exercise intervention for pancreas cancer patients increases tumor vascularity.

Claudia Alvarez Florez, Nathan Parker, Matthew Katz, An Ngo-Huang, Carol Ferreira Cardoso, Huamin Wang, Maria Petzel, David Fogelman, Keri Schadler. _MD Anderson Cancer Center, Houston, TX_.

We and others have demonstrated in mice that moderate aerobic exercise remodels tumor vasculature and improves chemotherapy delivery. The sparse, dysfunctional vasculature in pancreatic ductal adenocarcinoma (PDAC) is one major barrier to delivering chemotherapy to the tumor. Improved vascular function and chemotherapy delivery could substantially improve patient survival over the current 7% rate. We first evaluated whether exercise might improve survival for patients with PDAC using a patient-derived xenograft (PDX) mouse model and moderate treadmill running. Treatment of PDX-bearing mice with gemcitabine or gemcitabine plus exercise caused tumor regression. However, gemcitabine was more effective when combined with exercise and excitingly, regrowth of tumors after treatment cessation was significantly delayed in mice treated with exercise combined with gemcitabine compared to gemcitabine alone. Our animal data suggests that exercise may be beneficial for patients with pancreatic cancer. However, it is unclear whether a sufficient level of exercise can be performed by patients with advanced cancers to achieve improved vascular function as predicted by animal studies. Recently, we completed a pilot study delivering a home-based aerobic and strength training exercise program (EP) to patients with potentially operable pancreas cancer. Fifty-eight patients (48% female, median age 66) completed the EP concurrent with chemotherapy or chemoradiation over a mean duration of 15 weeks prior to pancreatectomy. Participants completed a mean of 145.8 minutes of weekly moderate to vigorous physical activity. Surgical tumor specimens were obtained from 28 patients who participated in the EP, and were evaluated for the total number of vessels, vessel density, average number of open lumens, and number of elongated vessels. We found that tumors of patients who participated in the EP had significantly increased vessel density and a significantly increased number of elongated vessels compared to tumors from control patient, similar to our observations in mice. This is the first demonstration that an EP is feasible during the chemotherapy/ chemoradiation phases for pancreatic cancer patients, and importantly, that moderate exercise changes human tumor vascular biology. There was also a trend toward increased immune cell infiltration in tumors from patients who exercised. Cell death in tumors is being evaluated. This data suggests that exercise is a low-cost, low risk way to remodel human tumor vasculature and therefore may be an adjuvant to chemotherapy. Remodeled vasculature may increase chemotherapy delivery to the tumor, improving survival, as predicted in animal models. Further studies evaluating the impact of exercise on patient outcome as well as angiogenic biomarkers are underway.

#5282

Implementing a colorectal cancer screening program: A navigation led effort in an urban community health center.

Kathryn M. Glaser, Carly Nichols, Alyssa Abrams, Deborah O. Erwin, Mary Reid. _Roswell Park Cancer Institute, Buffalo, NY_.

Background: Colorectal cancer (CRC) screening is effective in preventing and detecting cancer at an early stage. Yet populations served by community health centers (CHCs) are screened at lower rates than the general population, and screening rates are even lower in non-English speaking populations. Time constraints during office visits often impede providers from discussing CRC screening, insufficient access to colonoscopy resources and facilities, and patients' fear regarding colonoscopy preparation and procedure are just some of the challenges CHCs face when promoting CRC screening. These issues are only compounded by significant language and cultural barriers.

Methods: We conducted a quality improvement initiative that serves as a case study to measure the increase in CRC screening rates over a one year period at a CHC with two clinic locations (intervention and non-intervention site) providing primary care to diverse and low-income populations. A pilot site was selected by the CHC to test an intervention using patient education and navigation, while the second site received no direct navigation intervention. The patient navigator assigned to the intervention site provided patient education and developed new patient education materials (visual), in addition to assistance in scheduling and coordinating services (transportation, interpreters, obtaining prep solution etc.) for the actual colonoscopy, as well as distributing fecal immunochemical testing (FIT) for those refusing or ineligible for a colonoscopy. Our rationale for the project, supported by preliminary data was that CHC providers want to increase screening rates, particularly in non-English speaking patients, but need tools and support to implement change.

Results: Between August 2016 and August 2017, the intervention site increased from 32% to 42% of eligible patients screened for CRC, with the most notable change in the non-English speaking patients (12% increase), primarily Burmese, Nepali and Spanish speaking, while there was only a 1% increase at the non-intervention site. Patient navigation efforts at the intervention site focused primarily on educating and navigating non-English speaking patients due to the increased barriers to understanding CRC and accessing preventative screening services. In total, 224 additional patients were screened for CRC during the intervention period with 86% of patients received screening colonoscopies over a FIT test.

Conclusions: Specialized and tailored education and navigation can be effective to increase CRC screening rates at CHCs, particularly in non-English speaking populations. Understanding how different populations think about CRC screening would inform navigation strategies on how to better promote screening in diverse populations and develop more targeted interventions.

#5283

Ethnomedicinal practices and herbalists' perception of inflammatory bowel diseases as risk factor for colorectal cancer among patients with anorectal or gastrointestinal disorders in northern Nigeria.

Mubarak L. Liman,1 Sunday E. Atawodi,2 Uju E. Iliemene,3 Abdulrazaq Yusuf,2 Abduljalal Danbaba1. 1 _Nuhu Bamalli Polytechnic, Zaria, Nigeria;_ 2 _Ahmadu Bello University, Zaria, Nigeria;_ 3 _Bingham University, Karu, Nassarawa, Nigeria_.

Traditional alternative medicine providers are widely patronized in many parts of Africa. Inflammatory bowel diseases (IBD), anorectal and gastrointestinal disorders (AGD) usually designated as piles could be signs of early stage polyposis and are significant risk factors in colorectal cancers. A survey report reveals that over 20% of adults in Northern Nigeria have piles or symptoms of IBD or AGD and majority of them consume herbal preparations because they believed that such disorders are better managed using traditional medicine. Herbalist as such are important first contact for this particular group with IBD and AGD in Nigeria. Existing literatures have established that cultural practices and beliefs contribute to late-stage diagnosis of cancers in Africa. The study evaluated the ethno-medicinal practices used by herbalist in the management of piles and their knowledge and perceptions about colorectal cancers. Survey information including knowledge on colorectal cancer, herbal remedies used, methods of preparation and administration, as well as incidence of anal or bowel inflammation or hemorrhage in consulted patients were collected using a self-administered questionnaire. A total of 145 herbalists selling "pile" remedies in northern Nigeria were sampled for the study. The results identified 75 plants used for preparation of pile remedies, many of which contain toxic alkaloids and saponins; with those in the family leguminosae leading followed by euphorbiaceae and rubiaceae. Literature searches on these plants indicate that 31% are relatively safe for consumption, 15% contained quantified toxic phytochemicals with established toxicity status while the rest (46%) contained toxic phytochemicals with no evaluated toxicity. The main methods of preparation are infusion and decoction while main means of administration is by ingestion through oral route; 15% of respondents however indicated usage of herbs as suppositories and anal washes. Almost all the respondents (96%) are conversant with the term "Cancer" , 44% have an idea of what colorectal cancers are, while only 19% have correct knowledge of the sign and symptoms of colorectal cancers or believed that inflammation and hemorrhages are potential early signs of polyposis. Knowledge of association between IBD or AGD and colorectal cancers among herbalist showed weak correlation (r2 = 0.33). Findings concluded that there is a poor knowledge on colorectal cancer among herbalist and a general perception that AGDs are not risk factors for colon cancers. The study recommends for increased cancer education with particular attention at enlightenment of herbalist and patients with IBD and AGD on benefits of clinical treatments and importance of screening for early detection of colorectal cancers and possible intervention.

#5284

Public awareness of colorectal cancer in the Gaza Strip: A comparative cross-sectional study between adults and high school students.

MohamedRaed Elshami,1 Tamer Abdalghafoor,2 Maha Alfaqawi,1 Ayoub AbuNemer,2 Mohammed Ghuneim,2 Hussien Lubbad,2 Batool Almahallawi,2 Mosab Samaan,2 Abdallah Alwali,2 Ahmad Alborno,2 Deyaa Al-kafarna,2 Aseel Salah,2 Karam Shihada,2 Mohammed Abo Amona,2 Amira Al-Najjar,2 Rana Abu Subha,2 Basma Alhelu,2 Israa Abujayyab,2 Loai Albarqouni,3 Bettina Bottcher2. 1 _Palestinian Ministry of Health, Gaza, Palestinian Territory;_ 2 _Islamic University of Gaza School of Medicine, Gaza, Palestinian Territory;_ 3 _Bond University, Robina, Australia_.

Introduction: Colorectal cancer (CRC) is a major cause of cancer deaths worldwide. Raising the awareness of CRC symptoms for early recognition, prevention via reduction of lifestyle risk factors, and removing barriers to seek medical help could lower its mortality. This study aimed to assess the level of public awareness of CRC in the Gaza Strip, and compare it between adults and high-school students (HSS).

Methods: This is a cross-sectional study conducted at 3 major hospitals and 10 high schools (recruitment used stratified sampling) in the Gaza Strip between September and October 2017. A previously pilot-tested, Arabic version of the Bowel Cancer/CRC Awareness Measure (CAM) questionnaire, a validated standardized questionnaire to measure public CRC awareness, was used. It consists of four sections: sociodemographic data, awareness of CRC symptoms, awareness of CRC risk factors, and barriers to seek medical help. Adults (aged ≥18 years) visiting or admitted to any of the three hospitals, and high-school students (aged 15-17 years) in any of the ten schools were recruited for face-to-face interviews to complete the CAM. Patients and visitors to oncology departments were ineligible. Informed consent and ethical approval were obtained.

Results: A total of 3080 participants completed the CAM questionnaire (of 3236 approached; response rate was 97.3% in HSS and 92.8% in adults). 1502 (48.8%) were HSS (730 males; 48.6%) and 1578 (51.2%) adults (736 males; 46.6%). Overall, 1391 (45.2%) reported that the chance of getting CRC is not related to age. A persistent abdominal pain was the most commonly recognized cancer symptom (n=1899, 61.7%) while pain in back passage was the least (n=1056, 34.3%). 2177 (70.7%) reported that they are not confident in recognizing CRC symptom/sign. Having a bowel disease (e.g, ulcerative colitis) was the most frequently recognized CRC risk factor (n= 1456, 47.3%) and having diabetes was the least (n=591, 19.2%). In general, adults have better awareness than HSS in terms of: recognizing CRC symptoms (mean score = 20.8 vs 18.7 of 24 total score, p< 0.0001) and its risk factors (mean score= 27.2 vs 25.4 of 45 total score, p< 0.0001). Worry about what a doctor might find was the most reported barrier to seek medical help among adults (n= 773, 49%), whereas, lack of confidence to talk about symptoms with the doctor was the most commonly reported one in HSS (n= 777, 51.7%).

Conclusions:The public awareness of CRC is suboptimal in the Gaza Strip. Interventions to improve the public awareness of CRC (e.g., educational outreach) is warranted. School curriculums should include awareness sessions on cancer symptoms and risk factors, including CRC.

#5285

Evaluation of a postpartum human papillomavirus vaccination program.

Abbey B. Berenson, Jacqueline M. Hirth, Yong-Fang Kuo, Richard E. Rupp. _University of Texas Medical Branch, Galveston, TX_.

Evaluation of a postpartum human papillomavirus vaccination program

Berenson AB, Hirth JM, Kuo YF, Rupp RE

Purpose: To evaluate the effectiveness of a large vaccination program which offers the human papillomavirus (HPV) vaccine to women during their postpartum hospitalization.

Methods: Based on the success of a pilot which offered the HPV vaccine to a select group of women, a postpartum HPV vaccination program was expanded in 2016 to include all eligible mothers who gave birth in a large Texas hospital serving low income women. Women from 23 different counties were included. With funding from the Cancer Prevention Research Institute of Texas (CPRIT), patient navigators were hired to identify and personally counsel all eligible women who met catch-up recommendation guidelines prior to their hospital discharge. Women were informed about the need and availability of the HPV vaccine during their hospitalization and offered it free of charge. Follow-up doses are coordinated with postpartum visits as well as well-baby visits in collaboration with the Department of Pediatrics. With Institutional Review Board approval, records of women who were approached during the first 11 months of the expanded program were reviewed to determine the number who chose to participate.

Results: Baseline HPV vaccination rates were low. A total of 1,782 postpartum women were counseled about the HPV vaccine of which 168 (9.4%) were already vaccinated and 5 others were ineligible. Of the remaining 1,609 women, 71.2% received a dose of the HPV vaccine during their hospitalization. The refusal rate among eligible women was 24%. Another 4.3% (n-76) agreed to get vaccinated, but were released from the hospital before the injection was given. Of these, 48 (63.2%) received the dose at a later clinic visit.

Conclusions: Offering the HPV vaccine as part of postpartum care can increase catch-up vaccination of young women who did not have the opportunity to be vaccinated at a younger age.

#5286

Meharry Medical College medical student summer program in integrative science and cancer research.

Dana R. Marshall, Carol Freund-Taylor, Philip Lammers, Leon Dent, Samuel Adunyah, Billy Ballard. _Meharry Medical College, Nashville, TN_.

The American Cancer Society estimates that 1,688,780 new cases of cancer will be diagnosed in 2017 and that 600,920 people will die from their disease. The burden of cancer is disproportionately borne by the poor and underserved. Underrepresented minority physician scientists and clinician researchers are uniquely qualified to address these disparities as they have frequently experienced them in their own families and communities. As some portion of health disparities is rooted in socioeconomic status and lesser education, economically deprived and first generation college students of all races and ethnicities also experience these disparities. Unfortunately, the number of physicians who do research has been declining so the need for programs that educate students in the art and science of research is increasing. The Meharry Medical College Summer Program in Integrative Science and Cancer Research (MMC-SPiISCR) has provided Meharry medical students with the opportunity to participate in short-term cancer research experiences for eleven years. The program is unique in combining weekly half-day workshops at MMC, an historically black college or university, with a research experience supported by faculty mentors at the Vanderbilt University School of Medicine (VUSM),an institution that includes the Vanderbilt Ingram Cancer Center, one of only 69 NCI-Designated Cancer Centers in the United States and District of Columbia. The workshops include topics emphasizing bioethics and responsible conduct of research as well as presentations by Meharry faculty on their cancer research, cancer disparities, cancer and big data and more. The overarching goal of this program is to inspire Meharry medical students to aspire to medical careers in academic medicine and/or careers that will ultimately include cancer research. To date, 73 MMC students have started and completed the program and 94% are from underrepresented minority groups. The male to female ratio reflects the male to female ratio of the class. Over half of the participants presented their work at meetings outside of the program including AACR National Meeting, AACR Science of Cancer Disparities, Society of Black Academic Surgeons, Student National Medical Association, American Medical Association Research Symposium, American Society for Clinical Oncology, KBRIN Bioinformatics Summit and the Meharry-Vanderbilt-TSU Cancer Partnership Annual Retreat. The students are authors on 27 manuscripts and these numbers are still growing as is the interest of program participants in doing a year of research. Surveys of both mentors and program participant mentees overwhelmingly support the strength of this program and participation in the future. This comprehensive program will go far towards fueling the physician-scientist pipeline with researchers whose life experiences mirror those of minority and underserved patients.

#5287

Challenges and opportunities using mobile technology for data collection in biomedical research: An observational discovery science investigation with Spanish-speaking Latinas.

Kimlin T. Ashing, Alejandro Fernandez, Mayra Serrano, Marisela Garcia, Katty Nerio. _City of Hope, Duarte, CA_.

Mobile devices including phones offer significant contributions to improve reach and convenience of research participation and data collection. Technological advancements have enabled smartphone devices to track health behaviors such as physical activity and provide convenient feedback as well as survey data. High usage of mobile devices i.e., smartphones among both US born and immigrant Latinos may not similarly translate to mobile technology utility for research participation among Latinos and other ethnic minority populations. Among Latinos, smartphones are primarily used for communication as only 50% use the data application capabilities. The acceptability and application of mobile data harnessing tools can present both opportunities and challenges for engaging this population in research. We report our observations of the challenges Latinas face using mobile technology for data collection. Latina mothers were recruited from school to participate in a behavioral nutrition and physical activity study for cancer and chronic disease reduction. A total of 38 enrolled and 32 participated in the intervention. 63% were Spanish speakers, 59% were foreign-born, 66% had ≤high school education, 91% reported income <$40,000. Participants used a mobile device to perform two tasks: 1) complete a REDCap online survey on a tablet and 2) download and install a fitness tracker mobile application. Latinas rated their confidence in their ability to use mobile technology using a 4- point Likert scale. Participants with little/no confidence requested paper versions of the survey. 88% required individualized research staff assistance to complete the survey and set-up their fitness tracker. Monolingual Spanish-speakers were more likely to report little/no confidence connecting to Wi-Fi (χ2=13.175, p<0.01), downloading an app (χ2=11.277, p=0.01), creating an electronic account (χ2=7.882, p<0.05). Our findings suggest that lower-income, Spanish-speakers require more assistance and ongoing guidance with technology applications and devices for research data collection. In fact, we found that about 20% shared the device (Fitbit) with family members, especially children to facilitate the participants' use of the device. However, this threatened data accuracy. Therefore, studies using mobile technology should account for the added staffing and resources required to conduct studies with this population. Addressing this barrier may require a pre-study session to train research participants on the study's technology applications. Further research is needed to evaluate and improve the applicability of mobile technology for increasing reach and participation, as well as validation of data accuracy for medically underserved and understudied populations.

#5288

MPNST treatment and diagnosis in NF1: A health economic model.

Lydia Turkson,1 Hannah Mamuszka,2 Kyla Grimshaw,1 Erica Marie Marshall3. 1 _Locus Genetics LLC, Cambridge, MA;_ 2 _Alva10Dx, Cambridge, MA;_ 3 _Independent Consultant, Jacksonville, FL_.

Biomarkers have many applications along the care process in rare cancers and have potential to optimize treatment decisions. However, many diagnostic tests struggle to gain market access and appropriate value in terms of payer coverage because of a lack of evidence on their health economic benefit to patients. CMS reimbursement guidelines, of cost-plus pricing for diagnostics does not always work for niche small rare disease subpopulations. Nor does it reflect the lifelong value of effective patient care.

To address this, we have developed a cost benefit model for early diagnostic intervention, based on retrospective data to determine mutation status and monitor for disease progression in people diagnosed with neurofibromatosis type 1 (NF1) and soft tissue tumors. NF1 is an autosomal dominant hereditary cancer syndrome that affects around 1:2,500 people and is caused by mutations in the NF1 gene. 50% of patients can have few to many neurofibroma which vary in clinical behavior from benign to malignant. About 10% of people can develop malignant peripheral nerve sheath tumors (MPNSTs) thought to develop from pre-existing benign neurofibroma. Currently, there is no reliable way of knowing which individual and which tumors are malignant. MPNSTs are often difficult to diagnose in the early phases with current imaging methods like CT or MRI. Overall, these patients face a lifetime risk of cancer up to 16-26% higher than the normal population. Once metastasizes MPNSTs are associated with poor prognosis, less than 25% survival rate at 5 years.

We built a model used retrospective anonymized patient treatment records to demonstrate cost and risk based benefits to patients, payers and clinical practitioners, on the effect of early intervention versus late on treatment cost, and patient outcome in the long-term care process. For NF1 patients. In conclusion, our model proposes a new framework to engage healthcare insurers and managed care physician groups in the US to quantify the health economic benefits of prognostic biomarker tests in patient populations with a lifelong genetic predisposition to cancer.

#5289

Implementing cancer intervention programs in rural settings: A review.

Antoinette Percy-Laurry, David A. Chambers. _National Cancer Institute, Rockville, MD_.

INTRODUCTION While a decrease in overall cancer rates is found during the period, 1999-2013, geographic disparities still persist. Understanding the relationship between rurality and access to evidence-based interventions remain significant. This review investigates cancer control interventions developed to deploy in a rural environment and their common implementation strategies and approaches. METHOD We searched the National Cancer Institute's (NCI) Research-Tested Intervention Programs (RTIPs) database to identify interventions in rural settings. The interventions were reviewed for program focus, population, cultural relevance, physician influence, coordination of services, and implementation strategies. We compared the review in RTIPs to systematic reviews on breast cancer programs and obesity-related programs found in PubMed using keyword searches. RESULTS Of the 185 intervention programs provided by the RTIPs database, 36 were recommended for rural settings. Of the 36 programs, 12 were conducted in rural populations only. Cancer topics and related areas varied with most of the 12 interventions on nutrition/obesity-related (4) and breast cancer screening (3). In PubMed, we searched for systematic reviews on diet/nutrition and breast cancer screening in the US rural settings and found two systematic reviews specific to breast cancer screening interventions and one systematic review on lifestyle interventions focused on diet/nutrition and physical activity. Of the two breast cancer systematic reviews, one was focused on supportive care and the other review included only 5 interventions of a larger review of breast cancer studies. The lifestyle systematic review summarized 18 interventions, most of which were tailored to target a specific racial/ethnic group. Implementation strategies used were mainly education and building capacity in meal preparation and physical activity. The breast cancer review provided no detail on the implementation strategies and approaches of the 5 interventions since they were reviewed for differences in mammography uptake in rural vs urban. Only 5 RTIPs rural only interventions were found to target the underserved and an additional 2 interventions focused on minority women population. DISCUSSION More studies and interventions are needed on rural populations to contribute to the understanding and reduction of the high rates of cancer. Early detection is vital in advancing cancer efforts and NCI's RTIPs would benefit from having new interventions focused on messaging and physician guidance and knowledge to allow for effective comprehensive cancer control programs. The reviews also show there is a need for research on strategies and approaches used in rural settings. Furthermore, there needs to be a comprehensive evaluation on the effectiveness of breast cancer intervention programs and diet/nutrition-related programs in rural communities.

#5290

My crown or my health: Black women's knowledge of breast cancer and hair product-related risk.

Dede Teteh,1 Phyllis Clark,2 Lenna Dawkins-Moultin,1 Rick Kittles,1 Susanne Montgomery3. 1 _City of Hope, Duarte, CA;_ 2 _Healthy Heritage Movement, Riverside, CA;_ 3 _Loma Linda University, Loma Linda, CA_.

Introduction/Purpose: Black women now have higher breast cancer (BC) incidence and mortality than White women. They also use more hair products containing hormonally-active chemicals. Studies are beginning to link chemical ingredients in these products to BC risk. Little is known about community knowledge of BC and the potential association with hair product-related risk. The purpose of this study was to evaluate the hazard level of hair products used by Black women, and assess their knowledge about BC and hair product-related risks.

Methodology: We employed principles of community-based participatory research and conducted the study through partnership between community advocates and university scientists. We collected hair products from locations frequented by Black women and hair stylists and used the Environmental Working Group's Skindeep© database to evaluate ingredient toxicity. We then explored knowledge of BC and hair product-related risk using a 41-item survey. Survey data were analyzed using SPSS.

Results: All 54 products evaluated contained hazardous ingredients. Fourteen of the ingredients had an overall high hazard rating between 7-10, with 10 indicating very high levels of toxicity. The potential harmful effects of these chemicals include cancer, endocrine disruption and reproductive system toxicant. Survey data showed most women (62%) in the sample had a college or graduate degree. Participants (n=211) were more knowledgeable about the diagnosis and treatment of BC (46.3%), lifestyle-related risk factors (59.6%), and genetic risk factors (54.9%) than about hair product risk (40.8%).

Conclusion: Several hair products frequently used by black women contained ingredients that have potential links to BC. Though the women in this study were highly educated, they had limited knowledge about the association between BC and toxins found in hair products. Research should further clarify the link between hair products and BC risk. As knowledge is evolving, given our emerging risk understanding, health educators and community advocates can promote efforts for women to learn more about exposure reduction to help them make informed decisions about the risk beauty product chemicals may hold regarding cancer. There is also a need for policies on personal care products that aim to protect the health of our communities.

#5291

Professional nurses' awareness and practice with sexual concerns of the oncology patient.

Maurel C. Emeagi, Hester Rina De Swardt, Martjie De Villiers. _Tshwane University of Technology, Pretoria, South Africa_.

Sexuality is an important aspect of quality of life (QOL). QOL after cancer therapy is an important aspect of patient care of which sexual wellbeing forms part of the holistic nursing care although sexuality assessment is recognized as an important part of nursing care, in practice it has been observed that professional nurses may experience some difficulties to this aspect.

Purpose: The purpose of this study was to gain a deeper understanding of the awareness, practice and possible barriers of professional nurses when engaging in conversation about sexuality with patients receiving cancer treatment in a private oncology care setting in Pretoria, South Africa.

Objectives: The research objectives were: Ø To explore and describe the awareness and practice of professional nurses to engage in conversation about sexuality with the patient receiving cancer treatment in a private oncology setting in Pretoria. Ø To describe possible barriers that prohibit professional nurses to engage in conversation with patients receiving cancer treatment in a private oncology setting regarding sexuality Ø To make recommendations to improve nursing practice with regard to nursing care related to sexuality in the private oncology care setting.

Method: An exploratory, descriptive and qualitative design was followed. A convenient purposive sample consisting of nine participants who met the inclusion criteria participated. Data were gathered through semi-structured interviews, while Creswell's method of data analysis was applied. Trustworthy measures included credibility, transferability, dependability and confirmability. This study obtained ethical clearance from all stakeholders.

Results: The themes embarrassment, cultural beliefs and values emerged as barriers to engage in conversation about sexuality while, referring to a specialist in the field as suggested support to cancer treated patients arose. Limited knowledge about intensive therapy featured as the last theme. The data revealed that professional nurses themselves had restricted views on sexuality and very often would see the patient's body image and associated sexuality as not their responsibility. The concept intensive therapy was not fully understood which led to limited referral to various specialist.

Conclusions: Professional nurses should be sensitized and empowered with the skills and knowledge on how patients' perception of their body image changed when been treated with cancer and the effect thereof. Reflective conversations about this sensitive topic could assist in this regard.

#5292

Visceral adiposity-stimulated genotoxicity and malignant transformation of epithelial cells.

Debrup Chakraborty, Vladislav Jdanov, Vanessa Benham, Blair Bullard, Jamie J. Bernard. _Michigan State Univ., East Lansing, MI_.

Obesity is a global epidemic with a predicted rate of 42% in the U.S.A by 2050. Epidemiologic studies show that obesity is a risk factor for developing cancer; however, the molecular mechanism has not been fully elucidated. Our published data demonstrate that fibroblast growth factor-2 (FGF2) released from fat cells (adipocytes) in the visceral adipose tissue (VAT) induces transformation/tumorigenicity in the skin and mammary epithelial cells. Specifically, FGF2 released from VAT stimulates epithelial cell growth in soft agar by inducing the proto-oncogene c-Myc. Growth in soft agar is a measure of transformation/tumorigenicity; neither transformation nor c-Myc induction in epithelial cells was reversible. c-Myc overexpression can initiate a process of genetic instability linked to tumor initiation. Our discovery of this novel direct path of VAT-stimulated tumorigenesis adds mechanistic insight to our earlier discovery that VAT secretions promote UVR-induced nonmelamoma skin cancer. The objective of our current study was to determine the mechanism by which FGF2 stimulates malignant transformation. We hypothesized that FGF2 from VAT induces c-Myc and subsequent genomic instability in epithelial cells, leading to increased carcinogenesis. To test hypothesis we generated a filtered conditioned-medium from the human VAT treated MCF-10A (mammary epithelial) and JB6 P+ (skin epithelial) cells and measured several downstream mediators of FGF2 and activation of FGFR-1 (FGF2 receptor). Following VAT treatment, epithelial cells demonstrated induced c-Myc protein expression along with ROS accumulation, elevated γ-H2AX foci, and increased micronucleus (MN) formation. We found that inhibition of c-Myc attenuated VAT-induced neoplastic transformation of MCF-10A and JB6 P+ cells, while constitutive activation of the c-Myc-induced spontaneous neoplastic transformation of JB6 P+ cells. Collectively, our data suggested FGF2 released from VAT interacts with FGFR-1 and activates c-Myc. The role of c-Myc in the formation of MN and DNA damage is under investigation. Determining the impact of excess VAT on cancer will lead to strategies to help prevent adiposity-associated cancers and identify individuals at risk for disease or individuals who may be susceptible to compounded genotoxicity due to DNA-damaging environmental exposures.

## BIOINFORMATICS AND SYSTEMS BIOLOGY:

### New Algorithms

#5293

A method to identify somatic mutations from tumor samples in the absence of matched normal tissue.

Lijing Yao, Preeti Lal, Li-Tai Fang, John Lee, John Palma, Andre Rosenthal, Bernd Hinzmann, Alex Lovejoy, Hugo Y. K. Lam. _Roche Sequencing Solutions, Belmont, CA_.

Tumor-specific molecular profiling by next-generation sequencing (NGS) from tissue or blood (liquid biopsy) can have a tremendous potential to guide personalized healthcare for cancer treatments. However, based on the current clinical practices1, often tumor or blood may be sequenced without the matched germline normal tissue or buffy coat. Since the tumor cell content of the specimens can vary widely, as well as the fact that sequencing will identify both germline and somatic variations, confident identification of somatic mutations becomes very challenging in the absence of a matched normal. Identification of somatic variations in cell-free DNA becomes even more difficult due especially to the low yields of tumor-derived DNA in plasma samples.

An ensemble tree-based machine learning method was developed to classify tumor-specific somatic mutations in the absence of matched normal (CSMutan). This algorithm leverages multiple public databases and variant allele frequencies as features to classify somatic mutations and germline variants. Using The Cancer Genome Atlas (TCGA) and in-house datasets, models were built for each of the three different types of tumor specimens: i.e., fresh frozen, formalin-fixed, paraffin-embedded (FFPE), and plasma samples.

The performance of the models was evaluated using cross-validation and/or testing on independent sets of samples. Lung adenocarcinoma samples from TCGA were used to build the fresh frozen model, which achieved an area under the ROC curve (AUC) of 0.9968 on an independent data set of lung squamous cell carcinoma samples, also from TCGA. In-house sample sets were used to build the FFPE model and the plasma model. The FFPE model achieved an ROC AUC of 0.993, while the model trained on plasma samples was able to classify with an AUC of 0.997 based on ten-fold cross-validation.

Although sequencing matched normal tissue is the most preferred and accurate method for identifying high-confidence somatic mutations, this work demonstrates that it is possible to identify somatic mutations in tissue or blood with high accuracy when the matched normal samples may not be available. The future directions for this method are to add additional, and more diverse, cancer samples for both the training and independent validation so that potentially higher accuracy and generalizability can be obtained.

1. Meyerson, M., Gabriel, S. & Getz, G. Advances in understanding cancer genomes through second-generation sequencing. Nature Reviews Genetics 11, 685-696 (2010)

#5294

CIMerge: A machine learning approach for merging and genotyping complex indel calls from NGS data.

Tian Zheng, Yang Li, Yu Geng, Zhongmeng Zhao, Xuanping Zhang, Jiayin Wang. _Xi'an Jiaotong University, Xi'an, China_.

Complex insertion and deletion (complex indel) is a rare category of genomic structural variations, which is formed by inserting one or multiple DNA fragments into the genomic location where a deletion occurs. A recent study conducts a systematic analysis from over 8,000 pan-cancer cases, which reports hundreds of complex indels in cancer-associated genes, some of which are considered potentially druggable. Several approaches are proposed to detect complex indels from the new-generation sequencing data (both 2nd and 3rd generation). However, as different data-mining algorithms vary the preferences on capturing data patterns, different approaches may report the conflicted complex indel calls. Here, we propose a machine learning approach to correct the conflicted calls from different approaches and further estimate the genotype of each call. The proposed approach, implemented as CIMerge, adopts a relevance vector machine framework. For each candidate call, CIMerge first extracts a set of features on the candidate region, which includes the read depth, variant allelic frequency, number of the splitting/unmapped reads, number of the discordant paired-end reads, aligned contig, etc. CIMerge also considers another set of features on data-mining algorithm(s) that reported the candidate call, which includes the parameter settings, etc. Both sets of features are trained by the relevance vector machine framework, which outputs the probability of each candidate of the conflicted call. As a byproduct, it outputs the genotype of the candidate call with highest likelihood. We tested CIMerge on multiple datasets generated by different simulation configurations and compared it to several state-of-the-art approaches. The experiment results demonstrate that CIMerge outperforms the existing approaches. The average success rate of recognition is approaching 90%, while Pindel and Gindel are reported as 62.53% and 65.202%, respectively. The software package CIMerge is freely available for academic uses at https://github.com/xjtu712-lab/CIMerge.

#5295

Toward high throughput immune infiltrate analysis from H&E stained images.

Rishi R. Rawat, Daniel Ruderman, David B. Agus. _USC Keck School of Medicine, Los Angeles, CA_.

Our appreciation of the immune system in breast cancer is rapidly evolving. New methods to quantify the composition and spatial distribution of the immune infiltrate from pathology images are urgently needed to develop better strategies to activate the anti-tumor immune response. Existing methods to quantify the immune infiltrate are tedious (manual counting) or rely on immunohistochemistry (IHC) staining to identify immune cells. While significant efforts are underway to develop machine learning tools to categorize cells based on morphologic features in hematoxylin and eosin (H&E) stained histopathology images, all approaches are limited by the scarcity of large, annotated ground truth training sets. In this work, we propose a new approach, leveraging "style-transfer" algorithms from the computer vision community to generate large quantities of training data from IHC stains. Using style transfer, we generate synthetic H&E images from IHC stains for immune markers, such as CD45, CD8 and CD4. The generated images have pixel-perfect ground truth provided by IHC, and do not need manual labeling. We investigate the impact of training on increasing numbers of synthesized images and validate our algorithms on an independent test set annotated by a pathologist (n=100 patients, 3064 identified lymphocytes). Our baseline classifier achieves a 83% accuracy, compared to the pathologist ground truth. This work provides a novel, tractable, and efficient way to train data-hungry algorithms to identify multiple cell types from H&E stained images. This work is supported by a grant from the Breast Cancer Research Foundation (BCRF-17-002)

#5296

R2D2: An integrated analysis framework to infer the functional impact of single nucleotide variants (SNVs) using matched germline and tumor DNA and RNA sequencing data.

Alma Imamovic, Saud H. AlDubayan, Nathanael Moore, Celine G. Han, Brendan Reardon, Eliezer M. Van Allen. _Dana-Farber Cancer Inst., Boston, MA_.

Introduction: Single nucleotide variants (SNVs) are the most abundant genetic variation in the human genome. Unlike protein-truncating alterations, the functional impact of SNVs can be very difficult to infer from DNA sequencing alone. Although functional techniques such as RNA-Seq have been used to quantify protein expression in normal and tumor samples, up to date, there has not been a robust tool to systematically evaluate the transcriptional perturbations caused by SNVs in the germline and somatic spaces. Here, we describe R2D2, a computational algorithm that jointly analyzes matched germline and tumor DNA and RNA sequencing data to infer the functional impact of coding SNVs.

Methods: R2D2 was developed to evaluate the allelic fraction of called SNVs across matched normal and tumor genetic and transcriptomic data to classify these variants into predefined categories of potential functional and biological significance. Ideally, four sets of matched DNA and RNA variants from both normal and tumor tissues would be available to classify both germline and somatic SNVs. However, R2D2 is capable of analyzing various combinations of DNA and RNA sequencing data to infer the functional impact of detected SNVs. R2D2 has also been optimized to work with various commonly used variant calling and annotation tools such as the Genome Analysis Tool Kit (GATK) and Oncotator.

Results: We used R2D2 to evaluate the functional impact of germline and somatic SNVs of matched germline and tumor whole exome and transcriptome sequencing data of five patients with primary lung adenocarcinoma. On average, standard variant calling algorithms detected 34,227 (range: 33541-35698) SNVs across all sample types of each patient in our cohort. These variants were just classified as germline and somatic by these pipelines. However, surveying the allelic fraction (AF) of each variant across all sample types using R2D2 detected unexpected patterns of AF deviation, such as imbalanced allelic expression, somatic biallelic inactivation, and aberrant RNA splicing. For example, R2D2 identified somatic loss of heterozygosity of an average of 186 (range: 70-409) SNVs per patient, which can be important when studying potential mechanisms of tumorigenesis. In addition, 13.2% (range: 9.0-17.3) of all detected SNVs had an imbalanced RNA expression where one allele was more selectively expressed in the normal or tumor tissues. Lastly, we also identified a total of 1454 (range: 1349-1546) SNVs per patient that were only detected in the normal or tumor RNA-Seq data.

Conclusion: We developed R2D2 to help pinpointing functional genetic SNVs that are of potential clinical significance. R2D2 is an easy-to-implement and flexible module that can be integrated with various variant-calling pipelines to identify unexpected patterns of variant expression.

#5297

LCA: A robust and scalable algorithm to reveal subtle diversity in large-scale single-cell RNA-Seq data.

Changde Cheng,1 John Easton,1 Celeste Rosencrance,1 Yan Li,2 Bensheng Ju,1 Wenan Chen,1 Xiang Chen1. 1 _St. Jude Children's Research Hospital, Memphis, TN;_ 2 _University of Minnesota Twin Cities, Minneapolis, MN_.

Single-cell RNA sequencing (scRNA-seq) emerges as a powerful tool to characterize cell-to-cell variation and dynamics in a seemingly homogenous population. Efficient and affordable, scRNA-seq is gaining in popularity in both basic and translational biological research areas. However, significant challenges arise in the analysis of scRNA-seq data, including low signal-to-noise ratio with high data sparsity, rising scalability hurdles with hundreds of thousands of cells, and more. Due to inherent complexities in scRNA-seq data, the performance of currently available algorithms may not always be optimal even for fundamental tasks such as identifying heterogeneous subpopulations in the data. In this study, we developed Latent Cellular Analysis (LCA), a machine learning based analytical pipeline that combines similarity measurement by latent cellular states with a graph-based clustering algorithm. LCA features a dual-space model search for both the optimal number of subpopulations and the informative cellular states distinguishing them. LCA provides heuristic solutions for population number inference, dimension reduction, feature selection and confounding factor removal without explicit gene filtering. LCA has proved to be robust, accurate and powerful by comparison to multiple state-of-the-art computational methods on large-scale real and simulated scRNA-seq data. Importantly, LCA's ability to learn from representative subsets of the data provides scalability, thereby addressing a significant challenge for growing sample size in scRNA-seq data analysis.

#5298

The accurate noninvasive staging of liver fibrosis using deep learning radiomics: A prospective multicenter study.

Hui Zhou, Kun Wang, Jie Tian. _Institute of Automation Chinese Academy of Sciences, BeiJing, China_.

Purpose: We developed the deep learning Radiomics of elastography (DLRE) as a noninvasive method to assess liver fibrosis stages,which is essential for prognosis, surveillance of chronic hepatitis B (CHB) patients.

Methods and Materials: 402 patients were prospectively enrolled from 12 hospitals, and finally 2010 images were included into analysis randomly.DLRE adopted the CNN method, one of the deep learning radiomic techniques, for the automatic analysis of 2D-SWE images. This study was conducted to assess the accuracy of DLRE in comparison with 2D-SWE, transient elastography (TE), transaminase-to-platelet ratio index (APRI), and fibrosis index based on the four factors (FIB-4), by using liver biopsy as the gold standard. Analysis of receiver operating characteristic (ROC) curve were performed to calculate optimal area under it (AUC) for cirrhosis (F4), advanced fibrosis (≥F3), and significance fibrosis (≥F2) for all diagnostic approaches.

Results: AUCs of DLRE were both 0.98 for cirrhosis (95% confidence interval [CI]: 0.95-0.99) and advanced fibrosis (95% CI: 0.94-0.99), which were significantly better than other methods, as well as 0.76 (95% CI: 0.72-0.81) for significance fibrosis (significantly better than APRI and FIB-4).

Conclusion: DLRE shows the best overall performance in predicting liver fibrosis stages comparing with 2D-SWE, TE, and serological examinations. It is valuable and practical for popularized application of noninvasive diagnosis of liver fibrosis stages in HBV infected patients.

#5299

Machine learning approach to personalized medicine in breast cancer patients: Development of data-driven, personalized, causal modeling through identification and understanding of optimal treatments for predicting better disease outcomes.

Henry Kaplan,1 Anna Berry,1 Kristine Rinn,1 Erin Ellis,1 George Birchfield,1 Tanya Wahl,1 Xiaoyu Liu,1 Mariko Tameishi,1 J D. Beatty,1 Patricia Dawson,1 Vivek Mehta,1 Anna Holman,1 Mary Atwood,1 Shlece Alexander,1 Candy Bonham,1 Lauren Summers,1 Iya Khalil,2 Boris Hayete,2 Diane Wuest,2 Wei Zheng,2 Yuhang Liu,2 Xulong Wang,2 Thomas David Brown1. 1 _Swedish Cancer Institute, Seattle, WA;_ 2 _GNS Healthcare, Cambridge, MA_.

Background: In the era of personalized medicine, a major challenge is harnessing longitudinal data across the cancer care continuum, which includes multimodal data sets of biologic, molecular, and clinical information about patients (pts) and their tumors. There is a growing need for new computing analytics, such as machine learning--an important tool in healthcare bio-informatics. We report our approach to building cancer disease models in an unbiased manner through utilization of a causal machine learning and simulation platform.

Methods: The Swedish Cancer Institute (SCI) Personalized Medicine Research Program (PMRP) is a prospective registration protocol with the objective of establishing a centralized longitudinal, molecular, and phenotypic data repository. Since 2014, over 1,030 pts have been enrolled, having undergone next-generation sequencing (NGS) profiling of their tumors. Of these pts, we identified 100 breast cancer pts who also have detailed longitudinal clinical annotation within our SCI Breast Cancer Registry. All de-identified data, variables, and data points in the multimodal data types are integrated into normalized data frames to include demographics, cancer risks, tumor specifications, tumor sequencing, initial and subsequent cancer treatments, and outcomes data. A reverse engineering approach, via the Reverse Engineering and Forward Simulation (REFS) platform, is being utilized, focusing on discovering the complex causal mechanisms that determine which therapies will produce the best outcomes for an individual pt. This method goes beyond traditional approaches that rely on data correlations to match treatments to pts. The breast cancer causal model uncovers many of the possible combinations of causal relationships that drive outcomes and enables "what if?" simulations of a variety of interventions, across pts, to determine optimal therapies. Performance metrics and model robustness will be explored using a stratified, n-fold (e.g., 10-fold) cross-validation procedure, which is designed to provide an unbiased estimate of model generalization to new observations.

Results: The causal model and simulations can elevate the providers' abilities to better understand treatment responses based on pts' unique clinical data and mutational statuses; study different treatment options to optimize management; and understand the complex interactions among variables that lead to a range of treatment outcomes.

Conclusions: Knowledge generated from the simulations of the disease model can potentially streamline and support the clinical decision-making process, to include molecular tumor board deliberations, and ultimately assist providers in arriving at optimal treatment recommendations for pts.

#5300

smCounter2, a highly accurate UMI-aware variant caller and its applications in detecting 0.5-1% somatic mutations using targeted panel sequencing.

Chang Xu, Xiujing Gu, Zhong Wu, John DiCarlo, Yexun Wang. _Qiagen Sciences, Frederick, MD_.

We introduce smCounter2, our new version of low-frequency variant caller for targeted panel sequencing data. Upgraded from the previous version of smCounter (Xu et al, BMC Genomics, 2017), smCounter2 has a higher accuracy of detecting 0.5-1% variants and a more solid and scalable theoretical foundation. For 1% SNVs, smCounter2 achieved above 98% sensitivity and fewer than 10 false positives per megabase on a 680kb custom panel using a mixture of Genome in a Bottle reference samples. For 0.5% SNVs (including multi-allelic variants), smCounter2 achieved 87.5% sensitivity (7 out of 8) using a mixture of Horizon Tru-Q samples. We have also tested smCounter2 on other datasets with 5% and germline variants. On all these datasets except the germline variants, smCounter2 has demonstrated superior performance compared with other state-of-the-art UMI-aware variant calling algorithms. Currently, we are generating data on another custom panel (~900kb) to demonstrate the sensitivity and specificity of calling 0.5% variants.

smCounter2 models DNA polymerase errors for each substitution type, therefore more accurately distinguishes real variants from the first-cycle PCR errors, which cannot be corrected by UMIs. smCounter2 is equipped with model-based filters that can reduce false calls in repetitive regions such as homopolymers and micro-satellite sequences, where error rate is generally higher and UMIs tend to be less effective. These features, to some extent, overcome the intrinsic limitations of UMI-based variant calling and are essential to smCounter2's improved performance.

#5301

SubHap: An efficient algorithm for reconstructing clonal haplotypes of tumor sample from NGS data.

Rong Zhang, Yu Geng, Jianye Liu, Zhongmeng Zhao, Xuanping Zhang, Jiayin Wang. _Xi'an Jiaotong University, Xi'an, China_.

Clonal architecture is one of the important characteristics of tumor heterogeneity and tumor microenvironment. It often embodies the selective advantage along with the evolution and inheritance of subclones. Identifying and inferring subclonal heterogeneity is suggested, which may facilitate the comprehensive understanding of tumor progression and the interactions among microenvironment. Several state-of-the-art approaches are designed to estimate clonal architecture from the paired tumor-normal sequencing data. However, the existing approaches often suffer an accuracy loss when the loci with allelic imbalance interfere in the distribution of read depth. Some methods attempt to overcome this weakness by reconstructing the clonal genotypes, but still have difficulties to efficiently reach the haplotype resolution, where the latter one is considered to have greater values on both research and clinical implications. Here we propose a novel approach, implemented as SubHap, to reconstruct the clonal haplotypes of tumor sample. The input of the approach is two sets of mapped reads, which are sequenced from the tumor and normal samples, respectively. The outputs include the number of subclones and the possible haplotypes of each subclone. The proposed approach establishes a probabilistic model. It first clusters the somatic mutations according to the variant allelic frequencies, which is roughly proportional to the prior distribution of the proportion of each subclone. Then, an improved maximum spanning tree algorithm is designed. For any mutation site, this algorithm extracts the reads covered the site and iteratively strips the reads from each subclone, guided by both the prior distribution and the local read depth. Each group of the peeled reads is used to assemble the corresponding clonal haplotypes. During the assembly process, SubHap calculates and corrects the posterior distribution of the proportion of each subclone based on an inverse convolution algorithm, which solves the conflicts across some sites. We conduct a series of simulation experiments to test the performance of SubHap under different configurations. The given data include both the 2nd- and 3rd-generation sequencing data. Compared to some popular approaches, e.g., HapCompass, SubHap significantly improves the accuracy varying the coverages, numbers of preset subclones and proportions, etc. When the coverage is greater than 50X, the proposed algorithm achieves 85% on accuracy. Moreover, it requires less computational resources than the existing approaches. The software package SubHap is freely available for academic uses at https://github.com/xjtu712-lab/SubHap.

#5302

Phenotypic heterogeneity of patient-derived tumor cells visualized by unsupervised analysis in cell-based personalized drug testing.

Riku Turkki,1 Lassi Paavolainen,1 Piia Mikkonen,1 Päivi Östling,2 Peter Horvath,1 Vilja Pietiäinen,1 Olli Kallioniemi2. 1 _Institute for Molecular Medicine Finland, Helsinki, Finland;_ 2 _Science for Life Laboratory, Stockholm, Sweden_.

Patient-derived tumor cells are highly variable in their morphology and molecular phenotypes and after exposure to a library of drugs their heterogeneity further increases as the cells respond to the perturbations. The complete phenotypic diversity of the tumor cells and drug response patterns may remain undetectable if only few simple readouts, such as individual biomarkers in pre-defined tumor cells, are considered. Therefore, novel techniques that capture and visualize the entire spectrum of heterogeneous phenotypic changes in ex-vivo patient-derived tumor cells are needed.

Here, we applied unsupervised machine learning for comprehensive visualization of cell phenotypes with the aim to improve the analysis of image-based drug screening results. We performed high content screening of patient-derived renal clear cell carcinoma cells, exposed to 36 drugs in nine concentrations applied in 384-well format. The nuclei were stained with Hoechst and cell proliferation using Ki67, and images were acquired with a high-content imaging microscope (PE Operetta, 20x). To quantitatively describe the cell phenotypes, we used CellProfiler for cell segmentation and feature extraction and then applied a large-scale data embedding method (LargeVis) to cluster the cells based on their phenotypes. Using the clustering, we created an image-based phenotype-map to describe the phenotypic landscape. Overlaying the drug treatments on different concentrations on top of the map allowed for visualization of unique phenotypic fingerprints for each drug at the single cell resolution.

Visual inspection of the phenotype-map displayed visually similar cells clustering together. Likewise, a comparison with an expert trained supervised classification indicated consistency with the phenotype-map. As expected, Ki67-positive and -negative cells as well as dying cells formed clusters. Interestingly, the phenotype-map identified sub-clusters within the Ki67-positive and -negative cells. Furthermore, the map also allowed us to adjust for classification errors occurring in the supervised classification. In conclusion, we find that phenotypic signatures provide an intuitive way of linking and comparing phenotypes of distinct populations of drug-treated cells at a single-cell level. This will reveal additional systematic information of the drug responses, complementing traditional readout, such as cell viability, by highlighting the phenotypic changes over concentrations.

#5303

Identification of myelofibrosis from electronic health records with novel algorithms and JAKextractor.

Cosmin A. Bejan, Andrew Sochacki, Shilin Zhao, Yaomin Xu, Michael Savona. _Vanderbilt University, Nashville, TN_.

Myelofibrosis (MF) is a devastating myeloproliferative neoplasm (MPN) hallmarked by marrow fibrosis, extramedullary hematopoiesis, vascular thromboembolism, and ~50% incidence of JAK2V617F. MF is difficult to study in large EHR datasets due to clinical heterogeneity and unreliable ICD coding. The Synthetic Derivative is a cloned and de-identified research EHR with 2.9 million unique patients linked to BioVU, a DNA biorepository. To develop phenotype-genotype associations, we created an algorithm to classify MF, using NLP with negation detection of MF keywords, medications, and ICD coding. To enrich our cohort, we developed JAKextractor, an algorithm to identify patients tested clinically for JAK2V617F across all 248,000 BioVU patients.

For MF identification, we trained a supervised learning algorithm to learn decision rules that encode counts of MF-specific ICD codes, medications, text mentions, as well as the assertion status of MF and JAK2 mentions in patient notes. Experiments were evaluated using a 10-fold cross validation scheme. JAKextractor used pattern matching to extract the status (WT vs MUT) of each JAK2 text mention. Machine learning predicted a JAK2V617F patient based on the information extracted in the previous step from patient notes. We subsequently genotyped banked DNA on an enriched subset of MF cases via a Illumina® TruSight myeloid NGS panel to validate JAKextractor.

The top performing MF algorithm combined all sources of clinical information and achieved an F1-measure (F1) of 96% and identified 309 MF patients in BioVU. The extracted decision rule for predicting an MF patient was [JAK2V617F ^ ICD>1] v [JAK2WT ^ ICD>1 ^ TXT>3]. ICD is necessary but not sufficient to predict MF identification. Utilizing only ICD counts created a detrimentally lower F1 of 88% (P<0.001). Our MF cohort had a mean age at onset (60.3±12.6), last visit age (63.1±12.1), and JAK2V617F (46.1%). The mean age of MF onset was higher with JAK2V617F (64) compared to JAK2WT (57) (P<0.001). Survival was no different between JAK2V617F and JAK2WT MF cases via log-rank test (P=0.11) with median survival 108 months. 131 MF cases were genotyped with JAK2V617F in 71/131 (54.2%) compared to 66/131 (50.4%) via JAKextractor. Mean JAK2V617F allelic frequency was 0.569 with detection ranging 0.069-0.976. Ten cases displayed disagreement between JAKextractor and NGS. There were 2 FP and 4 FN JAKextractor predictions 6/131(4.6%); 2 true NGS failures, 1 incomplete chart and 1 loss of JAK2V617F over time. NGS detected JAKV617F on MF patients who had not been previously tested 7/131 (5.3%).

Our results demonstrated successful identification of MF and JAK2V617F within an EHR. We established the feasibility of creating a MPN database with retrospective genotyping of biobanked DNA. We plan for scaled implementation of similar algorithms across all myeloid disease within BioVU with the ability to retrospectively genotype each case.

#5305

DNA methylation and gene expression in melanoma: A large-scale integrated analysis.

Saverio Candido, Giuseppe A. Parasiliti Palumbo, Giulia Russo, Luca Falzone, Giulia C. Leonardi, Francesco Pappalardo, Massimo Libra. _University of Catania, Catania, Italy_.

Dysregulation of DNA methyltransferase activity, through global hypomethylation and localized hypermethylation phenomena, has been associated with cancer development. Even though the role of promoter methylation in gene regulation has been widely described, the regulatory function of methylation status of intergenic and intragenic regions has been not yet clarified. High-throughput technology and bioinformatics analysis represent useful tools to investigate the functional role of global methylation patterns in gene regulation. Therefore, a specific bionformatic tool was generated to identify methylation hotspots as well as extended genomic methylation regions in order to analyze specific genetic methylation patterns associated with cancer gene deregulation. In the present study, melanoma was selected as tumor model for its aggressive phenotype. Melanoma samples were obtained from the Cancer Genome Atlas (TCGA) to analyze DNA methylation and gene expression profiling data. We developed an algorithm, using R language, that performs cyclic correlation analysis between gene expression and methylation levels of all genes.

Our results show that in melanoma there are different methylation patterns that positively and negatively correlate with gene expression. In particular, the hypomethylation of the promoter (TSS1500, TSS200, 5'UTR) leads to the over-expression of selected gene (p<0.01) while the hypermethylation of gene body and 3'UTR regions were associated with gene over-expression. Furthermore, the frequency of genes showing a global hypomethylation status was higher compared to those that were hypermethylated (70% vs 30%). We have also identified the main individual CG probesets and methylated regions that are differentially correlated with gene expression. A further pathway analysis shows that the identified genes are implicated in central cellular processes associated with melanoma. Our algorithm allows the identification of methylation hotspots involved in the regulation of key genes for melanoma development. This may contribute to the development of new therapeutic strategies targeting methylome alterations in melanoma cells. Moreover, the identification of methylation hotspots associated to cancer initiation and progression can represent useful biomarkers for patients' risk stratification.

#5306

SAUCIE: Sparse autoencoder for unsupervised clustering, imputation, and embedding.

Matthew Amodio, Krishnan Srinivasan, David van Dijk, Hussein Mohsen, Kristina Yim, Rebecca Muhle, Kevin Moon, Ruth Montgomery, James Noonan, Guy Wolf, Smita Krishnaswamy. _Yale University, New Haven, CT_.

Handling the vast amounts of single-cell RNA-sequencing and CyTOF data, now being generated in patient cohorts, presents a computational challenge due to the noise, complexity, sparsity and batch effects present. Here, we propose a unified deep neural network approach to automatically process and extract structure from these massive datasets. Our unsupervised architecture, called SAUCIE (Sparse Autoencoder for Unsupervised Clustering, Imputation, and Embedding), simultaneously performs several key tasks for single-cell data analysis including 1) clustering, 2) batch correction, 3) visualization, and 4) denoising/imputation. SAUCIE is trained to recreate its own input after reducing its dimensionality in a 2-D embedding layer which can be used to visualize the data. Additionally, it uses two novel regularizations: (1) an information dimension regularization to penalize entropy as computed on normalized activation values of the layer, and thereby encourage binary-like encodings that are amenable to clustering and (2) a Maximal Mean Discrepancy penalty to correct batch effects. Thus SAUCIE has a single architecture that denoises, batch-corrects, visualizes and clusters data using a unified representation. We show results on artificial data where ground truth is known, as well as mass cytometry data from patients undergoing immunotherapy, dengue-infected patients, and single-cell RNA-sequencing data from embryonic mouse brain.

#5307

Inferring parsimonious migration histories for metastatic cancers.

Mohammed El-Kebir,1 Gryte Satas,2 Benjamin J. Raphael1. 1 _Princeton University, Princeton, NJ;_ 2 _Brown University, Providence, RI_.

Recent studies have attempted to infer the pattern of cellular migrations between a primary tumor and distant metastases using phylogenetic trees constructed from somatic mutations. Several of these studies have challenged the conventional view of monoclonal seeding of a metastasis, and reported complex patterns of migration between primary tumors and metastases, including polyclonal seeding and reseeding.

Most current analyses that attempt to infer migration patterns from somatic mutations rely on two key assumptions, sample homogeneity and mutation-migration concordance, that do not typically hold for cancer sequencing datasets. Consequently, standard phylogenetic analysis of mutations may result in incorrect or misleading patterns of metastasis.

We introduce a computational model to evaluate migration patterns in a rigorous manner. We use this model as a basis for Metastatic And Clonal History INtegrative Analysis (MACHINA), an algorithm that jointly infers parsimonious clone trees and migration histories of metastatic cancers from DNA sequencing data. We show that MACHINA accurately recovers clone trees and migration histories on simulated data. We apply MACHINA to sequencing data from metastatic ovarian, breast, prostate and skin cancer samples. In several cases, we find simpler migration histories than previously reported, and these alter conclusions regarding metastasis-to-metastasis spread, the anatomical site of the primary tumor, or the occurrence of polyclonal seeding and reseeding in individual patients. MACHINA enables researchers to rigorously assess the validity of different migration patterns in individual patients with metastatic disease and evaluate the prevalence of different migration patterns across large cohorts of patients and tumor types.

#5308

Using RNA expression data for splicing variant assessment and confirmation.

Jim Lund, Shannon Bailey, Sharvari Gujja, Jeffrey Gulcher. _WuXi NextCODE, Cambridge, MA_.

Nine percent of disease-causing mutations classified in HGMD have the potential to impact RNA splicing. Splicing variants called from DNA sequence data, especially variants outside the canonical splice site, are difficult to evaluate clinically as they often have no effect on protein expression. A method to utilize RNA-Seq data for splice site variant classification was developed. RNA reads aligned to the region of a splice site variant are examined to determine if RNA read through is present which indicates normal splicing at the site is disrupted.

Thousands of paired tumor and normal TCGA DNA and RNA samples were used to evaluate the algorithm and rule out alternate splicing in the tissue as explanation for RNA read through. Splicing variants can also alter exon usage and RNA isoform expression, and this can be identified by examining RNA isoform expression. An analysis of splicing variants in the TCGA project datasets has generated sets of confirmed functional and non-disruptive splicing varaints. This splicing dataset can be used to test and refine splicing defect prediction algorithms.

#5309

Determining EGFR and STK11 mutational status in lung adenocarcinoma histopathology images using deep learning.

Nicolas Coudray,1 Andre L. Moreira,1 Theodore Sakellaropoulos,2 David Fenyö,1 Narges Razavian,1 Aristotelis Tsirigos1. 1 _New York University, New York, NY;_ 2 _National Technical University of Athens, Athens, Greece_.

To characterize a tumor and suggest appropriate treatments, pathologists rely on the visual assessment of histopathology slides as well as molecular profiling of the tumors. This is especially true in lung adenocarcinoma cases where targeted therapies exist for tumors with EGFR mutations. In this work, we studied the use of deep learning approaches to classify lung cancer histopathology images and predict the mutational status of frequently mutated genes.

To achieve these tasks, more than 1600 scanned histopathology slides, obtained from TCGA, were separated into independent training, validation and test sets. They were then tiled into 512x512 pixel windows, and the training set was used to feed the inception v3 convolutional neural network developed by Google. The resulting model was evaluated on the test set, and the generated per-tile probabilities were aggregated to generate a per-slide classification score. We based our study on whole-slide images of adenocarcinoma, squamous cells carcinoma and normal lung tissues and the neural network was trained on several tasks: first, the goal was to identify normal from tumor regions, and then, once the tumor regions were identified, it was trained to identify adenocarcinoma versus squamous cell carcinoma. Finally, the neural network was trained predict the mutational status of the most frequently mutated genes in lung adenocarcinoma.

After verifying that the trained network properly identifies tumor slides compared to normal tissue (AUC=0.99) and distinguishes adenocarcinomas from squamous cell carcinomas (AUC=0.95), the network was trained to simultaneously identify different mutations. It achieved AUC scores of 0.82 and 0.86 for STK11 and EGFR respectively, showing that mutations in these two genes may confer specific macroscopic features, visible in histopathology images, and that a deep convolutional neural network can be trained to recognize those features.

Our results demonstrate that, given the availability of sufficient data, convolutional neural networks learn to perform complex diagnoses and thus assist pathologists in their work.

#5311

Integrated approaches for design of precision cancer immunotherapies: Selection of Class I and Class II T cell neo-epitopes and removal of Treg epitopes.

Guilhem Richard,1 Lenny Moise,1 Matthew Ardito,1 Frances Terry,1 Gad Berdugo,2 William Martin,1 Anne De Groot1. 1 _EpiVax Inc, Providence, RI;_ 2 _EpiVax Oncology, Inc., Providence, RI_.

Next-generation sequencing has opened the door to precision cancer therapies targeting mutations expressed by tumor cells. However, most neo-epitopes selected by traditional T cell epitope prediction algorithms prove to be non-immunogenic. Poor predictive performance may partially be due to inclusion of mutated epitopes cross-conserved with self-epitopes recognized by the T cell receptor of regulatory (Treg), anergic or deleted T cells. Vaccination with self-epitopes can lead to weak effector responses, active immune suppression, and toxicity due to immune-mediated adverse effects. We have developed Ancer, an advanced cancer T cell epitope identification and characterization tool, that streamlines the selection of Class I and Class II T cell neo-epitopes. Ancer leverages EpiMatrix and JanusMatrix, state-of-the-art predictive algorithms that have been extensively validated in prospective vaccine studies for infectious diseases [Moise et al., Hum. Vaccines Immunother 2015; Wada et al., Sci. Rep. 2017]. Distinctive features of Ancer are its ability to accurately predict Class II HLA ligands with EpiMatrix and its 82% positive predictive value, as estimated in previous prospective studies. Additionally, the application of JanusMatrix allows for the prioritization of neo-epitopes with reduced potential for Treg induction, that is responsible for diminished efficacy of current cancer therapies. We validated Ancer's predictive accuracy using datasets of HLA-bound peptides detected by mass spectrometry, which are independent of training sequence data used in model development. Analysis of sequences from Abelin et al., Immunity 2017 shows a 96% agreement between Ancer predictions and peptides eluted from common Class I HLAs, while only 86% of these sequences are accurately predicted by NetMHC or NetMHCpan. An additional retrospective analysis of a cancer immunogenicity study [Strønen et al., Science 2016] demonstrates that Ancer selects immunogenic neo-epitopes with 72% accuracy, as compared to 21% accuracy when using public prediction tools. These results demonstrate that Ancer may focus epitope candidate selection on higher value sequences than conventional algorithms. Class I and Class II neo-epitopes with low Treg activation potential may then be used to support the development of safer and more effective vaccines. 

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

### DNA Methylation

#5312

**The effect of** Dnmt1 **overexpression on intestinal tumorigenesis.**

Nicole A. Vander Schaaf,1 JinA Park,1 Oluwasei Dina,2 KwangHo Lee,1 Peter W. Laird1. 1 _Van Andel Research Institute, Grand Rapids, MI;_ 2 _University of Southern California, Los Angeles, CA_.

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in the United States. Strong genetic drivers of tumorigenesis, such as loss of function of the APC tumor-suppressor gene, have been well characterized, but the role of epigenetic alterations remains poorly understood. Upregulation of DNA methyltransferases (DNMTs), including DNMT1, has been reported in various human cancers, but its functional significance to cancer is unclear. Here we report a novel mouse model of inducible Dnmt1 upregulation, bypassing the embryonic lethality of Dnmt1 overexpression. We accomplish inducible control of Dnmt1 expression by targeting the tet transcriptional activator to the endogenous Dnmt1 promoter. We combine this model with the ApcMin allele to investigate the effect of Dnmt1 upregulation on intestinal tumorigenesis. Our preliminary data suggests that Dnmt1 overexpression increases the size and multiplicity of intestinal polyps in ApcMin/+ mice. Previous work in our lab showed that DNA methylation is critical for tumor formation, but it is not known which DNA methylation alterations have functional relevance. To investigate this, we will use the Dnmt1 overexpression model to identify DNA methylation events that are responsible for the observed phenotype in ApcMin/+ mice. In summary, we describe a novel mouse model of Dnmt1 overexpression and provide preliminary evidence to suggest that such upregulation exacerbates intestinal tumorigenesis. Our investigation may yield new insights into the role of epigenetics in CRC initiation and progression and into potential epigenetic therapies.

#5313

Occupational exposure to trichloroethylene and DNA methylation: a cross-sectional study.

Jinming Zhang,1 Luoping Zhang,2 Roel Vermeulen,3 Wei Hu,1 Bryan A. Bassig,1 Jason Y. Wong,1 Min Shen,1 Bu-Tian Ji,1 Boris Reiss,3 Mark Purdue,1 Martyn T. Smith,2 Qing Lan,1 Nathaniel Rothman1. 1 _National Cancer Insitute, Rockville, MD;_ 2 _UC Berkeley School of Public Health, Berkeley, CA;_ 3 _University Utrecht, UTRECHT, Netherlands_.

Trichloroethylene (TCE) is a chlorinated solvent that is often used in occupational settings for vapor degreasing of metal parts. In 2012, TCE was classified as a known human carcinogen (Group 1) by the International Agency for Research on Cancer, largely based on evidence for kidney cancer. Animal studies have suggested that TCE may modulate DNA methylation. We performed a cross-sectional study to examine the associations of DNA methylation of individual CpG sites with TCE exposure. We enrolled 80 workers from factories where TCE was used, as well as 96 unexposed controls from food and clothes manufacturing factories in the same region, and 69 exposed workers and 77 controls were available for analyses in this study. Personal exposure measurements (2-3 per subject) were collected for a full work shift using a 3MTM badge in both exposed workers and a subgroup of the control workers. Blood samples were collected from each subject and CpG methylation was assayed on the Illumina Infinium HumanMethylation450 BeadChip. After quality controls procedures, 485,512 CpG probes were available for analyses. We used separate multivariable linear regression models to examine the associations of TCE exposure and the methylation level at each single CpG site. All models were adjusted for age, sex, current smoking status, current alcohol consumption, recent infection, and body mass index. The median TCE exposure level in exposed subjects was 12 ppm (10th percentile: 2 ppm; 90th percentile: 45 ppm). We found 29 CpG probes that achieved genome-wide significance (p < 1.04x10-7) when comparing TCE exposed workers with controls. TCE exposure was associated with hypomethylation at nearly all (28/29) of those CpG probes. Of these 29 CpG probes, 6 CpGs showed a significant (p < 1.04x10-7) exposure-response relationship across the categories of controls and lower- (<12ppm) and higher-exposed (≥12 ppm) workers, and were mapped to 6 different chromosomes. To our knowledge, this is the first report that TCE exposure is associated with alterations in DNA methylation in a molecular epidemiologic study. Our findings may provide insight into potential biological mechanisms linking exposure to TCE and adverse health outcomes.

#5314

DNA methylation quantitative trait loci and breast cancer risk: Data from nearly 230,000 women of European descent.

Yaohua Yang,1 Qiuyin Cai,1 Xiang Shu,1 Lang Wu,1 Bingshan Li,2 Xingyi Guo,1 Kyriaki Michailidou,3 Manjeet K. Bolla,3 Qin Wang,3 Joe Dennis,3 Jacques Simard,4 Douglas F. Easton,3 Wei Zheng,1 Jirong Long,1 Breast Cancer Association Consortium (BCAC). 1 _Vanderbilt University Medical Center, Nashville, TN;_ 2 _Vanderbilt University, Nashville, TN;_ 3 _University of Cambridge, Cambridge, United Kingdom;_ 4 _Laval University, Québec City, Quebec, Canada_.

DNA methylation plays a critical role in the initiation and promotion of breast cancer. Previous studies have identified multiple promising methylation markers associated with breast cancer risk. However, these studies had very small sample sizes and potential confounding factors were not taken into consideration. In the present study, we used genetic variants as instruments to investigate DNA methylation in relation to breast cancer.

We extracted beta coefficients and standard errors for methylation quantitative trait loci of 30,477 CpG sites and applied them into the genetic data of 122,977 breast cancer cases and 105,974 controls of European descent included in the Breast Cancer Association Consortium (BCAC). Associations of genetically predicted methylation levels with breast cancer risk were evaluated using an inverse-variance weighted method. For CpG sites showing a significant association with breast cancer, we estimated the correlation between methylation with gene expression using data from the Framingham Heart Study. For genes with expression levels correlated to methylation, statistical models were built to predict gene expression levels via genetic variants using data from the Genotype-Tissue Expression project. These models were then applied to the BCAC to assess the association of predicted gene expression and breast cancer risk.

Of the 30,477 inspected CpG sites, significant associations with breast cancer risk were observed for 199 CpG sites at P < 1.64×10-6, a Bonferroni-corrected threshold. Among them, 22 CpG sites were located in genomic regions not yet reported as risk loci for breast cancer. Among the remaining 177 CpG sites located in breast cancer risk loci, significant associations were observed for 27 CpG sites after the breast cancer SNPs were adjusted. Among the 199 CpG sites, correlation was observed at 29 CpG sites with expression levels of 17 neighbor or enhancer target genes at P < 0.05. Among them, genetically predicted expression levels of 13 genes were significantly associated with breast cancer risk at a false discovery rate < 0.05, 11 of which showed consistent association directions with the associations between methylation and gene expression, and between gene expression and breast cancer. For example, the higher methylation level at cg00577578 was associated with an increased breast cancer risk (P = 1.31×10-7) and a

decreased expression level of GBAP1 (P = 5.07×10-15), and meanwhile, a lower expression level of GBAP1 was associated with increased breast cancer risk (P = 2.22×10-9).

Using genetic variants as instruments, we identified 199 CpG sites significantly associated with breast cancer risk. Our study demonstrates the potential of integrating genomics, DNA methylation and gene expression in identifying new biomarkers for breast cancer, and provides new insights into the etiology of this malignancy.

#5315

**Frequent downregulation of** SALL3 **by recurrent genetic and epigenetic alterations is involved in triple-negative breast cancers.**

Yosuke Matsushita,1 Masato Komatsu,1 Kazuma Kiyotani,1 Tetsuro Yoshimaru,1 Hiromu Suzuki,2 Yasuo Miyoshi,3 Mitsunori Sasa,4 Toyomasa Katagiri1. 1 _Tokushima University, Tokushima, Japan;_ 2 _Sapporo Medical University, Sapporo, Japan;_ 3 _Hyogo College of Medicine, Nishinomiya, Japan;_ 4 _Tokushima Breast Care Clinic, Tokushima, Japan_.

Triple negative breast cancers (TNBC), defined by the lack of estrogen and progesterone receptors and HER2 gene amplification, is a heterogeneous and clinically aggressive disease due to the lack of beneficial therapeutic targets. Even though one of the most major risk for the development of TNBC is carrying a deleterious germline and somatic mutations of BRCA1 are not frequently found in sporadic cases of TNBC. Therefore, we aimed to search genomic alterations in TNBC cases by whole-exome sequencing analysis of genomic DNAs from 36 Japanese patients with TNBC compared with their corresponding normal. We identified 36 genes that were recurrently mutated (>10% of cases) in TNBC cases, including TP53 and PIK3CA as described in the previous next generation sequencing analysis of TNBC cases. Remarkably, we identified a number of epigenetic-related genes involved in histone modification and DNA methylation, which were mutated in 20 out of 36 (55.6%) cases. Among them, we focused on spalt like transcription factor 3 (SALL3) gene which shows recurrent somatic mutations, and are most frequently downregulated in TNBC cases. Nonsense-mutation of SALL3 (E169X) completely abolish its binding to DNAMT3A. Ectopic overexpression of the wildtype SALL3, but not the somatically mutated SALL3 into BT549 breast cancer cells, which expresses low level of SALL3 gene, caused the significant suppression of cell growth. Importantly, SALL3 gene was frequently hypermethylated and transcriptionally silenced in only TNBC cases, but not in other types of breast cancer using TCGA data sets. Moreover, the expression of SALL3 was restored after treatments of 5-aza-2'-deoxycitidine (5-aza-dC) in TNBC cell line, HCC1937. Notably, siRNA-mediated knockdown of SALL3 expression in BT20 cells enhanced chemoresistance against paclitaxel and docetaxel, respectively. Moreover, low expression of SALL3 was associated with significantly shorter relapse free survival. Our findings provide the evidence of a pathophysiological role for SALL3 as a tumor suppressor which is possibly associated with carcinogenesis for TNBC.

#5316

DNA methylation index of lifetime estrogen exposure in breast cancer.

Annelie Johansson,1 Domenico Palli,2 Giovanna Masala,2 Sara Grioni,3 Claudia Agnoli,3 Rosario Tumino,4 Maria Concetta Giurdanella,4 Francesca Fasanelli,5 Carlotta Sacerdote,5 Salvatore Panico,6 Amalia Mattiello,6 Anthony Swerdlow,7 Minouk Schoemaker,7 Michael Jones,7 Nick Orr,7 Olivia Fletcher,7 Nichola Johnson,7 Katarzyna Tomczyk,7 Paolo Vineis,1 James M. Flanagan1. 1 _Imperial College London, London, United Kingdom;_ 2 _Cancer Research and Prevention Institute - ISPO, Florence, Italy;_ 3 _Istituto Nazionale dei Tumori, Milano, Italy;_ 4 _Azienda Sanitaria Provinciale di Ragusa, Ragusa, Italy;_ 5 _Città della Salute e della Scienza University-Hospital and Center for Cancer Prevention, Turin, Italy;_ 6 _University of Naples Federico II, Naples, Italy;_ 7 _Institute of Cancer Research, London, United Kingdom_.

Breast cancer is the most common female cancer worldwide and makes up 25% of all cancers diagnosed in women. There is a need for improved risk assessment methods to identify women at high risk before the disease develops. It is well established that estrogens are involved in estrogen receptor positive (ER+) breast cancer carcinogenesis, however the mechanisms are not fully understood. We hypothesize that lifetime estrogen exposure accumulates changes in DNA methylation detectable in blood as a surrogate for other tissues. Here, we have identified a methylation signature of estrogen exposure measured in blood DNA that could improve risk assessment for breast cancer.

Our Estimated Lifetime Estrogen Exposure (ELEE) model, taking into account reproductive time (age at menopause minus age at menarche), number of pregnancies and breastfeeding duration, shows a 5% increased risk of developing breast cancer per unit increase in ELEE (range 18 – 44) in the European Prospective into Cancer and Nutrition (EPIC)-Italy study cohort (30671 healthy controls, 1193 cases, age at diagnosis ≥ 50). An Epigenome-Wide Association Study (EWAS) of ELEE was conducted on 450K methylation data in EPIC-Italy (199 controls, 97 cases) and the Generations Study (GS) cohort (n=65 controls). For validation targeted bisulfite sequencing using the Fluidigm 48.48 Access Array was performed on independent DNA samples from the GS cohort (440 matched ER+ case-control pairs). The Methylation Index (MI) of ELEE was developed on 450K data using ridge regression and includes DNA methylation levels at selected CpG sites.

DNA methylation levels at 694 CpG sites show significant (false discovery rate q < 0.05) association with ELEE in the EWAS and of these 42 CpG sites from the top-probes were selected for validation. The MI was developed on 28 CpG sites passing quality control and shows high correlation with ELEE in 450K training data (r=0.69) and significant association with breast cancer risk in EPIC-Italy with an OR of 1.38 per unit MI (range 24 – 43, 95% CI: 1.23 – 1.57, P=2.0E-07). Validation step is yet to be finalized; initial analysis on the first 318 case-control pairs suggests a modest but significant association with ER+ breast cancer risk (unadjusted OR=1.04 per unit MI, 95% CI: 1.00 – 1.07, P=0.030, MI range 20 – 53).

In summary, we show that changes in DNA methylation following estrogen exposure are detectable in blood and we have developed a Methylation Index of ELEE that is significantly increased in breast cancer cases compared to controls in EPIC-Italy, and modestly increased in the GS validation cohort. This molecular measure of estrogen exposure could potentially improve risk assessment methods and be used to identify women at high risk of developing breast cancer.

#5317

JAK2 regulates oxidative damage-induced epigenetic alterations.

Ning Ding, Sam Miller, Heather O'Hagan. _Indiana University Bloomington, Bloomington, IN_.

Elevated levels of reactive oxygen species at sites of chronic inflammation cause oxidative DNA damage in epithelial cells, contributing to carcinogenesis. Aberrant silencing of key genes by promoter CpG island DNA hypermethylation occurs in many cancers that form at sites of chronic inflammation. Yet, it is not completely understood how these aberrant DNA methylation alterations are initiated. We hypothesize that the recruitment of epigenetic silencing proteins to sites of oxidative DNA damage is important for initiating DNA methylation. Here we demonstrate that after hydrogen peroxide (H2O2) treatment, mismatch repair proteins MSH2 and MSH6 become localized to sites of oxidative DNA damage, resulting in the recruitment of DNA methyltransferase 1 (DNMT1) and Polycomb repressive complex 2 (PRC2) members to chromatin. This recruitment results in the formation of repressive chromatin in the promoters of tumor-suppressor genes and the concomitant transient reduction in their expression. We sought to further understand the mechanism that drives H2O2-induced MSH2 and MSH6 chromatin binding. Janus kinase 2 (JAK2) is a kinase that is canonically known to be activated by cytokines and growth hormones and phosphorylate the transcription factor signal transducer and activator of transcription (STATs). Here, we demonstrate that in response to treatment with H2O2, but not interleukin 6 (IL-6), JAK2 translocates to the nucleus and interacts with MSH2 and MSH6. Nuclear JAK2 is important for H2O2-induced chromatin interaction of MSH2, MSH6, DNMT1 and PRC2 members. Interestingly, H2O2-induces a global and site-specific increase in trimethylation of histone H3 at lysine 27 (H3K27me3) that is also dependent on JAK2. Furthermore, by analyzing TCGA datasets, we demonstrate high JAK2 mRNA expression correlates with CpG island methylator phenotype (CIMP) status in gastric and colorectal cancer. All together, these findings suggest JAK2 plays a key role in initiating oxidative damage-induced epigenetic alterations during inflammation-driven tumorigenesis. Ultimately, understanding the mechanism of initiation of cancer-specific promoter DNA hypermethylation may lead to treatment strategies to reduce aberrant epigenetic changes induced by chronic inflammation and therefore reduce tumorigenesis.

#5318

High-grade serous ovarian cancer DNA methylation and survival in African-American women.

Lucas A. Salas,1 Lauren C. Peres,2 Sarah E. Abbott,2 Casey S. Greene,3 Jeffrey R. Marks,4 Anthony J. Alberg,5 Elisa V. Bandera,6 Jill S. Barnholtz-Sloan,7 Ann G. Schwartz,8 Michele L. Cote,8 Patricia G. Moorman,4 Ellen M. Funkhouser,9 Edward S. Peters,10 Melissa L. Bondy,11 Paul D. Terry,12 Jennifer A. Doherty,13 Brock C. Christensen,1 Joellen M. Schildkraut2. 1 _The Geisel School of Medicine at Dartmouth, Lebanon, NH;_ 2 _University of Virginia, Charlottesville, VA;_ 3 _Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA;_ 4 _Duke University Medical Center, Durham, NC;_ 5 _University of South Carolina, Columbia, SC;_ 6 _Rutgers Cancer Institute of New Jersey (CINJ), New Brunswick, NJ;_ 7 _Case Western Reserve University, Cleveland, OH;_ 8 _Wayne State University School of Medicine, Detroit, MI;_ 9 _University of Alabama at Birmingham, Birmingham, AL;_ 10 _Louisiana State University School of Public Health, New Orleans, LA;_ 11 _Baylor College of Medicine Houston, Houston, TX;_ 12 _The University of Tennessee, Knoxville, TN;_ 13 _University of Utah, Salt Lake City, UT_.

Ovarian cancer is the most lethal gynecologic cancer in the United States, and African-American (AA) women have the poorest outcomes compared to other racial/ethnic groups. Although several biomarkers have been proposed to establish prognosis in European ancestry (EA) patients, including some DNA methylation markers (FGF4, FGF21, MYLK2, MYLK3, MYL7, and ITGAE) (Phelps et al., 2017), it is unknown if these or other markers are applicable to AA patients. Using data from the African-American Cancer Epidemiology Study (AACES), we evaluated 1) if DNA methylation is associated with residual disease and survival, and 2) if previously reported CpG biomarkers for EA women are related to survival in AA women. 121 AA women with high-grade serous ovarian cancer (HGSOC) were randomly selected from 600 women enrolled in AACES. Clinical records and formalin-fixed, paraffin-embedded (FFPE) tumor tissue were retrieved, and a pathologist reviewed histopathologic slides to confirm diagnosis. 92 HGSOC had complete clinical information. DNA methylation was measured using Illlumina HumanMethylationEPIC. Beta-values were preprocessed (RELIC, Tost+BMIQ and ComBat). Low quality probes were filtered. Tumor purity was estimated using InfiniumPurify; <30% was considered low purity. Four cell estimates (RefFreecellmix) were used as a proxy of cell composition. To evaluate DNA methylation alterations, we used the top 100K most variable CpG sites and a semisupervised recursively partitioned mixture model (ssRPMM) approach to delineate the patients into RPMM classes. We also evaluated the six candidate CpGs from Phelps et al. The dataset was divided into training and validation subsets (50% each); if findings were consistent, a pooled statistic is reported. We used logistic regression to evaluate the association between DNA methylation and residual disease; Cox proportional hazard models were used for survival. Models were adjusted for age at diagnosis, low purity, cell types, neoadjuvant therapy, tissue source (adnexa vs. peritoneum), histology (serous vs. mixed), and residual disease. The RPMM classes were not associated with residual disease. For survival, four RPMM classes were delineated, which we collapsed into two classes. A lower risk of mortality was observed for one of the RPMM classes, HR: 0. 03[95% CI: 0.01-0.12]. This "low risk" RPMM class grouped five CpGs in genes PLEC1, AP5B1, DNAH7 and MAPK15. These genes have been associated with cell motility and ovarian cancer ascites. Among the candidate CpGs, we only observed a trend to better survival per every 10% increase in MYLK3 CpG methylation, HR: 0.51 [95%CI: 0.24-1.10]. These preliminary results suggest that some DNA methylation modifications may identify subgroups of AA women with better survival. Previously reported biomarkers in EA may not be as useful in AA women. Future studies with increased sample size may help to disentangle these associations.

#5319

Genetic regulation of DNA methylation in breast cancer.

Nan Lin,1 Aditi Shendre,2 JinPeng Liu,1 Chi Wang,1 Yunlong Liu,3 Chunyan He1. 1 _University of Kentucky, Lexington, KY;_ 2 _Indiana University, Indianapolis, IN;_ 3 _Indiana Univeristy, Indianapolis, IN_.

Aberrant DNA methylation plays an important role in breast cancer initiation and progression. It is believed that changes in DNA methylation occur early in breast cancer development or even in normal tissue before tumor occurrence, and these changes could be a useful source for early detection, prognosis, and targeted therapy. DNA methylation is tissue-specific and is influenced by inherited genetic variations in the human genome. Genome-wide association studies (GWAS) have identified more than 600 single nucleotide polymorphisms (SNPs) associated with breast cancer risk and outcome. However, how these germline genetic variants influence DNA methylation in breast tissue, especially in normal breast tissue, remains largely unknown. We performed a methylation quantitative trait loci (meQTL) analysis to examine the association between the GWAS identified breast cancer risk loci and DNA methylation in 48 women with paired blood and breast tissue samples. Genotyping of SNPs was performed using blood-derived DNA and the Illumina HumanOmni 2.5-4v1_D array chip. Genome-wide DNA methylation was measured using breast tissue derived genomic DNA and the new Illumina TruSeq Methyl Capture EPIC sequencing technology. We found that GWAS-identified breast cancer risk loci were associated with DNA methylation sites close to genes enriched in pathways related to cell differentiation and morphogenesis, DNA damage, cell movement, and hormonal signaling pathways. We identified more cis-acting than trans-acting meQTLs, and the peak enrichment for meQTLs was in close proximity to transcription start sites. We further compared our results with those from the meQTL studies conducted with lymphoblastoid cell lines and peripheral blood samples. We found more than 60% of the identified association between genetic variants and DNA methylation in our study are tissue-type specific. Our study may provide a post-GWAS functional characterization of breast cancer risk loci and shed light on epigenetic mechanisms that underlie the observed GWAS association.

#5320

Genome-wide DNA methylation analysis during nonalcoholic steatohepatitis-related multistage hepatocarcinogenesis: Comparison with hepatitis virus-related carcinogenesis.

Junko Kuramoto,1 Eri Arai,1 Ying Tian,1 Nobuaki Funahashi,2 Masaki Hiramoto,2 Takao Nammo,2 Yuichi Nozaki,3 Yoriko Takahashi,4 Nanako Ito,1 Ayako Shibuya,1 Hidenori Ojima,1 Aoi Sukeda,5 Yosuke Seki,6 Kazunori Kasama,6 Kazuki Yasuda,2 Yae Kanai1. 1 _Keio University School of Medicine, Tokyo, Japan;_ 2 _Diabetes Research Center, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan;_ 3 _National Center for Global Health and Medicine, Tokyo, Japan;_ 4 _Solution Center, Mitsui Knowledge Industry Co., Ltd., Tokyo, Japan;_ 5 _National Cancer Center Hospital, Tokyo, Japan;_ 6 _Yotsuya Medical Cube, Tokyo, Japan_.

Aim: The aim of this study was to clarify the significance of DNA methylation alterations during non-alcoholic steatohepatitis (NASH)-related hepatocarcinogenesis. Background: The incidence of NASH, a precancerous condition for hepatocellular carcinoma (HCC), has shown an alarming increase in recent years. Although significance of DNA methylation alterations in NASH has been studies in animal models, only a limited number of papers focusing on genome-wide DNA methylation analysis in a cohort of patients with NASH and NASH-related HCC have been reported. It would be informative to understand the significance of DNA methylation alterations in NASH-related hepatocarcinogenesis, especially at the precancerous stage. Methods: Single-CpG-resolution genome-wide DNA methylation analysis was performed on 264 liver tissue samples, i.e. 55 samples of normal liver tissue (NLT), 113 samples of non-cancerous liver tissue showing NASH (NASH-N), 22 samples of NASH-related HCC (NASH-T), 37 samples of non-cancerous liver tissue showing chronic hepatitis or cirrhosis associated with Hepatitis B Virus (HBV) or Hepatitis C Virus (HCV) infection (viral-N) and 37 samples of HCC associated with HBV or HCV infection (viral-T). Results: After Bonferroni correction, 3,331 probes showed significant DNA methylation alterations in NASH-N samples as compared with NLT samples. Principal component analysis using the 3,331 probes revealed distinct DNA methylation profiles of NASH-N samples that were different from those of NLT samples and viral-N samples. Receiver operating characteristic curve analysis identified 194 probes that were able to discriminate NASH-N samples from viral-N samples with area under the curve values of more than 0.95, again indicating that the DNA methylation status of NASH was clearly different from that of viral hepatitis and cirrhosis. Jonckheere-Terptsra trend test revealed that DNA methylation alterations in NASH-N samples from patients without HCC were inherited by or strengthened in NASH-N samples from patients with HCC, and then inherited by or further strengthened in NASH-T themselves. NASH- and NASH-related HCC-specific DNA methylation alterations, which were not evident in viral-N and viral-T samples, were observed in tumor-related genes, such as the WHSC1 gene which encodes a histone H3 lysine 36 methyltransferase and the WDR6 gene which encodes a WD repeat family protein implicated in cell growth arrest, and were frequently associated with mRNA expression abnormalities. Conclusion: These data suggested that NASH-specific DNA methylation alterations observed at the precancerous stage were shown to be inherited by NASH-T and may continuously participate in NASH-related multistage hepatocarcingenesis, at least partly via alterations in the expression of the affected genes.

#5321

Deciphering the DNA methylation signature of EpCAM-/CD49f- breast cancer stem cells.

Austin Y. Shull,1 Caroline E. Dyar,1 Maria Ouzounova,2 Nicole L. Hudson,1 Max S. Wicha,3 Hasan Korkaya4. 1 _Presbyterian College, Clinton, SC;_ 2 _Cancer Research Center of Lyon, Lyon, France;_ 3 _University of Michigan Comprehensive Cancer Center, Ann Arbor, MI;_ 4 _Georgia Cancer Center, Augusta, GA_.

The metastatic potential of breast cancer cells is believed to correspond with the preferential enrichment of a tumor population known as cancer stem cells (CSCs). Thus, evaluating the unique molecular characteristics of breast CSCs is of great interest from a prognostic and therapeutic standpoint. In order to provide a more detailed profile of breast cancer stem cells, we compared the 450K DNA methylation landscape of the EpCAM-/CD49f- cancer stem cell subpopulation from the isogenic MCF10A p53-/PTEN- breast cell line against the corresponding EpCAM+/CD49f+ and EpCAM-/CD49f+ subpopulations to determine how DNA methylation varies within the different genomic regions of CSCs. In addition, we also overlapped the 450K DNA methylation profiles from 16 established breast cancer cell lines of varying subtypes and aggressiveness to determine how these cell lines relate epigenetically with the isolated CSCs. Based on unsupervised PCA and matrix dissimilarity clustering, we were able to identify 3 distinct groups that appeared to independently cluster based on EpCAM-/CD49f- enrichment status. It is also interesting to note that the aggressive cell lines SUM149 and MDA-MB-231 would cluster away from the EpCAM-/CD49f- subset when utilizing strictly promoter probes for clustering, whereas SUM149 and MDA-MB-231 would cluster more closely with the EpCAM-/CD49f- subset when utilizing gene body probes, potentially indicating a more sensitive correlation between gene body methylation and CSC-associated aggressiveness. To further investigate the differing promoter and gene body DNA methylation patterns in CSCs, we performed differential methylation analysis between the 3 previously defined groups. Based on our results, we discovered 1432 differentially methylated promoter probes and 7243 differentially methylated gene body probes (ANOVA FDR p-value <0.001), with the majority of the promoter probes being hypermethylated in the CSC group and, inversely, the majority of the gene body probes being hypomethylated in the CSC group. Examples of genes that demonstrated significant hypomethylation throughout the gene body within CSCs included the guanine nucleotide exchange factor MCF2L, the synaptic-associated protein SHANK2, and the protein kinase C isoform PRKCZ. For the promoter regions, genes that were significantly hypermethylated in CSCs included the metabolic regulator GPD2, the e-cadherin protein CDH1, and the transcriptional regulator IRF6, all of which were transcriptionally suppressed in the CSC populations. Based on these findings, our work helps provide clarity to the stochastic nature of DNA methylation changes throughout the genome in EpCAM-/CD49f- breast CSCs and provide further motivation for determining the prognostic potential of these identified CSC-associated epigenetic events.

#5322

Normal breast tissue 5-hydroxymethylcytosine is enriched at DNA regulatory elements of breast cancer cells.

Owen Michael Wilkins,1 Kevin C. Johnson,2 E. Andres Houseman,3 Jessica E. King,4 Carmen J. Marsit,5 Brock C. Christensen1. 1 _Dartmouth College, Hanover, NH;_ 2 _The Jackson Laboratory for Genomic Medicine, Farmington, CT;_ 3 _Oregon State University, Corvallis, OR;_ 4 _University of Pennsylvania, Philadelphia, PA;_ 5 _Emory University, Rollins School of Public Health, Atlanta, GA_.

DNA methylation is an established modulator of gene expression in normal mammalian cells. Emerging evidence suggests that the oxidized 5-methylcytosine (5mC) product, 5-hydroxymethylcytosine (5hmC), has distinct biological functions and its dysregulation may contribute to cancer pathogenesis. While 5hmC levels in normal breast tissue have been shown to be elevated relative to several other tissue types, we lack a complete knowledge of the genomic distribution of 5hmC in breast tissue, as well as its potential biological functions. Here, we determined the genome-wide distribution of 5hmC and 5mC using methylation array data from 18 normal breast tissue samples. To disambiguate 5hmC from 5mC, we performed parallel processing of bisulfite and oxidative-bisulfite treated DNA, followed by statistical estimation of the 5mC and 5hmC proportions. Based on the distribution of 5hmC, we identified genomic loci with consistently elevated levels of 5hmC across normal breast tissues. In addition, we showed 5hmC is enriched within transcriptionally active chromatin in breast myoepithelial cells, marked by H3K4 trimethylation. Conversely, 5hmC was depleted within active promoters (H3K9ac-marked DNA) and transcriptionally inactive chromatin (H3K9me3 and H3K27me3-marked DNA). Importantly, the loci with high 5hmC in normal breast tissue were enriched among regulatory elements, including transcription factor binding sites, in transformed normal-like breast MCF10A cells, as well as in multiple breast cancer cell lines. These findings provide a genome-wide distribution of 5hmC in normal breast tissue. Furthermore, these findings identify DNA-regulatory regions in breast cancer cell lines that show statistical enrichment for those sites with elevated 5hmC in normal breast tissue, suggesting deregulation of 5hmC within these elements may contribute to breast carcinogenesis.

#5323

Gene-specific DNA methylation alterations in non-alcoholic fatty liver disease (NAFLD)-derived hepatocellular carcinoma in mice.

Barbara Borowa-Mazgaj,1 Aline de Conti,1 Mulugeta Seneshaw,2 Faridodin Mirshahi,2 Frederick A. Beland,1 Arun J. Sanyal,2 Igor P. Pogribny1. 1 _National Center for Toxicological Research, Jefferson, AR;_ 2 _Virginia Commonwealth University, Richmond, VA_.

Hepatocellular carcinoma (HCC) ranks as one of the most aggressive human cancers, with a steadily increasing incidence in the United States and worldwide. HCC is a disease characterized by the presence of multiple heritable cellular, molecular, and metabolic abnormalities driven by genetic and epigenetic alterations. Among these abnormalities, cancer-related gene-specific cytosine DNA methylation alterations, which are potentially reversible, are of special interest. In the vast majority of patients, HCC develops in association with an underlying chronic liver disease, such as non-alcoholic fatty liver disease (NAFLD). In the present study, we investigated the role of gene-specific DNA methylation alterations in the transcriptomic deregulation in HCC and adjacent liver tissue samples from mice subjected to a diet-induced animal model of non-alcoholic fatty liver disease (DIAMOND)-derived liver carcinogenesis. A combined transcriptomic and gene-specific methylation analysis showed that the number of differentially expressed protein-coding genes, which can be regulated by epigenetic mechanisms, was 2.6-times greater in full-fledged HCC than in adjacent liver tissue, 181 and 69, respectively. This indicates that the development of HCC is characterized by a progressive accumulation of epigenetic abnormalities in tumor tissue. The extent of gene-specific DNA methylation varied among epigenetically-regulated differentially expressed genes; however, the number of down-regulated genes was markedly greater in full-fledged HCC. This was evidenced by the fact that only eight epigenetically down-regulated genes in adjacent tissue were in common with HCC. In contrast, in addition to these eight common genes, the expression of additional 35 genes was reduced in HCC. Analysis of gene-specific DNA methylation demonstrated an increase in DNA methylation of several carcinogenesis-related genes down-regulated in HCC, including Gnmt, Cbs, Egfr, Fgfr2, and Esr1. These genes were hypermethylated also in adjacent liver tissue. The hypermethylation and down-regulation of these genes in HCC were confirmed independently in the Stelic Animal Model (STAM) of derived liver carcinogenesis, another mouse model of NAFLD-associated hepatocarcinogenesis. These results demonstrate that the altered expression of key genes is mediated through aberrant DNA methylation and can be a mechanism contributing to the development of NAFLD-related HCC.

#5324

**Intragenic DNA-hypomethylation promotes overexpression of** ITGB7 **in MF subgroup of multiple myeloma.**

Samrat Roy Choudhury, Cody Ashby, Ruslana Tytarenko, Yan Wang, Purvi H. Patel, Aneta Mikulasova, Michael Bauer, Shayu Deshpande, Faith E. Davies, Gareth J. Morgan, Brian A. Walker. _University of Arkansas for Medical Sciences, Little Rock, AR_.

Integrin-β7 is part of an extensive family of glycoproteins and is overexpressed in the MF subgroups in Multiple Myeloma (MM), contributing to drug resistance and poor survival. ITGB7 promotes adhesion and proliferation of myeloma cells in bone-marrow stroma by supplementing growth factors like VEGF. Here, we investigate the possible epigenetic mechanism of ITGB7 overexpression in the MF subgroup, comprising of t(14;16) and t(14;20) myeloma. Newly diagnosed MM (NDMM) patient bone marrow aspirates underwent CD138 cell selection to enrich tumor cells to >98%. Samples consisted of those with a t(14;16) (n=17), t(14;20) (n=7), t(4;14) (n=9), t(11;14) (n=10) and hyperdiploidy (n=19, separated into D1 (n=12) or D2 (n=7 subgroups). NDMM patient samples were compared to plasma cells isolated from age-matched healthy donors (n=4), to determine differential changes in epitranscripts. We performed unbiased genome-wide reduced representation bisulfite sequencing (RRBS) to identify differentially methylated regions (DMRs) in CpG islands, which were validated by Infinium MethylationEPIC arrays (Illumina) in conjunction with gene expression array data (U133 Plus 2.0, Affymetrix) to determine the epitranscriptomic profile in all samples. RRBS was performed using 75 bp reads to a minimum of 20 M reads per sample. Data from MM cells at the Blueprint consortium were also used for annotation of epigenetic marks. Using the RRBS data we identified 26 hypomethylated, overexpressed genes in the MF group samples, which were not present in the other subgroups. In comparison, only 1 gene, FUT7, was hypermethylated and down-regulated in the MF cluster. Most interestingly, we identified ITGB7 amongst the hypomethylated, overexpressed genes. With an in-depth analysis we identified 4 significant (p<0.05) DMRs across intragenic regions of ITGB7, within a 2.5 kb region. Mean methylation across the DMRs reduced from 58% to 22% (p<0.01) in t(14;16) and 39% (p=0.02) in t(14;20) with concomitant overexpression of ITGB7 in the t(14;16), 19.5-fold increase, and the t(14;20), 23.4-fold increase, subgroups. These 4 DMRs belong to CpG islands, annotated to be the part of possible intragenic enhancer site with promoter like activity, and enriched for open chromatin structures. The DMRs also align with the hotspot of H3K4me1 marks and a putative binding site of activating transcription factors such as AP1/2 or Sp1. This suggests that the identified DMRs could be within the enhancer that regulates ITGB7 overexpression through DNA-hypomethylation. ITGB7 is a known oncogenic factor in high-risk MM, contributing to cell adhesion, migration and homing. Here we show using combined DNA methylation and expression data that ITGB7 is regulated through hypomethylation of the an enhancer region in the MF subgroup in MM.

#5325

**5-hydroxymethylcytosine profiling identifies differential targeting in** IDH1 **mutant versus** IDH1 **wild-type high-grade gliomas.**

Wioletta Glowacka,1 Harshika Jain,1 Makiko Okura,2 Abulizi Maimaitiming,1 Romina Nejad,1 Mamatjan Yasin,1 Hamza Farooq,3 Kenneth Aldape,1 Paul Kongkham1. 1 _Univ. Health Network, Toronto, Ontario, Canada;_ 2 _Juntendo University Faculty of Medicine, Tokyo, Japan;_ 3 _Hospital for Sick Children, Toronto, Ontario, Canada_.

Glioblastoma demonstrates significant epigenetic dysregulation with subgroup differences highlighted by the Glioma CpG-Island Methylator Phenotype (G-CIMP) seen in IDH1 mutant tumors. 5-hydroxymethylcytosine (5hmC) has been implicated in IDH1 wild-type gliomas, however its role in IDH1 mutant tumors remains incompletely understood. We examined 5hmC profiles in high grade (WHO III/IV) IDH1 mutant (n = 12) and IDH1 wild-type (n = 9) tumors through parallel processing of samples using bisulfite (BS) and oxidative bisulfite (OxBS) conversion, with subsequent analysis on the Illumina MethylationEPIC Beadchip platform. Hydroxymethylation profiles were correlated with gene expression measured using the Affymetrix Human Gene 2.0 ST array platform. Cumulative density plots for mean 5hmC β-value across IDH1 mutant and wild-type tumors demonstrated 5hmC accumulation predominantly within the top 10th percentile of probes. No significant difference in global 5hmC levels between IDH1 mutant versus wild-type tumors was evident. Mean 5hmC β-values were 4.6%% and 3.8% for IDH1 mutant and wild-type tumors across all probes, respectively. To focus on regions with greatest 5hmC abundance, we assessed probes among the top 1% mean 5hmC β-values. In addition to probes with high 5hmC abundance, differentially hydroxymethylated (DHMR) regions were identified using the R package ChAMP, comparing IDH1 mutant to wild-type tumors. At the probe level, top 1% and DHMR probes demonstrated a relative increase in 5hmC among IDH1 mutant tumors versus IDH1 wild-type. Top 1% and DHMR probes were enriched for enhancer and super-enhancer regions in both IDH1 mutant and IDH1 wild-type tumors, however enhancer and super-enhancers targeted only partially overlapped. Pathway enrichment analysis for top 1% probe gene targets and DHMR-associated genes identified pathways implicated in glioma pathogenesis. Among genes characteristically 'hypermethylated' in G-CIMP + tumors, 28/50 were methylated while 22/50 were hypermethylated in our IDH1 mutant cohort, suggesting that 5hmC contributes to overall methylation of G-CIMP target genes. 5hmC marked the most highly expressed genes in our tumor cohort, with increased expression associated with gene body hydroxymethylation. Among 1367 genes differentially expressed between IDH1 mutant and wild-type tumors, 48 demonstrated a positive significant Spearman correlation between probe 5hmC β-value and gene expression (r ≥ 0.5, p < 0.05), including genes implicated in gliomagenesis as well as novel candidates. This correlation was most evident for genes upregulated in the IDH1 mutant cohort.

#5326

Differential methylation observed in cancer related genes between DNA samples from normal and obese individuals.

Ryan Drennan,1 Nicolas Culhane,1 Matthew L. Poulin,1 Zhenhua Liu,2 Liying Yan1. 1 _EpigenDx, Inc., Hopkinton, MA;_ 2 _University of Massachusetts, Amherst, MA_.

Based on research funded by The American Institute for Cancer Research, evidence is clear that weight can affect your risk of developing cancer. It is estimated that close to 1/3 of cancers that occur every year in the U.S. could be prevented by maintaining a healthy weight, being physically active and eating a nutritious diet. The body mass index (BMI) is defined as the body mass divided by the square of the body height and is typically expressed in kg/m2. BMI between 18.5 and 25 are considered normal while BMI of 25 to 30 is overweight and greater than 30 is considered obese. We have analyzed the DNA methylation levels of 18 individuals with BMI >30, and compared them to 7 individuals with BMI between 18.5 and 25 utilizing three targeted Next-Gen Bisulfite Sequencing (tNGBS) panels that we have developed as well as with individual pyrosequencing assays. The three panels, FOXP3, Immunology and Cancer Panels, cover regulatory regions of 44 genes that include over 400 CpG sites in addition to a global methylation assay to the LINE1 promoter. The results show that seven gene regulatory regions and the LINE1 global methylation assay show significant differential methylation between the obese and the normal BMI samples. ESR1, PAPR1, EN1, LXH2, TFF1, FOXP3 and LINE1 were shown to be hypermethylated and CD3G was hypomethylated, with p-values ranging between 0.08 and 0.005. Larger sample cohorts can now be examined more thoroughly with individual pyrosequencing assays to further validate the findings.

#5327

DNA methylation loss in late-replicating domains is linked to mitotic cell division.

Wanding Zhou,1 Huy Q. Dinh,2 Zachary Ramjan,1 Daniel J. Weisenberger,3 Charles M. Nicolet,3 Hui Shen,1 Peter W. Laird,1 Benjamin P. Berman2. 1 _Van Andel Research Inst., Grand Rapids, MI;_ 2 _Cedars-Sinai Medical Center, Los Angeles, CA;_ 3 _University of Southern California, Los Angeles, CA_.

DNA methylation loss occurs frequently in cancer genomes, primarily within lamina-associated, late-replicating regions termed Partially Methylated Domains (PMDs). We profiled 39 diverse primary tumors and 8 matched adjacent tissues using Whole-Genome Bisulfite Sequencing (WGBS), and analyzed them alongside 343 additional human and 206 mouse WGBS datasets. We identified a local CpG sequence context associated with preferential hypomethylation in PMDs. Analysis of CpGs in this context ("Solo-WCGWs") revealed previously undetected PMD hypomethylation in almost all healthy tissue types. PMD hypomethylation increases with age in both fetal and postnatal tissues, appearing to track accumulation of cell divisions beginning early in life. In cancer, the degree of PMD hypomethylation correlates with somatic mutation density and expression of cell-cycle dependent genes, reinforcing its mitotic clock-like role. We propose that late replication leads to progressive methylation loss throughout an individual's lifespan, potentially contributing to the onset of cancer and acting as a biomarker for cellular aging.

#5328

Intergenic DNA methylation differences in ER- breast tumors from African American versus European American women.

Matthew F. Buas, Jianhong Chen, Qiang Hu, Song Liu, Zhihong Gong, Michael J. Higgins, Christine B. Ambrosone. _Roswell Park Cancer Institute, Buffalo, NY_.

Incidence of estrogen receptor (ER)-negative breast cancer, a subtype with worse prognosis, is higher among African American (AA) versus European American (EA) women. The reasons for this gap remain poorly defined, but likely include differences in the prevalence and biological effects of known risk factors for ER- disease (eg. parity without breastfeeding and early menarche). These exposures may influence risk of ER- cancer via multiple mechanisms, one of which is epigenetic regulation of gene expression. Altered expression of genes controlling the proliferation and/or differentiation of luminal progenitor cells in the breast is likely to affect the propensity of such cells to give rise to ER- versus ER+ cancer. We previously conducted genome-wide DNA methylation profiling of 224 ER- tumor samples from 141 AA and 83 EA women in the Women's Circle of Health Study, and identified 374 differentially methylated loci (DML) by race. Our initial analysis focused on DMLs mapping to annotated genes, and revealed that a key pro-luminal transcription factor gene, FOXA1, was hypermethylated and repressed in AA compared to EA tumors. Moreover, parity without breastfeeding, more common among AAs, was associated with increased FOXA1 methylation. In this study, motivated by the biological importance of the non-coding genome, we focused on ~100 intergenic DML, and used paired 450K and RNA-seq data to assess the relationship between CpG methylation and RNA expression of genes located up to 50 kb away. Methylation at ten DML was significantly correlated with paired gene expression (FDR q<0.05). For seven of these loci, lower methylation tracked with higher expression of the paired gene. One such CpG, hypomethylated in AA versus EA tumors, mapped to a putative enhancer element located 19 kb upstream of, and predicted to interact with, the promoter of LEMD1, a pro-tumorigenic cancer-testis antigen. Analysis of an independent set of 207 ER- breast cancers from TCGA confirmed that LEMD1 RNA expression was higher in AA compared to EA tumors (P=0.01, log2FC=0.95). Our findings suggest that epigenetic differences between AA and EA ER- tumors are linked to altered gene expression and may hold functional significance in breast cancer pathogenesis. Further studies are needed to extend these results to normal breast tissues from cancer-free women, and explore a potential role for FOXA1/LEMD1 epigenetic regulation in luminal progenitor cell homeostasis.

#5329

Telomerase reverse transcriptase **promoter methylation in metastatic melanoma.**

Sumit Borah,1 Seung J. Lee,1 Yiping Fan,1 Raymond L. Barnhill,2 Reinhard Dummer,3 John M. Kirkwood,4 Armita Bahrami1. 1 _Saint Jude Children's Hospital, Memphis, TN;_ 2 _Institut Curie, Paris, France;_ 3 _University of Zurich, Zürich, Switzerland;_ 4 _University of Pittsburgh Medical Center, Pittsburgh, PA_.

Aberrant methylation of a particular region of the Telomerase Reverse Transcriptase (TERT) promoter, termed the upstream of transcription start site (UTSS), is associated with increased TERT expression and may be useful for differentiating malignant tumors, such as childhood brain tumors, prostate cancer, gastric cancer and melanoma, from their benign/low-grade histologic mimics. However, it is unclear whether UTSS methylation is actually responsible for transcription or is only associated with it. The purpose of this study was to test the hypothesis that UTSS methylation is an activating mechanism for transcription of this gene in melanoma.

Since the majority of melanomas express TERT mRNA, we predicted that i) the majority of these samples would have some detectable aberration in the TERT promoter that might account for expression and ii) UTSS methylation would preferentially occur in samples which lacked other recurrent TERT promoter aberrations (point mutation or rearrangement). To test this prediction, we used a cohort of 57 adult melanoma samples to catalog the presence of UTSS methylation (by high-throughput bisulfite-sequencing), promoter mutation (by Sanger sequencing) and/or promoter rearrangement (by fluorescence in situ hybridization) in each case. 5 cases of atypical Spitz tumors and congenital nevi served as negative controls, since these do not express TERT mRNA and were therefore not expected to have such aberrations. As predicted, 9 of the 10 melanoma samples which lacked promoter mutations or rearrangement had a high level of UTSS methylation. Unexpectedly, UTSS methylation sometimes overlapped with the presence of point mutations (9 of 40 cases) or promoter rearrangement (3 of 5 cases). None of the negative control samples expressed TERT mRNA or harbored any promoter aberrations.

We next predicted that, in samples with methylated UTSS, the actively transcribed TERT allele(s) would harbor the methylated UTSS while the transcriptionally silent allele(s) would not. Since methylation analysis of clinical samples is complicated by the presence of unmethylated UTSS from contaminating normal tissue, we tested this prediction in two relatively pure cancer cell lines. Although only one TERT allele was transcribed in each cell line, as measured by digital droplet PCR, we found that in each case both the transcriptionally active and transcriptionally silent alleles had methylated UTSS.

These results suggest that UTSS methylation is not sufficient for TERT upregulation, although it may still be useful as a biomarker for diagnosing melanoma and other cancers. Future studies will be aimed to determine if methylation of other regions of the TERT promoter marks the active allele in samples lacking mutations or rearrangement.

#5330

DNA hypomethylation promotes carcinogenesis by inducing cryptic transcription.

Dan Lou, Yanqiang Li, Suzanne M. Martos, Yan Zhu, Dana Freeman, Zhibin Wang. _Johns Hopkins University, Baltimore, MD_.

DNA methylation, regulate genomic processes and serve at the interface between genes and the environment, has long been recognized as the key event involved in cancer initiation and development. However, how exactly the role of DNA methylation and their epigenomic consequences underlying carcinogenesis remains to be elucidated. We previously reported that a division of labor between DNA methyltransferase (DNMT) (DNMT1 and DNMT3a/3b) to suppress distinct types of genome components. Additionally, allelic and biomodal methylation patterns have been revealed by using two bioinformatics methods. To determine how the DNA methylation affects the integrity of gene transcription, we applied the appropriate PRO-cap technique that detects 5'-capped RNA transcripts in wildtype (WT) mESCs and three mutant mESCs with altered DNMT: loss of DNMT1 (DNMT1-/- ; 1KO), loss of DNMT3a/3b (DNMT3a-/-/DNMT3b-/-; DKO) and loss of DNMT1 and DNMT3a/3b (DNMT1-/-/ DNMT3a-/-/DNMT3b-/-; TKO). With high quality of sequencing reads at high fold coverage and 150 millions of mapped reads for WT and mutant cells, our analyses revealed significant RNA transcripts alterations. By mapping global transcription start site (TSS) and chromatin dynamics, cryptic transcription of thousands of DNMT loss-induced non-annotated TSSs were observed. Some of the non-annotated TSSs were further confirmed in arsenic-treated malignant transformated cell line. Our results indicated that the alteration (completely loss or partially loss) of DNA methylation may promote the carcinogenesis by sabotaging the fidelity of gene transcription initiation.

#5331

**Repression of** TET **genes and enhancement of DNA methyltransferase activity are critical for induction of aberrant DNA methylation.**

Hideyuki Takeshima, Tohru Niwa, Harumi Yamada, Satoshi Yamashita, Mika Wakabayashi, Toshikazu Ushijima. _National Cancer Center, Tokyo, Japan_.

Chronic inflammation is deeply involved in the development of human cancers by inducing epigenetic alterations, such as aberrant DNA methylation. Among various inflammatory cytokines, the expression of Il1b, Tnf, and Nos2, is associated with induction of aberrant DNA methylation. However, the molecular mechanisms by which these cytokines induce aberrant DNA methylation remain unclear. Here, we show that the activation of the NF-kB signaling pathway, downstream of IL-1β and TNF-α, caused up-regulation of specific miRNAs, such as miR-20a, miR-26b, and miR-29c, and that these miRNAs caused repression of the Tet methylcytosine dioxygenases (Tet) genes, Tet1, Tet2, and Tet3. TET repression by overexpression of one of these miRNAs in cultured cells appeared to be insufficient for induction of aberrant DNA methylation as detected by an Infinium MethylationEPIC BeadChip array. However, triple knockout of TET genes induced strong DNA methylation at thousands of genomic loci. At the same time, exposure to nitric oxide, produced by Nos2, enhanced enzymatic activity of DNA methyltransferases (DNMTs). Treatment of cultured cells with nitric oxide induced weak DNA methylation at hundreds of genomic loci. These results show that both the repression of TET genes and enhancement of DNMT activity, induced by chronic inflammation, are critical for induction of aberrant DNA methylation. ''Vicious'' combination of such dysregulations might have a synergistic effect on induction of aberrant DNA methylation.

#5332

Prognostic value of aberrant promoter hypermethylation of tumor-related genes in early-stage head and neck cancer.

Atsushi Imai,1 Kiyoshi Misawa,1 Daiki Mochizuki,1 Hiroyuki Mineta,1 Takeharu Kanazawa2. 1 _Hamamatsu University School of Medicine, Hamamatsu, Japan;_ 2 _Jichi Medical University, Shimotsuke, Japan_.

Staging and pathological grading are useful, but imperfect predictors of recurrence in head and neck squamous cell carcinoma (HNSCC). Accordingly, molecular biomarkers that predict the risk of recurrence are necessary to improve clinical outcomes. The ability to distinguish between low- and high-risk HNSCCs at an early stage may reduce follow-up costs. We hypothesized that the quantitative methylation specific PCR (Q-MSP) assay could be used to define patterns of DNA methylation that differentiate low- and high-risk HNSCCs. The methylation statuses of the promoters of 11 tumor-related genes (p16, RASSF1A, E-cadherin, H-cadherin, MGMT, DAPK, DCC, COL1A2, TAC1, SST, and GALR1) were analyzed in 133 HNSCC cases using quantitative methylation-specific PCR. Frequent methylation of p16 (44%), RASSF1A (18%), E-cadherin (53%), H-cadherin (35%), MGMT (35%), DAPK (53%), DCC (42%), COL1A2 (44%), TAC1 (61%), SST (64%), and GALR1 (44%) in HNSCC were detected. Disease-free survival was lower in patients with 6-11 methylated genes than in those with 0-5 methylated genes (log-rank test, P = 0.001). In a multivariate Cox proportional hazards analysis, the methylation of E-cadherin, COL1A2, TAC1, and GALR1 was associated with poor survival, with hazard ratios of 4.474 (95% CI, 1.241-16.124). In a joint analysis of these four genes, patients with 2-4 methylated genes had a significantly lower survival rate than those with 0-1 methylated genes in early-stage HNSCC. Importantly, the methylation of some genes was closely related to poor prognosis in early-stage HNSCC, providing strong evidence that these hypermethylated genes are valuable biomarkers for prognostic evaluation. In conclusion, the methylation profiles of E-cadherin, COL1A2, TAC1, and GALR1 were the most powerful combination for predicting early-stage HNSCC. This demonstrates that molecular stratification may predict cancer progression. These findings can benefit HNSCC screening and surveillance algorithms. Although our study was retrospective, was conducted at a single institution, and the number of patients was small, it serves as a platform to establish optimal therapeutic strategies for early-stage HNSCC.

#5333

Methylation profiling identifies two subclasses of cutaneous squamous cell carcinoma related to distinct cell types of origin.

Manuel Rodríguez Paredes,1 Felix Bormann,1 Günter Raddatz,1 Julian Gutekunst,1 Carlota Lucena-Porcel,2 Florian Köhler,1 Elisabeth Wurzer,3 Katrin Schmidt,3 Joachim Röwert-Huber,4 Evgeniya Denisova,1 Lars Feuerbach,1 Esther Herpel,2 Ingo Nindl,4 Thomas G. Hofmann,1 Marc Winnefeld,3 Frank Lyko1. 1 _German Cancer Research Center, Heidelberg, Germany;_ 2 _Institute of Pathology, Heidelberg University, Heidelberg, Germany;_ 3 _Beiersdorf AG, Hamburg, Germany;_ 4 _Charité, University Hospital, Berlin, Germany_.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer type and arises from keratinocytes. Most cSCC progress from a UV-induced precancerous lesion termed actinic keratosis (AK). Despite various efforts to characterize these lesions molecularly, the etiology of AK and its progression to cSCC remain only partially understood. Here we have used Infinium MethylationEPIC BeadChips to interrogate the DNA methylation status of about 850.000 CpGs in epidermis samples from healthy skin, AK and cSCC. Importantly, we found that the premalignant AK samples displayed classical features of cancer methylomes and were highly similar to cSCC methylomes. Further analysis identified typical features of stem cell methylomes, such as a reduced DNA methylation age, non-CpG methylation and stem cell-related keratin and enhancer methylation patterns. Interestingly, this signature was detected only in one half of the AK and cSCC samples, while the other half showed keratin and enhancer methylation patterns that were more closely related to the control epidermis. These findings suggest the existence of two distinct subclasses of AK and cSCC that originate from distinct keratinocyte differentiation stages.

#5334

Spg20 (Spartin) promoter methylation identifies tumor B lymphocytes.

Raffaele Frazzi, Tonia De Simone, Mariaelena Pistoni, Francesco Merli. _Arcispedale S. Maria Nuova - IRCCS, Reggio Emilia, Italy_.

Introductory sentence. The layer of regulation represented by gene promoter methylation is being investigated in Non-Hodgkin lymphoma (NHL) cell lines, primary B lymphocytes and peripheral blood mononuclear cells (PBMCs). The aim is to assess whether the panel of differentially methylated genes we previously identified is able to distinguish normal B lymphocytes from lymphoma cells.

Experimental procedures. Quantitative, gene-specific methylation assays. Immunosorting of human B lymphocytes. Proliferation assays. 5-azacytidine treatment. NHL-derived cell lines.

New data. Here we show that NHL-derived cell lines display a characteristic profile of promoter methylation on specific tumor suppressors and target genes. A panel of 9 target promoters was selected previously and assessed on a series of healthy and tumor primary B lymphocytes. 4 out of 5 NHL-cell lines show a highly methylated profile on KLF4, DAPK1 and SPG20, at variance with normal B lymphocytes (lymph-node derived) and PBMCs where these targets are completely unmethylated. Interestingly, PBMCs from healthy donors display the same methylation pattern as the one shown by B lymphocytes immunologically sorted from follicular hyperplasias. Spartin (SPG20) emerges here as a differentially methylated gene associated to lymphocyte malignancy, at variance with other tumor suppressors in our panel that show variability among different cell lines. SPG20 is completely or highly methylated in all the tested NHL-cell lines. According to this observation, SPG20 results not methylated in PBMCs as well as sorted non-tumor B lymphocytes. On the contrary, the methylation status of three different BCL6 CpG islands (assessed with the same technique) may vary among different cell lines. In order to investigate what's the functional role of these specific promoters methylation, we treated Toledo and NU-DUL-1 cell lines with the demethylating agent 5-azacytidine (5AZA). We observe that the dose-dependent inhibition of proliferation upon 5AZA is accompanied by a small but reproducible decrease in SPG20 and MZB1 promoter methylation. Experiments aimed at assessing any involvement of DNA methyltransferases DNMT1 and DNMT3a are currently ongoing.

Conclusions. Our data show a consistent methylation profile of 9 gene promoters on 5 different NHL-derived cell lines. Spartin emerges as a completely methylated promoter in the tested tumor cells. PBMCs show a consistent unmethylated profile of Spartin and of all the target genes. The epigenetic regulation of these genes in B-derived lymphomas warrants further investigation.

#5335

A highly efficient targeted bisulfite sequencing method for accurate DNA methylation profiling.

Quan Peng, Daniel Kim, Mohammad Nezami Ranjbar, Sascha Strauss, Scott Winter, Yexun Wang. _QIAGEN, Frederick, MD_.

Background DNA methylation plays an important role in gene regulation. Bisulfite sequencing (BS), in which unmethylated cytosines are converted to uracil while methylated cytosines remain intact, is considered as the gold standard for DNA methylation detection with single nucleotide resolution. Due to the high cost of whole genome BS and the low throughput of amplicon BS, there remains the need for new targeted bisulfite sequencing methods with high efficiency and flexibility.

Results We developed a targeted bisulfite sequencing method, by incorporating single primer enrichment (SPE) and molecular barcoding (UMI) technology. This enables flexible and scalable targeted DNA methylation analysis with high efficiency. We first studied the ability of our assay to detect CpG methylation at various levels in reference materials. We also evaluated its utility in profiling CpG methylation in different cell line DNA, and compared the results with published data from alternative technologies (WBS, RRBS and microarray).

Conclusions We demonstrated the successful implementation of SPE and UMI technology on bisulfite converted DNA with high enrichment specificity and low bias. We showed that our method accurately detected CpG methylation at different levels, in concordance with expected titration mixture or published datasets. Comparing to reported amplicon BS, our technology enables much higher multiplexing scale and much simpler workflow. Comparing to probe capture based enrichment, our technology enables much lower DNA input, much higher sequencing efficiency (on-target) and shorter turnaround time.

#5336

Methylation patterns in hepatoblastoma: A report from the Children's Oncology Group.

Erin Marcotte,1 Logan G. Spector,1 Heather Nelson,1 Gail Tomlinson,2 Mark Krailo,3 Lauren Mills,1 Jenny Poynter1. 1 _Univ. of Minnesota, Minneapolis, MN;_ 2 _University of Texas Health Science Center, TX;_ 3 _University of Southern California, CA_.

Introduction. Hepatoblastoma (HB) accounts for over 80% of all liver cancer diagnosed in children under 15 years of age in the United States. Aberrant DNA methylation has been implicated in the pathogenesis of multiple types of cancer, including HB. Global hypomethylation and DNA methylation in the promoter region of selected tumor suppressor genes, including RASSF1A, p16, SOCS1, CASP8, and MT1G, has been observed in previous studies of HB. Methods. A total of 47 flash-frozen samples of HB tumors were available for analysis. These tumors include 26 tumors from cases enrolled in Children's Oncology Group study AEPI04C1 (the HOPE study) and 21 tumors obtained as part of the Children's Oncology Group P9346 HB biology protocol. The Cooperative Human Tissue Network Pediatric Division (CHTN; Columbus, OH) provided 23 normal liver tissue samples from children ages 0—6 to serve as controls in this analysis. Initial multi-dimensional scaling (MDS) plots showed that 8 samples, 2 control and 6 patients, did not cluster with the other samples of the same group and were considered outliers. These 8 samples were removed from the study leaving a total of 62 samples, 21 control and 41 patients, remaining. Differentially methylated regions (DMR) were identified between control and patient samples using the bumphunter algorithm included in the minfi package. We used Ingenuity Pathway Analysis (IPA) to identify pathways that were enriched in the group of CpG with a change in methylation beta value of ≥39% between patients and controls. Results. We found 4833 DMR, which are associated with 3543 genes, when comparing HB patients and controls. Pathway analysis revealed 76 pathways enriched among the DMR. Conclusions. Identification of methylation patterns may elucidate the developmental stage at which HB arose and the at-risk period when environmental exposures could be most harmful. Further, understanding the relevant genetic pathways could lead to the development of new targets for therapy.

#5337

**The epigenetic feedback loop of the CpG demethylase** TET **family genes in head and neck cancers.**

Kiyoshi Misawa, Atsushi Imai, Daiki Mochizuki, Takeharu Kanazawa. _Univ. of Hamamatsu Medical School, Hamamatsu, Japan_.

Purpose: The aim of this study is that clarifying the epigenetic regulation of TET family genes can provide insights into the mechanisms of tumorigenesis and the risk of disease recurrence in HNSCC.

Experimental Design: We generated methylation profiles of TET family genes in tumor samples obtained from 233 patients with head and neck squamous cell carcinoma (HNSCC); these included 57 hypopharynx, 44 larynx, 69 oral cavity, and 63 oropharynx tumor samples. TET family genes mRNA expression and promoter DNA methylation were examined via quantitative reverse transcription (Q-RT-PCR) and methylation-specific PCR (Q-MSP), respectively. Promoter methylation was compared with various clinical characteristics and methylation index.

Results: The TET methylation index (TE-MI) as the number of TET family genes methylation events was positively correlated with alcohol consumption (P = 0.004), HPV status (P = 0.004) and disease recurrence (P = 0.002). Simultaneous methylation analysis of TET family genes significantly correlated with reduced disease-free survival in unfavorable event groups (log-rank test, P = 0.026). In the multivariate Cox proportional hazards analysis, TET3 methylation of T1/2 patients, oropharyngeal cancer patients and oral cancer patients exhibited the high association with poor survival (hazard ratio: 2.64, P = 0.014; 3.55, P = 0.048; 2.63, P = 0.028, respectively). Joint analysis of tumor suppressor genes methylation index (TS-MI) showed a significant trend toward higher TE-MI increased.

Conclusions: TET family genes methylation status was independently associated with aggressive tumor behavior and forming a DNA methylation feedback loop in patients with HNSCC.

#5338

Comprehensive pan-cancer genomic analysis reveals link between 3'UTR DNA methylation and increased gene expression.

Michael H. McGuire. _MD Anderson Cancer Center, Houston, TX_.

Investigations into the function of non-promoter DNA methylation have yielded new insights into epigenetic regulation of gene expression. Previous studies have highlighted the importance of distinguishing between DNA methylation in discrete functional regions (e.g. promoter, gene body, enhancer); however, the relationship between DNA methylation outside of the promoter region and gene expression in cancer requires further exploration. Through integrated analysis of gene expression and DNA methylation profiles (>3,000 samples across multiple cancer types and corresponding normal tissues), we uncovered an enrichment of DNA methylation sites within the 3'UTR in which DNA methylation is strongly positively correlated with gene expression. In 5 of 10 tumor types examined, DNA methylation of the 3'UTR is associated with patient survival in a significant number of genes. Filtering for genes in which 3'UTR DNA methylation, relative to gene body DNA methylation, is more strongly correlated with gene expression yields a list of genes enriched for functions involving T cell activation. The immune checkpoint gene HAVCR2 (TIM-3) shows a substantial increase in 3'UTR DNA methylation, but not adjacent exonic/intronic, or promoter DNA methylation, upon T cell stimulation and upregulation of gene expression. Furthermore, this increase in HAVCR2 gene expression can be abrogated by treatment with demethylating agents. These findings implicate the 3'UTR as a functionally relevant DNA methylation site, particularly in regard to T cell activity. Additionally, they reveal a novel mechanism by which HAVCR2 is upregulated in T cells, providing a new means of targeting immune checkpoint blockade. Finally, these data offer a selective marker for choosing patients that could respond to the combination therapy of PD-1/PD-L1 inhibition and demethylating agents.

#5339

Epigenetic silencing of ANGPTL4 in tumor tissues and its overexpression in the urothelial carcinoma microenvironment.

Michael WY Chan,1 Hsiao-Yen Hsieh,2 Yeong-Chin Jou,2 Chun-Liang Tung,2 Yuh-Shyan Tsai,3 Yuan-Hung Wang,4 Chen-Lin Chi,5 Ru-Inn Lin,5 Shih-Kai Hung,5 Yu-Ming Chuang,1 Shu-Fen Wu,1 Chin Li,1 Cheng-Huang Shen,6 Cheng-Da Hsu6. 1 _National Chung Cheng University, Min Hsiung, Chia Yi, Taiwan;_ 2 _Ditmanson Medical Foundation Chiayi Christian Hospital, Chia Yi, Taiwan;_ 3 _National Cheng Kung University Hospital, Tainan, Taiwan;_ 4 _Taipei Medical University, Taipei, Taiwan;_ 5 _Buddhist Dalin Tzu Chi General Hospital, Chia Yi, Taiwan;_ 6 _Ditmanson Medical Foundation Chiayi Christian Hospital, Min Hsiung, Chia Yi, Taiwan_.

Urothelial carcinoma (UC) carcinogenesis has been hypothesized to occur through epigenetic repression of tumor suppressor genes (TSGs). By qRT-PCR array, we found that one potential TSG, ANGPTL4, was expressed at very low levels in all UC cell lines we examined. Previous studies had demonstrated that ANGPTL4 is highly expressed in some cancers, but downregulated, by DNA methylation, in others. Consequently, due to these seemingly conflicting functions in distinct cancers, the precise role of ANGPTL4 in the etiology of UC remains unclear. In this study, using methylation-specific PCR and bisulfite pyrosequencing, we show that ANGPTL4 is transcriptionally repressed by promoter hypermethylation in UC cell lines and primary tumor samples, as compared to adjacent noncancerous bladder urothelium. Functional studies further demonstrated that ectopic expression of ANGPTL4 potently suppressed UC cell proliferation, monolayer colony formation in vitro, and invasion, migration, and xenograft formation in vivo. As ANGPTL4 is a secreted factor, we therefore examined the circulating ANGPTL4 (cir-ANGPTL4) level in UC patients. Surprisingly, cir-ANGPTL4 was significantly higher in plasma samples from UC patients than normal control, suggesting it might be secreted from other cell types. More importantly, exogenous c-terminal fragment of ANGPTL4 (cANGPTL4) could promote cell proliferation and cell migration via activation of signaling through the Erk/FAK axis. We further confirmed that mouse xenograft tumor growth could be promoted by administration of exogenous cANGPTL4. Finally, immunohistochemistry demonstrated that ANGPTL4 was downregulated in tumor cells but overexpressed in tumor-adjacent stromal tissues including muscle and macrophages of muscle-invasive UC tissue samples. In conclusion, our data supports dual roles for ANGPTL4 in urothelial carcinoma progression, either as a tumor suppressor or oncogene, in response to microenvironmental context.

#5340

The methylation burden is determined by the duration of exposure to carcinogenic factors.

Harumi Yamada,1 Hideyuki Takeshima,1 Tohru Niwa,1 Takeshi Toyoda,2 Satoshi Yamashita,1 Toshikazu Ushijima1. 1 _National Cancer Center Research Institute, Tokyo, Japan;_ 2 _National Institute of Health Sciences, Tokyo, Japan_.

Aberrant DNA methylation accumulated in normal tissues, namely methylation burden, is closely associated with risk of carcinogenesis, especially in cancers related to chronic inflammation. Methylation burden is known to be influenced by multiple factors, such as genetic factors and strengths of carcinogens. However, the impact of the duration of exposure to a carcinogen is still unclear. Here, using a Mongolian gerbil model of Helicobacter pylori (H. pylori)-induced chronic gastritis, we aimed to clarify the impact of the duration of exposure on the methylation burden in normal gastric tissues (gastric mucosae). Following infection with H. pylori, DNA methylation levels at four CpG islands, HE6, SA9, SB5, and SD2, increased, depending upon the exposure duration. After eradication of H. pylori, DNA methylation levels decreased, but tended to be higher in gastric mucosae with a longer infection period. DNA molecules with dense DNA methylation, but not those with sparse DNA methylation, increased depending upon the infection period. DNA methylation levels at one of the four CpG islands, SA9, tended to be higher in the gastric mucosae of gerbils infected with H. pylori than in those of non-infected gerbils, even 50 weeks after eradication. These results showed, for the first time, that the methylation burden in normal tissues is influenced by the duration of exposure to a carcinogenic factor.

### Genomic Profiling of Tumors 4

#5342

Somatic bi-allelic loss of TSC genes in eosinophilic solid and cystic renal cell carcinoma (ESC RCC).

Nicole D. Lee,1 Pankaj Vats,1 Xuhong Cao,1 Fengyun Su,1 Robert Lonigro,1 Kumpati Premkumar,1 Kiril Trpkov,2 Jesse K. McKenney,3 Rohit Mehra,1 Saravana M. Dhanasekaran,1 Arul M. Chinnaiyan1. 1 _University of Michigan Health System, Ann Arbor, MI;_ 2 _University of Calgary, Calgary, Alberta, Canada;_ 3 _Cleveland Clinic, Cleveland, OH_.

Renal cell carcinoma (RCC) subtypes with overlapping histomorphologic features pose diagnostic challenges. For instance, a unique category of sporadic renal tumors with eosinophilic cytoplasm and solid and cystic growth pattern (ESC RCC) may mimic a RCC subtype usually encountered in patients with germline aberrations of tuberous sclerosis complex (TSC) genes (TSC RCC). Here, we used next-generation sequencing (NGS) technology to interrogate the clinicopathologic and molecular profiles of ESC RCC tumors. Mutational and copy number analysis of NGS data from ESC RCC tumors revealed a somatic bi-allelic loss of TSC family genes, specifically TSC1 or TSC2, in six out of seven profiled cases. However, the corresponding background kidney showed only wild type alleles, thus excluding any germline involvement and differentiating ESC RCC from TSC RCC. Furthermore, bi-allelic loss of the TSC genes occurred in a mutually exclusively manner in this cohort. Our study clarifies the molecular identity of ESC RCC, and can thus guide future therapeutic strategies and provide a basis for the revision of current RCC classification.

#5343

Mutational signatures in EGFR sensitizing mutation positive lung adenocarcinoma.

Eun Young Kim, Arum Kim, Yoon Soo Chang. _Yonsei Univ. College of Medicine, Seoul, Republic of Korea_.

Lung cancer has high mutational burden, which is featured by smoking signature. However, lung cancer with EGFR mutations is common in nonsmokers and women, which suggests that mutational stresses other than smoking are involved. We analyzed repeated deep sequencing data of EGFR sensitizing mutation positive lung adenocarcinoma tissue to identify characteristic mutational signatures, proposed by Alexandrov et al. Targeted next-generation sequencing was performed using Ion AmpliSeq Custom Panel of 0.6 Mb size containing 70 major genes including EGFR in 69 cases harboring sensitizing EGFR mutation. Publicly available data from EGFR sensitizing mutation were extracted from GDC data portal. Mutational signature was analyzed using maftools package. EGFR-TKI domain analysis revealed 53 mutations. C> T mutation was found in 26.4%, but C> T transition at CpG site that suggests aging effect was found in 2%, and C> T or C> G mutation in TCW motif reflecting APOBEC activity were found in 1.4%. C> A transversion corresponding to Signature 4 was also found in only one case, suggesting that the possibility of EGFR mutation by smoking effect is very low. The T> C substitution in ApT, indicating signature 5, was detected in 4%. The EGFR-TKI sensitizing mutation (E19del, L858R) has lower frequency of co-mutation while other EGFR-TKI domain mutations were frequently associated with multiple co-mutations. T>G substitution, indicating signature 28 and commonly found in Korean gastric cancer, was found in 39.1%. When the mutational signatures of a total 70 genes were analyzed, the C> T mutation was the most commonly detected (73.1%) and similarity with signature 5 was highest. These findings suggest that the mutational signature of EGFR sensitizing mutant lung cancer, which is frequent in Far East Asia, may be different from that of Western data. Additional data and analysis will be updated until the annual meeting.

#5344

Phylogenetic and signature analyses predict treatment response in high-grade serous ovarian cancer.

Jaana Oikkonen,1 Yilin Li,1 Amjad Alkodsi,1 Olli Carpen,1 Kaisa Huhtinen,2 Sakari Hietanen,2 Seija E. Grenman,2 Rainer Lehtonen,1 Sampsa Hautaniemi1. 1 _University of Helsinki, Helsinki, Finland;_ 2 _University of Turku, Turku, Finland_.

Background. High-grade serous ovarian cancer (HGSOC) is a heterogeneous cancer where majority of the patients will eventually relapse. Phylogenetic analysis of longitudinal samples can reveal mechanisms leading to chemoresistance in different subclones. Companied with signature analysis timing of major mutations can be estimated. These methods can reveal the origin of the resistance mechanisms and bring knowledge about the heterogeneity in the surviving subclones.

Methods. We employed whole genome sequencing in 48 tumor tissue samples from 9 patients with stage IIB -IVB HGSOC treated at the Turku University Hospital, Finland. DNA from 3-7 tumor tissues at diagnosis (debulking surgery or laparoscopy) and interval debulking surgery (NACT treated patients) and/or relapse from each patient was sequenced with coverage of 30-100x. The mutations were detected with MuTect2 and HaplotypeCaller and copy number alterations with Ascat, excluding germline variation using a blood control. The subclonal structures were identified with PyClone and ClonEvol using maximum of 3000 mutations in each patient. Phylogenetic trees explaining largest proportion of the variations were used. Signatures were fitted with the 30 consensus signatures from COSMIC for each tumor sample as well as for major subclones.

Results. Each patient was characterized with 7-15 variant clusters (subclones). Largest divergence was detected in ovaries (11-49% of the mutations were ovary spesific) and lymph nodes compared to other tissues. Even left and right ovary can be very different from each other and ovaries exhibit earlier spreading than other sampled sites. Other tissues were spread from clones detected in the ovaries. Constant mutation rate or higher mutation rate during the treatment could not be explained by the detected number of variants between time points.

Early clonal BRCA (COSMIC signature 3) with high contribution predicted better response to chemotherapy. Signature 3 has been associated with homologous recombination deficiency and sensitivity to platinum-based chemotherapy. High APOBEC (signature 2 and 13) associates with higher level of diversification of related branches. Patient level heterogeneity does not seem to predict outcome contrary to within sample heterogeneity which was higher in the patients with shorter survival times. Relapses contained late or early branching clones. In one poor outcome case a clone which was enriched in relapses dominated in the patient derived cell line indicating superior viability and cause of chemoresistance.

Conclusions. Cancer evolution trees improved mutation signature analyses and relieved clinically relevant differences between HGSOC patients. Early BRCA signature as well as similar signatures between subclones predicted good clinical outcomes.

#5345

Somatic mutation status in cfDNA between therapy responders and non-responders of oropharyngeal squamous cell carcinoma patients.

Alok R. Khandelwal,1 Adam Greer,1 Rhett Orgeron,1 Mickie Hamiter,1 Xiaohui Ma,1 Tara Moore-Medlin,1 Hong Yin,2 Glenn Mills,2 Cherie-Ann O. Nathan1. 1 _LSU Health-SHV, Shreveport, LA;_ 2 _Feist Weiller Cancer Center, Shreveport, LA_.

Circulating tumor DNA (ctDNA) could potentially be utilized as a biomarker in Oropharyngeal Squamous Cell Carcinoma (SCC) patients. The objective of this study was to determine if somatic variants could be detected in circulating cell-free DNA (cfDNA) of Oropharyngeal SCC patients using Next Generation Sequencing (NGS). Somatic mutations have been reported in Orophayngeal SCC cases. The identification and detection of somatic variants in circulating cell-free DNA in patient's plasma may predict response to therapy, help monitor disease progression, guide therapy, and determine likelihood of recurrence. In this study, we compared the somatic mutation status between therapy responders and non-responders among Orophayngeal SCC patients. IRB approval was obtained and 23 Oropharyngeal SCC patients were identified in our patient database. Genomic DNA was isolated from frozen tumor tissue using NucleoSpin Tissue kit by Clontech. Cell-Free (cfDNA) was then extracted from patient's plasma samples using Zymo's Quick-cfDNATM serum and plasma kit. The quality and size of DNA was determined by Agilent 2200 TapeStation. If genomic DNA contamination was identified it was removed with Zymo Select-a-Size DNA kit. Libraries were prepared with 10 to 25 ηg of cfDNA using Accel-Amplicon 56G Oncology Panel of Swift Biosciences. DNA sequencing was then performed on MiSeq platform with Miseq Reagent Kit. Variants were identified using Biomedical Genomic Workbench and Genialis's online data analysis platform for Swift Biosciences's Accel-amplicon panels. From the 23 matched samples, 8 somatic non-synonymous variants were found in both tissue and plasma, 7 of which were present in non-responders. The variants and their allele frequencies in the non-responder group were as follows (tumor DNA /cfDNA in %) HRAS Gly12Cys (36.54/0.45), TP53 Glu298* (5.48/0.21), CDKN2A Pro114Arg (48.11/1.52), TP53 Arg282Trp (46.47/1.77), FBXW7 Arg505Gly (30.17/0.58), FBXW7 Arg505Leu (30.46/0.64), TP53 Arg273Cys (38.91/2.51), and one variant in the responder group PTEN (21.07/0.67) Arg130Gln. In conclusion, somatic non-synonymous variants can be identified in cfDNA from Oropharyngeal SCC patients using NGS. The number of detected variants is far greater in the non-responder group suggesting somatic variant detection could be a prognostic marker for Oropharyngeal SCC patients.

#5346

Recurrent genomic copy alterations deregulate the transcriptome and key signaling networks in head and neck cancer cell lines and tumors with worse prognosis.

Hui Cheng,1 Xinping Yang,1 Han Si,2 Anthony Saleh,1 Wenming Xiao,3 Jamie Coupar,1 Susanne M. Gollin,4 Robert L. Ferris,5 Natalia Issaeva,6 Wendell G. Yarbrough,6 Mark E. Prince,7 Thomas E. Carey,7 Carter Van Waes,1 Zhong Chen1. 1 _National Insts. of Health, Bethesda, MD;_ 2 _MedImmune, Gaithersburg, MD;_ 3 _U.S. Food and Drug Administration, Jefferson, AR;_ 4 _University of Pittsburgh, Pittsburgh, PA;_ 5 _University of Pittsburgh Cancer Institute, Pittsburgh, PA;_ 6 _Yale University Medical School, New Haven, CT;_ 7 _University of Michigan, Ann Arbor, MI_.

Human cancer cell lines have been important tools for biological and preclinical investigation, and establishing their relationship to genomic alterations and subtypes found in tumors could further accelerate functional and therapeutic discoveries. We conducted an integrated analysis of the genomic and transcriptomic profiles of 15 human papillomavirus negative (HPV−) and 11 HPV(+) head and neck squamous cell carcinoma (HNSCC) lines, to identify models of important molecular subtypes uncovered among 279 HNSCC tumors from The Cancer Genome Atlas (TCGA). We developed analytic approaches to detect copy number alterations (CNAs) driving altered expression and predict deleterious mutations in HNSCC lines for comparison with TCGA. Strikingly, HNSCC lines displayed recurrent amplifications on chromosomes 3q22-29, 5p15, 11q13/22, and 8p11 that drive increased expression of a cluster of ~100 mRNAs encoding multiple known and understudied candidate oncogenes. These CNAs, together with loss and under-expression or mutations of putative tumor suppressor genes, recapitulate genomic alterations found in more aggressive HNSCC tumor subtypes. Among these, concurrent 3q26.3 amplification and TP53 mutation in HPV(−) HNSCCs is associated with worse overall survival. Common and distinctive CNAs, transcriptome, and mutations converge on biologically and therapeutically important signal pathways and functions in HNSCCs differing in HPV status, including PI3K, Hippo, TGF-β, Wnt/β-catenin, TP63, TP53/death, cell cycle/mitosis, stemness/differentiation, chromatin remodelling/DNA replication, post-transcriptional regulation, and mitochondrial biosynthesis. Our findings elucidate and validate genomic alterations underpinning numerous discoveries made with historic and recently derived HNSCC lines, and provide valuable models for future preclinical and therapeutic studies in subsets of HNSCCs with worse prognosis.

Supported by NIDCD/NIH intramural projects ZIA-DC-000073, ZIA-DC-000074.

This work utilized the computational resources of the NIH HPC Biowulf cluster. (http://hpc.nih.gov)

#5347

Somatic mutation and overexpression of anaplastic lymphoma kinase (ALK) are uncommon events in Asian head and neck cancers.

Wenying Piao,1 Peony Hiu Yan Poon,1 Lan Wang,1 Chin Wang Lau,2 Jason Ying Kuen Chan,1 Yuxiong Su,3 Amy Bik Wan Chan,4 Hoi Lam Ngan,1 Kwok Wai Lo,1 Vivian Wai Yan Lui1. 1 _The Chinese University of Hong Kong, Hong Kong;_ 2 _Yan Chai Hospital, Hong Kong;_ 3 _The University of Hong Kong, Hong Kong;_ 4 _The Chinese University of Hong Kong, Hong Kong, Hong Kong_.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) that is frequently translocated in anaplastic large cell lymphoma (ALCL) and non-small cell lung cancer (NSCLC), etc. Gain-of-function mutations, largely mutations associated with resistance to ALK inhibitors, have been identified in neuroblastoma, colon adenocarcinoma and prostate adenocarcinoma, etc. Pan-cancer analysis of whole-exome sequencing data of >25 cancer types showed a mutational rate ranging from 0-9.4% across pan-cancers (www.cbioportal.org). In cancers of the head and neck, the potential role of ALK on precision treatment has been demonstrated in an Asian head and neck sarcomatoid carcinoma patient bearing an ALK aberration (Kim et al., 2015).

In this study, we sought to examine the mutational rates of ALK in the US-TCGA HNSCC Provisional cohort, in a small Asian HNSCC cohort, as well as in Nasopharyngeal carcinoma (NPC, an Asian-prevalent HNC). Analysis of the whole-exome sequencing (WES) data of the US-TCGA HNSCC Provisional cohort (N=527; largely Caucasian ethnicity, 85.6%; www.cbioportal.org) revealed a somatic mutation rate of 4.0% (21 out of 527 patients, with 18 non-synonymous and 3 synonymous mutations) and a gene amplification rate of 0.4% (2 out of 522 tumors with copy number change data available). In contrast, the Asian prevalent Epstein-Barr Virus-associated NPC (N=102 micro-dissected tumors with paired blood samples analyzed by

WES), no somatic ALK mutation was identified. Using a small Asian HNSCC cohort (N=16), next-generation sequencing (NGS) analysis with an ALK exon coverage of ~90% revealed no somatic ALK mutation, but the presence of Asian-predilected SNPs with unknown clinical or biological significance. Lastly, ALK protein expression, analyzed by Western blotting, was found to be low or largely undetectable in 25 Asian HNSCC tumors analyzed thus far. Yet, we cannot exclude the possibility of focal ALK expression in HNSCC tumors, which can be largely obscured by Western blotting.

In conclusion, somatic mutation of ALK appears to be rare events in Asian HNSCC and EBV-associated Nasopharyngeal carcinoma.

Acknowledgements:

VWYL receives funding supports from the Research Grant Council, Hong Kong (General Research Fund: #1711484, #17121616; Theme-based Research: T12-401/13-R) and the Hong Kong Cancer Fund, Hong Kong.

KWL receives funding from the Research Grant Council (Theme-based Research: T12-401/13-R), Hong Kong.

WP is supported by Faculty Postdoctoral Fellowship Scheme from the Faculty of Medicine, Chinese University of Hong Kong.

JYKC receives funding support by the Dr Stanley Ho Medical Foundation.

Reference

Kim, S. M., Kim, M. J., Jung, H. A., Sun, J. M., Choi, Y. L., Ko, Y. H., . . . Ahn, M. J. (2015) Presence of anaplastic lymphoma kinase translocation in sarcomatoid carcinoma of head and neck and treatment effect of crizotinib: A case series. Head Neck, 37(5), E66-69

#5348

Single-cell analysis of mutational heterogeneity in acute myeloid leukemia tumors with high-throughput droplet microfluidics.

Dennis J. Eastburn,1 Maurizio Pellegrino,1 Adam Sciambi,1 Sebastian Treusch,1 Liwen Xu,2 Robert Durruthy-Durruthy,1 Kaustubh Gokhale,1 Jose Jacob,1 Tina X. Chen,1 William Oldham,1 Jairo Matthews,3 Hagop Kantarjian,3 Andrew Futreal,3 Keyur Patel,3 Keith W. Jones,1 James L. Zehnder,2 Koichi Takahashi3. 1 _Mission Bio, Inc., South San Francisco, CA;_ 2 _Stanford School of Medicine, Stanford, CA;_ 3 _MD Anderson Cancer Center, University of Texas, Houston, TX_.

To make possible the routine characterization of genetic diversity within cancer cell populations, we developed a novel two-step microfluidic droplet workflow that enables efficient and massively-parallel single-cell PCR-based genomic barcoding for single-cell DNA sequencing applications. We demonstrate that the two-step microfluidic approach is required for robust DNA amplification on thousands of individual cells per run with high coverage uniformity and low allelic dropout of targeted genomic loci. To apply our single-cell sequencing technology to human tumor samples, we developed a targeted panel to partially sequence 26 genes frequently mutated in acute myeloid leukemia (AML) including TP53, DNMT3A, FLT3, NPM1, NRAS, IDH1 and IDH2. Using this panel, we were able to sensitively identify SNV and indel-defined clones within AML samples and assess their distribution at the time of diagnosis, remission and relapse. We also used single cell SNVs to monitor host and donor cell populations during bone marrow transplantation (BMT), which allowed us to accurately evaluate engraftment and disease relapse. Collectively, our single-cell data indicates that clonal populations inferred from VAFs obtained from bulk sequencing data may not fully resolve the heterogeneity within tumors; moreover, the single-cell nature of our approach enabled the unambiguous identification of multiple co-occurring mutations within subclones that is not possible with bulk measurements. Collectively, our results show a greater degree of heterogeneity in AML tumor samples than is commonly appreciated with traditional sequencing paradigms and demonstrate the value of single-cell analysis for AML.

#5349

Research in correlation of ADAR and AIMP2 DX2 using pan-cancer analysis.

Seulki Song,1 Daeyoon Kim,1 Yoosik Kim,2 Sung-Soo Yoon,3 Sunghoon Kim,4 Youngil Koh3. 1 _Cancer Research Inst., Seoul, Republic of Korea;_ 2 _Department of Chemical and Biomolecular Engineering, Daejeon, Republic of Korea;_ 3 _Department of Internal Medicine, Seoul, Republic of Korea;_ 4 _Medicinal Bioconvergence Research Center, Seoul, Republic of Korea_.

AIMP2 is an interacting multifunctional protein complex of aminoacyl transferRNA synthase (ARS), a protein synthetase that binds to tRNA by activating amino acids. AIMP2 plays an important role as a tumor-suppressor gene, forming a complex with p53 and exon 2 deficient AIMP2 generated by alternative splicing mechanism (AIMP2-DX2) promotes carcinogenesis and loses its important role as a signaling modulator. On the other hand, the ADAR gene is a dsRNA-specific binding protein that triggers adenosine-to-inosine editing according to the hydrolytic adenosine deaminase activity and play an important role maintaining normal splicing activity. We studied the association between ADAR activity and AIMP2-DX2 generation. We used International Cancer Genome Consortium (ICGC) genomic data of cancer patients for this analysis: COAD-US (n=44), LUAD-US (n=42), and LUSC-US(n=48). Tophat and Cufflinks were used to map the raw RNA sequencing data of three carcinomas to a human reference and analyze for alternative transcription and splicing. The clustering algorithms-based Database for Annotation, Visualization and Integrated Discovery (DAVID) was used to find enrichment in pathway with lists of genes from each group. The difference in the genetic variation patterns between the two groups were determined using "deconstructSigs" package in R which best interprets the sample's mutational profile. In whole ICGC RNA sequencing data, the amount of AIMP2 DX2 negatively correlated with ADAR expression. Subgroup analysis was performed for lung cancer and colon cancer samples based on AIMP2 and AIMP2-DX2 expression levels. Inflection point of AIMP2-DX2 expression curve was used as a cut-off value, and this value divided samples into two groups (7 samples with high AIMP2-DX2 and 121 samples with low AIMP2-DX2). We could observe obvious contribution of downregulated ADAR in high AIMP2-DX2 samples. When we examined mutations enriched in each group and the mutational pattern differences, mutations of tumor-suppressor genes including TP53 (71.4%) and APC (57.1%) were enriched in group of high AIMP2-DX2. The oncogenic mutations of KRAS (42.9%) and PIK3CA (28.6%) were also enriched in AIMP2-DX2 high patient group. The mutations enriched in group of highly expressed AIMP2-DX2 were further analyzed by DAVID functional annotation analysis, and MAPK signaling pathway and BASE excision repair signaling was discovered. Furthermore, the difference in the genetic variation patterns between the two groups was inspected based on COSMIC database. Mutational signature 9 related to hyper-mutation in immunoglobulin was expressed higher in AIMP2-DX2 elevated samples. Signatures 17 and 18 were also expressed with DX2 high samples. We compared the mutational enrichment and mutational pattern differences between the two groups according to the expression of the ADAR gene and verified the effect of the relationship between ADAR gene and AIMP2 DX2 on cancer development.

#5350

Single cell RNA-seq reveals transcriptionally distinct subpopulations within patient-derived pancreatic cancer organoids.

Mohana Ray,1 Rajib K. Nandi,1 Isabel Romero Calvo,1 Kori Kirby,1 Christopher Weber,2 Hedy L. Kindler,2 Mary Buschmann,2 Kevin Roggin,2 Kevin P. White1. 1 _University of Chicago, Chicago, IL;_ 2 _University of Chicago Medicine, Chicago, IL_.

Advances in 3D cell culture over the last decade have enabled the establishment of organoid models from a multitude of organs in varying stages of development and disease. Organoids recapitulate the tissue-specific cell types and exhibit similar morphology and spatial organization as the organ it is derived from. However, to the best of our knowledge, tumor organoids have never been profiled at a single cell level.

In this study, we applied Drop-seq, a high throughput single cell RNA-seq platform, to characterize the different cell types present in pancreatic cancer organoids. We sequenced the transcriptomes of 5494 single cells from organoids derived from a patient undergoing a Whipple procedure for pancreatic ductal adenocarcinoma (PDAC). We then developed a novel clustering approach based on a class of probabilistic generative models called topic models, leading to the identification of two subpopulations of cells. One subgroup expressed marker genes known to play important roles in PDAC biology, such as MUC1, CLDN4 and GAS5. Interestingly, this subgroup also bore markers for cancer stem cells (HES1 and SQSTM1) that have been found by multiple studies to mediate chemoresistance in PDAC. The other subpopulation differentially expressed AGR2 and TMBIM6, which are associated with endoplasmic reticulum (ER) stress and correlates with invasiveness of PDAC cell lines. Functional studies are underway to determine the exact role of these subgroups, but our preliminary findings are suggestive of a subpopulation of cells that maybe involved in resistance to chemotherapy. This is consistent with the clinical progression of this patient's cancer, suggesting that rapid, inexpensive single cell transcriptomics might provide more precise molecular information to personalized medicine programs than traditional RNA-seq alone.

#5351

**Retrospective analysis of cancer exomes with** Roslin **, a portable and reproducible workflow infrastructure.**

Christopher Harris, Jaeyoung Chun, Cyriac Kandoth, Nikhil Kumar, Shweta Chavan, Ronak Shah, Ewa Reza, Aaron Gabow, Christopher A. Bolipata, Barry Taylor, Oliver A. Hampton, Nicholas D. Socci, David Solit. _Memorial Sloan Kettering Cancer Center, New York, NY_.

Reproducibility and portability are persistent problems in the analysis of genomic sequence data. Bioinformatics pipelines usually run only in the controlled computing environment in which they were built. But even when built for portability, it is increasingly complex and sometimes impossible to maintain versioned and reproducible pipeline components like reference genomes, sequence aligners, variant callers, and annotation sources. As a solution to these technical challenges, we present Roslin, a bioinformatics workflow system deployed at Memorial Sloan Kettering Cancer Center. We validate its precision and recall rates for detecting somatic alterations, microsatellite instability, and other relevant biomarkers from retrospective exome recapture of DNA from over 1000 patients, that previously underwent clinical sequencing using MSK-IMPACT. Roslin is written in the Common Workflow Language (CWL), a standard specification requiring tasks to be modularized and inputs and outputs be explicitly defined. The requirements of explicitness and modularization enable CWL workflows to be flexible, portable, scalable, and amenable to container technologies such as Docker and Singularity. Roslin utilizes the Toil workflow manager from the University of California at Santa Cruz, a portable, open-source workflow engine that supports CWL and is designed to securely and reproducibly run scientific workflows efficiently at scale. Roslin leverages Singularity, the Lawrence Berkley National Laboratory container system that is notionally similar to Docker. The Singularity container system is designed for mobility of compute and reproducibility of scientific analysis. Singularity containers are used to package complete scientific workflows, software and libraries, and data. Combining these makes Roslin well suited to run versioned bioinformatics workflows on cluster, cloud, and high-performance computing environments at scale. Roslin has been deployed and tested on multiple high performance computational clusters and cloud computing resources. It supports complete versioning of its workflows, the underlying software and libraries, and associated resource files. It offers end users GUI driven workflow logging, run reporting and real-time tracking. The Roslin CWL workflows are also suitable for deployment and execution on platforms without Toil, as Docker versions of every Singularity container are also provided. (https://github.com/mskcc/roslin)

#5352

Racial/ethnic differences in prostate cancer genomic alterations.

Albert Dobi,1 Gyorgy Petrovics,1 Shyh-Han Tan,1 Hua Li,1 Denise Young,1 Cara Schafer,1 Jesse Fox,1 Kevin Babcock,1 Heng-Cheng Hu,2 Gauthaman Sukumar,2 Yingjie Song,1 Lakshmi Ravindranath,1 Yongmei Chen,1 Joseph Cheng,3 Reinhard Ebner,3 Qingyu Xiao,4 Yidi Sun,4 Yixue Li,5 Yuan Ji,4 Jun Hou,5 Wendy Wang,6 Guo-Ping Zhao,5 Jacob Kagan,6 Sudhir Srivastava,6 Joel T. Moncur,7 Clifton L. Dalgard,2 Matthew Wilkerson,2 Inger L. Rosner,7 Jennifer Cullen,2 Matthew Freedman,8 Zoltan Szallasi,9 Isabell A. Sesterhenn,10 Shiv Srivastava1. 1 _Uniformed Services Univ. of the Health Sciences, Rockville, MD;_ 2 _Uniformed Services Univ. of the Health Sciences, Bethesda, MD;_ 3 _CytoTest Inc, Rockville, MD;_ 4 _Zhongshan Hospital, Fudan University, Shanghai, China;_ 5 _CAS-MPG Partner Institute for Computational Biology, Shanghai, China;_ 6 _National Cancer Institute/NIH, Bethesda, MD;_ 7 _Walter Reed National Military Medical Center, Bethesda, MD;_ 8 _Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA;_ 9 _Children's Hospital Informatics Program at the Harvard-Massachusetts Institute of Technology Division of Health Sciences and Technology, Boston, MA;_ 10 _Joint Pathology Center, Silver Spring, MD_.

Background: Distinct biology of prostate cancer (CaP) among men of different races and ethnicities have been recently highlighted. Along these lines higher frequencies of the most common CaP gene alterations, ERG proto-oncogene activation and deletion of the PTEN tumor suppressor, have been reported among Caucasian Americans (CA) when compared to other racial/ethnic groups. We have reported the cumulative analyses of CaPs from 435 patients (whole genome sequencing (WGS), FISH evaluations and SNP arrays) that highlighted the recurrent deletion and disease progression association of the Limbic System-Associated Membrane Protein (LSAMP) in CaP genomes of African American (AA) men. Further examination of these data indicated the AA CaP genome associated recurrent deletion of the Chromodomain Helicase DNA Binding Protein 1(CHD1) gene.

Methods: Combined evaluation of frequencies and prognostic associations of ERG oncoprotein by immunohistochemistry and deletions of LSAMP, CHD1 and PTEN genes by FISH were performed using a multi-core TMA (42 AA and 59 CA patients) with longitudinal follow up data (median 16 years). CaP and matched blood genomic DNA samples (N=60) from AA patients were analyzed by WGS. ERG frequencies were further evaluated in index tumors of Chinese CaPs (N=100) and were compared to ERG frequencies in index tumors of AA (N=336) and CA (N=594) patients.

Results: Frequent deletions of CHD1 (29% AA vs. 10% CA p=0.017) and LSAMP (26% AA vs. 7% CA, p=0.006) were found in AA CaPs by FISH assay. Both of these deletions were associated with rapid disease progression. Evaluation of CaPs by WGS further highlighted the recurrent deletion of CHD1 among AA men. Comparative evaluation of ERG (AA, N=336; CA, N=594, and Asian N=100) underscored highest ERG frequencies among CA patients (49.3%) followed by AA (23.2%) and Chinese (22%) men.

Conclusions: In light of distinct biology of CaPs in ethnically/racially diverse CaP patient populations there is a need for developing broadly applicable diagnostic, prognostic marker panels and therapeutic approaches. Recurrent CHD1 deletion in CaPs of AA patients may provide new therapeutic opportunities in light of recent reports suggesting that CaP patients harboring CHD1 deletion may benefit from PARP inhibitor or platinum agents therapies. Funding: This study was supported by CPDR, USUHS, HU0001-10-2-0002 to I.L.R., the NCI/EDRN IAA ACN12011-001-0 to S.S. and by the NCI R01CA162383 to S.S.

#5353

Single cell RNA sequencing of pancreatic ductal adenocarcinoma reveals tumor heterogeneity.

Pawan Noel,1 Wei Lin,2 Erkut Borazanci,3 Albert Amini,3 Ruben Muñoz,1 Emily Rodela,1 Serina Ng,1 Daniel Von Hoff,1 Haiyong Han1. 1 _TGen, Phoenix, AZ;_ 2 _Baylor Institute for Immunology Research, Dallas, TX;_ 3 _HonorHealth Research Institute, Scottsdale, AZ_.

Background: Pancreatic ductal adenocarcinoma (PDAC) accounts for ~95% of pancreatic cancer cases and is currently the 3rd leading cause of adult cancer deaths in the US. Due to the lack of methods for early detection and limited treatment options, PDAC has a dismal 5-year survival rate at just <9%. The desmoplastic PDAC tumor microenvironment (TME) is complex and heterogeneous. With recent advances in technology, the characterization of tumors to investigate cellular diversity and evolution in cancer is rapidly accelerating. Here we employed single cell RNA sequencing (scRNA-Seq) to profile transcriptomes of individual cells from dissociated pancreatic tumors isolated from either patients or transgenic mice developing spontaneous PDAC (KPC). This approach elucidated the cellular makeup of individual tumor to reveal distinct cell types like epithelial, endothelial and immune cells, cancer associated fibroblasts (CAFs) and cells undergoing epithelial-to-mesenchymal transition (EMT). Deregulated pathways specific to distinctly identified cell populations were also mapped.

Methods: Freshly harvested human or mouse PDAC tumors were mechanically and enzymatically dissociated to single cells using a Miltenyi gentleMACS Tissue Dissociator. The 10X Genomics Chromium Single Cell 3′ Solution was employed for capture, amplification and labeling of mRNA from single cells and for library preparation. scRNA-Seq was performed on Illumina HiSeq 2500. Custom R packages were used for clustering and bioinformatics data analyses.

Results: Viability of single cell suspensions used for library preps from all samples was >81%. By unsupervised clustering of the scRNA-Seq matrix and using known signature genes for various cell types we revealed the cellular heterogeneity in a human PDAC to identify distinct cell types. These included epithelial tumor cells (EPCAM+, KRT19+); CD133+ tumor stem cells; CAFs (COL5A1+, COL6A2+, SPARC+); endothelial cells CDH5+ VWF+; and CD45 (PTPRC)\+ immune cells also positive for CD3D, CD2, FCGR2A; and cells undergone EMT (CDH2+, ITGA5+, SNAI2+). Using a similar strategy, mouse PDAC diversity was represented by epithelial and EMT tumor cells, CAFs, endothelial cells, macrophages and MDSCs. KRAS and glycolysis signaling pathways in epithelial tumor cells, while hypoxia in the EMT cluster were up regulated. KPC tumors differed in the extent of immune cell infiltration, with a low influx in the smaller versus large tumor, both perhaps representing distinct stages of tumor progression.

Conclusions: Our data supports high throughput scRNA-Seq of solid tumors like PDAC as a powerful method to probe and elucidate the underlying tumor heterogeneity. Understanding diversity and complexity of the PDAC TME in individual tumors may help identify unique therapeutic targets and potentially inform treatment/maintenance strategies for patients with advanced disease.

#5354

Novel genomic alterations in renal medullary carcinoma tumors.

Jesse Fox,1 Denise Young,1 Yingjie Song,1 Heng Cheng Hu,2 Anthony Soltis,2 Matthew D. Wilkerson,2 Clifton L. Dalgard,2 Inger L. Rosner,1 Isabell A. Sesterhenn,3 Shiv Srivastava,1 Shyh-Han Tan1. 1 _Center for Prostate Disease Research, Uniformed Services University and the Walter Reed National Military Medical Center, Rockville, MD;_ 2 _Uniformed Services University, Bethesda, MD;_ 3 _Joint Pathology Center, Silver Spring, MD_.

Introduction: Renal medullary carcinoma (RMC) is an rare but aggressive non-clear cell carcinoma that occurs predominantly in young African American (AA) men who carry the sickle cell trait. Our knowledge of the molecular basis for the initiation and progression of this cancer remains inadequate to establish standard of care treatments. As a result, even with surgery and chemotherapy, outcomes have remained dismal, with a median overall survival of approximately 13 months. Most patients with RMC exhibit deletion of the SMARCB1 gene, which encodes for a subunit of the SWI/SNF chromatin remodeling complex. Subtypes of RMC without SMARCB1 loss but harbor a rare gene fusion between Vinculin and Anaplastic Lymphoma Kinase have also been reported. We hypothesize that whole exome sequencing (WES) of RMC tumors will identify novel genomic alterations responsible for RMC tumor initiation and progression, which may provide actionable targets for treatment.

Methods: Formalin fixed paraffin embedded (FFPE) RMC tumor and adjacent tissue specimens of 21 African American patients were obtained from the Joint Pathology Center. DNA were isolated from tumor and normal tissues with the Qiagen FFPE All Prep kit and measured by using the Qubit fluorometer. Integrity of the DNA samples were evaluated by using the Advanced Analytics Fragment Analyzer. WES DNA libraries were prepared from DNA samples that passed quality control using the Illumina TruSeq WES kit. Paired end sequencing (2x75) was performed on the Illumina HiSeq3000 platform. RMC tissues sections were analyzed for the expression of SMARCB1, VHL, HIF2A, AURKA, and EZH2 proteins by immunohistochemistry (IHC).

Results: Somatic copy number alterations were estimated from the WES of seven RMC cases. These include recurrent large scale DNA alterations involving chromosome 7 and 8 amplification and chromosome 15 deletion. Focal DNA copy number alterations were also identified in the cohort, including deletion of SMARCB1. Analysis of somatic sequence mutations detected a canonical hotspot mutation (R132C) in IDH1 and recurrent non-silent mutations on RBM47 and NF2. Evaluation by IHC showed the loss of SMARCB1 expression in all cases assayed. Upregulation of EZH2 and AURKA, which are negatively regulated by SMARCB1, were detected in 3 of 21 and 11 of 21 cases, respectively. Focal HIF2A staining was detected in 7 of 21 tumor cases.

Conclusions: The recurrent somatic sequence mutations, gene amplifications and deletions identified in this study reveal potential novel oncogenic drivers of RMC. Further validation of these genomic alterations may offer additional insight into the initiation and progression of RMC and provide actionable targets for establishing standard of care treatments.

Source of Funding: The work is supported a grant award (HU0001-14-2-0041) to ST from the Collaborative Health Initiative Research Program (CHIRP), a precision medicine-based collaborative effort between the NIH and the DOD.

#5355

Multiple mutations to the same gene are selected for and cooperate to drive cancer.

Alexander N. Gorelick, Ed Reznik, Yanyan Cai, Craig Bielski, Chaitanya Bandlamudi, Alexander Penson, Philip Jonsson, Konnor La, Maurizio Scaltriti, Nikolaus Schultz, Barry S. Taylor. _Memorial Sloan Kettering Cancer Center, New York, NY_.

Cancers accumulate oncogenic mutations whose biochemical impact depends upon other germline and somatic variants in the cell. It stands to reason that the consequence of a somatic mutation is mediated by the presence of additional genetic variants within the same gene, and by the allele on which they arise. We have undertaken integrative analyses of 27,534 human tumors, including 19,640 prospectively sequenced advanced and metastatic cancers, and revealed a startling rate of multiple non-synonymous somatic mutations in the same cancer gene and tumor, hereafter referred to as multiple mutations. Overall, 26% of primary and metastatic tumors had at least one cancer gene with two or more somatic mutations. A minority of multiple mutations arose from somatic hypermutation or as secondary mutations driving resistance to molecularly targeted therapy. Clonality analysis further indicated that the majority of multiple mutations were present in the same cell. Multiple cancer genes were enriched for a greater burden of multiple mutations than would be expected in the absence of selection, including APC, B2M, EGFR, and TERT. The allelic configuration of these mutations indicated that while multiple mutations in trans arose predominantly in tumor suppressor genes, consistent with the selective pressure for biallelic inactivation, the majority of phaseable mutations arose in cis in both tumor suppressors and oncogenes, implying a selective pressure exists for a biochemical function distinct from individual driver mutations in the same gene. Collectively, these data argue that multiple genetic variants within a gene and their allelic configurations can interact to drive cancers.

#5356

Robust gene fusion detection in formalin-fixed tissue sample with QIAseq RNAscan Oncology Panel.

Jeffrey Falk,1 Parth Sitlani,1 Claire Orosco,1 Maya Panjikaran,1 Dana Weiner,1 Song Tian,2 Raed Samara,2 John DiCarlo,2 Yexun Wang,2 Eric Lader,2 Frank Reinecke3. 1 _Genoptix, Inc, Carlsbad, CA;_ 2 _QiagenSciences, Inc, Fredrick, MD;_ 3 _QiagenGmbH, Hilden, Germany_.

Introduction: Gene fusions are frequently associated with tumorigenesismalignant tumors. Highly sensitive and reproducible detection of gene fusions in clinical samples, especially formalin-fixed paraffin-embedded (FFPE) specimens, is important for diagnosis, prognosis, and treatment guidance. Although many clinical tests utilize next-generation-sequencing (NGS) for detection of genetic alterations, reliable detection of gene fusions by NGS in FFPE samples remains a challenge.

Methods: Five FFPE non-small cell lung cancer samples confirmed by FISH to contain EML4-ALK or KIF5B-RET fusions were selected for testing along with the Seraseq™ FFPE Tumor Fusion RNA Reference Material, v1, which evaluates forcontains 12 known fusions. Four operators prepared NGS libraries for each sample (89-250ng input) using the commercially available QIAseq RNAscan Oncology Panel, which targets 223 fusion gene pairs or 576 unique breakpoints. The resulting libraries were sequenced on an Illumina® NextSeq 500 sequencer. Demultiplexed FASTQ files were analyzed using cloud-based QIAseq RNAscan Analysis pipeline v1.7.20.2. Detected fusions that are on the panel's target list were reported as curated gene fusions and assigned a score based on unique molecules supporting the transcript.

Results: For the Seraseq™ FFPE Tumor Fusion RNA Reference v1 sample, all 12 expectedknown fusions were detected in each of the libraries generated by four different operators. The analysis pipeline correctly called the expected fusion in all four replicates of the five clinical FFPE samples tested, which varied in both input quantityamount and quality. Reproducibility was evaluated by comparing the score values across replicate libraries for each sample; expected fusion detection was 100% consistent between users, and the average correlation coefficient for the fusion score was 0.995.

Conclusions: The commercially available QIAseq RNAscan Oncology panel is a highly sensitive and robust product for detection of gene fusions in FFPE samples. QIAseq RNAscan methodology provides a useful tool for studying gene fusions in tumor samples and with optimization, customization, and validation can further fit the specific needs for of clinical laboratories.

#5357

Mutational and transcriptomic profiling identify distinct angiogenic and inflammatory subtypes of angiosarcoma.

Jong Hyuk Kim,1 Kate Megquier,2 Aaron L. Sarver,3 Rachael Thomas,4 Chao Wang,5 Ingegerd Elvers,2 Elinor Karlsson,2 Matthew Breen,4 Kerstin Lindblad-Toh,2 Jaime F. Modiano1. 1 _University of Minnesota, St.Paul, MN;_ 2 _Broad Institute of Harvard and MIT, Cambridge, MA;_ 3 _University of Minnesota, Minneapolis, MN;_ 4 _North Carolina State University College of Veterinary Medicine, Raleigh, NC;_ 5 _Uppsala University, Uppsala, Sweden_.

Angiosarcoma is an aggressive, albeit rare cancer in humans. The cause of the vast majority of sporadic angiosarcomas is unknown, mortality is high, and no therapeutic targets have been identified to improve outcomes. Hemangiosarcoma (HSA) is a common cancer of dogs, and it shares histopathologic features with human angiosarcoma. In our previous work, canine HSAs were classified into angiogenic, inflammatory, and adipogenic subtypes based on transcriptional profiles. However, the genetic and molecular events that regulate transcriptional subtypes in angiosarcoma are not currently understood. Our goal was to use a comparative genomics approach to apply knowledge from appropriately powered canine studies to inform our research into human sarcomas. In this study, we identified recurrent mutations in RNASeq data from 93 HSAs and 16 nonmalignant controls, based on mutations first identified in exomes from 42 paired tumor and normal samples. In addition to identifying recurrent somatic mutations we also identified translocation fusions, allowing elucidation of oncogenic mechanisms for vascular endothelial growth factor receptors (VEGFR), phosphoinositide-3 kinase (PIK3) signaling pathways, and the p53 DNA damage repair pathway in canine HSA. Significantly, mutational signatures were associated with distinct molecular subtypes of canine hemangiosarcomas, and both the angiogenic and the inflammatory subtypes were apparent in RNASeq data from human angiosarcomas (n=14), suggesting that comparable etiologic mechanisms are operative in the canine and human disease. Our ongoing work seeks to understand how the molecular mechanisms give rise to molecular subtypes of angiosarcoma by defining the association between driver mutations, signaling pathway alterations and transcriptional patterns, which should allow us to identify rational therapeutic targets.

#5358

Multi-omics comparative analyses of pulmonary typical carcinoids, atypical carcinoids, and large-cell neuroendocrine carcinoma.

Noémie Leblay,1 Nicolas Alcala,1 David Hervás Marin,2 Tiffany M. Delhomme,1 Théo Giffon,1 Akram Ghantous,1 Amélie Chabrier,1 Cyrille Cuenin,1 Janine Altmueller,3 Geoffroy Durand,1 Catherine Voegele,1 Philippe Lorimier,4 Anne-Claire Toffart,4 Jules Derks,5 Odd Terje Brustugun,6 Joachim H. Clement,7 Joerg Saenger,8 John K. Field,9 Alex Soltermann,10 Gavin M. Wright,11 Luca Roz,12 Lucia Anna Muscarella,13 Paolo Graziano,14 Zdenko Herceg,1 Ernst-Jan Speel,5 Peter Nuernberg,3 James McKay,1 Nicolas Girard,15 Sylvie Lantuejoul,16 Juan Sandoval,2 Elisabeth Brambilla,4 Matthieu Foll,1 Lynnette Fernandez-Cuesta1. 1 _International Agency for Research on Cancer, Lyon, France;_ 2 _Instituto de Investigación Sanitaria La Fe, Valencia, Spain;_ 3 _Cologne Center for Genomics, Cologne, Germany;_ 4 _Centre Hospitalo-Universitaire Grenoble, Grenoble, France;_ 5 _Maastricht University Medical Centre, Maastricht, Netherlands;_ 6 _Vestre Viken Hospital Trust, Drammen, Norway;_ 7 _Jena University Hospital, Jena, Germany;_ 8 _Zentralklinik Bad Berka, Bad Berka, Germany;_ 9 _University of Liverpool, Liverpool, United Kingdom;_ 10 _University Hospital Zurich, Zurich, Switzerland;_ 11 _St Vincent's Hospital Melbourne, Fitzroy, Australia;_ 12 _Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy;_ 13 _Scientific Institute for Research and Health Care (IRCCS) "Casa Sollievo della Sofferenza", Italy;_ 14 _PathologyIRCCS Casa Sollievo della Sofferenza Hospital, San Giovanni Rotondo, Italy;_ 15 _Université Claude Bernard Lyon 1, EPHE, Lyon, France;_ 16 _Cancer Institute Léon Bérard, Lyon, France_.

Pulmonary grade-1 typical (TC) and grade-2 atypical (AC) carcinoids share molecular characteristics with grade-3 large-cell neuroendocrine carcinoma (LCNEC) despite the distinct clinical behaviors. Most carcinoids can be surgically resected, however, limited treatment options exist for metastatic disease, present in 10-23% of TC and 40-50% of AC. Comprehensive genomic studies could help identify better therapeutic opportunities, novel diagnostic markers, and provide insight on the mechanisms responsible for the increased aggressiveness of AC versus TC. Such studies are rare due to the limited availability of suitable material.

We have established a multi-center collaboration that has given us access to a unique collection of samples. We have already characterized 40 TC and 60 LCNEC genomes/exomes, and 61 TC, 8 AC and 69 LCNEC trancriptomes (published data). In the present study, we have performed whole-exome and transcriptome sequencing on 20 AC patients. Methylation data from 850K Illumina arrays were also generated for these samples, and for a subset of 20 TC and 20 LCNEC previously mentioned.

When comparing the mutational data on AC with that of TC and LCNEC, we have found that similar to TC, AC harbor recurrent alterations in chromatin remodeling genes (such as MEN1 and ARID1A). They also carry alterations in genes involved in other cancer-related pathways (based on STRING), such as cell motility and cell death explaining their more aggressive phenotype. Integrative clustering analysis (MOFA and iCLUSTER) based on expression and methylation data tends to classify carcinoids into four groups: groups 1 and 2 are mostly composed of females with TC, and differ by their age composition and smoking status (Fisher's exact test p=0.008 and 0.03, respectively). Groups 3 and 4 are mostly composed of males with AC (Fisher's exact test for tumor type p=8x10-5). When including the LCNEC data, the samples from group 3 cluster with LCNEC, suggesting that AC can display a variety of expression and methylation patterns that may be linked to aggressiveness. This result was supported by the better survival of groups 1 and 2 compared to groups 3 and 4 (log-rank p=0.02), for which survival was similar to that of patients with LCNEC.

Here, we present for the first time: (i) a multi-omics study on AC; (ii) the methylome characterization of TC, AC, and LCNEC; and (iii) the results of a comparative analysis of TC, AC, and LCNEC based on their molecular characteristics. We have identified the genes and pathways that might explain the progression from low-grade TC to intermediate-grade AC. Our expression and methylation data also supports the existence of a "super-AC" group, which clusters with LCNEC. Finally, we have identified a panel of molecular alterations that may help pathologist distinguishing between these three entities.

NL and NA contributed equally. LFC and MF jointly supervised this work.

#5359

Regulatory germline variants in 10,389 adult cancers.

Kuan-lin Huang,1 Amila Weerasinghe,1 Yige Wu,1 Wen-wei Liang,1 R. Jay Mashl,1 Sheila Reynolds,2 Kathleen E. Houlahan,3 Ninad Oak,4 The Cancer Genome Atlas, Alexander J. Lazar,5 Michael C. Wendel,1 Ekta Khurana,6 Sharon Plon,4 Feng Chen,1 Mark Gerstein,7 Ilya Shmulevich,2 Li Ding1. 1 _Washington Univ. St. Louis, Saint Louis, MO;_ 2 _Institute for Systems Biology, WA;_ 3 _Ontario Institute for Cancer Research, Ontario, Canada;_ 4 _Baylor College of Medicine, TX;_ 5 _The University of Texas MD Anderson Cancer Center, TX;_ 6 _Weill Cornell Medical College, NY;_ 7 _Yale University, CT_.

Previous studies of rare germline variants in cancer has largely been limited to the coding regions of known predisposition genes. The TCGA PanCanAtlas Germline Working Group is analyzing germline predisposing variants of 10,389 cases in 33 cancer types. We deployed more than 121,000 virtual machines running for over 600,000 hours on the ISB Cancer Genome Cloud to conduct massively parallel variant calling and analyses, and the resulting data are shared with scientists across institutions worldwide. Carriers of the functional regulatory variants add on to the 8.9% of cases carrying known pathogenic variants. Burden analyses reveal enrichment of rare variants in the 3'UTR region of NHP2 and POLH. Further, we observed variants aggregating in conserved regions of selected microRNA families that are also affected by somatic mutations, including mir-17 and mir-29. We nominate regulatory variants by using GWAVA and FunSeq2 corroborated with their enrichment in cancer. The prioritized variants are then further evaluated by further co-occurrence of two-hit events and expression changes in their respective tumor samples. Finally, we examine ancestries, familial history and age at onset for carriers of these variants. Overall, we aim to discover and establish the role of regulatory germline variants in oncogenesis.

#5360

Mutational profile and genomic instability according to response to therapy in rectal carcinomas.

Luisa Matos do Canto,1 Simon J. Larsen,2 Bruna E. Catin Kupper,1 Maria D. Ferreira de Souza Begnami,1 Cristovam Scapulatempo Neto,3 Jan Baumbach,2 Annabeth Høgh Petersen,4 Mads Malik Aagaard Jørgensen,4 Samuel Aguiar,1 Silvia R. Rogatto4. 1 _ACCamargo Cancer Center, Sao Paulo, Brazil;_ 2 _University of Southern Denmark, Odense, Denmark;_ 3 _Clinicas Oncologicas Integradas, Rio de Janeiro, Brazil;_ 4 _Vejle Hospital, Vejle, Denmark_.

Rectal cancer (ReCa) patients with locally advanced disease present a high risk of locoregional recurrence and death by the disease. Preoperative neoadjuvant chemoradiotherapy (nCRT) and total mesorectal surgery have been used to reduce these events. However, nCRT has resulted in significant morbidity and up to 30% of patients present pathologic incomplete response (pIR) and ~20% develop distant metastasis or minimal regression to stable disease. These findings reinforce the relevance of identifying predictive markers of response to therapy. Genomic instability (GI) is one of the cancer hallmarks. Patterns of genomic alterations (gains, losses, and cnLOH) and the mutational profile have resulted in prognostic and predictive signatures in several cancer types. Targeted next-generation sequencing (105 cancer-related genes panel, including 13 genes involved in the homologous recombination-HR and, 5 in the mismatch repair-MMR pathways) was performed in 31/33 pretreatment ReCa biopsies. 33 samples were evaluated by SNP array to identify the GI index and the HR deficiency (HRD) scores (LST: large-scale transitions, tAI: telomeric allele imbalance, HRD-LOH: loss of heterozygosity). The GI index represents the fraction of the altered genome and the HRD scores (LST, tAI, HRD-LOH) are reported as markers of deficiency in DNA repair by HR pathway. We found 161 mutations in 51 genes; TP53 (84%), APC (81%) and KRAS (45%) were more frequently mutated. No survival differences were observed among the subgroups studied according to the presence of two APC mutations, KRAS, and TP53 mutations or lacking APC mutations. Overall, a high burden of genomic alterations was observed in ReCa samples. The median GI index was 0.358, much higher than those observed in breast cancer. Patients with complete pathologic response (pCR) presented higher GI index (0.475) compared to pIR (0.294). A significant difference was observed grouping responders (TRG 0+1) and nonresponders (TRG 2+3) (p = 0.043). The GI index of chromosome 12 was higher in pCR (p=0.019). Deficient mismatch repair indicates ineffectiveness of 5-FU used in the nCRT. Three tumors presented mutations in MLH3 or MSH6. An additional analysis performed in HR pathway genes revealed that 7/31 cases presented mutations. Three of these seven ReCa showed high tAI scores, indicating sensitivity to platinum-based therapy. HR-defective tumors have been associated with better platinum response rates. In addition, five cases presented PTEN loss; five, PIK3CA mutations; and one case, BRAF mutation. These alterations have been reported as promising predictors for treatment response in colorectal cancer. The involvement of HR pathway or other driver mutations in the response to therapy in rectal cancers remains unclear. Comprehensive studies in a large set of cases are required to confirm the predictive value of these alterations in rectal cancer.

#5361

Spatial genome organization as a framework for somatic alterations in human cancer.

Kadir C. Akdemir, Andrew Futreal. _MD Anderson Cancer Center, Houston, TX_.

Genomic material within the nucleus is folded into successive layers in order to package and organize the long string of linear DNA. This hierarchical level of folding is closely associated with transcriptional regulation and DNA replication. Recent chromosome conformation studies have revealed that mammalian chromosomes are structured into tissue-invariant topologically associating domains (TADs) where the DNA within a given domain interacts more frequently together than with regions in other domains. Genes within the same TADs represent similar expression and histone-modification profiles. Therefore, regions separating different TADs (boundaries) have important roles in reinforcing the stability of these domain-wide features. Indeed, TAD boundary disruptions in human genetic disorders or human cancers lead to misregulation of certain genes, due to de novo enhancer exposure to promoters. Here, to understand effects and distributions of somatic structural variations across TADs, we utilized single nucleotide variations and genomic rearrangements from 2658 high-coverage whole genome sequencing data across various cancer types with paired normal samples. Our analysis revealed that deletions between repressed and active TADs result in up-regulation of genes on the repressed end of the deletions, whereas active domain genes remain unaffected. Interestingly, we further identified a strong correlation between the mutational distributions in human cancers and the spatial organization of the genome. Transcriptionally active TADs contain less mutation burden compared to inactive TADs, as a result regional mutation rates are drastically different around the boundaries delineating epigenetically distinct domains. However, mutation rates remain similar around the boundaries for samples with the DNA mismatch repair deficiency. Taken together, our analyses reveal new insights about genome architecture, aberrant gene expression and mutational distributions in human cancers.

#5362

Molecular subtyping and tumor markers in the outcome of Korean advanced/metastatic gastric cancer.

Jii Bum Lee,1 Sun Young Rha,2 Woo Sun Kwon,3 Sejung Park,4 Hyun Cheol Chung5. 1 _Division of Medical Oncology, Department of Internal Medicine, Yonsei Cancer Center, Yonsei University Health System, Seoul City, Republic of Korea;_ 2 _Songdang Institute for Cancer Research, Brain Korea 21 PLUS Project for Medical Science, Division of Medical Oncology, Department of Internal Medicine, Seoul City, Republic of Korea;_ 3 _Songdang Institute for Cancer Research, Department of Biostatistics, Yonsei University College of Medicine, Seoul City, Republic of Korea;_ 4 _Songdang Institute for Cancer Research, Yonsei University College of Medicine, Seoul City, Republic of Korea;_ 5 _Songdang Institute for Cancer Research, Brain Korea 21 PLUS Project for Medical Science, Division of Medical Oncology, Department of Internal Medicine, Yonsei Cancer Center, Yonsei University Health System, Seoul City, Republic of Korea_.

Much attention has been focused on utilizing targeted next-generation sequencing and to comprehensively characterize molecular profiles of gastric cancer (GC) adjunct to histopathology. In Asia, many of GC patients are diffuse type and classified as genome stable tumors, but limited efforts have been done to further investigate this specific subtype of cancer. Further assessment according to molecular subtypes is necessary to predict the outcome and determine the proper treatment of advanced/metastatic gastric cancer. Using targeted sequencing with 375 genes based on illumine platform, we divided 147 advanced/metastatic GC patients according to their molecular subtypes (EBV infected tumors (n=3, 2.0%), MSI tumors (n=3, 2.0%), chromosomally instable tumors (CIN, n=47, 32.0%), genomically stable tumors (GS, n=94, 64.0%) and level of CEA and CA 19-9 (cut off values <5 and <37 U/dL, respectively). Tumor specimens were subjected to GC specific histopathology panels to confirm genetic variations [EBV encoding RNA in-situ hybridization, IHC for mismatch repair proteins (ML1, MSH2, MSH6, PMS2), receptor tyrosine kinase (HER2, EGFR, C-met), and PTEN]. OS and PFS were calculated twice, corresponding to the respective 1st or 2nd line chemotherapy. Results show that there was no difference in OS, PFS among the molecular subgroups. Overall, there were no differences in tumor markers (CEA, CA 19-9) in relationship to molecular subtypes (p=0.254, 0.387, respectively). When CIN and GS tumors were further subdivided according to the level of tumor markers, CIN tumors expressing either CEA or CA 19-9 had the longest PFS2 (time from 2nd line chemotherapy until death), while both expression of CEA/CA 19-9 had the shortest PFS [p= 0.040; (median PFS of both positive, both negative, and either one positive tumor markers were 2.6, 3.7, 6.3 months, respectively)]. Amplification of RTKs were detected in a mutually exclusive pattern, and RTKs were amplified in 22 cases [ERBB2 (n=10), FGFR2 (n=4), EGFR (n=3), MET (n=2), ERBB3 (n=1), FGFR3 (n=1), IGF1R (n=1). ERBB4, FGFR1, FGFR4 were not amplified. In addition, 50 actionable (druggable) mutations and CNVs (copy number variations) were identified [EBV (n=1, 33.3%), MSI (n=2, 66.7%), CIN (n=22, 46.8%), GS (n=25, 26.6%)]. CIN tumors had more CNVs (13/22) while GS tumors had more mutations (23/25). In contrast with TGGA data, Korean GS tumors showed significant proportion of druggable targets than expected. In conclusion, genomic and molecular profiling is not prognostic but might be useful in conjunction with clinical parameters such as tumor markers. Moreover, we suggest genomic information could guide the proper treatment selection of advanced and metastatic GC.

#5363

Mutational landscape of non-small cell lung adjacent normal.

Kelly Quek, Vincent Lam, Tyler Hillman, Ming Tang, Xingzhi Song, Xizeng Mao, Junya Fujimoto, Chi-Wan Chow, Karina Eterovic, Edwin R. Parra Cuentas, Carmen Behrens, Xifeng Wu, Yuanqing Ye, Chang-Jiun Wu, Latasha Little, Curtis Gumbs, Paul Scheet, Humam Kadara, Mara Antonoff, Ara A Vaporciyan, Stephen Swisher, Roy Herbst, Jianhua Zhang, John Heymach, Waun Ki Hong, Ignacio I. Wistuba, P. Andrew Futreal, Jianjun Zhang. _UT MD Anderson Cancer Center, Houston, TX_.

Exome sequencing of 96 tumor adjacent morphologically normal lung tissues and paired tumors reveals different mutational processes between normal lung and cancer tissues

Most lung cancers are attributed to cigarette smoking and are thought to arise from airway epithelia that have been damaged through smoke exposure, a concept termed field cancerization. Understanding the genomic evolution from normal lung tissue to invasive cancer is critical in guiding prevention and early detection efforts.

We employed whole exome sequencing to fresh frozen tumor tissues and adjacent normal lung tissues (>2cm away from tumors, histology confirmed by lung cancer pathologists) from 96 patients with resected non-small cell lung cancer (NSCLC) (Table 1). DNA from matched peripheral blood mononuclear cells was used as germline control.

The median somatic mutation burden in adjacent normal lung tissues and tumor tissue was 2.13 mutations/megabase (Mb) (range: 53 – 2488) and 11.2 mutations/Mb (range: 26 – 2820) respectively. Tumors from ever smokers demonstrated significant higher mutation burden than never-smokers (11.6 mutations/Mb in ever-smokers versus 4.03 mutations/Mb in never smokers, p-value = 0.0001626). However, mutation burden is not significantly different in adjacent normal lung tissues between ever- and never-smokers (2.2 mutations/Mb versus 1.95 mutations/Mb, in ever- versus never smokers, respectively, p-value = 0.6874). A median of 16% (1.9% - 47.3%) mutations was shared between tumor and matched adjacent normal lung tissues. No canonical cancer gene mutations were identified in adjacent normal lung tissues. As expected, C>A nucleotide transversion was predominant single nucleotide variants in most of tumor tissues from smokers (47.1%) and mutation Signature 4 that is well known to be associated with tobacco smoking was the most predominant signature in tumor tissues. Surprisingly, 86% of adjacent normal lung tissues from smokers demonstrated predominately C>T transition.De novo mutation signature extraction identified 3 mutation signatures in adjacent normal lung tissues that were best match with COSMIC Signatures 1, 6, 5, 20 and 29 but Signature 4 was not identified in adjacent normal lung tissues. Two de novo mutational signatures were extracted from tumor tissues and one was best matched with COSMIC Signature 4 and the other was highly similar to COSMIC Signatures 2 and 13, both of which are associated with with APOBEC-mediated mutations.

Substantial somatic genomic aberrations are present in the morphologically normal lung tissues adjacent to NSCLC tumors with considerable proportion of somatic mutations shared between tumor and adjacent normal lung tissues supporting the concept of field cancerization. However, the mutational signatures appear to be different in tumors and adjacent normal lung tissues suggesting different mutational processes taking place at different stages of carcinogenesis.

#5364

Somatic mutations in African American and non-Hispanic White young-onset colorectal cancers.

Laura S. Rozek,1 Tingting Qin,1 Erika Koeppe,1 Peter Ulintz,1 Joel K. Greenson,1 Michele L. Cote,2 Elean M. Stoffel1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _Karmanos Cancer Center, Detroit, MI_.

The proportion of colorectal cancers (CRCs) diagnosed at age <50 years is two-fold higher among African-Americans (AAs) compared to non-Hispanic Whites (NHW). Additionally, despite the rising incidence of young-onset CRC, little is known about the molecular characteristics of these as only 23 young-onset tumors were profiled in the TCGA cohort. Studies of CRC diagnosed in older individuals (age >50) demonstrate that CRC tumors in AAs are more often right-sided and MMR proficient, with higher prevalence of KRAS mutations. Risk of death from CRC among AAs with MMR proficient tumors is 73% higher than for NHW, after adjusting for treatment and known prognostic factors. We analyzed archival FFPE CRC tumors diagnosed in individuals age <50 (NHW enrolled in the UM Cancer Genetics Registry N=38 and African American subjects ascertained through the Wayne State University EpiCore N=13) for somatic mutations. Mutation profiling of tumor DNA from 51 CRCs diagnosed age <50 was performed using NGS sequencing with multigene panels in 2 sets (Qiagen GeneRead Cancer panel of 124 genes [N=36 tumors], Qiagen Qiaseq Comprehensive Cancer Panel of 275 genes [15 tumors]). We observed differences in CRC somatic mutations by race and by age. There was marked heterogeneity among tumors with regard to numbers of somatic mutations, with an average of 659 (range 39-10,267) variants detected per tumor, with 19/51 (37%) tumors classified as hypermutated. Among the most frequently mutated genes in young CRC tumors were APC (98% of tumors), PIK3CA (92% of tumors), NOTCH2 (86% of tumors), PTCH1 (82%), BRCA2 (67%), BRCA1 (84%), HNF1A (100%), RAD50 (29%), POLE (27%), MSH6 (39%), and FGFR3 (39%). These mutation profiles differ markedly from those of TCGA older-onset, nonhypermutated tumors. We identified differences in somatic mutations by race, including mutations in PTEN (NHW: 21% vs. AA: 8%), TGFBR2 (NHW: 32% vs. AA: 15%), FBXW7 (NHW: 47% vs. AA: 8%), and KRAS (NHW: 45% vs. AA: 31%). The relatively high prevalence of hypermutated tumors in this racially diverse cohort highlights the critical gaps in knowledge about pathways involved in pathogenesis of young-onset CRCs and how genomic information could guide precision therapies.

#5365

Prospective patient stratification into robust cancer-cell intrinsic subtypes from colorectal cancer biopsies.

Matthew Alderdice,1 Susan D. Richman,2 Simon Gollins,3 Peter Stewart,1 Chris Hurt,4 Richard Adams,4 Amy M. McCorry,1 Aideen Roddy,1 Dale Vimalachandran,5 Claudio Isella,6 Enzo Medico,6 Tim Maughan,7 Darrgh G. McArt,1 Mark Lawler,1 Philip D. Dunne1. 1 _Queen's University Belfast, Belfast, United Kingdom;_ 2 _Leeds Institute of Cancer and Pathology, Leeds, United Kingdom;_ 3 _North Wales Cancer Treatment Centre, Rhyl, United Kingdom;_ 4 _Cardiff University, Cardiff, United Kingdom;_ 5 _Countess of Chester Hospital, Chester, United Kingdom;_ 6 _University of Torino, Torino, Italy;_ 7 _University of Oxford, Oxford, United Kingdom_.

Colorectal cancer (CRC) biopsies underpin accurate diagnosis, but are also relevant for patient stratification in molecularly-guided clinical trials. The consensus molecular subtypes (CMS) and colorectal cancer intrinsic subtypes (CRIS) transcriptional signatures have potential clinical utility for improving prognostic/predictive patient assignment. However, their ability to provide robust classification, in pre-treatment biopsies from multiple regions or at different time points remains untested.

In this study, we undertook a comprehensive assessment of the robustness of CRC transcriptional signatures, including CRIS and CMS, using a range of tumour sampling methodologies currently employed in clinical and translational research. These include analyses using (i) laser-capture microdissected CRC tissue, (ii) eight publically available rectal cancer biopsy data sets (n=543), (iii) serial biopsies (from AXEBeam trial, NCT00828672; n=10), (iv) multi-regional biopsies from colon tumours (n=29 biopsies, n=7 tumours) and (v) pre-treatment biopsies from the phase II rectal cancer trial COPERNCIUS (NCT01263171; n=44). Compared to previous results obtained using CRC resection material, we now demonstrate that CMS classification in biopsy tissue is significantly less capable of reliably classifying patient subtype (43% unknown in biopsy versus 13% unknown in resections, p=0.0001). In contrast, there was no significant difference in classification rate between biopsies and resections when using the CRIS classifier. Additionally, we demonstrated that CRIS provides significantly better spatially- and temporally- robust classification of molecular subtypes in CRC primary tumour tissue compared to CMS (p= 0.003 and p=0.02, respectively).

These findings may have potential to inform ongoing biopsy-based patient stratification in CRC, enabling robust and stable assignment of patients into clinically-informative arms of prospective multi-arm, multi-stage clinical trials.

#5367

Elucidating the factors determining P53 binding and transcriptomic response to DNA damage.

Varsha Sundaresan,1 Ying Li,2 Benedetto DiCiaccio,1 Victor T. Lin,1 Adrian A. Acuna Higaki,1 Nicole F. Pelletier,1 Jessica T. Cheung,1 Lei Zhou1. 1 _Univ. of Florida, Gainesville, FL;_ 2 _Macau University of Science and Technology, Taipa, Macau, China_.

Mutations in tp53 are thought to be one of the most common mechanisms by which cancer cells evade tumor suppression. Yet the exact mechanism of tumor suppression by P53 remains to be fully understood. The transcription factor P53 is activated in response to oncogenic stresses and exerts distinct anti-proliferative functions based on the stressor and cell type. Although numerous mammalian ChIP-Seq studies have identified thousands of P53 binding sites, the functionality of these binding sites remains to be established.

To study how P53 binding following DNA damage differs between cell types, we performed comparisons between P53 ChIP-Seq data from Drosophila embryos at different developmental stages and a Drosophila cell line (Kc167). Differential expression analysis using RNA-Seq indicated that at an early stem cell-like developmental stage of Drosophila embryos there is P53-dependent induction of pro-apoptotic genes in response to irradiation but not in the later differentiated stages. We aim to establish functionally significant P53 binding sites by combining ChIP-Seq and RNA-Seq data from Drosophila embryos as well as Kc167. The functional significance of these binding sites will be identified by CRISPR-Cas9–mediated genome editing. Preliminary genome-wide motif analysis studies also exhibited that homotypic clustering of P53 consensus motifs is associated with P53 binding. We will also perform comparison studies of DNA damage-induced P53 binding in humans, mice and flies to identify analogous patterns.

Preliminary studies revealed that a majority of the P53 binding sites are located at the repetitive regions of the Drosophila genome, especially the Long terminal repeats (LTRs) and Long interspersed nuclear elements (LINEs) as seen previously in mammals by other groups including Botcheva et al and Chang et al. P53 has been known to regulate the expression of retrotransposons and maintain genetic stability by keeping the number of repeats in control. We further aim to see if the functions carried out by P53 differ between repetitive vs. non-repetitive binding regions in Drosophila.

This comparative genomics study will help identify the functionally significant P53 binding sites thus further our understanding of tumor suppression by P53. The knowledge obtained from this study will also be valuable in devising novel strategies to restore P53 function in cancers. 

### Genomic Profiling of Tumors 5

#5368

The evolutionary history of human colitis-associated colorectal cancer.

Ann-Marie Baker,1 William Cross,1 Kathleen Curtius,1 Chang-ho Ryan Choi,1 Ibrahim Al-Bakir,1 Daniel Temko,1 Pierre Martinez,1 Marc Williams,1 Hayley Davis,2 Sujata Biswas,2 Nicholas A. Wright,1 Morgan Moorghen,3 Stephen J. Hayes,4 Manuel Rodriguez-Justo,5 Andrew Silver,6 Lai Mun Wang,7 Marnix Jansen,5 Ailsa L. Hart,3 Simon J. Leedham,2 Trevor A. Graham1. 1 _Barts Cancer Institute, London, United Kingdom;_ 2 _University of Oxford, Oxford, United Kingdom;_ 3 _St Mark's Hospital, London, United Kingdom;_ 4 _University of Manchester, Manchester, United Kingdom;_ 5 _University College London Hospital, London, United Kingdom;_ 6 _Blizard Institute, London, United Kingdom;_ 7 _John Radcliffe Hospital, Oxford, United Kingdom_.

Introduction: Inflammatory bowel disease (IBD) confers an increased lifetime risk of developing colorectal cancer (CRC). Our study aimed to compare the molecular and genetic features of colitis-associated CRC (CA-CRC) to the more common sporadic CRC (S-CRC), and to dissect the evolutionary history of CA-CRC using multi-region next generation sequencing.

Methods: Fresh frozen colectomy specimens were collected from 12 patients with CA-CRC. For each case, whole exome sequencing was performed on multiple regions of tumor, adjacent normal mucosa and blood. Variants in key genes were validated by Sanger sequencing and BaseScope in situ hybridization, and copy numbers were validated by FISH. Copy number profiling (by SNP array or low-pass whole genome sequencing) was performed on low grade dysplasia (LGD; n=28), high grade dysplasia (HGD; n=13), mixed LGD/HGD (n=7) and CA-CRC (n=15). Phylogenetic trees were reconstructed for each case, and evolutionary analysis was used to reveal the temporal sequence of events leading to CA-CRC.

Results: The majority of sequenced tumors (10/12) were microsatellite stable, and these had a median mutation rate of 3.0 single nucleotide alterations (SNAs) per Mb, ~20% higher than that of S-CRC. Mutational signatures identified 'accelerated ageing' in the colitic bowel, likely a consequence of repeated inflammation and regeneration cycles. There was considerable mutational burden in non-dysplastic IBD mucosa (median 47 SNAs), with a median of 24% of these SNAs also clonal within the neighboring CA-CRC, indicating a mutational field. In CA-CRC the most commonly mutated gene was TP53, occurring more frequently than in S-CRC (80% vs. 58%), whereas APC mutations were significantly less common (40% vs. 75%, p=0.03). We analyzed the genetic heterogeneity of CA-CRCs and found that the number of clonal SNAs per tumor was not significantly different to S-CRCs, however CA-CRCs had significantly more SNAs that were unique to one region (p=0.04); this increase in diversity is likely due to an elevated mutation rate. CA-

CRCs were often near-triploid (42%) or near-tetraploid (21%), with many other recurrent copy-number alterations that were distinct from those observed in S-CRC. Phylogenetic analysis revealed that copy number alterations (CNAs) accrue in non-dysplastic bowel, but the transition from LGD to HGD involves a punctuated 'catastrophic' increase in CNA burden.

Conclusions: Multi-region sequencing of CA-CRC has revealed a distinct pathway of colon carcinogenesis, with an increase in mutational burden and heterogeneity compared to S-CRC. Copy-number profiling indicated extensive genomic alterations, with dramatic accrual at the LGD to HGD transition. The significant mutational burden in surrounding normal mucosa indicates field cancerization, which is an encouraging prospect for screening programs; however the likelihood of punctuated evolution may offer a limited window for early detection.

#5369

Tumorigenesis in Lynch syndrome: Somatic mutation profiles compared to sporadic counterparts.

Noora Porkka,1 Alisa Olkinuora,1 Satu Mäki-Nevala,1 Samuli Eldfors,2 Päivi Peltomäki1. 1 _University of Helsinki, Helsinki, Finland;_ 2 _Institute for Molecular Medicine Finland, Helsinki, Finland_.

Lynch syndrome (LS) is the most prevalent cancer predisposition syndrome in which germline mutation in one of four DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6, and PMS2 causes significantly increased lifetime risks of colorectal, endometrial, ovarian, and other cancers. Whether or not breast cancer is part of LS tumor spectrum is currently debated. Moreover, the reasons for organ selectivity in germline mutation carriers are unknown. We have reported that breast carcinoma from LS individuals resembles common breast carcinoma in many respects, but differs with respect to genomic instability (Lotsari et al. 2012, Cancer Res.). To extend these preliminary observations, we are now carrying out an in-depth investigation into the spectra of somatic mutations in LS and sporadic breast carcinomas. All available breast carcinomas among 300 LS families from the nation-wide Hereditary Colon Cancer Registry of Finland have been identified and the tumors investigated for MMR status, DNA methylator phenotype, mechanisms of two-allele inactivation for MMR genes, and somatic mutation status. For the latter, we use massive parallel sequencing as described in our recent publication (Porkka et al. 2017, Oncotarget). To define the unique vs. shared molecular features of LS breast tumorigenesis, profiles of somatic alterations in LS breast carcinomas are compared to those of established LS spectrum carcinomas (colorectal and ovarian) as well as sporadic breast carcinomas with sequence information available from large electronic data-sets. Of 14 LS breast carcinoma samples, 9 (64 %) were MMR-deficient and 5 (36 %) MMR-proficient. This contrasts with LS colorectal and ovarian carcinomas, all of which were MMR-deficient (p=0.0012). MMR-deficient LS breast carcinomas harbored an average of 783 non-synonymous mutations and MMR-proficient tumors 555 mutations (statistically non-significant). In contrast to colorectal and ovarian carcinomas, in which loss of heterozygosity (LOH) or somatic MMR gene mutation provided a second hit consistent with two-hit inactivation in 89 % (25/28) of the tumors, a detectable second hit (mainly LOH) was present in only 43 % (6/14) of breast carcinomas (p=0.0025). Genes harboring high-frequency mutations in at least one-third of tumors were regarded candidates for driver genes. There were 133 such genes in LS colorectal carcinomas, 10 in ovarian carcinomas, and 18 in breast carcinomas. Genes involved in epigenetic regulation were significantly enriched in all three tumor types. In addition, a significant enrichment of NOTCH signaling associated genes characterized LS breast carcinomas. This research is likely to shed light to the mechanisms of organ-specific cancer susceptibility in germline carriers of MMR gene mutations. Our results are expected to guide the surveillance and other clinical management of LS individuals with breast carcinoma.

#5370

Targeted sequencing of actionable mutations to enhance the prognostication of concurrent genes-defined high-risk breast cancers.

Chi-Cheng Huang,1 Chao-Chiang Tu,2 Ching-Shui Huang3. 1 _Fu-Jen Catholic University, New Taipei, Taiwan;_ 2 _Fu-Jen Catholic University Hospital, New Taipei, Taiwan;_ 3 _Cathay General Hospital, Taipei, Taiwan_.

Purpose:

The aim of the study is to perform targeted sequencing to enhance the prognostic power of breast cancer concurrent genes. Somatic mutations with clinical significance will be identified.

Materials and Methods:

Our previous study had identified the concurrent genes signature from the coherent patterns between genomic and transcriptional profiles among Taiwanese breast cancers, and those predicted into the high-risk group were furthered assayed by targeted sequencing. Target-enrichment sequencing was performed with Illumina SolexaTM technology with read length of 150 bp and was analyzed with Agilent SureCallTM software.

Results:

In total, 109 breast cancer samples were collected during surgery and analyzed with AffymetrixTM U133 plus 2.0 microarrays. Breast cancer risk prediction was performed with the published concurrent genes signature. Targeted sequencing of 61 high-risk breast cancers revealed 1061 variants, including 76 pathogenic and 545 likely pathogenic variants based on ACMG classification. The most impacted genes were NOTCH, BRCA1, AR, ERBB2, FANCA, ATM, and BRCA2, harboring 57, 36, 30, 27, 27, 26, and 26 variants. Table 1 showed that the most frequent pathogenic deletions were FGFR1 , ATM, and WT1 (47, 47, and 37 patients) while BRCA1 (rs1799965, non-sense mutation), FGFR2 (missense mutation), and BRCA1 (rs1799949, non-sense mutation) were the most frequent pathogenic SNPs (44, 35, and 11 breast cancers, respectively). The distributions of deletions and pathogenic SNPs were similar across ER+, HER2+, and triple-negative breast cancers.

Conclusion:

This two-stage predictive model, concurrent genes signature risk stratification followed by targeted sequencing of actionable genes, is believed to provide clinical applicability and substantial benefits for Taiwanese breast cancer patients.

Table 1. Pathogenic mutations affecting more than 10 high-risk breast cancers.

---

Gene | Mutation type | Effect | Affected

subjects | Pathway | Potential therapy

FGFR1 | Deletion | FRAME_SHIFT|NM_001174063 | 47 | PI3K, FGF signaling | Dovitinib

ATM | Deletion | FRAME_SHIFT|NM_000051 | 47 | DNA damage response | ATR inhibitor, PARP inhibitor, Chk 1/2 inhibitor

WT1 | Deletion | FRAME_SHIFT|NM_000378 | 37 | Mutated in Wilm's tumor

|

PDGFRA | Deletion | FRAME_SHIFT|NM_006206 | 18 | PDGF signaling | Sorafenib, imatinib, sunitinib

PIK3R | Deletion | FRAME_SHIFT|NM_181523 | 16 | PI3K signaling | Buparlisib

RUNX1 | Deletion | FRAME_SHIFT|NM_001001890 | 15 | Transcription factor, mutated in AML

|

BRCA1

(rs1799965) | SNP | nonsense | 44 | DNA damage response | PARP inhibitor, cisplatin

FGFR2

(chr10:123298158:T:C ) | SNP | missense | 35 | PI3K, FGF signaling | Brivanib, nintedanib, dovitinib

BRCA1

(rs1799949) | SNP | nonsense | 11 | DNA damage response | PARP inhibitor, cisplatin

#5371

The Metastatic Breast Cancer Project: Partnering with patients to accelerate progress in cancer research.

Nikhil Wagle,1 Corrie Painter,2 Elana Anastasio,2 Michael Dunphy,2 Mary McGillicuddy,2 Rachel Stoddard,2 Esha Jain,2 Dewey Kim,2 Simona Di Lascio,1 Brett N. Tompson,2 Sara Balch,2 Beena Thomas,2 Priti Kumari,1 Shawn Johnson,3 Jamie Holloway,2 Ofir Cohen,2 Erik H. Knelson,1 Katie Larkin,2 Sam Pollock,2 Alicia Wong,2 Samira Bahl,2 Simone Maiwald,2 Andrew Zimmer,2 Esme O. Baker,2 Jen Hendry Lapan,2 Scott Sutherland,2 Scott Sassone,2 Viktor Adalsteinsson,2 Eric S. Lander,2 Todd R. Golub2. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Broad Institute of MIT and Harvard, Cambridge, MA;_ 3 _Harvard Medical School, Boston, MA_.

The Metastatic Breast Cancer Project (MBCproject) is a research study that directly engages patients (pts) through social media and advocacy groups, and empowers them to share samples, clinical data, and experiences. The goal is to create a publicly available database of genomic, molecular, clinical, and patient-reported data to enable research. Working with pts and advocates, a website (MBCproject.org) was developed that allows pts with metastatic breast cancer (MBC) to register. Registered pts are sent an online consent form that asks for permission to obtain and analyze their medical records and samples. Once enrolled, pts are sent a saliva kit and asked to mail back a saliva sample, which is used to extract germline DNA. We contact participants' medical providers and obtain medical records and a portion of their stored tumor biopsies. Pts may be asked to mail in a blood sample, which is used to extract cell free DNA (cfDNA). Whole-exome sequencing (WES) is performed on tumor DNA, germline DNA, and cfNDA; transcriptome sequencing is performed on tumor RNA. Clinically annotated genomic data are used to study specific pt cohorts (including outliers) and to identify mechanisms of response and resistance to therapies. All de-identified data are shared via public databases. Study updates are shared with participants regularly. From 10/2015-11/2017, 4237 MBC pts registered, representing over 1,000 institutions. 95% answered the 16-question survey about their cancer, treatments, and demographic information. 2471 (58%) completed the consent form. 2,136 saliva kits were mailed to pts and 1,523 saliva samples were sent in (71%). 408 blood kits were mailed to pts and 175 blood samples have been received for cfDNA analysis. To date, we have obtained medical records from 311 pts and 190 tumors from 127 pts. In 10/2017, all data generated so far were publicly released on cbioportal.org, including WES for 103 tumors from 78 pts linked to clinical data including pathology (22 elements), medical record abstraction including all treatments and timelines/durations (67 elements), and patient-reported data (11 elements). 81% of biopsies included in this release were from the breast and 19% from metastatic sites. 75% were obtained prior to any therapy, 24% following therapy. New data will be released 4/2018 and every six months thereafter, as they are generated. Additional patient-reported data, including treatments, side effects, quality of life, family history, pregnancies, and sites of metastasis, will also be collected and shared. In summary, a patient-driven approach enabled rapid identification of thousands of MBC pts willing to share samples and clinical data. Remote acquisition of medical records, saliva, blood, and tumor tissue for pts across the U.S. is feasible. This shared clinico-genomic database should enable research in MBC and may serve as a model for patient-driven research in other cancers.

#5372

The mutational landscape of primary cutaneous melanoma.

Sofia Y. Chen,1 Rashid Mamunur,1 Jon Laye,2 D. Timothy Bishop,2 Julia A. Newton-Bishop,2 David J. Adams1. 1 _Wellcome Trust Sanger Institute, Cambridge, United Kingdom;_ 2 _University of Leeds, Leeds, United Kingdom_.

Large-scale melanoma genome profiling studies to date have mainly focused on metastatic tumors. To better understand the link between genomic changes and outcome, primary tumors must be studied.

We have designed a comprehensive targeted genomic panel to study genetic alterations in melanoma, and applied this to a large collection of 536 primary melanomas, part of the Leeds Melanoma Cohort. Patients were invited to participate 3 months after diagnosis of primary melanoma. 1% received targeted therapies or immunotherapy and thus, our analysis is reflective of the landscape of primary melanoma in the treatment naïve setting. The sequencing panel allowed us to study genetic alterations in over 500 genes, including more than 150 genes frequently mutated in melanoma and an additional 250 genes linked to other solid tumor types. Both tumor and germline DNA were analyzed for most samples.

The average somatic mutation rates we observed was 15 mutations/MB (0 to 236 mutations/MB), while the nonsynonymous mutational load ranged from 0 to 57 mutations/MB (average 4 mutations/MB). We found that a high mutational burden (highest 20%, >45 nonsynonymous mutations/Mb) positively correlated with better survival (Cox proportional hazards model with age, sex and AJCC stage as additional covariates). Among the top recurrently mutated genes were well established melanoma driver genes such as BRAF (44%), NRAS (28%), NF1 (10%), CDKN2A (15%), TP53 (11%) and ARID2 (8%). We also found hotspot mutations in less well documented melanoma genes, including RQCD1 p.P131L (4.5%).

Altogether, our preliminary data shows a strong correlation between survival and high mutational load in primary melanomas. Our data also suggests a similar landscape of driver mutations in primary and metastatic melanoma, with some variants/genes possibly reflecting the differences in disease stage.

We will further investigate the mutational landscape in our cohort of primary melanomas, including analyses of gene co-occurrence and mutual exclusivity. Additionally, we will use extensive clinical parameters and transcriptomics to examine the possibility of predicting survival and other clinically relevant parameters in our cohort based on patient mutational profiles.

#5373

The Oxford Ovarian Cancer Predict Chemotherapy Response (OXO-PCR) study: Understanding the genomic drivers of primary chemotherapy-resistant microscopic residual disease.

Mara Artibani, Garry Mallett, Dhar Sunanda, Mohammad KaramiNejadRanjbar, Matteo Morotti, Salma ElSahhar, Tatjana Sauka-Spengler, Ahmed A. Ahmed. _University of Oxford, Oxford, United Kingdom_.

In spite of excellent clinical response to primary chemotherapy, more than 70% of patients with high grade serous ovarian cancer (HGSOC) suffer from recurrence within 12 months of completion of treatment. Moreover, clinical and biochemical response are poor surrogates for survival outcomes: patients with complete clinical response following primary treatment usually suffer from recurrence, which can occur as early as within the first six months after treatment. The reason for this paradoxical disconnect between response and survival is unclear. Results from patients treated with neoadjuvant chemotherapy (NACT) suggest that, while complete clinical response following primary treatment is common, complete pathological response is highly unusual in HGSOC. The characterization of microscopic residual disease (MRD) following NACT is required to advance our understanding about the key mechanisms of primary chemotherapy resistance. Accurate and comprehensive characterization of MRD, however, has been very difficult because of the minute nature of these samples. Here we report our results from a prospective phase 0 translational clinical study (OXO-PCR) for the comprehensive genomic characterization of MRD. Twenty women with at least stage IIIC HGSOC were prospectively recruited to this study. All patients had paired samples collected at the time of diagnostic laparoscopy prior to chemotherapy and following at least three cycles of NACT. Chemotherapy response was evaluated using CT and CA125, while site-specific response was assessed through laparoscopy, which was conducted before and after chemotherapy. Laser capture microdissection was then used to obtain MRD which were analyzed through RNA-seq and whole genome sequencing. Our results show that, in contrast to sites where only partial clinical response was observed, complete clinical response and MRD are characterized by differential expression of specific gene signatures related to cell adhesion, actin binding and RNA processing. Most notably, MRD is characterized by the upregulation of a large number of ATP-binding cassette transporters (n = 12). For one patient, recurrence samples were also collected and analyzed: PCA of RNA-seq data from this patient shows that the recurrence resembles the pre-chemotherapy samples, suggesting that MRD represents a distinct cell state that is capable of resisting chemotherapy. Such state is lost following repopulation and emergence of recurrence. Our results show that the approach of comprehensive analysis of MRD is a powerful method for the early characterization of tumor resistance. This has important implications for future design of appropriate maintenance therapies to prevent recurrence.

#5374

Uterine sarcomas: A preliminary study for characterization of a specific molecular signature for Brazilian patients.

Leonardo T. Costa, Giovana T. Torrezan, Claudia A. Paula, Glauco B. Neto, Edmund C. Baracat, Dirce M. Carraro, Kátia C. Carvalho. _Univ. of Sao Paulo Faculty of Medicine, São Paulo, Brazil_.

Background: Uterine sarcomas are the most common gynecological sarcomas, comprising around 90% of all of the cases. These tumors can arise either from the endometrial stroma or from the muscle itself. They are genetically heterogeneous tumors and have no specific molecular and radiological diagnosis, or treatment.

Histologically, uterine sarcomas are classified according to their cell of origin and are divided into: carcinosarcomas (CCS), leiomyosarcomas (LMS), endometrial stromal sarcoma (ESS) and adenosarcomas (ADS). There are many factors that can be attributed to uterine sarcomas etiopathogeny such as ethnic, hormonal and hereditary factors. However, few studies were performed searching for specific molecular signature in these tumors, especially concerning a singular population. Objective: To assess the oncogenic profile of Brazilian patients with different histological types of uterine sarcomas. Methods: Until now, we performed the Next Generation Sequencing for 13 samples of uterine sarcomas (comprising: 5 CCS, 3 LMS, 4 SEE and 1 ADS) using the Ion Proton System (ThermoFisher) with the Comprehensive Cancer Panel (containing 409 genes related to carcinogenesis). Results: The genetic sequencing profile analysis showed 140 specific mutations in our samples. Among them, the most common were TP53 (58%), JAK3 (33%), NF1 (33%), KMT2D (33%), MTOR (25%) and PIK3CA (25%). Some findings corroborate the literature on uterine sarcomas, with an exception of the KMT2D gene, that despite of being known and described in other tumors, including the ones of the genital tract, such as vulva, ovary and endometrium, has never been described in uterine sarcomas Conclusion: We found the most frequent mutations in 6 genes in our samples. Only one mutation had not been described before for uterine sarcomas (KMT2D). These are preliminary results, and further analyzes are ongoing.Key words: Uterine Sarcomas, Carcinosarcomas, Next Generation Sequencing

#5375

Whole-genome sequencing reveals genomic signatures associated with the immunologic microenvironments in Chinese NSCLC patients.

Cheng Wang, Juncheng Dai, Yayun Gu, Zhibin Hu, Hongbing Shen. _Nanjing Medical University, Nanjing, China_.

Over the past decades, next-generation sequencing has accelerated the systematic characterization of genomic events, including single base substitutions and small insertions/deletions (indels), and has yielded substantial insights into the unique and shared genomic features of the two NSCLC subtypes. Genomic studies of lung cancer have mainly been conducted in patients from Western countries; however, Chinese lung cancer patients have distinct epidemiologic and genomic features, highlighting the presence of specific etiologic mechanisms in addition to smoking. To reveal the underlying mechanisms, we presented a comprehensive genomic landscape of 149 Non-Small Cell Lung Cancer (NSCLC) cases. We identified 15 potential driver genes and three mutational signatures, two of which have been connected with APOBEC enzymes and smoking. In addition, we revealed that Chinese patients are specially characterized by not only highly clustered and functional EGFR mutations but also a mutational signature, that is associated with inflammatory tumor-infiltrating lymphocytes (P = 0.001). The EGFR mutation rate is significantly increased with the proportion of the MS3 signature (P<0.001). As a classic tumor-enabling hallmark, inflammatory infiltration may contribute to the accumulation of EGFR mutations, especially in never-smokers. Additionally, patients with a higher proportion of the MS3 signature carried a higher contribution of characterized chromosomal rearrangements (P<0.001). The results revealed specific genomic features in the inflammatory microenvironments in Chinese patients with potential implications for understanding the etiology, prevention and therapy of NSCLC.

#5376

Whole-exome sequencing of acquired nevi identifies novel mechanisms for development and maintenance of benign neoplasms.

Mitchell S. Stark,1 Jean-Marie Tan,1 Lisa Tom,1 Kasturee Jagirdar,1 Duncan Lambie,2 Helmut Schaider,1 H. Peter Soyer,1 Richard A. Sturm1. 1 _Univ. of Queensland Diamantina Institute, Brisbane, Australia;_ 2 _IQ Pathology, Brisbane, Australia_.

Background: Acquired melanocytic nevi (AMN) are often mimicking melanoma and ~30% of all melanomas arise within a pre-existing nevus. However, the melanoma transformation rate of each nevus is rare despite the detection of oncogenic mutations BRAF or NRAS mutations in 100% of nevi.Objective: To identify the underlying genetic mechanisms for nevus development.Methods: Nevi, adjacent normal skin, and saliva were sampled from a cross-sectional study. All nevi were clinically, dermoscopically, and histopathologically documented. Using whole-exome sequencing (WES) we assessed the somatic mutational landscape, mutation signatures and copy-number aberrations (CNA) in 30 acquired melanocytic nevi and matching normal skin.Results: In addition to identifying somatic mutations, we confirm the presence of mutational signatures relating to age and ultra-violet radiation (UVR) which mirrors those observed in melanomas of the skin. In matching normal skin of all nevi, we rarely observed the presence of a UVR mutation signature (10% vs. 97% in nevi) despite being exposed to the same amount of UVR as the associated naevus. Instead, we have identified the predominance of defective DNA repair mutation signatures (93%) which we postulate creates the environment for melanocyte transformation sufficient for nevi and perhaps de novo melanoma growth. In copy number aberration (CNA) analysis, in nevi with copy number loss of tumour suppressor genes (TSG), these were balanced by loss of potent oncogenes. Moreover, reticular and non-specific patterned nevi revealed an increased (p<0.0001) number of CNA as compared with globular patterned nevi. Conclusion: The mutation signature data generated in this study confirms that UVR and defective DNA repair mechanism strongly contributes to nevogenesis. The observed copy number changes reflect at a genomic level, the dermoscopic differences of AMN. Lastly, we propose that the balanced loss of TSGs and oncogenes is a protective mechanism of AMN.

#5377

Tracking genomic and epigenomic evolution from preneoplastic lesions to lung adenocarcinoma by multiregion sequencing.

Xin Hu,1 Junya Fujimoto,1 Lisha Ying,2 Runzhe Chen,1 Marcos Roberto Estecio,1 Chi-Wan Chow,1 Jaime Rodriguez Canales,1 Xingzhi Song,1 Xizeng Mao,1 Paul Scheet,1 Humam Kadara,1 Edwin R. Parra Cuentas Cuentas,1 Carmen Behrens,1 Chang-Jiun Wu,1 J. Jack Lee,1 Mara Antonoff,1 Ara A Vaporciyan,1 Stephen Swisher,1 Jianhua zhang,1 John Heymach,1 Waun Ki Hong,1 Ignacio I. Wistuba,1 Wenyong Sun,2 Jinlin Hu,2 P. Andrew Futreal,1 Dan Su,2 Jianjun Zhang1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Zhejiang Cancer Hospital, Hangzhou, China_.

Background: Carcinogenesis may result from accumulation of genomic and epigenomic aberrations. It has been postulated that atypical adenomatous hyperplasia (AAH) represents lung preneoplastic lesion that may progress to adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) and further to frankly invasive adenocarcinoma (ADC). Yet the pathologic definition and management of these lesions remain controversial due to lack of sufficient molecular evidences. This aim of this study is to delineate the temporal molecular carcinogenetic events and evolutionary process during the evolution from preneoplastic lesions to early-stage adenocarcinoma.

Methods: We have collected 116 resected pre- and early neoplastic lung lesions including AAH (N=22), AIS (N=27), MIA (N=54) and ADC (N=13) from 53 patients, including 39 patients presenting with multifocal disease and 23 patients carrying more than two different types of lesions. Two to five spatially separated regions were subjected to whole-exome sequencing and reduced representation bisulfite sequencing.

Results: Mutation burden progressively increases from AAH (average SNVs 0.57/Mb) to AIS (2.04/MB), to MIA (2.98/MB) and ADC (5.4/MB), p <2.2e-16 with evidence of positive selection of non-silent mutations (60.2% in AAH, 69.7% in AIS, 74.4% in MIA, 84.4% in ADC, p = 0.009). APOBEC signature also progressively increases with APOBEC enrichment scores of 0.94 in AAH, 0.99 in AIS, 1.04 in MIA and 1.28 in ADC (p = 0.011). In addition, genomic heterogeneity becomes more complex with neoplastic evolution with tumor allelic frequency-derived median Shannon index of 1.24 in AAH, 1.52 in AIS, 1.64 in MIA and 1.79 in ADC (p = 0.0004). On the other hand, the proportion of trunk mutations (detected in all regions within the same lesion) progressively increased (35.6% in AAH, 73.6% in AIS, 73.1% in MIA, 70.8% in ADC and p = 1.758e-07). Phylogenetic analysis revealed varying evolutional processes in different pre- and early neoplastic lung lesions with progressive increase in perturbance of genes involved MAPK pathway. Certain key driver mutations were found to be early molecular events occurring at the stage of AAH (e.g., KRAS), while others tend to occur at a later stage (e.g., EGFR). Copy number alterations and genomic doubling were observed in AIS, MIA and ADC, but not in AAH. In addition, DNA methylation profiling revealed that epigenome aberrations fuel preinvasive progression from AIS to MIA and ADC, with parallel phylogenic evolution pattern of the genome.

Conclusions: We provide molecular evidence supporting the pathologic model of early lung carcinogenesis from AAH, to AIS, MIA and ADC. With disease evolution, both genomic and epigenomic landscape of lung neoplastic lesions has become progressively more complex along with sequential acquisition of molecular events with concomitant selective sweep of subclone in preneoplasias.

#5378

Whole exome and RNA sequencing identify novel somatic mutations in gangliogliomas.

Suely K. Marie,1 Sueli M. Oba-Shinjo,1 Antonio M. Lerario2. 1 _University of Sao Paulo, Sao Paulo, Brazil;_ 2 _University of Michigan, Ann Arbor, MI_.

Gangliogliomas (GG) are rare, well differentiated, slow growing neuroepithelial tumors, composed of neoplastic mature ganglion cells and neoplastic glial cells. GG comprises 0.4% of all CNS primary tumors, and 70% of GG cases are diagnosed in patients with long-term temporal lobe epilepsy. The molecular pathogenesis of GG is poorly understood. Few groups have described a high frequency of a hotspot BRAF p.V600E mutation. More recently, a co-occurrence of histone H3 p.K27M and BRAF p.V600E mutations was reported in a pediatric midline grade I GG. However, the rarity of these tumors poses challenges to uncovering a more comprehensive landscape of somatic alterations. In this study, we describe the somatic mutational profiles of 12 GG from the University of Sao Paulo Medical School in one of the largest GG case series reported to date. We performed paired germline and somatic whole exome sequencing (WES) and RNA-Seq in all cases: 9 males and 3 females with ages ranging from 1 to 49 years (mean 17 ± 13 years). These tumors were located in the temporal lobe (8), frontal lobe (1), occipital lobe (1), intraventricular region (1), and intramedullary region (1). We used the Nextera Exome Enrichment Kit (Illumina) to perform WES, and the TruSeq Stranded Total RNA (Illumina) to perform RNA-Seq. Sequencing was performed in the Illumina HiSeq 2000. We aligned reads from WES and RNA-Seq libraries to the reference genome (hg19) using BWA and STAR, respectively. We used freebayes to perform variant call from germline WES paired with the respective tumor library (WES or RNA-Seq). We used the vcfcompare tool from vcflib to filter out germline variants and annovar to annotate the final list of variants. We identified mutations in cancer-related genes in 6/12 GGs: hotspot BRAF p.V600E in 4/6 cases, biallelic NF1 mutations (p.Y333X/p.R2258X) co-occurring with hotspot H3F3A (p.K27M) and FGFR1 (p.N546K) mutations in a 13-yo female with intraventricular GG, and, finally, biallelic hotspot TP53 mutations (p.V173M, p.P172T) combined with a hotspot IDH1 (p.R132H) mutation in a 26-yo male with frontal GG. Interestingly, the same combination of mutations observed in one of our patients (FGFR1 p.N546K/H3F3A p.K27M) was recently described in a diffuse leptomeningeal tumor with glial and neuronal markers. In summary, we identified novel mutations in cancer-related genes in 6 GG (50% of our cohort), including cases with mutations in more than one known cancer driver gene. In one of the largest GG case series reported to date, this study expands the spectrum of somatic alterations in GGs to include mutations in genes recurrently altered in higher-grade gliomas, such as IDH1, NF1, and TP53.

#5379

Stratification of colorectal cancer patients based on various sequencing platforms and tumor mutational burden.

Fang Yin Lo,1 Claire Olson,1 Kerry Deutsch,1 Tuuli Saloranta,1 Inah Golez,1 Timothy Yeatman,2 Steven Anderson,3 Anup Madan1. 1 _Covance Genomics Laboratory, Redmond, WA;_ 2 _Gibbs Cancer Center, Spartanburg, SC;_ 3 _Covance Genomics Laboratory, Durham, NC_.

Colorectal cancer (CRC) is the third most common type of cancer in the United States. Although chemotherapy, radiation and targeted therapies can improve survival rates, recent studies have shown the potential benefit of immunotherapies to improve outcomes for patients with advanced CRC. Targeted therapies that use monoclonal antibodies (mAbs) to EGFR have been shown to benefit some CRC patients. Until recently, KRAS has been the only predictive biomarker for anti-EGFR therapy for metastatic CRC. However, 40% to 60% of patients with wild-type KRAS do not respond to anti-EGFR therapy. Therefore, to accurately predict patients' response to treatments and improve clinical outcomes, additional prediction and treatment methods are imperative. One of the many efforts to improve prediction for CRC patient's response to the anti-EGFR therapy is the development of gene expression based RAS signature scores for identification of RAS activated tumors independent of mutations in the KRAS gene. There is also considerable effort being placed on combinations of targeted therapy and immunotherapies to improve responses for these cancers. Previously, we have stratified 55 CRC samples by applying a RAS gene signature score which measures MEK pathway functional output independent of tumor genotype. We showed that samples that have RAS activating mutations such as KRAS and BRAF have significant higher RAS scores (p<0.001). Here we investigate 120 colorectal cancer patients using exome sequencing, targeted panel sequencing, and RNA sequencing. Specifically, we acquired 40 normal-tumor pair samples and performed GATK tumor-normal pair mutation calling. Among these 120 samples, 65 of them are from patients treated with either cetuximab or panitumumab. Continued from our previous study, we stratified these samples based on calculated RAS scores, and showed how the results correlate with clinical outcome. Tumor mutational burden (TMB) can help predict the likelihood that a patient will benefit from certain immunotherapies; however, clinical applicability is limited due to cost and computing requirements of whole-exome sequencing to detect variants with high degree of accuracy. Therefore it would be helpful if TMB can be identified based on targeted sequencing. Here we investigate how TMB calculated based on different sequencing platform correlates with each other and whether targeted sequencing panel can be used instead of exome sequencing for stratifying patients. In addition, we investigated if Illumina's global screening array can be used for TMB analysis. Furthermore, to investigate the potential immune reactivity in these CRC samples, and thereby the potential benefit of immunotherapy, we performed in-silico prediction of neo-antigens and peptide binding affinity between tumor antigens derived from mutations and human HLA alleles.

#5380

Whole-exome sequencing of Brazilian non-small cell lung cancer.

Leticia Ferro Leal, Adriane Feijo Evangelista, Pedro R. de Marchi, Ysadhora Christiane Camargo Rodrigues, Eduardo Caetano Albino da Silva, Rui Manuel Reis. _Barretos Cancer Hospital, Barretos, SP, Brazil_.

Background: Non-Small Cell Lung Cancer (NSCLC) is the most common type of lung cancer with low survival rates. NSCLC patients harboring EGFR mutations are benefited by targeted therapy with tyrosine kinase inhibitors. The mutational profiling and the knowledge about recurrent mutations in Brazilian NSCLC is limited. Therefore, the detection of the mutation landscape of Brazilian NSCLC tumors is crucial for tailored therapy. Aim: To evaluate the mutational profiling of NSCLC of a Brazilian series.

Methods: We analyzed a series of 40 NSCLC patients from Barretos Cancer Hospital. Clinical staging and exposure data were collected from all patients. Exome sequencing was performed in paired tumor/blood tissue DNA using the Illumina HiSeq2500™ System. Germline variations were excluded and the Genome Analysis Toolkit and VarScan were used for variants annotation and mutation detection.

Results: Overall, 29 (72.5%) of cases were adenocarcinomas and 11 (27.5%) were squamous cell carcinoma (SCC) and the series was enriched by initial stages cases (I, n=16; II, n=12; III, n=6; IV, n=6). Regarding tobacco exposure, 33 patients were current smokers or former smokers and 7 patients were never smokers. In accordance, most of the cases presented the mutation signature 4, related to tobacco exposure, which is characterized by C>A substitutions and signature 29, related to tobacco chewing habit, which is characterized by C>A and C>T substitutions. All SCC patients were current or former smokers. TP53 gene was the most recurrently mutated gene observed in 48% (n=14) of the adenocarcinomas and 100% (n=11) of SCC cases (p=0,003). In adenocarcinomas, besides of TP53 gene, recurrent mutations were also found in KRAS (28%; n=8), STK11(21%; n=6), USP36 and APOB (14%; n=4/each), POLQ and CHD5 (10% n=3/each), EGFR and ANO3 (7%; n=2/each) and PDGFRA, BRAF, ERBB2, ERBB4, NRAS, RB1, CD22, CTNNA2 and ATR mutations were identified in only one adenocarcinoma case (3%). In SCC, besides of TP53 gene, recurrent mutations were also found in RB1, CD22, CTNNA2 and CHD7 (27%; n=3/each), ATR and APOB (18%; n=2/each), KRAS, PDGFRA, ERBB2, ERBB4, POLQ, CHD5 and ANO3 were identified in only one SCC case (9%). EGFR, STK11, BRAF, NRAS and USP36 mutations were exclusively found in adenocarcinomas and CHD7 mutations were exclusively found in SCC.

Conclusions: The mutational landscape of lung adenocarcinoma and SCC are clearly different. A noteworthy number of pathogenic mutations was identified in this initial stages-enriched series. Our results may contribute to improve the knowledge about the molecular pathogenesis of NSCLC to better guide the clinical management with targeted therapies.

#5381

Understanding the genesis and oncogenic consequences of tandem duplicator phenotypes in human cancers.

Francesca Menghi,1 Floris Barthel,1 Vinod Yadav,1 Ming Tang,2 Bo Ji,3 Gregory Carter,3 Jos Jonkers,4 Roel Verhaak,1 Ralph Scully,5 Ed Liu3. 1 _The Jackson Laboratory for Genomic Medicine, Farmington, CT;_ 2 _MD Anderson Cancer Center, Houston, TX;_ 3 _The Jackson Laboratory, Bar Harbor, ME;_ 4 _Netherlands Cancer Institute, Amsterdam, Netherlands;_ 5 _Beth Israel Deaconess Medical Center, Boston, MA_.

Genome-scale instability configurations in cancer genomes are commonly found across different types of cancers. We and others have described an enrichment of head-to-tail somatic segmental tandem duplications (TDs) in primarily breast and ovarian cancers, a configuration known as the Tandem Duplicator Phenotype (TDP). Here, we perform a meta-analysis of 2,717 human cancer genomes, representing 30 different tumor types and refine the TDP classification based on the identification of highly specific and recurrent TD span profiles. Overall, we identify the TDP as a highly recurrent chromosomal instability configuration, occurring in 14% of the cancer genomes examined. The TDP is remarkably prevalent in triple negative breast cancer (50%), ovarian cancer (55%), and uterine cancers (45%), but is infrequent in most other cancer types. In TDP tumors, TD span sizes can be classified into three major discrete intervals with modal values of 10 Kb, 230 Kb, and 1.7 Mb. TDPs with a dominant TD modal span size of 10 Kb are strongly associated with conjoint TP53 mutations and BRCA1 deficiency across different tumor types. TDPs with larger span sizes (230 Kb and 1.7 Mb) are associated with CCNE1 pathway activation and CRKRS/CDK12 gene disruptions. We proved the driver role of the conjoint abrogation of the BRCA1 and TP53 genes for the induction of TDP cancers with the 10 Kb predominant span peak by generating mammary cancers with a Tandem Duplicator Phenotype of the ~10 Kb TD span configuration type in genetically modified mouse models harboring deleterious mutations in only these same two genes. Lastly, we describe how heterogeneous combinations of tumor suppressors and chromatin topologically associating domains are disrupted, and oncogene and tissue-specific super enhancers are duplicated as a consequence of TD formation in TDP tumors. In summary, our work unifies a number of observations around a specific cancer genomic signature, shows how a number of genetic drivers converge on creating the tandem duplicator phenotype, and how the downstream consequences of the TDs generate oncogenic diversity that allows for further formation of genetic subgroups. The implications of TDP-based stratification for therapy response are currently under investigation.

#5382

The evaluation of the DEPArray NXT™ as a clinical application for somatic tumor profiling from FFPE tissue.

Jasmina Uvalic,1 Kevin Kelly,1 Daniel Bergeron,1 Yadwinder Deol,2 Claudio Forcato,2 Honey Reddi1. 1 _The Jackson Laboratory, Farmington, CT;_ 2 _Menarini Silicon Biosystems, Inc., Italy_.

Introduction:

Current methods of somatic tumor profiling involve the use of FFPE tissues from tumor biopsies, which present challenges in clinical testing due to poor qualities and overall availability of tissue. The use of the DEPArray allows one to address some of the issues that come with the use of FFPE samples by allowing for isolation of pure tumor and pure stromal populations for downstream tumor analysis. Here we evaluate the use of the DEPArray NXT™ to complement current clinical somatic testing workflows as a means to reduce the tumor content requirement for clinical samples as well as to isolate pure tumor populations to accompany and build on our current clinical offerings.

Methods:

Thirty micron sections were prepared from 10 FFPE samples from varying tumor types. The samples had tumor contents ranging from 20-75% used to determine clinical specificity. Immunostaining was performed on individual cell suspensions of the samples, with vimentin and cytokeratin to be able to isolate and recover pure populations of stromal and tumor cells respectively, using the DEPArray NXT workflow. Pure populations were recovered and libraries were prepared using an amplicon panel for NGS, targeting over 60 genes that are known to be associated with cancer. Samples were sequenced using the Illumina MiSeq and NextSeq.

Results:

On average two hundred pure stromal and pure tumor cell populations were recovered from each of the samples evaluated and an average of 2.5 million reads were generated per sample. A subset of the sequenced samples underwent in-depth comparative analysis of stromal, tumor and unsorted cell populations. Results show that even from ~200 cells recovered, CNA's and SNV's were still able to be called within the pure tumor population compared to that of the stromal. The unsorted cell population mimicked more of the stromal population, further highlighting the need for cell selection prior to library preparation.

Conclusion:

The DEPArray NXT was successfully able to isolate pure tumor and stromal cells which generated adequate libraries for sequencing and downstream analysis which allows for greater reliability and precision in making accurate variant calls compared to current best practices. The DEPArray aids in lowering the tumor content sample requirements as we are able to target and recover pure cell populations as opposed to current methods of extraction which extract all cells present, resulting in analysis of DNA that is not relevant to the tumor. Overall, cells isolated purely from the underlying tumor will provide the physician and the patient clearer test results to help manage and treat the patient with a more specific course of treatment based on the identified variants. This also enables the offering of testing for those specific cases that would have been turned away due to poor quality and quantity of tissue obtained from the tumor biopsies.

#5383

Tumor evolution of uterine leiomyosarcoma from a benign leiomyoma precursor.

Netta Mäkinen,1 Terhi Ahvenainen,1 Pernilla von Nandelstadh,1 Ralf Bützow,2 Pia Vahteristo1. 1 _University of Helsinki, Helsinki, Finland;_ 2 _Helsinki University Hospital, Helsinki, Finland_.

Uterine leiomyosarcoma (ULMS) is a highly aggressive smooth muscle tumor with poorly understood pathogenesis. Although ULMS is believed to develop as a de novo tumor, it has been suggested that in some cases uterine leiomyoma (UL) could serve as a precursor lesion for the pathogenesis. ULs are extremely common tumors affecting up to 70% of women. Whereas 90% of ULs are classified as conventional ULs, 10% of the tumors represent subtype histopathologies, some of which have features associated with malignancy. These subtypes include tumors with bizarre nuclei and increased cellularity. In this study, we investigated the genetic and molecular background of one ULMS showing simultaneously mixed histopathologies of UL with bizarre nuclei and cellular UL.

The study material consisted of four formalin-fixed paraffin-embedded samples having distinct areas of ULMS, UL with bizarre nuclei, and cellular UL. Fallopian tube sample was used as a normal control. Libraries for exome sequencing were prepared with KAPA Library Preparation Kit and subjected to exome capture with NimbleGen SeqCap EZ System (Roche). Paired-end sequencing was performed with HiSeq2500 (Illumina). SNP genotyping data was produced with Infinium Omni2.5-8 Kit (Illumina). Immunohistochemistry was performed to proteins commonly displaying aberrant expression in UL and/or ULMS. Telomere-specific fluorescence in situ hybridization was conducted to detect alternatively lengthened telomeres (ALT), a feature often associated with ULMS pathogenesis.

Exome sequencing data and protein expression analyses revealed that all tumor compartments were negative for UL driver alterations, including MED12 mutations, HMGA2 overexpression and biallelic FH inactivation. Data from exome sequencing indicated that ULMS shared the largest number of variants with UL with bizarre nuclei, including the pathogenic splice site mutation c.994-1G>A in tumor protein 53 (TP53). SNP genotyping data showed identical and large chromosomal aberrations in 6q, 9p, 10q and 19q in ULMS and UL with bizarre nuclei. No such aberrations were shared between ULMS and cellular UL. All tumor compartments shared changes in chromosomes 1q, 2p, 12q, 13q, 17q and 17p. ULMS showed prominent ALT, whereas UL with bizarre nuclei had a weaker ALT phenotype. Cellular UL had normal telomere lengths.

In conclusion, these results suggest that the genome, including telomeres, of UL with bizarre nuclei resembles that of ULMS. Large genomic events have probably occurred in UL with bizarre nuclei during tumor evolution, leading to unstable chromosomes, which in turn favored the development of ULMS. Also, all tumor compartments displayed shared genetic variation, suggesting a common evolutionary origin. Understanding the pathogenesis of ULMS and the malignant potential of ULs is clinically highly relevant, as it may improve the diagnosis, prevent malignant transformation and enable the design of new treatments.

#5384

The Angiosarcoma Project: Generating the genomic landscape of an exceedingly rare cancer through a nationwide patient-driven initiative.

Michael Dunphy,1 Esha Jain,1 Elana Anastasio,1 Mary McGillicuddy,1 Rachel Stoddard,1 Beena Thomas,1 Sara Balch,1 Kristin Anderka,1 Katie Larkin,1 Niall Lennon,1 Yen-Lin Chen,2 Andrew Zimmer,1 Esme O. Baker,1 Simone Maiwald,1 Jen Hendrey Lapan,1 Jason Hornick,3 Chandrajit Raut,3 George Demetri,4 Eric Lander,1 Todd Golub,1 Nikhil Wagle,4 Corrie Painter1. 1 _Broad Institute of MIT and Harvard, Cambridge, MA;_ 2 _Massachusetts General Hospital, Boston, MA;_ 3 _Brigham and Women's Hospital, Boston, MA;_ 4 _Dana-Farber Cancer Institute, Boston, MA_.

Angiosarcoma (AS) is an exceedingly rare soft tissue sarcoma, with an incidence of 300 cases/yr and a 5-year disease-specific survival of 30%. The low incidence has impeded large-scale research efforts that may lead to improved clinical outcomes. To address this, we launched a nationwide clinical-genomics study in order to empower patients to accelerate research by sharing their normal and tumor samples and clinical information remotely. Patients can access the study through an online portal (ASCproject.org). Enrolled patients are mailed saliva and blood draw kits. The study team obtains medical records and stored FFPE tumor samples. All received FFPE samples are examined by an expert pathologist to confirm a diagnosis of angiosarcoma. In order to validate that our processes would enable the generation of a robust dataset from tissues acquired from multiple institutions, we sought to characterize previously described genes known to be altered in angiosarcoma (e.g., TP53, NF1, KDR, BRCA2, MET, ARID1A, POT1, BRCA1, ASXL1, KDM6A, BRAF, SETD2, PTPRB, NRAS). A total of 251 patients have enrolled since the project launched in March of 2017. Primary locations of AS are primary breast 59 (25%), breast with prior radiation 45 (19%), head/face/neck/scalp 52 (22%), bone/limb 26 (11%), abdomen 5 (2%), heart 5 (2%), lung 2 (1%), liver 1 (1%), lymph 1 (0.4%), multiple locations 25 (11%), and other locations 12 (5%); 107 (52%) reported being disease free at the time of enrollment. To date, we have received 129 saliva kits, 106 medical records, 19 blood samples, and 36 tissue samples. Whole-exome sequencing (WES) was performed on 21 FFPE/saliva matched pairs with a goal mean target coverage of 150x for tumors. Ultra-low pass whole-genome sequencing (0.1x) was performed on cell free DNA (cfDNA) from plasma in order to determine tumor fraction. Of 10 cfDNA samples sequenced, 4 samples met criteria to perform WES. Additionally, transcriptome sequencing was performed on 9 FFPE samples. Sequence data processing and analysis has been completed on the first 10 samples and is in progress for the subsequent samples. Alterations were detected in genes previously described to be affected in angiosarcoma. Recurrent mutations in TP53 were detected in 50% (5/10) of analyzed samples, comprising 3 missense mutations, 1 frameshift deletion, and 1 frameshift insertion. Alterations were seen in at least one sample in all other genes selected for this initial analysis. This initiative demonstrates the feasibility of studying tissues from geographically dispersed patients and serves as proof of concept that patient-driven genomics efforts can democratize research for exceedingly rare cancers. Enrollment is still in progress, and additional samples will be sequenced and analyzed at scale. The data generated from these studies will be deposited into the public domain in six-month intervals.

#5385

Supporting neoantigen discovery and monitoring in plasma through analytical validation of a deep Augmented Content Enhanced (ACE) exome.

Ravi K. Alla, Robin Li, Sean Michael Boyle, Shujun Luo, Rena McClory, Rob McCord, John West, Richard Chen. _Personalis, Inc., Menlo Park, CA_.

Neoantigens are increasingly critical in immuno-oncology as a therapeutic target for neoantigen-based personalized cancer vaccines and as a potential biomarker for immunotherapy response. An important step in identifying neoantigens is comprehensive exome and transcriptome sequencing of a tumor biopsy sample and the matched normal to enable identification of putative neoantigens derived from mutations in any gene in the genome. However, as tumor biopsy samples cannot always be obtained, and because tumor heterogeneity can result in an incomplete set of neoantigens from a single biopsy, we developed our Accuracy and Content Enhanced (ACE) circulating tumor DNA (ctDNA) Exome to (1) identify neoantigens in cell free DNA (cfDNA) as a complement to tumor biopsy derived neoantigens and (2) track neoantigens in the cfDNA post immuno-therapy treatment. Our ACE ctDNA Exome covers >20,000 genes as neoantigens can occur in any gene across the genome. This is in contrast to most current tumor cfDNA tests which are designed to assess a small number of variants or genes (often <100), and as such will miss many putative neoantigens. Our ACE ctDNA Exome is performed at very high sequencing depth to accurately identify lower allele frequency variants that are candidate neoantigens.To demonstrate the utility of the ACE ctDNA Exome for both identification and monitoring of neoantigens directly from cfDNA, we show preliminary results from two studies. Firstly, to monitor colorectal cancer (CRC) tumor-derived variants in cfDNA, we sequenced 8 late stage paired patient tumor, normal and cfDNA samples. We identify an average of 380 somatic events ranging from 10% to 100% Allele Frequency (AF) in each tumor and, through site-specific interrogation, were able to corroborate ~60%-98% of these events in cfDNA. This high concordance demonstrates strong sensitivity for tumor derived mutation monitoring using our platform. Secondly, to understand our de novo variant Limit of Detection (LOD), we assessed the sensitivity of our ACE ctDNA Exome for detecting single nucleotide variants (SNVs) down to 1% AF. We chose 3 Seracare cfDNA standards as our "Gold Set". These samples harbor 25 SNVs at various AFs (2%, 1%, 0% control). We analyzed these data in a tumor-normal fashion using the cell line GM24385 as matched normal, identifying 24/25 (96%) variants in the 1% and 2% standards. To perform a genome wide assessment of sensitivity, we obtained cfDNA from 2 healthy donors, mixed them to create somatic variants with AFs down to 0.625% with analytical sensitivity calculated against >10,000 variants. Initial results show that our deep ACE ctDNA Exome is able to identify low allele frequency SNVs with high accuracy. These results show the potential of using ACE ctDNA Exome to identify and monitor neoantigens as a complement to the results from sequencing of the tumor biopsy alone.

#5386

The Seven Bridges Cancer Genomics Cloud: Enabling reproducible and cost-effective analysis in the cloud.

Milos Jordanski,1 Robert Bierman,2 Erik Lehnert,3 Ana Damljanovic,4 Eric Freeman,2 Gillian Hsieh,2 Julia Salzman2. 1 _Seven Bridges Genomics, Belgrade, MA;_ 2 _Stanford University, Stanford, CA;_ 3 _Seven Bridges Genomics, Cambridge, MA;_ 4 _Seven Bridges Genomics, Belgrade, Serbia_.

Next-generation sequencing has led to the generation of petabytes of public data with the potential to significantly advance biomedical research. The Cancer Genome Atlas (TCGA) network alone, for example, has produced more than 2.5 petabytes of data. The logistical difficulties that researchers face while accessing such large datasets continue to present challenges, however. Downloading the complete TCGA dataset to a local data store can take several weeks or more, and, traditionally, integrated analysis has required resources available only to a limited number of researchers with access to large institutional compute clusters. In 2015, the National Cancer Institute (NCI) launched three Cancer Genomics Cloud Pilots, including the Seven Bridges Cancer Genomics Cloud (CGC; cancergenomicscloud.org), to democratize access to datasets such as TCGA by colocalizing data and computational resources in the cloud. In 2017, NCI expanded this effort to the development of an NCI Cancer Research Data Commons in which the CGC and other Cloud Pilots, now known as Cloud Resources, continue to deliver cloud-based access to petabyte-scale data and analysis resources.

The Seven Bridges CGC is a customizable and scalable data access and analysis platform that connects users via the web to extensive public datasets, including multi-omic data from TCGA, the Simons Genome Diversity Project, the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative, the International Cancer Genome Consortium (ICGC), the Cancer Cell Line Encyclopedia, The Cancer Imaging Archive (TCIA), and the Clinical Proteomic Tumor Analysis Consortium (CPTAC). The CGC enables collaborative, reproducible analysis across both public and private cohorts through access to customizable workspaces, a public toolkit containing more than 300 common analytical tools and workflows, and additional resources including an open-source Software Development Kit known as Rabix. Since the launch of the CGC in early 2016, more than 2500 researchers from more than 150 institutions in 30 countries have used the platform to deploy more than 5,000 applications to perform analyses representing more than 100 years of computation time.

To illustrate the potential of the CGC to provide a customizable and scalable research environment, we present a collaborative project that enables unprecedented precision in detection of gene fusions and splice variants using novel statistical algorithm called Machete. We describe how this software was refactored in order to optimize deployment to the cloud for cost-effective analysis of thousands of samples at scale. We also provide the results of benchmarking that demonstrates the substantial savings in wall-clock time that can be obtained by processing large datasets on the cloud.

#5387

Novel targeted single-cell TCR sequencing method used for analysis of a T cell infiltrate from a glioblastoma patient.

Shuqiang Li,1 Jing Sun,2 Annabelle Anandappa,2 Oliver Spiro,1 Ignaty Leshchiner,1 Krishnalekha Datta,3 Yun Bao,3 Zhuting Hu,2 Nir Hacohen,4 Nathan D. Mathewson,2 Itay Tirosh,1 Kai Wucherpfennig,2 Aviv Regev,1 Mario Suva,4 Derin B. Keskin,2 Catherine J. Wu,2 Kenneth J. Livak2. 1 _Broad Institute of MIT and Harvard, Cambridge, MA;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _Integrated DNA Technologies, Redwood City, CA;_ 4 _Massachusetts General Hospital, Boston, MA_.

Obtaining paired alpha- and beta-chain information of the T cell receptor (TCR) enables identification of which TCR binds to a particular antigen, providing critical knowledge for understanding how a tumor interacts with its immune microenvironment and for monitoring responses to immunotherapy. We have developed a rapid, cost-effective single-cell TCR sequencing protocol (scTCRseq) that processes single T cells flow sorted into 384-well plates. Exploiting the added specificity provided by RNase H-dependent PCR (rhPCR; Dobosy et al., BMC Biotechnology 11:80, 2011), our scTCRseq protocol mixes primers for all known productive alpha and beta TCR alleles and obtains specific amplification of only the alleles present in a single-cell lysate (typically one alpha allele and one beta allele) without the use of nested PCR. Applying this protocol to T cell samples from normal donors, we detect TCRs arising from all productive TRAV and TRBV genes, and observe the expected highly diverse repertoires. Our method also includes targeted sequencing of 96 (or more) marker transcripts in order to obtain cell phenotype information. Our scTCRseq protocol has been adapted to analyze low-input samples (100 pg-10 ng) of PBMC RNA for characterization of the TCR repertoire and to improve the sensitivity of paired TCR detection in Smart-seq2 libraries prepared from single-cell RNA.

We recently applied this approach to analyze T cell clonality in the T cell infiltrate from a glioblastoma patient receiving neoantigen-specific peptide vaccination. Fresh tumor obtained post-vaccination at the time of recurrence was dissociated and single CD3+ T cells were isolated by flow cytometry. Smart-seq2 analysis suggested the presence of clones, but this was based largely on inference from one-chain data. Using our scTCRseq protocol to re-amplify the TCR segments, paired alpha- and beta-chain information was obtained from 277 of 363 (76%) single cells that passed quality control. Out of 231 distinct clonotypes detected, 25 were identified as clones, where a clone is defined as two or more cells with identical alpha- and beta-CDR3 segments. GLIPH analysis (Glanville et al., Nature 547:94-98, 2017) was used to cluster infiltrate TCRs by specificity. A representative TCR from each specificity cluster was cloned in a TCR-negative Jurkat cell line. Cellular assays were used to determine which specificity groups react with epitopes from peptides used in the vaccination. TCRs specific for neoantigens included in the vaccine were detected.

We have demonstrated a robust workflow that combines sensitive detection of paired alpha- and beta-chain TCR sequences with TCR cloning and expression to determine which TCRs in a tumor infiltrate have neoantigen specificity.

#5388

RNA in situ detection and characterization of ALK rearrangement in NSCLC FFPE tissues.

Na Li,1 Shafei Wu,2 Mindy Wang,1 Hongzhe Sun,1 Zhifu Zhang,1 Emily Park,1 Xiao-Jun Ma,1 Xuan Zeng,2 Robert Monroe1. 1 _Advanced Cell Diagnostics, Newark, CA;_ 2 _Peking Union Medical College Hospital, Beijing, China_.

Anaplastic lymphoma kinase (ALK) rearrangements are involved in ~4-7% of non-small cell lung cancers (NSCLCs). About 80% of ALK rearrangements generate a fusion gene between EML4 and ALK, resulting in overexpression of the fusion protein and constitutive kinase activity. Due to the sensitivity of tumors harboring ALK rearrangements to crizotinib, the identification of these rearrangements is clinically important. In this study, we developed an RNAscope® RNA ISH assay to specifically detect ALK rearrangement leading to upregulation of the 3' region of the ALK gene encoding the kinase domain in FFPE tissues from NSCLC. Two ALK-specific probes targeting ALK exons 1-18 (Hs-ALK E1-E18) and exons 19-29 (Hs-ALK E19-29) were tested in seven NSCLC FFPE samples. In three FISH-confirmed ALK rearrangement positive NSCLC cases, no signal was detected with the Hs-ALK E1-E18 probe, as expected. However, the Hs-ALK E19-29 probe showed positive signals (score 1 or 2) in all three FISH-confirmed cases of NSCLC. Among four ALK rearrangement negative samples (FISH and IHC confirmed), two samples exhibited no positivity with either probe. The other two cases showed low positivity with both probes in parts of the tumors, presumably representing baseline ALK mRNA expression (see table). Our results demonstrate that RNAscope® is a reliable method for the detection of ALK rearrangements that lead to the upregulation of the exons encoding the ALK kinase domain. The RNAscope® assay is both sensitive and specific for the detection of ALK rearrangement in FFPE tissues. Furthermore, the method provides information on the spatial distribution and morphologic context of cells associated with ALK rearrangements in NSCLC. | |  | RNAscope HD 2.5 Red assay result score | RNAscope HD 2.5 Red assay result score | RNAscope HD 2.5 Red assay result score | RNAscope HD 2.5 Red assay result score | |

|

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

sample label | ALK-FISH | IHC ALK-D5F3 | probe-PPIB (positive control) | probe-DapB (negative control) | probe-Hs-ALK E1-E18 | Probe-Hs-ALK E19-29 | Pathologic Diagnosis | Age | Gender (M/F)

1 | + | N/A | 2 | 0 | 0 | 1 (50%) | Highly-moderately differentiated invasive mucinous lung adenocarcinoma | 77 | F

2 | + | N/A | 2 | 0 | 0 | 2 (>90%) | Moderately-poorly differentiated lung adenocarcinoma | 32 | M

3 | + | N/A | 3 | 0 | 0 | 2 (>90%) | Highly-moderately differentiated lung adenocarcinoma, partly mucinous adenocarcinoma | 49 | M

4 | - | - | 3 | 0 | 0 | 0 | Lepidic predominant lung adenocarcinoma | 58 | F

5 | - | - | 4 | 0 | 0 | 0 | Lepidic predominant lung adenocarcinoma | 63 | M

6 | - | - | 4 | 0 | 0 (90%), 2(10%) | 0 (90%), 2(10%) | Invasive lung adenocarcinoma (papillary and alveolar predominant, partly micropapillary) | 48 | M

7 | - | - | 3 | 0 | 0 (98%), 2(2%) | 0 (98%), 2(2%) | Invasive lung adenocarcinoma (alveolar and lepidic predominant) | 52 | M

### MicroRNAs as Biomarkers

#5389

Circulating miRNAs as potential targets for the protection of radiation-induced immunologic injury.

Heng Zhou, Wenjun Wei, Jufang Wang. _Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, China_.

MicroRNAs (miRNAs) are non-coding RNAs of 19-22 nucleotides which regulate a diverse set of physiological processes including immune cell development by post-transcriptional gene regulation. Circulating miRNAs in blood have been assessed as biomarkers of disease diagnosis and are capable of modulating a wide-range of biological functions through targeting the molecules of recipient cells. In our previous work, some radiosensitive circulating miRNAs have been identified, and most of them were closely associated with immune and hematopoietic system. Here, we further investigated the effects of radioprotector on the changes of miRNA expression levels after exposure of mice to X-rays or carbon ion beam, aiming to explore novel potential therapeutic targets.

Kunming mice were whole-body exposed to 2 and 4 Gy of X-rays generated by Faxitron RX650 or 0.1 and 0.5 Gy of carbon ion beam generated by the HIRFL (Institute of Modern Physics, Lanzhou, China). Angelica sinensis polysaccharide (ASP) which is able to promote the hematopoietic function in vivo and GANRA agent which can scavenge the ROS were administrated to mice by gavage for 7 days before exposure. The expression of circulating miRNAs were detected by qRT-PCR analysis. Our results confirmed that ASP significantly protected the immune and hematopoietic systems of irradiated mice by recovering the numbers of bone marrow hematopoietic

cells, peripheric leucocytes, lymphocytes and atrophy of the spleen. GANRA increased the number of lymphocytes, leucocytes and the ratio of CD4+/CD8+ cells in the spleen of irradiated mice. Moreover, GANRA could eliminate ROS which was produced by X-rays and carbon ion beam, and increased the cell viability. Meanwhile, the expression levels of circulating miRNAs such as let-7a, miR-34a, miR-223, miR-150 and miR-574 were distinctively different between ASP or GANRA treated and the control group. ASP or GANRA treatment could rescue the up-regulation of let-7a, miR-34a and miR-223, and the down-regulation of miR-150 and miR-574 that were induced by radiation, indicating that circulating miRNAs could be possible targets of radioprotector responding to radiation exposure.

In conclusion, ASP and GANRA treatments have significant protective effects on the immune and hematopoietic system. The differential expressions of circulating miRNAs in radioprotector treated and non-treated groups indicate that miRNAs are potential targets for the protection of radiation-induced immunologic injury. By further exploring the function of these miRNA and their influence on the immune and hematopoietic systems, it will accelerate the development of new biomarkers and drug targets.

#5390

microRNA signature of T cell exhaustion.

Lydia M. Greenlees, Michael Kuziora, Yinong Sebastian, Todd Creasy, Young S. Lee, Fernanda Pilataxi, Nick Holoweckyj, Li Cheng, Brandon W. Higgs, Koustubh Ranade, Katie Streicher. _MedImmune, Gaithersburg, MD_.

Background: T cell exhaustion is driven by persistence of antigen and inflammation, common features of cancer. The success of a checkpoint inhibitor blockade may depend upon reactivation of pre-existing tumor-specific CD8+ T cells in the tumor microenvironment. Using an in vitro model and tumor infiltrating lymphocytes (TILs) isolated from multiple tumors, we explored microRNAs (miRNA, miR) involved in T cell dysfunction in order to identify pathways that may be important for altering this phenotype following immunotherapy.

Methods: We used an established in vitro model of T cell exhaustion: healthy donor CD8+ T cells were stimulated with anti-CD3/CD28 for six days. We used RNASeq and quantitative PCR to evaluate genomic (mRNA and microRNA (miR)) changes associated with T cell function and paired differentially expressed microRNAs with predicted target genes. Exhaustion of CD8+ TILs isolated from melanoma (n = 2), NSCLC (n = 3), renal (n = 3), bladder (n = 10), and colorectal tumors (n = 2) was measured by flow cytometry analysis of PD-1/TIM-3. miRNA and mRNA relationships identified by the in vitro model were evaluated in a subset of TILs from NSCLC, renal, and bladder tumors (n = 6).

Results: Results showed expected phenotypic and functional changes across 6 donors stimulated chronically with anti-CD3/CD28: 2-4-fold increased PD-1 and TIM-3 surface expression with a 3-5-fold loss of intracellular IFNg production (p < 0.05). Comparing exhausted T cells with unstimulated T cells revealed ~1300 differentially expressed genes and ~100 differentially expressed miRs (p < 0.05). A set of differentially expressed genes/miRs from the in vitro exhaustion model was then confirmed in TILs from NSCLC, bladder, and renal tumors. Differentially expressed miRs included microRNAs-155 and 181a, previously implicated in regulating IFNg signaling and T cell proliferation, respectively. Focusing on miRs that were altered between activation and exhaustion revealed a novel miR exhaustion signature, which was increased ≥4-fold relative to activated T cells. Evaluating this miR exhaustion signature in TCGA demonstrated a 16-fold range in expression across indications, with melanoma, head and neck, and NSCLC, among the highest median expression, while renal, HCC and rectal cancers were among the lowest. In HCC, GITR and OX40 pathway activation signatures were increased >2-fold in tumors with high miR signature, while in renal cancer, B cell and CD40 pathway signatures were enriched. In melanoma, high miR signature showed a trend toward increased myeloid-derived suppressor cell (MDSC) signature expression (>1.5-fold change), while the opposite trend was observed in head and neck cancer.

Conclusions: We identified a novel miRNA exhaustion signature associated with immune-related pathways in multiple tumor types. This signature may help generate hypotheses to guide prioritization of specific IO combination treatments.

#5391

Evaluation of seq-based biofluid miRNA profiling platforms.

Beihong Hu, Desai H. Keyur, Patrik Vitazka, Zhenhao QI. _Bristol Myers Squibb, Pennington, NJ_.

Background: Due to relatively high stability in serum/plasma, urine and other biofluids, miRNA profiling is currently being established as an exploratory tool to identify noninvasive biomarkers for human disease. Because of the small size of mature miRNAs, the high degree of homology between miRNA family members, and the low abundance of miRNAs in biofluids, miRNA expression profiling is technically challenging. Several Seq-based platforms have emerged and are attractive because of high sensitivity and whole miRNome survey but a rigorous platform evaluation is necessary.

Methods: We expand the miRQC study of Mestdagh et al. to evaluate newly advanced Seq-based platforms QIAGEN QIAseq, HTG EdgeSeq, Exiqon mirSeq with Taqman based Exiqon array and focus on more challenging biofluid miRNA profiling for objectively assess the performance of titration response, reproducibility, specificity, sensitivity, differential expression, and accuracy. We prepared identical 20 samples at Phase I and additional 9 samples at phase II for accuracy for QIAseq only.

Results: For specificity assessment, we spiked in 4 different miRNAs in liver and phage samples with 1 or 2 nucleotide differences. QIAseq ranked the highest for specific by cross reactivity calculation. The detection rate in serum RNA were much more variable among platforms with up to 6 fold differences. QIAseq shows the highest, while Exiqon mirSeq is at the low end for detection rate. We calculated raw UMI from phage spike in samples, the difference between the expected versus observed FC are range from 0.98 to 1.14 and demonstrated high accuracy for QIAseq platform. The reproducibility appears to be comparable with >80% corrections among serum samples. We applied the product rank sum method and identified 443 out of 2524 miRNAs as differentially expressed for QIAseq, 345 out of 2096 for EdgeSeq and 175 out of 794 for mirSeq at 0.05 cut-off p-value. For titration response, we computed the % of correctly titrating miRNAs as the function of Fold-change (FC) from Universal Human miRNA Reference RNA and Human Brain RNA. QIAseq is close to Exiqon Taman platform on the top performance with > 90% of the top half FC-ranked miRNAs titrated correctly.

Conclusion: QIAGEN QIAseq platform performed better in specificity, serum miRNAs detection rate, titration response among the three Seq-based platforms and outperforms the bench mark Exiqon Taqman, while QIAGEN QIAseq platform performed comparably in reproducibility, differential analysis among the four platforms. QIAseq also demonstrates high accuracy at phase II study and has been chosen the platform for biofluid miRNA clinical studies.

References: Ø P. Mestdagh et al "Evaluation of quantitative miRNA expression platforms in the microRNRNA quality control (miRQC) study" Nature Methods doi:10.1038/nmeth.3014

#5392

Exosomal miRNA expression profiling in triple-negative breast cancer.

Patricia M. Ozawa,1 Débora S. Lemos,1 Evelyn Vieira,1 Ingrid L. Souza,1 Silvio M. Zanata,1 Vânia C. Pankievicz,1 Thalita R. Tuleski,1 Emanuel M. Souza,1 Rosiane V. Silva,2 Pryscilla F. Wowk,2 Rodrigo C. Almeida,1 Iglenir J. Cavalli,1 Danielle M. Ferreira,1 Luciane R. Cavalli,3 Enilze M. Ribeiro1. 1 _Universidade Federal do Paraná, Curitiba, Brazil;_ 2 _Instituto Carlos Chagas - Fiocruz, PR, Curitiba, Brazil;_ 3 _Georgetown Lombardi Comprehensive Cancer Center, Washington, DC_.

Exosomes are extracellular vesicles that are present in body fluids and known to play key roles in intercellular signaling communication. Exosomes carry several types of molecules of their tumor of origin, including miRNAs, and therefore present potential as biomarkers for cancer prognosis and treatment. Triple-negative breast cancer (TNBC) is a clinically aggressive subtype of breast cancer, associated with treatment resistance, recurrence and high mortality rates. These tumors are molecularly heterogeneous, which impairs the identification of effective prognostic molecular markers and target therapies. The main objective of this study was to characterize the miRNA expression profile of exosomes derived from TNBC cells in comparison to normal controls and to the luminal A (LA) breast cancer subtype. The exosomal miRNAs were isolated from 19 serum samples of breast cancer patients (10 with the LA and 9 with the TNBC subtype) and 10 serum samples of healthy women (control group) using the exosome precipitation method. Exosome characterization and quantification was assessed by Western blot using the exosomal surface markers, CD6 and CD9, and Nanotracking analysis. The tumor and normal exosomal samples were combined into 6 different pools (of at least 4 samples in each group) and sequenced in the Ion Torrent Proton platform (Applied Biosystems). RNA-seq analysis revealed differentially expressed (DE) levels of exosomal miRNAs, with log2FC ranging from 1.2 to 3.6, among the four groups studied (control, cancer (TNBC+LA), TNBC and LA groups): 22 DE miRNAs were observed in the cancer vs. control, 19 in the LA vs control, 30 in the TNBC vs. control and 7 in the LA vs. TNBC groups. Eleven miRNAs were commonly DE in the cancer, LA and TNBC groups when compared to the control group, which included miRNAs, such as miR-103a-3p, miR-107 and miR-423-5p, involved in the biosynthesis of unsaturated fatty acids and neutrophin signaling pathways. Nine miRNAs were only observed DE in the TNBC group (vs. control), with the majority of them being downregulated. The top 10 significant functional enriched pathways affected by these miRNAs included proteoglycans, pathways in cancer and TGF-beta signaling pathways. Only three miRNAs were observed DE in the TNBC vs. LA groups, all with downregulated expression in the TNBC group. These miRNAs were observed mostly involved in fatty acid biosynthesis, proteoglycans in cancer and TGF-beta signaling pathways. In conclusion, our results indicate that expression alterations of exosomal miRNAs affect critical cancer-associated signaling pathways, suggesting their potential use as noninvasive cancer biomarkers. Additional studies should be performed to determine whether the expression patterns of these exosomal miRNAs reflect the ones observed in the corresponding patients' TNBC tissue.

Funding: CAPES, PPSUS-Fundação Araucária, Brazil.

#5393

Exosomal microRNA profiles in peritoneal fluids as a therapeutic biomarker for peritoneal metastasis of gastric cancer.

Hideyuki Ohzawa, Yuko Kumagai, Hironori Yamaguchi, Yoshinori Hosoya, Naohiro Sata, Joji Kitayama. _Jichi Medical University, Shimotsuke, Tochigi, Japan_.

Background: Repeated intraperitoneal chemotherapy (IPC) using taxane is highly effective for peritoneal metastasis. Peritoneal fluids are supposed to contain various factors derived from tumor cells seeded in peritoneal cavity. This study aimed to explore possible biomarkers for suitable IPC using exosomal miRNA profiles derived from malignant ascites or peritoneal lavages.

Methods: Peritoneal fluid samples were obtained from 11 patients with peritoneal metastasis (PM+) and 14 patients who received gastrectomy for early gastric cancer (PM-). Exosomal fractions were isolated using ultracentrifuge method and total RNAs were extracted. The pooled RNA samples in both groups were mixed and expression of miRNAs were analyzed using miRNome miScript miRNA PCR Array (QIAGEN). Based on these results, we constructed the custom miRNA PCR panel and evaluates expression of miRNAs in individual samples.

Results: The comprehensive PCR analysis showed that total of 56 and 54 miRNAs were strongly expressed in exosomes in PM+ and PM- peritoneal fluid sample, respectively. In analysis of individual samples, 7 upregulated miRNAs (miR-150-5p, miR-223-3p, miR-204-5p, miR-720, and miR-92a, miR-21-5p, miR-4301, and miR-342-3p) and 3 downregulated miRNAs (miR-29a-3p, miR-29b-3p, and miR-29c-3p) were identified in PM+ samples.

Conclusion: In patients who receive IPC, peritoneal fluids can be repeatedly obtained from peritoneal access port. Exosomal miRNA profiles of peritoneal fluid samples might be a useful biomarker to determine the therapeutic efficacy as well as to predict the outcome in those patients with peritoneal metastasis.

#5394

Initial results from TRANSCAN ERA-NET BREMIR project: MicroRNAs expression profiling for identification of breast cancer patients at high risk to develop distant metastases.

Paola Parrella,1 Raffaela Barbano,1 Ella Evron,2 Barbara Pasculli,1 Orazio Palumbo,1 Michelina Rendina,1 Raffaella Stallone,1 Michela Coco,1 Andrea Fontana,1 Tommaso Mazza,1 Massimiliano Copetti,1 Vanna Maria Valori,1 Maria Morritti,1 Paolo Graziano,1 Roberto Murgo,1 Evaristo Maiello,1 Massimo Carella,1 Vito Michele Fazio,1 Manel Esteller3. 1 _IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy;_ 2 _Assaf Harofeh Medical Center, Zerifin, Israel;_ 3 _Bellvitge biomedical Research Instute (IDIBELL), Barcelona, Spain_.

The mortality of breast cancer (BC) is primarily caused by metastatic spread to distant organs, rendering the ability to predict, detect and eliminate metastases one of the most important challenges in patients' management. This study is aimed to evaluate the potential role of several candidate miRNAs as predictive markers of the metastatic processes. The TRANSCAN ERA-NET BREMIR project has been designed to identify miRNA biomarkers able to predict distant metastases development. The correlation of miRNAs expression with distant metastasis free survival is evaluated in a large retrospective cohort (n=302). The best candidate markers will be validated in the independent prospective cohorts which currently include 505 patients. Additionally, miRNA potentialities as circulating biomarkers for treatment monitoring and early metastases detection will be evaluated in plasma samples. The analysis of the retrospective cohort has identified miR-10b-5p, miR-10a-5p, miR-210-3p, miR-30a-5p, miR200a-3p,miR200b-3p,miR200c-3p, miR141-3p, miR-429, miR-155-5p, miR-9-3p and miR-9-5p as crucially involved in BC progression. For miR-9-5p and miR-9-3p we also found a strong inverse correlation with ER and PgR expression, suggesting that they may be directly involved in hormone regulated pathways. To identify novel miRNAs associated with tumour metastases potentials, we also executed a global miRNA profiling on a selected number of breast cancer cases by using the Affymetrix GeneChip® miRNA 4.0. In particular, we analysed 34 BC cases characterized by different outcomes: 17 non metastatic after 10-years follow-up and 17 who developed metastatic disease within 5-years follow-up. As controls, 3 Normal Breast Tissues from reductive mammoplasty, 3 in situ carcinomas and 3 breast cancer cases with synchronous metastases were also evaluated. This analysis identified 8 miRNAs differentially expressed between metastatic and non metastatic cases. The association with study endpoints is currently under evaluation on the entire retrospective cohort. Our hope is that this study will lead to the identification of novel biomarkers allowing a better management of breast cancer patients.

#5395

MicroRNA-196b-3p identified from serum microRNA expression profiling is a potential diagnostic biomarker for gastritis and early gastric cancer.

Atsushi Fujii,1 Sho Nambara,1 Yosuke Kuroda,1 Shuhei Ito,1 Hidetoshi Eguchi,1 Takaaki Masuda,1 Takao Otsuka,2 Masafumi Nakamura,2 Koshi Mimori1. 1 _Kyushu University Beppu Hospital, Beppu, Japan;_ 2 _Kyushu University, Fukuoka, Japan_.

Background: Gastric cancer is one of the most common forms of cancer worldwide. The progression of chronic atrophic gastritis (CAG) is involved in gastric carcinogenesis. With the progression of chronic atrophic gastritis, the risk of gastric cancer increased in a stepwise fashion. Non-invasive tools for early gastric cancer are required to improve the prognosis. MicroRNAs (miRNAs), a class of small non-coding RNAs of 19-22 nucleotides in length, function as post-transcriptional regulators by directly cleaving target messenger RNA or translational repression. MiRNAs in serum have been shown to be potential biomarkers in various type of cancers and several miRNAs have been reported in gastric cancer. The purpose of present study was to identify a serum miRNA expression profile that can serve as a novel diagnostic biomarker for early gastric cancer.

Materials and Method: We defined a normal gastric mucosa as grade0, CAG classified C-1, 2 in Kimura-Takemoto classification as grade1, CAG classified C-3, O-1 in Kimura-Takemoto classification as grade2, and CAG classified O-2, 3 in Kimura-Takemoto classification as grade3. We obtained 344 serum samples, which gathered from 19 health people (grade0), 125 patients with chronic atrophic gastritis (grade1: 54, grade2: 36, grade3: 35), and 200 patients with early gastric cancer. The miRNAs were extracted from serums and miRNA microarray analysis was performed. In the discovery phase, we analyzed 71 cases (grade0: 11, grade1: 20, grade2: 20, grade3: 20). Next, in the training phase, we analyzed 73 cases (grade0: 8, grade1: 34, grade2: 16, grade3: 15). Then, we analyzed 200 early gastric cancer serum samples.

Result: The microarray analysis identified a profile of miRNA (miRNA-196b-3p) as a biomarker for early gastric cancer. The analysis results showed that the expression level of the serum miRNA was correlated to degree of CAG and carcinogenesis. When compared with grade0,1,2 and grade3, the area under the receiver operating characteristic (ROC) curve of this serum miRNA signature was 0.730 and 0.763 for the two sets of serum samples. There was significant difference in serum value of miRNA-196b-3p between grade1-3 and early gastric cancer (p=0.003). Conclusion: Serum miRNA-196b-3p is good biomarker for early gastric cancer.

#5396

Identification of meningioma patients in high risk of tumor recurrence using microRNA profiling.

Josef Srovnal,1 Vladimir Balik,1 Hanus Slavik,1 Magdalena Houdova Megova,1 Miroslav Vaverka,2 Lumir Hrabalek,2 Jiri Ehrmann,1 Katerina Staffova,1 Marian Hajduch1. 1 _Palacky University, Faculty of Medicine and Dentistry, Olomouc, Czech Republic;_ 2 _University Hospital Olomouc, Olomouc, Czech Republic_.

Introduction: Meningioma represents one of the most common intracranial tumors. They are generally thought to progress from low to high-grade lesions and relapse. However, the molecular mechanisms underlying their pathogenesis remains to be ellucidated. Identification of meningioma patients with higher risk of recurrence may have significant impact for future clinical management. Methods and patients: Formalin-fixed paraffin embedded tumor samples were obtained from 45 meningioma patients (15 recurrent patients, 30 patients without recurrence) and 5 healthy controls (dura mater). Comprehensive clinical-pathological data were mined. There were 15 males and 30 females; median age was 54 years, range 28 - 99 years. Total RNA was purified from FFPE samples after pathological verification using miRNeasyMini kit (QIAGEN). Microarray analysis was performed using the MiRNA 4.0 Array and FlashTagTM Biotin HSR(Affymetrix). Affymetrix CEL files have been read with read.celfiles function from oligo package and preprocessed with rma function (Bioconductor). The expression dataset was analyzed with Wilcoxon exact two-sample test (exactRankTestsR package) in order to discover significantly altered miRNAs. Results: We revealed different miRNA profiles in primary meningioma tumors that will relapse in comparison with non-recurrent tumors. 54 differentially expressed miRNAs were identified in meningeoma patients at high risk of recurrence. High-risk patients had significantly higher expression of miR-572 and miR-320c, and lower expression of miR-140 and miR-16-5p. Sixteen candidate miRNAs (including miR-107, miR-16-5p, miR-320c, miR-371b-5p and miR-15a-5p) were chosen for further qPCR validation on independent dataset. Moreover, miRNA expression levels were also compared in paired samples (primary and recurrent tumors) of 15 meningioma patients. However, only small differences in miRNA expression were detected. Conclusion: We have found different miRNA profiles in meningioma patients identifying patients in high risk of recurrence. Sixteen candidate miRNAs (including miR-107, miR-16-5p, miR-320c, miR-371b-5p and miR-15a-5p) were further validated. Acknowledgment: This work was financially supported by Ministry of Health of the Czech Republic, grant nr. 15-29021A, IGA UP LF 2017_013, NPU LO1304 and NCMG LM2015091.

#5397

ExomiRs can distinguish tumor-associated from normal stroma: Potential biomarkers in colorectal cancer.

Rahul Bhome,1 Rebecca Goh,1 Nir Pillar,2 Noam Shomron,2 Emre Sayan,1 Alex Mirnezami1. 1 _University of Southampton, Southampton, United Kingdom;_ 2 _Tel Aviv University, Tel Aviv, Israel_.

There is urgent need for prognostic markers to stratify intermediate-stage colorectal cancer (CRC) patients for the benefit of adjuvant therapy. Tumor heterogeneity is a major obstacle in stratification attempts as rare cancer cell populations may display stem cell properties and allow generation of therapy resistance. Tumor stroma, on the other hand, is less susceptible to genetic alterations than cancer cells, providing signals that are more reproducible across patient samples. Exosome encapsulated microRNAs (exomiRs) are particularly amenable to biomarker development because they circulate, closely resemble the tissue of origin and are inherently stable. We sought to identify a stromal exomiR signature in colorectal cancer with biomarker potential. Paired primary tumor-associated and normal fibroblasts were extracted from human colonic tissue. Exosomes were isolated by differential ultracentrifugation, validated according to International Society for Extracellular Vesicles recommendations, and profiled for over 800 microRNAs (miRNAs) by NanoString. Data preprocessing and normalization was conducted prior to differential expression analysis. Candidate miRNAs were validated by qPCR. Pathway and network analyses were conducted using miRPath and Ingenuity Pathway Analysis. Forty one exomiRs were differentially present in tumor-associated and normal fibroblasts. Of these, 21 were more abundant in tumor-associated fibroblasts. Thirty-six KEGG pathways were found to be regulated by this exomiR signature, such as "PI3K-Akt" and "colorectal cancer." A potential target of these exomiRs is PHLPP2, which encodes a phosphatase acting on AKT. In keeping with this, exposure to fibroblast exosomes increased AKT phosphorylation and that of its direct target Bad, in CRC cells, potentially due to a decrease in PHLPP2 protein abundance. Consequently, these cells were protected from drug-induced apoptosis. For the first time, stromal exomiRs have been profiled in colorectal cancer. Tumor-associated and normal stroma can be distinguished by a specific exomiR signature, which is potentially functionally important. Further mechanistic characterization is now required to identify the clinical significance of stromal exomiRs.

#5398

Companion diagnostic for predicting the efficacy of mTOR inhibitor in neuroendocrine tumor.

I-Hua Liu, Jia-Ming Chang. _Development Center for Biotechnology, New Taipei City, Taiwan_.

Neuroendocrine tumor (NET) is having both characterizations of hormone and neurotransmitter secretion. The mTOR, a serine-threonine kinase inhibitor, was used for treating the NET tumors by affecting angiogeneic blood vascular vessels as a disrupting agent and killing the tumor cells by inhibiting the mTOR signalling. Everolimus is a U.S. FDA-approved drug for treating advanced breast cancer, which is investigated for use in NET treatment. The efficacy will be evaluated by exosome reflecting the tumor microenvironment secreted into blood circulation. NCI-H727 cells are a kind of NET tumor that show the secretion of chromogranin A (CgA) and serotonin. By profiling of exosome with miRNA, the efficacy may be predicted by the level of miRNA. Here, we demonstrated the pattern of miRNA profile in everolimus-treated NCI-H727 cells. MIR192 and MIR375 were consistently secreted in exosomes with a high level. A panel of miRNA, MIR24-2, MIR3131, and MIR708, was increased and showed a change greater than 0.5 fold compared with untreated cells. In conclusion, by analyzing the pattern of miRNA in exosomes, the efficacy of drug can be monitored during the treatment. A new therapeutic strategy can be tailored according to the miRNA pattern.

#5399

Correlation of a microRNA expression profile and the prognosis of penile cancer: A prospective study using microarray data analysis.

Tatiane K. Furuya,1 Claudio B. Murta,2 José Pontes Jr,2 Miyuki Uno,1 Alexis Carrasco,1 Laura C. Sichero,1 Luisa L. Villa,1 Rafael F. Coelho,2 Giuliano B. Guglielmetti,2 Mauricio D. Cordeiro,2 Kátia R. Leite,3 Miguel Srougi,3 Roger Chammas,1 William C. Nahas1. 1 _Faculdade de Medicina da Universidade de São Paulo (FMUSP) / Instituto do Câncer do Estado de São Paulo (ICESP), São Paulo, Brazil;_ 2 _Instituto do Câncer do Estado de São Paulo (ICESP), São Paulo, Brazil;_ 3 _Faculdade de Medicina da Universidade de São Paulo (FMUSP), São Paulo, Brazil_.

Introduction and objective: Penile cancer is a rare disease that has high morbidity and mortality rates. While a few biomarkers related to prognosis have been previously described to date none of them was adopted in clinical practice. Our aim was to identify a molecular signature based on miRNA expression levels that could identify those patients with high risk of metastatic penile carcinoma.

Methods: We prospectively collected fresh samples of primary tumors from 11 patients with squamous cell penile carcinoma who underwent surgical treatment between July/2015 and June/2017. Five patients had localized disease (non-metastatic group) and 6 had inguinal lymph node metastases (metastatic group). RNA was purified and microarray analysis was performed using miRNA 4.0 Genechip (Affymetrix). We identified differentially expressed miRNAs (DEmiRs) comparing metastatic in relation to non-metastatic groups using TAC Software (Affymetrix), fold change (FC) > 1.5 and p<0.05. Their validated/predicted targets were investigated using miRTarBase 7.0, mirWalk 2.0 and/or MetaCore 6.32 softwares. Enrichment Pathways analysis was performed using Enrichr. Up/downstream interaction networks were identified by MetaCore v6.32 software.

Results: Twenty two DEmiRs (17 up- and 5 downregulated) were identified when comparing metastatic in relation to non-metastatic patients. Hierarchical clustering analysis showed a set of 7 DEmiRs (miR-181c-5p, 744-5p, 196b-5p, 200a-5p, 152-3p, 421, 149-5p) which clustered samples according to prognosis. Network interaction analyses identified transcriptional factors (NANOG, Oct-3/4, SOX2 and c-Jun) that commonly regulate those DEmiRs. Moreover, TGFB, CMYC, RICTOR, ADAM-17 and PTEN are some of the common targets of those DEmiRs and are more likely to be deregulated. Validated and predicted targets of these 7 DEmiRs were found enriched in relevant pathways such as "Proteoglycans in cancer", "Pathways in cancer", "miRNAs in cancer" and "Signaling pathways regulating pluripotency of stem cells". Among their targets, CMYC, SP1, ESR1 and AR are examples of hubs which presented the highest number of network interactions.

Conclusions: We found 7 DEmiRs correlated with prognosis and capable of discriminating metastatic from non-metastatic tumors. In addition, their targets are enriched in cancer-related pathways and could be potential biomarkers for prognosis in penile cancer.

#5400

A comprehensive analysis of the interactome of miR-21, an established oncomir, by Argonaute-CLIP analysis identifies novel conserved and species-specific targets of miR-21 in human liver and hepatocellular carcinoma.

Aaron B. Koenig,1 Juan M. Barajas,2 Kalpana Ghoshal2. 1 _The Ohio State University College of Medicine, Columbus, OH;_ 2 _Comprehensive Cancer Center, The Ohio State University, Columbus, OH_.

MicroRNAs are ~22 nucleotide RNAs that regulate gene expression at the post-transcriptional level by binding messenger RNA transcripts. MicroRNA (miR)-21 participates in carcinogenesis in hepatocellular carcinoma (HCC) and other malignancies. Here we analyze the miR-21 interactome in HCC by high-throughput sequencing of RNA isolated by Argonaute cross-linking immunoprecipitation (Argonaute-CLIP) in nine HCC cases with matched livers. Argonaute-CLIP identified miR-21 bound to 580 target sites on coding transcripts, of which 332 were located in the coding sequences, 214 in the 3'-untranslated region, and 34 in the 5'-untranslated region, introns or downstream sequences. Overall, 403 target sites (69.5%) enriched for miR-21 in HCCs relative to liver. We compared expression of miR-21 targets in 377 patients with liver cancer registered in The Cancer Genome Atlas. Ago-CLIP miR-21 enrichment score in HCC distinguished expression of miR-21 targets by their correlation with miR-21 expression. Comparison of the miR-21 interactome in mouse and human livers identified common and species-specific targets. Among targets of miR-21, 27.6% were downregulated in HCC. The miR-21 interactome was predicted to regulate tumor metabolism by inhibiting PPAR alpha/RXR alpha and LXR/RXR signaling. To identify targets of miR-21, we matched Ago-CLIP targets to genes previously reported as aberrantly expressed after anti-miR-21 overexpression in HCC. RMND5A, encoding an E3 ubiquitin ligase, was identified as a potential target of miR-21 regulation and was variably expressed in HCC. Three predicted miR-21 targets, CAMSAP1, DDX1 and MARCKSL1, correlated with HCC patient survival. Collectively, our analysis biochemically identified novel targets of miR-21 likely to play a causal role in hepatocarcinogenesis.

#5401

Dysregulated microRNAs in aflatoxin-induced hepatocellular carcinoma: Serum miR-182 as a potential predictive biomarker.

Merricka C. Livingstone,1 Natalie M. Johnson,2 Bill D. Roebuck,3 Thomas W. Kensler,4 John D. Groopman1. 1 _Johns Hopkins Univ. School of Public Health, Baltimore, MD;_ 2 _Texas A &M School of Public Health, College Station, TX; _3 _Dartmouth Medical School, Hanover, NH;_ 4 _University of Pittsburgh School of Medicine, Pittsburgh, PA_.

Hepatocellular carcinoma (HCC) continues to be a major cause of cancer death globally, with aflatoxin B1 (AFB1) as a prevalent risk factor in low- and middle-income countries. MicroRNAs have been shown to be differentially expressed in HCC and may serve as predictive biomarkers. In this study, we analyzed tumor and non-tumor tissue from rats dosed with AFB1 (200 µg/kg BW) for 28 consecutive days that received vehicle only or AFB1 plus the chemopreventive agent CDDO-Im (30 µmol), and control animals (Johnson et al., CaPR, 2014). Total RNA was isolated from tumor or non-tumor tissue at the time of sacrifice and sequenced. MicroRNA transcriptomic analysis revealed 17 miRNAs significantly upregulated (> 5 fold) in tumors compared to non-tumor tissue. The top ten dysregulated miRNAs determined by fold change and biological significance were selected for further investigation: rno-miR- 205, 200b-3p, 182, 429, 31a-5p, 10b-5p, 141-3p, 132-3p, 802-5p. Validation of sequencing results by quantitative PCR (qPCR) confirmed the upregulation of the majority of candidate miRNAs in tumors and rno-miR-224-5p as the most dysregulated miRNA (over 400 fold). We also examined the levels of these candidates in terminal sera of the same animals by qPCR. Circulating miRs-224-5p, 182, and 122-5p were increased (> 1.5 fold) in animals diagnosed with HCC compared to untreated animals and those previously dosed with AFB1 plus CDDO-Im. Analysis of tracking of serum miR-182 by generalized estimating equations (GEE) revealed significantly increased levels (5 fold; CI: 2.17- 2.50) in animals that developed HCC. This sustained increase in serum miR-182 is seen months before any tumors or other symptoms are present, and highlights this miRNA as a potential predictive biomarker in AFB1-induced HCC. Supported by T32ES007141-31A1 and CA197222.

#5402

A panel of miRNAs for diagnosis of wild-type thyroid nodules with pre-surgical indeterminate cytology.

Maria Denaro,1 Vincenzo Condello,1 Clara Ugolini,2 Elisabetta Macerola,1 Anello Marcello Poma,1 Gabriele Materazzi,1 Paolo Vitti,1 Fulvio Basolo1. 1 _University of Pisa, Pisa, Italy;_ 2 _Azienda Ospedaliero-Univarsitaria Pisana (AOUP), Pisa, Italy_.

Fine-needle aspiration (FNA) cytology is the gold standard for the evaluation of thyroid nodules, but about 30% are cytologically indeterminate. The inability to resolve preoperative thyroid nodules as benign or malignant is therefore a challenge. Molecular tests in FNA are useful to assist the diagnosis and the management of patients. However, if a thyroid nodule is indeterminate and genetic tests are inconclusive, surgical excision may be considered for definitive diagnosis. To overcome these limitations, novel diagnostic procedures and molecular tests are required. Among these, miRNAs have been reported as promising molecular markers in thyroid cancer.

The aim of this study was to evaluate the potential diagnostic utility of miRNA expression in preoperative diagnosis of thyroid nodules with indeterminate cytology and wild type in RAS family genes and BRAF gene.

The miRNA expression profile was evaluated in a small cohort of 12 wild-type Follicular Adenomas (FAs) and 4 wild-type Infiltrative Follicular Variant of Papillary Thyroid Carcinomas (IFVPTCs) with pre-surgical indeterminate cytology or in patients with cytological diagnosis not available. The two groups have been selected with similar characteristics in terms of age of patients, nodules size and with a ratio Male-Female 1:3. The expression of 798 miRNAs has been evaluated by using nCounter Nanostring technology, a sensitive and reproducible method. The differential miRNAs expression between FAs and IFVPTCs was investigated by applying the non-parametric Mann-Whitney U-test. In addition, an unsupervised hierarchical clustering analysis by Pearson correlation was performed.

We found a set of 13 miRNAs differentially expressed between malignant and benign lesions. miR-222-3p, miR-30d-5p, miR-100-5p, miR-1972, miR-125b-5p, miR-155-5p were significantly up-regulated, whereas miR-574-5p, let-7f-5p, miR-148a-3p, miR-598-3p, miR-195-5p, miR-301a-3p, miR-5196-3p+hsa-miR-6732-3p were significantly down-regulated in IFVPTCs compared to FAs (p-value<0,05). The unsupervised hierarchical clustering analysis has identified 2 main clusters: the first one consisted of 9 FAs, whereas the second one consisted of 4 IFVPTCs and 3 FAs. The 4 IFVPTCs are grouped in an unique sub-cluster with a correlation of 0,74. This miRNA expression profile could distinguish malignant from benign lesions. If these results will be confirmed in a wide cohort of patients and in FNA cytology, this strategy could increase the diagnostic yield and improve the preoperative diagnosis of wild-type thyroid nodules with indeterminate cytology.

Although this is a preliminary study, the deregulation of this set of miRNAs might help identify malignant nodules that are negative for well-known gene alterations in preoperative FNAs.

#5403

Discovery of high metastatic potential microRNAs in human colorectal cancer.

Jee Hyun Lee, Gyeonghwa Kim, Hye Jin Kim, Gyu-Seog Choi, Keun Hur. _Kyungpook National Univ. School of Med., Daegu, Republic of Korea_.

Background and Aim: Colorectal cancer (CRC) is the third most common cancer worldwide. More than half of the CRC patients have liver metastasis at the time of diagnosis, which is the major cause of cancer-related death in CRC. MicroRNAs (miRNAs) have been identified as modulators of the epithelial-mesenchymal transition (EMT)-mesenchymal to epithelial transition (MET) process by regulating transcription factors. However, it is not clear which specific miRNAs are critical drivers of CRC metastasis development. The aim of this study was to discovery novel high metastatic potential miRNAs in matched CRC and metastasis tissues, serum-exosome, and CRC cells with high metastasis property.

Materials and Methods: We isolated total RNAs from pairs of normal colon (NC), primary CRC (pCRC), liver metastasis (LM), normal liver (NL) tissue specimens, and pre-OP/post-OP serum-exosome sample from 15 each CRC patients. We also prepared total RNAs from 4 CRC cell lines (SW480, LS174T, COLO 205, and EMT-induced SW480) as well as exosome from their culture medium. To induce EMT process, epithelial phenotype CRC cell (SW480) was treated with epidermal growth factor (EGF). Next, miRNAs discovery step was conducted via NanoString analysis. Further miRNAs expression was determined by quantitative real-time PCR (qRT-PCR), and miRNAs expression was normalized relative to U6 and cel-miR-39 expression for tissue and serum-exosome samples, respectively.

Results: We successfully generated miRNAs expression profiles to distinguish NC vs. pCRC, pCRC vs. LM, LM vs. NL, and epithelial phenotype cells vs. high metastatic phenotype cells. Interestingly, a subset of miRNAs, including miRNA-100-5p, -339-3p, -549a, -1304-5p, and -1972, was commonly enhanced in LM, EMT-induced SW480, and high metastatic phenotype CRC cells compared to pCRC, SW480, and low metastatic phenotype CRC cells. In our further miRNAs validation analysis using pairs of pCRC and LM tissues from 15 CRC patients, miRNA-100-5p (P<0.0001), miR-339-3p (P=0.0022), and miR-1972 (P<0.0001) were significantly upregulated in pCRC than matched LM tissues. We also observed discordance expression patterns of miRNAs between CRC tissues and serum-exosome specimens.

Conclusions: Our findings suggest that a unique subset of miRNAs is involved in CRC metastasis development, which may associate with EMT process and acquiring of high metastatic potential in CRC cells. Moreover, our metastasis specific miRNAs profile might be useful for development of markers predicting and/or treatment of CRC patients with metastasis.

#5404

RNA sequencing-based discovery and validation of a noninvasive microRNA signature for the detection of early-stage ovarian cancer.

Raju Kandimalla,1 Wei Wang,2 Michael Hsieh,1 Gerald C. Gooden,3 Monique Spillman,1 Xin Wang,2 Bodour Salhia,3 Ajay Goel1. 1 _Baylor Scott &White Research Institute and Charles A. Sammons Cancer Center, Dallas, TX; _2 _City University of Hong Kong, Hong Kong;_ 3 _University of Southern California, Los Angeles, CA_.

Purpose Ovarian cancer (OC) is one of the most lethal gynecological malignancies with a 5-year survival rate of less than 50%. While only 15% of OCs are diagnosed at an early stage (stage 1), more than 75% of these neoplasms at first presentation are already advanced (stage 3 or 4), where a cure is very unlikely and recurrent disease is quite common. Previous attempts at discovering early detection biomarkers for OC have largely failed because of reliance on sub-optimal discovery approaches and the lack of comprehensive patient cohorts. MicroRNAs (miRNAs), which belong to the family of small non-coding RNAs, are frequently dysregulated in cancers including OC. Although several miRNA biomarkers have previously been reported for OC patients, lack of systematic biomarker discovery and validation approach has hampered their clinical translation. To address some of these limitations, we performed a comprehensive and systematic biomarker discovery and validation to identify miRNAs dysregulated in early stage OCs, with the ultimate aim of developing a blood-based early diagnostic assay. Experimental design We performed miRNA expression profiling using a high-throughput small RNA-sequencing in stage 1 ovarian cancer (N=23) and matched normal (N=10) tissues. Differentially expressed miRNAs between cancer and normal tissues were identified using the limma package, which were subsequently validated in a cohort of plasma samples (N=32, healthy individuals and N=32 stage 1 OC patients) using Taqman-based RT-PCR assays. Results Our genome-wide systematic discovery approach led to the identification of eight miRNAs which were significantly upregulated in OC vs. adjacent normal tissues. Of note, even individual miRNAs achieved AUROC values ranging from 0.80 to 0.89; however, the combined panel of these 8 miRNAs achieved an even greater AUROC of 0.90 in detecting stage 1 OC patients (p<0.001). Subsequently, we analyzed this 8-miRNA panel in a set of 64 plasma (N=32, Healthy subjects and N=32, stage 1 OC patients) using RT-PCR assays. We performed binary logistic regression and ROC analysis to evaluate the diagnostic accuracy of these biomarkers. Interestingly, in accordance with the tissue findings, we achieved AUROC values ranging from 0.70 to 0.90 for individual biomarkers, while the combination panel achieved an impressive AUROC of 0.99 (95% CI: 0.98-1.00, p<0.0001) for the identification of early stage ovarian cancer patients. Conclusions In conclusion, our RNA sequencing based comprehensive biomarker discovery identified an 8-miRNA panel that demonstrated a robust diagnostic accuracy in not only tissue specimens, but also in the plasma specimens from early stage OC patients. Further evaluation of these miRNAs in multicenter retrospective and prospective cohorts might lead to the development of a non-invasive diagnostic, as well as a population-screening assay for OC patients.

#5405

**Multiplex profiling of miRNA biomarkers for discovery and verification using the FirePlex** ® **platform.**

Elnaz Atabakhsh, Ivona Kelley, Michael Tackett, Izzuddin Diwan, Gensis Tejada, Conor Rafferty, Daniel Pregibon. _Abcam, Inc., Cambridge, MA_.

To address the needs for microRNA (miRNA) biomarker discovery and verification, we developed the FirePlex® miRNA assay. This assay enables the detection of 5-400 miRNA targets per sample in 96-well format, with readout on standard flow cytometers and analysis with an included bioinformatics software package. The FirePlex miRNA assay combines particle-based multiplexing, using patented FirePlex® hydrogel particles, with single step RT-PCR signal amplification using universal primers. Thus, the FirePlex miRNA assay leverages PCR sensitivity while eliminating the need for separate reverse transcription reactions and mitigating amplification biases introduced by target-specific qPCR. The miRNA assay can reliably detect as few as 1000 miRNA copies per sample with a linear dynamic range of ~5 logs, and without the need of prior RNA purification, making the assay ideally suited for profiling in serum, plasma, exosomes, urine, and directly from FFPE tissues. Furthermore, the ability to multiplex targets in each well eliminates the need to split valuable samples into multiple reactions. Results from the FirePlex miRNA assay are displayed and interpreted using the integrated FirePlex Analysis Workbench, which allows visualization, normalization, and export of experimental data with only a few mouse clicks. To aid biomarker discovery studies, we offer pre-designed panels of 400 miRNAs based on a research-curated list of miRNAs published to be found in circulating biofluids. For studies focused on specific research areas of interest, we offer pre-designed focus panels for Oncology, Cardiology, Neurology, Immunology, and Organ Toxicity. These carefully curated panels include hemolysis markers to assess sample quality, as well as critical normalization factors. Here we present the data from several studies investigating circulating miRNA profiles, as well as miRNA profiles obtained directly from FFPE tissues, using the FirePlex miRNA Assay Panels. Together, this novel combination of bioinformatics tools and multiplexed, high-sensitivity assays enables rapid discovery and verification of miRNA biomarker signatures from biofluid samples.

#5406

Predictive value of tumor microRNAs in head and neck cancer patients treated with intensity-modulated radiation therapy.

Parwez Ahmad,1 Jiri Sana,1 Marek Vecera,1 Jaroslav Juracek,1 Tana Machackova,1 Natalia Anna Gablo,1 Marek Svoboda,2 Marketa Hermanova,3 Marek Slavik,4 Pavel Slampa,4 Pavel Smilek,5 Ondrej Slaby1. 1 _Central European Institute of Technology, Masaryk University, Brno, Czech Republic;_ 2 _Department of Comprehensive Cancer Care, Masaryk Memorial Cancer Institute, Brno, Czech Republic;_ 3 _Department of Pathological Anatomy, St. Anne's University Hospital, Brno, Czech Republic;_ 4 _Department of Radiation Oncology, Masaryk Memorial Cancer Institute, Brno, Czech Republic;_ 5 _Department of Otorhinolaryngology and Head and Neck Surgery, St. Anne's University Hospital, Brno, Czech Republic_.

Introduction: Head and neck cancers are the sixth most common cancers worldwide mainly represented by the squamous cell carcinoma (HNSCC). Very important treatment modality in HNSCC is radiotherapy (RT). Closed as well as ongoing clinical trials are evaluating especially potential for reduced-dose RT in less aggressive radiosensitive HNSCC defined by human papillomavirus (HPV) positivity, with promise of less acute and late toxicity. To this end, variety of different biomarkers with promising predictive value is currently investigated in HNSCC. MicroRNAs (miRNAs) are short endogenous RNAs that post-transcriptionally modulate gene expression and their deregulated expression has been observed in many cancers including HNSCC. Specific expression patterns of miRNAs have been also shown to predict prognosis and therapeutic response in HNSCC. Aim of our study was to identify tumor tissue miRNAs enabling of predict locoregional control (LRC) in HNSCC patients who underwent intensity-modulated RT.

Materials and methods: We have analyzed global miRNA expression profiles in 43 FFPE tumor biopsies collected from HNSCC patients treated with intensity-modulated radiation therapy, who were divided into two groups according to their LRC as follows: short LRC [n = 22; median 5.1 months (min 1.3, max 18.6)] vs. long LRC [n = 21; 60.4 (46.8, 98.8)]. This analysis has been performed using the hybridization Affymetrix GeneChip miRNA 4.0 array. Validation of miRNA candidates was performed in independent cohort of 64 HNSCC patients. MiRNA determination was carried out by RT qPCR technology using the miRNA-specific RT stem-loop primers according to the Taq-Man MicroRNA Assay protocol (Thermo Fisher Scientific).

Results: We identified 24 miRNAs with significantly different expression between both examined groups (p < 0.05; average log(Fold Change) = 0.42). Based on pre-defined critera, 12 miRNAs were selected for independent validation, from which miR-421 and miR-1228 were differentially expressed between groups of patients with short and long LRC. Moreover, risk-score based on combination of these 2 miRNAs expression and CD44 status enabled to predict 3-years LRC with 82% sensitivity and 82% specificity (AUC = 0.798).

Conclusion: Our results suggest that miR-421 and miR-1228 are promising predictive biomarkers in HNSCC patients treated with intensity-modulated RT. This work was supported by Ministry of Health of the Czech Republic, grant nr. 15-34553A, 15-33158A, 15-31627A, 15-34678A, 16-31314A and 16-31765A.

#5407

MicroRNA expression analysis of advanced colorectal cancer reveals a microRNA signature with prognostic and predictive value.

Zhaolei Zeng, Jiahua Lu, Zhixiang Zuo, Qi Zhao, Ruihua Xu. _Sun Yat-sen University Cancer Center, Guangzhou, China_.

Background

Prognostic and predictive markers are needed to predict the clinical outcomes of patients with advanced colorectal cancer (CRC) who receive standard first-line treatments. We performed a prospective cohort study in patients with advanced CRC to identify a miRNA signature that could predict the benefit of receiving first-line chemotherapy for these patients.

Methods

Twenty-one paired tumours and adjacent normal tissues were collected from patients with advanced CRC and analysed by miRNA microarrays. Between tumour and normal tissues, 33 miRNAs were differentially expressed. The differential expression of these miRNAs was confirmed by qRT-PCR using paraffin-embedded specimens from another group of 76 patients from a prospective cohort study. A two-miRNA-based signature was obtained using the least absolute shrinkage and selection operator (LASSO) Cox regression model based on the association between the expression of each miRNA and the progression-free survival (PFS) time of individual patients. Internal and external validation cohorts, including 45 and 50 patients with advanced CRC, respectively, were performed to validate the prognostic and predictive accuracy of this signature.

Results

A signature based on two miRNAs, miR-125b-2-3p and miR-933, was built by the LASSO model. CRC patients were classified into high- and low-risk groups for disease progression based on this tool. The patients with high risk scores generally had worse PFS than those with lower risk scores. In the training set, the average PFS was 23.906 months for the low-risk group and 9.010 months for the high-risk group (hazard ratio [HR] 2.535, 95% CI 1.478-4.348, p=0.001). In the internal validation set, the average PFS was 14.029 months for the low-risk group and 6.334 months for the high-risk group (HR 3.173, 95% CI 1.415-7.116, p=0.005). In the external validation set, the average PFS was 16.815 months for the low-risk group and 7.657 months for the high-risk group (HR 2.498, 95% CI 1.281-4.871, p=0.007). Furthermore, we detected miR-125b-2-3p associated with CRC cell sensitivity to first-line chemotherapy. The two-miRNA-based signature was an independent prognostic factor for predicting the outcome of patients with metastatic CRC receiving first-line chemotherapy.

#5408

Elevated level of serum miR-1290 is correlated with high-grade serous ovarian epithelial ovarian cancer and might be a potential biomarker.

Masaki Kobayashi, Sawada Kenjiro, Yoshimura Akihiko, Miyamoto Mayuko, Nakatsuka Erika, Kodama Michiko, Hashimoto Kae, Mabuchi Seiji, Kimura Tadashi. _Osaka University Hospital, Osaka, Japan_.

Objective: There is a critical need for improved diagnostic markers to detect ovarian high grade serous ovarian cancer (HGSOC). MicroRNAs (miRNAs) stably exist in circulating blood, reflecting tissue or organ conditions and present in circulating microvesicles such as exosomes. Recent studies have confirmed the potential use of miRNAs profiling as a novel non-invasive biomarker for diagnosis of HGSOCs. The aim of this study is to identify which miRNAs are highly produced from HGSOCs and analyze whether serum miRNA can discriminate patients with HGSOC from healthy controls. Methods: Secreted exosomes from ovarian cancer cell lines were collected and exosomal miRNAs extracted. miRNA microarray was performed and several elevated miRNAs specific to HGSOCs were picked up. Among these, we focused on miR-1290. Serum from 71 pre-operative, 46 post-operative ovarian cancer patients and 13 healthy controls were gathered and its expression levels were detected by quantitative Real Time PCR. Results: In HGSOC patients, miR-1290 emerged overexpressed compared to healthy controls (3.52-fold). ROC analysis showed that at the cut-off of 1.61(healthy controls ; 1), the sensitivity and specificity were 63 % and 85 % respectively for detecting HGSOC (AUC = 0.71). The AUC of CA125 + miR-1290 combination was 1.00. Its expression singnificantly decreased after operation(5.87 → 1.17 ; P < 0.01). In advanced stage HGSOC patients, moreover, it expressed marginally higher than early stage ones(4.23 VS 1.58 ; P = 0.23). Conclusions: Serum miR-1290 can be a potential diagnostic biomarker for HGSOC.

#5409

Deep sequencing of circulating microRNAs in rectal cancer patients undergoing neoadjuvant chemoradiotherapy.

Tana Machackova,1 Dominika Brchnelova,1 Zdenek Kala,2 Vladimir Prochazka,2 Tomas Grolich,2 Lukas Fiala,3 Beata Hemmelova,2 Jaroslav Juracek,1 Marek Vecera,1 Jiri Sana,1 Natalia Gablo,1 Parwez Ahmad,1 Marek Svoboda,3 Ondrej Slaby1. 1 _Central European Institute of Technology, Brno, Czech Republic;_ 2 _University Hospital Brno, Brno, Czech Republic;_ 3 _Masaryk Memorial Cancer Institute, Brno, Czech Republic_.

Introduction: Rectal cancer accounts for approximately one third of all colorectal cancers (CRC), which belong among leading causes of cancer deaths worldwide. Standard treatment for locally advanced rectal cancer (cT3/4 and/or cN+) includes neoadjuvant chemoradiotherapy (CHRT) with fluoropyrimidines followed by radical surgical resection. MicroRNAs (miRNAs) are small non-coding RNAs playing significant roles in the pathogenesis of many cancers including rectal cancer. MiRNAs could present the new predictive biomarkers of response to CHRT in rectal cancer patients. Materials and Methods: In prospective one-center study, 48 patients diagnosed with rectal cancer who underwent neoadjuvant chemoradiotherapy followed by surgical treatment were included. Blood plasma samples were collected before the neoadjuvant chemoradiotherapy (in 24 cases) and after the therapy at the time of clinical restaging (in 48 cases). Total RNA was isolated from 72 blood plasma samples of 48 patients. cDNA libraries were prepared using CleanTaq Small RNA Library Prep Kit (TriLink BioTechnologies). The final sequencing analyses were performed by Next 500/550 High Output v2 Kit - 75 cycles using the NextSeq 500 instrument (both Illumina). For miRNA mapping and analysis, an online tool Chimira was used. Obtained data were statistically evaluated using the Bioconductor edgeR and DESeq2 package. Results: When miRNA profiles of samples collected before and after therapy were compared, 10 miRNAs showed higher levels and of 2 miRNAs lower levels in pre-treatment specimens (P<0.05). Samples collected at the time of clinical restaging were divided into groups accordingly to tumor regression grade (Dworak score) evaluated after surgical resection of the tumor. Pre-treatment miRNA profiles of patients from CHRT responsive group (Dworak 3+4) and non-responsive group (Dworak 1+2) were compared and 4 miRNAs were upregulated and 4 miRNAs were downregulated in patients with good response to CHRT. Finally, samples collected after the therapy, at the time of clinical restaging, were divided into groups accordingly to the nodal metastatic involvement and 10 miRNAs were identified to be significantly deregulated between groups (P<0.025). Conclusion: Our findings suggest that circulating miRNAs could serve as potential predictive biomarkers of the response to neoadjuvant CHRT in rectal cancer. This work was supported by Ministry of Health of the Czech Republic, grant nr.15-33158A, 15-34553A, 15-31627A, 15-34678A, 16-31314A, 16-31765A and by grant of Czech Grant Agency nr. 16-18257S.

#5410

An miRNA signature selects patients at risk for Barrett's esophagus progression to dysplasia and cancer.

James Saller,1 Kun Jiang,1 Kevin Neill,1 Zachary Mayer,2 Jae Lee,1 Luis Pena,1 F. Scott Corbett,3 Jose Pimiento,1 Mokenge Malafa,1 Anthony Magliocco,1 Domenico Coppola1. 1 _H. Lee Moffitt Cancer Center, Tampa, FL;_ 2 _Bradenton Christian High School, Bradenton, FL;_ 3 _Gastroenterology Associates of Sarasota, Bradenton, FL_.

Background. Esophageal adenocarcinoma (EAC) is an aggressive malignancy with increasing incidence in the US. Progression of Barrett's Esophagus (BE) to EAC adenocarcinoma occurs via a stepwise process. BE is monitored with periodic esophageal biopsies. While a small percent of BE patients will progress to dysplasia and cancer, the majority of them will continue to have long-standing BE without progression. In the absence of dysplasia, the behavior of BE cannot be predicted based on evaluation of histologic features alone. MicroRNA studies have differentiated between the specific evolutionary events in the progression of BE to dysplasia and cancer. There has yet to be a comparison between BE that had failed to progress to dysplasia or carcinoma (BEN), to BE that had progressed to dysplasia and/or carcinoma (BEP). We propose the introduction of a reliable miRNA based assay that differentiates BEN from BEP.

Methods. Fifty cases of BE were profiled with two different miRNA profiling techniques: HTG EdgeSeq miRNA WT Assay and nanoString Assay. There were 15 cases of BEN (follow up >7 years) as well as of 11 cases of BEP (progression to dysplasia and/or EAC within 3 years). We used these samples for our miRNA discovery and miRNA prediction model training. We then used another independent patient data set of total 24 BE cases (13 cases of BEN and 11 cases of BEP; namely Normalized Nano), which was profiled with nanoString miRNA Assay. This set was used as an independent validating cohort.

Results. Using and confirming with two different statistical methods--Limma and Wilcoxen rank sum test, we identified 6 significant miRNAs, controlling for FWER (family-wise error rate for type I error) less 0.05 to avoid a high chance of discovering false positives. The final miRNA model showed a high prediction performance at the optimal cutoff with - specificity 80% and 50% and sensitivity 100% and 75% for the two sets in order to capture a high proportion of the progressors. We then independently validated this miRNA signature with the Normalized Nano set, which showed sensitivity 70% and specificity 67.5% in this independent validation. While the prediction performance was weaker, we consistently validated its prediction power and clinical utility on an independent patient cohort with a completely different miRNA profiling technique.

Conclusion: These favorable findings support further investigation in a larger population of patients, and may have potentially prognostic utility in the evaluation of BE patients with the goal of early detection of BE progression.

#5411

Micro-RNA expression in cemento-ossifying fibroma.

Thais S. Pereira,1 André Luiz S. Guimarães,2 Carolina C. Gomes,1 Ricardo S. Gomez,1 Marina G. Diniz1. 1 _Federal Univ. of Minas Gerais, Belo Horizonte, Brazil;_ 2 _University of Montes Claros, Montes Claros, Brazil_.

Cemento-ossifying fibroma (COF) is a benign fibro-osseous neoplasm of uncertain pathogenesis and its treatment is associated with significant morbidity. MicroRNAs (miRNA) are small non-coding RNAs that regulate gene expression and may represent therapeutic targets. The purpose of the study was to generate a comprehensive miRNA profile of COF compared to normal bone. Additionally, the most relevant pathways and target genes of differentially expressed miRNA were investigated by in silico analysis. Nine COF and ten normal bone samples were included in the study. miRNA profiling was carried out by using TaqMan® OpenArray® Human MicroRNA panel containing 754 validated human miRNAs. We identified the most relevant miRNAs target genes through the leader gene approach, using STRING and Cytoscape software. Pathways enrichment analysis was performed using DIANA-miRPath. Eleven miRNAs were downregulated (hsa-miR-95-3p, hsa-miR-141-3p, hsa-miR-205-5p, hsa-miR-223-3p, hsa-miR-31-5p, hsa-miR-944, hsa-miR-200b-3p, hsa-miR-135b-5p, hsa-miR-31-3p, hsa-miR-223-5p, hsa-miR-200c-3p) and five were upregulated (hsa-miR-181a-5p, hsa-miR-181c-5p, hsa-miR-149-5p, hsa-miR-138-5p, hsa-miR-199a-3p) in COF compared to normal bone. Eighteen common target genes were predicted, and the leader genes approach identified the following genes involved in human COF: EZH2, XIAP, MET and TGFBR1. According to the biology of bone and COF, the most relevant Kegg-pathways revealed by enrichment analysis were Proteoglycans in cancer, miRNAs in cancer, Pathways in cancer, p53, PI3K-Akt, FoxO, and TGF-beta signalling pathways, which were previously found to be differentially regulated in bone neoplasms, odontogenic tumours and osteogenesis. Dysregulation of miRNAs occurs in COF and EZH2, XIAP, MET and TGFBR1 are potential targets for functional analysis validation. Supported by: CNPq, CAPES and FAPEMIG (Brazil).

#5412

miR155 and miR9 expression in human prostate cancer cell lines and prostate cancer tissue.

Ines Benedetti, Juan Rebollo, Niradiz Reyes. _Universidad de Cartagena, Cartagena, Colombia_.

Background: Differential expression of miRNAs between malignant, precancerous lesions, and nonmalignant prostate tissues may indicate that these molecules play a role in cancer biology. Several miRNAs have been commonly reported to be differentially expressed in prostate cancer, compared to benign prostate tissue, but some results have been contradictory. Expression patterns of specific miRNAs across prostate cancer, precancerous lesions, and benign tissue have not been determined.

Design: The expression patterns of 20 miRNAs were determined in the metastatic prostate cancer cells lines LNCaP and PC3, using the nontumorigenic prostate cell line PWR-1E as reference. Differentially expressed miR-9 and miR-155 were subsequently evaluated in epithelial cells obtained by laser capture microdissection from cancerous lesions, high-grade prostatic intraepithelial neoplasia, proliferative inflammatory atrophy, and benign tissue from radical prostatectomy and transurethral prostatectomy tissue specimens, from patients diagnosed with prostate cancer. Total RNA was extracted from the cell lines and microdissected epithelial cells and expression of miR-9 and miR-155 was evaluated by qPCR; miRNA levels in individual samples were normalized to corresponding RNU62 levels. Fold-change values were calculated, and p value < 0.05 was considered statistically significant. The Ethical Review Board of the academic institution approved this study.

Results: Differential expression of several miRNAs was found between cell lines. Four miRNAs showed a similar expression profile in the cancer cell lines, two were overexpressed in metastatic cell lines LNCaP and PC3 compared to the nontumorigenic PWR-1E cells (hsa-miR-195, hsa-miR-9), and two were underexpressed (hsa-miR-143, hsa-miR-155). Evaluation of miRNA expression in laser capture microdissected epithelial cells showed a trend towards higher expression of miR-9, and lower expression of miR-155 in microdissected epithelial cells derived from prostate tissue with cancer and proliferative inflammatory atrophy compared to epithelial cells from benign tissue.

Conclusions: Our results are in agreement with the description of miR-9 as oncomiRNA. miR-155 was underexpressed in LNCaP and PC3 cells in relation to nontumorigenic PWR-1E cells. Also, this miRNA showed a trend towards underexpression in epithelial cells microdissected from prostate tissue with cancer and proliferative inflammatory atrophy compared to epithelial cells from benign tissue. Further studies of these deregulated miRNAs will help clarify their role in prostate tumorigenesis.

#5413

MicroRNA profiling in AML.

Diana Gilligan, Aakriti Pandita, Poornima Ramadas, Aarati Poudel, Nibal Saad, Ankit Anand, Alina Basnet, Frank Middleton. _Upstate Medical University, Syracuse, NY_.

Differences in microRNA (miR) expression levels provide clues to the disruption of normal hematopoiesis that leads to the emergence of a leukemic clone. Analysis of miR expression levels may be useful to understanding normal cytogenetic acute myeloid leukemias (AMLs) and developing novel treatments for them. We collected peripheral blood samples from 10 adult patients with newly diagnosed AML, prior to induction chemotherapy, and 9 controls. Two and a half ml of whole blood was collected in Paxgene RNA tubes. miRNA was purified using standard Trizol method, followed by RNeasy mini column (Qiagen). Quality of the RNA samples was assessed using the Agilent Bioanalyzer prior to library construction using the Illumina TruSeq Small RNA Sample Prep protocol (Illumina; San Diego, CA). Multiplexed samples of RNA that exceeded quality control metrics (RIN &gt 6.0) were run on an Illumina NextSeq500 instrument at a targeted depth of 10 million reads per sample. After filtering and trimming of index and adapter sequences, whole genome alignment of the miR FASTQ reads was performed using the Homo sapiens/hg19 reference genome in the SHRiMPS aligner included in the miRNAs analysis application available in BaseSpace (Illumina), as well as the sRNA Toolbox application suite. Quantification and normalization of aligned reads to the miRBase 21 database was performed, and differential expression between AML and control groups was performed using DESeq2, NOIseq, and EdgeR algorithms. We sequenced approximately 800 miRs from each of 10 patients with AML and 9 controls. We identified 9 miRs that showed a statistically significant increase in expression in AML patients versus controls and 4 miRs that showed a statistically significant decrease in expression in AML patients versus controls, with adjusted p-value less than 0.05. Among these 13 differentially expressed miRs, only 4 were previously described in leukemia, including miR 181a-3p and 181a-2-3p, miR 409-3p, and miR 126-5p. Finding these 4 miRs differentially expressed confirmed the validity of our approach. The remaining 9 of the miRs that showed differential expression in our study have not been described in relation to AML (miR-328-3p, miR106b-3p, let-7i-5p, miR10b-5p, miR-24-3p, miR-3200-5p, miR-23a-3p, miR-323b-3p, miR-652-3p). In subset analysis, patients with NPM1 and FLT3 mutations showed lower levels of miR 181a-2-3p and higher levels of miR 10b-5p compared to patients without NPM1 and FLT3 mutations. We are continuing to accrue patients to this study and we are following them over time in order to analyze miR expression in peripheral blood as a biomarker that may predict relapse. Our approach of global sequencing of miRs as opposed to microarray analysis removes the bias regarding which miRs to assay and has demonstrated discovery of new associations of miRs with AML. Our study provides further information about the molecular changes that lead to evolution of the leukemic clone and offers new targets for development of treatments.

#5414

The correlation between the expression of microRNA hsa-miR-101 and the prognosis of the patient with hepatocellular carcinoma.

Go Nakajima, Kazuhiko Hayashi. _Tokyo Women's Medical Univ., Tokyo, Japan_.

Aim: MicroRNAs (miRNA) are small RNAs that regulate gene expression at the post-transcriptional level, and they play a various role in cancer cell such as proliferation, migration and metastasis. Our previous study identified forty-nine miRNAs that are differentially expressed by comparing 26 pair samples of hepatocellular carcinoma (HCC) tumor and adjacent liver tissue using next-generation sequencer system. One of them, hsa-miR-101 is chosen in this validation study. The hsa-miR-101 expression in HCC tumor samples and its adjacent liver tissues were measured by real-time PCR, and were examined the correlation between the miRNA expression and clinical data.

Patients and Methods: Forty-three patients diagnosed as HCC had undergone surgical removal from 1991 to 1997 at the Department of Surgery, Institute of Gastroenterology, Tokyo Women's Medical University, Japan. The formalin-fixed paraffin embedded samples were prepared using the standard protocol. The paraffin blocks were cut into 10 micrometer sections, and the tumor and its adjacent non-tumor liver tissue were collected using laser-captured microdissection and total RNA including miRNA fraction were isolated. After synthesized cDNA using miRNA specific primer, real-time PCR was performed using miRNA specific primer/probe.

Results: The expression of hsa-miR-101 in tumor tissue was downregulated compared to adjacent liver tissue (P=0.003). No significant differences were obtained in survival time between higher or lower intratumoral expression level (P=0.8039). The patients were divided into two groups, higher/lower hsa-miR-101 expression in the tumor or in the adjacent liver tissue, the group of the higher expression in adjacent liver tissue and the lower in tumor was longer survival time compared to the other groups (P=0.0184).

Conclusion: The hsa-miR-101 expression may be related to tumorigenesis, and both the expression in the tumor and in the adjacent liver tissue may be potential prognostic factor for HCC patients.

### Signaling and Hormonal Inputs to Transcription Factor Regulation

#5415

STAT3 and GLI1/tGLI1 oncogenic transcription factors interact to promote the aggressiveness of triple-negative and HER2-enriched breast cancers.

Sherona R. Sirkisoon,1 Richard L. Carpenter,2 Rimkus Tadas,1 Anderson Ashley,1 Alexandria Harrison,1 Allison M. Lange,1 Guangxu Jin,1 Kounosuke Watabe,1 Hui-Wen Lo1. 1 _Wake Forest Univ. School of Medicine, Winston Salem, NC;_ 2 _Indiana University, Bloomington, IN_.

Patients with triple-negative (TN) and HER2-enriched (HER2) breast cancers have high rates of metastasis, which accounts for 90% mortality for these patients, highlighting the need for improved therapeutic targets. The oncogenic transcription factors, STAT3, GLI1, and truncated GLI1 (tGLI1) are associated with poor clinical outcomes for breast cancer patients. However, physical or functional interactions between STAT3 and GLI1/tGLI1 have never been investigated. The purpose of this study is to determine whether the JAK2/STAT3 and GLI1/tGLI1 pathways are concurrently activated in TN and HER2 breast cancers. We examined 47 node-positive breast tumor samples using immunohistochemical staining and found that p-STAT3 (Y705), GLI1, and tGLI1 were co-overexpressed in 64% of TN breast cancers, 68% of HER2 breast cancers, and in 65% of lymph node metastases. Gene Set Enrichment Analysis of 710 breast tumors revealed that the activation signatures for STAT3 and GLI1/tGLI1 are co-enriched in TN and HER2 subtypes, but not in the luminal subtypes. Furthermore, Kaplan-Meier and log-rank analyses revealed that breast tumors with high levels of STAT3 and GLI1/tGLI1 co-activation were associated with shortened metastasis-free survival compared to those with lower levels. To determine whether these two pathways functionally cooperate, we examined whether STAT3 and GLI1/tGLI1 formed complexes by IP-WB and observed that STAT3 interacted with both GLI1 and tGLI1. ChIP assay showed that these complexes bind to both consensus GLI1- and STAT3-binding sites. We further observed that co-overexpression of STAT3 and GLI1/tGLI1 resulted in significant activation of a promoter controlled by GLI1-binding sites. We next analyzed three ChIP-Seq datasets to identify gene promoters that can be occupied by the STAT3-GLI1/tGLI1 complexes, and found 36 potential target genes. Validation of these target genes by RT-qPCR and Kaplan-Meier analysis resulted in three genes that are upregulated by STAT3-GLI1 and/or STAT3-tGLI1, namely, R-Ras2, Cep70, and UPF3A, and correlated with poor survival. These novel transcriptional targets of STAT3 and GLI1/tGLI1 are implicated in breast tumorigenesis, angiogenesis, and metastasis suggesting that co-activation of STAT3 and GLI1/tGLI1 may promote aggressive breast cancers. Lastly, co-overexpression of STAT3 and GLI1/tGLI1 increased the mammosphere-forming ability of breast cancer cells and interestingly, only the STAT3-tGLI1 complex allowed mammosphere formation of immortalized mammary epithelial cells. In conclusion, our study reports, for the first time, that the physical and functional interactions between STAT3 and GLI1/tGLI1 oncogenic transcription factors lead to gene co-activation, stem-like phenotype of breast cancer cells, and unfavorable prognosis for patients with triple-negative and HER2 breast cancers.

#5416

Isolation of the PAX8 transcriptional complex to identify novel therapeutic vulnerabilities for ovarian cancer.

Daniele Chaves-Moreira, Marilyn Mitchell, Simone Sidoli, Benjamin Garcia, Ronny Drapkin. _University of Pennsylvania, Philadelphia, PA_.

A central problem in the treatment of ovarian cancer remains the heterogeneity among ovarian tumors. DNA and RNA sequencing studies have demonstrated both intertumoral and intratumoral genetic variation. Despite efforts to elucidate common signaling pathways among various ovarian cancer subtypes, few have led to meaningful patient stratification or to truly individualized targeted molecular therapies. Our proposed set of experiments is a radical departure from the conventional approach to treating ovarian cancer. PAX8, a transcription factor that identifies nearly all high-grade serous ovarian cancers (HGSOCs), is also the master regulator of fallopian tube development. Building off observations pointing to the fallopian tube epithelium as a major site of origin for HGSOCs, we propose targeting the fallopian tube developmental program as the basis for new therapies. This possibility is supported by the observation that knockdown of PAX8 leads to apoptosis in ovarian cancer cells. We hypothesize that blocking the ability of PAX8 to influence its gene targets, either by interrupting PAX8 protein-protein interactions or by inhibiting the products of PAX8-driven signaling, has the potential to eliminate the primary growth stimulus for high-grade serous tumors. In order to achieve that goal, we have identified candidate PAX8-interacting partners using gel filtration chromatography and tandem-affinity immunopurification coupled with mass spectrometry analyses. Interactions have been validated by Western blot, immunofluorescence, and proximity ligation assay. We used ovarian cancer (KURAMOCHI, OVSAHO and OVCAR4) and fallopian tube (FT189, FT194 and FT246) cell lines to determine whether PAX8 protein partners changed between benign and malignant cells. Our preliminary results suggest that PAX8 interacts with other transcription factors such as TEAD4 and components of chromatin-remodeling complexes (RUVBL1, RUVBL2 and CHD4). Many of these factors are known to mediate the effects of oncogenes such as YAP, β-catenin and c-Myc. Further studies will be done in order to decipher the functional contribution of each candidate PAX8-interacting protein. Our ultimate goal is to determine how PAX8 regulates expression of its target genes and how PAX8-interacting proteins influence that function. Inhibition of certain protein-protein interactions may provide novel avenues to abrogate PAX8 function in ovarian cancer cells.

#5417

The role of PPAR beta/delta in melanoma development and progression.

Xiaomeng Wang, Chee Woei Lim, Yuet Ping Kwan, Xuan Rui Ng, Walter Wahli. _Nanyang Technological University, Singapore, Singapore_.

Peroxisome proliferator-activated receptor β/𝛿;, a member of the nuclear receptor/ligand-activated transcription factor family, is involved in diverse biological functions including promoting terminal differentiation, inhibiting inflammatory signaling and increasing skeletal muscle fatty acid catabolism. Despite its well established functions in intermediary metabolism and developmental processes, the role of PPARβ/𝛿; in cancer remains controversial. It is well established that cancer angiogenesis and metastasis play significant role in cancer development and progression. Our studies showed that PPAR β/𝛿; antagonist, 10H, leads to a reduced cell proliferation but increased EMT, migration and invasion of B16F10 cells. To better understand PPARβ/𝛿;'s role in tumour development and progression, tail vein assay of tumour metastasis and tumour xenograft were performed in PPARβ/𝛿;-/- mice. Our study showed that the tumour growth is much slower whereas the incidence of lung metastasis is significantly higher in mice deficient of PPAR β/𝛿; as compared to age-matched wild type controls. Interestingly, we demonstrated that TGFβ1 is a direct target gene of PPARβ/𝛿; and controls the expression of PPARβ/𝛿; target genes, Angptl4 and CPT1a. Furthermore, TGFβ1-mediated PPARβ/𝛿; target gene expression is completely abolished upon the treatment with 10H. In summary, we demonstrated that PPARβ/𝛿; exerts a context-dependent role in tumour growth and progression, possibly through TGFβ1.

#5418

GATA3 zinc-finger mutation induces transcriptional reprogramming in breast cancer through gain and loss of function.

Motoki Takaku,1 Sara A. Grimm,1 John D. Roberts,1 Kaliopi Chrysovergis,1 Page Myers,1 Charles M. Perou,2 Paul A. Wade1. 1 _National Institute of Environmental Health Sciences, Research Triangle Park, NC;_ 2 _Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC_.

Recent large-scale breast cancer genomic profiling identified frequent mutations in a pioneer transcription factor, GATA3. The expression of GATA3 is a prominent marker of luminal breast tumors, and loss of GATA3 expression is associated with aggressive tumor phenotypes. In addition, GATA3 mutations are frequently found in these luminal tumors. However, the clinical and molecular outcomes of GATA3 mutations are poorly understood. Our novel classification scheme of GATA3 mutations defines distinct clinical features of breast tumors with mutations in the second GATA3 zinc-finger (ZnFn2): frequent observation in luminal B subtype and worse prognosis than other GATA3 mutant cases. To dissect the molecular impacts of these ZnFn2 mutations, we generated a GATA3 mutant breast cancer cell clone by CRISPR-Cas9 gene editing. An engineered ZnFn2 mutant cell line manifested increased tumor growth as xenografts and more aggressive phenotypes in vitro. The ZnFn2 mutation led to loss of GATA3 binding and decreased expression at a subset of genes, including progesterone receptor (PR). The mutant cells also exhibited gain of GATA3 binding at other loci, correlated with increased expression of mesenchymal marker genes. Decreased expression of PR and impaired response to progesterone were crucial for cancer-promoting functions in GATA3 ZnFn2 mutant cells. Downregulation of PR and its downstream genes was also observed in the clinical gene expression data. These results illuminate tumor-promoting functions of GATA3 ZnFn2 mutations in breast cancer.

#5419

HDAC1 regulates RUNX1 activity in inv(16) acute myeloid leukemia.

Lisa Richter, Yiqian Wang, Michelle Becker, Jake Williams, R. Katherine Hyde. _University of Nebraska Medical Center, Omaha, NE_.

RUNX1 and CBFβ form a transcription factor dimer that regulates normal hematopoiesis and leukemogenesis. Inversion of chromosome 16 (inv(16)) is one of the most common mutations in acute myeloid leukemia (AML), fusing CBFβ with the gene encoding smooth muscle myosin heavy chain (MYH11). The fusion protein encoded by CBFB-MYH11 (CM), retains the ability to bind to RUNX1, and together they cause changes in gene expression leading to leukemogenesis. Recently, we found that Histone Deacetylase 1 (HDAC1) is part of the RUNX1:CM complex, that all three proteins co-localize on the promoters of target genes, and that HDAC1 is required for target gene expression. By deacetylating histones, HDAC1 can act as a transcriptional repressor. However, HDAC1 can also deacetylate non-histone proteins, which may explain its unexpected role in gene activation. We hypothesized that HDAC1 acts by deacetylating proteins in the RUNX1:CM complex.

To test this, we immunoprecipitated the RUNX1:CM complex and probed for acetyl-lysine. The only visible band was at the expected size for RUNX1, suggesting that RUNX1 is the target of HDAC1 activity. Indeed, cells transfected with RUNX1 and HDAC1 showed significantly less acetylation than cells with RUNX1 alone. To test if HDAC1 deacetylates endogenous RUNX1, we treated leukemia cells from CM-expressing knockin mice with the HDAC1 selective inhibitor entinostat. The level of RUNX1 acetylation was significantly increased in treated cells compared to control, providing further support that HDAC1 deacetylates RUNX1.

Acetylation at lysines 24 and 43 of RUNX1 was previously shown to increase its activity. To determine the effect of deacetylation on RUNX1 activity, we used the M-CSFR promoter fused to luciferase. Cells transfected with RUNX1, CBFβ, and HDAC1 showed significantly decreased promoter activity compared to cells with RUNX1 and CBFβ. Importantly, in cells expressing RUNX1 acetylation and deacetylation mimetics, promoter activity was unchanged with the addition of HDAC1, indicating that lysines 24 and 43 are the relevant HDAC1 targets. To test if RUNX1 acetylation status affects the activity of the CM complex, we transfected a CM+ cell line with WT or mutated RUNX1 and examined target gene expression. In CM\+ cells over-expressing WT RUNX1, we found similar target gene expression as untransfected CM+ cells. However, expression of either of RUNX1 mutant blocked the CM-induced changes in gene expression. This implies that regulation of RUNX1 acetylation is important for the activity of the RUNX1:CM complex. The observation that both RUNX1 mimetics had the same effect on CM activity may imply that alternate rounds of RUNX1 acetylation and deacetylation is required for continuous RUNX1:CM transcriptional activity. This work provides a possible explanation for HDAC1's role in gene activation in inv(16) AML, and may have implications for other RUNX1 dependent leukemias as well.

#5420

Nuclear PTEN directly regulates androgen receptor activity in prostate cancer.

Yen T. Nguyen, Jose A. Rodriguez-Nieves, Qi Yang, David VanderWeele. _NCI, Bethesda, MD_.

The deletion of PTEN, a negative regulator of the PI3K pathway, is among the most frequent genomic alterations in prostate cancer. Attempts to target this alteration using PI3K pathway inhibitors, however, have yielded little clinical benefit for patients, and the function of PTEN loss in prostate cancer progression is not fully understood. Prostate cancers lacking PTEN are less responsive to therapy targeting the Androgen Receptor (AR), a dominant driver of prostate cancer growth, but how PTEN function interacts with AR activity is not clear. We used immunoblotting, immunoprecipitation, proximity ligation assay, chromatin immunoprecipitation, and transcriptome sequencing in cells with endogenous PTEN or PTEN knocked down to evaluate the interactions between these major factors in prostate cancer progression. We demonstrate that PTEN interacts with the Androgen Receptor in the nucleus, and PTEN can bind chromatin at known Androgen Response Elements. Stimulation of cells with androgen and other media components leads to accumulation of phosphorylated (inactive) PTEN, loss of nuclear PTEN, and decreased PTEN-AR interaction. Transcriptome analysis of cells with PTEN knocked down demonstrates increased expression of AR pathway genes, even in the absence of androgen stimulation. These results suggest nuclear PTEN may play a direct role in negative regulation of the Androgen Receptor, with implications for management of PTEN-negative prostate cancers.

#5421

Roles of sex hormone pathways in the gender disparity of esophageal squamous-cell carcinoma.

Furong Huang,1 Hongyan Chen,1 Qianben Wang,2 Zhihua Liu1. 1 _State Key Laboratory of Molecular Oncology, National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China;_ 2 _Department of Pathology, Duke University School of Medicine, Durham, NC_.

Objectives: Esophageal squamous-cell carcinoma (ESCC) incidence has been increasing rapidly, with an imbalanced ratio of occurrence in men more often than in women. The expression of sex hormone receptors is associated with the prognosis of ESCC. However, the exact roles of sex hormones and hormone receptors in ESCC remain unclear. We systematically evaluated the effects and the underlying mechanism of sex hormones and their receptors on esophageal carcinogenesis and malignant proliferation to clarify the extent to which sex hormones pathways contribute to the gender disparity of ESCC.

Methods: To determine the roles of hormones in vivo, we used a 4NQO-induced esophageal carcinogenesis model, a xenograft model and the castrated BALB/c Nude mouse model. To observe the function of hormones on the proliferation of ESCC cells in vitro, we performed colony formation, Cell Counting Kit-8, and EdU assays. To elucidate the precise molecular mechanism by which hormone receptors exert their functions, we performed ChIP-seq and RNA-seq to comprehensively identify the ESCC-specific hormone responsive genes and regulatory networks.

Results: There is a large male predominance in the 4NQO-induced esophageal carcinogenesis C57BL/6J mouse model. AR-positive esophageal cancer cells grow more rapidly in the male BALB/c Nude mice than the female mice. ERα-positive esophageal cancer cells possess faster proliferative capability in the female BALB/c Nude mice compared to the male mice, whereas ERβ-positive cells exhibit the opposite gender disparity in phenotypes. Moreover, the above proliferative effect is attenuated in castrated or ovariectomized mice. Furthermore, exogenous supplementation of estrogen pellet in ovariectomized female mice enhances ERα-positive cell growth but suppresses ERβ-positive cell growth. In vitro experimental data show that dihydrotestosterone treatment promotes the growth of AR positive ESCC cells, while 17-β estradiol (E2) exerts opposite functions depending on the estrogen receptor subtypes. Integrative analysis of ChIP-seq and RNA-seq data reveals that AR/ER is transactivated upon hormone treatment and binds to the promoter or enhancer of hormone responsive genes to induce differential gene expression. These genes are responsible for the malignant development of ESCC and may provide a new explanation of the gender disparity of ESCC.

Conclusions: Androgen promotes the proliferation of ESCC cells via AR while estrogen exerts dual functions dependent on the specific estrogen receptor subtypes, which may provide new insight into the etiology, prevention, and hormone-based treatment of ESCC.

#5422

**TMPRSS2-ERG fusions confers efficacy of enzalutamide in an** in vivo **bone tumor growth model.**

Sreenivasa R. Chinni, Louie Semaan, Michael Cher. _Wayne State Univ. School of Medicine, Detroit, MI_.

Castrate Resistant Prostate Cancer (CRPC) is an advanced disease resistant to systemic anti-androgen therapies and resistance is primarily attributed to reactivation of AR through multiple mechanisms. TMPRSS2-ERG fusions have been shown to regulate AR signaling, interfere with pro-differentiation functions, and mediate oncogenic signaling. In fact, cooperation between AR and ERG drives invasive adenocarcinoma even in the castrate environment. We have recently shown that ERG regulates intra-tumoral androgen synthesis thereby facilitates AR function in CRPC disease. We hypothesize that enzalutamide treatment will be more effective in cells/tumors with TMPRSS2-ERG translocations because these tumors have increased AR signaling. In other words, AR blockade is likely to induce a greater treatment response in tumors with ERG gene fusions than in tumors without such fusions. ERG knockdown was performed with VCaP cells using lentiviral infections to generate VCaP ERGshRNA cells and control VCaP scr cells with scrambled shRNA. Cell-growth analysis was performed to determine the effect of enzalutamide. Luciferase tagged VCaP scr and shRNA infected cells were used in intra-tibial animal model for bone tumor growth analysis and enzalutamide treatment used to inhibit AR signaling in bone tumors. Reverse transcription, quantitative real-time PCR (RT-qPCR) was used to determine the expression of AR responsive genes and androgen biosynthetic enzyme genes; Western blotting (WB) samples, for ERG, AR and AKR1C3 in VCaP bone tumors. Enzalutamide inhibited VCaP scr more effectively than shERG cells. Analysis of AR responsive genes show that Enzalutamide treatment at 5 micro molar concentration inhibited 85-90% in VCaP Scr cells whereas these genes inhibited to lesser extent in VCaP shERG cells. In bone tumor growth experiment, VCaP ERG shRNA cells grown at slower than VCaP scr shRNA cells. Enzalutamide treatment resulted in a sever growth inhibition in VCaP scr shRNA cells compared to VCaP shERG cells. Androgen biosynthetic enzyme expression is lower VCaP shERG bone tumors compared to VCaP scr shRNA bone tumors and enzalutamide inhibited the enzyme expression in both types of tumors. These data suggest that ERG transcription factor regulates androgen biosynthetic enzyme expression and enzalutamide treatment is effective against VCaP bone tumors with an intact ERG expression, and knocking down ERG in VCaP cells leads lesser response for enzalutamide therapy.

#5423

**Lysine-52 stabilizes the MYC oncoprotein through a SCF** FBXW7 **-independent mechanism.**

Jason De Melo,1 Sam S. Kim,1 Corey Lourenco,2 Diana Resetca,2 Maria Sunnerhagen,3 Brian Raught,1 Linda Z. Penn1. 1 _University Health Network, Toronto, Ontario, Canada;_ 2 _University of Toronto, Toronto, Ontario, Canada;_ 3 _Linköping University, Linköping, Sweden_.

The oncogenic transcription factor c-MYC (MYC) is deregulated, and often overexpressed, in more than 50% of cancers. MYC deregulation is associated with poor prognosis and aggressive disease, suggesting that the development of therapeutic inhibitors targeting MYC would dramatically impact patient care and outcome. MYC is a highly regulated transcription factor, with a protein and mRNA half-life of approximately 30 min. The most extensively studied pathway regulating MYC protein stability involves ubiquitylation and proteasomal degradation mediated by the E3-ligase, SCFFBXW7. Here we provide evidence for a SCFFBXW7-independent regulatory mechanism centered on Lysine 52 (K52) within MYC Box I (MBI) of the MYC protein. This residue has been shown to be post-translationally modified by both ubiquitylation and SUMOylation, hinting at the interplay of post-translational modifications at this site, and the importance of this residue. We demonstrate that mutation of K52 to arginine (R) renders the MYC protein more labile. Mechanistically, we show that the degradation pathway regulated by K52 is independent of the Cullin-Ring-Ligase (CRL) family of E3-ligases, which includes not only the canonical SCFFBXW7, but also a number of other known MYC-targeting E3-ligases, such as SCFSKP2, SCFβTCRP, SCFFBXO28 and DCXTRUSS. To characterize this degradation pathway further we will utilize unbiased and targeted experiments to elucidate the proteins involved. Taken together, our data identifies a novel regulatory pathway centred on K52 that may be exploited for the development of anti-MYC therapeutics.

#5424

Improved anticancer activity of clotam as a copper-complex.

Umesh T. Sankpal,1 Rafid Patel,1 Bianca Arechiga,1 Abigail Hunter,1 Jaya Chhabra,2 Deondra T. Brown,2 Raj K. Gurung,2 Alvin A. Holder,2 Riyaz M. Basha1. 1 _UNT Health Science Center, Fort Worth, TX;_ 2 _Old Dominion University, Norfolk, VA_.

Clotam (tolfenamic acid or TA) is a nonsteroidal anti-inflammatory drug used to treat migraine headaches. Research from our laboratory and other groups has demonstrated that TA also acts as an anticancer agent in several adult and pediatric cancer models. It is well established that the anticancer activity of TA is partly mediated by its ability to downregulate the specificity protein (Sp) family of transcription factors and their downstream targets, notably the inhibitor of apoptosis protein family member survivin. Both Sp1 and survivin are upregulated in cancer cells and are associated with poor prognosis, making them ideal therapeutic targets. It was recently suggested that the therapeutic potential of TA could be enhanced by forming a complex with copper(II). In this study we synthesized a copper(II) complex of TA (Cu-TA) and tested its anticancer activity using cell lines representing various cancers such as breast, prostate, pancreatic, brain, colon, neuroblastoma, medulloblastoma, and sarcoma. The compound was synthesized and its purity was assessed by UV-visible spectrophotometry. Purified compound (>98%) was dissolved in DMSO and its antiproliferative activity was determined using the CellTiter-Glo cell viability assay kit. In our initial screening various cancer cells were treated with increasing concentration of Cu-TA or TA, and cell viability was assessed at 24 and 48h post-treatment. The effect of Cu-TA on expression of Sp1 and survivin was determined by Western blot analysis of lysates prepared using cells treated with Cu-TA or TA for 48h. We also studied the effect of Cu-TA on cell cycle and apoptosis using medulloblastoma cell lines. Both Cu-TA and TA decreased cell viability in a dose-dependent manner; however, Cu-TA consistently had lower IC50 values in all the cell lines tested, suggesting its higher efficacy compared to TA. In line with the viability results, Western blot analysis of survivin expression demonstrated that Cu-TA was more effective in inhibiting its expression compared to TA. Also, treatment of medulloblastoma cells with Cu-TA resulted in cell cycle arrest in G0/G1 phase accompanied by an increased apoptosis as determined by annexin-V staining. This preliminary screening demonstrates the potential of Cu-TA as an effective anticancer agent with the ability to target survivin. Further studies to delineate the mechanism of action of Cu-TA are under way.

#5425

Investigating the effects of p53 activation and HDAC inhibition on genes associated with poor prognosis prostate cancer.

Gemma M. Gregg, Fiammetta Falcone, Andrea Lees, Gerard Quinn, Peter Gallagher, Richard Kennedy, Ian I. Mills, Simon S. McDade. _Queens University Belfast, Belfast, United Kingdom_.

Background

Prostate cancer (PCa) is now the most commonly diagnosed malignancy in men worldwide. SOC combines radiotherapy with ADT, however recurrence rate remains unacceptably high and many men present with late stage aggressive and metastatic disease, with poor prognosis and limited treatment options.

Despite tumor suppressor p53 being mutated in approximately 50% of human tumors, it has been reported that p53 alteration occurs in only 12% of primary prostate tumors. Emerging data links epigenetic-mediated gene silencing as a major differentiator between aggressive and indolent disease that is associated with up-regulation of key cell cycle regulator FOXM1, which is known to be suppressed by p53. HDAC inhibitors (HDACi) are an emerging class of epigenetic modifying anti-cancer agents, which we hypothesises will enhance p53 activity, cell death and alter patterns of gene expression to targeting p53 dependent and independent vulnerabilities in PCa.

Methods

We used basally p53 deficient/proficient PCa models (PC3/LNCaP) and novel C4-2B and LNCap p53 CRISPR-Cas9 knockout models to investigate the combination effects of Nutlin 3A or radiation (IR) with class 1 HDACi Entinostat by Annexin V/PI Flow Cytometry and Western Blot. Transcriptional effects were evaluated by RT-PCR and RNA-seq analysis. TCGA mutational and RNA-seq data were used to link p53 mutation with altered gene expression and outcome.

Results

We have demonstrated that there is a synergistic induction of cell death when we combine N3A/IR with Entinostat that is p53-dependent. In addition, we see that p53 acts as a barrier to genes associated with poor prognosis including FOXM1 which is phenocopied by single-agent Entinostat independent of WT-p53. This suppression may potentially be downstream of p21 via the DREAM complex. Importantly p53 mutation in TCGA primary PCa patients is associated with poor outcome and elevated FOXM1 expression.

Conclusion and Future Work

Results to date suggest that Entinostat enhances p53 dependent cell death but also has p53 dependent and independent mechanisms to suppress genes associated with poor prognosis PCa. The mechanism looks to be dependent of p21 and may involve DREAM complex.

#5426

Regulation of mTOR signaling pathway by Ikaros and CK2 in high risk leukemia.

Chandrika S. Gowda,1 Chunhua Song,1 Mary McGrath,1 Charyguly Annageldiyev,1 Kimberly payne,2 Sinisa Dovat1. 1 _Penn State Univ. Hershey Medical Ctr., Hershey, PA;_ 2 _Loma Linda University, Loma Linda, CA_.

Acute Lymphoblastic Leukemia (ALL) is the most common cancer in children. Alterations in IKZF1 have proven to be an indicator of inferior outcome in patients with high-risk ALL. Casein Kinase II (CK2) is a pro-oncogenic protein which is overexpressed in various cancers including leukemia. CK2 phosphorylates Ikaros and impairs its tumor suppressor functions. Targeted inhibition of CK2 restores Ikaros tumor suppressor function in high-risk B-ALL even in cases with single allele Ikaros deletion. Global chromatin immunoprecipitation (ChIP) coupled with the next-generation sequencing (ChIP-seq) studies in primary human B-ALL cells and in cell lines, demonstrated Ikaros occupancy of the promoter of genes involved in mTOR pathway. mTOR (mechanistic target of rapamycin) is the key component of the PI3K/AKT/mTOR pathway - a major signaling pathway that stimulates cellular proliferation in B-ALL. Several specific inhibitor of mTOR (rapamycin, Everolimus, sirolimus) are currently being used for the treatment of relapsed/refractory leukemia.

Objective of this study is to understand the mechanisms of transcriptional regulation of mTOR in B- ALL. We hypothesize that Ikaros negatively regulates the mTOR pathway by repressing transcription of mTOR and PI3K genes.

Ikaros binding to promoters of mTOR and genes that promote the PI3K pathway was confirmed using quantitative ChIP. Functional experiments such as overexpression of Ikaros in B-ALL cells results in transcriptional repression of mTOR and PI3K genes whereas Ikaros silencing using shRNA resulted in increased transcription. These results suggest that Ikaros negatively regulates the mTOR pathway by repressing transcription of mTOR and PI3K genes. Molecular inhibition of CK2 with shRNA targeting the CK2 catalytic subunit, as well as pharmacological targeting of CK2 with CX4945 resulted in transcriptional repression of mTOR and PI3K genes. CK2 inhibition was associated with increased Ikaros DNA-binding to the promoters of mTOR and PI3K genes. However, the ability of CX4945 to repress mTOR and PI3K genes is lost or severely reduced, in cells with shRNA silencing of Ikaros, as compared to cells with intact Ikaros. Moreover, similar results were noted following treatment with CX4945 in leukemia cells obtained from high risk B-ALL patients with deletion of one IKZF1 allele. Ikaros binds poorly to promoters of mTOR and PI3K genes in these cells. Treatment with CX4945 restores Ikaros DNA-binding to the promoters of both mTOR and PI3K genes, which is associated with strong repression of mTOR and PI3K genes.

In conclusion, these results suggest that CK2 inhibition with CX4945 represses the mTOR pathway by enhancing Ikaros-mediated transcriptional repression of mTOR and PI3K pathway genes. Results provide the rationale for the use of CK2 inhibitors in combination with mTOR inhibitors in high risk leukemia with dysregulation of mTOR/PI3K pathway.

#5427

Inhibition of hypoxia-inducible factor 1 (HIF-1) transcriptional activity and cell mobility via G9a/GLP-mediated methylation of HIF-1α.

Lei Bao,1 Yan Chen,1 Hsien-Tsung Lai,1 Cheng-Ming Chiang,1 Gregg L. Semenza,2 Yingfei Wang,1 Weibo Luo1. 1 _UT Southwestern Medical Center, Dallas, TX;_ 2 _Johns Hopkins University School of Medicine, Baltimore, MD_.

Hypoxia-inducible factor 1 (HIF-1) is a main regulator of cellular response to hypoxia and its transcriptional activity is crucial for cancer cell migration and invasion. Post-translational modifications of the α subunit of HIF-1 (HIF-1α) play dominant roles in regulating its function. In this study, we demonstrated that the lysine methyltransferases G9a and GLP catalyzed mono- and di-methylation of HIF-1α at lysine (K) 674 in vitro and in vivo. Functionally, K674 methylation suppressed HIF-1 transcriptional activity as well as expression of the HIF-1 target genes including PTGS1, NDNF, SLC6A3, and Linc01132 in human glioblastoma U251MG cells. Moreover, mutation of K674 of HIF-1α significantly enhanced U251MG cell migration under hypoxia. Importantly, we found that G9a level was downregulated by hypoxia in glioblastoma, which was negatively correlated with PTGS1 expression and survival rate in glioblastoma patients. These findings reveal a novel naturally existing negative feedback mechanism that maintains high activity of HIF-1 and cell mobility in human glioblastoma.

#5428

Effect of amentoflavone on Nrf2 signaling in HaCaT cells.

Lilik Duwi Wahyudi,1 Jiwon Jeong,1 Heejung Yang,2 Jung-Hwan Kim1. 1 _Gyeongsang National University, Jinju, Republic of Korea;_ 2 _Kangwon National University, Chuncheon, Republic of Korea_.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key mediator of cell response to oxidative stress and xenobiotics. Multiple lines of evidence suggest that Nrf2 activation protects against many diseases, aging, inflammation, and cancer. To date, Nrf2 activators from natural products have been widely studied. In this study we investigated the potential effect of Amentoflavone (AFN), a biflavonoid in many natural plants, on Nrf2 signaling in human keratinocytes cells (HaCaT). As results, AFN significantly increased ARE-luciferase activity and nuclear accumulation of Nrf2. Furthermore, a Nrf2 target protein, NQO-1, was significantly increased by AFN in dose- and time-dependent manner. To verify the Nrf2 signaling mechanism by AFN, firstly we measured reactive oxygen species (ROS) using DCFDA because mild intracellular ROS could stimulate Nrf2 activation. Surprisingly AFN triggered the production of ROS in 1 h and AFN-induced Nrf2 was inhibited by N-acetyl cysteine (NAC). Thus, we studied the ROS-related signaling on Nrf2 by measuring the activation of Akt and mitogen-activated protein (MAP) kinase family such as extracellular signal-regulated kinase (ERK) and p38. As results, pharmacology inhibitor of PI3K/Akt (LY294002) or p38 (SB 203580), but not ERK (U0126), abrogated AFN-activated Nrf2. Furthermore, silencing p38 using p38siRNA or inhibition using its pharmacology inhibitor decreased phosphorylation of Akt and attenuated Nrf2 activation. We also confirmed that AFN stabilized Nrf2 by inhibiting the ubiquitination. Taken together, AFN activates Nrf2 through ROS-mediated phosphorylation of p38 and followed by phosphorylation of Akt.

#5429

Targeting Nrf2 anti-oxidative pathway as a novel strategy to suppress IDH1-mutated cancer.

Yang Liu. _NIH, NCI, Bethesda, MD_.

Background: Reactive oxygen species are recently recognized as a second messenger that affects many aspects in cell biology. In cancer cells, ROS has been proof closely related to metabolic reprogramming, tumor proliferation, progression and metastasis. In tumors with isocitrate dehydrogenase (IDH) mutations, the neomorphic enzyme results in unusual accumulation of ROS in cancer cells, suggesting distinctive vulnerabilities may be established in these types of cancer. However, the biologic function of ROS in IDH1-mutated cancers remains elusive.

Methods: In this study, we explored the ROS homeostasis in IDH1 mutated glioma. We investigated the correlation between ROS scavenging genes and overall survival in 530 IDH1-mutated glioma cases. Mechanistically, we focused on nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway and seek for novel therapeutic strategy by targeting ROS burden in cancer cells. We further investigated the therapeutic value of Nrf2 inhibitor, Brusatol, in IDH1 mutated cancer in vitro and in vivo.

Results: We found IDH1 mutation leads to substantial reprogramming in ROS homeostasis, highlighted with prompted ROS generation and detoxification. Stronger expression of ROS scavenging gene predicts drug resistance and poor disease outcome. Enhanced Nrf2-associated scavenging pathway was found essential to maintain reasonable intracellular ROS level during cancer progression. Targeting Nrf2 scavenging pathway not only led to ROS overload, DNA/protein oxidative damage, but also reduced tumor cell proliferation and xenograft growth in vivo.

Conclusions: IDH1 mutant glioma highly depend on Nrf2 scavenging pathway to maintain ROS homeostasis. Targeting ROS scavenging might be a novel approach to treat gliomas with IDH1 mutation.

#5430

Evaluating a novel phytobiologic mixture against breast cancer cell lines: Effect on glycolytic enzymes and EMT gene expression profile.

Aliaa A. Alamoudi,1 Afnan A. Alqarni,1 Ghada Ajabnoor,1 Aleksandra Niedwiecki,2 Matthias Rath,2 Steve M. Harakeh,1 Ahmed M. Al-Abd3. 1 _King Abdulaziz University, Jeddah, Saudi Arabia;_ 2 _Dr. Rath Research Institute, Santa Clara, CA;_ 3 _National Research Ctr. of Egypt, Cairo, Egypt_.

Background: Recently, there has been a growing evidence proposed regarding role of phytobiological compounds against various malignancies. One of the novel phytobiological mixture (PB), which is a mixture composed of the following: quercetin, resveratrol, cruciferex, epigallocatechin-3-gallate and curcumin at different proportions. To the best of our knowledge, this is the first study to evaluate the effects of a PB on breast cancer cell lines and its impact on the expression profile of these cells.

Aim: Herein, we investigated the anti-proliferative effects of PB against two breast cancer cell lines: MDA-MB-231 and MCF-7, in addition, to investigating whether PB may alter the expression profile of glycolytic enzymes and epithelial mesenchymal transition (EMT) genes in these cell lines.

Method: The anti-proliferative effect of PB was evaluated in vitro against MCF-7 and MDA-MB-231 cell lines using SRB assay. The effect of PB on the relative expression profile of some of the glycolytic enzymes and EMT genes was conducted using TaqMan gene expression assays via reverse transcription-quantitative PCR (RT-qPCR).

Results: At 72 hrs, treatment with PB showed cytotoxic effects against MDA-MB-231 and MCF-7 cells with an IC50's of 25.9±3 µg/ml and 29.5± 0.9 µg/ml, respectively. At 24 hrs post PB treatment, there was a significant increase in the gene expression of most glycolytic enzymes such as; Phosphofructokinase muscle type (PFKM) (1.4± 0.1, p<0.01) and Pyruvate dehydrogenase kinase isoenzyme 1 (PDK-1) (1.6± 0.2 fold, p< 0.001). PB did not seem to alter the EMT gene expression profile with both cadherin 1 and 2 showing no significant changes. Interestingly, however, a significant up-regulation was seen with ZEB2 member of the zinc finger E-box binding homeobox 2, post PB treatment.

Conclusion: In conclusion PB showed a promising anti-proliferative effect against two breast cancer cell lines. However, we postulate that the drug could be modulating glycolytic enzymes expression, which ultimately can affect key molecules in cell differentiation. Further studies are needed to elucidate the exact underlying mechanism and to better understand the most beneficial drug combinations that can work synergistically with PB to interfere with different tumor hallmarks.

#5431

BRD4 regulates cellular senescence in gastric cancer cells via E2F/miR-106b/p21 axis.

Xingchen Dong. _University of Illinois at Urbana-Champaign, Urbana, IL_.

Small molecules targeting bromodomains of BET proteins possess strong anti-tumor activities and have emerged as potential therapeutics for cancer. However, the underlying mechanisms for the anti-proliferative activity of these inhibitors are still not fully characterized. In this study, we demonstrated that BET inhibitor JQ1 suppressed the proliferation and invasiveness of gastric cancer cells by inducing cellular senescence. Depletion of BRD4, which was overexpressed in gastric cancer tissues, but not other BET proteins recapitulated JQ1-induced cellular senescence with increased cellular SA-β-Gal activity and elevated p21 levels. In addition, we showed that the levels of p21 were regulated at the post-transcriptional level by BRD4-dependent expression of miR-106b-5p, which targets the 3'-UTR of p21 mRNA. Overexpression of miR-106b-5p prevented JQ1-induced p21 expression and BRD4 inhibition-associated cellular senescence, whereas miR-106b-5p inhibitor up-regulated p21 and induced cellular senescence. Finally, we demonstrated that inhibition of E2F suppressed the binding of BRD4 to the promoter of miR-106b-5p and inhibited its transcription, leading to the increased p21 levels and cellular senescence in gastric cancer cells. Our results reveal a novel mechanism by which BRD4 regulates cancer cell proliferation by modulating the cellular senescence through E2F/miR-106b-5p/p21 axis and provide new insights into using BET inhibitors as potential anti-cancer drugs.

#5432

Cancer-induced hypercalcemia promotes breast cancer progression via AP-1 transcription factor mediated upregulation of malignancy-associated genes.

Heather K. Beasley, Sarrah E. Widatalla, Stephen D. Williams, Diva S. Whalen, Olga Y. Korolkova, Amos M. Sakwe. _Meharry Medical College, Nashville, TN_.

Cancer-induced hypercalcemia (CIH), is common in breast cancer patients with metastatic disease and in up to 30% of cases with solid tumors without evidence of metastasis. However, its role in the development of large and aggressive breast tumors remain poorly understood. In this study, we show that prolong exposure of triple negative breast cancer (TNBC) cells to high Ca2+ is accompanied by reduced sensitivity of the calcium sensing receptor to high Ca2+, increased growth and tumor cell motility. Gene expression profiling of TNBC cells cultured at high Ca2+ (5.0 mM) revealed that in addition to early response genes (FOS/FOSB), high extracellular Ca2+ also modulated the expression of MAGEC2/CT10 and PAI-2 and other malignancy associated genes (MAGs). To determine whether the expression of MAGs is regulated by FOS/FOSB we show that down regulation of FOSB strongly inhibited the high Ca2+-induced expression of MAGEC2 and that FOSB, as a component of the AP-1 transcription factor bound to the MAGEC2 promoter at high Ca2+. We also show that down regulation of MAGEC2 or FOSB affected the migration and proliferation of TNBC cells at high Ca2+. These data suggest that high Ca2+-induced expression of malignancy promoting genes such as MAGEC2/CT10 underlie the successful tumor cell survival, growth and/or motility in calcium-rich microenvironments and presumably, during the hypercalcemia prone late stages of the disease. This research is supported by NIH/NIGMS 5SC2 CA170244 and 1SC1 CA211030, NIH/NCI 2U54CA 163069-07

#5433

XRCC5 promotes tumor growth and progression via PDK-1 signaling and predicts poor prognosis in melanoma.

Ge Qin, Tianze Liu, Wenbin Li, Yixin Li, Changlin Zhang, Qian Long, Dingbo Shi, Miao Chen, Shuting Huang, Wuguo Deng. _Sun Yat-sen University Cancer Center, Guangzhou, China_.

Melanoma is one of the most malignant and aggressive cancers with high cancer-related deaths. Patients with melanoma are often diagnosed at an advanced stage that has developed local or even remote metastasis due to its inapparent symptoms at early stages. Melanoma is often insensitive to chemotherapies, and most patients that respond well to targeted therapies or immunotherapies at first will develop resistance within several months. Therefore, it is of urgency to better understand the mechanisms of melanoma tumorigenesis, and to discover novel molecular targets for its effective treatment. In this study, we identified XRCC5 as a potential therapeutic target by siRNA library screening in A375 melanoma cells. We found that knockdown of XRCC5 inhibited cell growth, migration and invasion, and induced apoptosis in melanoma cells. Conversely,overexpression of XRCC5 significantly promoted the growth, migration and invasion of melanoma cells. We next analyzed the global transcriptome in A375 cells with XRCC5 knockdown, and identified PDK-1 as a candidate gene regulated by XRCC5. Further investigation revealed that XRCC5 cooperated with HIF-1α to bind at the promoter of PDK-1, and enhanced the expression of PDK-1. Consistently, overexpression of PDK-1 rescued the cell growth inhibition caused by XRCC5 knockdown. Our clinical data showed that the expression of XRCC5 and PDK-1 were positively correlated and elevated in melanoma samples, and high expression of XRCC5 predicted poor prognosis in melanoma patients. Collectively, our study discovered that XRCC5 bound to the promoter of PDK-1 to activate its expression, which promoted melanoma growth and progression. Our results indicated that the XRCC5/PDK-1 pathway could be a potential therapeutic target for melanoma.

#5434

Screen for immune-related transcriptional targets of the Hippo pathway in human breast cancer cells.

Helena J. Janse van Rensburg, Taha Azad, Min Ling, Yawei Hao, Lori M. Minassian, Brooke Snetsinger, Prem Khanal, Michael J. Rauh, Charles H. Graham, Xiaolong Yang. _Queen's University, Kingston, Ontario, Canada_.

The Hippo signaling pathway has recently emerged as a cellular network that is dysregulated in cancer. In breast cancer cells, aberrant activation of the Hippo transducers (and transcriptional co-activators) TAZ and YAP leads to altered expression of their downstream gene targets and endows cells with multiple "hallmarks of cancer". While several transcriptional targets of TAZ and YAP underlying their pro-tumorigenic activity have been described (e.g. CTGF, CYR61), gene targets of TAZ and YAP that modulate immune cell behavior in the tumor microenvironment are poorly understood. We have performed a comprehensive screen for immune-related transcriptional targets of TAZ and YAP in human breast cells. We have identified many candidate genes that are potentially regulated by TAZ and YAP including the immune checkpoint molecules PD-L1 and PD-L2, the lymphocyte regulator S1PR1 and the inflammasome component NLRP3. We have further validated PD-L1 as a bona fide transcriptional target of TAZ and YAP. These findings reveal new functions for the Hippo pathway in modifying immune responses and implicate TAZ and YAP in cancer immune evasion.

#5435

Role of the WT1 transcription factor in high-grade serous ovarian cancer.

Norma I. Rodriguez-Malave,1 Justyna E. Kanska,1 Kevin C. Vavra,1 Dennis Hazelett,1 Rosario I. Corona,1 Ji-Heui Seo,2 Matthew Freedman,2 Simon R. Knott,1 Sandra Orsulic,1 Beth Y. Karlan,1 Kate Lawrenson,1 Simon A. Gayther1. 1 _Cedars-Sinai Medical Center, Los Angeles, CA;_ 2 _Dana-Farber Cancer Institute, Boston, MA_.

The Wilms Tumor protein 1 (WT1) transcription factor (TF) is a ubiquitous biomarker of high-grade serous ovarian carcinoma (HGSOC), which is the most common and aggressive subtype of epithelial ovarian cancer (EOC). This TF interacts with a host of cellular factors including the tumor suppressor P53, which is mutated in virtually all HGSOC cases. Studies have suggested a role for WT1 in HGSOC development, but little is known about the underlying mechanism. We have generated unique catalogues of epigenomic landscapes (specifically H3K27ac) in primary HGSOC using chromatin immunoprecipitation sequencing (ChIP-seq). Using Bayesian analyses of H3K27ac profiling in HGSOCs, we identified significant tissue specific enrichment of active chromatin at the WT1 locus in HGSOC when compared to all other EOC tumor histotypes. Furthermore, this locus contains a superenhancer in HGSOCs and in normal fallopian tube secretory epithelial cells (FTSECs). This locus features a bidirectional promoter that is shared by WT1-AS lncRNA and that contains a WT1 binding site. LncRNAs are essential in many cellular processes, and have been shown to play pivotal roles in tumorigenicity. The WT1-AS lncRNA is implicated in other diseases but little is known of its function in EOC. We have investigated the mechanisms of interaction between WT1 and WT1-AS. We observed that both protein and transcript expression for WT1 and WT1-AS are positively correlated in HGSOC cell lines. Analysis of data for >400 primary HGSOCs from TCGA further supported this correlation. We hypothesized that WT1 is a critical TF in the development of HGSOC, and that WT1-AS plays a key role in its regulatory pathway. We evaluated the regulatory role of WT1 first by analyzing cellular phenotypes and target gene expression changes in HGSOC cell lines after shRNA mediated knockdown (KD) of WT1. HGSOC cells with stable WT1 knockdown had impaired proliferation and concomitant reduction of WT1-AS expression. We also identified a decrease in expression of SHMT1 in WT1 KD cells, which is a known WT1 target gene. Integrated genome-wide analysis of the WT1 transcriptome by RNA-seq and the WT1 cistrome by ChIP-seq, in parallel with ChIP-MS, is being used to establish the architecture of the WT1 regulome, specifically to establish the regulatory mechanisms and relationships between WT1 and WT1-AS. Ultimately, these studies aim to establish the mechanisms of action for the WT1 TF in neoplastic transformation of HGSOC.

#5436

Novel single-domain antibody (sdAb), SBT-100, localizes in the cytoplasm to inhibit IL-6 mediated P-STAT3 nuclear translocation in cancer cells.

Sunanda Singh,1 Genoveva Murillo,2 Avani Singh,1 Samara Singh,1 Meenakshi Parihar,1 Rajendra Mehta,2 Anjali Singh,1 Ashutosh Parihar1. 1 _Singh Biotechnology, Tampa Bay, FL;_ 2 _IIT Research Institute, Chicago, IL_.

BACKGROUND: Interleukin-6 (IL-6) is a multifunctional cytokine that is involved in immune defense and plays an important role in biologic activities of cells including tumor cells. These effects are mediated by several signaling pathways, including the signal transducer and transcription activator 3 (STAT3), which plays a critical role in the development, growth, and metastasis of cancer. Inside cancer cells within the tumor microenvironment IL-6 activates (phosphorylates) STAT3. The P-STAT3 dimers translocate into the nucleus where it turns on many genes (e.g., BCL-XL, MCL-1, Myc, Cyclin D1/D2, VEGF, MMP-2, etc.) that are necessary for tumorigenesis. Since there are no commercially available IL-6 or STAT3 inhibitors, blocking IL-6/STAT3 signaling is a potential therapeutic strategy for cancer. SBT-100 is a novel sdAb which binds P-STAT3 to inhibit its translocation into the nuclei of cancer cells, thereby suppressing the cancers growth.

METHODS: Human cancer cell lines obtained from ATCC. The STAT3 reporter assay (Promega, WI). IHC staining with IL-6 stimulation, the primary antibody (Ab) was STAT3 (124H6) mouse mAb (Cell Signaling), secondary Ab was anti-mouse IgG (H&L), Alexa Fluor 488 (Cell Signaling), blocking Ab was SBT-100, and recombinant human IL-6 was from Peprotech. In vitro cancer cell suppression via MTT assay (3 day). Xenograft study, athymic nude mice were obtained from Envigo.

RESULTS: The STAT3 reporter assay demonstrates that IL-6 activation of STAT3 is completely inhibited by 100ug/ml of SBT-100 (p<0.0012). SBT-100 shows significant (p<0.001) suppression of ten different human cancers in vitro which have constitutive expression of P-STAT3. Immunohistochemical (IHC) staining demonstrates that SBT-100 crosses the cell membrane and localizes within the cytoplasm of MDA-MB-231 cells. IHC staining of STAT3 shows IL-6 stimulation drives P-STAT3 into the nuclei of HEp-2 and PANC-1 cells and this translocation is inhibited by SBT-100. In vivo xenograft studies demonstrate that SBT-100 gives significant (p<0.001) tumor growth suppression of MDA-MB-231.

CONCLUSION: SBT-100 inhibits IL-6 mediated function of P-STAT3 as demonstrated by two different assays. IHC staining demonstrates SBT-100 crosses the cell membrane to localize inside cancer cells. Based on its mechanism of action, SBT-100 can be a potential antibody targeted IL-6/STAT3 therapy for human cancer.

#5437

MESP1 cooperates with loss of ARF in malignant transformation.

Neha Tandon, Benjamin Soibam, Robert Schwartz, Yu Liu. _University of Houston, Houston, TX_.

Cancer is a highly complex disease involving multiple genetic and epigenetic abnormalities. Inactivation or expression of key regulators can be critical for the maintenance and survival of tumor. My research aims at characterizing one such novel gene, Mesoderm Posterior Basic Helix-Loop-Helix transcription factor-1 (MESP1), in lung cancer initiation and progression. MESP1 is transiently expressed during embryonic development and is the first sign of nascent mesoderm. It is a master regulator of cardiovascular development. Our lab has discovered through genome-wide identification of MESP1 targets that it is required in mesendoderm lineage development. However, whether MESP1 plays any role in adult physiology, diseases and cancer, has never been investigated. The Cancer Genome Atlas (TCGA) data show that MESP1 expression is upregulated in cancers of all mesendoderm-derived tissues. Bioinformatic gene expression analysis of clinical lung adenocarcinoma patient samples (396) showed that MESP1 was highly overexpressed in lung cancer. Similarly, RT-qPCR analysis of patient RNA samples from various stages of lung cancer showed that MESP1 expression was induced in early stages of lung cancer. It was found that MESP1 stable overexpression in ARF-null MEFs leads to oncogenic transformation of cells. This was evidenced by changes in various hallmarks of cancer such as increased cell proliferation, colony formation and anchorage-independent growth (about 6-fold increase in no. of colonies per field). In order to determine if the MESP1-induced oncogenic transformation was p53 dependent, we overexpressed MESP1 in p53-null MEFs. Interestingly, we saw no significant change in cell proliferation, colony formation and anchorage-independent growth in MESP1-overexpressing p53 null MEFs, suggesting that these effects are p53-independent. Upon performing global transcriptomic analyses in MESP1-overexpressing ARF-null MEFs, we found that cell adhesion and cell migration were among the top pathways that were significantly altered. Future studies will focus on in vivo studies to check for effect of MESP1-overexpression and/or knockdown on tumor formation and investigate the mechanism of MESP1-induced oncogenic transformation linked with increased cellular adhesion and migration. Lung cancer is the leading cause of cancer-related deaths in both men and women. More than 50% of cases are diagnosed only at an advanced stage, for which the survival rate of the patient is only 4%. Hence, knowledge of new driver oncogenes of lung cancer is imperative. These results suggest that MESP1 plays an important role in cancer initiation and progression. Ultimately, this research would pave the way for therapeutic intervention of key processes regulated by MESP1 involved in lung cancer.

#5438

XPNPEP3: A novel transcriptional target of canonical Wnt/β-catenin signalling.

Raju Kumar,1 Ashmala Naz,1 Viswakalyan Kotapalli,1 Swarnalata Gowrishankar,2 Satish Rao,3 Jonathan R. Pollack,4 Murali Dharan Bashyam1. 1 _Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India;_ 2 _Apollo hospitals, Hyderabad, India;_ 3 _Krishna Institute of Medical Sciences, Hyderabad, India;_ 4 _Stanford University School of Medicine, Stanford, CA_.

Canonical Wnt/β-catenin signalling plays pivotal roles during embryonic development and adult tissue regeneration. Its aberrant activation however drives expression of a panoply of genes to facilitate colorectal tumorigenesis. Hence, it is imperative to delineate the complete β-catenin transcriptome. We performed genome-wide mRNA profiling of sporadic rectal cancer samples stratified by Wnt status. Results revealed significant up-regulation of XPNPEP3 transcript levels along with that of canonical Wnt targets AXIN2 and EPHB2 in Wnt+ samples. The differential expression of XPNPEP3 was further validated by quantitative PCR (Q-PCR) in additional samples. Nuclear stabilization of β-catenin achieved through LiCl treatment in three colorectal cancer (CRC) cell lines followed by Q-PCR and promoter-luciferase assays confirmed up-regulation of XPNPEP3. XPNPEP3 encodes X-prolyl aminopeptidase 3 which functions to remove the penultimate N-terminal Proline residue from nascent proteins and appears to play a role in ciliary function. Immunohistochemistry based expression analysis using a CRC tissue microarray revealed significant correlation between XPNPEP3 levels and β-catenin nuclear localization as well as increased XPNPEP3 expression in tumor compared to matched normal samples. Survival analysis of The Cancer Genome Atlas (TCGA) breast invasive carcinoma, skin cutaneous melanoma and lung adenocarcinoma data sets revealed poor survival among patients having perturbed XPNPEP3 expression. Characterization of possible oncogenic function of XPNPEP3 in CRC cells and in mice tumor xenografts is currently underway. Altogether, our results suggest XPNPEP3 to be a novel transcriptional target of Wnt/β-catenin signalling having a possible significance in CRC tumorigenesis.

#5439

Study of polymorphisms in stra6 in Mexican population with non-small cell lung cancer.

Sae Muniz-Hernandez, Laura-Alejandra Ramírez-Tirado, Oscar Arrieta. _National Cancer Inst. of Mexico, Mexico City, Mexico_.

STRA6 mutations have been associated with severe pathologic phenotypes in multiple human organs, including the eye, brain, heart and lung. In this study, we analyzed the presence of two polymorphisms in patients with diagnosis of lung cancer in advanced stage. Patients consecutive, nonselected, pathologically confirmed with advanced NSCLC, who visited the Thoracic Oncology Unit at the Instituto Nacional de Cancerología from January 2014 to December 2015, were enrolled. Genomic DNA was obtained from peripheral blood. RT-PCR was used to genotype the different allelic variant. The SNPs, NC_000015.10:g.74194695T>A (rs351224) and NC_000015.10:g.74194103C>T (rs974456) were genotyped with commercially available TaqMan-validated SNP assays (C_3152256_10 and C_3152257_20, respectively). An allelic discrimination plot was used to identify individual genotypes. Subsequently, each allelic variant was confirmed by direct sequencing in 5% of the samples. All statistical analyses were carried out using the SPSS software package, v. 15 (SPSS Inc, Chicago, IL). This study included 145 patients, of whom 55.2% (80/145) were female. The mean age at diagnosis was 60.8 + 12.9 years. Around half of the patients had a tobacco history (52%) and 47% wood smoke exposure; most patients had a good performance status (≤ 1) (68%). Adenocarcinoma was the most common histologic subtype (83%). Approximately 15% of patients were known to have diabetes and at least 25% had a fasting glycemia upper of 126 mg/dL. Seventy-seven percent of patients received chemotherapy (QT) and 35.8% received treatment with TKIs, with a global response rate of 13% and 46%, respectively. We analyzed the frequency of the STRA6 SNPs in our population; we found a frequency of SNP1 (rs351224) of 0.503, 0.393 and 0.103, for wild-type homozygous patients (AA), heterozygous (AT), and variant homozygous (TT), respectively, whereas the frequency found for SNP2 (rs974456) was 0.352 for wild-type homozygous patients (CC), 0.531 for heterozygous (CT) and 0.117 for variant homozygous (TT). We analyzed the relationship between clinical characteristics and the polymorphism presence. According to our data, the absence of SNP2 was associated with response rate at QT (33% vs. 0%, p= 0.022), and patients with this polymorphism had glycemia below to 120 mg/dL (27.1% vs. 6.3%, p=0.068). No other clinicopathologic characteristics were found associated with the presence of any SNP. The overall survival of our patients was 15.5 months (10.6-20.3 months). We found a bordering difference in relation to SNP2 presence (presence vs. absence) (23.1 vs. 12.9 months, p=091). Other factors associated with a better OS were female gender (18 vs. 11 m, p=0.025), age <60 y (18 vs. 12 m, p=0.05), no tobacco history (23 vs. 10 m, p =0.021), and ECOG <2 (23 vs. 6 m, p < 0.001). We can suggest that the presence of SNP2 could be related to a better response to QT and better OS in our population.

#5440

Role of tyrosine phosphorylation of β-catenin in EGFR-mutant lung cancer.

Masanori Fujii, Sohei Nakayama, Kohei Shimizu, Hisashi Takei, Mariko Ando, Eunyoung Heo, ikei Kobayashi, Gilbert Pan, Hiroyuki Inuzuka, Susumu Kobayashi. _Beth Israel Deaconess Medical Ctr., Boston, MA_.

Wnt/ β-catenin signaling plays a key role in the pathogenesis of colon and other cancers. Emerging evidence indicates that oncogenic β-catenin regulates several biological processes essential for cancer initiation and progression. The role of β-catenin in lung cancer with epidermal growth factor receptor (EGFR) activating mutations has not been fully understood; however, recent studies suggest that co-occurring genetic alterations in CTNNB1 might cooperate with mutant EGFR in promoting tumor progression and treatment resistance to EGFR-tyrosine kinase inhibitors (EGFR-TKIs). This sheds new light on this pathway in EGFR-mutant lung cancer. We previously demonstrated that β-catenin plays an essential role in lung tumorigenesis driven by EGFR mutants, particularly EGFR-T790M. We also found that β-catenin is activated through tyrosine-phosphorylation by EGFR mutants. In this study, we identified five tyrosine phosphorylation sites (Y5) of β-catenin by mass spectrometry. Tyrosine phosphorylation at these sites may be critical in β-catenin stability, nuclear translocation and its role as a transcriptional switch in oncogenesis. Firstly, we demonstrated that tyrosine phosphorylation of β-catenin disrupts the binding of a main ubiquitin ligase β-Trcp1, leading to an escape from degradation. Secondly, in contrast to known transcriptional activity with TCF/LEF, β-catenin that is tyrosine phosphorylated by mutant EGFR was found to form a complex with a transcriptional regulator, YAP1, and a transcriptional factor, TBX5. Immunoprecipitation experiments suggested Y5 tyrosine phosphorylation of β-catenin plays a critical role in the interaction between β-catenin and TBX5. Moreover, this complex may regulate anti-apoptotic genes, including BCL2L1, which encodes Bcl-xL. Combination treatment with osimertinib, a 3rd generation EGFR-TKI, and dasatinib suppressed Y5 tyrosine phosphorylation of β-catenin, leading to reduced Bcl-xL expression. Subsequent TBX5 knockdown in vitro decreased Bcl-xL expression while Bcl2l1 knockout in vivo mouse models suppressed lung tumor development. These observations suggest that β-catenin-YAP1-TBX5 complex contributes to EGFR-T790M lung cancer and may serve to explain the underlying mechanism for EGFR-TKIs resistance and for the effectiveness of combination therapy with osimertinib and dasatinib.

#5441

Regulation of AKT phosphorylation by GSK3β and PTEN to control chemoresistance in breast cancer.

Chunyi Gao, Guohua Wang, Zhenglin Jiang. _Nantong Univeristy, Nantong, China_.

Background Chemotherapy is an important element in the treatment paradigm for breast cancers. However, the resistance of cancer cells to chemotherapeutic agents frequently results in the subsequent recurrence and metastasis. AKT phosphorylation is highly expressed or over-expressed in chemoresistant tumor samples. However, the precise molecular mechanism involved in AKT phosphorylation-related chemoresistance in breast cancer remains unknown. The aim of the present study was to assess whether the role of AKT phosphorylation in the cell viability and chemoresistance of breast cancer. Methodology/Principal Findings We developed two taxol resisted breast cancer cells: TAX Resis HepG2 and TAX Resis HepG2 SMMC7721. Western blot and immunofluorescence analysis indicated that glycogen synthase kinase 3 beta (GSK3β), phosphorylated phosphatase and tension homologue (p-PTEN) and phosphorylation of AKT (p-AKT) were highly expressed in taxol resisted HepG2 and SMMC7721 cells. The increase of GSK3β phosphorylated and inactivated PTEN, thereby enhanced phosphatidylinositide 3-kinases (PI3K)/Akt signaling. The increase in GSK3β, p-PTEN and p-AKT was associated with cell viability by MTT assay. Annexin-V-PI assay and transwell analysis were indicated that GSK3β knockdown with lentiviral shRNA (shRNA-GSK3β) promoted the apoptosis and suppressed the migration ability of Taxol resisted HepG2 and SMMC7721 cells, and both effects were reversed by activating p-AKT via PTEN inhibitor bpV(pic). Conclusions In conclusion, AKT phosphorylation manipulated by GSK3β and PTEN were correlated with cell viability, migration and apoptosis in Taxol resisted HepG2 and SMMC7721 cells, which may promote chemoresistance of breast cancer. Furthermore, functions of GSK3β can modulate cell viability via the PTEN/PI3K/Akt signaling pathway and induce chemoresistance, serving as a valuable molecular target for treatment of breast cancer. 

### Signaling and Therapy

#5442

Dose-dependent toxicity of FTY720, a sphingosine-1-phosphate receptor agonist, on double hit lymphoma cells via PP2A-mediated dephosphorylation of c-myc and bcl-2.

Geoffrey P. Shouse,1 Rosalia de Necochea-Campion,2 Saied Mirshahidi,2 Chien-Shing Chen,3 Kimberly J. Payne3. 1 _Loma Linda University Medical Center, Loma Linda, CA;_ 2 _Biospecimen Laboratory, Loma Linda, CA;_ 3 _Loma Linda University School of Medicine, Loma Linda, CA_.

High grade B cell lymphoma with translocations involving MYC and rearrangements involving BCL2 and/or BCL6 define an aggressive subtype of diffuse large B cell lymphoma (DLBCL) with poor prognosis and high rates of relapse. The standard treatment for DLBCL is not considered effective and targeted molecular therapy has yet to be identified for this disease. The mechanisms that make this type of lymphoma particularly aggressive and refractory to treatment include cell growth signals from the c-myc transcriptional regulator working together with the anti-apoptotic activity of the bcl-2 protein.

Protein Phosphatase 2A (PP2A) is a ubiquitous cellular enzyme that acts as a tumor suppressor through dephosphorylation of specific cellular phosphoproteins. In fact, PP2A has been shown to dephosphorylate bcl-2 at ser70 leading to inhibition of its function. Further, PP2A-mediated dephosphorylation of c-myc at ser62 promotes its degradation. In many cancers, PP2A activity is inhibited due to overexpression of binding proteins such as SET and CIP2A. Small molecules can act as PP2A activators by releasing PP2A from these inhibitory binding proteins. In the present study we investigated the efficacy of FTY720, a high-affinity agonist of the sphingosine 1-phosphate receptor-1 and PP2A activator, on the double hit lymphoma derived cell line, WSU DLCL2.

WSU DLCL2 cells were treated with various concentrations of FTY720, a derivative of myriocin, a fungal metabolite of the Chinese herb Iscaria sinclarii known to promote PP2A activation. Cell viability and PP2A activity were measured 24 hours after FTY720 treatment. In addition, cell lysates were assayed by Western blot using antibodies to detect PP2A subunits, as well as total and phosphorylated c-myc and bcl-2 proteins.

A dose-dependent decrease in cell viability was observed over the range of FTY720 concentrations from 1 to 10 micromolar with concomitant increase in in vitro PP2A activity. Western blots showed decreases in c-myc protein levels with FTY720 treatment as well as decreases in ser70 phosphorylation levels of bcl-2 and ser62 phosphorylation levels of c-myc.

The results indicate that PP2A activation via FTY720, a small molecule activator of PP2A, leads to a dose-dependent induction of cell death in double hit lymphoma. The data are consistent with an underlying cell death mechanism that involves the targeted dephosphorylation of c-myc and bcl2. Taken together, these data suggest that FTY720 may act as a targeted treatment with therapeutic potential as a single agent or as a combination with other therapies in the treatment of this subtype of lymphoma. There is also the potential of a therapeutic role in other lymphoma subtypes with poor prognosis including transformed follicular lymphoma and relapsed burkitt's lymphoma that rely individually on bcl-2 and c-myc, respectively, for their growth.

#5443

Methylation-dependent suppression of Tissue Factor is a key contributor to the less aggressive phenotype in IDH1 mutant versus IDH1 wild-type gliomas.

Dusten J. Unruh,1 Snezana Mirkov,1 Seamus Caragher,1 Jann Sarkaria,2 Atique Ahmed,1 C. David James,1 Craig Horbinski1. 1 _Northwestern Univ., Chicago, IL;_ 2 _Mayo Clinic, Rochester, MN_.

Gliomas with mutations in isocitrate dehydrogenase 1 (IDH1mut) have global genomic hypermethylation, are less aggressive than IDH1 wild-type (IDH1wt) gliomas, and generally grow poorly in vitro and in vivo. Yet little data exist that connect specific hypermethylation events to this unique phenotype. We previously reported that the gene encoding Tissue Factor (TF), F3, is among the most hypermethylated and downregulated genes in IDH1mut gliomas relative to IDH1wt gliomas. TF was originally described in blood coagulation, but also can enhance tumor malignancy via protease-activated receptor 2 (PAR2) signaling, though its role in gliomas is not as well understood. In this study, we further explored the significance of TF on malignancy in IDH1wt and IDH1mut gliomas, and showed that receptor tyrosine kinases (RTKs) and β-catenin are downstream effectors of TF-PAR2 signaling. Studies were performed using 6 patient derived glioma cell lines, 3 IDH1wt (GBM6, GBM12, GBM43) and 3 IDH1mut (TB09, BT142, GBM164). DNA methylation was assessed by Illumina Human 850K. TF procoagulant activity (PCA) was determined by FXa generation, using conditioned medium. Markers of malignancy included BrdU incorporation for cell proliferation, transwell invasion, and soft agar colony formation. Nude mice (N≥6/group) were transplanted with glioma cells and monitored for growth using bioluminescence imaging. The TF gene, F3, was significantly hypermethylated in IDH1mut cells compared to IDH1wt cells, with corresponding lower TF protein in IDH1mut cells. Treatment of IDH1mut glioma cells with a demethylating agent, decitabine, increased F3 transcription 5-fold and PCA 19-fold in IDH1mut cells. TF increased the in vivo "take rate" of IDH1mut GBM164 from 0% to 100% (P=0.0001, Fisher's exact test). Conversely, TF knockdown in IDH1wt cells greatly reduced cell proliferation, invasion, colony formation, and extended median survival of engrafted mice by 86% (P=0.001). In both IDH1wt and IDH1mut cells, TF-PAR2 activated β-catenin, ERK1/2, and Akt, and was associated with multiple pathways that increase high-grade behavior. Disrupting TF-PAR2 signaling had the greatest impact on tumor growth in IDH1wt gliomas driven by epidermal growth factor receptor (EGFR), a major receptor tyrosine kinase (RTK) involved in glioma malignancy. TF-PAR2 transactivated EGFR through a Src-dependent intracellular pathway, even when extracellular stimulation of EGFR was blocked, and further enhanced phosphorylation of the constitutively activated EGFRvIII. In IDH1mut cells, TF-PAR2 signaled through another RTK, PDGFR. In contrast, TF-PAR2 stimulated invasion in all glioma cell lines through an RTK-independent, β-catenin-dependent mechanism. These results demonstrate the importance of TF-PAR2 in gliomas, and show that its suppression is a critical component of the IDH1mut phenotype.

#5444

APIO-EE-9 is a novel Aurora A and B antagonist that suppresses esophageal cancer growth in a PDX mouse model.

Guoguo jin. _Zhengzhou University Hospital, Zhengzhou, China_.

Esophageal cancer (EC) is one of the most aggressive malignancies of the upper aerodigestive tract. Over the past three decades, with advances in surgical techniques and treatment, the prognosis of esophageal cancer has only slowly improved. Thus identifying novel molecular targets and developing therapeutic agents are critical. Aurora kinases play a crucial role in mitosis and selective inhibitors might provide an effective therapeutic treatment for cancer. However, the role of Aurora kinases in EC is still inadequately studied. Here, we identified a novel compound, referred to as APIO- EE-9, which inhibits growth and colony formation and induces apoptosis of esophageal cancer cells. Using computer modeling, we found that APIO-EE-9 interacted with both Aurora A and B in the ATP-binding pocket. APIO-EE-9 inhibited both Aurora A and B kinase activities in a dose-dependent manner. Treatment with APIO-EE-9 substantially reduced the downstream Aurora kinase phosphorylation of histone H3 (Ser10), resulting in formation of multiple nuclei and centrosomes. Additionally, esophageal cancer cells expressing shAurora A or shAurora B kinase exhibited a dramatic reduction in proliferation and colony formation. Injection of these cells as xenografts in mice reduced tumor formation compared to wildtype cells. Importantly, APIO-EE-9 significantly decreased the size of esophageal patient-derived xenograft (PDX) tumors implanted in SCID mice. These results demonstrated that APIO-EE-9 is a specific Aurora kinase inhibitor that could be developed as a therapeutic agent against esophageal cancer.

#5445

A novel mechanism of inhibition of ovarian cancer growth by Compound C, dorsomorphin.

Alia Ghoneum, Hesham M. Afify, Neveen Said. _Wake Forest University, Winston Salem, NC_.

High grade serous ovarian carcinoma (HGSC) is the most lethal gynecologic malignancy with a five-year survival rate of 15%. As a highly aggressive cancer, HGSC has elevated energy demands to ensure its survival and proliferation in its unique peritoneal environment. Compound C or Dorsomophin is of particular interest as it is the only available cell permeable molecule that works selectively and competitively to inhibit Adenosine Monophosphate Protein-activated protein kinase or AMPK, a sensitive sensor of ATP levels. It has long been established that Compound C has a role as a BMP1 inhibitor during embryogenesis and as an inhibitor of AMPK activity in different contexts. However, the mechanism by which Compound C exerts anti-tumorigenic effects remains unknown. The purpose of this study is to understand how Compound C suppresses growth of HGSC. Our preliminary studies indicate that Compound C inhibits ovarian cancer cell proliferation and survival via a novel mechanism not only through inhibition of AMPK but also through the inhibition of upstream PI3K-Akt as well as TGFβ1/ TGFβ1/2 receptors/Smad 3,4 activation. These findings elucidate a novel mechanism of inhibition of ovarian cancer by Compound C, suggesting its potential use as a novel therapeutic agent inhibiting oncogenic and metabolic pathways.

#5446

Serotonin modulates AKT-mTOR and Notch signaling pathways, promotes liver cancer cell steatosis and cell survival.

Suresh Niture, Deepak Kumar. _North Carolina Central University, Durham, NC_.

The hormone serotonin is known as 5-hydroxytryptamine. It is a precursor of melatonin, derived from the amino acid tryptophan. Serotonin acts as a neurotransmitter and modulates several key functions in the human body, such as appetite, mood and sleep. Apart from its important roles in the central nervous system (CNS), serotonin regulates various extra-neuronal pathophysiological functions. Serotonin is synthesized by Enterochromaffin cells of the gut, mostly stored in platelets and about 2% of free serotonin released in the blood. The role of peripheral serotonin in adipocytes, pancreatic cells, macrophages and liver cells is not fully understood. In the current study, we investigated the role of serotonin in liver cancer cell signaling and liver cancer cell growth. Treatment of serotonin in liver cancer cells HepG2 and SK-Hep1 induced autophagy markers and effectors such as LC3 I/II, phospho-4EBP1, ATG3 and Beclin1, and, increased cell proliferation and cell survival. AKT/mTOR pathway controls cellular autophagy and our results demonstrated that exposure of serotonin to liver cancer cells inactivates AKT/mTOR signaling and induced autophagy. Also, we demonstrate that serotonin also activates Notch signaling pathway as evident by induction of Notch target gene Hes1. Notch inhibitors Avagacestat 2 and FLI-06 inhibited serotonin-mediated Notch signaling and cellular autophagy. Furthermore, by the activation of Notch signaling, serotonin-induced oleic acid-mediated cell steatosis in both HepG2 and SK-Hep1 liver cancer cells. In addition, treatment of serotonin also induced autophagy modulator proteins such as SIRT1 and Tumor Necrosis Factor Alpha Inducing Protein 8 (TNFAIP8) which participate in the modulation of cellular autophagy and cell survival. Taken together our data suggested that peripheral serotonin regulates liver cancer cell steatosis, cells survival and may promote liver carcinogenesis by the modulation of AKT/mTOR and Notch signaling pathways.

#5447

Overexpression of DARPP-32 promotes activation of STAT3 through IGF1R-SRC axis in gastric cancer.

Shoumin Zhu, Mohammed Soutto, Zheng Chen, Wael El-Rifai. _University of Miami, Miami, FL_.

Background: Dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32), is a novel cancer gene overexpressed in 2/3 of gastric cancer patients. DARPP-32 promotes cancer cell survival, drug resistance, and invasion. Activation of STAT3, signal transducer and activator of transcription 3, is important for tumorigenesis and drug resistance. Among factors that regulate STAT3, SOCS3 acts as a negative regulator of STAT3 to prevent its uncontrolled oncogenic activation. In this study, we aimed to investigate if DARPP-32 regulates STAT3 and determine the underlying molecular mechanisms.

Methods: The mRNA and protein levels were evaluated by quantitative real-time PCR and Western blot analysis. The association between DARPP-32 and STAT3 was evaluated by immunofluorescence. SOCS3 degradation was analyzed by Ubiquitination Assay. The interaction between DARPP-32 and IGF1R was evaluated by co-immunoprecipitation assays. Luciferase reporter and immunohistochemistry stain were used in this study.

Results: Overexpression of DARPP-32 in AGS gastric cancer cells increased phosphorylation of STAT3 (Y705) and activation of STAT3 luciferase reporter (P<0.01), and decreased SOCS3 protein expression. DARPP-32 expression increased phosphorylation and ubiquitination of the SOCS3 protein, and subsequently shortened the SOCS3 protein half-life. We provide evidence showing that DARPP-32 promotes activation of IGF1R-SRC axis, critical for phosphorylation and degradation of SOCS3, a negative regulator of STAT3 activity. Proximity ligation and co-immunoprecipitation assays indicated that DARPP-32 and IGF1R co-exist in the same protein complex where DARPP-32 binding to IGF1R promotes its phosphorylation with activation of downstream SRC and STAT3. We obtained double knockouts mice (Darpp32KO/Tff1KO) to determine the histological changes at 3 age groups (3, 6, 9- 12 month). Using the different age groups, we found that the double knockout mouse model have significantly lower incidence of LGD at the age of 3 month, as compared to TFF1 KO (P<0.05) mice. The Darpp32KO/Tff1KO gastric mucosa demonstrated lack of phosphorylation and nuclear localization of STAT3 with significant delay in the onset of tumor lesions. Immunohistochemistry analysis confirmed overexpression of DARPP-32 and nuclear localization of STAT3 in human gastric cancers.

Conclusion: The in vitro studies indicate that DARPP-32 plays a critical role in activation of STAT3 signaling through regulation of IGF1R and SRC, leading to SOCS3 degradation. In vivo results suggest that lack of DARPP-32 delays the development of tumors in the TFF1 knockout mouse model but is not sufficient alone to abrogate the full carcinogenesis cascade. These novel findings add to the oncogenic functions of DARPP-32 and highlight its potential role in the pro-inflammatory gastric carcinogenesis cascade.

#5448

The role of CLCA2 in cell proliferation and survival of HNSCC—potential biomarker for sensitivity to EGFR inhibitors.

Yufang Yin, Randolph C. Elble. _Southern Illinois Univ. School of Medicine, Springfield, IL_.

Head and neck squamous cell carcinoma (HNSCC) develops in mucous membranes that line the mouth, throat and sinuses. Late stage disease has a poor prognosis, with 5 year survival less than 50%. The epidermal growth factor receptor (EGFR) is overexpressed in more than 90% of HNSCC, and the inhibition of EGFR is an established therapeutic strategy for HNSCC. However, sensitivity to EGFR-targeted therapy cannot be predicted from the expression level of EGFR. Only a subset of HNSCC patients benefit from anti-EGFR targeted therapy, suggesting that additional unknown pathways are involved in EGFR activation. We found that chloride channel accessory 2 (CLCA2) is frequently overexpressed in HNSCC and that its expression level predicts sensitivity to EGFR inhibitors in HNSCC cell lines. Knockdown of CLCA2 in HNSCC cancer cell line Fadu inhibited cell proliferation and sensitized cells to anoikis. Mechanistically, CLCA2 knockdown reduced EGFR signaling, which subsequently attenuated extracellular signal-regulated kinase (ERK). In addition, surface localization of cell-cell adhesion molecule E-cadherin was inhibited, corresponding to reduced cell aggregation. CLCA2 has recently been identified as a positive regulator of store operated calcium influx, and both EGFR activation and E-cadherin surface localization are known to depend on intracellular calcium release. We propose that CLCA2 overexpression in HNSCC allows the cell to better exploit EGFR proliferative signaling and protects from anoikis by promoting cell-cell aggregation. Thus, CLCA2 may serve both as a biomarker for sensitivity to EGFR inhibitors and as a target for inhibition itself.

#5449

Characterization of clinically relevant mechanism of resistance to angiogenic inhibitors in different growth patterns of human colorectal cancer liver metastases (CRCLM) by studying the angiopoietins-Tie2 mechanisms.

Nisreen Samir Ibrahim, Anthoula Lazaris, Stephanie Petrillo, Abdellatif Amri, Zu-Hua Gao, Peter Metrakos. _RI-MUHC, McGill University, Montreal, Quebec, Canada_.

Colorectal carcinoma (CRC) remains the third leading cause of cancer death in the Western world. Over 50% of CRC patients develop liver metastases (LM) and 90% will die from metastatic disease. Angiogenic inhibitors AIs (bevacizumab, Bev) were introduced with chemotherapy as first-line therapy for CRCLMs. While some patients respond well to this therapeutic strategy, the outcomes have fallen short of expectations. Recently we have reported the outcomes of resected CRCLM patients who have received neoadjuvant chemotherapy with or without AIs stratified by histologic growth patterns (HGPs): Desmoplastic HGP (DHGP), Pushing HGP (PHGP) and Replacement HGP (RHGP). The majority of metastases were either DHGPs, which derive the majority of their blood supply via sprouting angiogenesi,s or RHGP, which derive their blood supply from vessel co-option. When the neoadjuvant regimen included Bev, the DHGP patients more than doubled their 5-year overall survival (OS) compared to RHGP patients receiving the same Bev regimen. This OS difference was lost when both groups of patients were treated with chemo alone (no Bev). Although sprouting angiogenesis has been studied extensively and there is some understating of those mechanisms, we have no understanding of the mechanism behind co-option, and furthermore, apart from cytotoxic chemotherapy we have no targeted therapies for these patients. Theoretical Model: Cancer cells that arrive to the liver have to vascularize in order to increase in size, or as evidence has recently suggested, can grow avascularly in vascularized tissue by co-option of existing mature vessels. We hypothesize that there are several steps required for this process to occur. In our recent work, we have shown that cancer cell motility is essential for co-option. The angiopoietins-Tie2 mechanisms are critically involved in angiogenesis. Using human samples (chemonaïve, chemo-only and chemo plus Bev), we have evidence that Ang1-Tie2 mechanism is differently expressed in RHGP vs. DHGP metastases. The ratio of Ang2: Ang1 expression in DHGP tumors was higher compared to RHGP tumors, whereas VEGF appears to be equally expressed in both. Furthermore, a significant expression of Ang1 was detected in the hepatocytes at the interface region of the tumor and liver in RHGP. Thus, vascular quiescence maintained by Ang1-Tie2 mechanism prevails in RHGP verses DHGP metastasis; therefore this mechanism may be important in the development of vessel co-option vs. angiogenesis. Interestingly, Tie2 expression was expressed not only by vessels and tumor cells but also found in the leucocytes that were more abundant in DHGP vs. RHGP. This study will allow us to characterize the factors and mechanisms by which vessel co-option occurs in a metastatic setting and will stratify patients in terms of treatment.

#5450

**Anticancer effects of picrasidine Q from** Angelica keiski **on esophageal squamous cell carcinoma by downregulation of FGFR2 signaling pathways.**

Xuejiao Liu,1 Yuanyuan Shi,1 Mengqiu Song,1 Ran Zhao,1 Hanyong Chen,2 Jung Hyun Shim,3 Zigang Dong,2 Mee-Hyun Lee1. 1 _China-US (Henan) Hormel Cancer Institute, Zhengzhou, China;_ 2 _The Hormel Institute, University of Minnesota, Austin, MN;_ 3 _College of Pharmacy and Natural Medicine Research Institute, Mokpo National University, Mokpo, Republic of Korea_.

Fibroblast growth factor receptor (FGFR) 2 and its downstream signaling cascades, PI3 K/AKT/mTOR is playing an important role in cell survival and proliferations. In this study, we firstly found that picrasidine Q (PQ), an alkaloid component extracted from Angelica keiskei species, has the capacity of anti-cell transformation and anticancer. After ligand shape similarity approach of PQ, we found that PQ targeted FGFR 2 and verified by FGFR2 kinase assay as well as computational docking model. FGFR2 highly expressed in esophageal cancer tissues and PQ inhibited fibroblast growth factor (FGF)-induced cell transformation. Furthermore, PQ inhibited cell proliferation and induced G1 phase arrest of cell cycle and apoptosis in KYSE30, KYSE410, and KYSE450 esophageal squamous cell carcinoma (ESCC) cells. It was confirmed by detecting of biological markers such as cyclinD1, cyclinD3 and cyclinB1 for cell cycle or cleaved caspase-7, caspase-3, and PARP for apoptosis. PQ targeting of FGFR2 kinase activities suppressed downstream target proteins including phosphorylation of AKT and mTOR but not MEK/ERK signaling pathways. Taken together, our results are the first to identify that PQ might be a chemopreventive and chemotherapeutic agent by direct targeting FGFR2 and inhibiting cell proliferation of ESCC cells.

#5451

Testing a combination of AKT inhibitor (AZD5363) with PARP inhibitor olaparib plus carboplatin in TNBC.

Jennifer H. Carlson,1 Mark J. O'Connor,2 Casey Williams,1 Pradip De,1 Nandini Dey,1 Brian Leyland-Jones1. 1 _Avera Research Inst., Sioux Falls, SD;_ 2 _AstraZeneca, Cambridge, United Kingdom_.

Background: AKT, a hallmark of cancer in a wide range of solid tumors, is a serine/threonine protein kinase. Inhibition of AKT is known to cause the perturbation of cell proliferation signals and induction of apoptosis in several preclinical in vitro and in vivo models. AKT inhibitor AZD5363 shows promise in different solid tumors in patients with activating mutations of AKT (E17K) (Hyman D. M. et al., JCO 2017), the efficacy is not substantial in patients with PIK3CA mutations (Banerji. U, CCR 2017). PI3K-PTEN-AKT pathway alterations are very common in TNBC. Alterations of AKT1/2/3 (15%), PTEN (29%) and PIK3CA (25%) mediated upregulation of the PI3K pathway are common in TNBC (De et al., 2016; cBioPortal). In our Avera cohort (January 2014-June 2017) of TNBC patients (n=93), we have recorded a total of 16.5 % alteration in AKT1/2/3. We have also identified an integral role of the PI3K-AKT pathway in the DDR-mediated antitumor action of PARP inhibitor in TNBC (De et al., 2014).

Aim: Here we tested the role of genetic background in determining the effectiveness of a combination of AZD5363 with PARP inhibitor Olaparib (O, Lynparza) plus carboplatin (C) in TNBC.

Methods: Using cell line based models of TNBC with defined genetic backgrounds, we studied proliferation (Incucyte) and cell cycle by PI (Accuri). Mechanistic induction of apoptosis was evaluated by live-cell triple immuno-fluorescence (IF) (MitoView Blue+NucView488 Casp3 substrate + CF 594 AnnexinV) with a simultaneous quantification of Annexin (Accuri). Long-term clonogenic response to triple combination was tested using 3D On-Top Matrigel assay.

Result: AZD5363 treatment in PTEN-null SUM149 cells with BRCA1 mutation [2288delT (fs>735X)] responded synergistically to O and C with a dose-dependent increase apoptosis but with limited G1 increase. AZD5363 treatment in PTEN-null MDA-MB468 cells with wt BRCA1 led to an additive effect in slowing proliferation when combined with O and C and a very strong G1 increase but limited induction of apoptosis. AZD5363 as a single agent slowed proliferation and increased G1 in PIK3CA (P539R/H1074R) mutated BT20 cells with a concomitant increase in apoptosis. Mechanistically AZD5363 inhibited pophoyrlation of AKT substrates PRAS40 and GSKβ in TNBC cell lines. As expected KRAS/BRAF mutated (G13D/G464V), BRCA1 wt MDA-MB231 cells showed no effect on either proliferation or apoptosis. Best effect on the inhibition of clonogenic growth (reducing 3D colony formation and size as compared to control over single or double treatment) was recorded in SUM149 compared to MDA468 > BT20, > HCC1937.

Summary: Here we present the effect of the combination of AZD5363 and O plus C in several TNBC cell line based models. Our data demonstrate that maximum effect of the triple combination is seen in PTEN-null cell line harboring BRCA1 mutations followed by PTEN-null cell BRCA1wt. Mechanistic work utlizing AKT1-E17K transfected cells is ongoing.

#5452

Pimozide suppresses the growth of brain tumor by targeting oncogenic pathways.

Itishree Kaushik, Alok Ranjan, Blake Schwettmann, Sanjay Srivastava. _Texas Tech University, Amarillo, TX_.

Brain tumor is considered as one of the most aggressive and incurable form of cancer. Majority of the brain tumors have a median survival rate of only 12%. Even though advanced treatments such as surgical removal, chemotherapy and radiotherapy are available, brain tumor persists to be lethal. Obstacles associated with the current treatment options are: recurrence, development of resistance and inability to cross blood brain barrier (BBB). BBB restricts majority of drugs to reach the brain thus resulting in an ineffective treatment. Pimozide (PMZ) is an antipsychotic drug used for the treatment of schizophrenia and chronic psychosis. In this study, pimozide has shown significant reduction in the viability of U-87MG, U-251MG, DAOY and T98G cell lines with an IC50 ranging from 11µM to 20µM after 24 h of treatment. Pimozide induced apoptosis in these cell lines as evaluated by FITC/Annexin assay and further validated by the cleavage of caspase 3 as well as PARP by western blot analysis. Pimozide treatment resulted in the concentration dependent decrease in the expression of GLI1, OCT4, SOX2 and NANOG in U87MG, U251MG, DAOY and T98G brain cancer cell lines, depicting a reduction in the stem cell like property of these cell lines. In addition, pimozide treatment inhibited the expression of proto-oncogene c-Myc. Inhibition of Gli1 and c-Myc demonstrates that pimozide might also inhibit the growth of cancer cells by targeting SHH signaling. Our results further demonstrated that oral administration of pimozide (25mg/kg) inhibits the brain tumors in intracranial tumor model with no significant difference in average weight of critical organs. It is important to note that pimozide is an FDA approved drug with no considerable toxicity. Overall, this study depicts that pimizode is a potential candidate to target brain tumors.

#5453

Novel pan-PI3K inhibitor WX008 demonstrates significant antitumor efficacy in three xenograft tumor models.

Tao Yu,1 Lingwei Kong,1 Yong Wang,1 Peipei Jiang,1 Zhe min Rong,1 Chang jun Wang,1 Jing jie Huang,1 Dan Yao,1 Yu xin Qin,1 Cheng de Wu,1 Shu hui Chen,1 Xin Tian,2 Fei Liu,2 Ping Dong,2 Zhi Lin Chen2. 1 _WuXi AppTec, Shanghai, China;_ 2 _Chia Tai Tianqing Pharmaceutical Group Co., Ltd., Nanjing, China_.

Background: PI3K/Akt signaling pathway plays a key role in a series of cellular functions related to cell growth, proliferation, survival and differentiation. Dysregulation of the PI3K pathway is implicated in many human cancers. Thus we intended to discover novel pan-PI3K inhibitors to treat various solid tumors, such as colorectal cancer, gastric carcinoma, lung carcinoma and breast cancer, etc.

Methods: We explored SAR based on a PI3K/mTOR dual inhibitor series to selectively improve the PI3K activity over that of mTOR. PI3Kα/mTOR enzymatic activity assays, MCF-7 cellular (p-AktS473) activity assay and mouse in vivo pharmacokinetic analyses were used to direct compound optimization. Compounds with superior in vitro activity and in vivo pharmacokinetic properties were then tested for in vivo antitumor efficacy in patient derived xenograft (PDX) tumor models.

Results: WX008 was identified as a potent PI3K inhibitor with PI3Kα/mTOR IC50 of 0.07 nM/233 nM (selectivity 3328×) and superior MCF-7 cellular (p-AktS473) activity (IC50 of 5.91 nM). For its excellent pharmacokinetic properties WX008 was evaluated for in vivo antitumor efficacy. As results, WX008 displayed equivalent efficacy at much lower dose than BKM-120 in several PDX tumor models (Table 1).

Table 1 In vivo efficacy of BKM-120 and WX008 | |

---|---|---

Cmpd_ID | BKM-120 | WX008

CO-04-0032 PDX | 72% TGI @30 mpk | 86% TGI @8 mpk

ST-02-0013 PDX | 50% TGI @8 mpk

77% TGI @15 mpk | 53% TGI @4 mpk

90% TGI @8 mpk

LU-01-0010 PDX | 74% TGI @30 mpk | 74% TGI @8 mpk

Conclusions: WX008 is a novel potent pan-PI3K inhibitor with high selectivity over mTOR and inhibits tumor growth in several PDX tumor models.

#5454

New insights into the mode of action of cytotoxic alkyl-phospholipids.

Elisabetta Argenzio, Wouter H. Moolenaar, Marcel Verheij. _Netherlands Cancer Inst., Amsterdam, Netherlands_.

Background: Synthetic alkyl-phospholipid analogs (APLs), such as Edelfosine and Perifosine, are a class of anti-tumor agents that target cell membranes to induce growth arrest and apoptosis. In addition, APLs enhance the cytotoxic effect of radiation in preclinical models making these compounds attractive candidates as radiosensitizers. We previously found that different mechanisms determine ALP accumulation and consequent apoptotic toxicity in lymphoma versus carcinoma cells. Yet, despite decades of research, it is still unclear how ALPs are transported across the plasma membrane and how they affect, either positively or negatively, key intracellular signaling pathways to alter tumor cell behavior. Here we examine (i) the possible role of lipid flippases in mediating transbilayer transport of ALP-like lysophospholipids (notably lysoPC and lysoPE), and (ii) how Edelfosine affects gene expression in human breast cancer cells. Methods: The uptake of fluorescent lysophospholipids (TopFluor-LPC and -LPE) was monitored over time in wild-type HAP1 and CDC50A -/- cells using single-cell live-imaging. For RNA-seq analyses, MDA-MB-231 and Hs5788T triple-negative breast cancer (TNBC) cells were used. Results: Ad (i). Knockout of the flippase subunit CDC50a slows and attenuates the uptake of fluorescent lysoLPC and lyoPE. This suggests that CDC50a mediates, at least in large part, the active uptake of cytotoxic ALPs, similarly to natural lysophophoslipids.

Ad (ii). Whole genome RNA-seq analysis reveals that Edelfosine rapidly induces a unique gene expression program in breast cancer cells. When compared to bioactive lysoPA (LPA), Edelfosine upregulated about 25 genes after 4 hrs, whereas LPA upregulated about 300 genes under the same conditions. Strikingly, the Edelfosine- and LPA-induced early genes showed significant overlap, including transcription factors (FOS, EGR1, EGR3), CSF2 and the EGFR/HER2 inhibitor ERRFI1 (MIG6). Increased MIG6 expression by Edelfosine was confirmed by Western blot. Basal expression of MIG6 was high in MDA-MB-231 cells, but undetectable in Hs5788T TNBC cells. The latter cells were more sensitive than MDA-MB-231 cells to Edelfosine-induced apoptosis. Conclusions: We conclude that Edelfosine "hijacks" LPA receptor signaling pathways at early time points. Edelfosine-induced upregulation of the anti-proliferative gene MIG6, a negative regulator of the EGFR family, is of particular interest. Since MIG6 is induced by the G-actin-MAL transcriptional pathway, these results suggest that Edelfosine affects tumor cell behavior in part by influencing F-actin polymerization, a new scenario that warrants further studies.

#5455

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

Xiaojuan Wu,1 Jiayuh Lin2. 1 _Huazhong Univ. of Science & Technology, Wuhan, China; _2 _University of Maryland School of Medicine, Baltimore, MD_.

Objective: IL-6/IL-11/GP130/STAT3 signaling is crucial for osteosarcoma cells and tumor survival, growth and progression. Here, we show a novel IL-6/GP130 and IL-11/GP130 inhibitor, Bazedoxifene which was reported as a FDA-approved selective estrogen receptor modulator, in osteosarcoma therapy.

Methods: We treated osteosarcoma cells with different concentrations of bazedoxifene and tested expression of GP130/STAT3 signaling and the downstream genes at PCR and protein levels in different cell lines. Furthermore, cell migration and colony formation experiments were done in Bazedoxifene-treated osteosarcoma cells. To determine the in vivo activity of Bazedoxifene, we further tested the efficacy of Bazedoxifene combination treatment with temsirolimus in tumor xenografts generated from the SJSA osteosarcoma cells.

Results: Bazedoxifene appeared specific to IL-6/IL-11 as phosphorylation of STAT1 and STAT3 by IFN-γ and LIF respectively were not affected by the compound. Bazedoxifene inhibited P-STAT3, P-AKT, and induced apoptosis in human osteosarcoma cell lines expressing IL-6 and IL-11. In addition, Bazedoxifene can inhibit colony formation after the drug treatments in cancer cells. This inhibition by Bazedoxifene was partially rescued by IL-6. IFN-γ and IL-4 which do not use GP130 as a receptor and do not induce P-STAT3, did not rescue Bazedoxifene-mediated inhibition. Furthermore, IL-6 but not IFN-γ could rescue the Bazedoxifene-mediated inhibition of cell viability in osteosarcoma cells. Bazedoxifene plus temsirolimus had greater inhibition in growth of SJSA tumor xenografts than single group.

Conclusions: Bazedoxifene is novel and potent IL-6/GP130 and IL-11/GP130 inhibitor and has synergistic effect with temsirolimus treatment in osteosarcoma therapy. Repositioning a drug such as Bazedoxifene already approved for safety by the FDA as a novel inhibitor of IL-6/GP130 and IL-11/GP130 signaling should provide an easier path to clinical trials and have a potential to improve the outcome of osteosarcoma.

#5456

Mechanism of epithelial-mesenchymal transition in erlotinib resistance in NSCLC cells containing both wild-type and mutant EGFR.

Sanjana Singh, Tsatsral Iderzorig, Pragya Kumar, Neelu Puri. _University of Illinois at Chicago, College of Medicine, Rockford, IL_.

EMT is a vital process in development of metastasis and occurs when epithelial cells lose their polarized structure, by downregulation of adherens junction proteins E-cadherin, Claudin and ZO-1 on the membrane. Cells with EMT are elongated spindle like structures due to upregulation of mesenchymal markers Vimentin, N-cadherin and transcription repressor of E-cadherin, ZEB1. EMT may be responsible for Tyrosine kinase inhibitor (TKI) resistance to epidermal growth factor receptor (EGFR) in patients with activated EGFR mutations. This acquired resistance to TKIs is due to a secondary T790M mutation in the kinase domain which could be responsible for inducing EMT. An EMT regulator, p120-catenin when it is no longer bound to membranous E-cadherin forms a complex with Kaiso factor suppressing its transcription repressor activity, promoting oncogenesis. PRMT1, another EMT inducer is also overexpressed in NSCLC. Cells undergoing EMT also acquire cancer stem-cell (CSC) like characteristics by expressing a CSC marker ABCB1. Thus, we investigated EMT characteristics in wild type EGFR TKI resistant H358, H2170 cells and TKI resistant EGFR mutant H1975 cells (L858R and T790M mutation) and TKI sensitive EGFR mutant H3255 cells (L858R mutation). The modulation of EMT biomarkers was determined by immunoblotting and qPCR. Expression of ABCB1 and E-cadherin was measured using flow cytometry. Key EMT regulators such as PRMT1 and p120-catenin were upregulated 4.0 and 3.2-fold, respectively. Hence, knockdown of p120-catenin and PRMT1 were performed using siRNA or an inhibitor and examined by immunoblotting and MTT. EMT transcription factors such as Slug, Snail and Twist were also upregulated 3.18, 6.2 and 1.68-folds and E-cadherin, Claudin and ZO-1 were downregulated by 8.6, 11.6 and 15.2-folds in H1975 cells compared to H3255 cells. Also, N-cadherin, ZEB1 and Vimentin were upregulated by 4.9, 3.0 and 10.7-fold. Immunofluorescence studies with Vimentin showed that H1975 cells were elongated and have a mesenchymal phenotype compared to the H3255. 90% colocalization of p120-catenin and Kaiso factor was seen in H1975 cells whereas 10% colocalization was seen in H3255 cells. In resistant NSCLC cells with wild type and mutated EGFR, flow cytometry studies showed significant increase in expression of CSC marker ABCB1 whereas E-cadherin expression was decreased. Knockdown of PRMT1 by Furamidine, increased erlotinib efficacy by 27% in H1975 cells. Similarly, knockdown of p120-catenin by siRNA increased erlotinib efficacy by 40% in H1975 cells. In conclusion, EMT maybe mediated through biomarkers such as PRMT1, which methylate's Twist, upregulates transcription factors and p120-catenin which represses E-cadherin and Kaiso factor, activating EMT in H1975 cells.

#5457

The relationship between ADAR1 and STAT3 and its relevance in multiple myeloma.

Phaik Ju Teoh,1 Tae Hoon Chung,2 Henry Yang,2 Polly Chen,1 Wee Joo Chng3. 1 _National University of Singapore, Singapore, Singapore;_ 2 _Cancer Science Institute, Singapore, Singapore;_ 3 _National University Cancer Institute, Singapore, Singapore, Singapore_.

Multiple myeloma (MM), the second most common hematological cancer, is characterized by the accumulation of malignant plasma cells in the bone marrow (BM). Survival of MM cells is highly dependent on the bone marrow microenvironment, which is enriched with an array of growth factors secreted by the BM stromal cells. Amongst these growth factors, interleukin-6 (IL6) has been reported to be a major driver for MM disease progression. In line with the mechanism of action of IL6, MM cells are associated with constitutive activation of the oncogenic STAT3 signaling. ADAR1, an RNA editing enzyme, exists in two distinct isoforms, namely the P110 and P150. Aberrant ADAR1 expression and activity have been reported in various cancer types, however, this remains elusive in MM. Herein, we analyzed the publicly available myeloma patient datasets and identified that ADAR1 is overexpressed in MM and its expression level is closely associated with patients' overall survival. In vitro and in vivo assays revealed that ADAR1 can drive the growth and proliferation of MM cells, underlining its potential oncogenic effects in MM. Till date, much studies have reported the association of aberrant ADAR1 expression and its canonical functions of RNA editing, however, the knowledge on the upstream regulators of ADAR1 is still largely limited. To elucidate the cause of ADAR1 overexpression in MM, human myeloma cell lines were stimulated with IL6 at different time points. Besides potently activating the STAT3 signaling, IL6 stimulation also renders an increased expression of ADAR1 P150 isoform, at both the protein and mRNA level, with minimal effects seen on the P110 isoform, suggesting a transcriptional regulation of P150 by STAT3. Downstream validations with ChiP-qPCR and Luciferase Reporter Assay revealed that ADAR1 P150 is a direct target of STAT3 transcription factor. Specific STAT3 inhibition caused a downregulation of P150 and its oncogenic properties. Likewise, ectopic introduction of P150 rendered a more proliferative profile in the cells. Interestingly, we also observed that IL6-induced-ADAR1 expression could in turn mediate the activity of STAT3 signaling to enhance the oncogenic signaling within MM. Cells with high ADAR1 demonstrated a more rapid IL6-induced-STAT3 activation. In summary, we identified that ADAR1 overexpression is clinically relevant in MM and is driven by the hyperactivity of IL6-STAT3 signaling. Our data suggests that the P150 isoform, instead of the P110, is the more critical driver of MM disease aggressiveness in the patients with constitutively activated STAT3 pathway. The potential interplay between ADAR1 and STAT3 may represent a novel mechanism by which IL6 confers oncogenicity in MM.

#5458

Antiproliferative activity of a novel 8-hydroxyquinoline ruthenium II complex against human breast cancer cell lines.

Amr M. Khalifa,1 Ahmed S. Sultan2. 1 _Faculty of Allied Medical Sciences, Pharos University and Faculty of Science, Alexandria University, Alexandria, Egypt;_ 2 _Faculty of Science, Alexandria University, Alexandria, Egypt_.

Ruthenium (Ru) complexes are recognized as the most perspective chemotherapeutic agents available, because of their low general toxicity and variety of modes of action. Ru(II)-based complexes show a higher tendency for ligand exchange reactions and therefore interact with target molecules more rapidly and there cytotoxicity often correlates with the DNA binding ability. The present study was aimed to test the anti-tumor effect of a new formulated ruthenium (II) complex Bis(quinolin-8-olato)bis(triphenylphosphine)ruthenium(II) complex (Ru(quin)2) that was prepared by reaction of the precursor RuCl2(PPh3)3 with Quinolin-8-ol on two human breast cancer cell lines, estrogen receptor-positive (ER+), T-47D, and MDA-MB-231, triple negative breast cancer cell line that display different phenotypic and differentiation patterns ranging from poor to high differentiation.

The two tested breast cancer cells showed a significant growth inhibition after 24 h of treatment, but the response of T47D cells to treatment was much higher than that of MDA-MB231 cells. In contrast, the near-normal mammary cell lines, MCF-10A, showed no response to Ru(quin)2 at higher doses. In tested cells, Ru(quin)2 significantly inhibited cells proliferation after 24 h treatment as evaluated by MTT assay, cell morphology alteration, and clonogenic assay. In addition, treated cells showed significant morphological and biochemical signs of apoptosis induction through releasing of histone protein, caspase-3 activation, and G2/M phase cell cycle arrest in a dose-dependent manner compared to control.

In T47D cells, Ru(quin)2 significantly downregulated protein expression of STAT3, and S6 kinase1 (S6K1) and significantly upregulated LC3B expression protein, a hallmark of autophagosomes formation, after 24 h of treatment as detected by western blot analysis and immunocytochemistry.

Furthermore, in established xenograft tumor model of MDA-MB231 cells in nude mice, the growth of MDA-MB231 tumors that treated with Ru(quin)2 was inhibited by 46.88% compared to the mock group (t=2.821, P<0.05) after three weeks of treatment. Besides, there was no cytotoxicity on both liver and kidney function tests. Collectively, our observations support the anticancer role of the novel Ru(II)- macrocyclic complex that might be through apoptosis and autophagy induction in human breast cancer cell lines.

#5459

Regulation of IGF2 by TGF-β signaling in liver cancers and stem cell homeostasis.

Shuyun Rao,1 Sobia Zaidi,1 Kazufumi Ohshiro,1 Jian Chen,2 Shoujun Gu,1 Wilma Jogunoori,3 Jon White,1 Bibhuti Mishra,1 Shulin Li,2 Rehan Akbani,2 Lopa Mishra1. 1 _George Washington University, Washington, DC;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 3 _Veterans Affairs Medical Center, Washington, DC_.

Development of hepatocellular carcinoma (HCC), which remains lethal, is associated with alterations in multiple factors including the transforming growth factor beta (TGF-β) signaling pathway. Previously we have uncovered a unique role for TGF-β signaling molecules, Smad3 and its adaptor β2SP, in suppressing stem cell transformation into cancer. Yet, while TGF-β plays a pleiotropic role including regulating stem cell differentiation, proliferation, and inflammation, mechanistic insight into the dichotomy of TGF-β, and its role in stem cell transformation remains poorly understood for these cancers. Here, we took an integrated approach to identify and validate effects of changes in this pathway in HCC and identify potential therapeutic targets such as IGF2. We extended our mechanistic studies associated with the regulation of insulin-like growth factor 2 (IGF2) in the context of the TGF-β-pathway, by utilizing both human liver cancer cell lines and TGF-β signaling-deficient mice (β2SP+/- and β2SP+/-/Smad3+/-) that develop liver cancers. We found the following: 1) IGF2 promotes stemness, overexpression leading to increased expression of stem cell genes that include SOX2 and ALDH1A1, and sphere formation in liver cancer cells; 2) A marked increase in IGF2 mRNA and protein in HepG2 cells and Huh7 cells with β2SP stable knockdown; 3) TGF-β signaling through β2SP/Smad3 significantly inhibits IGF2 transcription; ChIP assays validated binding of Smad3 at the human IGF2 promoter region between -859bp to -688bp upstream of the IGF2 transcriptional start site; 4) TCGA based integrated analyses identify specific HCC patient groups with altered TGF-β members and high expression of IGF2 that could be potentially targetable for IGF2; 5) TCGA analyses of 33 cancers also revealed that potentially "switching off" TGF-β-pathway (through decreased expression levels) leads to increased stemness validating findings of our TGF-β deficient mouse models that phenocopy a human cancer stem cell syndrome. In summary, TGF-β negatively regulates IGF2 transcription: loss of TGF-β signaling through β2SP/Smad3 leads to IGF2 activation, contributing to tumor progression. TGF-β-β2SP/Smad3 are important for IGF2 repression and maintaining stem cell homeostasis, which may result from TGF-β/β2SP/Smad3 and/or CTCF-dependent regulation of IGF2. Our studies provide a framework for new animal models of liver and gastrointestinal cancers and future new therapeutics.

#5460

Discovery and evaluation of WX047, a potent and oral active phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) dual inhibitor.

Ning Li,1 Amy Guan,1 Lei Huang,1 Dongling Hao,1 Bo Gao,1 Jikui Sun,1 Nengyang Shih,1 Lingwei Kong,1 Peipei Jiang,1 Yi Li,1 Dan Yao,1 Yuxin Qin,1 Tao Yu,1 Chengde Wu,1 Shuhui Chen,1 Hongyu Yuan,2 Xiulian Lu2. 1 _WuXi AppTec Co Ltd, Shanghai, China;_ 2 _Cisen Pharmaceutical Co Ltd, Jinan, China_.

Background: The PI3K/AKT/mTOR signaling pathway is important in regulating cell cycle, which is directly related to cellular quiescence, proliferation and longevity. Dysfunctional signaling of the PI3K/AKT/mTOR pathway in cancer, and its crucial role in cell growth and survival have made it a much desired target for cancer therapeutics.

Methods: By using structure-based design, the 4H-pyrido[1,2-a]pyrimidin-4-one derivative as an early lead was discovered. Further SAR studies optimized the in vitro and PK properties and led to the identification of WX047 as a potent dual inhibitor of PI3K and mTOR. Antitumor efficacy of WX047 was evaluated in the PC-3M xenograft tumor model.

Results: A series of 7-(pyridin-3-yl)-4H-pyrido[1,2-a]pyrimidin-4-one derivatives were synthesized and evaluated for in vitro and in vivo activities. Among them, WX047 was identified as a potent dual PI3K and mTOR inhibitor with IC50 for mTOR of 19.17 nM and IC50 for PI3Kα of 3.13 nM. The cellular activity of WX047 was determined in PC-3M cells by western blot analysis of p-Akt and p-70S6K with p-Akt and p-70S6K inhibition IC50 of 16 nM and 65 nM respectively. WX047 had low clearance and high oral bioavailability relative to those of the reference compound GSK2126458. In addition, WX047 also demonstrated significant antitumor efficacy in the PC-3M xenograft tumor models (79% TGI@10mpk, PO, QD).

Conclusions: WX047 has been identified as an orally active PI3K and mTOR dual inhibitor. It displays significant antitumor efficacy in the PC-3M xenograft tumor model. These support moving the compound forward to clinical investigation.

#5461

B7-H3 regulates NF-kB signaling in cancer cells by modulating the degradation and re-synthesis of IkBα.

Sarah Glazer, Seth Gammon, David Piwnica-Worms. _MD Anderson, Houston, TX_.

B7-H3, a B7 homologue, is highly expressed on tumors and tumor vasculature. Although its role as a T cell inhibitor or co-stimulator is still under investigation, high expression of membranous B7-H3 in the tumor compartment is correlated with poor overall survival. NF-kB activation, standing at the intersection between inflammation and cancer, can lead to both the production of cytokines that engage the innate immune system, as well as pro-proliferative signals in cancer. B7-H3 has been shown to enhance and suppress inflammatory signaling by modulating NF-kB. However, the mechanism by which this occurs in cancer cells remains unknown. In this study, we aim to understand how B7-H3 modulates the NF-kB pathway in order to regulate incoming inflammatory signals in cancer cells. The human colon cancer cell line, HCT116, was stably transfected with a transcriptionally-coupled post-translational NF-kB luciferase reporter comprising of the inhibitor of NF-kB, IkBα, fused to firefly luciferase and placed downstream of an NF-kB responsive promoter, kB5. This construct allows for the real-time readout of the degradation of IkBα and NF-kB driven transcriptional re-synthesis of IkBα in response to a stimulus. The reporter cell line was treated with two stimulants, TNFα and flagellin, that activate NF-kB through different pathways and to different degrees with or without treatment with human B7-H3. Treatment of the reporter cell line with B7-H3 alone did not induce IkBα degradation or re-synthesis. However, stimulation of the reporter cell line with TNFα or flagellin in combination with B7-H3 attenuated the degradation of IkBα and its re-synthesis compared to stimulation with TNFα or flagellin alone. Maximal re-synthesis of IkBα when stimulated with TNFα was 8.25 ± 1.51 fold (S.D.) and significantly increased compared to cells additionally treated with B7-H3 (5.83 ± 1.23 fold, p = .01). In addition, peak degradation time of IkBα when cells were stimulated by TNFα was 45 ± 5 min (S.D.), and significantly shorter than when cells were also treated with B7-H3 (61.67 ± 2.89 min, p = .01). Similar trends were observed when cells were treated with both flagellin and B7-H3 compared to stimulation with flagellin alone. These studies show that B7-H3 suppresses the response of colon cancer cells to incoming inflammatory stimuli and attenuates NF-kB signaling by tempering the kinetics and dynamics of IkBα.

#5462

Functional characterization of potential gain-of-function p53 mutants in esophageal squamous cell carcinoma.

Valen Z. Yu, Maria L. Lung. _The University of Hong Kong, Hong Kong, Hong Kong_.

One of the most frequently mutated genes reported in human cancers is TP53. Esophageal squamous cell carcinoma (ESCC) has an extremely high frequency of p53 mutations of 60-90% in ESCC patients. Evidence of gain-of-function (GOF) p53 mutations that exert wild-type (WT) function-independent oncogenic effects are emerging. We aim to determine the functional role of several candidate GOF p53 mutations in ESCC and explore their clinical utility for prognostication and as potential treatment targets. In our recent study of Hong Kong ESCC specimens, we detected TP53 alterations in over 90% of ESCC tumors, ~36% of which are candidate GOF mutations verified in other cancers. By analyzing published ESCC sequencing data, we showed that patients from the high-risk ESCC region show frequent candidate GOF p53 mutants. We applied the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technique to functionally knockout (fKO) mutant p53 protein expression in various ESCC cell lines with different endogenous p53 mutants and subject them to in vivo and in vitro functional assays. Interestingly, only KYSE150 cells with an endogenous p53R248Q mutation show reduced tumor growth in nude mice after p53-fKO, suggesting that there is a specific mutant p53 that confers oncogenic features in ESCC. Consistently, only KYSE150 p53-fKO cells show reduced colony formation in three-dimension in vitro culture. Tyrosine phosphorylation of Signal Transducer and Activator of Transcription 3 (STAT3) is significantly reduced in KYSE150 p53-fKO cells, suggesting that regulation of the STAT3 pathway contributed to the oncogenic feature of the GOF p53 mutant. p53R248Q exhibits concentrated subcellular localization to the membrane fraction (including ER, mitochondria, etc.) and shows increased resistance to HSP90/HDAC inhibition-induced protein degradation, as compared to other p53 mutants. Together, these results suggest a GOF role of certain mutant p53 proteins with specific characteristics. Targeting such mutants and/or related oncogenic pathways may be of therapeutic benefit. Acknowledgement: We acknowledge the Research Grants Council of Hong Kong Special Administrative Region for General Research Fund support (17109515 to M.L.L).

#5463

Free fatty acids augment radiation-induced cytotoxicity in cervical cancer cells via mTOR/Akt//PPARg/p53-mediated pathway.

Naoshad Muhammad, Ramachandran Rashmi, Fiona Ruiz, Michael C. Zahner, Julie K. Schwarz. _Washington University in St. Louis, St. Louis, MO_.

Purposes of the study: Our clinical results show that obese patients with cervical cancer who are treated with definitive chemoradiation have favorable outcomes, and this effect is independent of diabetes or metformin use. PI3K/AKT pathway signaling is decreased in cervical tumors from obese hosts, even in the context of genomic mutations that would otherwise activate PI3K signaling, such as PTEN deletion. The objective of the present study is to determine whether other factors related to obesity, such as circulating free fatty acids, influence radiation responses in vitro.

Experimental procedures: Radiation sensitivity experiments were performed in cervical cancer cells with and without PI3K pathway mutations (CaSki (mutant), ME180 (mutant), SiHa (WT). To determine whether combination treatment of free fatty acids (palmitic, oleic and linoleic) plus radiation increases cytotoxicity in cervical cells, we performed cell viability assay, long term colony formation assays (CFA) and morphological examination by vertical microscope. To determine whether the addition of free fatty acids to radiation influences cell survival signaling, a series of Western blots were performed.

Summary: We observed that combination treatment of free fatty acids and radiation significantly increased cytotoxicity in all the cervical cancer cell lines tested. Mechanistic investigations revealed that free fatty acids induced potentiation of radiation-induced cytotoxicity through downregulation of p-mTOR, p-Akt and PTEN. Furthermore, we observed that this combination regimen upregulates the expression PPARƔ and p53.

Conclusions: Free fatty acids potentiated the effect of radiation in PI3K-activated cervical cancer cell lines through mTOR/Akt//PPARg/p53 mediated pathway. Future studies will characterize the mechanism of free fatty acid potentiation of radiation-induced tumor cell death in detail, including the potential contribution of lipid peroxidation and alterations in glucose metabolism.

#5464

Pharmacologic activation of REV-ERBs is lethal in cancer and oncogene-induced senescence.

Gabriele Sulli,1 Amy Rommel,1 Xiaojie Wang,2 Matthew J. Kolar,1 Francesca Puca,3 Alan Saghatelian,1 Maksim V. Plikus,4 Inder M. Verma,1 Satchidananda Panda1. 1 _Salk Institute, San Diego, CA;_ 2 _University of California, Irvine, San Diego, CA;_ 3 _The University of Texas MD Anderson Cancer Center, Houston, CA;_ 4 _University of California, Irvine, Irvine, CA_.

The circadian clock drives daily rhythms in cell proliferation, metabolism, inflammation and DNA damage response. Perturbations of these processes are hallmarks of cancer, and circadian rhythm disruption predisposes to tumor development in animal models and humans. This raises the hypothesis that pharmacologic modulation of the circadian machinery may be an effective therapeutic strategy for combating cancer. The nuclear hormone receptors REV-ERBα and REV-ERBβ (REV-ERBs) are essential components of the circadian clock. Here we show that SR9009 and SR9011, two different agonists of REV-ERBs, are specifically lethal to cancer cells and oncogene-induced senescent (OIS) cells, including melanocytic naevi, while having no effect on viability of normal cells or tissues. Anticancer activity of SR9009 and SR9011 affects several oncogenic drivers (such as H-RAS, BRAF, PIK3CA, and others), and persists in the absence of p53 and under hypoxic conditions. The regulation of autophagy and de novo lipogenesis by SR9009 and SR9011 plays a critical role in evoking an apoptotic response in malignant cells. Importantly, the selective anticancer properties of these REV-ERB agonists impair glioblastoma growth in vivo and improve survival without causing any overt toxicity in mice. These results indicate that pharmacologic modulation of circadian regulators is an effective novel antitumor strategy, identifying the existence of a previously unknown class of anticancer agents with a wide therapeutic window. We propose that REV-ERB agonists are novel autophagy and de novo lipogenesis inhibitors with selective activity towards malignant and benign neoplasms.

#5465

Resveratrol inhibits cell proliferation via crosstalk between integrin αvβ3 and IGF-1R in primary cell cultures of human uterine fibroids.

Yi-Ru Chen,1 Ya-Jung Shih,1 Heng-Yuan Tang Tang,2 Hung-Yun Lin,1 Paul J. Davis2. 1 _Taipei Cancer Center, Taipei Medical University, Taipei, Taiwan;_ 2 _Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Albany, NY_.

A uterine fibroid is the most common benign smooth muscle cell tumor of the myometrium. Resveratrol, a stilbene, has been used as an anti-inflammatory and antitumor agent. We aimed to determine the expression of growth factor molecules for growth inhibition after treatment of resveratrol in the primary cell lines from human uterine fibroid. In terms of growth rate, of the 24 uterine fibroid primary cell culture cells, 5 were mildly sensitive, 13 were medium-, and 6 were sensitive to resveratrol treatment. 19 out of the 24 primary cell lines responded to treatment of resveratrol from middle to sensitive ranges. Resveratrol arrested cell proliferation and also induced apoptosis, indicating that an intact apoptotic pathway was present in these cells by resveratrol. Reduction of integrin αvβ3 expression diminished resveratrol-induced antiproliferative effect in uterine fibroid cells. Resveratrol induced the expression of proapoptotic genes such as COX-2 and p21. On the other hand, the expression of proliferative (antiapoptotic) genes was either inhibited in such as BCL2 and CDKN2 or not changed in Cyclin D1 and PCNA. The expression of mRNA and protein of insulin-like growth factor-I (IGF-I) was inhibited by resveratrol in primary uterine fibroid cells. The proapoptotic proteins such as caspase 3 and caspase 9 were activated after resveratrol treatment and demonstrated that resveratrol induced apoptosis in the resveratrol-sensitive primary cell cultures, which was consistent with studies of the flow cytometry. In addition, resveratrol blocked the expression of integrin αvβ3 and protein. Concurrently, constitutive AKT phosphorylation in uterine fibroid cells was inhibited by resveratrol. Therefore, growth modulation of uterine fibroid cells occurs via mechanisms dependent on crosstalk between integrin αvβ3 and IGF-1R. Our findings suggest that resveratrol can be considered as an alternative therapeutic agent for uterine fibroid.

#5466

CXCL14 suppresses the aggressive phenotype in triple-negative breast cancer (TNBC) through CXCR4.

Carla R. Gibbs,1 Rosa Mistica C. Ignacio,1 Ann Richmond,2 Deok-Soo Son1. 1 _Meharry Medical College, Nashville, TN;_ 2 _Vanderbilt University, Nashville, TN_.

The chemokine network is involved in cancer metastasis by modulating cell migration and angiogenesis. CXCL12-CXCR4 is a common signaling axis that is associated with cancer metastasis. Metastatic breast cancers often overexpress the CXCR4 receptor, whereas lung tissues express high levels of CXCL12; consequently, this is a proposed mechanism for breast cancer metastasis to the lung. CXCL14, an anti-inflammatory chemokine, has been postulated to be an alternative ligand for CXCR4, though a formal assignment of CXCR4 as the receptor for CXCL14 has not been made. Interestingly, CXCL14 competes with CXCL12, a main ligand for CXCR4, to modulate CXCL12-CXCR4 downstream activation of ERK. CXCL14 expression is downregulated in oral, colon, prostate, lung, pancreatic, cervical, kidney cancers, and in TNBC when compared to other subtypes of breast cancer. Overexpression of CXCL14 in cancer cell lines in vivo and in vitro leads to decrease in tumor metastasis. CXCL14 overexpression in the MDA-MB-231 breast cancer cell line leads to a decrease in cell growth, invasion and migration. To further explore a role for CXCL14 in breast cancer aggressiveness, we examined the effects of CXCL14 on the cell migration and metastasis of additional breast cancer cell lines. Here we performed cell migration and MTT cytotoxicity assays using MDA-MB-231 and BT-549 TNBC cell lines and the MCF-7 estrogen receptor+ cell line. Our results demonstrate TNBC cells are significantly more sensitive to CXCL14 compared to non-TNBC MCF7 cells (in Boyden chamber migration assay). Also, in our experiments CXCL14 did not alter proliferation in either TNBC cells or non-TNBC cells. However, when we generated CXCR4-overexpressing breast cancer cell lines using parental BT-549, MDA-MB-231, and MCF-7 cells, we observed the changes in the phosphorylation of ERK1/2 (MAPK targets) and pAKT (PI3K downstream effector protein). Based on these data, we propose CXCL14 as a negative modifier of the CXCL12-CXCR4 axis in TNBC. Based on these data, we propose a negative role for CXCL14 in the modulation of TNBC aggressiveness.

#5467

**Efficacy of the ERK inhibitor LY3214996 and the PI3K/mTOR inhibitor LY3023414 in patient-derived** RAS- **mutant NSCLC models.**

Jens Köhler,1 Prafulla C. Gokhale,2 Jiaqi Li,1 Margaret K. Wilkens,2 Hong Tiv,2 Shripad V. Bhagwat,3 Greg Donoho,3 Patrick Brueck,4 Ramon V. Tiu,3 Amanda J. Redig,1 Emily S. Chambers,1 Atsuko Ogino,1 Jihyun Choi,1 Man Xu,2 Paul Kirschmeier,2 Pasi A. Jänne1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Belfer Center for Applied Cancer Sciences, Boston, MA;_ 3 _Eli Lilly and Company, Indianapolis, IN;_ 4 _Eli Lilly and Company, Bad Homburg, Germany_.

Introduction: RAS mutations are present in 30% of non-small cell lung cancer (NSCLC) and, despite activated MAPK signaling, MEK inhibitors have disappointed in clinical trials. MAPK pathway reactivation and activation of anti-apoptotic signaling via the PI3K/mTOR axis are major resistance mechanisms, among others. ERK inhibitors are believed to overcome some of the restrictions of MEK inhibitors, and combinations with PI3K/mTOR inhibitors could potentially increase therapeutic efficacy. Past trials of MEK and PI3K inhibitor combinations, however, showed that toxicities and side effects are likely to be expected also for the ERK-PI3K/mTOR inhibitor combination. Therefore, more specific and potent drugs as well as alternating dosing schedules may overcome toxicities and improve treatment outcome.

Methods: We so far established five patient-derived RAS-mutant NSCLC cell lines (1/5 NRASQ61K, 1/5 KRASQ61K, 2/5 KRASG12C, 1/5 KRASG12D) and xenograft models (PDX), characterized them by next-generation sequencing (DFCI Oncopanel) and tested the antiproliferative (CellTiter-Glo® assay) and apoptosis-inducing (Caspase3/7 activity, IncuCyte® technology) efficacy of LY3214996 (ERK inhibitor) and LY3023414 (PI3K/mTOR inhibitor) in vitro. Effects on signal transduction were investigated by Western blot analysis.

Results: The ERK inhibitor LY3214996 and the PI3K/mTOR inhibitor LY3023414 exhibit single-agent antiproliferative and apoptosis-inducing activity in patient-derived RAS-mutant NSCLC cell lines (for LY3214996: 1/5 sensitive (IC50<1μM), 2/5 intermediate sensitive (IC50<5μM), 2/5 resistant (IC50>10μM); for LY3023414: all IC50 <3μM, 3/5 <1μM). Despite reactivation of MAPK signaling (pMEK) upstream of ERK during LY3214996 treatment, ERK downstream targets (cMyc, DUSP4, SPRY2) were strongly suppressed. In sensitive cell lines, LY3214996 induced PARP cleavage and Bim accumulation in a dose- and time-dependent manner. Sensitivities to ERK and PI3K/mTOR inhibition varied between cell lines, indicating different pathway dependencies for proliferation and cancer cell survival.

Conclusions and Outlook: Both LY3214996 and LY3023414 have single-agent in vitro efficacy in patient-derived NSCLC models with different RAS mutations and co-mutational landscapes. Single-agent in vivo studies and tolerability studies of drug combinations are ongoing, and treatment predictors and intrinsic/adaptive resistance mechanisms will be investigated via RNAseq/GSEA and phospho-receptor tyrosine kinase arrays.

#5468

Neratinib significantly inhibits responses to androgen in human prostate cancer cells.

Dafydd A. Dart,1 Alshad S. Lalani,2 Francesca Avogadri-Connors,2 Richard Bryce,2 Wen G. Jiang1. 1 _Cardiff University, Cardiff, United Kingdom;_ 2 _Puma Biotechnology, Inc., Los Angeles, CA_.

Background: Prostate cancer (PCa) is the most commonly diagnosed male cancer in the Western world. Tumor growth is initially androgen dependent - and driven by the androgen receptor (AR). The mainstays of prostate cancer treatment are androgen ablation and antiandrogen treatment, which block AR signalling. However, PCa often relapses to an androgen- independent disease. Androgens can transactivate genes directly via the AR-mediated transcription factor and indirectly via less well understood signal transduction pathways. These signal transduction pathways become increasingly relevant as prostate cancer cells progress to anti-androgen resistance, with HER2 being associated with higher relapse rates. Neratinib is an orally available tyrosine kinase inhibitor that irreversibly binds and inhibits EGFR, HER2 and HER4 receptor tyrosine kinases. This study aimed to examine the effect of neratinib on androgen signalling and on the expression of androgen-regulated genes in prostate cancer cells.

Methods: Changes in protein phosphorylation after androgen treatment of hormonally starved prostate cancer cells (LNCaP) was assessed using protein microarrays (Kinexus, Canada). Changes in gene expression after neratinib or androgen treatment were ascertained using AmpliSseq® technology or standard qPCR, and analysed via IPA Ingenuity software.

Results: Androgen treatment of hormonally starved prostate cancer cells (LNCaP) caused phosphorylation of several members of the signal transduction cascade including HER2 and Src within 2 hours, indicating a role for HER2 in rapid androgen signalling. Additionally, from over 1000 genes upregulated by androgen treatment (>2fold within 2 hours), 87% showed downregulation with neratinib treatment. Ingenuity pathway analysis indicated that STAT3, ETS-family and NF-κB transcription factors may be responsible for the rapid androgen-induced gene upregulation observed, and that these pathways were inhibited by neratinib treatment. Q-Quantitative PCR analysis of PSA expression in LNCaP cells stimulated with androgen in the presence of increasing concentrations of neratinib resulted in a dose-dependent inhibition of androgen activity.

Conclusions: These results show that neratinib is able to inhibit the responses of prostate cancer cells to androgens, and that a strong potential signaling cross-talk exists between the androgen receptor and the certain signal transduction pathways - pathways known to be involved in the progression of androgen-independent prostate cancer.

#5469

RAS and STAT5 pathway lesions are mutually exclusive in B-cell malignancies through mechanisms of biochemical cross-inhibition.

Lai N. Chan,1 Seyedmehdi Shojaee,2 Christian Hurtz,2 Rebecca Caeser,3 Gang Xiao,1 Huimin Geng,2 Steven Kornblau,4 Markus Muschen1. 1 _Beckman Research Institute of City of Hope, CA;_ 2 _University of California San Francisco, CA;_ 3 _University of Cambridge, Cambridge, United Kingdom;_ 4 _University of Texas MD Anderson Cancer Center, TX_.

Activation of STAT5- and RAS-signaling are segregated to early and later stages of normal B cell development, respectively. Studying B-lineage acute lymphoblastic leukemia (ALL; n=578), we found that STAT5 (e.g. BCR-ABL1, JAK2, cytokine receptors) and RAS (NRAS, KRAS, PTPN11, NF1) lesions were mutually exclusive, with only 9 cases (1.6%) carrying lesions in both pathways. Reverse phase protein array measurements revealed that phosphorylation of MEK and ERK1/2 were inversely correlated with STAT5-phosphorylation (MDACC, 1983-2007; P<0.001). These findings prompted us to study mechanisms of cross-inhibition between RAS and STAT5 pathways. Inducible NRASG12D activated ERK at the expense of STAT5 phosphorylation. This was due to stabilization and increased activation of the STAT5-phosphatase PTPN6 (SHP1). Inducible ablation of Ptpn6 elevated phospho-STAT5 levels, while genetic inactivation of Stat5 strongly increased ERK activity. Constitutively active STAT5 suppressed phosphorylation of ERK. Interestingly, STAT5 negatively regulated BCL6, which marks the transition from cytokine receptor-dependent pro-B cells (Stat5+) to pre-BCR dependent stages of development (ERK+). While oncogenic RAS suppressed STAT5, we also found that induction of RAS induced BCL6 expression at both the mRNA and protein levels. Increases in BCL6 expression in response to NRASG12D were abrogated upon treatment with a MEK kinase inhibitor or activation of STAT5. Studying a matched patient-derived pre-B ALL sample at diagnosis and at relapse (acquired KRASG12V mutation) revealed activation of ERK in association with increased BCL6 and decreased STAT5 levels in the KRASG12V relapsed ALL sample. With engagement of BCL6 and the STAT5-inhibitory phosphatase PTPN6 downstream of RAS and ERK signaling, these findings suggest that occupancy of either RAS or STAT5-pathway represents a commitment step that renders cells non-permissive to the respective alternative pathway. To test this hypothesis, we induced B cell transformation with either RAS or STAT5-pathway oncogenes and then subsequently transduced with either empty vectors (EV) or vectors carrying the alternative oncogene. While EVs were easily transduced, RAS- and STAT5-transformed B cells did not tolerate the alternative oncogene. Reflecting early (STAT5) and later (RAS) stages of B cell development, oncogenic activation of these pathways occurs in a mutually exclusive way, owing to biochemical cross-inhibition between STAT5 and RAS.

#5470

IKK-epsilon supports anchorage independent growth via alternative NF-kB signaling in triple-negative breast cancer.

Carrie D. House, Valentina Grajales, Michelle Ozaki, Elizabeth Jordan, Helmae Wubneh, Danielle Kimble, Marianne Kim, Christina M. Annunziata. _National Cancer Institute, Bethesda, MD_.

Metastatic breast cancer carries a poor prognosis despite the success of newly targeted therapies and treatment options remain especially limited for the subtype of triple negative breast cancer (TNBC). Several signaling pathways, including NF-κB, are altered in TNBC. Given that ΙΚΚε behaves as an oncogene in breast cancer we hypothesized that IKKε regulates classical or alternative NF-κB signaling to control diverse oncogenic functions in TNBC. Vector expression and RNA interference were used to investigate the functional role of IKKε in triple-negative breast cancer cells. Viability, protein expression, NF-κB binding activity, invasion, anoikis, and spheroid formation were examined in cells expressing high or low levels of IKKε in conjunction with alternative NF-κB p52 RNA interference or MEK inhibition. Our data show that p52 protein levels and binding activity are inversely proportional to ΙΚΚε. Moreover, knockdown of IKKε leads to increased expression of p52 and the p52-regulated gene CXCL1. This interaction was confirmed by CHiP-PCR experiments showing increased binding of p52 in the CXCL1 promoter of cells with IKKε knockdown relative to control. We further found that IKKε and MEK were required for growth in anchorage supportive conditions whereas p52 was required in anchorage resistant conditions. Western blots confirm diminished IKKε expression and enhanced p52 processing in anchorage resistant conditions relative to anchorage supportive conditions. In this model, IKKε and MEK cooperate to support growth of cancer cells in the attached, anchored environment, whereas p52 is required for growth in unattached 3-D environment. These studies reveal novel information about the role of IKKε in TNBC and highlight the adaptability of NF-κB signaling in maintaining cancer cell survival under different growth conditions. Current studies are underway to clarify the mechanism of IKKε regulation of p52. A better understanding of the diversity of NF-κB signaling may ultimately improve the development of novel therapeutic regimens for TNBC.

### Targets Affecting Metabolism

#5471

EWS-FLI1 reprograms the metabolism of Ewing sarcoma cells via positive regulation of glutamine import and serine-glycine biosynthesis.

Nirmalya Sen,1 Allison M. Cross,1 Philip L. Lorenzi,2 Javed Khan,1 Berkley E. Gryder,1 Suntae Kim,1 Natasha J. Caplen1. 1 _Center for Cancer Research, National Cancer Instiute, Bethesda, MD;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Ewing sarcoma (EWS) is a soft tissue and bone tumor found primarily in adolescents and young adults. In most cases of EWS, the chimeric transcription factor, EWS-FLI1 is the primary oncogenic driver. The expression profile of EWS cells reflects EWS-FLI1 binding and activation or repression of transcription. Recent studies have shown EWS-FLI1 deregulates proteins that function in tryptophan metabolism (Mutz et al., 2016, FEBS Lett. 590, 2063) or serine-glycine biosynthesis (Tanner et al., 2017, Mol. Cancer Res., 11, 1517). In our study, we extend these findings by establishing that EWS-FLI1 transcriptionally regulates the expression of enzymes required for serine-glycine biosynthesis and the mitochondrial one-carbon cycle, and the uptake of the alternative nutrient source glutamine, directly. To identify metabolic pathways regulated by EWS-FLI1, we interrogated the expression profiles of EWS-FLI1-silenced TC32 EWS cells. We observed that silencing of EWS-FLI1 results in the decreased expression of genes required for serine-glycine biosynthesis specifically, PHGDH, PSAT1, PSPH, and SHMT2. Using ChIP analysis, we demonstrated that EWS-FLI1 binds specific regulatory regions within these four serine-glycine biosynthesis genes. Also, ectopic expression in 293T cells results in increased expression of PHGDH, PSAT1, PSPH, and SHMT2. Consistent with the ability of EWS cells to synthesize serine and glycine de novo, EWS cells grown in medium depleted of these amino acids exhibit no change in viability. However, EWS cells were sensitive to the depletion of glutamine. Analysis of the expression profiles of EWS-FLI1-silenced TC32 EWS cells showed EWS-FLI1 positively regulates expression of the glutamine transporter, SLC1A5 and that EWS cells require glutamine for glycine synthesis. Next, we examined metabolic processes downstream of serine-glycine biosynthesis and showed that enzymes associated with the mitochondrial one-carbon cycle, MTHFD2 and MTHD1L, are transcriptional targets of EWS-FLI1, but not their cytosolic counterpart MTHFD1. Our data suggests EWS-FLI1 favors activation of enzymes associated with the mitochondrial one-carbon cycle, which provides the reducing equivalents GSH and NADPH. Depletion of EWS-FLI1 or the inhibition of PHGDH reduced the GSH/GSSG and NADPH/NADP ratios in EWS cells resulting in increased production of reactive oxygen species, inducing DNA damage and apoptosis. Importantly, analysis of EWS primary tumor transcriptome data confirmed that the aforementioned metabolism genes we identified as regulated by EWS-FLI1 exhibit increased expression compared with normal tissues. In summary, our study demonstrates that EWS-FLI1 reprograms the metabolism of EWS cells and that serine-glycine metabolism and glutamine uptake are potential vulnerabilities in this tumor type.

#5472

Basal-like breast cancer subtype is characterized by deregulated glutamine metabolism and is sensitive to GLS inhibition.

Christophe Henry,1 Michael Lampa,2 Timothy He,2 Beatriz Ospina,2 Bailin Zhang,2 Gejing Deng,2 Claude Barberis,3 Dietmar Hoffmann,3 Jack Pollard,2 Adrian Francisco,2 Heike Arlt,2 Jason Reeves,2 Joshua Murtie,2 Christopher Winter,2 Victoria Richon,2 Hong Cheng,2 Carlos Garcia-Echeverria,1 Dmitri Wiederschain,2 Lakshmi Srinivasan2. 1 _Sanofi, Vitry-sur-Seine, France;_ 2 _Sanofi, Cambridge, MA;_ 3 _Sanofi, Waltham, MA_.

Tumor cells display enhanced requirements for energy and building blocks such as amino acids, nucleic acids and lipids to sustain their proliferation. Metabolic demands of cancer cells are often met in the context of limited access to nutrients and the need to maintain redox balance. Glutamine catabolism is considered critical to cancer cell survival by not only delivering carbon and nitrogen atoms needed for nucleic acid and amino acid precursors but also by serving as the source of antioxidants such as NADPH and glutathione. A vital step in the catabolism of glutamine is its conversion to glutamate by the mitochondrial enzyme glutaminase (GLS).In this study, we confirmed the significance of GLS and enhanced glutamine utilization in the basal subtype of breast cancer as evidenced by higher GLS to GLUL (Glutamine synthetase) ratio in a panel of breast cancers in The Cancer Genome Atlas (TCGA) database. Using inducible shRNA mediated gene knockdown, we discovered that loss of GLS function in triple-negative breast cancer (TNBC) cell lines led to profound tumor growth inhibition in vitro and in vivo. This anti-tumor effect of GLS knockdown was rescued either using shRNA resistant cDNAs encoding both GLS isoforms or by addition of an a-ketoglutarate analog thus confirming the critical role of GLS in TNBC. Along with these observations, we have found that pharmacological inhibition of GLS with the small molecule inhibitor CB-839 reduced cell growth and led to a decrease in mammalian target of rapamycin (mTOR) coupled with an increase in the stress response pathway driven by activating transcription factor 4 (ATF4). In line with the observed impact on mTOR pathway we found that GLS inhibition synergizes with mTOR inhibition. In conclusion, our preliminary investigation revealed that glutaminolysis is deregulated in a majority of TNBC cancer patients. Our target credentialing studies confirmed that GLS is essential for the survival of TNBC cell lines and xenografts tumors. The synergistic activity of GLS and mTOR inhibitors in this specific breast cancer subtype suggests a new therapeutic modality that may bring benefit to TNBC patients with high unmet need.

#5473

Identification of compensatory pathway for glutamate production upon glutaminase 1 inhibition.

Karim B. Nabi,1 Jimmy Kirsch,2 Tu Nguyen,1 Addison Quinones,1 Jessica Tan,1 Felipe Camelo,1 Marjorie Antonio,1 Jin Jung,1 Anne Le1. 1 _Johns Hopkins School of Medicine, Baltimore, MD;_ 2 _Johns Hopkins University, Baltimore, MD_.

Rationale and objectives: A current clinical trial is testing a drug inhibitor for glutaminase 1 (GLS1), the enzyme responsible for glutamine's conversion into glutamate, for cancer therapy. However, the results indicated that monotherapy is not ideal. While metabolic profiling of tumors after treatment with a GLS1 inhibitor revealed numerous upregulated pathways which could explain the resistance, the specific pathways involved were not identified. In this study, using stable isotope-resolved metabolomics, we aim to detail the specific mechanism by which cancer cells are capable of circumventing GLS1 inhibition and compensating for glutamate production.

Measurement and Methods: Patient-derived pancreatic tumors were implanted through orthotopic model in the pancreas of mice. After using our innovative delivery method through nanoparticle encapsulation of a selective GLS1 inhibitor, BPTES, we then injected 13C5, 15N2-glutamine via intraperitoneal administration, 3 times, 15 minutes apart. Tumors were harvested 2 hours post first glutamine injection. Metabolites were then extracted from tumors, and analyzed using Agilent 6520 Q-TOF mass spectrometer and 1H-NMR. Metabolite intensities were later normalized to protein concentration following analysis.

Results: Our results showed that total glutamate levels were lower in BPTES-NP treated tumors as compared to vehicle control ones. Interestingly, we found an increase in (m+5) labeled glutamate (mass of the parent ions (m) and 5 more mass units due to 13C415N1-glutamate or 13C5-glutamate labelling) in BPTES-NP treated tumors as compared to the vehicle control tumors. Moreover, we found that (m+5) glutamate is a product of (m+7) glutamine being converted to (m+5) alpha-ketoglutaramate (KGM) via glutamine-pyruvate transaminase and further on into alpha-ketoglutarate (aKG) by omega-amidase, which can finally produce the identified (m+5) glutamate through glutamate dehydrogenase. Further analysis using 1H-NMR detailed a significant increase in overall KGM intermediate intensity in treatment groups compared to the control groups, confirming the upregulation of compensatory pathway (glutamine-KGM- aKG-glutamate) to produce glutamate upon treatment of GLS1 inhibitor.

Conclusion: These results explain the reasons behind the limited clinical outcomes for single therapy with a GLS1 inhibitor, and provide potential therapeutic targets: glutamine-pyruvate transaminase, for combination treatments with GLS1 inhibitors to prevent the compensation for glutamate production amid GLS1 inhibition.

#5474

Activity of the S1P pathway promotes ovarian cancer and serves as a novel metabolic target of metformin.

Peter C. Hart,1 Tatsuyuki Chiyoda,1 Marion Curtis,1 Xiaojing Liu,2 Chun-Yi Chiang,1 Stephanie McGregor,1 Ricardo Lastra,1 Jason Locasale,2 Ernst Lengyel,1 Iris L. Romero1. 1 _University of Chicago, Chicago, IL;_ 2 _Duke University, Durham, NC_.

Sphingosine-1-phosphate (S1P), a metabolite of the phospholipid sphingosine, is a bioactive lipid that directly interacts with specific G-protein-coupled receptors to transduce signals that regulate growth, proliferation and motility. Sphingosine kinases (SPHK1 and 2) catalyze the formation of S1P from sphingosine, thereby regulating S1P homeostasis and the sphingolipid rheostat. Recently, using TCGA data, we noted SPHK1 amplification in several gynecologic cancers and that high expression of SPHK1 was associated with increased mortality in high-grade serous ovarian cancer (HGSOC). The findings reported here show that exposure of HGSOC tumor cells to exogenous S1P, or overexpression of SPHK1, induced migration, proliferation and clonogenecity in multiple ovarian cancer cell lines in vitro. Likewise, in a xenograft mouse model of ovarian cancer, overexpression of SPHK1 markedly enhanced tumor growth. In prior pre-clinical studies, we have demonstrated a strong protective effect of the diabetes medication metformin in ovarian cancer. Interestingly, we found that patients with HGSOC that use metformin for diabetes have reduced serum S1P levels compared to controls, suggesting that the sphingolipid rheostat may be a novel metabolic target of metformin in ovarian cancer. Supporting this, we identified that SPHK1 expression in ovarian cancer cell lines is reduced by treatment with metformin, and further that the reduction of SPHK1 by metformin was mediated through inhibition of transcriptional activity of hypoxia-inducible factors (HIF1α and HIF2α). Finally, we show that overexpression of SPHK1 in HGSOC cell lines enhanced the cytotoxic effects of metformin. Taken together, our data indicates that hypoxia-induced SPHK1 expression and downstream S1P signaling promote tumor progression in HGSOC, and that tumors utilizing this pathway may be particularly vulnerable to the anti-cancer effects of metformin.

#5475

Enhanced lipid uptake: A novel target to fight the adaptive response to androgen-targeted therapies in prostate cancer.

Kaylyn D. Tousignant. _Queensland University of Technology, Brisbane, Australia_.

Prostate cancer is the most commonly diagnosed cancer among men and is the second leading cause of cancer-related death in men worldwide. Current mainstay treatments of advanced PCa target the androgen receptor (AR), a transcription factor that controls the expression of a large set of genes associated with metabolism, differentiation, survival and tumor growth. Despite initial disease regression following AR-targeted therapies (ATT) through androgen-deprivation therapy (ADT) or AR antagonism (enzalutamide), almost all PCa patients develop recurrent disease within 2-4 years, which progresses to incurable and lethal castrate-resistant PCa (CRPC). Increasing evidence suggests that ATT induces adaptive responses within the tumor and microenvironment that provide a survival advantage and treatment resistance, facilitating progression to CRPC. While increased de novo lipogenesis, the synthesis of fatty acids and cholesterol, is a well-described hallmark of prostate cancer, little is known about lipid uptake in this context. Furthermore, targeting lipogenesis alone has had little clinical success, which we hypothesize is due to the ability for cells to be rescued by exogenous lipids. Using novel quantitative high-content fluorescent imaging assays, we show enhanced lipid uptake as an adaptive response to ATTs as well as increased mRNA and protein expression of lipid transporters. Additionally, we present tunneling nanotubes (TNTs) as a novel lipid supply pathway utilized by PCa cells to acquire lipid droplets and mitochondria from neighboring cells in response to metabolic stress. Shotgun lipidomics revealed extensive remodeling of cellular lipid content, including a significant increase in essential fatty acids and phospholipids as well as elongation of very-long chain fatty acids in response to ATTs. Co-treatment with lipogenesis inhibitors and inhibitors of exogenous lipid supply pathways resulted in synergistic growth inhibition in PCa cells. These findings suggest enhanced lipid uptake is an adaptive response to androgen-targeted therapies and that co-targeting uptake and lipogenesis may serve as a novel therapeutic strategy to improve efficacy of current treatments to delay progression to lethal CRPC.

#5476

Inhibiting lactate transporters MCT-1 and MCT-4 target hypoxic HNSCC cells and sensitize them to metformin.

Pedro H. Boasquevisque,1 Verena Schoeneberger,2 Laura Caporiccio,3 Ravi Vellanki,3 Marianne Koritzinsky,3 Bradly G. Wouters3. 1 _University of Toronto, Toronto, Ontario, Canada;_ 2 _Johannes-Gutenberg-University Mainz, Mainz, Germany;_ 3 _University Health Network, Toronto, Ontario, Canada_.

Head and neck squamous cell carcinoma (HNSCC) is treated primarily through a combination of surgery and radiation therapy. The problem of radioresistance, however, persists and requires new approaches to overcome it. Tumor hypoxia has been shown to be a driver of radioresistance, thereby prompting the targeting of the hypoxic niche, known to be highly glycolytic. Two key components of the hypoxic metabolic profile are lactate transporters MCT-1 and MCT-4, which sustain hypoxia-driven lactate production in cells. We hypothesize that inhibition of MCT-1 and MCT-4 will selectively target the growth of hypoxic tumor cells and potentially exert a synergistic effect with the antidiabetic drug metformin, a known inhibitor of mitochondrial respiration that has been shown to improve the radiation response. To study this, we employed CRISPR-Cas9 to genetically ablate both MCT-1 and MCT-4 in an HNSCC cell line. Validation of double knockout (DKO) cells was done through immunoblotting and sequencing. Cells were grown under conditions of 21% O2 (normoxia) in a regular CO2 incubator or 0.2% O2 (hypoxia) in an H45 HypOxystation® hypoxia chamber. Cell proliferation was measured under normoxia and hypoxia through the use of IncuCyte automated imaging while the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of cells were measured using a Seahorse™ XF analyzer. Metabolic profiling of the DKO cells showed a higher rate of oxygen consumption and a decreased ability of exporting lactate, indicating a metabolic shift when lactate transport is impaired. The loss of MCT-1 and MCT-4 by themselves did not significantly alter cell proliferation under normoxic conditions, but disruption of both transporters concurrently significantly altered growth under normoxia. When exposed to hypoxia, proliferation of the DKO cells was completely halted. In addition, MCT-1/MCT-4 double knockouts showed greater inhibition of cell growth under metformin treatment than either single knockouts or wild-type cells. Significantly lower doses of metformin, which had no effect on the proliferation of wild-type or single knockout cells, were capable of impairing the growth of DKO cells under hypoxia. In conclusion, our study shows that inhibition of lactate export has a profound effect on cell growth under hypoxic conditions in vitro. Moreover, loss of lactate export enhances the sensitivity of HNSCC cells to metformin and could constitute a new way of targeting the hypoxic niche with the purpose of leading to better treatment outcomes.

#5477

Targeting nicotinamide phosphoribosyltransferase (NAMPT) with OT-82 in Ewing sarcoma.

Anna E. Gibson,1 Arnulfo Mendoza,1 Lioubov Korotchkina,2 Olga Chernova,2 Christine M. Heske1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _OncoTartis, Inc., Buffalo, NY_.

EWS is an aggressive malignancy affecting adolescents and young adults, for which novel therapies are critically needed. Recent insights have revealed that NAMPT is a potential therapeutic target, mediating the rate limiting step of the NAD salvage pathway which converts nicotinamide (NAM) to nicotinamide mononucleotide (NMN). This pathway is preferentially utilized in tumor cells where it is essential for maintaining cellular bioenergetics and provides the necessary substrate for NAD-dependent proteins. OT-82 (OncoTartis) is a novel NAMPT inhibitor expected to enter early phase clinical trials in the near future. The purpose of this study was to evaluate the efficacy and mechanism of action of OT-82 as a single agent and in rational combinations in EWS.

Proliferation of EWS cells lines was assessed after treatment with OT-82 as a single agent and in combinations with PARP inhibitors and DNA damaging agents using IncuCyte live cell analysis and MTS assays. Targeted activity of OT-82 was evaluated via NAD/NADH-Glo assays and rescue experiments with metabolic intermediates. Protein expression of targets was assessed with Western blot. For in vivo studies, SCID mice underwent orthotopic injection of EWS cells from established cell lines and PDX cells. When tumors reached a desired size, mice were randomized and treated with vehicle, OT-82, irinotecan, niraparib or combinations of OT-82 plus niraparib or irinotecan. Tumors were measured semiweekly with calipers and harvested at midpoints and at study endpoint for biology studies.

EWS cell lines were sensitive to OT-82 in a dose-dependent manner, with IC50 values in the low nanomolar range for all cell lines tested, consistent with the known activity of NAMPT inhibitors in EWS cell lines. While NAMPT expression in EWS cell lines was high, the level of expression did not correlate with sensitivity to OT-82. OT-82 activity was on-target, demonstrated by dose-dependent reduction in NAD/NADH levels in EWS cells after treatment. This effect could be rescued through the addition of NMN in the majority of cell lines tested, supporting the proposed mechanism of OT-82 as a NAMPT inhibitor. When combined with either niraparib or SN-38, anti-proliferative activity was synergistic in vitro. In vivo, treatment with OT-82 resulted in tumor regression or growth inhibition in multiple xenograft models of EWS treated at doses between 25 and 50 mg/kg three days per week. When combined with low dose niraparib (50 mg/kg five days per week), tumor growth was suppressed, and survival time was significantly increased as compared to either single agent. When OT-82 was combined with low-dose irinotecan (2-5 mg/kg two days per week), the combination exhibited enhanced efficacy in multiple models. Biology studies of tumor tissue are ongoing and will be reported. OT-82 may be a novel targeted approach for the treatment of EWS and the addition of niraparib or irinotecan may enhance its efficacy.

#5478

Targeting kynurenine pathway for the treatment of cisplatin-resistant lung cancer.

Medhi Wangpaichitr,1 Chunjing Wu,2 Dan JM Nguyen,1 Ying-Ying Li,1 Lynn G. Feun,3 Niramol Savaraj1. 1 _Univ. of Miami/VA Medical Ctr., Miami, FL;_ 2 _VA Medical Ctr., Miami, FL;_ 3 _Univ. of Miami, Miami, FL_.

We have found that increasing ROS (reactive oxygen species) level is a specific and yet common feature found in cisplatin resistant (CR) lung cancer cells. We also showed that manipulation of ROS levels can selectively killed CR cells. Here, we have further identified the underlying mechanism for increasing ROS and its relationship with metabolic reprogramming and immune evasion. CR cells no longer follow classic aerobic glycolysis (Warburg effect). They do not primarily utilize glucose, but rather consume amino acids such as glutamine and tryptophan for survival. CR cells take up twice as much L-[G-3H] glutamine (n=4, p<0.05) and tryptophan (TRP) (n=4, p<0.04) when compared to its parental counterpart. These fundamental understandings have led us to study another unique mechanism where we found that CR cells activate kynurenine pathway (TRP catabolism) when they encounter excessive oxidative stress in order to maintain a finite level of ROS for their growth and proliferation. Importantly, kynurenine (KYN) which is oxidized through indoleamine 2,3-dioxygenase (IDO) via kynurenine pathway, plays a key role in reprogramming naïve T-cells to immune suppressive regulatory T-cell (T-reg) phenotype. Inhibiting IDO using Epacadostat (Incyte Corp.), a known IDO inhibitor which is currently in phase 3 trial, led to elevated ROS and resulted in the demise of CR cells. ROS levels were increased (2-3X) more in resistant cells after Epacadostat treatment while no significant change occurred in parental cells. The ID50 dosage of parental cells was 2-4 fold more than their CR cell counterparts and no cytotoxicity was found in normal lung fibroblast (n=6; p<0.05). To further determine whether IDO correlated with ROS levels and sensitivity to Epacadostat, we inhibited IDO expressions in CR cell lines (ie. ALC and H460CR) using siRNA. Knocking down IDO enhanced ROS production and more importantly suppressed CR cells' growth which further implicates the important role of kynurenine pathway (KP) in proliferation of CR tumors. Moreover, the treatment of knocked down cells with Epacadostat did not result in further cell death, indicating the specificity of this compound for the IDO enzyme. FoxP3, a lineage-specific marker of T-reg cells, was higher in CR cells when compared to their parental cell counterparts. Treatment with Epacadostat significantly suppressed FoxP3 expression which further support the relationship between metabolic reprogramming and immune evasion. Overall, we believe that our work will have potential impact on (i) how one can effectively exploits the KP pathway to treat CR tumors, and (ii) improved understanding on how CR cells evade immune surveillance. Supported by Department of Veterans Affairs, CDA2 award (1K2BX001289).

#5479

Targeting fructose-induced metabolic reprogramming in liver metastasis.

Xiling Shen. _Duke Univ., Durham, NC_.

Primary tumors gradually accumulate genetic alterations and are influenced by their microenvironment until they acquire the ability to metastasize to distant organs. Typical of this process, colorectal cancer (CRC) progresses through an adenoma-to-carcinoma sequence that eventually leads to metastasis, preferentially (~70% patients) to the liver. At this phase, the disease becomes challenging to treat and eventually develops resistance to most forms of combination therapy, making CRC metastasis a leading cause of cancer-related deaths. Patients with inoperable liver metastasis respond poorly to chemotherapeutic intervention and have a median survival of 6 to 9 months. Liver lesions have also been shown to seed tertiary tumors in the lungs of patients. Current chemotherapy for advanced CRC does not target liver metastases specifically. This is partly based on observations that CRC metastases are not consistently associated with any specific genetic mutations and they generally resemble cells in the primary tumor. However, emerging evidence suggests that nongenetic alterations, such as epigenetic and metabolic reprogramming, may promote cancer metastasis, including CRC. Targeting such mechanisms may provide a way to enhance therapeutics against metastasis.

In this study, pair-wise analysis of 90 matched samples (normal colon, primary CRC, liver metastasis) from 30 Stage IV CRC patients and an in vivo CRC metastasis model via cecum transplantation suggests that CRC liver metastases alter activity levels of certain metabolic pathways. LC-MS based metabolomics systematically mapped the altered metabolic pathways in CRC liver metastasis. In particular, via hypoxia-responsive GATA6 and fructose-responsive ChREBP, liver metastases upregulate ALDOB, an enzyme involved in fructose metabolism, given that 70% of fructose is metabolized in the liver. Intrahepatic implantation indicates that the liver environment causes CRC cells to upregulate ALDOB. Metabolomics and 13C-labeled fructose tracing studies indicate that ALDOB promotes fructose metabolism to fuel glycolysis, gluconeogenesis and the pentose phosphate pathway. Fructose also triggered downstream signaling to enhance lipid synthesis. ALDOB silencing or dietary fructose restriction suppresses growth of CRC liver metastases, but not primary tumors or lung metastases, highlighting the importance of tumor environment. Our findings suggest that metastatic cells can take advantage of abundant metabolites in their new microenvironment, and manipulation of involved pathways impacts the course of metastatic growth.

#5480

Colorectal cancer cell lines harboring mutation in Wnt/β-catenin pathway shows enhanced sensitivity to the stearoyl-CoA desaturase (SCD) inhibitor, DSP-0692.

Yudai Furuta,1 Yuichi Fukunaga,1 Satoshi Ikeda,1 Erina Koga,2 Hiroki Umehara,1 Tsuguteru Otsubo,1 Manabu Watanabe,1 Futoshi Hasegawa,1 Hitoshi Ban,1 Eiji Sugaru1. 1 _Sumitomo Dainippon Pharma Co.,Ltd., Osaka, Japan;_ 2 _Boston Biomedical, Inc., Cambridge, MA_.

Colorectal cancer (CRC) is one of the most frequent malignant diseases worldwide. Recent studies suggest that a small subpopulation of cells known as cancer stem cells (CSCs) may promote the high metastasis and relapse associated with CRC. It has been reported that Wnt/β-catenin signaling is aberrantly activated in a variety of cancer including colorectal cancer, and plays a critical role in CSC maintenance.

Here it is shown that a novel SCD inhibitor, DSP-0692, may inhibited Wnt/β-catenin signaling pathway through two different mechanisms in CRC. The effect of SCD inhibition on Wnt3A secretion, in which process post-translational fatty acylation (with palmitoleic acid) is required, was investigated. DSP-0692 led to reduced Wnt3A protein levels secreted in the media in Wnt3A-overexpressing cell line. This result was further confirmed by the TCF/LEF reporter assay in HCT116 incubated with conditional media from L-Wnt3A cells, with or without DSP-0692. DSP-0692 also inhibited exogenous Wnt3A-induced reporter activity. This indicates DSP-0692 may inhibit the β-catenin/Tcf transactivation by additional mechanism in addition to inhibiting Wnt secretion. To clarify this additional mechanism, the effect of DSP-0692 on nuclear translocation of β-catenin was investigated. SCD inhibition led to a decrease of β-catenin amounts within the nucleus. Currently, how SCD inhibition affects nuclear translocation of β-catenin is being investigated. It was confirmed that DSP-0692 exhibited lower IC50 value to sphere cells carrying mutations related to Wnt/β-catenin signaling pathway than those without these mutations. Next, the effect of SCD inhibition on Wnt/β-catenin signaling pathway in vivo was investigated. Treatment with DSP-0692 in a SW620 colorectal cancer xenograft model suppressed mRNA expression of the Wnt/β-catenin target genes, as well as CSC marker gene.

Taken together, these results suggest fatty acid desaturation in tumor cells may have an impact on Wnt/β-catenin signaling pathway and involve in CSC maintenance. The approaches targeting SCD could be a new therapeutic strategy for anti-CSC therapy in CRC.

#5481

Targeting the kynurenine pathway as a novel metabolic treatment for head and neck cancer.

Chitra Subramanian, Thekkelnaycke M. Rajendiran, Tanu Soni, Mark S. Cohen. _Univ. of Michigan, Ann Arbor, MI_.

Introduction: Head and neck squamous cell carcinoma (HNSCC) still claims 330,000 lives globally each year, indicating a critical need for novel therapies. Recently, we identified high levels of kynurenine (K) in HNSCC patients through global metabolic profiling of HNSCC tumor tissues via mass spectrometry (MS). Indoleamine-2,3-dioxygenase (IDO) and tryptophan-2,3- dioxygenase (TDO) are enzymes that degrade tryptophan to K. Elevated K allows tumor cells to evade immune response more easily, promoting cancer progression, while increased IDO levels are associated with poorer prognosis. We hypothesize that restoring immune response to tumors by blocking IDO1 will be an effective therapeutic strategy for sensitizing HNSCC.

Methods: Validated human HNSCC cell lines were grown in 2D culture. Kynurenine levels were measured by MS. IDO1, IDO2 and TDO levels in tissue samples were measured by Western blot (WB) analysis. siRNAs for IDO1, IDO2, and TDO were used to knock down tryptophan pathway enzymes (non-targeting siRNA controls). Knockdown efficiency was evaluated by immunocytochemistry. After knockdown of IDO1, RNA was isolated, reverse transcribed and cancer associated pathways were analyzed using RT2 cancer pathway profiler (data analyzed using GeneGlobe; Qiagen).

Results: Evaluation of the expression levels of enzymes that play a critical role in kynurenine pathway by WB analysis indicated upregulation of IDO1, IDO2 and TOD in both primary and metastatic tumor samples. siRNA-mediated knockdown of these enzymes indicated that only IDO1 knockdown was able to significantly reduce the levels of kynurenine by more than 80% (p<0.001), whereas knockdown of IDO2 and TDO reduced the kynurenine levels only by 10-25% (p>0.05%). Regulation of IDO1 function evaluated by the RT2 cancer pathway finder indicated downregulation of several genes involved in angiogenesis (ANGPT1, MCP-1, FGF2, TEK, VEGF, SERPINF1), pro-survival (c-IAP2 and XIAP), EMT transition (CDH2, OCLN and SNAI1), and DNA repair (LIG4, POLB, ERCC3 and DDB2), whereas genes involved in glycolysis (ACLY, G6PD,COX5A,LPL and PFKL), DNA damage (GADD34, GADD45G etc) and apoptosis (CASP7, CASP9, BCL2L11) were upregulated.

Conclusion: Kynurenine is highly upregulated in HNSCC with checkpoint inhibition of IDO1 leading to increased apoptosis in vitro as well as increases in several glycolytic enzymes. Further translational analysis of glycolytic influences on the tryptophan pathway and immune checkpoint inhibition of IDO1 will provide mechanistic support for development of novel metabolic therapies for HNSCC.

#5482

L-2HG/ L2HGDH axis as therapeutic target for kidney cancer.

Sandeep Balu Shelar,1 Eun-hee Shim,1 Garrett Brinkley,1 Anirban Kundu,1 Hyeyoung Nam,1 Francesca Carobbio,1 Tyler Poston,1 Jubilee Tan,1 Daniel Benson,1 Dinesh Rakheja,2 Richard Kirkman,1 Yusuke Sato,3 Seishi Ogawa,3 Shilpa Dutta,1 Sadanandan E. Velu,1 David Crossman,1 Anja Becker,4 Conrad Kunick,4 Sunil Sudarshan1. 1 _University of Alabama at Birmingham, Birmingham, AL;_ 2 _University of Texas Southwestern Medical Center, Dallas, TX;_ 3 _Kyoto University, Kyoto, Japan;_ 4 _Technische Universität Braunschweig, Braunschweig, Germany_.

Background: The D-enantiomer of 2-hydroxygultarate (D-2HG), along with fumarate and succinate, are considered oncometabolites that promote tumorigenesis. Our lab has reported elevated levels of the L-enantiomer of 2-hydroxyglutare (L-2HG) in clear cell renal cell carcinoma (ccRCC), in part due to reduced expression of the enzyme L-2HG dehydrogenase (L2HGDH).

Objectives: Here we evaluated the underlying biochemical mechanisms of L-2HG accumulation and characterized the contribution of the L-2HG/L2HGDH axis to tumorigenesis.

Experimental strategy: We assessed the role of raised L-2HG in renal carcinogenesis via both in vitro and in vivo means. Mutagenesis and knock down approaches were applied to study involvement of loss of L2HGDH in accumulation of high L-2HG levels and associated tumor phenotypes. Metabolomics coupled with 13C tracer labeling studies were utilized to dissect the biochemical axis that promotes L-2HG accumulation in RCC cells.

Results: Translational based studies demonstrate that loss of L2HGDH expression is associated with both cancer progression and worsened outcomes. In connection with our previous findings, we demonstrate that raising cellular L-2HG levels by treatment with cell permeable octyl ester of L-2HG as well as by shRNA-mediated knock down of L2HGDH in renal epithelial cells (HK-2) promotes in vitro tumor phenotypes. Concurrently, the epigenetic mark 5hmc was significantly decreased under high L-2HG levels. Further investigation with restoration of L2HGDH in RCC cells (RXF393 and A498) shows decreased L-2HG levels and suppression of in vivo tumor growth in nude mice (NU/NU). Interestingly, expression of loss-of-function mutant of L2HGDH was unable to decrease L-2HG levels and failed to suppress in vitro and in vivo tumor phenotypes. In addition, high L-2HG levels were found to upregulate epithelial-mesenchymal-transition (EMT) marker SNAIL1 and correspondingly downregulate E-cadherin. Biochemical studies demonstrate that the predominant carbon source for L-2HG in RCC is glutamine through the activity of glutaminase and malate dehydrogenase (MDH). Pharmacological inhibition of the glutamine/MDH axis by treatment of glutaminase inhibitor (CB-839) and MDH inhibitor (4k) in RCC cells reduced L-2HG levels and mitigates in vitro tumor phenotypes. Furthermore, suppression of in vitro phenotypes by CB-839 and shRNA-mediated MDH2 knockdown was rescued by concurrent treatment with octyl ester of L-2HG. Finally, restoration of L2HGDH promoted the expression of genes targeted by the polycomb repressor complex 2 (PRC2), whereas inhibition of PRC2 in high L-2HG cells suppressed tumor phenotypes.

Conclusion: Collectively, our data demonstrate the biologic relevance of high L-2HG to renal carcinogenesis and reveal novel therapeutic opportunities for L-2HG driven kidney tumors.

#5483

SIRT1 stabilization provides a therapeutic opportunity for reversing cachexia in pancreatic cancer.

Aneesha Dasgupta, Enza Vernucci, Surendra K. Shukla, Scott E. Mulder, Ryan J. King, Jaime Abrego, Nina V. Chaika, Kyla Buettner, Pankaj K. Singh, Pankaj K. Singh. _Univ. of Nebraska Medical Ctr., Omaha, NE_.

Cancer-associated cachexia is a complex metabolic syndrome which leads to excessive loss of skeletal muscle and adipose deposits. Up to 80% of pancreatic cancer patients suffer from cachexia and nearly one third die due to complexities related to the syndrome. Treatment of cachexia will not only improve the standard of living of pancreatic cancer patients but would also improve the patient survival. In the clinic, majority of the cancer patients are diagnosed at the refractory phase of cachexia, which cannot be reversed by dietary interventions. Till late therapies have not shown promising results in clinical trials. Hence, there is an urgent need to identify new targets to combat cancer cachexia. Sirtuins are protein deacetylases which affect the activity of various transcription factors and signaling components. The expression of all the sirtuins were analyzed after culturing mouse myotubes with tumor cell-conditioned media. The expression of SIRT1 decreased consistently upon treatment of myotubes with conditioned media from multiple human pancreatic cancer cell lines. Resveratrol, which is an activator of SIRT1 rescued the wasting phenotype in the myotubes. However, treatment of pancreatic cancer cells with resveratrol also decreased cell proliferation. Hence, we designed in vivo experiments to examine the effect of resveratrol on muscle wasting irrespective of its effects on tumor burden. We generated SIRT1 knockdown pancreatic cancer cell lines and orthotopically implanted them into the pancreas in athymic nude mice. The tumor-bearing mice were subjected to an oral delivery of resveratrol. These studies demonstrated that while resveratrol had limited effect on cancer cells due to SIRT1 knockdown, resveratrol treatment still protected the mice from muscle wasting by stabilizing SIRT1 in muscles. Hence, our results indicate that resveratrol-mediated stabilization of SIRT1 in muscles can revert wasting irrespective of its effects on tumor cells. Overall, our studies demonstrate the therapeutic potential of SIRT1 stabilization by resveratrol or related class of drugs in managing cancer-associated cachexia.

#5484

Pyruvate kinase isozymes M2 and glutaminase might be promising molecular targets for the treatment of gastric cancer.

Masakazu Yashiro, Kishu Kitayama. _Osaka City Univ. Grad. School of Medicine, Osaka, Japan_.

Glycolysis is the primary mode of glucose metabolism, not only in normal cells but also in cancer cells. The Warburg effect, known as anaerobic glycolysis even in the presence of ample oxygen, might contribute to tumor development in such hypoxic environments. It has been suggested that some kinds of glucose metabolism-related enzymes—including pyruvate kinase isozyme M2 (PKM2), PKM1, enolase 1 (ENO1), and glucose-6-phosphate dehydrogenase (G6PDH)—might be associated with the anaerobic glycolysis of cancer cells. Glutamine is also associated with the proliferation of cancer cells by an alternate source of biosynthesis from the TCA cycle, namely glutaminolysis. In the present study we evaluated the significance of the enzymes for glycolysis and glutaminolysis in relation to the proliferation of gastric cancer cells in hypoxic environments. The aim of this study was to analyze the significance of glucose metabolism-related enzymes in the proliferation of gastric cancer under hypoxia. Four hypoxia-resistant gastric cancer cell lines and 4 parent cell lines were used. RT-PCR was used to evaluate the mRNA expression levels of the following metabolism-related enzymes: PKM2, GLS, ENO1, G6PDH, and PKM1. The effects of these enzymes on the proliferation of gastric cancer cells were examined using siRNAs, Shikonin as a PKM2 inhibitor, or BPTES as a GLS inhibitor, in vitro and in vivo. Levels of both PKM2 and GLS mRNA were significantly high in all hypoxia-resistant cell lines, compared with those of their parent cells. Knockdown of PKM2 and GLS significantly decreased the proliferation of all of hypoxia-resistant cells. The combination of siPKM2 and siGLS significantly decreased proliferation compared with treatment by siPKM2 or siGLS alone. The knockdown of ENO1, G6PDH, or PKM1 did not decrease the proliferation of all hypoxia-resistant cells. Combination treatment by Shikonin and BPTES inhibited the proliferation of all hypoxia-resistant cancer cells more than that by either one of the two agents. The in vivo study indicated that the tumor size treated by the combination of Shikonin and BPTES was significantly smaller than that of vehicle-treated group. These findings suggested that PKM2 and GLS might play important roles in the proliferation of hypoxic gastric cancer cells. A combination of PKM2 and GLS inhibitors might be therapeutically promising for the treatment of gastric cancer.

#5485

A promising pathway in immuno-oncology: CD73-adenosine axis highlighted by quantitative mass spectrometry imaging.

Lauranne Poncelet,1 Rima Ait-Belkacem,1 Bruno Gomes,2 Stefan Linehan,3 Jonathan Stauber1. 1 _Imabiotech, Loos, France;_ 2 _Roche, Basel, Switzerland;_ 3 _Imabiotech, Billerica, MA_.

Introduction The CD73-Adenosine (CD73-ADO) axis constitutes one of the most promising pathways in immuno-oncology. CD73 catalyzes the conversion of adenosine monophosphate (AMP) to ADO. CD73 is believed to play a role in mediating the inhibitory function of regulatory B and T cells. It is widely expressed and up-regulated in many cancerous tissues. The presence of extracellular ADO within tumor microenvironment has been described as an immunosuppressive halo surrounding the tumor, permeating the tumor microenvironment and preventing antitumor immunity. In this study, the endogenous level of AMP and ADO were analyzed by quantitative mass spectrometry imaging (QMSI) and LC-MS/MS in CT26 tumor mouse model. Clones with different CD73 expression levels were selected (low, medium & high), which mimed different CD73-inhibitors efficacies. Then, ADO metabolic pathway was targeted in order to highlight the tumor microenvironment response linked to a mimic immunotherapy effect.

Methods Undifferentiated colon carcinoma CT26 cell line was implanted into C57BL/6 mice. Then, tumors were harvested and frozen at -80°C until use. 1,5-diaminonaphthalene (1,5-DAN) and 2,5-dihydroxybenzoic acid (2,5-DHB) matrices mixed to internal standards were sprayed onto tumor sections of 10 µm thickness with the automatic TM Sprayer (HTX Technologies, LLC) prior analysis. Data acquisition was performed using 7T MALDI-FTICR (SolariX XR, Bruker Daltonics) and analyses were set at 120µm spatial resolution in full scan positive and negative modes. Acquired data were treated with MultimagingTM software (ImaBiotech). All quantitative results were confirmed by LC-MS/MS analysis. Histological and immunohistochemistry staining anti-CD73 were applied on adjacent section.

Results A validation of CT26 mouse model occurred since an increase of ADO/AMP ratio of 77% was relative to the high CD73 expression. In other words, CT26 mouse models with high CD73 expression showed higher ADO/AMP ratio (2.1) than CT26 medium (1.7) and low (1.6) clones. Then, as an immunosuppressive halo, the impact of ADO expression on tumor microenvironment was also characterized using MSI analysis. A large number of metabolites that are implicated in this pathway were highlighted (ADP, IMP, GMP, Guanosine, Inosine, Hypoxanthine, Xanthine, Uric acid⋯) and differences were also observed.

Conclusion The combination of all these data extended our understanding on the relative contribution of ADO signaling in suppressing antitumor immunity. Because the development of immunotherapies such as CD73 inhibitor requires a deep understanding of the interplay between the immune system and cancer cells in the tumor microenvironment, these immune/tumor metabolites and nutrients can now be followed by QMSI as biomarkers of response to treatment towards enhancing immunotherapies efficacy.

#5486

The role of the GABA shunt in prostate cancer metabolic reprogramming and aggressive phenotype.

Erika L. Knott, Sumitra Miriyala, Hyung Nam, Manikandan Panchatcharam, Nancy Leidenheimer. _LSU Health Sciences Center, Shreveport, Shreveport, LA_.

The GABA (γ-aminobutyric acid) shunt, a bypass mechanism for a portion of the tricarboxylic acid (TCA) cycle, is responsible in the mammalian central nervous system for the synthesis and degradation of the inhibitory neurotransmitter GABA. Glutamic acid decarboxylase (GAD67 and GAD65; gene names GAD1 and GAD2, respectively) synthesizes GABA from glutamate, while GABA-transaminase (GABA-T; gene name ABAT) metabolizes GABA into succinic semialdehyde, which is dehydrogenated into succinate. Although poorly studied in mammals, the GABA shunt plays a protective role against pH, thermo-, and oxidative stresses in plants and bacteria. We hypothesize that both cell non-autonomous GABA from the tumor microenvironment (TME), and cell-autonomous production of GABA contribute to cancer progression. Cell types within the TME such as endothelial and T-cells, are known to secrete GABA. To investigate the utilization of cell non-autonomous GABA by prostate cancer cells, we determined via LC-MS/MS that although the prostate adenocarcinoma LNCaP cell line does not contain significant amounts of GABA, it is capable of taking up exogenous GABA. The effect of exogenous GABA treatment on the metabolic capacity of LNCaP cells was examined using the SEAhorse XF24 Bioanalyzer. GABA treatment increased oxygen consumption rate while decreasing the rate of glycolysis and glycolytic reserve. This effect was blocked by co-treatment with GABA-T inhibitors, suggesting the ability of GABA to increase mitochondrial respiration occurs through the GABA shunt. GABA treatment did not affect the metabolic phenotype of a normal epithelial cell line control. Regarding cell-autonomous production of GABA, recent studies show dysregulation of genes encoding GABA shunt enzymes in various cancers; specifically relevant to this study, GAD1 is upregulated in castration-resistant prostate cancer cell lines (Ippolito 2014). Consistent with the involvement of GAD1 in aggressive prostate cancers, our analysis of the TCGA Provisional prostate cancer dataset reveals that GAD1 mRNA expression level increases with increasing Gleason Score (Pearson r = 0.19; p < 0.00005). Additionally, analysis of the Beltran (2016) data set reveals that GAD1 transcripts are upregulated in treatment-induced neuroendocrine prostate cancer compared to prostate adenocarcinoma (unpaired t-test, p < 0.0005). We hypothesize that both cell non-autonomous and autonomous GABA play a role in cancer progression. Uptake of GABA from the TME may support metabolic reprogramming of primary tumor cells, while upregulation of GAD1 and subsequent increase in cell-autonomous GABA production may provide a malignant advantage to late-stage cancer. The GABA shunt may offer a novel therapeutic target for prostate cancer treatment by exploiting a poorly understood metabolic pathway linked to the TCA cycle. We thank the LSUHSC-S Feist-Weiller Cancer Center for financial support.

#5487

Targeting glycolysis for the treatment of cancer.

Jason Chesney, Aubrey Mojesky, Yoannis Imbert-Fernandez, John Trent, Sucheta Telang. _Univ. of Louisville, Louisville, KY_.

Tumors exhibit markedly increased glucose uptake and glycolysis and this metabolic phenotype serves to satisfy their increased requirement for energy and biosynthetic intermediates. Tumor glucose metabolism is stimulated in part by fructose-2,6-bisphosphate (F26BP), which is the product of a family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) and allosterically activates a key glycolytic enzyme, 6-phosphofructo-1-kinase (PFK-1). Previous studies have found the PFKFB3 family member to be a dominant source of F26BP in cancer cells due to a high kinase activity, expression in multiple tumor types and the inhibition of tumor growth by specific PFKFB3 inhibitors including a compound, PFK158, that is currently under evaluation in a phase I trial. Recent data, however, indicate that the PFKFB4 enzyme plays a significant role in regulating glucose metabolism in neoplastic cells. We have found that PFKFB4 is highly expressed in multiple cancers, strongly induced by HIF-1α, and that its suppression markedly reduced F26BP and glycolytic flux, increased apoptosis and inhibited growth of xenograft tumors in vivo, thus providing strong rationale for the development of antineoplastic agents selectively targeting PFKFB4. Using virtual screening, we have discovered a first-in-class small molecule inhibitor of PFKFB4, termed 5MPN, that selectively inhibits recombinant PFKFB4 activity with a resultant decrease in F26BP, glycolytic flux and cancer cell growth in vitro and markedly reduces the growth of established tumors without systemic toxicity. During our studies, we unexpectedly observed that PFKFB4 expression is increased by PFKFB3 inhibition, suggesting that PFKFB4 may compensate for decreased PFKFB3 expression and activity. We examined the effects of simultaneous silencing of PFKFB4 and PFKFB3 on cancer cell lines and found that co-knockdown led to a significant decrease in cell viability and glycolysis and to a near-complete abrogation of anchorage-independent growth in vitro. We additionally examined the effect of simultaneous inhibition of PFKFB4 and PFKFB3 using 5MPN and several PFKFB3 inhibitors in vitro and found a synergistic increase in cell death. Taken together, our data indicate that targeting PFKFB4 may be a viable therapeutic option against cancer and strongly support the further development of PFKFB4 small-molecule inhibitors as well as the co-targeting of PFKFB4 and PFKFB3 as potential strategies for the treatment of cancer.

#5488

Serine racemase is a new therapeutic target for colon cancer.

Kenji Ohshima, Satoshi Nojima, Naoki Wada, Yumiko Hori, Shinichiro Tahara, Masako Kurashige, Keisuke Kawasaki, Junichiro Ikeda, Eiichi Morii. _Osaka University, Suita, Osaka, Japan_.

Cancer cells have an altered metabolic state to meet the requirements of rapid proliferation, and altered amino acid metabolism is known to be significant for cancer cell growth. Amino acids consist of L- and D-amino acids, and humans are known to metabolize L-amino acids. D-amino acids have been considered not to exist in human body except in intestinal flora. Recently, D-serine was reported to be synthesized and catabolized by serine racemase (SRR) and D-amino acid oxidase, respectively, and it has an important function as co-activator of the N-methyl-D-aspartate receptor in the human brain. However, whether D-amino acids function in other human organs has not been elucidated. Moreover, the role of enzymes metabolizing D-amino acids in the context of cancer metabolism has not been studied. Here, we present a novel role for SRR in colon cancer. First, we analyzed SRR expression levels in colon adenoma, colon adenocarcinoma, and normal colon mucosa in ONCOMINE datasets. We found that SRR expression levels were significantly elevated in colon adenoma and colon adenocarcinoma compared to normal colon mucosa in several datasets, including that of the Cancer Genome Atlas (TCGA). To determine whether SRR has a functional role in colon cancer cells, we disrupted its expression using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) system in two colon cancer cell lines, HCT116 and DLD-1, using two distinct targets of short guide RNAs and examined the effects on cell proliferation. We found that SRR knockout resulted in reduced cell proliferation in both HCT116 and DLD-1 cells, which was recovered by exogenous SRR expression. In addition, a positive correlation was found between SRR expression levels and cell proliferation rate in four colon cancer cell lines, HCT116, DLD-1, WiDr, and Lovo. Moreover, phenazine methosulfate and L-aspartic acid β-hydroxamate, which have been previously reported to inhibit SRR, suppressed in vitro colon cancer cell proliferation. In conclusion, SRR is highly expressed in colon adenoma and colon adenocarcinoma compared to normal colon mucosa, and it enhances colon cancer cell proliferation. SRR is expected to be a potential new target for the development of novel therapies for colon cancer.

#5489

Characterization of mitochondrial STAT3 (mitostat3) function and the mitoStat3 interactome as a therapeutic strategy for multiple myeloma.

Serges P. Tsofack, Aaron Botham, Zhihua Li, Ellen Nong Wei, Danielle Croucher, Ioulia Jitkova, Neil MacLean, Aaron D. Schimmer, Brian Raught, Suzanne Trudel. _Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada_.

The oncogenic transcription factor STAT3 is an appealing therapeutic target in cancer including multiple myeloma (MM) where its inhibition has the potential to downregulate aberrant signaling from various upstream molecules. STAT3 has been studied extensively as a transcription factor however, much less is known about its non-classical functions in the mitochondria and in turn, the role of mitochondrial STAT3 (mitoStat3) in cancer biology. Data suggests that mitoStat3 is critical for mediating Ras-induced oncogenic transformation. As activating mutations of the MAPK pathway are reported in over 50% of myeloma patients, we sought to characterize the function of mitoStat3 in MM.

We first confirmed the presence of STAT3 in the mitochondria of myeloma cell lines, AMO1 and XG6 by Western blot analysis. We next used CRISPR/CAS9 to knockdown STAT3 in XG6 and AMO1 cells, which resulted in inhibition of cell proliferation (XG6 and AMO1), apoptosis (XG6) and reduced oxygen consumption rate as measured by extracellular flux analysis (Seahorse assay). To better define the role of mitoStat3 and to identify indirect strategies to interfere with STAT3 activity, we employed a proximity-dependent Biotin Identification (BioID) method to discover mitoStat3 interacting proteins. BioID is a unique method to screen for physiologically relevant protein interactions that occur in living cells. First, wild-type STAT3 was fused to a mitochondrial localization Signal (MLS-Stat3), cloned into pcDNA5 FRT/TO [MCS]-BirAR118G-FLAG plasmid and transfected into HEK293 TRE-x Flp-In cells. BioID identified 225 high confidence mitoStat3-interacting partners in MLS-Stat3 transfected cells. The most abundant proteins in the mitoStat3 interactome included those with roles in mitochondrial translation and mitochondrial electron transport. Futhermore, mitoStat3 was found to interact with the translocase of inner membrane protein Tim44, which together with Tim23 complex facilitates translocation of proteins into the mitochondrial matrix. Genetic knockdown of Tim23 in XG6 cells resulted in decreased mitoStat3 suggesting that this complex may be involved in the import of STAT3 into the mitochondria. Additional mitoStat3 interacting proteins identified by BioID are currently being validated and will be presented.

Taken together, results support the further exploration of the role of mitoStat3 in myeloma cells. Further, identification of mitoStat3 interacting proteins including Tim44 and others may inform alternative strategies to therapeutically target oncogenic STAT3.

#5490

Targeting mitochondrial function in lung tumor growth and progression.

Sarada Preeta Kalainayakan,1 Poorva Ghosh,1 Sancharika Dey,1 Li Liu,2 Li Zhang1. 1 _UT Dallas, Richardson, TX;_ 2 _University of Texas Southwestern Medical Center, Richardson, TX_.

Contrary to Warburg's hypothesis, mitochondrial oxidative phosphorylation contributes significantly to fuel cancer cells. Besides oxidative phosphorylation, mitochondria are also involved in heme synthesis. Heme serves as a prosthetic group for several proteins that constitute the complexes of mitochondrial electron transport chain. Therefore, heme plays a pivotal role in mitochondrial oxidative phosphorylation. Recently, our lab demonstrated that non-small cell lung cancer (NSCLC) cells exhibit intensified mitochondrial respiration. Targeting increased mitochondrial respiration with therapeutic agents effectively hampers proliferation of NSCLC cells in vitro. Previous results from our lab also demonstrated that NSCLC cell lines exhibited elevated heme synthesis, heme uptake, and expression of hemoproteins. These results suggest that increased mitochondrial respiration is facilitated by excess intracellular heme that is incorporated into hemoproteins. Our study aims to assess whether targeting mitochondrial function and heme synthesis affects NSCLC tumor growth and progression in vivo. To this end, luciferase-expressing NSCLC cells were implanted in NOD/SCID mice to generate lung orthotopic xenografts. The mice bearing orthotopically implanted NSCLC tumors were treated with various agents that target mitochondrial function alone (MTA1 or MTA2) and in combination with heme synthesis inhibitor. Tumor growth was monitored by noninvasive bioluminescence imaging (BLI). Our BLI data demonstrated that there was a significant reduction in radiance in mice that received the mitochondria-targeting agent alone (MTA1) and a combination of mitochondrial-targeting agents (MTA1 and MTA2). BLI data also demonstrated that there was a considerable reduction in radiance in mice that received combination of MTA and heme synthesis inhibitor. Treatments with combination of MTAs and heme inhibitor are under way. Our Immunohistochemistry data indicate that there was a reduction in expression of proteins involved in heme transport as well as hemoproteins involved in mitochondrial respiration in mice treated with the mitochondria-targeting agent. Our results suggest that targeting mitochondria significantly inhibits lung tumor growth by affecting proteins involved in heme synthesis and degradation, and consequently decreasing mitochondrial respiration.

#5491

Blocking acyl-CoAs synthesis inhibits prostate cancer progression.

Houjian Cai, Yongjie Ma. _University of Georgia, Athens, Athens, GA_.

Fatty acid metabolism is essential for the biogenesis of cellular components and ATP production to sustain proliferation of cancer cells. Bioconversion of exogenous or de novo synthesized fatty acids to their corresponding fatty acyl-CoAs, which is required for the fatty acids to participate in metabolic processes, is catalyzed by long chain fatty acyl-CoA synthetases (ACSLs). However, the influence of individual ACSL isoforms on the progression of prostate cancer remains unknown. The goal of this study is to analyze how ACSLs regulates the biosynthesis of acyl-CoAs, thereby prostate cancer progression. In this study, lentivirus carrying shRNA of ACSL1 was constructed and produced in house. Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) were measured by Seahorse. The amount of protein and acyl-CoAs was analyzed by immunoblotting and LC-MS/MS, respectively. Additionally, the amount of triglyceride and neutral lipid were measured by gas Chromatography and Oil red O staining. TCGA database was also used for analysis of ACSL1 expression levels in different stages of prostate cancer. Finally, inhibition of cell proliferation, migration, invasion assays and cell cycle analysis were examined in numerous cancer cells. Prostate xenograft tumor models were applied for in vivo studies. Prostate tumor tissue array was utilized for the analysis of ACSL1 expression. Based on the systematic analysis on the correlation of ACSL expression levels and the amount of intracellular acyl-CoAs, we demonstrate that expression levels of ACSL1 were significantly associated with the levels of a variety of acyl-CoAs, and were elevated in human prostate tumors. ACSL1 increased the biosynthesis of fatty acyl-CoAs including C16:0-, C18:0-, C18:1- and C18:2-CoA, triglycerides and lipid accumulation in cancer cells. In addition, ACSL1 modulated mitochondrial respiration through regulation of carnitine palmitoyltransferase 1A (CPT1A) activity. Knockdown of ACSL1 expression inhibited the cell cycle, and suppressed the proliferation and migration of prostate cancer cells in vitro, and growth of prostate xenograft tumors in vivo. Our study demonstrates ACSL1 plays an important role in prostate tumor progression. Our results provide a therapeutic strategy of targeting fatty acid metabolism for the treatment of prostate cancer.

#5492

Inhibition of ERK MAPK signaling increases pancreatic cancer dependency on autophagy.

Kirsten Bryant,1 Sen Peng,2 Andrey Tikunov,3 Mariaelena Pierobon,4 Venugopal Gunda,5 Garima Tomar,1 Pankaj Singh,5 Emanuel Petricoin,4 Jeffrey Macdonald,3 Nhan Tran,6 Alec Kimmelman,7 Channing Der1. 1 _UNC Lineberger Comp. Cancer Ctr., Chapel Hill, NC;_ 2 _Translational Genomics Research Institute, Phoenix, AZ;_ 3 _School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 4 _School of Systems Biology, George Mason University, Fairfax, VA;_ 5 _Eppley Institute for Cancer Research, University of Nebraska, Omaha, NE;_ 6 _Mayo Clinic, Phoenix, AZ;_ 7 _Perlmutter Cancer Center, NYU Langone Medical Center, New York, NY_.

Pancreatic ductal adenocarcinoma (PDAC) cell growth is dependent on altered biochemical and cellular activities that facilitate increased metabolic dependencies. We determined the role of mutationally activated KRAS, found in ~95% of PDAC, in supporting autophagy. Surprisingly, acute KRAS suppression, which blocks proliferation, was associated with increased rather than decreased autophagic flux. Similarly, KRAS transformation of immortalized human pancreatic epithelial cells showed decreased autophagic flux. Pharmacologic inhibition of ERK MAPK phenocopied the genetic silencing of KRAS and also increased autophagic flux. Addressing a mechanism for ERK suppression increased autophagy, we found increased AMPK activity, decreased mTOR activity, and increased expression of autophagic proteins. We speculated that increased autophagy may be a compensatory mechanism caused by the loss of KRAS- and ERK-dependent metabolic processes. To address this possibility, we performed RNA-seq analyses to monitor gene expression changes in response to ERK inhibition. We observed increased transcription of genes responsible for the metabolic processes of autophagy and β-oxidation, with concurrent decreased transcription of glycolysis and mitochondrial-associated genes. These changes were validated in metabolomic analyses following either genetic silencing of KRAS or ERK inhibition. Thus, the loss of ERK-driven metabolic processes may induce compensatory mechanisms to increase autophagy. We then addressed whether ERK inhibition increased PDAC dependence on autophagy. Supporting this possibility, we found that cotreatment with the autophagy inhibitor chloroquine synergistically enhanced ERK inhibitor mediated antiproliferative activity. Similarly, genetic or pharmacologic inhibition of specific regulators of autophagy also enhanced ERK inhibitor activity. We conclude that concurrent suppression of multiple metabolic processes, to block compensatory rebound activities, will be needed for effective PDAC treatment.

#5493

Targeted inhibition of glutaminase in GRM1-expressing melanoma cells inhibits cell proliferation by reducing glutamate bioavailability.

Raj Shah,1 Mengying Zhu,1 Andrew Boreland,1 Fabian Filipp,2 Suzie Chen1. 1 _Rutgers University, Piscataway, NJ;_ 2 _University of California, Merced, CA_.

Aberrant glutamatergic signaling has been implicated in many cancer types. Our laboratory has previously illustrated the role of metabotropic glutamate receptor 1 (GRM1) in neoplastic transformation of melanocytes in vitro and spontaneous metastatic melanoma in vivo. Glutamate, the natural ligand of GRM1 is also the predominant excitatory neurotransmitter in the central nervous system. We have demonstrated significant upregulation of glutaminase (GLS) expression in GRM1 expressing melanoma cells, resulting in excess glutamate production and the establishment of autocrine loops in vitro. GLS catalyzes the first step in glutamine metabolism--the conversion of glutamine to glutamate. Comparison of glutamate levels in circulating blood plasma between melanoma prone and wild type mice showed elevated glutamate levels in melanoma prone GRM1-transgenic mice, suggesting that aberrant GRM1 expression also promotes an increase in circulating glutamate, to ensure constitutive activation of the GRM1 receptor and its downstream signaling affecters in vivo. Inclusion of CB-839, a potent, selective, and orally bioavailable inhibitor of GLS, resulted in significant inhibition of GRM1+ melanoma cell proliferation compared to GRM1\- ones. We also demonstrated that treatment with Riluzole, an inhibitor of glutamatergic signaling by decreasing the available ligand, glutamate, led to significantly reduced melanoma cell proliferation in vitro and tumor progression in vivo. We found that simultaneous targeting of glutamate production (CB-839) and release (Riluzole) in GRM1+ melanoma cells led to synergistic suppression of cell proliferation in vitro. Furthermore, we found that CB-839 altered the function not the expression of GLS in melanoma cells. Additionally, we also discovered that manipulation of GRM1 expression by genetic means led to robust parallel changes in gene expression levels and intracellular glutamate concentrations by mass spectrometry analysis, as well as the endogenous levels of GLS in melanoma cells, suggesting association between GRM1 and GLS. The sensitivity of GRM1+ melanoma cells to modulation of GLS point to the dependency of these cells to glutamate amounts via GLS activity. We are continuing to investigate the mechanisms and regulation of GLS in our experimental system, with the goal of developing a rational approach for the treatment of GRM1 expressing tumors.

#5494

Targeting deregulated expression and function of Mitofusin 1 in glioblastoma.

Maheedhara Reddy Guda, Swapna Asuthkar, Collin M. Labak, Chase P. Smith, Andrew J. Tsung, Kiran Velpula. _Univ. of Illinois College of Med. at Peoria, Peoria, IL_.

Glioblastoma (GBM) is the most common form of adult primary malignant brain tumor with a poor prognosis. Despite advances in standard treatments, such as surgery, radiation, and adjuvant chemotherapy, outcome remains poor. To further develop effective therapeutic approaches, it is essential to garner a better understanding of GBM progression. Substantial research has shown that GBM's progression is associated with metabolic remodeling, that includes the use of aerobic glycolysis as the main source of energy. Here, we report the novel role of Mitofusin 1 (MFN1), a key regulator of mitochondrial outer membrane fusion in glycolysis. We first established that MFN1 is highly expressed in GBM when compared to its isoform MFN2 using TCGA (The Cancer Genome Atlas) and OncomineTM glioma datasets. Silencing MFN1 using MFN1-shRNA in GBM10 cells showed decreased expression levels of PDK1 and HIF1-alpha as observed in western blotting and RT-PCR analysis. Interestingly, c-myc expression was significantly reduced in the shMFN1 treated GBM 10 cell. In addition, RNASeq analysis conducted on shPDK1 treated GBM-10 cells revealed reduced MFN1 levels. Further silencing of MFN1 showed increased OCR and decreased ECAR using seahorse XFp bio analyzer. Collectively, our preliminary data points to MFN1 as a candidate metabolic oncogene in GBM, and targeting MFN1 will be a novel GBM treatment.

#5495

Inhibiting ERRα blocks liver steatosis and tumorigenesis induced by PTEN loss.

Chien-Yu Chen, Yang Li, Jingyu Chen, Lina He, Bangyan Stiles. _University of Southern California, Los Angeles, CA_.

Mitochondrial dysfunction has been attributed to be a major cause for fatty liver development and also underlies tumor development. Using a liver-conditional Pten deletion model where the activation of its downstream PI3K/AKT signaling led to fatty liver, steatohepatitis, fibrosis and finally liver cancer development, we reported previously that the fatty liver and liver cancer is accompanied by elevated mitochondrial bioenergetics and a dramatic induction of estrogen-related receptor α (ERRα), a master regulator that orchestrate mitochondria response to modulate metabolism. In this project, we intended to investigate whether blocking ERRα expression and inhibiting mitochondrial respiration can attenuate tumor growth and lipid accumulation in the Pten null livers and human liver cancer cells. Using a genetic knockdown approach with siERRα and chemical genomic approach with a small molecular polyamide compound that binds to the ERRα consensus binding site, we showed that ERRα knockdown suppressed mitochondrial function and impeded cell proliferation in human liver as well as prostate cancer cell lines lacking Pten. In addition, blocking ERRα transcriptional activity with ERRα specific polyamide err-PA significantly reduced tumor growth in xenograft models. Furthermore, err-PA administration in vivo in 1.5-month old liver- specific Pten null mice remarkably prevented the development of fatty liver morphology and quantitatively reduced the hepatic triglyceride level. In summary, our study highlights ERRα's crucial role in regulating mitochondrial bioenergetics and underscores its therapeutic potential in cancer and lipid disorders.

#5496

A predictive model for selective targeting of the Warburg effect through GAPDH inhibition with a natural product.

Maria V. Liberti,1 Ziwei Dai,2 Suzanne E. Wardell,2 Joshua A. Baccile,1 Xiaojing Liu,2 Xia Gao,2 Robert Baldi,2 Mahya Mehrmohamadi,1 Marc O. Johnson,3 Neel S. Madhukar,4 Alexander Shestov,5 Iok I. C. Chio,6 Olivier Elemento,4 Jeffrey C. Rathmell,3 Frank C. Schroeder,1 Donald P. McDonnell,2 Jason W. Locasale2. 1 _Cornell University, Ithaca, NY;_ 2 _Duke University, Durham, NC;_ 3 _Vanderbilt University, Nashville, TN;_ 4 _Weill Cornell Medicine, New York, NY;_ 5 _University of Pennsylvania, Philadelphia, PA;_ 6 _Cold Spring Harbor Laboratory, Cold Spring Harbor, NY_.

Cancer cells undergo numerous adaptive processes to sustain growth and survival. One notable mechanism is by rewiring metabolism, most prominently through a phenomenon known as the Warburg effect (WE). The WE is defined by increased glucose consumption and lactate excretion in the presence or absence of oxygen. Although the WE has been extensively studied, efforts to develop successful glycolytic inhibitors have been largely unsuccessful. Targeting cancer metabolism has remained a challenge due to the lack of obvious metabolic biomarkers and difficulties achieving full enzyme inhibition without inducing toxicity in normal tissue. Although targeted cancer therapies that use genetics have been largely successful, principles for selectively targeting tumor metabolism that also depend on the environment remain unknown. In the present study, we employ metabolic control analysis to reveal that glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the sixth enzyme in glycolysis, exhibits differential control properties during the WE and can be used to predict response to targeting glucose metabolism. Using high-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC-HRMS), we conducted comparative metabolomics to establish a natural product produced by Trichoderma fungi, koningic acid (KA), as a selective inhibitor of GAPDH. We expressed a fungal-derived resistant-GAPDH allele in human cells to show that KA is highly specific for GAPDH. With machine learning, integrated pharmacogenomics, and metabolomics, we demonstrate that KA efficacy is not determined by the status of individual genes, but by the quantitative extent of the WE, leading to a therapeutic window in vivo. Thus, the basis of targeting the WE can be encoded by molecular principles that extend beyond genetic status. Current work focuses on elucidating acquired resistance mechanisms of KA in cancer cells undergoing the WE. Together, these data importantly demonstrate that a complete understanding of pharmacogenomics for cancer therapy likely requires information encoded at the metabolic level.

#5497

Effective combination therapy for breast cancer targeting BACH1 and mitochondrial metabolism.

Jiyoung Lee,1 Ali Yesilkanal,1 Casey Frankenberger,1 Mohamad Elbaz,1 Daniel Rabe,1 Jielin Yan,1 Felicia Rustandy,1 Peter Hart,2 Christie Kang,2 Elizabeth Grossman,3 Jason Locasale,4 Daniel Nomura,3 Marcelo Bonini,2 Marsha Rosner1. 1 _University of Chicago, Chicago, IL;_ 2 _University of Illinois at Chicago, Chicago, IL;_ 3 _University of California at Berkeley, Berkeley, CA;_ 4 _Duke University, Durham, NC_.

Oxidative phosphorylation is an attractive target for cancer therapy. Reprogramming metabolic pathways by promoting oxidative phosphorylation could improve the ability of metabolic inhibitors to suppress cancers with limited treatment options like triple negative breast cancer (TNBC). Here we show that BACH1, a heme-binding transcription factor whose expression is enriched in patients with TNBC, inhibits oxidative phosphorylation through direct transcriptional regulation of electron transport chain (ETC) gene expression. Treatment of cells with hemin, which induces BACH1 degradation, mimics BACH1 depletion with shRNA. Pretreatment of TNBC tumors with BACH1 shRNA or hemin overcame resistance to metformin, an anti-diabetic drug, and abolished the growth of both cell line and patient-derived tumor xenografts. BACH1 gene expression inversely correlated with ETC gene expression in breast cancer patients as well as other tumor types, highlighting the clinical relevance. This study demonstrates that oxidative phosphorylation represents an Achilles heel that can be exploited through targeting BACH1 to sensitize breast cancer and potentially other tumor tissues to mitochondrial inhibitors.

### Tumor Suppressor Genes 2

#5498

Natural product-derived carbon nanodots overcome drug resistance through regulation of Hippo pathway effectors.

Ayan A. Nurkesh,1 Mannix P. Balanay,1 Tleubek Yeleusizov,1 Darkhan Tursynkhan,1 Qing Yang,1 Xiaoling Wan,2 Limara Manarbek,1 Aisulu Maipas,1 Bakhyt Matkarimov,3 Yan Zhang,2 Haiyan Fan,1 Li-Xia Miao,4 Si-Pian Li,4 Zhenbang Chen,5 Yingqiu Xie1. 1 _School of Science and Technology, Nazarbayev University, Astana, Kazakhstan;_ 2 _Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China;_ 3 _National laboratory of Astana, Nazarbayev University, Astana, Kazakhstan;_ 4 _School of Basic Medical Sciences, Wuhan University, Wuhan, China;_ 5 _Meharry Medical College, Nashville, TN_.

The yes-associated protein 1 (YAP) is a nuclear downstream effector of Hippo pathway that plays a critical role in various cancers. Literature shows that, in addition to nucleus, YAP also localizes in cytosol and membrane of cells. We and our collaborators previously reported that elevation of YAP associates with Arf in the genetically-engineered mouse (GEM) model of Pten/Trp53, implicating the possible correlation between Hippo/YAP and ARF in human cancers. We recently found ARF is indeed involved in dysregulation of Hippo pathway in prostate cancer. In details, ARF inhibited nuclear YAP translocation in cancer cells, and ARF knockdown suppressed the stability of YAP protein with upregulated active AKT and dysregulated phosphorylation of YAP through ARF/AKT/mTOR/YAP pathway. Here we further report that carbon nanodots (c-dots) can induce DNA damage through an increase of ROS by upregulation of γH2AX and DNA tails in PC3 and DU145 prostate cancer cells. We found that c-dots derived from natural products such as tea or beet can enter into cancer cell nucleus to interact with ARF for the regulation of the ARF-mediated signaling such as the nuclear translocation of YAP. Mechanistically, c-dots bind ARF to induce cell cycle arrest, apoptosis, and PARP cleavage in prostate cancer cells. Most importantly, addition of c-dots into cultured PC3 cells leads to the reversal of drug resistance to mTOR inhibitor Rapamycin and MET inhibitor Crizotinib by enhancing DNA damage. Moreover, c-dots suppress the tumor size and growth of PC3 cells in xenograft mouse model in vivo. Our data suggest that the nano-scale of c-dots derived from natural food may be efficient in overcoming drug resistance via augmentation of DNA damage linked cell death. Thus we reported a new approach of abolishing drug resistance of aggressive malignancy using nanoparticles, implicating an innovative avenue for precision cancer treatment.

#5499

**A potent BH3 mimetic targeting BCL-X** L **induces apoptosis regulated by PTEN loss and integrin alpha 5 in prostate cancer cells.**

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

Background: PTEN-loss is a highly prevalent genomic event associated with higher risk of metastatic progression and resistance to hormonal therapy in prostate cancer. Akt inhibitor therapy in combination with abiraterone demonstrates early promise in the clinical control of PTEN-deficient prostate cancers. We have previously implicated BCL-XL in the survival of PTEN-null PC-3 prostate cancer cells in vitro, regulated by integrin alpha 5 (ITGA5) (Ren, MCR 2016). Toward advancing therapeutic strategies in PTEN-deficient prostate cancer we further assessed the link between PTEN and BCL-XL mediated prostate cancer cell survival.

Methods: Isogenic PTEN knockdown stable cell lines (VCAP and DU145) were generated by lentiviral transduction of pLKO-control-shRNA and two pLKO-PTEN-shRNAs, respectively. PTEN was exogenously expressed in PTEN-null prostate cancer cell lines (PC-3, LNCaP and C4-2B) by 1) transfecting cells with pCMV-Flag-vector or pCMV-Flag-PTEN 2) transfecting cells with pcDNA3-GFP-PTEN and then sorting GFP-PTEN-negative and GFP-PTEN-positive cells by flow cytometry. Cells were then treated with a single agent BCL-XL inhibitor (A1331852 or navitoclax (ABT263); AbbVie), PI3K (buparlisib; Selleck Chem) or Akt (ipatasertib, Selleck Chem) inhibitors or combined PI3K and BCL-2 family inhibitors. Cell survival was assessed by cell viability assay. Expression of pAkt, ITGA5 and apoptotic markers was assayed by Western blot analysis and/or flow cytometry.

Results: Notwithstanding the reduction of phospho-bad expression with PI3K inhibition, the potent BCL-XL-targeting BH3 mimetic, A1331852, compared to single agent PI3K or Akt inhibitors in titrated doses was alone sufficient to induce apoptosis and loss of cell viability in all PTEN-null prostate cancer lines tested. PTEN expression reversed the apoptotic response to BCL-XL targeting and blocked the anti-proliferative effect of PI3K or Akt inhibition in PTEN-mutant cells. PTEN knockdown induced membrane expression of ITGA5 whereas PTEN overexpression diminished ITGA5 expression.

Conclusions: PTEN regulates the apoptotic threshold in prostate cancer cells via BCL-XL independent of the PI3K/Akt pathway. The induction of ITGA5 membrane localization by PTEN loss defines a potential upstream signaling mechanism for the BCL-XL regulated apoptotic threshold in these cells. Highly potent BCL-XL inhibition requires further investigation as a therapeutic strategy in PTEN-deficient prostate cancer.

#5500

Nitric oxide donor DETA/NO inhibits the growth of endometrial cancer cells by upregulating expression of RASSF1 and CDKN1.

Hannah Taitz,1 Ana Paucarmayta,1 Robert Cheng,2 George L. Maxwell,3 Chad A. Hamilton,1 David A. Wink,2 Viqar Syed1. 1 _Uniformed Services Univ. of the Health Sci., Bethesda, MD;_ 2 _National Cancer Institute, National Institutes of Health, Frederick, MD;_ 3 _Inova Fairfax Hospital, Falls Church, VA_.

Nitric oxide (NO), a small signaling molecule, is implicated in several biological processes including cancer progression. At low concentrations, it promotes cell survival and tumor progression, and at high concentrations it causes apoptosis and cell death. To date, the effects of NO donor on human endometrial cancer are largely unknown. Four endometrial cancer cell lines (Ishikawa, AN3CA, KLE and HEC-1B) with varying degrees of genetic complexity were treated with different concentrations (50-500 μM) of DETA/NO for 24 to 120 h. The effects of treatment on cell viability and invasion was determined using MTS, and Boyden chamber assays respectively. Treatment of endometrial cancer cells with DETA/NO induced a dose and time-dependent decrease in cell viability. Ishikawa and AN3CA cells were more responsive to DETA/NO induced growth inhibition compared to HEC-1B and KLE cells. Furthermore, DETA/NO effectively inhibited invasive potential of endometrial cancer cells. To understand the mechanisms by which DETA/NO elicits anti-cancer effects, RNA sequencing (RNA-seq) was used to ascertain alterations in the transcriptomes of human endometrial cancer cells. AN3CA, KLE, Ishikawa, and HEC-1B were treated with DETA/NO for 24 h and RNA was extracted. RNA-seq analysis revealed that of the twenty-one top differentially expressed genes, fourteen were upregulated and seven were downregulated in endometrial cancer cells with DETA/NO. The genes that were upregulated in all four cell lines with DETA/NO were tumor suppressors, RASSF1 and CDKN1A. The expression patterns of these genes were confirmed by Western blotting. Taken together, the results provide the first evidence in support of the anti-cancer effects of DETA/NO in endometrial cancer.

#5501

The role of G-protein coupled receptor 64 (GPR64) as a tumor suppressor in endometrial cancer.

Jung-Yoon Yoo,1 Jong Il Ahn,2 Tae Hoon Kim,3 Russell R. Broaddus,4 Jeong Mook Lim,2 Ho-Geun Yoon,1 Jae-Wook Jeong3. 1 _Yonsei University, Seoul, Republic of Korea;_ 2 _Seoul National University, Seoul, Republic of Korea;_ 3 _Michigan State University, Grand Rapids, MI;_ 4 _University of Texas M.D. Anderson Cancer Center, Houston, TX_.

Endometrial cancer is the most common gynecologic cancer. G-protein coupled receptor 64 (GPR64) belongs to a family of adhesion GPCRs and plays an important role for male fertility. However, GPR64 function has not been reported in endometrial cancer. Our objective is to investigate the role of GPR64 in endometrial cancer. We examined the levels of GPR64 in human endometrial cancer tissue by immunohistochemistry analysis. GPR64 levels were significantly lower in 10 of 21 (47.62%) of endometrial carcinoma samples compared to control. To test whether GPR64 has a role of tumor suppressor in endometrial cancer, we used an siRNA loss-of-function approach in human endometrial adenocarcinoma cell lines. Depletion of GPR64 by siRNA transfection in Ishikawa and HEC1A cells showed an increase of colony formation ability and cell proliferation. Depletion of GPR64 also significantly increased the migration and invasion activity of Ishikawa and HEC1A cells. Furthermore, the phosphorylation of AMP-activated protein kinase (AMPK) and expression of Connexin 43 (Cx43), a member of the large family of gap junction proteins, were regulated in GPR64-deficient Ishikawa cells. These results suggest that GPR64 plays an important role for cell proliferation, migration, and invasion as a tumor suppressor in endometrial cancer.

#5502

**Characterization of a hotspot** PTEN **mutation in endometrial cancer.**

Ye Wang,1 Charles Jakubik,2 Rachel Peterson,2 Cyril Benes,2 Wilhelm Haas,2 Jeffrey Engelman,1 Carlotta Costa1. 1 _Novartis Institutes for BioMedical Research, Cambridge, MA;_ 2 _Massachusetts General Hospital Cancer Center, Boston, MA_.

Endometrial cancer is the most common gynecologic malignancy in the developed world. These cancers harbor the highest rates of PI3K pathway alterations reported, with PTEN being the most frequently altered gene. Differently from other cancer types, in endometrial cancer a specific hotspot mutation in the lipid phosphatase domain of PTEN is present at residue 130. This hotspot mutation significantly co-occurs with other mutations in the PI3K pathway, such as PIK3CA and PIK3R1. Interestingly, in the TCGA data we also found a positive correlation between this mutation and the expression level of the estrogen receptor alpha. Although it has been reported that the PTEN-R130 mutant loses its lipid phosphatase activity in vitro, little is known regarding the other unconventional functions of PTEN, such as its protein phosphatase activity. We performed unbiased phospho-proteomics experiments in order to explore the impact of this specific mutation in multiple endometrial cancer cell models.

#5503

Loss of MED12 induces tumor dormancy in human ovarian cancer.

Xiao-Lin Luo, Cheng-Cheng Deng, Ji-Hong Liu, Li-Wu Fu. _Sun Yat-Sen Univ. Cancer Ctr., Guangzhou, China_.

Epithelial ovarian cancer (EOC) is the leading cause of death among all gynecological malignancies due to high rate of disease relapse. Disease relapse in cancer patients after clinical remission are often referred to tumor dormancy. Here we identify MED12 as an important molecular regulator of tumor dormancy. We found that MED12 knock-out could induce dormancy of EOC cells in vitro and in vivo. Mechanistically, microarray analysis showed that MED12 knock-out decreased the expression of EGFR. Restoration of EGFR expression in MED12 knock-out cells restored the proliferation ability. Additionally, we found that MED12 could bind to the promoter of EGFR, and correlation studies showed that MED12 expression positively correlated with the level of EGFR. Clinical data demonstrated that chemotherapy-resistant patients showed a lower expression of MED12. In conclusion, MED12 plays an important role in regulating dormancy of EOC through regulation of EGFR.

#5504

TRAF3 acts as a tumor suppressor through modulation of TP53 and RB in human papillomavirus associated head and neck squamous cell carcinomas.

Tony Wayne Chen, Jialing Zhang, Xinping Yang, Zhong Chen, Carter Van Waes. _NIDCD/NIH, Bethesda, MD_.

The number of human papillomavirus-associated (HPV+) head and neck squamous cell carcinoma (HNSCC) cases has increased sharply in recent years, especially among tonsillar and oropharyngeal cancers. The Cancer Genome Atlas (TCGA) network identified novel loss-of-function genomic alterations of TNF receptor-associated factor 3 (TRAF3) in a subset of HPV+ HNSCC specimens. Previously, most studies of TRAF3 function are from the immune system and lymphoid malignancies, where TRAF3 has been implicated as a ring-finger E3 ubiquitin ligase that promotes anti-viral immunity, while inhibiting alternative NF-κB signaling by which DNA viruses can transform lymphoid cells. However, the function of TRAF3 and the mechanisms by which TRAF3 deficiency contributes to tumorigenesis are largely unknown. To explore the potential mechanisms of TRAF3 defects observed in the tumor subset identified by TCGA, we surveyed a panel of HPV+ HNSCC cell lines, and identified lines with lower steady-state protein levels of TRAF3 than normal oral keratinocytes. Functional studies show that TRAF3 expression leads to suppression of oncogenic phenotypes, including decreased alternative NF-κB signaling, cell proliferation, colony formation, and cell migration. Additionally, restoring TRAF3 increased anti-viral cytokine interferon alpha (IFNA) expression, and sensitivity to chemotherapy agent cisplatin. The anti-proliferative activity of TRAF3 was partially mediated by restoration of expression of classical tumor suppressor genes, TP53 and RB, which are degraded in HPV+ cancers. TRAF3 re-expression increased the steady-state protein levels of TP53 and gene expression of TP53AIP, which mediates cell cycle arrest and apoptosis. Knockdown of TP53 in TRAF3 transfected cell lines partially reversed the slowed cell growth rate, supporting the contribution of TP53 to TRAF3 mediated growth inhibition. TRAF3 expression also increased RB protein expression and decreased RB regulated transcription factor E2F1, which regulates cellular genes involved in cell proliferation and DNA synthesis. In conclusion, we have revealed a novel role whereby TRAF3 loss leads to enhanced HPV mediated repression of the TP53 and RB tumor suppressors that control cell growth and cell death in HPV+ HNSCCs.

(Supported by NIDCD intramural project ZIA-DC-000016, 73 and 74)

#5505

GAN gene exon 8 SNP is related to gigaxonin expression and increased expression of e-cadherin in head and neck cancer.

Albert Ko,1 Natarajan Venkatesan,2 Jenna Chatoff,2 Marco Morselli,1 Gregory Fishbein,1 Pascale Bomont,3 Matteo Pellegrini,1 Marilene Wang,1 Eri Srivatsan2. 1 _UCLA David Geffen School of Medicine, Los Angeles, CA;_ 2 _VA Greater Los Angeles Healthcare System, Los Angeles, CA;_ 3 _Institute for Neurosciences Montpellier, Montpellier, France_.

Gigaxonin is the protein product of Giant Axonal Neuropathy (GAN) gene and is involved in the processing of intermediate filaments of neural cells and vimentin fibers in fibroblasts. We have shown earlier that gigaxonin is associated with p16 in NFkB ubiquitination of cisplatin sensitive tumor cells. Our studies have shown that there is a direct relationship between the presence of exon 8 SNP (C>T) and expression of giagxonin in cancer cell lines. We have also seen an inverse relationship between exon 8 SNP and tumor recurrence and metastasis. RNA-seq studies of cancer cell lines and primary tumors have shown a direct relationship in the expression of gigaxonin to the expression of e-cadherin and inverse relationship to the expression of Vimentin and Zeb1. Immunohistochemical (IHC) studies of primary head and neck cancers have confirmed the inverse relationship between e-cadherin and vimentin. We hypothesize therefore that gigaxonin is involved in the ubiquitination of vimentin and cancer stem cell markers such as zeb1 resulting in the chemo sensitization of human tumor cells.

#5506

NOTCH1 activation in head and neck squamous cell carcinoma leads to growth inhibition, changes in gene expression associated with early differentiation, and acquisition of stem cell-like properties.

Chenfei Huang,1 Shhyam Moorthy,2 Qiuli Li,2 Rami Saade,2 Jiping Wang,2 Xiayu Rao,2 Noriaki Tanaka,2 Jiexin Zhang,2 Lin Tang,2 Curtis R. Pickering,2 Patrick A. Zweidler-McKay,2 Abdullah A. Osman,2 Tong-Xin Xie,2 Eve Shinbrot,1 Liu Xi,1 David Wheeler,1 Adel K. El-Naggar,2 Jing Wang,2 Jeffrey N. Myers,2 Mitchell J. Frederick1. 1 _Baylor College of Medcine, Houston, TX;_ 2 _University of Texas MD Anderson Cancer Center, Houston, TX_.

Background: The NOTCH1 gene functions as either an oncogene or tumor suppressor in cancer depending upon the tumor type. Our group previously characterized the genomic alterations in head and neck squamous cell carcinoma (HNSCC), discovering that NOTCH1 is frequently altered with a pattern of inactivating mutations suggesting it is a tumor suppressor in this cancer type. However, recent work by others suggests NOTCH1 signaling plays a more complex role, possibly promoting a more aggressive phenotype or cancer stem cell-like properties in HNSCCs with wild type NOTCH1. Our present study aimed to systematically compare the phenotypic consequences of NOTCH1 signaling in HNSCC to better understand its function in cancer, and identify targets downstream of NOTCH1 signaling. Methods: Established HNSCC cell lines wild type for NOTCH1 (PJ34, FADU) or harboring an inactivating mutation (UMSCC22A) were engineered to express activated cleaved NOTCH1 (cl-NOTCH1) from a doxycycline-inducible promoter. In vitro cell growth was measured with clonogenic assays. Stem cell-like properties were measured by orosphere formation and anoikis resistance. Stem cell markers for HNSCC including Aldehyde dehydrogenase activity (ALDH), CD133, and CD44 expression were measured by flow cytometry. NOTCH1-regulated downstream gene expression changes were examined by RNA-seq and qRT-PCR. Results: Activation of NOTCH1 inhibited clonogenic growth of all three cell lines, regardless of original NOTCH1 gene status. Growth inhibition was frequently accompanied by spontaneous formation of spheroid-like structures, characteristic of stem cells. NOTCH1 activation in UMSCC22A and FADU cells promoted orosphere formation and anoikis resistance, conveying some stem cell-like properties. However, classical stem cell markers including ALDH activity, CD133, and CD44 expression were not affected by NOTCH1 activation. Furthermore, RNA-seq demonstrated that critical cancer-associated pathways, including proliferation, differentiation, and migration, were regulated by NOTCH1. NOTCH1 activation downregulated gene expression of ITGA3, ITGA4, ITGB1, ITGB6, and LAMC2, which are key adhesion molecules that human basal keratinocytes use for attachment to the basement membrane and maintenance of the stem cell compartment. Concomitantly, NOTCH1 activation increased the basal/superbasal marker SOX2, but also the early differentiation markers KRT4 and KRT13. SiRNA-mediated SOX2 silencing blocked NOTCH1-promoted anoikis resistance. Conclusion: NOTCH1 activation inhibits in vitro growth regardless of mutational status. We hypothesize that stem cell-like properties associated with NOTCH1 activation in HNSCC may be a consequence of pathways that recapitulate early differentiation, rather than true stem cell maintenance.

#5507

RAG1 **high expression associated with** IKZF1 **dysfunction in adult B-cell acute lymphoblastic leukemia.**

zheng Ge,1 Qi Han,1 Jinlong Ma,1 Yan Gu,1 Huihui Song,1 Malika Kapadia,2 Sinisa Dovat,2 Chunhua Song2. 1 _Zhongda Hospital, Medical School of Southeast University, Nanjing, China;_ 2 _Penn State Health Milton S. Hershey Medical Center, Hershey, PA_.

The recombination activating gene (RAG)-mediated recombination is the dominant mutational process and the predominant driver of oncogenic genomic rearrangement in acute lymphoblastic leukemia (ALL). This then leads to further leukemic clonal evolution. IKZF1 encodes a kruppel-like zinc finger protein, IKAROS that is essential for normal hematopoiesis and acts as a tumor suppressor in ALL. The genetic defects of a single IKZF1 allele are linked to the development of human ALL, characterized by an increased risk of relapses and poorer prognosis. We observed that RAG1 is significantly increased in subsets of B-ALL patients. High RAG1 expression correlates with high proliferation markers. IKAROS directly binds to the RAG1 promoter in B-ALL cells by quantitative chromatin-immunoprecipitation assay. IKAROS suppresses RAG1 promoter activity by luciferase reporter assay. Lentiviral IKAROS expression significantly suppresses RAG1 expression, but IKAROS shRNA promotes RAG1 expression in B-ALL cells. CK2 inhibitor by restoring IKAROS activity, significantly suppresses RAG1 expression in an IKAROS-dependent manner in B-ALL cells. RAG1 expression is significantly higher in patients with IKZF1 deletion, as compared to patients without IKZF1 deletion. Treatment with CK2 inhibitor also results in an increase in IKZF1 binding to the RAG1 promoter and suppression of RAG1 expression in primary B-ALL cells. Taken together, our results demonstrate that high expression of RAG1 correlates with high proliferation markers in B-ALL, and are the first to demonstrate that IKAROS directly suppresses RAG1 expression. Our data suggest RAG1 high expression works together with IKAROS dysfunction to drive oncogenesis of B-ALL, which have significance in an integrated prognostic model for adult ALL.

#5508

ANKRD52 inhibited tumor metastasis in lung adenocarcinoma.

Ting-Fang Lee, Yen-Fan Lin, Ying-Pu Liu, Cheng-Wen Wu. _National Yang-Ming Univ., Taipei, Taiwan_.

Cancer metastasis is an ultimate challenge in our effort to fight cancer as a life-threatening disease. A member of ankyrin repeat proteins, ankyrin repeat domain 52 (ANKRD52), is studied here to be involved in the regulation of lung adenocarcinoma metastasis. Ankyrin repeat proteins are known to function as a platform for protein-protein interactions, and ANKRD52 was shown to interact with protein phosphatase 6 (PP6). Immunohistochemistry staining in lung adenocarcinomas tissues revealed low ANKRD52 expressing in stage-IV patients compared to those in stages I to III. Overexpressed ANKRD52 prevented lung cancer formation in the xenograft model. Using lung cancer cell lines, ANKRD52 knockdowns showed significantly increased cell mobility while forced expression decreased cell mobility. Using mass spectrometry analysis, p21-activated kinases (PAK1) was identified to interact with ANKRD52. The regulation of PAK1 phosphorylation by the ANKRD52-PP6 complex is essential to cell mobility. Our model demonstrated a novel anti-metastatic protein, ANKRD52, that regulates cell mobility through interacting with PP6 leading to PAK1 dephosphorylation.

#5509

**Early suppression of the** TBX2 **subfamily of transcription factors during in vivo tobacco carcinogen-mediated lung adenocarcinoma development.**

Athar Khalil,1 Junya Fujimoto,2 Tina McDowell,2 Paul Scheet,2 Georges Nemer,1 Humam Kadara1. 1 _American University of Beirut, Beirut, Lebanon;_ 2 _The University of Texas MD Anderson Cancer, Houston, TX_.

Targets for early treatment of lung adenocarcinoma (LUAD), the leading cause of cancer mortality among smokers, are far and few largely due to the paucity in our knowledge of the molecular pathogenesis of the malignancy. In our efforts to address this glaring gap in knowledge, we previously surveyed the expression of members of the T-box 2 subfamily of transcription factors (TBXs 2, 3, 4 and 5) in normal and malignant lung tissues owing to their evolutionarily conserved expression and function in normal lung organogenesis,. We found that all four members were profoundly suppressed in human premalignant lesions and malignant LUADs. While these findings showed that the TBX2 subfamily of transcription factors is suppressed in overt LUADs, their role in the molecular and temporal oncogenesis of the malignancy in smokers is still elusive. To fill this void, we first surveyed the expression of these genes in a mouse model of LUAD we had developed comprised of knockout of the airway-lineage specific gene Gprc5a (Gprc5a-/-) and exposure to the tobacco-specific carcinogen nicotine-derived nitrosamine ketone (NNK). We had previously found that NNK-exposed Gprc5a-/- mice developed adenomas and LUADs that harbored somatic activating mutations (p.G12D and p.Q61R) in the Kras oncogene -- the same variants posited to function as drivers of LUAD in human smokers. In ongoing RNA-Seq efforts cataloging expression in frozen-fixed temporally collected (up to seven months post-NNK) Gprc5a-/- normal tissues, adenomas and LUADs, we found that all four TBX genes were markedly down-regulated (all P < 0.05) in all lesions and across all time points following exposure, relative to normal lung tissues. Concurrent analysis of canonical genes directly activated or repressed by the TBX2 subfamily were found to be consistently down-regulated or elevated, respectively, in the same NNK-exposed lung lesions relative to normal lung. We employed a mouse LUAD cell line (mF471 cells) we had previously derived from an NNK-exposed Gprc5a-/- mouse tumor to further functionally probe the role of TBX2 subfamily in LUAD oncogenesis. Transient over-expression of each of the four TBX2 subfamily members in the mF471 LUAD cells significantly inhibited cell proliferation and growth evidenced by trypan blue dye exclusion, MTT and cell cycle analyses. Over-expression of each of the four TBX2 subfamily members yielded similar anti-growth effects with significant increases in sub-G0 phase of the cell cycle indicating cell cycle arrest (up to 35%) 48 hours following transfection. These findings suggest that the TBX2 subfamily genes are suppressed at early stages in the development of smoking-associated Kras-mutant LUAD and play key tumor suppressor roles in the molecular pathogenesis of this common and fatal malignancy.

#5510

Loss of epithelial membrane protein 2 enhances Kras-driven lung tumorigenesis via FAK/AKT pathways.

Ji Yun Jang,1 Mi Kyung Park,1 Chang Hoon Lee,2 Ho Lee1. 1 _National Cancer Center Korea, Goyang-si, Republic of Korea;_ 2 _College of Pharmacy, Dongguk University, Goyang-si, Republic of Korea_.

Epithelial membrane protein-2 (EMP2) belongs to the growth arrest specific-3 (GAS3)/PMP22 family of tetraspan proteins. These family members are involved in the pathogenesis of human diseases, including malignancy. EMP2 is highly expressed in normal lung alveoli; however, its functional role in lung tumor is largely unknown. In this study, we focus a novel function for EMP2 in lung tumor progression. To investigate this possibility, EMP2 knock-out mice were crossed with KrasG12Dmice that carry an oncogenic mutation of the Kras gene (G12D) and develop lung adenocarcinoma. Incidence of lung tumor in KrasG12DEMP2-/- mice was higher than that in KrasG12DEMP2+/+ mice. We also found that loss of EMP2 accelerated lung tumor progression. Then, we investigated the role of EMP2 in lung cancer cell line. Expression of EMP2 was knocked out by CRISPR/Cas9 editing system in H1299 (KrasWT), H358 (KrasMut) and A549 cells (KrasMut). Loss of EMP2 induced that expression of integrin α5 is increased but expression of integrin α6 is decreased. Alteration of integrin expression activated FAK/AKT pathway. These results support the role of EMP2 loss in the control of lung cancer progression with oncogenic Kras. Taken together, these findings establish that EMP2 is a potent suppressor of Kras-driven lung tumorigenesis via FAK/AKT pathway.

#5511

**Characterization of molecular and functional consequences of somatic** NF1 **mutations in non-small cell lung cancers.**

Camille Tlemsani,1 Nicolas Pecuchet,2 Aurélia Gruber,1 Audrey Mansuet-Lupo,1 Diane Damotte,1 Marco Alifano,1 Jennifer Varin,1 Ingrid Laurendeau,1 Armelle Luscan,1 Benoit Rousseau,3 Beatrice Parfait,1 Ivan Bieche,4 Audrey Briand,1 Benoit Terris,1 Helene Blons,2 Karen Leroy,1 Dominique Vidaud,1 Michel Vidaud,1 Pierre Laurent-Puig,2 Eric Pasmant1. 1 _Cochin Hospital, Paris, France;_ 2 _HEGP Hospital, Paris, France;_ 3 _Mondor Hospital, Paris, France;_ 4 _Curie Institute, Paris, France_.

Driver molecular alterations are found in >20% of non-small cell lung cancers (NSCLCs). They specifically target the RAS-MAPK pathway, including the EGFR, KRAS, and BRAF oncogenes. NF1 is a tumor suppressor gene that encodes neurofibromin, an inhibitor of the RAS-MAPK pathway. NF1 mutation detection is challenging owing to its large size, the presence of numerous pseudogenes, and the absence of mutation hotspot. According to The Cancer Genome Atlas data (TCGA), NF1 somatic mutations are found in ~15% of lung cancer. However, NF1 mutations in NSCLCs are not extensively explored in NSCLC to date. We hypothesized that NF1 alterations could define a specific NSCLC population with distinct clinical and molecular profiles. We performed NF1 analysis using next-generation sequencing in NSCLC surgical specimens with known KRAS, EGFR, TP53, BRAF, HER2, and PIK3CA status. We evaluated the specificities of NF1-mutated NSCLCs. Then, we established of NF1-mutated cellular models with different NF1 wild-type (WT) cell lines. We chose two NSCLC cell lines (A549 and NCI H-1703, ATCC), and one nontumorigenic human bronchial epithelial cell line (HBE4-E6/E7-C1, ATCC). Mono- and biallelic NF1 mutations were established using CRISPR-Cas9 and nickase CRISPR-Cas9 technologies. In vitro functional tests and drug screening were performed using these isogenic cell models. In our series of 138 lung adenocarcinoma specimens, 25 tumors showed NF1 mutations (18%) and 11 showed NF1 deletions (8%). NF1 mutations were rarely associated with other mutations. Most of patients with NF1 alterations were males (72%) and smokers (75%). Overall survival and disease-free survival were statistically better in patients with NF1 alteration patients (N=35) than in KRAS mutated patients (N=30) in univariate analysis. There were more NF1 mutations in patients treated by neoadjuvant chemotherapy (p = 0.01). Then, we established cellular models of NF1-mutated NSCLC, using nickase and CRISPR-Cas9 technology. In HBE4-E6/E7-C1 cells, mono- and biallelic NF1 mutations were generated. Loss of NF1 expression was confirmed by Western blot: partial and total loss-of-expression of neurofibromin was found in monoallelic and biallelic NF1 mutated cell lines, respectively. Using Western blot, we showed that pERK/ERK ratio was higher in NF1-mutated cell lines versus WT cell lines, confirming that NF1 loss-of-function triggered RAS-MAPK pathway activation. Transcriptome analysis confirmed this activation. Pharmacologic screen (including MEK inhibitors) in this isogenic NSCLC model will enable us to assess specific vulnerabilities due to NF1 mono- or biallelic mutations. Our results confirm that NF1 is frequently mutated and represents a distinct subtype of NSCLCs. A better comprehension of functional consequences of NF1 mutations, including mono- and biallelic alterations, may open new avenues for NSCLC therapy.

#5512

Dual inhibition of AXL and FN14 reverses cisplatin resistance in non-small cell lung carcinoma by inducing higher caspase 3 cleavage.

Soumavo Mukherjee, Dhananjay Suresh, Ajit P. Zambre, Anandhi Upendran, Raghuraman Kannan. _Univ. of Missouri, Columbia, MO_.

Background and Objectives: NSCLC patients undergoing Cisplatin therapy often develop resistance within 9-12 months that decreases treatment efficacy and is the major reason for the low median survival of 10-14 months among stage IV patients. Therefore, resistance is the main therapeutic limitation in the later part of the progressive disease. It has been suggested that suppressed Caspase-3 is one of the major reasons for failure to induce apoptosis by the cell during cisplatin resistance. We hypothesized this resistance to be related to EMT and other survival pathways. Recently, AXL and TWEAK/FN14 pathways have been shown to be upregulated in cisplatin resistant cells causing activation of EMT and survival pathways. Therefore, we examined whether dual inhibition of AXL and TWEAK/FN14 successfully reverses the cisplatin resistance in NSCLC.

Materials and Methods: Drug resistant EGFR mutant H1975 and KRAS mutant A549 cells were tested for cisplatin desensitization following knock down of AXL and FN14 inhibition. We studied resensitization by determining the IC50 values in the drug resistant cell lines by treatment with both AXL and FN14 siRNA. We investigated detailed cellular mechanisms imparting resistance by detecting relative levels of Caspase 3, cleaved Caspase 3, PARP, cleaved PARP, p-SRC, p-EGFR, AXL and FN14 by western blotting. Cytochrome-C induced apoptosis was detected by monitoring cytochrome-c released from mitochondria using fluorescence microscopy.

Results: Suppression of AXL and FN14 by siRNA treatment resensitized NSCLC to cisplatin and induced apoptosis. IC50 values were decreased significantly following dual inhibition (by more than 200%). A marked increase in cleaved caspase 3 levels was also observed in both mutant cell lines. A subsequent increase in PARP and cleaved PARP levels verified the fact that caspase 3 is indeed responsible for the apoptosis in these cells. Upregulation of p-SRC and eventually p-EGFR failed to suppress the apoptosis through PI3K/AKT pathway. Cytochrome c release from mitochondria further confirmed cellular apoptosis.

Conclusion: Our results show that dual inhibition of AXL and TWEAK/FN14 can reverse cisplatin resistance by inducing higher caspase 3 cleavage that can enhance survival of NSCLC patients undergoing chemotherapy.

#5513

Role of the evolutionarily conserved TBX2 subfamily of transcription factors in the molecular pathogenesis of human lung adenocarcinoma.

Athar Khalil,1 Nehme El-Hachem,2 Batoul Dekmak,1 Humam Kadara,1 Georges Nemer1. 1 _American University of Beirut, Beirut, Lebanon;_ 2 _Institut de Recherches Cliniques de Montréal, Quebec, Canada_.

T-box (TBX) transcription factors are evolutionary conserved genes and master regulators of transcription repression and activation. In mammals, 18 members were described functionally and structurally, of which the TBX2 subfamily (TBX2, TBX3, TBX4, TBX5) genes were shown to be expressed early on in the developing lung bud and tracheae. Despite these insights into the role of the TBX2 subfamily in normal lung organogenesis, little is known about the role of these genes in pathological pulmonary conditions in humans; particularly lung cancer, an aggressive malignancy that is the leading cause of cancer-deaths worldwide. To fill this void, our group previously surveyed the expression of TBX2 subfamily in various publicly available datasets and found that all four members were preferentially and highly expressed in human normal lung, but markedly and consistently suppressed in lung adenocarcinoma (LUAD) the most common histological subtype of lung cancer. We also showed that the subfamily was also suppressed in preneoplastic lesions preceding the development of LUADs. Following the above and to further elucidate the role of the TBX2 subfamily in LUAD pathogenesis, we first probed and confirmed abundant expression of protein products of the four members by immunostaining in adult human normal lung tissues. On the other hand, quantitative real-time PCR and western blotting analyses demonstrated overall suppressed expression of the genes and corresponding proteins in a panel of human LUAD cell lines. Transient over-expression of each of the four genes in human LUAD cell lines (H1299 and H1944) was found to overall significantly inhibit cancer cell growth and proliferation. Additionally, over-expression of the four genes induced apoptosis, evidenced by sub-G0/G1 accumulation following cell cycle analysis, in both cell lines (ranging from 40% to 90% compared to control). To understand genome-wide effects of TBX2 subfamily in LUAD, we interrogated global expression programs downstream of these transcription factors by RNA-Seq in H1299 cells engineered to over-express the four members separately. We unraveled novel signaling cues signifying canonical pathways found in our analysis to be directly regulated by members of the TBX2 subfamily. These included, among others, inhibition of cell cycle progression and glycolysis, suppression of pathways mediated by epidermal growth factor (EGFR) and WNT signaling and activation of the major anti-tumor immune marker interferon gamma (IFNG). All in all, our findings point to tumor suppressor roles for TBX2 subfamily in human LUAD pathogenesis and suggest "oncophenotypes" downstream of these factors as putative targets for lung cancer therapy.

#5514

Loss of hepatocyte growth factor activator inhibitor type-1 (HAI-1) in human lung adenocarcinomas promotes RON receptor phosphorylation and increased sensitivity to crizotinib.

Austin J. McHenry,1 Gautam Sondarva,2 Vijayalakshmi Ananthanarayanan,1 Ashley Hess,1 Patricia E. Simms,1 Alhareth Alsayed,3 Hiroaki Kataoka,4 Jan Marusarz,5 Sandeep Kumar,2 Ravi Salgia,6 Arkadiusz Dudek,7 Stanley Borowicz,1 Ajay Rana2. 1 _Loyola University Chicago, Maywood, IL;_ 2 _University of Illinois at Chicago, Chicago, IL;_ 3 _Rosalind Franklin University, North Chicago, IL;_ 4 _, University of Miyazaki, Miyazaki, Japan;_ 5 _St. Olaf College, St. Olaf, MN;_ 6 _City of Hope, Duarte, CA;_ 7 _HealthPartners Institute, Minneapolis, MN_.

Introduction: Non-small cell lung cancer (NSCLC) is the top cause of cancer-related mortality. We have identified a role for the tumor suppressor hepatocyte growth factor activator inhibitor type-1 (HAI-1) in human lung adenocarcinoma. HAI-1 loss results in unregulated downstream MET/RON tyrosine kinase receptor signaling. We hypothesized that HAI-1 is lost in human lung adenocarcinomas and could serve as a biomarker for therapy with crizotinib, a tyrosine kinase inhibitor (TKI) with activity against both MET and RON. We also hypothesized that unregulated RON signaling in the tumor microenvironment, via HAI-1 loss, may promote tumor metastasis by skewing tumor associated macrophages (TAMs) from an anti-tumor (M1) to a pro-tumor (M2) phenotype.

Methods: Immunohistochemical staining with HAI-1 antibody (1N7) was performed on human tissue microarray containing 20 normal and 20 lung tumor specimens. Intensity grading (0, 1+, 2+, 3+) was performed, and percent cell expression was calculated for each group. For all in vitro assays, H358 lung adenocarcinoma cells, which express high levels of endogenous HAI-1, were stably transfected with mock shRNA or HAI-1 shRNA. Cell protein expression and phosphorylation was assessed via western blot. Cell viability after exposure to crizotinib was assessed by trypan blue staining. For flow cytometric analysis of macrophage phenotype, undifferentiated (M0) macrophages were exposed to conditioned medium from transfected H358 cells and macrophage phenotype was assessed by flow for surface markers CD68 (M0), CCR7 (M1), and CD206 (M2).

Results: HAI-1 expression is significantly decreased in human lung adenocarcinoma compared to normal lung with overall HAI-1 IHC positivity ~20% compared to ~60% respectively. HAI-1 knockdown in H358 cells caused increased phosphorylation of RON and increased sensitivity to crizotinib. Culturing M0 macrophages in conditioned media from H358 shHAI-1 knockdown cells caused a decrease in the CCR7 positive M1 fraction from 8.4% to 1.9%, and an increase in the CD206 positive M2 fraction from 0.77% to 2.09%.

Conclusion: We have demonstrated that loss of HAI-1 occurs in human lung adenocarcinoma consistent with previous studies in human NSCLC cell lines. Furthermore, we showed significantly increased RON signaling activity after HAI-1 knockdown. We have also demonstrated that HAI-1 loss can skew tumor associated macrophages (TAMs) from an anti-tumor (M1) to a pro-tumor (M2) phenotype. We have shown that loss of HAI-1 in vitro can increase sensitivity to crizotinib, suggesting that loss of HAI-1 may predict for tumor sensitivity to MET/RON inhibition. Our findings show that dysregulation of the HAI-1/MET/RON pathway exerts effects both on lung tumor cells as well as TAMs, making this pathway a potentially powerful therapeutic target.

#5515

Ras-like estrogen-regulated growth-inhibitory gene (RERG): A novel biomarker and potential therapeutic target for women with lung cancer.

Michelle Van Scoyk,1 Sreedevi Avasarala,1 Pei-Ying Wu,1 Yanlin Su,1 Lane Lerner,2 Alicia Hulbert,1 Kamesh Bikkavilli,1 Robert A. Winn2. 1 _Univ. of Illinois at Chicago, Chicago, IL;_ 2 _Univ. of Illinois Cancer Center, Chicago, IL_.

Background: Lung cancer is the second most common cancer diagnosed in women. In fact, twice as many women will die from lung cancer compared to breast cancer, the key risk factor being tobacco use. Hence, there is a need to identify new molecular biomarkers to aid in early detection and develop new therapeutic strategies in tobacco-related lung cancer. Ras-like Estrogen-Regulated Growth-Inhibitory Gene (RERG) is a member of the Ras superfamily of GTPases that play an important role in cellular processes such as proliferation, differentiation, and apoptosis. Unlike other members of the Ras superfamily members of proto-oncogenes, RERG functions as a tumor suppressor.

Objective: To investigate the utility of RERG expression as a novel biomarker and a potential therapeutic target in lung cancer.

Results: In silico analysis of The Cancer Genome Atlas (TCGA) lung cancer datasets revealed a significant downregulation of RERG expression in lung tumors. Furthermore, reduced expression of RERG in lung tumors was also observed to be correlated with poor prognosis as determined by Kaplan-Meier survival analysis. Evaluation of formalin-fixed, paraffin-embedded (FFPE) human lung cancer tissues and fresh frozen lung tumor tissues corroborated our in silico observations. Most notably, tobacco smoke was identified to be associated with decreased RERG expression in lung tumors. The most intriguing observation from this study is the significant decrease of RERG expression in female smokers as a result of promoter hypermethylation.

Conclusion: Taken together, these findings suggest that RERG may represent a potential biomarker in lung cancer, particularly among smokers. Further investigation would focus on the mechanism of RERG loss and its influence on lung tumor development, particularly in female smokers.

#5517

The correlation of AIMP2-DX2 expression and oncogenic signaling pathways in lymphoma.

Yungyeong Park,1 Heejoo Han,1 Seulki Song,1 Dongchan Kim,1 Youngil Koh,2 Sung-Soo Yoon2. 1 _Seoul National University, Seoul, Republic of Korea;_ 2 _Seoul National University Hospital, Seoul, Republic of Korea_.

Introduction: ARS-interacting multifunctional protein 2 (AIMP2) has been known to play an important role in the regulation of cell fate, and acts as a tumor-suppressive agent. Unlike normal AIMP2, AIMP2-DX2, a splicing variant of exon 2 deletion, promotes carcinogenic stress. We recently discovered that AIMP2-DX2 is an important poor prognostic factor in acute myeloid leukemia. Likewise, we conjectured AIMP2-DX2 would have a potential role in lymphoid hematologic malignancy. The aim of this study is to investigate the biologic role of AIMP2-DX2 in lymphoma.

Materials and Methods: We studied the composition of genetically categorized variation in lymphoma using 38 diffuse large B cell lymphomas (DLBC) and 56 germinal center B cell lymphoma (MALY) of RNA sequencing data shared by International Cancer Genome Consortium (ICGC). The Differentially Expressed Gene (DEG) analysis method was applied to the RNA data aligned to the human reference, and the different expression pattern in each group was confirmed by nonbiased clustering. We observed whether clustering identifies certain subset of high AIMP2-DX2 level tumors. The Gene Set Enrichment Analysis (GSEA) was performed to confirm the statistical differences or similarities in the number of genes in each subgroup with adjusted p-value <0.05 to identify the representative pathway. Malignant lymphoma-based NAMALWA and HS-sultan cell lines were used and expression level of DX2 was quantitatively analyzed by quantitative real-time PCR. The relationship between AIMP2-DX2 and oncogenic signaling pathway components was verified with Western blotting.

Results: As a result of DEG using unbiased clustering, we could successfully cluster patients into two groups in both DLBC and MALY. Interestingly, unbiased clustering of tumors resulted in a distinct cluster composed of AIMP2-DX2 high-expressing tumors. This phenomenon was observed both in DLBCL and MALY. GSEA analysis revealed that, in DLBC, the expression of JAK-STAT, MAPK, KRAS, and MYC-related genes was increased while the expression of the spliceosome-related genes was reduced as DX2 expression increased. MALY also showed high expression of JAK-STAT, MAPK and KRAS-associated genes and high expression of WNT signaling pathway in high AIMP2-DX2 samples. Using cell lines, we confirmed the expression of AKT MEK, and ERK correlates in malignant lymphoma cell line with expression of AIMP2-DX2 than in cell line with low AIMP2-DX2 expression.

Conclusion: Thus, our data suggest that differences in expression of DX2 cause lymphomagenesis by affecting other oncogenic signaling pathway, such as JAK-STAT, MAPK and KRAS signaling pathways are related to AIMP2 DX2-induced lymphoid hematologic malignancy.

#5518

Bi-allelic loss of CDKN2A initiates melanoma invasion and metastasis via E2F1-BRN2 axis.

Hanlin Zeng,1 Aparna Jorapur,1 A. Hunter Shain,1 Ursula E. Lang,2 Rodrigo Torres,1 Yuntian Zhang,1 Thomas Botton,1 Jue Lin,3 Andrew S. Mcneal,1 Matthew Donne,4 Ingmar N. Bastian,1 Jeffrey North,5 Laura Pincus,5 Richard Yu,1 Beth S. Ruben,1 Nancy Joseph,2 Iwei Ye,1 Boris C. Bastian,1 Robert L. Judson1. 1 _Helen Diller Family Comprehensive Cancer Center. University of California San Francisco, San Francisco, CA;_ 2 _Department of Pathology, University of California San Francisco, San Francisco, CA;_ 3 _Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA;_ 4 _Department of Anatomy, University of California San Francisco. San Francisco, San Francisco, CA;_ 5 _Department of Dermatology, University of California San Francisco, San Francisco, CA_.

CDKN2A acts as a critical tumor suppressor in melanoma, as evidenced by frequent loss of function mutations and deletion. Loss of CDKN2A is believed to permit escape from senescent pre-neoplastic cell populations through relieve of a cell cycle block mediated by its two gene products. We performed a comprehensive analysis of CDKN2A gene status, mRNA and protein expression levels of p16 and p14 in a cohort of melanomas and their adjacent pre-neoplastic lesions and observed that bi-allelic CDKN2A loss coincides with the progression stage when primary melanomas become invasive. In melanoma lines, p16INK4A, one of the protein products of the CDKN2A locus, is a potent barrier to metastasis, independent of its known role inhibiting cell proliferation. We genetically engineered primary human melanocytes to harbor CDKN2A deletions and/or BRAF V600E mutation at their endogenous BRAF locus. Using this physiologic model for the early phases of neoplastic transformation, we found no evidence for BRAF-induced senescence, rather observing that p16INK4A loss activates a master regulator of melanoma invasion, BRN2, through Rb-E2F1 pathway. These results demonstrate that one of the most frequently altered genes across human cancers, CDKN2A, has an unexpected novel role in inhibiting cellular invasion through lineage specific transcription factors and acts as an essential gatekeeper of early metastatic dissemination.

#5519

BAP1 modulates gene-environment interaction in carcinogenesis.

Michele Carbone,1 Angela Bononi,1 Carlotta Giorgi,2 Simone Patergnani,2 Harvey I. Pass,3 Wei Jia,1 Paolo Pinton,2 Haining Yang1. 1 _Univ. of Hawaii Cancer Center, Honolulu, HI;_ 2 _University of Ferrara, Ferrara, Italy;_ 3 _NYU Langone Medical Center, New York, NY_.

We discovered that heterozygous germline BAP1 mutations (BAP1+/-) cause a novel cancer syndrome that we named "The BAP1 cancer syndrome" characterized by development of malignant mesothelioma (MM), uveal and cutaneous melanoma, and other cancers. We demonstrated how germline BAP1 mutations are transmitted over the centuries across generations and found that 100% of individuals who inherited germline BAP1 mutations have developed one or more malignancies in their lifetime. Very recently we demonstrated that the potent BAP1 tumor-suppressor activity is linked to its dual function in the nucleus, where it regulates DNA repair, and in the cytoplasm, where it regulates apoptosis by deubiquitylating and stabilizing the IP3R3 endoplasmic reticulum (ER) Ca2+ channel. In parallel we discovered that cells carrying heterozygous BAP1 mutations derive energy largely via aerobic glycolysis, Warburg effect. Now we have found the mechanisms regulated by BAP1 that are responsible for the changes in metabolism. We discovered that reduced BAP1 levels lead to reduced release of Ca2+ from the ER into the cytoplasm and reduced intra-mitochondrial Ca2+ levels. Reduced mitochondrial Ca2+ levels directly impair aerobic respiration and cells switch to glycolysis even in the presence of oxygen. We demonstrate that aerobic glycolysis can be induced in apparently any human cell type by reducing BAP1 levels using siRNAs or other strategies, and that in these cells glycolysis can be reverted to aerobic respiration upon re-establishing normal Ca2+ levels. In conclusion, our findings indicate how BAP1 deficiency regulates DNA repair and apoptosis and how low BAP1 levels induce the Warburg effect, an effect seen in all "normal" cell types from individuals born with inherited germline heterozygous BAP1 mutations. In these individuals aerobic glycolysis predates by many years malignant transformation.

#5520

Oncogenic RTK signaling inhibits Spred1/NF1 to sustain constitutive Ras/MAPK signaling.

Evan Markegard,1 Ellen L. Mercado,2 Pau Castel,1 Jillian Silva,1 Jacqueline Galeas,1 Kathy Li,3 Anatoly Urisman,4 Frank McCormick1. 1 _UCSF Helen Diller Family Comp. Cancer Ctr., San Francisco, CA;_ 2 _Health Interactions, San Francisco, CA;_ 3 _UCSF Pharmaceutical Chemistry, San Francisco, CA;_ 4 _UCSF Pathology and Laboratory Medicine, San Francisco, CA_.

Spred proteins negatively regulate Ras/MAPK signaling following growth factor stimulation. Inhibition of Ras primary occurs through Spreds ability to bind and localize NF1, a RasGAP and major tumor suppressor, to the plasma membrane. Spred1 and NF1 loss-of-function mutations occur across multiple cancer types including non-small cell lung carcinoma, glioblastoma, melanoma, stomach carcinoma, and uterine carcinosarcoma. Here we demonstrate that oncogenic RTK signaling leads to phosphorylation of Spred1(S105) in the EVH domain, which disrupts Spred1-NF1 binding and function. Phosphomimetic Spred1 is unable to suppress Ras-GTP following growth factor stimulation and cancer cell proliferation. The Spred1(S105) kinase is likely to be a CDK based on in vitro kinase and in vivo cell line assays. Our findings provide one potential mechanism by which oncogenic RTK signaling disrupts negative feedback to sustain constitutive Ras signaling. Furthermore, this work may elucidate a novel therapeutic target for restoring NF1-mediated inhibition of Ras.

#5521

**Functional characterization of a candidate tumor suppressor gene,** Mirror image polydactyly 1 **, in nasopharyngeal carcinoma.**

Merrin Man Long Leong, Arthur Kwok Leung Cheung, Tommy Chin Tung Kwok, Maria Li Lung. _The University of Hong Kong, Hong Kong, Hong Kong_.

Introduction: Nasopharyngeal carcinoma (NPC) is a malignancy with a high incidence in southern China. Previously, a NPC candidate tumor suppressor gene (TSG), MIPOL1, was identified by utilizing a microcell-mediated chromosome transfer (MMCT) approach. MIPOL1 was found to be down-regulated in NPC cell lines and tumors. Promoter hypermethylation and loss of heterozygosity were confirmed to be critical mechanisms to induce MIPOL1 down-regulation. Functionally, MIPOL1-re-expression suppressed in vivo tumor growth of NPC cells via the up-regulation of p21 and p27. These results provide the impetus for further studying this candidate gene in NPC.

Aim: The current study aims to perform comprehensive signaling and functional studies of MIPOL1 in NPC to elucidate its mechanistic functions in NPC development.

Methodologies: A total of 119 pairs of matched NPC tumors and normal tissues with different clinical outcomes were used to investigate the level of MIPOL1 in NPC clinical samples. MIPOL1 wild type (WT) and two truncated mutants were expressed in two NPC cell lines, HONE1 and HK1, and the changes in the cancer-related signaling pathways were examined by protein array and western blot. Yeast 2-hybrid (Y2H) and co-immunoprecipitation (CoIP) were performed to identify the MIPOL1 interaction partners. A RhoB activity assay was used to determine the role of MIPOL1 re-expression in the regulation of tumor suppressor, RhoB, in NPC cells. Furthermore, subcutaneous injection and intrasplenic injection were used to examine the suppressive role of MIPOL1 in both in vivo tumor growth and metastasis.

Results: MIPOL1 showed down-regulation in 75% of the 119 NPC tumors. In the pathway level, the protein array and western blot revealed that re-expression of MIPOL1 can suppress the cancer-related pathways, including angiogenesis, cell survival (Akt/NFκB), and invasion and migration (FAK/Src). Furthermore, RhoB was confirmed to be a potential interaction partner with MIPOL1 and re-expression of MIPOL1 in NPC cells resulted in increased RhoB activity. The MIPOL1 truncation study shows that ΔN100 and ΔC200-442 could reduce the expression of pAKT, pIκBα, and pFAK in HK1. Also, suppression of the angiogenesis markers could not be observed, when ΔN100 or ΔC200-442 were over-expressed in both HONE1 and HK1. Finally, the in vivo study showed that re-expression of the MIPOL1 WT could suppress the tumor growth in the nude mice. Likewise, the MIPOL1 WT re-expression resulted in inhibition of metastasis in the nude mice.

Conclusion: Re-expression of the WT MIPOL1 inhibits tumor growth and metastasis in vivo with the evidence of MIPOL1/RhoB interaction, up-regulation of RhoB activity, and inhibition of the AKT/NFkB, Fak/Src pathways.

Acknowledgement: This study was supported by the Research Grants Council of the Hong Kong Special Administrative Region, People's Republic of China: Grant number 17101715.

#5522

Cancer-associated point mutant in the START domain of DLC1 encodes a protein attenuated for tumor suppressor activity and for binding both Caveolin-1 and PLCD1 but not for Rho-GAP activity.

Beatriz Sanchez-Solana, Xiaolan Qian, Dunrui Wang, Marian Durkin, Brajendra Tripathi, Alex Papageorge, Douglas R. Lowy. _National Cancer Institute, NIH, Bethesda, MD_.

The tumor suppressor gene DLC1 encodes a multi-domain protein, including a Rho-GAP domain (Rho GTPase activating protein domain) that negatively regulates the activity of RhoA, RhoB, and RhoC, and has been hypothesized to be the basis of its tumor suppressor functions. Other well recognized DLC1 domains are an N-terminal sterile alpha motif (SAM) domain, and a C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain. DLC1 is down-regulated in several malignancies by gene deletion, epigenetic promoter methylation, or post-translational mechanisms. In addition, we have recently determined, by analyzing the TCGA database that point mutations in the DLC1 gene occur frequently, and have provided evidence that most of the mutant proteins display attenuated tumor suppressor function. Moreover, the mutations are distributed along the entire coding region, implying that regions of DLC1 in addition to its RhoGAP domain contribute to its full tumor suppressor activity.

Several DLC1 binding partners, in addition to Rho-GTP, have been identified. They include Caveolin-1, the principal structural component and marker of caveolae, which interacts with the START domain of DLC1. The distribution of caveolae is closely related to intracellular events, such as MAPK activation or the control of intracellular Ca2+ levels. Phospholipase C delta 1 (PLCD1) transforms PtdIns(4,5)P2 (PIP2) into inositol triphosphate (IP3) and diacyglycerol (DAG), which participate in intracellular Ca2+ mobilization and protein kinase C activation, respectively. p122RhoGAP, the rat homolog of DLC1, was initially cloned as a novel PLCD1-interacting protein, but the interacting region of PLCD1 with p122RhoGAP was not identified. In addition, others have concluded the function of PLCD1 is not affected by human DLC1.

In the current study, we have characterized a colon cancer-associated DLC1 mutant (R947C) whose mutation lies in the START domain. The mutant has an attenuated tumor suppressor phenotype, as determined by deficiency in reduction of growth in soft agar and cell migration. Compared with the wild type, the binding of the mutant to PLCD1 and to Caveolin-1 is reduced, but its RhoGAP activity is intact. In addition, phosphorylation of Caveolin-1 by Src at Tyr 14 reduces its interaction with wild type DLC1, and Caveolin-1 and PLCD1 interact in vivo more efficiently with each other in the absence of DLC1. Moreover, wild type DLC1, Caveolin-1 and PLCD1 possess cooperative tumor suppressor activities in cells expressing all three genes. Overall, this study provides new insight into the role of the START domain of DLC1, and reinforces the Rho-GAP-independent regulation of tumor growth by DLC1.

#5523

Allele-specific restoration of biological function of the mutant p53 by Baylis-Hillman adducts.

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

p53 is the most frequently mutated gene in human cancer, and the p53R175 mutant is the third most frequently found missense mutant that confers resistance to anticancer drugs and apoptosis. Therefore, small-molecule reactivation of mutant p53 function(s) represents an important and novel anticancer strategy. We have previously reported that induction of oxidative protein modifications primarily underlies the mechanistic conversion of mutant to wt or wt-like p53 forms (Eur J Medchem 107:233, 2016; Int J Oncol 48:1426, 2016). Here, we describe the identification of Baylis-Hillman (BH) adducts as potent and novel mutant p53 reactivators through cell-based, high-throughput small-molecule screening procedures. An extensive structure-activity relationship studies indicated that carbon-carbon double bond and the presence of an electron-withdrawing substituent at the aromatic ring were essential for the activity. Cell lines used in this study includes SKBR3, AU565 (p53R175H), MCF7 (p53 wt), MDA-MB-231 (p53del), MDA-MB-468 (p53R273H) and MCF10a normal breast epithelial cells. BH adducts displayed preferential cytotoxicity against cancer cells expressing the allele-specific R175H mutation (SKBR3 and AU565) in comparison with the wt p53 or p53-null cancer cell lines. Additionally, the BH adducts did not show significant cytotoxicity against normal cells (MCF10a). Further, we developed isogenic cell lines expressing the p53R175H in the p53-null H1299 lung cancer cells and evaluated their sensitivities towards BH adducts; these models again verified the greater drug efficacy. To prove the hypothesis the higher efficacy of BH adduct stems from the reactivation of mutant p53, the conformation-specific antibodies (Pab1620 for wt and Pab420 for mutant) were employed. Immunoprecipitation and immunostaining using confocal microscopy showed that BH adduct treatment of p53R175H cells resulted in a marked reciprocal loss of mutant protein and gradual increase of wt-like protein. EMSA revealed a time-dependent restoration of DNA-binding of the mutant p53, which was accompanied by a distinct upregulation of pro-apoptotic signaling pathways mediated by PUMA, Bax, p21cip1 and MDM2 proteins. A strong G2/M arrest and significant apoptosis were observed with BH adduct in a time and concentration-dependent manner. Other data showed that BH adduct restores the wt-structure and function by covalently binding with the mutant p53R175H. In conclusion, BH adduct is an attractive lead compound for further development because of its potent p53 reactivation of nonfunctional mutant protein (supported by CPRIT grants RP130266 and RP170207 to KSS).

#5524

Tumor suppressor Numb represses monocarboxylate transporters to reduce the glycolysis in oral carcinoma.

Chung-Hsien Chou, Chun-Yu Fan Chaing, Shu-Chun Lin, Kuo-Wei Chang, Hsi-Feng Tu. _National Yang-Ming University, Taipei, Taiwan_.

Oral squamous cell carcinoma (OSCC) is one of the most common malignancies worldwide. Our previous studies have shown that Numb pluripotent gene drives suppressor activity against OSCC, and it is co-targeted by multiple oncogenic miRNAs. In this study, we further identified that Numb mRNA expression was down-regulated in a large fraction of OSCC tumors. We established the Numb-knockout (KO) OSCC cell subclones using CRISPR-Cas9 system. We also generated lentiviruses for exogenous expression of Numb 1 and Numb 4 isoforms. The anchorage independent growth potential was found increased in Numb-KO cell subclones. However, such eligibility was repressed after the rescue of the Numb expression by lentiviral infection. In addition, the level of lactate production and the ratio of ECAR/OCR in the Numb-KO cell subclones were higher than parental cells. The knockout of Numb was able to up-regulate the expression of monocarboxylate transporter (MCT) family members MCT1 and MCT4, but not other metabolism genes, to increase the lactate production. Knockdown of both MCT1 and MCT4 significantly decreased the anchorage independent growth and the lactate production of OSCC cells. This study provides new evidences denoting that Numb represses MCTs to attenuate the transformative capability and the glycolysis in oral carcinoma.

#5525

Rgs8 **and** Rgs16 **protect against pancreatitis and PDA progression.**

Shreoshi Pal Choudhuri,1 Yalda Zolghadri,1 Luke Mascarenhas,2 Thomas Wilkie1. 1 _UTSW, Dallas, TX;_ 2 _UT Dallas, Dallas, TX_.

Pancreatic ductal adenocarcinoma (PDA) has the highest death rate among major cancers, and new treatments are desperately needed. The mice we developed in this study, KCR8-16, are an excellent mouse model for identification, characterization, and in vivo validation of novel PDA therapeutics. Kras oncogenic mutations (e.g., KrasG12D) are found in over 90% of human PDA. Kras can be activated by protein kinase and G-protein coupled receptor (GPCR) signaling. Regulator of G-protein signaling (Rgs) proteins regulate GPCR signaling by accelerating the GTPase activity of Gq- and Gi class alpha subunits. Activating alleles of Gq that are resistant to Rgs inhibition are found in benign precursors of PDA in humans. We previously reported that Rgs8 and Rgs16 are in vivo reporters of Kras activity in pancreatic intraepithelial neoplasia (PanIN), intraductal papillary mucinous neoplasm (IPMN), and PDA progression in KIC;Rgs16::GFP mice (p48::Cre/+; KrasG12D/+; Cdkn2af/f; Rgs16GFP ) (DMM 8, 2015). To identify the role of Rgs 8 and 16 in PDA, we crossed the Rgs8-16 double knockout into pancreas-specific KrasG12D (KC) mutant mice (termed KCR8-16). We found that deletion of Rgs8 and Rgs16 in KC accelerated PDA progression. Additional pancreatic stress evoked by caerulein treatment caused immediate and pancreas-wide progression to PDA in KCR8-16 mice. Our study suggests that Rgs8 and Rgs16 act as tumor-suppressor genes in PDA initiation and progression. Moreover, KCR8-16 and KIC;Rgs16::GFP mice can be used as an excellent model for identification and rapid in vivo validation of PDA therapeutics.

#5526

**Mapping signaling networks in** Nf1 **mutant tumors.**

Daniela Pucciarelli, Ganesh Krishnamurthi, Steve Braunstein, Jean L. Nakamura. _University of California, San Francisco, San Francisco, CA_.

Introduction: The tumor suppressor gene Neurofibromatosis type 1 (NF1) encodes the Ras-GTPase activating protein neurofibromin, which negatively regulates Ras signaling. Loss of NF1 increases signaling through Raf/MEK/ERK and PI3K/AKT/mTOR. Inhibition of each of these pathways can produce variable responses, suggesting compensatory mechanisms and heterogeneity in signaling mechanisms in cancers. To study the biochemical sources of signaling we sought to functionally characterize Nf1-mutant tumors based on their variable drug sensitivities and development of drug resistance.

Methods: We previously established Nf1 null tumor cell lines from malignancies arising in irradiated Nf1+/- mice. We functionally profiled this panel of tumor cell lines using a custom 94 compound drug library targeting core signaling pathways commonly activated in cancers. Phosphorylated and total ERK, AKT, and S6 were assessed by western blotting. Cell lines resistant to treatment with MAPK and mTOR inhibitors were generated from parental cell lines. Parental and resistant cell lines were compared using MTS, exome and transcriptome sequencing, western blotting and kinome analysis.

Results: Nf1 mutant tumor cell lines demonstrated heterogeneous drug responses and, based on this observation, they could be organized into 3 functional sub-classes sharing similar drug sensitivities. All 12 Nf1 null cell lines demonstrated heterogeneous basal Ras pathway activation, as indicated by phosphorylated ERK, AKT, and S6. This variability was associated with variable sensitivity to treatment with MAPK and mTOR inhibitors. Acquired resistance to MAPK or mTOR inhibition (MEKi and mTORi) revealed differences in Ras activation that distinguished resistant from parental lines. For example, in contrast to parental lines, Ras-GTP levels in mTORi resistant cell lines demonstrated stability after serum stimulation compared to the parental counterpart, suggesting a reprogrammed role for Ras activation when mTORi resistance is acquired. These data highlight the plasticity of Ras-driven signaling in Nf1 tumors, which likely must be addressed to optimize therapy and counter the emergence of resistant clones.

Conclusion: Our analysis indicates significant variability in Ras pathway signaling in the context of Nf1 loss, which has implications for the choice of specific kinase inhibitors to use therapeutically. Cancers, including Nf1 null tumors, develop resistance to therapy and our data points to specific reprogramming of signaling pathways that distinguish resistance mechanisms.

#5527

Characterizing the role of WWOX dysregulation in canine osteosarcoma.

Justin Breitbach,1 Feng Xu,1 F. Kay Huebner,1 Cheryl A. London,2 Joelle M. Fenger1. 1 _Ohio State University, Columbus, OH;_ 2 _Tufts University, New Grafton, MA_.

Introduction: The WW domain-containing oxidoreductase (WWOX) is a tumor suppressor gene that is frequently deleted or reduced in human osteosarcoma (OS) tumors and loss of WWOX in OS cells promotes a highly tumorigenic phenotype. The purpose of this study was to investigate the functional consequences of WWOX deficiency in canine OS, a well-established spontaneous large animal model of human OS. Methods: Real-time PCR and Western blotting was performed to evaluate WWOX expression was in primary canine OS tumors and canine OS cell lines. Full-length canine Wwox cDNA was amplified from normal canine testes and cloned into the pCDH-cop-GFP lentiviral vector (Systems Biosciences). Canine OS cell lines expressing low basal levels of WWOX were transduced with either empty or WWOX lentiviral vectors and stably transduced cells were sorted based on GFP positivity. Overexpression of canine WWOX lentiviral constructs was confirmed by Western blotting and OS cell lines were evaluated for differences in proliferative capacity, expression of bone differentiation markers, and the ability to migrate through matrigel. Transgenic mice (Tg) carrying a conditional allele of Wwox (Wwoxfl/fl-Tg) were crossed with mice expressing Cre recombinase under the osteoblast-specific Collagen1α1 promoter (Col1a1-Cre-Tg), restricting deletion of WWOX to osteoblasts. Calvarial osteoblast cultures were generated from Wwoxfl/fl and Col1a1-Cre; Wwoxfl/fl mice and the effects of WWOX deletion on osteoblast functions was evaluated using assays described above. Results and Conclusions: Western blotting demonstrated that WWOX protein is absent or reduced in primary canine OS tumors and canine OS cell lines. To assess the functional consequences of WWOX deletion on normal osteoblast behavior, the canine OSA8 and Abrams OS cell lines which express low levels of WWOX were transduced with WWOX lentiviral constructs resulting in high levels of WWOX expression as demonstrated by real time PCR and Western blotting. Restoration of WWOX in canine OS cells resulted in decreased cellular proliferation and invasion. To more critically evaluate the consequences of Wwox deficiency in normal osteoblast biology without the confounding influence of altered Wwox expression in other tissues, Col1a1-Cre; Wwoxfl/fl-Tg mice were generated in which Wwox deletion is restricted to osteoblasts. Consistent with our findings in canine OS cell lines, deletion of WWOX in osteoblasts derived from Col1a1-Cre; Wwoxfl/fl-Tg mice resulted in enhanced cellular proliferation. Studies are underway to identify WWOX binding partners and investigate regulatory pathways altered by WWOX that promote cell proliferation and invasion in normal and malignant osteoblasts. In summary, these data provide evidence supporting a role for WWOX in canine OS cell proliferation and invasion, and suggest that dysregulation of WWOX may be fundamental to the disease process in both human and canine OS.

## CLINICAL RESEARCH:

### Diagnostic and Prognostic Biomarkers in Clinical Trials

#5528

**Evaluation of tumor mutation burden as a biomarker for immune checkpoint inhibitor efficacy: A calibration study of whole exome sequencing with FoundationOne** ® **.**

Joseph D. Szustakowski, George Green, William J. Geese, Kim Zerba, Han Chang. _Bristol-Myers Squibb, Princeton, NJ_.

Background: Tumor mutation burden (TMB) is emerging as a potential predictive biomarker for the efficacy of immune checkpoint inhibitors. TMB has traditionally been evaluated using whole exome sequencing (WES), an established method for analysis of genomic alterations that requires both tumor and germline DNA. In the phase 3 CheckMate 026 study (NCT02041533) of nivolumab vs chemotherapy in first-line non-small cell lung cancer (NSCLC), exploratory analyses were performed to determine the value of TMB as a predictive biomarker in 312 tumor samples using WES. Patients with baseline TMB in the upper tertile were associated with an increased objective response rate to nivolumab and longer median PFS, relative to patients with TMB in the lowest 2 tertiles and compared with chemotherapy-treated patients in the upper tertile (Carbone et al. N Engl J Med. 2017). Increasingly, targeted cancer gene panels, including the FoundationOne® (F1) assay, are being assessed as an alternative to WES for clinical use. To determine the feasibility of transitioning from WES to a potential in vitro diagnostic next-generation sequencing (NGS) platform for TMB analysis, we performed a study comparing values generated by WES with those generated by the F1 assay.

Methods: Using the 2 hybridization-capture/NGS methods (WES and F1), TMB was assessed in formalin-fixed, paraffin-embedded NSCLC tumor samples. For WES, coding regions of 21,522 genes were analyzed, with TMB defined as the total number of missense mutations in the tumor exome. The F1 assay is a targeted gene panel of 315 cancer-related genes (Frampton et al. Nat Biotechnol. 2013), with TMB defined as the number of somatic mutations per megabase of sequenced tumor genome.

Results: TMB data from 44 NSCLC samples were available on both F1 and WES platforms. We determined an empirical method to convert TMB values derived from WES into those derived from F1 to establish calibration between the 2 methodologies. TMB assessed by WES and by F1 was highly correlated (Spearman's r=0.9). As an additional test of concordance between the platforms, we examined agreement between the assays around the median WES value of 148 mutations. This value projects to 7.64 mutations per megabase on F1. The overall agreement between F1 and WES around these values was 86% (95% Wilson CI, 73-94). Positive and negative agreements between the 2 hybridization-capture/NGS methods were both 86% (95% Wilson CI, 67-95).

Conclusion: Bridging TMB analysis by WES to F1 facilitates the transition of WES-derived biomarker data to the clinical in vitro diagnostic F1. This study demonstrates the feasibility of harmonization of TMB results across testing platforms, which provides alternative TMB testing options.

#5529

PD-L1 and CD8 IHC dual assessment in NSCLC using the Halioseek® assay: A multicentric study.

Luciana Batista,1 Nicolas Brandone,2 Stéphane Garcia,2 Yanis Boumber,3 Essel Dulaimi,4 Philippe Tanière,5 Paul Hofman,6 Marius Ilié,6 Julien Adam,7 Jérôme Galon,8 Catherine Gerbon,1 Jacques Fieschi1. 1 _HalioDx, Marseille, France;_ 2 _Service de pathologie, Hôpital Nord, Marseille, France;_ 3 _Department of Hematology/Oncology, Fox Chase Cancer Center, Philadelphia, PA;_ 4 _Department of Pathology, Fox Chase Cancer Center, Philadelphia, PA;_ 5 _Queen Elizabeth Hospital - Histopathology - University Hospitals Birmingham NHS Foundation Trust, Birmingham, United Kingdom;_ 6 _Laboratory of Clinical and Experimental Pathology, University Côte d'Azur, Nice, France;_ 7 _Department of Pathology, Institute Gustave Roussy, Villejuif, France;_ 8 _INSERM, UMRS1138, Laboratory of Integrative Cancer Immunology, Cordeliers Research Centre, Paris, France_.

Background

Immune checkpoint inhibitors (ICI) have revolutionized the treatment of NSCLC, but only a small proportion of treated patients exhibit a long term response. Improved selection of the patients that will respond to ICI is a major unmet need. Despite its relatively limited predictive value, the percentage of PD-L1+ tumor cells supports their application. It is proposed that NSCLC patient stratification might be improved by additional biomarkers, in particular, the presence of tumor infiltrating lymphocytes (TILs) which play a crucial role in tumor immune response. Halioseek® PD-L1 staining allows accurate detection of positive tumor cells in NSCLC, is comparable to other IVD assays, and is CE-IVD marked (In Vitro Diagnostic Medical Device compliant with European Directive 98/79). This standardized assay also provides complementary characterization of the tumor micro-environment without requiring additional precious tumor samples. The dual staining of Halioseek® allows simultaneous evaluation of PD-L1+ and CD8+ cells on the same tissue section. The assay also includes a Digital Pathology (DP) analysis module to determine CD8+ cell density and a proximity index between CD8+ and PD-L1+ cells.

Material and methods

A multi-centric study including 5 laboratories was performed in order to 1) Further demonstrate the inter-laboratory reproducibility of the Halioseek® assay; 2) Compare Halioseek® results to routine PD-L1 laboratory tests; 3) Explore Halioseek® Analyzer DP tools to further characterize NSCLC samples. 10 NSCLC FFPE samples (50 slides) were stained using Halioseek® by 5 independent laboratories and 66 NSCLC FFPE samples were assessed using both Halioseek® and the laboratory's routinely used PD-L1 assay by 4 independent centers. The Halioseek® assay staining was performed according to manufacturer's instructions. Percentage of PD-L1+ tumor cells was estimated by pathologists; DP analysis was performed using Halioseek® Analyzer.

Results

For the 50 slides stained using Halioseek® and evaluated independently by 5 different laboratories, a concordance of 94% and 90% was observed using the 1% and 50% cut-offs of PD-L1 positive tumor cells, respectively. When analyzed by the same pathologist, concordance increased to 100% and 94%, respectively. For the 66 samples analyzed using both Halioseek® and laboratory PD-L1 routine test, concordances were 92% and 97%. Digital pathology tools allowed further characterization of patient samples in terms of CD8+ cells density and proximity indexes.

Conclusion

Halioseek® is a new robust assay leveraging the advantages of DP to combine TILs and PD-L1 quantification within the tumor and its microenvironment. Halioseek® gives reproducible and accurate results in independent routine laboratories and its DP solution allows better sample characterization. The predictive performance of Halioseek® for the response to ICI needs to be further investigated.

#5530

PD-L1 expression in patients screened for phase 2 head and neck squamous cell carcinoma clinical studies (HAWK and CONDOR).

Sophie Wildsmith,1 Marietta Scott,1 Anita Midha,1 Craig Barker,1 Jessica Whiteley,1 Marianne Ratcliffe,1 Marlon Rebelatto,2 Jill Walker,1 Dan Zandberg,3 Lillian L. Siu4. 1 _AstraZeneca, Cambridge, United Kingdom;_ 2 _MedImmune, Gaithersburg, MD;_ 3 _University of Maryland Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, MD;_ 4 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada_.

PD-L1 expression by immunohistochemistry (IHC) has proven to be a useful biomarker in determining patient response to anti-PD-1/PD-L1-directed therapeutics in several cancers, including HNSCC. Understanding the impact of sample type and demography on PD-L1 expression will inform the suitability of tumor biopsies for testing.

Patients entering the HAWK and CONDOR studies were screened for PD-L1 expression (n=669) in a tumor tissue sample using the Ventana PD-L1 SP263 IHC assay as part of the inclusion criteria. PD-L1 assessment was performed centrally using fully validated test procedures and scoring by trained pathologists. Patients were classified as PD-L1 high if ≥25% of tumor cells (TCs) showed membrane staining. Recently acquired or archival samples ≤3 years old were permitted. Baseline demographic and clinical factors were obtained from the clinical database. Prevalence of PD-L1 expression was assessed according to various clinical and demographic characteristics, sample type (biopsy or resection) and location of biopsy (primary tumor vs metastatic site). Comparisons of PD-L1 expression between subgroups of samples were undertaken using a test for equality of proportions.

PD-L1 prevalence was significantly higher in females, patients who never smoked, and those with oral cavity primary. Prevalence was similar in archival and recently acquired samples, by HPV status, and between primary and metastatic sites and for biopsies compared with resections (Table). A separate internal study using 40 pairs of matched samples from a commercial source confirmed strong concordance in PD-L1 status between primary and metastatic sites.

These results indicate that various sample types can be reliably used for the determination of TC PD-L1 status in HNSCC. Similar prevalence of PD-L1 expression was found between primary and metastatic lesions and archival compared to recently acquired tissue, with important implications in everyday clinical practice.

Table | |

|

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

|  | % patients with tumor cell PD-L1 ≥25% (n) | P Value

Sex | Male

Female | 24.5% (534)

36.3% (135) | 0.00817

Ethnicity | Asian

Black/African American

White | 23.8% (21)

20.7% (29)

28.7% (561) | 0.584

Age | >65

≤65 | 24.0% (183)

28.0% (486) | 0.354

Smoking status* | Current smoker

Ex-smoker

Never smoked | 20.8% (48)

24.1% (245)

50.0% (86) | 1.27e-05

Disease classification* | Locally advanced

Metastatic | 28.9% (135)

29.9% (244) | 0.926

HPV status* | HPV+

HPV- | 29.3% (116)

26.9% (242) | 0.720

WHO status at baseline* | Normal activity

Restricted activity | 29.6% (115)

29.3% (263) | >0.999

Site of disease at study entry* | Hypopharynx

Larynx

Oral cavity

Oropharynx

Other | 17.3% (52)

20.0% (75)

46.5% (101)

27.2% (147)

25.0% (4) | 0.000258

Site of biopsy/resection | Primary tumor

Metastatic site | 26.0% (338)

27.8% (331) | 0.670

Sample type | Recently acquired

Archival | 27.0% (285)

26.4% (363) | 0.941

|

Biopsy

Excision/resection | 25.9% (544)

30.6% (124) | 0.337

*Data available only for randomized patients

HPV, human papillomavirus; WHO, World Health Organization

#5531

**Detection of co-occurring and potential resistance mutations in cell-free, circulating tumor DNA from patients with** BRAFmutant **metastatic melanoma undergoing treatment with BRAF-targeted therapies.**

Broderick Corless,1 Greg Chang,1 Weihua Liu,2 Jin Li,2 Andre Marziali,3 Laura Mai,3 Matthew Wiggin,3 Melissa Wilson,1 Anna Pavlick,1 Iman Osman,1 George Karlin-Neumann,4 Cindy Spittle,5 David Polsky1. 1 _NYU Langone Health, New York City, NY;_ 2 _MolecularMD, Cambridge, MA;_ 3 _Boreal Genomics, Vancouver, British Columbia, Canada;_ 4 _Bio-Rad, Pleasanton, CA;_ 5 _MolecularMD, Portland, OR_.

Purpose Melanoma patients with BRAFmutant tumors often develop resistance to BRAF-inhibitor therapies. Co-occurring mutations, present at the time of treatment initiation, have been identified in patients with primary treatment resistance, and NRAS mutations have been associated with secondary resistance. We tested the ability of multiplex mutation detection assays to identify possible co-occurring and resistance mutations in cell-free, circulating tumor DNA (ctDNA) from patients undergoing treatment with BRAF-targeted therapies.

Methods Purified ctDNA samples remaining from a previous longitudinal study of metastatic melanoma patients, which measured BRAFmutant and NRASmutant ctDNA using droplet digital PCR (ddPCR) duplex assays, were used for this study. Twelve samples from 6 patients with BRAF V600E mutant (V600E) tumors, who were receiving BRAF-inhibitor therapy, were separately analyzed in different laboratories using the two different multiplex assays (Oncomine and OnTarget). Oncomine used approximately 20ng DNA; OnTarget used approximately 30ng DNA. Patient samples were chosen based on clinical response or disease progression at the time of blood draw.

Results Ten of 12 samples had V600E ctDNA detected by ddPCR. The On-Target detected V600E in 9/10 samples; the Oncomine detected V600E in 8/9 samples (1 sample was not analyzed due to limited sample availability). The 1 sample with a ddPCR detected mutation that was not detected by either OnTarget or Oncomine had a 0.01% fractional abundance, which is below the detection limit of these multiplex assays. Neither assay detected V600E DNA when it was not detected by ddPCR. One patient had 2 samples with a co-occurring p53 (R273H) mutation detected by OnTarget and Oncomine. This mutation was present at the time of partial response, and at disease progression with an associated increased fractional abundance as determined by both assays. The OnTarget detected NRASmtant ctDNA (Q61K x 2, Q61H x1) in 3 of the V600E samples; the Oncomine detected the Q61K in 1 of these 2 samples (the third sample was not analyzed as noted above). The NRAS mutations arose at times of disease progression. Oncomine also detected additional p53, GNAS, and FBXW7 mutations at generally low fractional abundances that were not detected by OnTarget.

Conclusion Co-occurring and potential resistance mutations are detectable in the plasma of metastatic melanoma patients using OnTarget or Oncomine assays. The assays demonstrate high sensitivity, as evidenced by their ability to identify V600E ctDNA. These findings suggest that studies analyzing patient ctDNA for resistance mutations while they are undergoing treatment are feasible. Results of such studies may eventually help inform treatment choices, such as switching therapies when resistance mutations emerge.

#5532

Serum progranulin (GP88) level correlates with change in RECIST and survival in metastatic breast cancer.

Ginette Serrero,1 David Hicks,1 Douglas Hawkins,2 Binbin Yue,1 Paula Rosenblatt,3 Nancy Tait,3 Katherine Tkaczuk3. 1 _A &G Pharmaceutical, Inc., Columbia, MD; _2 _University of Minnesota, Minneapolis, MN;_ 3 _University of Maryland Greenebaum Comprehensive Cancer Center, Baltimore, MD_.

Monitoring of disease status in metastatic breast cancer (MBC) patients is a necessary step for optimal clinical management of patients during and post-therapy. While imaging technologies are the methods of choice in the standard of care (SOC), they are expensive, time-consuming, and have limited sensitivity. Measurements of circulating tumor markers CA15-3, CA125 and CEA have provided, albeit with limitation, minimally invasive methods for MBC disease management. An ideal approach would be to measure biomarkers involved in tumor biological processes, as they may provide better evaluation of the disease state and thus aid in real-time clinical management of MBC patients. The 88kDa glycoprotein Progranulin (GP88/PGRN) fit these criteria. GP88/PGRN is expressed in tumor tissue and not in normal mammary tissue counterpart and is secreted into the circulation of BC patients. Biological studies have established GP88/PGRN as a critical driver of BC cell proliferation, survival, invasiveness and drug resistance. Clinical studies have demonstrated that high tumor GP88/PGRN expression was prognostic for recurrence and that BC patients had a statistically elevated GP88/PGRN serum level compared to healthy individuals. In the present study, we examined whether GP88/PGRN serum levels were elevated in MBC patients and whether GP88 serum levels were correlated to patient survival and to a change in disease status as measured by RECIST 1.1.

An IRB approved protocol at the University of Maryland Greenebaum Comprehensive Cancer Center consented and enrolled 101 stage 4 BC patients undergoing SOC therapy. Patient demographics, together with clinical and disease characteristics by RECIST were collected. Blood samples were collected from each patient at follow-up visits during and post-therapy and tested for GP88 using A&G's GP88 enzyme linked immunoassay.

Statistical analyses established a GP88 cut-off value associated with overall survival as well as determined that GP88 level was associated with disease status measured by RECIST criteria. Kaplan-Meier functions established a correlation between GP88 serum level and overall survival in MBC patients that was independent from age, race, tumor characteristics, receptor status and metastatic burden (number and sites of metastasis). Additionally, we examined the data to investigate if change in GP88 serum levels correlated with a contemporaneous change (Progression vs Response) in RECIST/ Clinical findings. Statistical analysis determined that contemporaneous GP88 is significantly associated with disease progression (p=0.0101) and response (p=0.0194).

We conclude that circulating levels of GP88/PGRN in MBC patients are correlated with overall survival and disease status and that monitoring circulating GP88/PGRN levels would provide additional information and valuable insight into real-time MBC disease status.

#5533

Cell-free DNA sequencing in ERBB2-mutant breast cancer patients treated with neratinib and fulvestrant: Exploratory analysis from the Phase 2 SUMMIT 'basket' trial.

Alison M. Schram,1 S. Duygu Selcuklu,1 Rebecca J. Nagy,2 Lillian M. Smyth,1 Lisa D. Eli,3 Richard E. Cutler,3 Alshad S. Lalani,3 David M. Hyman,1 Richard B. Lanman2. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Guardant Health, Redwood City, CA;_ 3 _Puma Biotechnology, Los Angeles, CA_.

Background: Mutations in ERBB2, encoding the HER2 protein, occur in up to 4% of breast cancers. A subset of ERBB2 mutations have been shown to have oncogenic potential through constitutive activation of the HER2 kinase. SUMMIT (NCT01953926) is a phase II 'basket' study investigating the use of the irreversible pan-HER tyrosine kinase inhibitor neratinib, in combination with the estrogen degrader fulvestrant, for the treatment of patients with advanced hormone-positive breast cancers harboring ERBB2 mutations. We collected and sequenced serial samples of circulating cell-free DNA (cfDNA) from participating patients in order to identify biomarkers of response and resistance.

Methods: Patients were treated with neratinib 240 mg daily and fulvestrant at the labelled 500 mg dose. Plasma was collected and cfDNA was extracted from patients at baseline and on Day 1 of each cycle (occurring every 4 weeks). Next generation sequencing (NGS) was performed on cfDNA samples collected from 12 patients using the Guardant360 test (Guardant Health, CA), which identifies select point mutations, small insertions and deletions, copy number changes, and structural rearrangements in 73 genes. Paired samples were sequenced from 9 of the 12 patients, including baseline and progression samples from 7 patients. cfDNA NGS results were compared to pretreatment tissue NGS results obtained 0-6 years prior (median: 6 months).

Results: 90% (9/10) baseline cfDNA NGS detected the locally reported ERBB2 mutation identified by tissue NGS. In one patient with only a sample obtained at progression, the ERBB2 mutation was identified. No ERBB2 mutation was identified in a singular on-treatment sample obtained from a patient with continued response to therapy. Eight ERBB2 mutations were identified in the baseline cfDNA sample of one patient with a hypermutated tumor, 6 of which were not identified by tissue NGS. Emergence of at least one additional ERBB2 mutation was seen in 3/7 patients with paired baseline and progression samples, including patients with 2 and 5 acquired ERBB2 mutations. The ERBB2 mutant allele fraction (MAF) increased compared to baseline in 4/7 of patients at the time of progression and declined in 2/2 patients with continued response to therapy. cfDNA NGS detected mutations, unidentified by tissue NGS in 9/10 baseline samples and in 11/12 patients, including 5 ESR1 mutations in 3 patients.

Conclusion: cfDNA NGS was sensitive for the detection of ERBB2 mutations. MAF dynamics typically tracked response to, or progression on therapy, at the time of cfDNA collection. The emergence of additional ERBB2 mutations was common at the time of progression; however, no T798 gatekeeper mutation was observed. Many subclonal mutations were identified on cfDNA NGS that were not observed on tissue NGS.

#5534

**Analysis of** TERTmutant **circulating tumor DNA as a potential biomarker of disease activity in patients with unresectable stage III/IV melanoma receiving immuno-oncology therapies.**

Mahrukh M. Syeda,1 Broderick Corless,1 Melissa Wilson,1 Yesung Lee,1 Jeremy Tchack,1 Todd Wechter,1 Una Moran,1 George Karlin-Neumann,2 Anna C. Pavlick,1 Iman Osman,1 Yongzhao Shao,1 David Polsky1. 1 _NYU Langone Health, New York City, NY;_ 2 _Bio-Rad Laboratories, Pleasanton, CA_.

Purpose: Our goal is to develop response predictive biomarkers for melanoma patients that are more sensitive than standard measures of disease activity. TERT promoter mutations occur frequently in many cancers, especially melanoma. We developed droplet digital PCR assays that detect the 2 most frequently occurring TERT promoter mutations, C228T and C250T. We previously demonstrated that these assays could detect TERTmutant cell-free, circulating tumor DNA (ctDNA) in the plasma of metastatic melanoma patients. In the current study we sought to compare the clinical sensitivity and specificity of these blood-based assays to the current blood biomarker, lactate dehydrogenase (LDH), in detecting the presence of metastatic melanoma and disease progression among patients with unresectable Stage III/IV disease.

Methods: We analyzed 71 patients' tumors with unresectable stage III/IV metastatic melanoma undergoing treatment with immuno-oncology therapies, prospectively acquired by the NYU Interdisciplinary Melanoma Cooperative Group. TERT mutations were identified in tumors using droplet digital PCR (ddPCR) or SNaPShot assays (prior to the development of the ddPCR assays). Patients with TERT mutant tumors were selected for further study based on the availability of serial blood collections and radiographic scan data. ddPCR assays used to analyze ctDNA from these patients were chosen based on the tumor mutation detected. Plasmas used for ctDNA analysis ranged from 1-5 mL (73% having at least 4 mL). The ctDNA results were compared with LDH levels and disease status based on radiographic scans and clinical notes.

Results: We identified TERT mutations in 31/71 (44%) tumors (18/31 C228T and 13/31 C250T). Our final data set consisted of 26 patients with 85 plasmas. Among patients with available pretreatment samples, ctDNA and LDH were elevated in 14/24 (58%) and 5/24 (21%) patients, respectively (p-value=0.012, McNemar exact test). Among patients whose disease progressed, ctDNA and LDH were elevated at the time of progression in 11/16 (69%) and 4/16 (25%) patients, respectively (p=0.039). Among the patients who had either a complete response (n=2), partial response (n=3), or stable disease (n=3), 1/8 had a slightly elevated ctDNA (3.8 copies/mL with normal level <3 copies/mL). None of the patients had an elevated LDH. One patient had a mixed response associated with an elevated ctDNA (13.2 copies/mL) and RECIST score equal to 3.4 cm, and then the ctDNA dropped below baseline when the RECIST measurement fell to 2.8 cm on a follow-up scan.

Conclusion: These data demonstrate that ddPCR assays detecting TERT C228T or C250T mutations in ctDNA of metastatic melanoma patients have a greater sensitivity to detect the presence of metastatic disease and disease progression than the currently used standard, LDH.

#5535

**C-EDRN and US-EDRN collaboration on systematic review and meta-analysis of the association between** RAD51 **135G/C genetic biomarker and cancer risk.**

Run Chen,1 Wendy Wang,2 Wenqiang Wei,1 Sudhir Srivastava,2 Jie He1. 1 _National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College (NCC, CAMS), Beijing, China;_ 2 _National Cancer Institute (NCI), National Institutes of Health (NIH), Bethesda, MD_.

Introduction: The Early Detection Research Network (EDRN) is an initiative of the National Cancer Institute (NCI) to support the translation of biomarker research into clinical applications. The US-EDRN and China-EDRN (C-EDRN), led by CICAMS, China, have strong collaborations on discovering and validating molecular biomarkers for early detection, diagnosis, and prevention. Our collaboration projects promote scientific exchanges, visiting scientists, biomarker discoveries and validations, as well as system review. Here, we report the system review of a genetic marker, RAD51 135G/C polymorphism and cancer risk.

Background: The RAD51 gene is essential for the repair of damaged DNA related to tumor development. Although some studies have investigated the association between RAD51135G/C polymorphism and the risk of different cancers, the results are conflicting rather than conclusive. Therefore, it is necessary to further perform the system review of the polymorphism related to cancer risk.

Method: The PubMed, Embase, and Cochrane library databases were searched to identify eligible studies that were published in English up to July 2017. Two investigators independently screened, extracted the data, and evaluated the quality of all eligible studies using Newcastle-Ottawa Scale (NOS). Crude odds ratios (ORs) together with their corresponding 95% confidence interval (CI) were calculated to assess the strength of association under dominant and recessive models. Subgroup analyses were performed based on ethnicity and cancers. Begg's test was used to measure publication bias.

Result: A total of 75 studies from 71 articles were finally qualified and enrolled in this meta-analysis. The publication year of selected studies ranged from 2002 to 2017. The pooled results involving 24,284 cases and 27,649 controls showed that RAD51 polymorphism was associated with increased cancer risk under both dominant and recessive models. In subgroup analysis, the association varied among different ethnicities and cancers. Significantly elevated cancer risk was observed in Caucasians under both models, and in Asians under dominant model. Breast cancer, hematologic malignances, ovarian cancer, endometrial cancer, prostate cancer also showed significant associations with RAD51 polymorphism. Begg's test results showed there were no publication bias in the study.

Conclusion: This meta-analysis indicated that the RAD51135G/C polymorphism was significantly associated with the susceptibility of cancer and it may be utilized as a valuable biomarker in early diagnostics and risk assessment. Further efforts are needed to identify and validate this finding in prospective studies and to explore the potential function of the variants in different cancers for clinical applications.

#5536

Simultaneous characterization and quantification of immune cell subpopulations and PD-L1 expressing CTCs in peripheral blood of cancer patients.

Rachel Krupa, David Lu, Adam Jendrisak, Angel Rodriguez, Nadia Ebrahim, Robin Richardson, Sean Nisperos, Ryon Graf, Jiyun Byun, Yipeng Wang, Mark Landers, Ryan Dittamore. _Epic Sciences, San Diego, CA_.

Background: Expression of PD-L1 on tumor and immune markers in tumor tissue is associated with improved response to PD-1 and PD-L1 checkpoint inhibitors. However, each alone has limited predictive utility. Multimodal characterization of both the tumor and host immune system is an unmet medical need for the improved prediction of response to immunotherapy. Metastatic lesions are likely to be undersampled and require a liquid biopsy, given tumor heterogeneity and evolution and temporal changes in the host immune system. We sought to examine expression of PD-L1 on circulating tumor cells (CTCs) as well as characterize rare immune cell populations with a noninvasive liquid biopsy. Examining dynamic biomarker changes in longitudinal samples could enable the development of novel diagnostic tools for response prediction and pharmacodynamics studies related to immunotherapy.

Methods: Blood samples from lung cancer patients were collected and shipped to Epic Sciences. Contrived samples were also developed, using cancer cell lines spiked into healthy donor (HD) blood. Red blood cells were lysed and nucleated cells were plated onto glass slides. Slides were stained with DAPI and immune cocktails, then imaged. Targets included pan-CK, CD45, PD-L1, CD4, CD8, Ki-67, and TIM-3. Approximately 3 million nucleated cells per slide were examined through advanced digital pathology pipelines to detect and quantify changes in T-cell populations and to assess circulating tumor burden.

Results: Epic Sciences' rare cell detection platform has an analytically validated limit of detection of 2 cells/mL of blood. Immuno-panels were developed to profile leukocyte subpopulations and CTCs from a single blood sample and feasibility was demonstrated in cell lines, HD and lung disease patient blood. Quantitation experiments showed that immunomagnetically purified CD8+ and CD4+ cells spiked in 3:1, 1:1, and 1:3 ratios were detected as 3.1:1, 1:1.1, and 1:3.1. Purified CD4 cells were also spiked into healthy donor WBCs at target ratios of 1-10%. Percentages of detected spike-in CD4 cells were linearly correlated with the target ratios with correlation coefficient (r) = 0.96. 22.8% (18/79) lung cancer patients had PD-L1+ CTCs detected, and the majority (91%) of patients had PD-L1+ leukocyte populations.

Conclusions: The low limit of detection of the Epic Sciences CTC platform coupled with the ability to archive patient blood samples allowed for retrospective, precise quantification of leukocyte subpopulations and PD-L1 expression on CTCs. Development of a liquid biopsy-based platform that can simultaneously measure immune biomarkers in CTCs as well as on leukocytes will allow for real-time assessment and monitoring of response to immune checkpoint inhibitors.

#5537

Circulating tumor DNA analysis for ESR1 mutations in patients with hormone receptor-positive metastatic breast cancer.

Sung Hoon Sim, Su Yeon Jeon, Kyoung Hee An, Sun Young Kong, Min Jung Kwon, Keun Seok Lee, In Hae Park. _National Cancer Center, Gyeonggi-do, Republic of Korea_.

Background: ESR1 mutations are known as a major mechanism of resistance to antihormonal therapy in breast cancer. ESR1 mutation frequency is high in metastatic disease when the tumor is refractory to aromatase inhibitor, in contrast to nonmetastatic setting. We tried to detect ESR1 mutations (Y537N, Y537S and D538G) in circulating tumor DNA (ctDNA).

Method: Patients who had hormone receptor-positive breast cancer were enrolled in this study. Plasma samples were collected and ctDNA was analyzed by ddPCR and digital targeted DNA panel. Primers and probes for ddPCR were designed for Y537N, Y537S and D538G mutations using sequences of wild-type normal human DNA.

Results: A total of 40 patients were enrolled. Histologic type was invasive ductal carcinoma (IDC) in 32, invasive lobular carcinoma (ILC) in 4 and others in 4. Thirty-four patients received endocrine treatment and 6 patients had no prior exposure to endocrine treatment. Using ddPCR, overall mutation rate was 52.5% (n=21). D538G was the most frequent mutation (n=16), followed by Y537N (n=9), and Y537S (n=6). Nine patients showed multiple ESR1 mutations. In patients without endocrine exposure (n=6), 2 patients showed ESR1 mutations (Y537N, Y537S). The mutation detection rate was higher in patients with prior use of aromatase inhibitor (AI) than in prior use of tamoxifen (TMX) only (prior AI, 17/29=58.6%; prior TMX, 2/5= 40%). In 9 cases, we conducted both ddPCR and targeted DNA panel assay using ctDNA at the same time. Overall concordance rate between two platforms was 55.6% (5/9). Among discordant cases (n=4), ESR1 mutations were detected by ddPCR in 3 cases, which were not found in DNA panel assay.

Conclusions: ESR1 mutations can be robustly identified with ctDNA analysis. Overall mutation detection rate was comparable to other previous reports. However, there is a significant discordance between clinically available NGS panel assay and ddPCR method in detection of ESR1 mutations. Further study will be needed for clinical implementation of ESR1 mutation analysis using ctDNA.

#5538

**Arv7 validation in castrate resistant prostate tumors utilizing Rnascope** in situ **hybridization assay.**

Tanesha Cash-Mason, Jackson Wong, Martinez Martinez, Michael Sharp, Jayaprakash Karkera, John D. Alvarez, Gerald C. Chu, Shibu Thomas, Weiman Li, S. Ken Tian. _Janssen Research & Development, LLC, Spring House, PA_.

Background

Androgen receptor splice variant 7 (ARV7) is a ligand-independent transcription factor associated with resistance to androgen deprivation therapy. There are efforts in development to monitor ARV7 expression during the treatment of patients with prostate cancer.

Methods

We evaluated ARV7 expression in primary and metastatic prostate adenocarcinomas tissues utilizing automated RNAScope (Advanced Cell Diagnostics) ISH assay, Immunohistochemistry (IHC) and quantitative PCR (qPCR). The novel RNAscope ISH assay was used for direct chromogenic visualization of RNA transcripts and to confirm positivity as an orthogonal approach to validate IHC and quantitative qPCR assay findings. We screened a total of 44 CRPC FFPE tumor samples by IHC and a subset of 28 (excluding controls) with the ISH and qPCR assays. Samples for qPCR were enriched for tumor by macrodissection. RNAScope Scoring Criteria was used to score ISH expression and raw Ct values were normalized to the RPL19 housekeeping gene for qPCR. Thresholds for each assay was established by comparing the findings with the other 2 assays. The data was calculated as ARV7 (+) or ARV7 (-) based on thresholds and compared to the other 2 assays to establish assay performance statistics.

Results

The ISH screening of 28 CRPC tumors resulted in 16 tumors with some level of positivity and approximately 10 tumors with significant positivity. Of the 44 CRPC tumors stained by IHC, 16 tumors showed various levels of ARV7 nuclear positivity and 8 tumors with strong positivity.

Conclusions

The detection of ARV7 was achieved in three independent assays. The correlation of three orthogonal data sets were used to identify positive and negative tissues. ISH has the potential for greater sensitivity compared to traditional IHC. The development of automated RNAscope ISH assay allows for more samples to run in a standardized manner with minimal inter-operator variably and requires less hands on-time gives consistently reproducible results.

#5539

An integrated multidisciplinary approach for prediction of HCC development in patients with liver disease utilizing genomics, elastography, imaging and circulating tumor cells.

Emmanuel Thomas, Eugene Schiff, Deukwoo Kwon, Beatrice Madrazo. _University of Miami Miller School of Medicine, Miami, FL_.

Background: HCV induced HCC is higher in minority populations. To serve these communities, it is imperative that we understand the genetic and cellular based mechanisms underpinning the linkages between HCV and HCC. The aims of our study were to assess changes in transient elastography (TE) and fibrosis-4 (FIB-4) score in patients with chronic hepatitis C (CHC) in a large cohort of patients. Patients were also stratified for HCC risk based on genomic and imaging data with inclusion of screening for circulating tumor cells. Methods: Our cohort included 1,400 patients and approximately 500 had liver biopsies. Our retrospective prospective study included 60 patients with CHC and a baseline liver biopsy who achieved SVR after treatment with DAA regimens and had a pretreatment TE study and at least one follow up TE measurement at 24 weeks or later post end of treatment response (EOTR). The estimated stage of liver fibrosis based on TE was categorized as F0-F2 (<9.4kpa), or F3 (9.5 - 12.4 Kpa), or F4/cirrhotics (TE >12.5 kpa). Results: Approximately 1,400 patients were successfully assessed by fibroscan and comparisons made with clinical parameters of liver disease. The median baseline TE for the 60 patients who achieved SVR was 11.9 Kpa (range 3.8 to 65.2) and at follow up, TE decreased to 7.35 Kpa (range 2.9 to 34.8) with a median change in TE of -3.4 Kpa (range -35.3 to +1, p=7.355e- 11). Follow up median TE done in the cirrhotic population after median time of 39 weeks post EOTR decreased to 11.7 Kpa and FIB4 was 2.3. The median change of TE in cirrhotic patients was -6.5 kpa (range -35.3 to +1, p=1.043e-7) and for FIB4 was -1.97 (range -17.47 to -0.33, p=1.49e-8). Non-cirrhotic patients (TE<12.4) comprised 55% of the entire cohort and their median change of TE was -2.4 Kpa (range -6.4 to 0.7, p=1.539e-6) and FIB4 was -0.68 (range -2.8 to 0.41, p=2.987e-6). 48% of the entire cohort down-staged their liver fibrosis as determined by TE. In a multiple logistic regression analysis for factors associated with down-staging in liver fibrosis, we found that patients who were treatment naïve were more likely to improve their fibrosis stage (OR 5.73, p=0.033). Conclusions: Liver fibrosis stage, as determined by TE, improved after achieving SVR with DAA treatments in most patients. Although cirrhotic patients had a more significant drop in their median TE when compared to non-cirrhotic patients, they had a lower probability of improving their fibrosis stage and maintained a continued risk to develop HCC. In addition, we identified a group of patients whose TE score did not improve post SVR also representing a cohort of patients whose HCC risk remained. The patients who remain at risk for HCC will be further stratified for increased propensity to develop HCC utilizing genomics, advanced imaging and screening for circulating tumor cells.

#5540

Clinical significance of soluble forms of immune checkpoint molecules in advanced esophageal cancer.

Juichiro Yoshida, Takeshi Ishikawa, Takayuki Ohta, Tomoyo Yasuda, Osamu Dohi, Tetsuya Okayama, Naohisa Yoshida, Kazuhiro Kamada, Kazuhiko Uchiyama, Osamu Handa, Tomohisa Takagi, Hideyuki Konishi, Yuji Naitoh, Yoshito Itoh. _Kyoto Prefectural University of Medicine, Kyoto, Japan_.

Background: Immune checkpoint molecules such as PDL1 and CD155 are expressed on cancer cells and regulate tumor immunity by binding to ligands on immune cells. Although soluble forms of immune checkpoint molecules have been detected in the blood, the roles of these soluble forms have not been fully elucidated. In this study, the clinical significance of soluble forms of immune checkpoint molecules in advanced esophageal cancer was examined. Methods: Soluble PDL1 (sPDL1), PD1(sPD1), CD155(sCD155), and LAG3 (sLAG3) were measured by ELISA in 47 patients who were diagnosed with advanced esophageal cancer and planned to receive multidisciplinary treatment in our department from April 2015 to March 2017. The levels of these molecules in cancer patients were compared with those of 24 control subjects. Results: The mean age of the tumor group was 66 years (41 years to 78 years), the ratio of males and females was 42: 5. Based on TNM-UICC stage grouping, 8, 24 and 15 patients were in clinical stage II, III and IV, respectively. All patients underwent chemotherapy, 25 patients underwent radiation therapy in combination with chemotherapy, and 26 underwent surgical therapy after chemotherapy. There was no difference in age between two groups, but the proportion of male was significantly higher in the cancer group than in the tumor group (P = 0.014). Regarding sPDL1 and sLAG3, there was no significant difference between two groups, but the levels of sPD1 and sCD155 were significantly higher in the tumor group than the control group (sPD1: P = 0.0225; sCD155: P = 0.0006). In subgroup analysis of patients with esophageal cancer, there was a tendency for higher levels of sPD1 in patients with lymph node metastasis (P = 0.150), large tumor diameter (P = 0.189), and higher levels of serum SCC (P = 0.078). The level of sPD1 was related to neither response rate nor overall survival. On the other hand, we did not find any association between clinical characteristics and the level of sCD155. However, the level of sCD155 tended to be higher in patients with response to chemotherapy (CR / PR) (P = 0.111) and in patients with a longer survival (P = 0.183). Discussion: We confirmed that sPD1 and sCD155 are elevated in advanced esophageal cancer. In addition, results in this study suggested that sCD155 may be a biomarker for therapeutic effect or prognostic prediction of esophageal cancer. Large-scale studies are needed to confirm our findings.

#5541

Clinical impact of hypothyroidism and PD-L1 SNPs in patients having non-small cell lung cancer treated with nivolumab.

Tomoko Funazo, Hiroaki Ozasa, Takashi Nomizo, Takahiro Tsuji, Yuto Yasuda, Hironori Yoshida, Yuichi Sakamori, Hiroki Nagai, Toyohiro Hirai, Young Hak Kim. _Kyoto University Hospital, Kyoto, Japan_.

Although Nivolumab is one of the immune-checkpoint inhibitors that improves the prognosis of lung cancer, it is known to cause immune-related adverse events (irAEs). It has recently been reported that patients with irAEs have a longer progression-free survival (PFS) than those without irAEs. However, there are few detailed reports on irAEs in patients with NSCLC. We previously reported the association between single nucleotide polymorphisms (SNPs) of programmed death ligand 1 (PD-L1) and PFS for nivolumab treatment. Therefore, we hypothesized that the SNPs of programed death 1 (PD-1) and PD-L1 were associated with irAEs and PFS.

Between January 2016 and March 2017, 95 consecutive patients with advanced NSCLC were treated with nivolumab and 80 of these patients participated in this study. Patients who did not provide informed consent, patients without follow-up blood tests, patients with double cancer history, and patients diagnosed with disease progression within 15 days were excluded. The prevalence of genotypes between patients with adverse events and those without adverse events was evaluated. PFS was calculated in 59 out of 80 patients excluding liver metastasis at baseline imaging.

The response rate was 15% and the median PFS was 85 days in this cohort. For all adverse events, hypothyroidism, which is defined as a low free T4 level, was associated with SNPs of PD-L1: rs1411262 and rs822339. Moreover, patients with hypothyroidism had a significantly longer PFS than those without hypothyroidism (67 days vs N.R.; P= 0.0055). The T/T genotype of rs1411262 and the A/A genotype of rs822339 were significantly associated with longer PFS than the C/T and C/C genotypes of rs1411262, and the A/G and G/G genotypes of rs822339, respectively (82 versus 175 days; P = 0.0468).

In conclusion, the T allele of rs1411262 and the A allele of rs822339 were significantly associated with hypothyroidism and longer PFS. SNPs of PD-L1 may be associated with functions of the PD-1 and PD-L1 pathway.

Hypothyroidism and SNPs of PD-L1

---

|

hypothyroidism (-) | hypothyroidism (+) | P value

|

(n=7) | (n=73)

|

rs1411262 | 0 | 19 | 0.0386

CC | 3 | 36

|

CT | 4 | 18

|

TT | |

|

rs822339 | |

|

AA | 0 | 19 | 0.0386

AG | 3 | 36

|

GG | 4 | 18

|

#5543

Low ATM expression is associated with improved progression-free and overall survival in advanced gastric cancer patients treated with platinum-based chemotherapy.

Munveer S. Bhangoo,1 Hubert Y. Luu,2 Seung Tae Kim,3 Jeeyun Lee,3 Joseph Chao,4 Samuel J. Klempner5. 1 _Scripps Clinic, San Diego, CA;_ 2 _University of California, San Francisco, San Francisco, CA;_ 3 _Samsung Medical Center, Seoul, Republic of Korea;_ 4 _City of Hope Cancer Center, Duarte, CA;_ 5 _The Angeles Clinic and Research Institute; Cedars-Sinai Medical Center, Los Angeles, CA_.

Introduction: Platinum-based combination chemotherapy regimens represent standard approaches for locally advanced and metastatic gastric adenocarcinoma. Ataxia-teleangiectasia mutated protein (ATM) plays a critical role in repairing DNA damage to double-stand breaks. Tumors deficient in ATM (defined as pathologic loss measured by IHC) are hypothesized to demonstrate increased susceptibility platinum chemotherapy.

Methods: We retrospectively reviewed patients from a single center (Samsung Medical Center, Seoul, South Korea) with advanced, unresectable gastric cancer (stage 3b or higher) receiving capecitabine/oxaliplatin (XELOX) from 2013-2017. Clinicopathologic data, treatment response, progression-free survival (PFS), and overall survival (OS) were analyzed according to level of ATM expression by immunohistochemistry (IHC). Low-expression of ATM was defined as greater than 50% loss by IHC. Two-tailed, unpaired t-tests and Fisher's exact tests were used to compare outcomes between patients with preserved ATM versus low ATM expression. Kaplan-Meier survival analysis was used to compare disease-free survival and overall survival; the log-rank test was used to determine statistical significance.

Results: During the study period, 138 patients underwent treatment with XELOX for a diagnosis of advanced gastric cancer. Median (IQR) follow-up time was 11.8 mo. (6.8-17.3). Between patients with ATMintact or ATMlow, there were no significant differences in mean age, sex, final pathologic diagnosis, or mean number of cycles of chemotherapy received. Kaplan-Meier analysis demonstrated that patients with ATM low were associated with a longer PFS (ATMlow 14.9 mo versus ATMintact 6.8 mo.) (HR 0.24, [0.18-0.34, 95% CI], p<0.0001), and longer OS (ATMlow 30.0 mo. versus ATMintact 14.7) (HR 0.23, [0.17-0.33, 95% CI], p<0.0001). There was no difference in response rates between the two groups.

Conclusions: This retrospective, case-control study in patients with advanced gastric cancer shows improved survival in patients with ATMlow tumors compared with ATMintact tumors after treatment with platinum-based chemotherapy. Findings are suggestive and require prospective validation in cohorts of patients with ATMlow/ATMintact tumors.

#5544

Project Survival: Year 3 update on a 7-year prospective clinical study driven by quality metrics, multi-omic analysis and artificial intelligence to develop translational biomarkers for pancreatic cancer.

Eric Michael Grund,1 Michael A. Kiebish,1 Viatcheslav R. Akmaev,1 Rangaprasad Sarangarajan,1 John Crowley,2 Amy Stoll-D'Astice,2 Tori Singer,3 Corrine Decicco,3 Wendy Hori,3 Valerie Bussberg,1 Karl Diedrich,1 Leonardo Rodrigues,1 Emily Chen,1 Vivek Vishnudas,1 Robert Najarian,4 Tomislav Dragovich,5 Manuel Hidalgo,4 Niven Narain,1 A. James Moser4. 1 _BERG, Framingham, MA;_ 2 _Cancer Research and Biostatistics, Seattle, WA;_ 3 _Beth Israel Deaconess Medical Center, Boston, MA;_ 4 _Beth Israel Deaconess Medical Center, Framingham, MA;_ 5 _Banner MD Anderson Cancer Center, Gilbert, MA_.

Pancreatic adenocarcinoma is expected to be the second leading cause of cancer-related death by 2020 and has a dismal 5-year survival rate of 8%. The high mortality rate is in part due to the lack of available methods to detect the disease at an early stage or stratify patient populations into more effective treatment regiments in meaningful time frames. To accomplish this, robust quality-controlled OMIC molecular profiling platforms and analytic solutions need to be developed and incorporated into discovery precision medicine protocols to align with translation goals and ensure utility of each sample type. Project Survival, a multisite prospective longitudinal study, is in year 3 of enrolling subjects within 6 categories: healthy volunteers with a relative with pancreatic cancer (N=50), pancreatitis (N=50), pancreatic cystic neoplasm (N=50), suspicious pancreatic masses with pathology other than pancreatic cancer (N=50), early stage (N=200), and metastatic pancreatic cancer (N=200). This study utilizes a systems medicine approach for translational biomarker discovery by performing analysis of matched subject sera, plasma, buffy coat, saliva, urine, and tumor/adjacent normal tissues and integrating them with the respective full clinical annotation using the BERG Interrogative Biology® platform. Multiple longitudinal time points are taken over the course of the six-year timeline, enabling dynamic modeling. Proteomic, signaling lipidomic, structural lipidomic, and metabolomic analysis are performed on all samples and stringent quality control metrics are engaged to ensure quantitative accuracy of the platforms over time. Additionally, several analytic normalization tools are employed to align longitudinal OMIC datasets for quantitative comparisons. We utilized bAIcis™ (BERG Artificial Intelligence Clinical Information System) to align multi-omic profiles with longitudinal clinical information to infer probabilistic cause-and-effect relationships among molecular and clinical variables in a network-based model. We will be presenting updated enrollment and preliminary quality metrics across the study and sites, along with initial molecular stratification markers for clinical endpoints.

### Immunomodulatory Agents and Interventions 3

#5545

Heterodimeric interleukin 15 (hetIL-15) treatment decreases primary breast cancer 4T1 tumors and alleviates the metastatic burden.

Dimitris Stellas, Konstantinos Dimas, Bethany A. Nagy, Antonio Valentin, Barbara K. Felber, George N. Pavlakis. _National Cancer Institute at Frederick, Frederick, MD_.

Introduction: In this preclinical study, we tried to model the therapeutic strategies often used in the clinic to treat breast cancer patients. We analyzed the role of hetIL 15 immunotherapy alone and in combination with chemotherapy and surgery using the 4T1, a triple-negative breast cancer cell line, murine breast cancer model.

Experimental Procedures: We evaluated the therapeutic potential of combining surgery, chemotherapy (doxorubicin) and immunotherapy (hetIL-15) in the murine 4T1 breast cancer model. Cancer cells were orthotopically implanted into the mammary fat pad of Balb/c mice, and the efficacy of hetIL-15 in combination with doxorubicin on the growth of primary tumors and metastatic disease was assessed. In addition, we monitored the impact of surgery alone in the development of metastatic disease, and, finally, we evaluated the effects on the metastatic burden of the three different approaches combined.

Results: hetIL-15 monotherapy and combination of doxorubicin chemotherapy with hetIL-15 immunotherapy inhibited the growth of primary tumors. Interrogating the role of these treatments on lung metastatic disease, the results were unexpected. Doxorubicin alone did not affect the numbers of the metastatic lesions in the lungs. In contrast, hetIL-15 monotherapy reduced the metastatic burden. The combination strategy did not show advantage as compared to hetIL-15 monotherapy. Surgical removal of the growing tumor also decreased metastatic burden, especially when performed at an early point. Chemotherapy with doxorubicin prior to and post-surgery had no impact on the metastatic disease. In contrast, hetIL-15 treatment strongly diminished or eliminated the metastatic burden.

Conclusion: Our data strongly suggest that treatment with hetIL-15 as an adjuvant therapy after surgery, or as a neoadjuvant agent before surgery, significantly impacts metastatic disease.

#5546

eFT508, a potent and highly selective inhibitor of MNK1 and MNK2, regulates T-cell differentiation promoting an antitumor immune response.

Rajesh K. Sharma, Vikas K. Goel, Jocelyn Staunton, Maria Barrera, Ana Parra, Eric Sung, Gary G. Chiang, Kevin R. Webster. _eFFECTOR Therapeutics, San Diego, CA_.

An effective and durable T-cell response is a cornerstone of current immunotherapies. We show that eFT508, a potent, selective inhibitor of MNK1 and MNK2, establishes a regulatory program that promotes multiple steps in the cancer immunity cycle including expansion of memory T cells and prevention of T-cell exhaustion. Using OT-I and OT-II transgenic systems, we show that eFT508 shifts the distribution of T cells towards a CD62LhighCD44high central memory (CM) phenotype in both CD4 and CD8 T cells upon activation with SIINFEKL peptide in vitro without adverse effects on T-cell proliferation, interferon-γ production or cytotoxic function. Similar effects are seen in vivo, where eFT508 treatment also enriches the CM T-cell pool in a SIINFEKL vaccine-induced OT-I adoptive T-cell transfer model, which results in increased persistence as demonstrated by a higher memory-recall T-cell response upon rechallenge. In addition, the CM bias elicited by eFT508 remains dominant when combined with agonists of co-stimulatory molecules, such as 4-1BB, OX-40 and GITR, or checkpoint inhibitors, such as PD-1, PD-L1 and CTLA-4, suggesting that eFT508 can affect the rate of T-cell differentiation in these combinations. eFT508 treatment also reduces the expression of exhaustion markers such as PD-1, LAG3 and TIM3, leading to increased cytotoxic T-cell function. eFT508 is currently under evaluation as a single agent in two phase 1/2 clinical trials for patients with advanced solid tumors and patients with advanced lymphoma. In addition, a phase 2 study evaluating eFT508, alone or in combination with avelumab, a PD-L1 immune checkpoint inhibitor, in microsatellite stable relapsed or refractory CRC patients is ongoing. The preclinical studies presented here provide further evidence that eFT508 may combine well with additional immunotherapies beyond checkpoint blockade.

#5547

Durable remission with nivolumab, anti-PD-1 monoclonal antibody, in a patient with advanced thymic carcinoma.

Young Kwang Chae, Anderson Cho, Kyunghoon Rhee, Andrew Davis, Wade Iams, Manali Bhave, Marcelo Cruz, Lee Chun Park. _Northwestern Univ., Chicago, IL_.

Thymic carcinoma is a rare neoplasm with poor prognosis that forms on the outside surface of the thymus. Due to the rarity of the disease, there are no randomized clinical trials that guide therapy. Currently, there are clinical trials under way to assess the efficacy of anti-programmed death 1 (PD-1) monoclonal antibodies for the treatment of advanced thymic carcinomas. However, the role of PD-1 inhibitors in treating thymic carcinoma remains mostly unknown. Here we present a case of a patient with thymic carcinoma who showed durable remission when treated with nivolumab. 61-year-old male was diagnosed with thymic carcinoma during an episode of COPD exacerbation in April 2015. The patient was treated with neoadjuvant chemotherapy regimen consisting of four cycles of carboplatin and paclitaxel every three weeks from June to September 2015. The mass was resected in October 2015. Margins were found to be grossly positive. The patient was deemed a poor candidate for adjuvant chemotherapy, and in February 2016 the decision was made to begin 3 mg/kg nivolumab therapy considering the poor prognosis of disease and high risk of recurrence. Close to two years after initiating therapy, he currently has no measurable disease and tolerates the medication without side effects. This is the first report demonstrating durable remission of thymic carcinoma with nivolumab treatment. Our case suggests that prospective trials evaluating the use of nivolumab in patients with thymic carcinoma are warranted.

#5548

The therapeutic potential of AMV564, a novel bispecific bivalent (2x2) T-cell engager, for the treatment of CD33-expressing hematologic malignancies.

Tae H. Han,1 Jeanmarie Guenot,1 William S. Denney,2 Eric J. Feldman1. 1 _Amphivena Therapeutics, South San Francisco, CA;_ 2 _Human Predictions, Cambridge, MA_.

AMV564 is a novel bispecific bivalent (2x2) immunotherapy that initiates an immunological synapse between T cells and CD33-expressing cells by binding both CD33 and CD3 with strong avidity. CD33 (Siglec-3) is a clinically-validated antigen target that is broadly expressed on the surface of malignant cells in acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and other hematologic malignancies, as well as on cells that potently suppress immune function, such as myeloid-derived suppressor cells (MDSC). AMV564 has been shown to eliminate MDSCs and restore hematopoiesis, induce potent cytolysis of leukemic cells in patient blood and bone marrow samples, and produce robust antitumor activity in a disseminated model of AML. In vitro studies, including cytotoxicity assays with AML patient samples, human T-cell activation assays, and cytokine-release assays, as well as animal safety studies were performed to characterize the pharmacokinetics, pharmacodynamics, and potential toxicities of AMV564. AMV564 demonstrated potent in vitro antitumor activity with EC50 values of approximately 0.7 - 3 pM. In vitro studies also demonstrated that leukemic cells are more sensitive to T-cell engagement using AMV564 than peripheral blood mononuclear cells by approximately 10-fold. In animal safety studies, AMV564 was tolerated at exposures that exceed those expected to have clinical antitumor activity by > 10-fold. Toxicological findings noted in these studies were anticipated based on the pharmacological activity of AMV564; observed findings included events related to CD3-mediated T-cell activation, cytokine release, and decreases in neutrophil and monocyte counts with rapid recovery. Results from a pharmacokinetic/pharmacodynamic model integrating the preclinical data suggest the potential for a wide therapeutic index for patients with AML. Based on the in vitro and in vivo preclinical data, AMV564 is expected to have a wide therapeutic index, with robust antitumor activity in AML and MDS at doses well below those predicted to be associated with dose-limiting toxicities, and a predictable and monitorable adverse event profile.

#5549

Antitumor efficacy of the oncolytic peptide LTX-315 in genetic mouse models that resist conventional chemo- and immunotherapeutic treatments.

Mikael J. Pittet,1 Hsin-Wei Liao,1 Baldur Sveinbjørnsson,2 Øystein Rekdal2. 1 _Massachusetts General Hospital, Boston, MA;_ 2 _Lytix Biopharma, Tromso, Norway_.

LTX-315 is an oncolytic peptide that induces immunogenic cancer cell death and is currently evaluated in phase 1 clinical trials for its ability to trigger recruitment of tumor-specific T cells to tumors. Previous experimental studies identified LTX-315's ability to control cancer in mice grafted with various tumor cell lines. Here we extend our analyses to conditional genetic mouse models of both melanoma (driven by Braf and Pten alterations) and soft tissue sarcoma (driven by Kras and P53 alterations). In these genetic models, cancer cells are derived from somatic cells that are transformed in their normal tissue microenvironment and progress to high-grade tumors that are poorly infiltrated by T cells and typically resist prescribed chemo- and immunotherapeutic treatments. We report that LTX-315 not only delays tumor progression substantially in both models, but also profoundly alters the tumor microenvironment, most notably characterized with CD8+ T cell, NK cell and dendritic cell infiltration. Transition from a 'cold' to a 'hot' tumor microenvironment was also observed in patients with melanoma, sarcoma and breast cancer. Furthermore, CD8+ T cell depletion in mice abrogated long-term antitumor efficacy of LTX-315, indicating that these cells are required for drug-induced tumor control. Importantly, the ability to convert non-T-cell-infiltrated tumors into ones that display antitumor T cell immunity opens the possibility to prime and make unresponsive tumors sensitive for systemic immune treatments.

#5550

Translational biomarkers for SAR439459, an anti-TGFβ antibody for cancer immunotherapy.

Joachim Theilhaber,1 Jean Cavallo,2 Stephen L. Madden,2 Charlene Manning,2 Sherry Cao,2 Parminder Mankoo,1 Robert Pomponio,3 Hongjing Qu,1 Natalia Malkova,1 Gary Shapiro,1 Christopher Winter,1 Dmitri Wiederschain,1 Michele Sanicola-Nadel,1 Fangxian Sun,1 Tun Tun Lin,1 Richard C. Gregory,1 Jack Pollard1. 1 _Sanofi Oncology, Cambridge, MA;_ 2 _Sanofi Translational Sciences, Framingham, MA;_ 3 _Sanofi Translational Medicine, Cambridge, MA_.

TGFβ is a potent immunosuppressive cytokine, acting on multiple cell types of both the innate and adaptive immune systems. Emerging preclinical and clinical data implicate TGFβ in tumor immune evasion, poor prognosis and resistance to PD1/PDL1 directed checkpoint therapy. Thus, neutralization of TGFβ is suggested to relieve immunosuppression via mechanisms that are distinct yet complementary to checkpoint inhibitors such as PD1. SAR439459 is a potent human pan-neutralizing anti-TGFβ antibody that has entered first-in-human studies in advanced solid tumors (NCT03192345*).

I. Biomarkers for indication selection: A gene expression signature of TGFβ pathway activation was derived from analysis of TGFβ-stimulated versus naïve cancer cell lines. Queries of the signature against The Cancer Genome Atlas (TCGA) revealed that primary head and neck, ovarian, and colorectal cancers are enriched for activation.

II. Biomarkers for patient selection: An analysis of the patients enriched for activation revealed that mesenchymal tumors predominate. Machine learning methods were applied to panels of gene expression data for colorectal (CRC), ovarian serous and head and neck squamous cell carcinoma to derive a two-gene biomarker for selection of patients with mesenchymal tumors. Implementation in a logistic regression model achieves good for prediction in CRC, and similar performance in other indications. Patient cohorts are enriched over three-fold for the mesenchymal subtype and false-negative rates are acceptable. Implementation of the two-gene biomarker on a low complexity platform and its validation on an independent panel of samples is described.

III. Biomarkers for assessment of the tumor microenvironment: In partnership with NeoGenomics, the immune contexture of patient tumors was evaluated using MultiOmyx, a multiplex IHC assay, on CRC and melanoma. Multiplexing was conducted with 12 biomarkers (jointly describing 22 immune cell types) on single FFPE section from each tumor sample. Pilot studies included a range of inflammation to evaluate how well the analytics assess each tumor type and correlate to possible treatment effects. Statistical methods were developed to assess differences at the cell population level, including replicate concordance, volcano plots for analyses of variance, and correlation matrices. The MultiOmyx assay demonstrated excellent technical reproducibility and precision, a favorable dynamic range, inflammation status differences in select immune cells and regions of interest, and included both positive and negative correlations between cell populations.

* https://clinicaltrials.gov/ct2/show/NCT03192345

#5551

SGN-2FF: A small-molecule inhibitor of fucosylation modulates immune cell activity in preclinical models and demonstrates pharmacodynamic activity in early phase 1 analysis.

Nicole M. Okeley,1 Ryan A. Heiser,1 Weiping Zeng,1 Shawna Mae Hengel,1 Jason Wall,1 Peter C. Haughney,1 Timothy Anthony Yap,2 Francisco Robert,3 Rachel E. Sanborn,4 Howard Burris,5 Laura Q. Chow,6 Khanh T. Do,7 Martin Gutierrez,8 Karen Reckamp,9 Amy Weise,10 D Ross Camidge,11 John Strickler,12 Conor Steuer,13 Zejing Wang,1 Megan M. O'Meara,1 Stephen C. Alley,1 Shyra J. Gardai1. 1 _Seattle Genetics, Bothell, WA;_ 2 _MD Anderson Cancer Center, Houston, TX;_ 3 _University of Alabama at Birmingham Hospital, Birmingham, AL;_ 4 _Robert W. Franz Cancer Center, Providence Cancer Insititute, Portland, OR;_ 5 _Sarah Cannon Research Institute, Nashville, TN;_ 6 _University of Washington, Seattle, WA;_ 7 _Dana-Farber Cancer Institute, Boston, MA;_ 8 _John Theurer Cancer Center, Hackensack, NJ;_ 9 _City of Hope, Duarte, CA;_ 10 _Karmanos Cancer Institute, Detroit, MI;_ 11 _University of Colorado Hospital, Aurora, CO;_ 12 _Duke Cancer Center, Durham, NC;_ 13 _Emory Winship Cancer Institute, Atlanta, GA_.

SGN-2FF, an orally bioavailable small molecule inhibitor of glycoprotein fucosylation, demonstrates encouraging preclinical antitumor activity in mouse models with suggested multiple mechanisms of action, including direct and indirect effects on immune cells, tumor cells, and the tumor microenvironment. The effects of SGN-2FF were evaluated on tumors implanted in multiple strains of mice to determine how differences in the immune repertoire affect the antitumor activity. SGN-2FF treatment of nude mice, which maintain functional B cells and antibody production, resulted in a delay in LS174T tumor growth compared with untreated mice, while LS174T tumors in SCID mice, which lack B cells, were unaffected by SGN-2FF. These data suggest that activity of SGN-2FF in nude mice may be dependent on residual B cells and circulating antibodies. The antitumor effect of SGN-2FF in syngeneic mouse models with intact immune systems also appears to be dependent on T cell activity. Transfer of T cells isolated from SGN-2FF-treated tumor-bearing mice to naïve tumor-bearing mice was sufficient to delay tumor growth. T cells isolated from untreated tumor-bearing mice did not have the same effect. These results demonstrate that afucosylated immune cells play a key role in the preclinical activity of SGN-2FF.

Various preclinical assays were used to detect SGN-2FF-mediated changes in cellular and IgG fucosylation important for biological activity. These assays are being applied in evaluating patient samples in the ongoing phase 1, multicenter, dose-escalation study investigating the safety, tolerability, PK, and biomarkers of antitumor activity of SGN-2FF administered orally to adult patients with advanced solid tumors (NCT# 02952989). Changes in peripheral IgG fucosylation, absolute neutrophil count, and immune cell surface fucosylation were identified as initial biomarkers for proof of pharmacodynamic activity. Preliminary data following daily doses of SGN-2FF demonstrate that cell surface fucosylation on granulocytes was significantly reduced and neutrophil count was significantly increased in 6 of 7 treated subjects; additionally, IgG fucosylation was significantly decreased in 7 of 7 subjects. PK have been characterized, and preliminary results are within the expected range as predicted from preclinical studies. Following daily administration of SGN-2FF, accumulation of the active metabolite, GDP-2FF, was observed intracellularly, while no accumulation of SGN-2FF was observed in plasma. Collectively, these data demonstrate robust biological effects of SGN-2FF. The pharmacodynamic biomarkers and PK analysis are informing next steps in identifying an optimal dose and dosing schedule for SGN-2FF.

#5552

Novel, heterocyclic small molecule inhibitors of PD-1 and PD-L1 pathway.

Sivanandhan Dhanalakshmi,1 Shivani Garapaty,1 Chandregowda Venkateshappa,1 Guru Pavan Seerapu,1 Reshma Das,1 Pradeep Nagaraj,1 Ronodip Kar,1 Anuj Kumar Singh,1 Venkatesha Ashokkumar Venkatesubbiah,1 Ramakishore VP Putta,1 Muralidhar Pendyala,1 Girisha Lokesh,1 Hari Madaka,1 Harikrishna Reddy Thummuru,1 Shikas AP,2 Prateeksha Anchan,1 Prathima Bhat,1 Rudresha G,1 Mohd Zainuddin,1 Krishnakumar V,1 Ramachandraiah Gosu,1 Rajendra Kristam,1 Jeyaraj DA,1 Sriram Rajagopal1. 1 _Jubilant Biosys Ltd., Bangalore, India;_ 2 _Jubilant Biosys Ltd., Bengaluru, India_.

The PD-1/PD-L1 molecular pathway is one of the primary mechanisms of immune evasion deployed by cancer cells. Induction of PD-L1 expression on cancer cells is associated with inhibition of immune responses against cancer, thus permitting cancer progression and metastasis. Activation of PD-1/PD-L1 pathway induces apoptosis of activated T-cells, inhibits their proliferation, facilitates T-cell anergy and exhaustion and enhances the function of regulator T-cells. Therefore, blocking this pathway restores the proliferation and cytotoxicity of CTLs, inhibits the function of Tregs and results in decreased T-cell apoptosis. A number of cancer immunotherapy agents targeting PD-1/PD-L1 have been developed and approved for a number of malignancies (PD-1: Nivolumab, Pembrolizumab, PD-L1: Atezolizumab, Avelumab, Durvalumab). However, there is a still a need for potent, selective small molecule inhibitors of the PD-1/PD-L1 pathway. The approved therapies require bolus intravenous injections, are administered in high dose and have a long half life. The long residence time of these mAbs could contribute to the well-documented drug-related adverse effects. Small molecule inhibitors, therefore, can provide increased oral bioavailability, increased bio-efficiency and shorted half life activity for a more controllable treatment, particular in the case of auto-immune or other adverse effects. Rational and structure guided de novo design approaches were used to design novel small molecule PD-1/PD-L1 pathway inhibitors; potency of these inhibitors was assessed in an in-vitro TR-FRET assay. Checkpoints signaling reporter assays as well as ex-vivo co-culture assays were used to assess the ability of the compounds to restore T-cell proliferation and function. Three novel chemical series as potent PD-1/PD-L1 pathway inhibitors are being developed for the treatment of cancer. Compounds from these series showed strong in vitro potency of 0.01 to 0.2 µM against PD-1/PD-L1. JBI-426 exhibited an IC50 of 0.04 µM and no cytotoxicity against cancer cell proliferation per se. JBI-426 showed good in vitro ADME properties in terms of aqueous solubility, metabolic stability, permeability and excellent oral bioavailability in mouse pharmacokinetics. In a RENCA syngeneic model, oral administration of JBI-426 at 50 mg/kg resulted in a strong tumor growth inhibition, comparable (or better) than the PD-L1 mAb, and was well tolerated. The effect of JBI-426 on tumor infiltrating lymphocytes was also assessed; a significant increase in CD8+ cytotoxic lymphocytes was observed. Further studies to assess additional compounds from the three chemical series are underway. The oral administration route of these PD-1/PD-L1 inhibitors would provide an attractive alternate to the currently available antibodies in treating cancer either as a stand-alone therapy or in combination with other immuno-modulatory agents, as well as other standard of care agents.

#5553

A novel immunocytokine for the treatment of cancer.

Mattia Matasci,1 Emanuele Puca,2 Tiziano Ongaro,1 Sarah Wulhfard,1 Alessandra Villa,1 Dario Neri2. 1 _Philochem AG, part of the Philogen Group, Otelfingen, Switzerland;_ 2 _ETHZ, Zurich, Switzerland_.

Interleukin-12 (IL12) is an immunomodulatory cytokine, which offers unique opportunities for cancer therapy due to its stimulatory function on cell-mediated immunity and its anti-angiogenic activity. IL12 was shown to polarize CD4+ T cells into a TH1 type and to be a key activator of NK cells and CD8+ T cells. It also induces the production of interferon-gamma by T cells and NK cells and subsequently of the anti-angiogenic chemokines CXCL10/IP-10, and CXCL9/Mig. However the potent anti-tumor IL12 activity, which has been reported in mice, has not yet been successfully translated into clinical development, mainly because of early reports of severe toxicity and low response rates in human. Following our pioneering work which started in 2002 with the first description of an IL12 based antibody-cytokine fusion protein (i.e. Immunocytokine), we have extensively explored and perfected alternative molecular formats, with the aim to further improve biodistribution properties and therapeutic activity of IL12-based immunocytokines. Here we describe the development and evaluation of new targeted variants of both murine and human IL-12 with enhanced therapeutic efficacy and improved safety profile. To this end we combined the immunomodulatory properties of the IL12 payload with the tumor-homing activity of the L19 antibody. L19 is a clinical grade fully human antibody, which recognizes with identical affinity in both mouse and human, the alternatively spliced EDB domain of fibronectin. EDB represent an optimal target for anti-cancer pharmacodelivery, due to its pan-tumoral over-expression nature combined with very low expression levels in normal tissues. This has also been confirmed by extensive Nuclear Medicine work in which radiolabeled L19 has been administered to more than 150 patients, making L19 one of the best validated tumor-targeting agent. Our novel immunocytokine, termed IL12-L19-L19, relies on the L19 antibody in tandem diabody format, with a monomeric IL12 moiety expressed as single-chain polypeptide at the N-terminal extremity. Recombinant IL12-L19-L19 proteins based either on human or murine IL12, were efficiently expressed in CHO cells and purified to high quality. The tumor-targeting properties of both variants were validated in tumor-bearing mice, using radioiodinated protein preparations. In preclinical therapy studies IL12-L19-L19 showed potent anti-cancer activity when used either as single agent or in combination with other anticancer agents. PK studies in Cynomolgus Monkey using the fully human IL12-L19-IL19 product, revealed a favorable profile, which is compatible with other clinical-stage immunocytokines based on antibody-fragments. These results strongly support the further development of the fully human IL12-L19-L19 product for future clinical investigations.

#5554

Preclinical characterization of GSK532, a novel STING agonist with potent anti-tumor activity.

Jingsong Yang, Michael Adam, Jim Clemens, Katrina Creech, Jess Schneck, Kishore Pasikanti, Jean-Luc Tran, Devika Joglekar, Chris Hopson, Scott Pesiridis, Joshi Ramanjulu, Yiqian Lian, Jerry L. Adams, James Smothers, Axel Hoos. _GlaxoSmithKline, Collegeville, PA_.

The cGAS-STING pathway is the major cytosolic DNA sensing pathway to induce downstream innate immune responses against viral infection as well as tumorigenesis. Recently it has been demonstrated that cGAS-STING pathway takes central stage in the immune sensing of tumor. STING activation induces the production of type I interferons (IFNα and β) and pro-inflammatory cytokines that collectively stimulate tumor antigen cross presentation to CD8 T cells and primes the adaptive immune response against tumor. In order to evaluate the potential of STING agonism to activate the anti-tumor immune response, we developed GSK532, a novel cyclic dinucleotide (CDN). Unlike cGAMP, the endogenous CDN ligand for STING, GSK532 potently activates STING both in vitro and in vivo. In vitro functional assays demonstrated the ability of GSK532 to induce cytokine responses in a panel of human PBMC samples with various STING haplotypes. Furthermore, GSK532 was shown to activate STING orthologs from preclinical species including cyno, minipig, dog, rat and mice. GSK532 demonstrated high stability in human whole blood with minimal degradation observed over 52 hrs. When dosed intratumorally, GSK532 induced a strong anti-tumor effect in the CT26 murine syngeneic model in both the injected (primary) and uninjected (distal) tumors. The cured mice were resistant to rechallenge with same tumor cell line indicating the involvement of an adaptive immune response. Further evidence of the development of anti-tumor adaptive immune response comes from the observation that the efficacy of GSK532 was significantly attenuated when CD8+ T cells were depleted in the murine model. Consistent with the anti-tumor efficacy, pharmacokinetic analysis demonstrated high exposure and long retention of GSK532 in both injected and distal tumors and pharmacodynamic studies showed induction of IFNβ and several inflammatory cytokines (TNFα, IL-6, etc) in both tumors. Currently, GSK532 is being progressed towards clinical investigation. All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed.

#5555

Discovery of DN-016: A highly potent, selective and orally available IDO1 inhibitor for treating cancers.

Shoujun Chen, Fengtao Liu, Hongli Guo, Shuwen Ren, Yikun Zeng, Wei Yang, Ping Qing, Tao Chen, Feng Zhou, Guocheng Li, Mingliang Sun, Xiaogang Ye, Xingzhong Zhang, Panhu Zhu, Hui Xu, Pei Li, Donghai Li, Zang Jie, Huanping Li, Shuda Zhao, Jiangjing Tan, Heping Yang, Longsheng Wang, Fang Liu, Guangliang Fu, Jianggang Du, Hongye Yang, Wenting Xue, Pei Wang, Lan Guo, Lu Wang, Qun Li, Yuxun Wang, Daxin Gao. _Shanghai Denovo Pharmatech, Shanghai, China_.

Indoleamine-2,3-dioxygenase (IDO1) is well-recognized as an important target for immunotherapeutic intervention. Growing interests on this target have been demonstrated by the recent expansion of enthusiasms to inhibit the Trp-to-Kyn pathway as a means to control immunosuppression for cancer treatment. Clinical validation of IDO1 inhibitors for treating various tumors including glioblastoma, melanoma, non-small cell lung, pancreatic, and/or breast cancer, as well as metastatic diseases are currently pursued by pharmaceutical companies and other sponsors. These IDO1 inhibitors include several pioneering clinical compounds such as INCB024360 (epacadostat), GDC-0919, indoximod, and BMS-986205, etc. At the 2017 Annual Meeting of the American Society of Clinical Oncology (ASCO), it was reported that patients with squamous cell carcinoma of head and neck were responded well to the combinations of epacadostat plus pembrolizumab and epacadostat plus nivolumab. In addition, epacadostat also demonstrated very promising efficacy in combination with PD-1 checkpoint inhibitors for treating various kind of solid tumors. Most recently, at the 32nd SITC annual meeting held in this month, very exciting and encouraging results were just reported by BMS from an early clinical trials for the combo studies of BMS-986205 with Opdivo. Previously we reported a moderately potent IDO1/TDO dual inhibitor DN131 for cancer immunotherapy (AACR2015 Abstract# 4877), herewith we wish to present a much more potent and selective IDO1 inhibitor DN-016 with superior drug-like properties. DN-016 is a novel heterocyclic compound that was discovered through structure-based drug design and medicinal chemistry approaches. In in vitro studies, DN-016 showed very high potency in inhibiting hela IDO1 cell with an IC50 of 0.71 nM. It exhibited good ADME properties and safe hERG parameter with IC50 over 30 μM. Compared to the reference compounds in clinical stages, DN-016 exhibited much superior permeability with excellent efflux ratio (A-B/B-A: 22 × 10-6 cm.s-1 / 24 × 10-6 cm.s-1). In in vivo rat PK studies, DN-016 showed very high oral bioavailability (100% F) when dosing at 10 mg/kg via po. Currently DN-016 is under preclinical evaluation as a single agent and together with a PD-1 inhibitor. In summary, as a new anticancer agent, DN-016 demonstrated a remarkable in vitro potency and selectivity with favorable pharmacokinetic properties. Detailed preclinical evaluation of DN-016 will be presented.

#5556

Combining adenosine receptor inhibition with AB928 and chemotherapy results in greater immune activation and tumor control.

Matt J. Walters, Dana Piovesan, Joanne Tan, Daniel DiRenzo, Fangfang Yin, Dillon Miles, Manmohan R. Leleti, Tim Park, Ferdie Soriano, Eshan Sharif, Ulrike Schindler, Jay P. Powers. _Arcus Biosciences Inc., Hayward, CA_.

INTRODUCTION: Extracellular adenosine triphosphate (ATP) is efficiently hydrolyzed to adenosine by ecto-nucleotidases CD39 and CD73, which converts adenosine-monophosphate (AMP) into adenosine (ADO). ADO suppresses immune responses including those of T cells, natural killer (NK) cells, and dendritic cells (DC) through activation of A2aR and A2bR receptors. Treatment of cancer cells with platinum-based and anthracycline chemotherapy has been shown to induce immunogenic cell death (ICD), characterized by increased extracellular ATP levels, and upregulation of CD39 and CD73, leading to the enhanced generation of adenosine.

METHODS: The ability of AB928 to inhibit adenosine-mediated suppression of immune cell function was assessed using human CD4 and CD8 cells. BALB/c mice were inoculated with AT-3-OVA tumors, which express the model antigen ovalbumin, and subsequently treated with chemotherapy or AB928 alone, or a combination of both. The growth rate and immune composition of the tumors were assessed (flow cytometry).

RESULTS: Consistent with the ability of adenosine to suppress immune function, AB928 inhibited the ability of adenosine to suppress CD4 or CD8 T cell activation. Tumor-bearing mice that have been treated with either doxorubicin (DOX, an anthracycline) or oxaliplatin (OX) exhibit increased expression of CD39 and CD73. Established AT-3-OVA tumors treated with AB928 alone had a small but significant decrease in their growth rate; similarly, tumors treated with chemotherapy exhibited a reduction in growth rate. Consistent with the ICD hypothesis, treatment of AT-3-OVA-bearing mice with DOX or OX resulted in an increase in OVA-specific CD8 T cells in the tumor. Concurrent treatment with AB928 and chemotherapy resulted in significantly reduced tumor growth rates, when compared to chemotherapy alone. Analysis of the tumor-infiltrating cell populations showed a significant increase in OVA-specific CD8 T cells, relative to those treated with chemotherapy alone.

CONCLUSIONS: Treatment of tumor-bearing mice with a combination of AB928, a dual A2aR and A2bR antagonist, and ICD-inducing chemotherapy results in increased tumor antigen-specific CD8 T cell responses and significantly reduced tumor growth. AB928 is currently undergoing clinical evaluation.

#5557

EnanDIM, a family of potent enantiomeric TLR9 agonists, modulate the tumor microenvironment and show single-agent antitumor effects in various syngeneic murine models.

Barbara Volz,1 Kerstin Kapp,1 Detlef Oswald,1 Burghardt Wittig,2 Manuel Schmidt1. 1 _Mologen AG, Berlin, Germany;_ 2 _Freie Universitaet Berlin, Berlin, Germany_.

Introduction: TLR9 agonists, being recognized via non-methylated CG-motifs, target the Toll-like receptor 9 (TLR9). This way, they activate the innate and the adaptive immune system. Two different families of TLR9 agonists are currently used in preclinical and clinical studies: Covalently-closed dumbbell-shaped DNA molecules with natural phosphodiester backbone which are protected from degradation by their conformation (dSLIM®) and single-stranded, oligodeoxynucleotides (CpG-ODN) commonly stabilized by phosphorothioates (PTO). However, chemical PTO-modifications have resulted in off-target effects and an unfavorable risk-to-benefit ratio in clinical trials. EnanDIM® are a new family of TLR9 agonists. EnanDIM® molecules consist of linear single-stranded ODN. Protection against exonucleases is achieved through L-deoxyribose nucleotides instead of the naturally occurring D-deoxyribose nucleotides at their 3'-ends. EnanDIM® lead to pronounced induction of cyto- and chemokines, like IFN-alpha and IP-10, as well as broad activation of immune cells, like monocytes, NK cells and dendritic cells, within PBMC. Therefore, EnanDIM® were evaluated in various syngeneic murine tumor models for their anti-tumor effects.

Methods: Mice were subcutaneously inoculated with either CT26 (colon carcinoma), MC38 (colon carcinoma), EMT-6 (breast cancer), B16F10 (melanoma) or A20 (lymphoma) cells for solid tumor growth. EnanDIM® (250 µg per dose, intratumoral) was used to treat established tumors. In addition, the capability of EnanDIM® to modulate the tumor micro-environment (TME) was analyzed via immunohistochemistry in a syngeneic tumor model.

Results: Mice in all models showed clear reduction in tumor growth (TGI: 53 to 85%) and, therefore, an increased survival (p ≤0.001, logrank). Notably, in the EMT-6 tumor model 8/10 mice showed complete tumor regression after EnanDIM® injection. Those 8 mice rejected EMT-6 tumor cells in a subsequent re-challenge study, indicating a sustained immune memory against the tumor, which was in contrast to age-matched naïve mice. When analyzing the TME of mice from the CT26 model an increased immune cell infiltration into the tumor by CD3+, especially CD8+ T cells was detected, showing the ability of EnanDIM® to beneficially modulate the TME.

Conclusions: The enantiomeric TLR9 agonists EnanDIM® show promising anti-tumor effects in a variety of syngeneic murine tumor models and a beneficial modulation of the TME towards an increase of anti-tumor effector cells. These data provide the basis for a further development of EnanDIM® in cancer.

#5559

Agonist-redirected checkpoint (ARC), TIM3-Fc-OX40L, for cancer immunotherapy.

George Fromm, Suresh de Silva, Kellsey Johannes, Arpita Patel, Josiah C. Hornblower, Taylor H. Schreiber. _Shattuck Labs, Inc., Durham, NC_.

Current attempts at combination immunotherapy with bispecific antibodies, linked scFv's or T cell engagers have not demonstrated that both checkpoint blockade and TNF receptor activation can be achieved with a single molecule. This is likely due to the fact that these molecules lose target avidity when engineered to bind multiple targets with monovalent antigen binding arms. Fusion proteins incorporating the extracellular domain (ECD) of type I membrane proteins (eg. Enbrel, Orencia) or type II membrane proteins (eg. OX40L-Fc, GITRL-Fc), linked to the hinge-CH2-CH3 domain of antibodies are both functional, despite the fact that the ECDs are in opposite orientation. Here we report the generation of a two-sided fusion protein incorporating the ECD of TIM3 and the ECD of OX40L, adjoined by a central Fc domain.

Initial confirmation of the structural integrity of TIM3-Fc-OX40L was performed using functional ELISA assays, wherein simultaneous binding to GAL9 and OX40 was observed. A potential advantage of utilizing the intact ECD of TIM3 is the ability to bind all candidate TIM3 ligands, and the TIM3 end of the fusion protein binds both GAL9 and phosphatidylserine (PS) on the surface of human tumor cells. The OX40L end of the fusion protein binds OX40 on the surface of primary T cells. TIM3-Fc-OX40L activates NFκB signaling in cells engineered to overexpress OX40 and an NFκB-luciferase reporter in the absence of Fc receptor cross-linking. Additionally, the TIM3-Fc-OX40L ARC added to primary human PBMCs along with the super-antigen Staphylococcal enterotoxin B, induced secretion of the cytokines IL2 and TNFα. In vitro human T cell/tumor cell co-culture studies indicated that TIM3-Fc-OX40L stimulated enhanced T cell mediated killing of GAL9 positive human tumor cells as compared to OX40L-Fc controls. In vivo, TIM3-Fc-OX40L stimulated significant expansion of antigen-specific CD4 and CD8 T cells in mice adoptively transferred with OT-I/OT-II cells and vaccinated with Ova/Alum. Time-lapse high content live cell imaging has provided direct mechanistic support for TIM3-Fc-OX40L functionally tethering T cells to tumor cells and eliciting cytotoxic activity (tumor killing). Finally, the therapeutic activity of TIM3-Fc-OX40L in established murine MC38, B16.F10 and CT26 tumors was significantly superior to either TIM3 blocking antibody, OX40 agonist antibody or combination antibody therapy. Importantly, a pharmacodynamic biomarker of tumor rejection was identified by the elevation in serum cytokines post-treatment.

These data demonstrate feasibility and functional activity of a novel chimeric fusion protein platform, providing checkpoint blockade and TNF superfamily costimulation in a single molecule, which is uniquely advantageous because the construct links those two signals in the same microenvironmental context, at the time in which T cells are engaging cognate tumor antigen.

#5560

PD-1/PD-L1 blockade enhances MDM2 inhibitor activity in p53 wild-type cancers.

Hui Qin Wang, Jinsheng Liang, Iain Mulford, Fiona Sharp, Swann Gaulis, Yan Chen, Gina Trabucco, David Quinn, Joseph D. Growney, Matthew J. Meyer, Juliet Williams, Peter Hammerman, Francesco Hofmann, Glenn Dranoff, Jeffrey Engelman, Jennifer Mataraza, Ensar Halilovic. _Novartis Insts. for BioMedical Research, Cambridge, MA_.

p53 is a transcription factor that plays a central role in guarding genomic stability of the cell through cell cycle arrest or induction of apoptosis. It has also been reported that p53 participates in the regulation of tumor immunity and in homeostatic regulation of immune responses. However, the immunomodulatory effect of p53 in tumor and the tumor microenvironment is not well understood. From gene expression and immunohistochemical analysis of pre- and post-treatment biopsies from patients treated with the MDM2 inhibitor NVP-CGM097, we observed upregulation of immune checkpoint transcripts (PD1 and PDL1), and an increase in the number of CD8+ tumor infiltrating lymphocytes. To investigate the immunomodulatory effect of p53 in more detail, we studied murine syngeneic models treated with NVP-HDM201, a potent and selective second generation MDM2 inhibitor. We performed multi-color FACS analysis on tumors and tumor draining lymph nodes. With NVP-HDM201 treatment, we observed increase in numbers of CD103+ DC cells, capable of antigen cross-presentation. Furthermore, NVP-HDM201 increased the percentage of Tbet+EOMES-T cells in tumors as well as tumor draining lymph nodes and also resulted in increased ratios of CD8+ Tcells/Treg in tumor. In addition, levels of both PD-L1 on CD45- cells and PD-1 on CD4+ T cells were increased. Importantly, PD-1 and PD-L1 blockade enhanced HDM201 activity in p53 WT syngeneic mouse models but not in p53 mutated models. The rate of complete tumor regressions (CR) was significantly increased with combination treatment as compared to either treatment alone. The animals that

achieved complete regressions also developed long lasting anti-tumor memory against the specific tumor cells as evidenced by re-challenge experiments. Taken together, these results demonstrate that MDM2 inhibition triggered adaptive immunity which was further enhanced by blockade of PD-1/PD-L1 pathway, thereby providing a rationale for combining MDM2 inhibitors and checkpoint blocking antibodies in cancer patients with wildtype p53.

#5561

High-affinity antisense oligonucleotides targeting Foxp3 inhibit immunosuppressive function of regulatory T-cells and produce antitumor effects in syngeneic tumor models.

Charles Sinclair,1 Alexey S. Revenko,2 Alison Peter,1 Robert B. Johnson,2 Lisa A. Hettrick,2 Molly Taylor,1 Anna Staniszewska,1 Adina Hughes,1 Lisa Sandin,1 Stephanie Klein,2 Andrew Watt,1 Simon Barry,1 Brett T. Monia,2 Paul Lyne,3 A Robert Macleod,2 Mark Edbrooke1. 1 _AstraZeneca, Cambridge, United Kingdom;_ 2 _Ionis Pharmaceuticals, Calsbad, CA;_ 3 _AstraZeneca, Waltham, MA_.

Regulatory T-cells (Treg) contribute to cancer progression by suppressing anti-tumor immuity. Tregs specifically require expression of the lineage defining transcription factor Foxp3 for their development and function, but this protein cannot be targeted with conventional small molecule or biologic drugs. We employed next-generation antisense inhibitors (Gen 2.5 cEt-modified ASOs) in an attempt to selectively inhibit Foxp3 expression in mouse Treg cells, and evaluated consequences of ASO-mediated Foxp3 knock-down in vitro and in vivo. Mouse Foxp3-specific ASOs promoted potent dose-dependent reductions in Foxp3 mRNA and protein in vitro, without the use of transfection reagents. Foxp3 knockdown also resulted in loss of immunosuppressive markers and conferred Tregs with a reduced immunosuppressive capacity. Whilst genetic ablation of FOXP3 leads to manifestation of autoimmunity due to a complete loss of Treg function, we observed that the systemic delivery of unformulated mouse Foxp3 ASOs to WT mice resulted in Foxp3 knockdown in vivo, but spared mice from an autoimmune phenotype. Nevertheless, when syngeneic tumor-bearing mice were treated with mouse Foxp3 ASOs tumor growth was significantly attenuated, with a fraction of animals (25%-50%) achieving complete regressions. Anti-tumor activity of mouse Foxp3 ASOs was associated with immunophenotypic changes consistent with an increased anti-tumor immune response. Overall these data demonstrate the therapeutic capacity of mouse ASO to directly target regulatory T-cells, and suggesting that targeting of Foxp3 represents an attractive opportunity in cancer immunotherapy.

#5562

A 3D image-based quantification of T cell-mediated killing of CRC organoids in the presence of immuno-modulators.

Gera Goverse, Lidia Daszkiewicz, Kuan Yan, Mohamed Tleis, Mariusz Madej, Lucia Salinaro, Leo Price. _OcellO, Leiden, Netherlands_.

BACKGROUND

Cancer immunotherapy has already yielded promising clinical results but most patients still do not respond. The mechanisms of action of these treatment modalities are also not fully understood and the progress in this direction is hampered by a lack of appropriate pre-clinical testing models. To investigate the role of novel therapeutics targeting the immune cell compartment to kill tumor cells, we developed an in vitro assay based on 3D cultures and image based analysis in a 384-wells plate format. Immune cells are added to recapitulate the tumor micro-environment and its complex interactions between different cellular players. Specifically, infiltration of immune cells into the tumoroids and their killing are visualized and measured, enabling a better understanding of the immune-modulatory profile of different treatments.

METHODS

Autologous patient derived colon organoids from normal and tumor tissue from several patients were cultured in a 3D environment. HLA-matched PBMCs with and without activation were added and immune-cell infiltration and killing of the CRC organoids was visualized in 3D using automated microscopy. Quantification of immune cell effects was achieved with morphometric analysis with OMinerTM software.

RESULTS

3D image data analysis enabled the discrimination of immune-tumor cell interactions and revealed a higher immune cell infiltration and tumoroid killing upon activation. In addition, we were able to compare both normal and tumor colon tissue from the same patient and compare between patients, demonstrating patient related differences and elucidating the effect of immune cell targeting on normal colon tissue.

CONCLUSIONS

The 3D assay presented here allows the analysis of different cell types that engage in a more realistic setting than when culturing them in traditional 2D cultures. Using image-based analysis, immune-tumor interactions can be visualized and quantified. The 3D environment, both for the cell culture and image analysis, allows for measurement of spatially resolved information, not accessible by monolayer cultures or biochemical assays. This new and innovative platform can empower immunotherapy drug developers to select the most promising candidates and better understand their mechanism of action.

#5563

Kinetically-controlled release of blocking molecules for the targeted enhancement of therapeutic activity in antibody-cytokine fusion proteins.

Tiziano Ongaro,1 Martina Bigatti,1 Filippo Sladojevich,1 Etienne Donckele,1 Alessandra Micaela Villa,1 Dario Neri2. 1 _Philochem AG, part of the Philogen Group, Otelfingen, Switzerland;_ 2 _ETH Zurich, Switzerland_.

Cytokines are able to mediate a potent anticancer activity but have significant side effects that prevent dose escalation to therapeutically-active regimens. Targeting the cytokines to the site of disease helps to improve their therapeutic index while decreasing associated toxicities. However, peak concentrations in blood are still associated with side-effects. We have developed a novel technology (termed "CORK Technology"), which consists in masking the undesired activity of a cytokine in circulation. The "cork" can be either an antibody fragment or a small molecule, which specifically binds to the cytokine thus preventing the interaction with its receptor. Once the complex accumulates in the tumor, the cork dissociate from the cytokine allowing it to exert its function. Capitalizing on the fact that the kinetic dissociation constant koff of the complex is identical in different species, this technology can be easily translated from mouse to man with comparable results. In this poster we describe the experimental implementation of CORK Technology to improve the therapeutic index of the clinical stage L19-IL2 (L19 is an antibody fragment specific to the alternatively-spliced EDB domain of fibronectin, a marker of tumor angiogenesis). Two classes of IL2 inhibitors were discovered and characterized: (i) a fully human scFv fragment, and (ii) a small organic ligand, featuring a modified methylindole moiety. Both types of agents reduced IL2 activity in vitro, were able to dissociate from the cognate cytokine in a kinetically-tuned process and allowed the administration of the L19-IL2 fusion protein at higher doses, both in normal and in tumor-bearing mice. The technology is applicable to other cytokine payloads and facilitates the development of targeted cytokine products with "activity on demand" (i.e., with increased activity at the tumor site, promoting an activation and proliferation of tumor-resident lymphocytes). Albeit still in its infancy, this elegant approach provides promising results and a strong rationale to further investigate this therapeutic power of the CORK Technology.

#5564

Agonist redirected checkpoint, VSIG8-Fc-OX40L, for cancer immunotherapy.

George Fromm, Suresh de Silva, Kellsey Johannes, Arpita Patel, Josiah Hornblower, Taylor H. Schreiber. _Shattuck Labs, Inc., Durham, NC_.

Combination immunotherapy with bispecific antibodies, linked scFv's or T cell engagers have not demonstrated that both checkpoint blockade and TNF receptor activation can be achieved with a single molecule, because these molecules lose target avidity when engineered to bind multiple targets with monovalent antigen binding arms. Fusion proteins incorporating the extracellular domain (ECD) of type I membrane proteins (eg. Enbrel, Orencia) or type II membrane proteins (eg. OX40L-Fc, GITRL-Fc), linked to the hinge-CH2-CH3 domain of antibodies are both functional, despite the ECDs being in opposite orientation. We report the generation of a two-sided fusion protein incorporating the ECD of VSIG8 and the ECD of OX40L, adjoined by a central Fc domain. It has been suggested that the VSIG (V-set and immunoglobulin domain containing) family of proteins play important yet diverse roles in cancer, however little is known about their MOAs or therapeutic functions.

Each of the three individual domains of VSIG8-Fc-OX40L were identified using conformation-dependent antibodies by Western blots, and binding to OX40 was demonstrated in functional ELISA assays. Although VSIG8 has been described as a putative binding partner for VISTA, no binding was detected between VSIG8-Fc-OX40L and recombinant VISTA using ELISA, Octet or a retroviral membrane display platform. Interestingly, binding was observed to VISTA positive mouse and human tumor cell lines. Membrane preparations of these cell lines were generated in order to identify the putative binding partner(s) of VSIG8, which were then re-confirmed using recombinant protein in ELISA assays. VSIG8-Fc-OX40L stimulated potent activation of NFkB signaling in reporter cells lines, and stimulated IL2 and TNFa secretion from PBMCs primed with the super-antigen SEB. Time-lapse live cell imaging demonstrated that VSIG8-Fc-OX40L led to functional tethering of primary human T cells to human tumor cells and stimulated tumor cell killing (as evidenced by cleavage of caspase 3/7) to a greater extent than antibody controls. In tumor bearing mice, VSIG8-Fc-OX40L expanded both effector CD4 and CD8 T cell subsets (without activating Tregs) in the spleen, lymph node and tumor, including a significant increase in AH1-tetramer+ cells (antigen-specific CD8+ T cells) in the isolated TIL. Finally, the therapeutic activity of VSIG8-Fc-OX40L in established murine MC38 and CT26 tumors was significantly superior to either VISTA blocking antibody, OX40 agonist antibody or combination antibody therapy.

The V-set and immunoglobulin domain containing family of proteins, including TIGIT, have potent checkpoint-like activity in cancer immunotherapy but are not well understood. These data demonstrate feasibility and functional activity of a novel chimeric fusion protein platform, which provided potent anti-tumor immunity and also facilitated the de-orphanization of the poorly understood protein, VSIG8.

#5565

Potency-reduced IL15/IL15Rα heterodimeric Fc-fusions display enhanced in vivo activity through increased exposure.

Matthew J. Bernett, Rajat Varma, Christine Bonzon, Rumana Rashid, Liz Bogaert, Ke Liu, Suzanne Schubbert, Kendra N. Avery, Irene W. Leung, Nicole Rodriguez, Seung Y. Chu, Umesh S. Muchhal, Gregory L. Moore, John R. Desjarlais. _Xencor, Inc., Monrovia, CA_.

IL15 and IL2 are two similar cytokines that stimulate the proliferation of lymphocytes, and their therapeutic potential has been well established in animal models and human trials. Both cytokines exert their cell signaling function through binding to a trimeric complex consisting of two shared receptors, the common gamma chain (γc) and IL2Rβ, as well as an alpha chain receptor unique to each cytokine: IL2Rα or IL15Rα. Both cytokines share a similar biology, with the exception that IL2 has a greater preference for Tregs due to their high constitutive expression of IL2Rα. IL15 functions as a stabilized heterodimeric complex with membrane-bound IL15Rα on the surface of monocytes and DCs, and this IL15/IL15Rα complex is presented in trans to lymphocytes expressing IL2Rβ and γc. It has been shown that recombinant IL15/IL15Rα heterodimer is stable and highly active. As potential drugs, both IL2 and IL15 are extremely potent and suffer from low tolerability and very fast clearance that limits therapeutic window. Seeking to engineer a more druggable version of IL15, we created various IL15/IL15Rα heterodimeric Fc-fusions (IL15/IL15Rα-Het-Fc) with reduced potency to improve tolerability, slow receptor-mediated clearance, and prolong half-life.

We engineered IL15/IL15Rα-Het-Fc by fusing IL15 to one side of a heterodimeric Fc-region, and the sushi domain of IL15Rα to the other side, or by creating a single-chain IL15/IL15Rα that was attached to one side of a heterodimeric Fc-region. These Fc-fusions were tuned for optimal activity by engineering amino acid substitutions in IL15 - at the IL2Rβ or γc interface - that reduced overall in vitro potency. In vitro proliferation of lymphocytes in normal human PBMCs was monitored by counting Ki67+ cells after incubation with Fc-fusions for 4 days and by measuring signaling in a STAT5 phosphorylation assay for 15 minutes. In vivo activity was evaluated using a huPBMC-NSG mouse model by measuring the extent of human leukocyte engraftment by flow cytometry and IFNγ. Tolerability, immune stimulation, and pharmacokinetics were evaluated in non-human primates (NHP).

IL15/IL15Rα-Het-Fc were produced with good yield and purity. The Fc-fusions enhanced proliferation of CD8+ T and NK cells in vitro, and potency of variants with substitutions at the IL2Rβ and/or γc interface was reduced up to ~700-fold compared to wild-type IL15/IL15Rα-Het-Fc. Treatment of huPBMC-NSG mice with IL15/IL15Rα-Het-Fc promoted enhanced T cell engraftment and elevated IFNγ in a dose dependent manner. NHP studies indicated half-lives of several days for IL15/IL15Rα-Het-Fc, which are significantly longer than the <1 hr half-life of IL15. In both in vivo settings, a marked inverse correlation of pharmacodynamics and clearance was observed, with weaker potency variants allowing higher doses and enhanced in vivo lymphocyte proliferation as a result of more sustained exposure.

#5566

LYC-55716, a first-in-class RORγ agonist: Rationale and preclinical data to support clinical combinations with established immunotherapies.

Xiao Hu,1 Xikui Liu,1 Hongxiu Li,1 Garry Weems,2 Elizabeth Zawidzka,1 Yilin Gao,1 H. Jeffrey Wilkins,2 Laura Carter1. 1 _Lycera Corp., Ann Arbor, MI;_ 2 _Lycera Corp., Plymouth Meeting, PA_.

The retinoic acid-related orphan receptor γt (RORγt) is a nuclear receptor transcription factor that acts as an immune cell master control switch driving the generation and function of Th17 (helper) and Tc17 (cytotoxic) T cells. Preclinical data show that synthetic RORγ agonists modulate immune cell gene expression to both increase immune activity and decrease immunosuppressive mechanisms in the tumor microenvironment. Because RORγ agonists impact multiple antitumor mechanisms, these compounds have the potential to combine with other agents to enhance tumor immunity. In addition to preclinical data supporting a biologic rationale, bioinformatic and machine learning approaches based on the RORγ pathway were undertaken to prioritize possible clinical combinations. This work highlighted other immuno-oncology agents as well as targeted therapies and more conventional treatments. Preclinical, syngeneic mouse cancer models were used to test some of the hypotheses. Based on the finding that RORγ agonists decrease PD-1 expression and that anti-CTLA4 induces ICOS, which stabilizes RORγ expression, anti-PD1 or anti-CTLA4 agents were combined with the RORγ agonist LYC-54143 in 12 syngeneic murine models. Compared with individual agents, combination treatments elicited superior tumor growth inhibition in 6 of the models (Table). RORγ agonist treatment was also able to augment antitumor activity of doxorubicin. In preclinical and phase 1 clinical testing, the first-in-class, investigational oral small-molecule RORγ agonist LYC-55716 has demonstrated a favorable safety profile supporting combination with other immuno-oncology agents. Ongoing clinical trials include a phase 2a trial of LYC-55716 in patients with select solid tumors (NCT02929862) and a phase 1b trial of LYC-55716 combined with pembrolizumab in patients with non-small cell lung cancer.

Percentage of tumor growth inhibition induced in syngeneic murine models of cancer.

---

Murine model | Anti-PD1 alone | Anti-CTLA4 alone | RORγ agonist alone | RORγ agonist + anti-PD1 | RORγ agonist + anti-CTLA4

H22 (Liver) | 30-50% | >50%* | 10-30% | >50%* | >50%*

Pan02 (Pancreas) | <10% | 10-30% | <10% | 10-30%* | 10-30%*

CT26 (Colon) | 10-30% | <10% | <10% | <10% | 30-50%

B16F10 (Melanoma) | 10-30% | 10% | 10-30% | 10-30% | 30-50%

A230 (Lymphoma) | <10% | <10% | 10-30% | 30-50% | 10%

Renca (Renal) | <10% | 10-30% | 30-50%* | 30-50%* | 30-50%

*P<.05 vs vehicle.

#5567

Agonist redirected checkpoint (ARC), SIRPα-Fc-CD40L, for cancer immunotherapy.

George Fromm, Suresh de Silva, Kellsey Johannes, Arpita Patel, Josiah C. Hornblower, Taylor H. Schreiber. _Shattuck Labs, Inc., Durham, NC_.

Current attempts at combination immunotherapy with bispecific antibodies, linked scFv's or T cell engagers have not demonstrated that both checkpoint blockade and TNF receptor activation can be achieved with a single molecule. This is likely because these molecules lose target avidity when engineered to bind multiple targets with monovalent antigen binding arms. Fusion proteins incorporating the extracellular domain (ECD) of type I membrane proteins (eg. Enbrel, Orencia) or type II membrane proteins (eg. SIRPα-Fc, GITRL-Fc), linked to the hinge-CH2-CH3 domain of antibodies are both functional, despite the ECDs being in opposite orientation. We report the generation of a two-sided fusion protein (ARC) incorporating the ECD of SIRPα (CD172a) and the ECD of CD40L, adjoined by a central Fc domain.

The SIRPα end of the ARC binds immobilized CD47 at 3.59 nM affinity and binds CD47 on the surface of human tumor cells both in vitro and in vivo, but does not bind human platelets or RBCs. Further, no hemolytic activity was observed with the human SIRPα-Fc-CD40L ARC in vitro as compared to CD47 mAbs. The CD40L end of the ARC binds immobilized CD40 at 756 pM affinity and binds CD40 on primary macrophages. The SIRPα-Fc-CD40L ARC stimulates functional activity (independent of Fc receptor cross-linking) in NFκB-luciferase reporter cells (CD40 driven activation of NFκB). Addition of SIRPα-Fc-CD40L to an ex vivo super-antigen (SEB) cytokine release assay stimulated secretion of IL2 and TNFα from human PBMC. Furthermore, time-lapse high content live cell imaging has shown that when activated human macrophages were co-cultured with CD47 positive human tumor cells, SIRPα -Fc-CD40L was shown to enhance phagocytosis. Finally, the therapeutic activity of SIRPα-Fc-CD40L in established murine MC38 and CT26 tumors was superior to either CD47 blocking antibody, CD40 agonist antibody or combination antibody therapy. Notably, the superior anti-tumor immunity observed with SIRPα-Fc-CD40L was not accompanied by significant mucosal inflammation and weight loss stimulated by murine CD40 agonist antibodies. Finally, treatment of cynomolgus macaques with SIRPα-Fc-CD40L was safe, and no evidence of hemolysis or thrombocytopenia was observed.

These data demonstrate feasibility and functional activity of a novel chimeric fusion protein platform, providing checkpoint blockade and TNF superfamily costimulation in a single molecule. Signal replacement of CD47 by CD40L may uniquely poise macrophages in the tumor microenvironment for activation and cross-presentation of tumor antigens following enhanced tumor cell phagocytosis.

#5568

Clinical immunostimulatory activity of imipridone ONC201, a selective DRD2 antagonist, in advanced solid tumor patients.

Rohinton S. Tarapore,1 Mark N. Stein,2 Andrew Zloza,2 Lorna Rodriguez,2 Jenna Newman,2 Charles Cheeson,2 Martin Stogniew,1 Wolfgang Oster,1 Joshua E. Allen1. 1 _Oncoceutics Inc, Philadelphia, PA;_ 2 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ_.

Background: Dopamine receptor D2 (DRD2) is a G protein-coupled receptor that is overexpressed in several malignancies and is also expressed in a variety of immune cells including Natural Killer (NK) cells. Interestingly, pharmacological antagonism of DRD2 results in induction of apoptosis in tumor cells and proliferation of immune cells. ONC201 is an anti-cancer, small molecule DRD2 antagonist that is in clinical trials for various cancers that overexpress DRD2 following completion of the first-in-human trial that established its recommended phase II dose of 625mg.

Methods: A Phase I trial previously investigated single agent oral ONC201 in adult advanced solid tumor patients on a once every one (Q1W) or three weeks (Q3W) administration schedule. Twenty-eight evaluable patients received ONC201 on a Q3W schedule while 20 evaluable patients received ONC201 on a Q1W schedule. Levels of serum immune cytokines and effector molecules were evaluated throughout treatment using the Legendplex Assay. Peripheral blood mononuclear cells (PBMCs) and tumor biopsies from patients were also probed for immunomodulatory effects following treatment initiation.

Results: Early serum immune cytokine induction occurred in the majority of patients on the trial, whereas delayed induction of immune effectors was exclusive to patients who experienced at least stable disease by RECIST criteria. Analysis of PBMCs revealed an increase in the number of circulating NK cells, as well as their expression of granzyme B, which occurs with NK cell activation, up to 3 days after initiating ONC201. Intratumoral infiltration of granzyme B+ NK cells was evident in one patient who underwent an on-treatment biopsy 10 days after beginning ONC201. A statistically significant increase in the induction of immune cytokines and effectors was apparent in patients who received ONC201 once every week versus once every three weeks.

PK analyses in patients dosed on a Q1W schedule revealed therapeutic Cmax, AUC, and half-life values that were consistent with that of Q3W dosing and similar between the first (cycle 1) and fourth (cycle 2) dose. Prolonged stable disease for >6 months was observed in several prostate and endometrial cancer patients. Patients who had stable disease for >12 weeks had a significantly higher induction of cytokines and effector molecules compared to patients that progressed rapidly.

Conclusions: ONC201 exhibits immunostimulatory activity in advanced cancer patients that is well tolerated and may be associated with clinical benefit. Activation of immune cells by ONC201 is another dimension of its efficacy that should accounted for when considering its clinical evaluation as a single agent or in combination for the treatment of advanced cancers.

### Liquid Biopsy 5

#5569

Donor-derived circulating cell-free DNA (ccfDNA) reference materials for concordance studies.

Yves Konigshofer,1 Matthew G. Butler,1 Jessica Dickens,1 Katherine Bianco,2 Karl G. Sylvester,2 Bharathi Anekella,1 Russell K. Garlick3. 1 _SeraCare Life Sciences, Inc., Gaithersburg, MD;_ 2 _Stanford University School of Medicine, Stanford, CA;_ 3 _SeraCare Life Sciences, Inc., Milford, MA_.

The limited quantities of ccfDNA in plasma can make it difficult to assess the sensitivities and specificities of circulating tumor DNA (ctDNA) assays because there is often insufficient material to confirm the presence or absence of particular mutations by orthogonal assays. Additionally, there have been reports of discordance between different ctDNA assays with patient samples at lower variant allele frequencies (VAF), but the limited amount of material is a bottleneck to investigating the causes of discordance. To overcome this, we developed a method to amplify nanograms of ccfDNA from donors to microgram quantities. As an initial proof of concept, we amplified ccfDNA from pregnant female donors that were known to be carrying either a normal fetus or one with chromosomal abnormalities. These samples were analyzed externally at commercial Non-Invasive Prenatal Testing (NIPT) laboratories and were found to be compatible with both chromosome counting and SNP-based testing, and fetal fraction could also be assessed. In order to evaluate the suitability of amplified ccfDNA for ctDNA assays, we split ccfDNA samples so that part was analyzed as-is by a given ctDNA assay and part was analyzed after amplification. The same amplified material was also analyzed with four different ctDNA assays on different NGS platforms in order to assess concordance between assays in shared regions. Good concordance was seen between variants and their VAFs identified in native and amplified ccfDNA, although increasing amounts of discordant background errors were observed at lower VAF. With the Archer Reveal ctDNA 28 assay, obtaining more unique fragments was associated with obtaining more low VAF background errors that could be offset by obtaining more replicate reads of a given unique fragment. Discordant variants were often C>T (or the corresponding G>A) that can be generated by cytosine deamination, which can occur by heating the sample during PCR for library construction. In conclusion, we have developed a method for amplifying limited amounts of ccfDNA that generates sufficient donor ccfDNA-like material for analysis by multiple assays. This should enable the development, improvement and validation of assays that analyze ccfDNA - and especially ctDNA - because sufficient material can be generated in order to assess sensitivity and specificity using orthogonal assays. Additionally, this should also allow for concordance studies that attempt to analyze the same material at multiple sites and with multiple assays.

#5570

Analytical and clinical validation of blood-based NGS panel for detection of genomic alterations in DNA repair pathways.

Xiaohong Wang, Jianjun Yu. _Predicine, Inc, Hayward, CA_.

Response to DNA-damaging agents, such as PARP inhibitors (PARPi), is associate with homologous recombination deficiency (HRD). In fact, patients with advanced breast, prostate, and ovarian cancers who carry harmful mutations in BRCA1/2 have benefited greatly from olaparib, niraparib, and rucaparib as reported in multiple clinical trials. However, current available assays either require tumor biopsy which may be of insufficient quantity or unobtainable or only examine very limited genes such as BRCA1, BRCA2, and ATM from patient blood. We recently developed a blood-based NGS panel to accurately detect genomic alterations in 51 genes related to DNA damage and repair. With in-house proprietary ultra-sensitive DeepSeaTM(Deterministic, Efficient, Precise, Sensitive Algorithm) variant detection algorithm, analytical performance was rigorously assessed via serial dilutions of reference materials and also further validated by clinical plasma samples in prostate, ovarian, lung and breast cancer.

Key words: cfDNA, DNA damage repair, PARP, BRCA1, BRCA2, liquid biopsy, biomarker

#5571

Changes in plasma genomic abnormalities at chromosome 8 and 20 predict treatment response and monitor disease progression in advanced gastric cancer.

An D. Do, Charles D. Baudo, Samir Seshadri, Xiaoji Chen, Rebecca Suttmann, David S. Shames, Mark R. Lackner, Shih-Min A. Huang, Teiko Sumiyoshi. _Genentech, Inc., South San Francisco, CA_.

Gastric cancer (GC) is the fifth most common cancer diagnosis with roughly 60% of patients with metastatic disease. Tumor heterogeneity represents a major challenge to personalized cancer therapy. Intratumor heterogeneity can result in missing critical DNA genomic alterations using conventional tumor biopsies. Circulating cell-free DNA (cfDNA) in the peripheral blood has the potential to capture DNA alterations in a more dynamic manner in particular types of advanced tumors where feasibility of repeat tissue biopsies is limited. Using low-pass whole-genome sequencing (LP-WGS) data from 329 blood plasma samples collected from 63 1L metastatic (mGC) patients enrolled in phase II trial NCT01590719, receiving either onartuzumab or placebo in combination with mFOLFOX6, we evaluated the global percent genome change (%GC) and copy number (CN) gain at baseline and throughout the course of treatment. cfDNA samples were isolated with the automated QIAsymphony system and analyzed on the Illumina NextSeq 500 system with a novel LP-WGS protocol at 0.5x coverage. Seven chromosomes (chr1, 7, 8, 14, 15, 20, and 21) showed the most frequent CN gains of evaluated patients (≥15%). Of these seven chromosomes, chr8 and 20 featured the largest number of genes at baseline with CN gains with 30.2% (19/63) at chr8 and 25.4% (16/63) at chr20. 81% of patients (13/16) with chr20 CN gain also showed an increase in CN at chr8. Patients with gains at chr8 and/or 20 have higher %GC compared to patients with normal copy status. Recurrent CN gains at chr8 and 20 in tumor tissues were previously reported in GC patients and correlated with tumor progression. Copy number gains at chr8 and 20 in cfDNA from mGC patients, reported here for the first time, suggests a potential utility for early detection of disease progression. Moreover, patients with CN gains at chr8 and/or 20 had higher circulating tumor cells detected at baseline (p=0.003) and also showed the most decrease in %GC post-treatment. 75% (9/12) of the patients with CN gains at chr8 and/or 20 were responders and showed a mean 3-fold decrease (range of 1.6-4.7 fold change) in tumor size post-treatment. This suggests CN gain at chr8 or 20 in cfDNA could be used as a noninvasive measure to identify a novel subset of mGC patients sensitive to chemo-based treatment. When comparing %GC pre- and post-treatment, the %GC decreased significantly at C2D1 (p=0.006). This decrease in %GC correlates with a decrease in tumor burden in patients with response, while %GC and tumor burden remain unchanged or increased in nonresponders. Monitoring %GC pre- and post-treatment demonstrates the ability to assess the depth of the treatment and early detection of disease progression. Findings from this study suggest that plasma genomic abnormality monitoring may be used for treatment decision-making in mGC and should be evaluated further in clinical trials.

#5572

Circulating tumor cells: The tumor trail left in the blood.

Arutha Kulasinghe,1 Chris Perry,2 Liz Kenny,3 Tony Blick,4 Majid Warkiani,5 Ian Vela,2 Ken O'Byrne,2 Jean-Paul Thiery,6 Erik Thompson,4 Colleen Nelson,4 Chamindie Punyadeera4. 1 _Institute of Health and Biomedical Innovation, QUT, School of Biomedical Sciences, Brisbane, Australia;_ 2 _Princess Alexandra Hospital (PAH), Brisbane, Australia;_ 3 _Royal Brisbane and Women's Hospital, Brisbane, Australia;_ 4 _Institute of Health and Biomedical Innovation, QUT, Brisbane, Australia;_ 5 _University of Technology Sydney, Sydney, Australia;_ 6 _CNRS, Paris, France_.

Background: Metastasis in HNC patients is reflected by measurable levels of circulating tumor cells (CTCs) in the peripheral blood of cancer patients. CTCs represent cancer cells from the primary and metastatic sites, thereby providing a comprehensive representation of the tumor burden of an individual patient. For patients without CTCs at presentation, the detection of CTCs in the blood and analysis of biomarkers within them provide an opportunity to identify patients "at risk" of developing overt metastasis, accelerating targeted treatment in addition to routing care with the clear aim of improving cure.

Methods: Our study aimed to assess whether CTCs from the blood of HNC patients attending the Princess Alexandra Hospital and Royal Brisbane and Women's Hospital provided early cues of distant metastases (n=250).

Results: With significant advances in CTC isolation technologies, we could demonstrate a higher CTC capture efficiency using epitope-independent platforms. By assessment of single and clustered CTCs, our data showed that HNC patients can be identified 4-6 months prior to developing clinical/radiographically evident metastasis. In these patients, a window for treatment escalation could become a possibility. In a proof-of-principle study, using novel culture formulations and hypoxic conditions (1-2% O2), we were able to demonstrate, for the first time, short-term patient-derived CTC cultures ex vivo from 7/18 HNC samples (4/7 HPV-positive, oropharyngeal) in a clinically relevant time period. Recent advancements have shown that PD-1 immune checkpoint therapies have durable responses in metastatic HNC patients that fail 1st- and 2nd-line therapy. Our preliminary data suggest PD-L1 is frequently expressed on HNC CTCs, and an immunoscore may be able to stratify patients likely to respond to immunotherapy.

Conclusion: Expanding CTCs outside the patient's body allows for the recapitulation of the molecular diversity present within the tumor, understanding the disease progression and testing of therapies. Patients with a high percentage of PD-L1+ CTCs could be potential candidates for anti-PD-L1 therapy, a promising new immunotherapy.

#5573

Highly multiplexed diagnostic test for the detection of extended EGFR del19 variants in patients with NSCLC.

Hestia S. Mellert, Leisa Jackson, Kristin Alexander, Amanda Weaver, Dianna Maar, Dawne Shelton, Gary Pestano. _Biodesix, Inc., Boulder, CO_.

The detection of nucleic acids in circulation has become an increasingly important part of the clinical workup and testing for cancer patients, particularly those with non-small cell lung cancer (NSCLC). The utilization of blood-based PCR platforms for detection of somatic variant mutations addresses several limitations of previously established molecular testing approaches such as an inability to access tissue, insufficient tissue quantity or quality, and slow turnaround time for results. However, the number of variants detected may be limited by use of some rapid PCR technologies. We have focused in this study on the analytic and clinical development of a highly multiplexed assay for the detection of EGFR del19 while maintaining a rapid turn-around time. The test system includes the prospective collection of whole blood into a cfDNA BCT and the reproducible detection of del19 variants out of plasma using ddPCR-based technology. Analytic specificity and sensitivity was examined using in vitro DNA designed to mimic the EGFR del19 variants in the assay. Analytic sensitivity was performed and demonstrates that the theoretical limit of detection of the test was below 0.02% minor variant frequency when using high concentrations of synthetic standards. Precision studies were conducted with both analytic and clinical samples at high, medium and low mutation frequencies, over three consecutive days, three runs in one day and with two operators. All test parameters were successfully met. Normal healthy donor (n = 11) and reference ddPCR positive and negative NSCLC donor (n = 80) samples were tested. EGFR del19 variants were not identified in the plasma from the normal healthy donors or the EGFR del19 reference negative NSCLC donors. All reference positive cases were accurately detected above our required threshold of 2 variant copies with detection ranging from 14 to 1584. For all donor specimens the wild-type copies were well above the predefined clinical QC threshold of 300, ranging from 380 to 760 copies. Of note, no negative reference samples contained more than two mutant copies. We conclude that the developed test is highly suited for the sensitive and reproducible detection of multiple EGFR del19 variants from blood specimens in the clinical laboratory.

#5574

A comprehensive, targeted next-generation sequencing method that rapidly and accurately detects circulating tumor DNA variants at 0.1% frequency in plasma samples.

Jessica L. Larson,1 Liangjing Chen,1 Lando Ringel,1 Blake Printy,1 Farol L. Tomson,2 Yves Konigshofer,2 Sarah Statt,1 Joseph K. Kaplan,1 Shobha Gokul,1 Jeffrey Shelton,1 Gary J. Latham,1 Brian C. Haynes1. 1 _Asuragen, Inc., Austin, TX;_ 2 _SeraCare Life Sciences, Milford, MA_.

Introduction: Mutation analysis of circulating tumor DNA (ctDNA) in blood-based liquid biopsies provides a minimally invasive approach to detect and monitor disease. Existing next-generation sequencing (NGS) liquid biopsy techniques have laborious and/or inefficient workflows, heuristic error-correction algorithms, and variable performance with clinical tumor-plasma samples. We describe a method that combines a kittable and efficient wet-bench workflow with accurate dry-bench analytics to reduce costs and turnaround time, and is relevant to clinical research and patient testing. Methods: We developed a comprehensive, targeted NGS technology to enable the ultra-sensitive detection of variants from liquid biopsy samples. Input DNA molecules from Seraseq™ ctDNA v2 reference materials (SeraCare) and >50 matched FFPE and plasma samples were uniquely tagged with a random molecular barcode (MBC), amplified in an efficient PCR protocol, and sequenced using a targeted panel covering >500 somatic mutation hotspots. Sequence-ready libraries were prepared from input DNA within 9 hours. Data were analyzed with a robust bioinformatics pipeline tailored to the library chemistry to correct for multiple background errors. Variants were identified with a site-specific model, which effectively eliminated recurring non-biological aberrations that remained despite MBC-facilitated error-suppression. We verified variant calls in plasma by Droplet Digital™ PCR (Bio-Rad). To evaluate the limit of detection, healthy control and mutation-positive plasma admixtures were prepared and sequenced. Results: Input template molecules were efficiently recovered. The median number of unique MBCs across all amplicons was >90% of expectation based on input DNA quantities for both the tumor-associated plasma and Seraseq™ ctDNA v2 material. SNVs and indels were recognized with >90% sensitivity and PPV at 0.5% allele frequency (AF) in the Seraseq™ ctDNA mutation mixes. In both neat plasma samples and plasma admixtures, we correctly identified tumor mutations down to ~0.1% AF while maintaining a low false-positive rate (sensitivity and PPV remained high for AF ≥0.1%). Reference ctDNA material and tumor-associated plasma had analogous template amplification and variant frequency rates. Conclusions: A fast, efficient, and sensitive NGS panel approach was developed and evaluated, and it demonstrated the reliable and specific detection of rare variants in liquid biopsy specimens. The method is able to distinguish a low frequency ctDNA signal from the overwhelming background noise in plasma cell-free DNA using a streamlined workflow and purpose-built bioinformatics pipeline. This technology may provide an easy-to-use, high-performance, and adaptable NGS diagnostic framework for disease detection and therapeutic intervention monitoring.

#5575

A multiplexed RE-qPCR cell-free DNA assay to assess response and resistance to cancer therapy.

Sudhir K. Sinha,1 Hiromi Brown,1 Zhide Fang,2 Mathilde Couetoux,3 Karen Gambaro,3 Gerald Batist4. 1 _InnoGenomics Technologies,LLC, New Orleans, LA;_ 2 _Louisiana State University Health Science Center-NO, New Orleans, LA;_ 3 _Exactis Innovation, Montreal, Quebec, Canada;_ 4 _Segal Cancer Centre-Jewish General Hospital-Mc Gill University, Montreal, Quebec, Canada_.

A qPCR multiplex that utilizes two types of multi-copy retrotransposable elements (RE) in the human genome, as target loci, has been developed and validated. The single-well, qPCR reaction amplifies 80bp, 265bp and an internal-positive-control simultaneously, within 2 hours and with high PCR efficiencies. The selected RE targets comprise over 1500 copies in the genome and allow robust detection of low level cell-free (cf) DNA pg/µL) from <0.5mL plasma. The 80bp target measures total cfDNA. The 265bp target is used to assess cfDNA fragmentation and is presented as the DNA Integrity Index (DII=265/80) which is a ratio of the 265bp quantity divided by 80bp quantity. cfDNA concentration and integrity assessment has shown great promise as a highly sensitive, minimally invasive blood biomarker for multiple cancer types. InnoGenomics has developed an accurate, extremely sensitive liquid biopsy blood test for improved management of cancer patient care. Utilizing standard qPCR platforms for rapid and cost-effective processing, this test can reliably obtain results from small blood volumes (<0.5 mL of plasma), and possesses exceptionally high analytical sensitivity with a detection capability of < 0.1 pg of DNA (LOD). The developed assay demonstrated high predictive capacity in discriminating a group of 39 CRC stage IV patients from 40 healthy controls using cfDNA concentration and DNA Integrity Index (DII-265/80) values. All markers displayed high Area Under the receiver operating characteristic Curve (AUC) values - 80bp target: 0.9891, 265bp target: 0.9859, and DII-265/80: 0.8603. The DNA integrity ratio (DII) showed great potential as a prognostic marker for progression free survival (P=0.019). A retrospective cohort study of 3 patients demonstrated the potential use of this RE-qPCR cfDNA assay for cancer treatment monitoring. NCI SBIR Phase I studies showed high predictive capacity in discriminating metastatic colorectal cancer (CRC) patients from non-cancer healthy controls, with a significant increase in sensitivity and specificity compared to the CEA test, a serum biomarker currently used for CRC patient monitoring. Retrospective patient case studies demonstrated the test's ability to detect resistance to therapy and disease progression more accurately than CEA results and earlier than CT imaging. These results suggest strong potential clinical utility in several applications: (1) treatment monitoring for CRC stage IV patients, (2) recurrence surveillance for CRC stage I-III patients, (3) assessment of residual disease and (4) as a prognostic tool to help predict CRC patient outcomes which can serve as a tool to guide personalized/effective treatment regimens. A detailed prospective study will be undertaken during Phase II research, in collaboration with MD Anderson Cancer Center, to clinically validate the test. ACKNOWLEDGEMENT: This project was funded by an SBIR Phase I contract from NCI.

#5576

Isolation and detection of breast cancer cells in blood samples using antibody cocktails selected by large-scale genome-wide screening.

Lucas Delmonico, Edward C. Goodwin, Marcia Vasconcellos Fournier. _Bioarray Genetics Inc., Farmington, CT_.

Screening and prevention is critical to reducing breast cancer deaths, and mammography is not an ideal tool. Mammography has not reduced invasive breast cancer incidence, it has high numbers of false positives and it detects many non-invasive cancers that are not life threatening. Metastatic breast cancer is the second leading cause of cancer death for women. The development of a new blood test for breast cancer screening could offer significant improvements with the potential to be more accurate, less costly, less intimidating, not subject to age differences, and able to discriminate between cancers subtypes. Detection of breast cancer cells (BCC) in blood samples offers a valuable opportunity for screening. However, BCC in blood are rare and must be identified against a background of millions of other blood cells. Although many technologies have been developed for the isolation and detection of circulating tumor cells using EpCAM and cytokeratins, these tests do not provide levels of sensitivity and specificity required for a cancer screening tool. To address this critical unmet need, we developed a panel of cell surface markers whose expression patterns specifically distinguish breast cancer cells in blood samples. These genes were selected based on a discovery platform tailored to detect early stage breast cancers and include 20 novel putative cell surface molecules that are candidates for breast cancer cell detection and isolation. We prioritized these molecules by specificity of expression in tumor cells including: 1) Ratio >25:1 average breast tumor biopsy expression versus average normal blood expression, 2) High expression relative (>3 fold) in >20% of tumor biopsy samples, 3) High expression (>3 fold) in >40% of breast cancer cell lines, and 4) Expression significantly associated with 10-year outcomes (Kaplan Meier analysis, p<0.05). We combined the top 10 ranking genes in each of these categories, resulting in a list of 20 candidates that represent high priority targets. Antibodies for all 20 genes are commercially available. Expression in micro dissected biopsies was markedly higher than non-breast cancer blood for most. Also, most were expressed at lower levels than EpCAM in normal breast tissue and had background level expression in isolated normal blood cells. The innovation of this strategy is twofold. First, it uses a panel of molecules instead of an individual molecule to detect and isolate breast cancer cells and, second, it relies on molecules that are clinically relevant to breast cancers, rather than relying on generic epithelial cell markers. Current studies include the validation of antibody cocktails with specificity and sensitivity that outperforms the current EpCAM enrichment method and provides a proof of feasibility for screening of breast cancer.

#5577

Single cell mutation and gene expression co-analysis of lung circulating tumor cells.

Sarah Owen, Ting-Wen Lo, Shamileh Fouladdel, Ebrahim Azizi, Sunitha Nagrath. _University of Michigan, Ann Arbor, MI_.

With the aim of personalized medicine, there have been many targeted therapies developed against cancer-specific antigens. The efficacy of targeted therapies was first shown in lung cancer patients containing activating epidermal growth factor receptor (EGFR) mutations who responded to tyrosine kinase inhibitors. Despite the promise of these EGFR targeted therapies, lung cancer patients routinely acquire an additional EGFR mutation, T790M, which causes treatment resistance. In order to determine if a patient qualifies for targeted therapy, they must get an invasive tumor biopsy, and may require a second biopsy if they stop responding to treatment. Circulating tumor cells (CTCs) are shed from the primary tumor into the bloodstream, a fraction of which lead to the development of metastases. CTCs can be isolated from a minimally-invasive blood draw, termed a "liquid-biopsy", and their increased presence in the blood correlates with poor patient prognosis. CTCs present an approach to track and screen patients during treatment to determine if they acquire new mutations or other aggressive cell phenotypes. CTCs have been shown to be highly heterogeneous, existing in phenotypic states across the entire epithelial to mesenchymal transition spectrum as well as contain both primary tumor matched and unmatched mutations. We have previously established a workflow for the isolation and single cell characterization of CTCs. CTCs are isolated from the blood using a novel high-throughput inertial microfluidic device, the Labyrinth. This device has been optimized using cancer cell lines across many cancer types and has been used to process hundreds of blood samples from breast, lung, and pancreatic cancer patients for CTC enumeration. After CTC isolation using the Labyrinth, single cell suspensions are generated for lysis and RT-qPCR for up to 96 single cells using a 96-gene panel. Here we present initial results for the incorporation of mutation screening on the same single cells using digital PCR (dPCR). dPCR relies on small sample quantity to load one target molecule per reaction droplet for end-point PCR analysis. Due to its single molecule resolution, it has increased sensitivity compared to other traditional PCR methods, making it an ideal platform for single cell mutation screening. This enables us to determine if the cell is homo- or heterozygous, as well as quantify the relative expression in heterozygous mutations. We have validated the technology for single cell mutation detection of EGFR mutations, L858R and T790M, in addition to other genes such as TP53 and KRAS. The specificity of the EGFR mutation detection assays was confirmed using lung cancer cell line H1975, which is known to carry these two heterozygous mutations. Ultimately, the ability to couple single cell mutation and gene expression analysis using CTCs may allow oncologists to better monitor treatment response to guide the use and duration of targeted therapies.

#5578

Genomic and expression profiling reveals molecular heterogeneity of disseminated tumor cells in early breast cancer.

Mark Jesus M. Magbanua, Hope Rugo, Louai Hauranieh, Ritu Roy, Janet Scott, Jen Chieh Lee, Feng Hsiao, Eduardo Sosa, Denise Wolf, Laura van't Veer, Laura Esserman, John Park. _University of California San Francisco, San Francisco, CA_.

BACKGROUND: The presence of disseminated tumor cells (DTCs) in bone marrow of early breast cancer (EBC) patients is a strong predictor of poor prognosis. We assessed heterogeneity in copy number, PIK3CA mutation, and gene expression in DTCs to shed light on the molecular biology of these cells.

METHODS: We isolated EPCAM-positive DTCs in 71 EBC patients using immunomagnetic enrichment and fluorescence-activated cell sorting (IE/FACS). Isolated DTCs, along with corresponding primary tumors (n=16), were subjected to genome-wide copy number profiling (n=47) by array comparative genomic hybridization, and mutation screening of the PIK3CA gene (n=53) by Sanger sequencing. The expression of 64 cancer-related genes in DTCs was analyzed by microfluidic-based multiplexed RT-QPCR (n=30). DTC expression profiles were compared with available gene expression data from circulating tumor cells (CTCs) of metastatic breast cancer patients.

RESULTS: Copy number profiles of DTCs were less aberrant and distinct from their corresponding primary tumors. PIK3CA mutations detected in 26% of DTCs were mutually exclusive to those found in matched primary tumors. Expression profiles of DTCs were distinguishable from marrow leukocytes, and displayed up-regulation of oncogenes MYC and CCNE1. Unsupervised hierarchical clustering analysis revealed two subtypes of DTCs: (1) luminal with dual epithelial-mesenchymal properties (high ESR1 and VIM/CAV1 expression), and (2) basal-like with proliferative/stem cell-like phenotype (low ESR1 and high MKI67/ALDH1A1 expression). DTCs possessed gene expression signatures that were unique from those of CTCs. ALDH1A1, CAV1 and VIM were up-regulated in DTCs relative to CTCs. ESR1/ER and ERBB2/HER2 status in DTCs vs. corresponding primary tumors showed high discordance (40% and 43%, respectively), suggesting shift in biomarker status in micrometastatic cells in the bone marrow.

CONCLUSIONS: We demonstrate the feasibility of isolation and comprehensive molecular characterization of DTCs from bone marrow of EBC patients. Comparative genomic analysis suggests that DTCs disseminate early and acquire genomic aberrations independently of the primary tumor. Expression profiling revealed two distinct subpopulations of DTCs. Validation in larger cohorts is needed to confirm the presence of these molecular subtypes and to evaluate their biological and clinical significance.

#5579

Increased number of EMT-like CTCs relative to epithelial CTCs reveals signatures of poor outcomes in pancreatic cancer.

Mina Zeinali,1 Vasudha Murlidhar,1 Shamileh Fouladdel,1 Mathias Hafner,2 Shimeng Shao,1 Lili Zhao,1 Heather Cameron,1 Armand Iii Bankhead,1 Jiaqi Shi,1 Kyle C. Cuneo,1 Vaibhav Sahai,1 Ebrahim Azizi,1 Max S. Wicha,1 Diane M. Simeone,1 Sunitha Nagrath1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _Institute for Medical Technology of Heidelberg University & University of Applied Sciences Mannheim, Mannheim, Germany_.

Objective: Although circulating tumor cells' (CTCs) isolation from pancreatic adenocarcinoma (PaCa) patients is feasible, investigation of their clinical utility has proven less successful than other cancer types, due to poor technology sensitivity and EpCAM-only based CTC assays.

Design: We present an integrated technology and biology-based approach using a microfluidic "Carpet chip" to study the biologic relevance of rare CTCs. We isolate both circulating epithelial CTCs (EpCs) and EMT-like CTCs (EMTCs) simultaneously from whole blood of PaCa patients (n=35), targeting two different surface markers: anti-EpCAM and anti-CD133. Gene expression profiling of CTCs was performed with 96 genes (n=17).

Results: Recovery of cancer cell lines spiked into whole blood is ≥97% with >76% purity. Of the 35 PaCa patients analyzed, 34 had ≥5 EpCs/mL and 35 had ≥15 EMTCs/mL. Higher numbers of EMTCs were observed in patients with lymph node involvement (N1) compared to patients without. The ratio of EpCs to total number of CTCs marginally decreased with advanced stage, while the ratio of EMTCs increased. Gene expression profiling showed CXCR1 was significantly upregulated in EpCs whereas POU5F1/Oct-4 and MYC were upregulated in EMTCs, demonstrating inherent phenotypic differences. Early-stage patients with significantly better overall survival and/or progression-free survival showed significant higher expression of genes including ERCC1 and TTF1.

Conclusions: We demonstrated successful isolation of heterogeneous population of CTCs (EpCs and EMTCs). We recovered significantly higher number of EMTCs in PaCa patients compared to EpCs, reflecting aggressive nature of PaCa. Profiling of CTCs revealed genetic signatures relevant to predicting patients' outcomes.

#5580

Identification and monitoring of somatic mutations in circulating cell-free DNA by next-generation sequencing in patients with lung adenocarcinoma.

Ilaria Francaviglia,1 Gilda Magliacane,1 Greta Grassini,1 Salvatore Girlando,2 Daniela Medicina,3 Chiara Lazzari,1 Alessandra Bulotta,1 Lorenza Pecciarini,1 Claudio Doglioni,1 Maria Giulia Cangi1. 1 _San Raffaele Scientific Institute, Milano, Italy;_ 2 _Santa Chiara Hospital, Trento, Italy;_ 3 _University of Brescia, Spedali Civili, Brescia, Italy_.

The analysis of circulating cell-free tumor DNA (ctDNA), which can be obtained from plasma by non-invasive procedures, was proven useful to provide biomarkers in the management of non-small-cell lung cancer (NSCLC) patients. Several studies assessed ctDNA prognostic and predictive value as source of key data for therapeutic target selection and drug resistance in such patients. The purpose of the present study was to compare the assessment of NSCLC common hot spot mutations in ctDNA samples using the NGS Oncomine cfDNA Lung Cancer assay (ThermoFisher) to the standard clinical tests (i.e. real-time quantitative PCR and droplet digital PCR), performed in both FFPE tumor and ctDNA samples. In particular we aimed to evaluate the feasibility and accuracy of this assay for (1) the detection of EGFR/KRAS mutations in 50 patients with newly diagnosed lung cancer and no possibility to obtain tissue samples and (2) the identification of anti-EGFR treatment acquired resistance mutations in 50 NSCLC EGFR-mutated patients with disease progression. The Oncomine cfDNA Lung assay is a multiplexed sequencing test, designed to detect 150 hotspot mutations in 11 lung cancer-related genes (ALK, BRAF, EGFR, ERBB2, KRAS, MAP2K1, MET, NRAS, PIK3CA, ROS1, and TP53) with a limit of detection as low as 0.1%. We analyzed a total of 100 ctDNAs which were sequenced using the Ion S5 system. Libraries were templated using the Ion Chef and multiplexed as 8 libraries on a 520 chip. Data analysis was performed in Torrent Suite using the variant Caller plugin. The total process time, from ctDNA isolation to result reporting, was as short as 4 days, supporting a workflow where blood samples are received early on day 1 and final variant calls are available on day 4. ctDNA NGS analysis for the newly diagnosed patients with no available tumor sample identified KRAS mutations (7%) and more importantly, targetable EGFR mutations (10%). For the patients with progression disease, EGFR acquired resistance mutations were found in 78% of the cases. Overall, the most frequently mutated genes were EGFR (85%), TP53 (26%), PIK3CA (18%), ALK (10%), and MET (8%). Taken the EGFR mutation detected by routine methods as the gold standard, the concordance with EGFR variants detected by NGS was 97%. Interestingly, the NGS approach detected the same EGFR mutations with the same allelic frequency of standard methods. These preliminary data confirm the potential of the Oncomine cfDNA lung assay for plasma genotyping which allows both noninvasive multiplexed detection of targetable genomic alterations and monitoring of acquired resistance mutations in lung cancer.

#5581

Clinical validation of a highly sensitive assay to detect mutations in plasma from advanced lung adenocarcinoma patients.

Harriet Wikman,1 Malte Mohme,1 Melanie Janning,1 Alexander Sartori,2 Katrin Lamszus,1 Sabine Riethdorf,1 Darryl Irwin,3 Manfred Westphal,1 Sonja Loges,1 Klaus Pantel1. 1 _Univ. Medical Ctr. Hamburg-Eppendorf, Hamburg, Germany;_ 2 _Agena Bioscience, Hamburg, Germany;_ 3 _Agena Bioscience, Inc., Herston, Australia_.

The use of different liquid biopsy tools including circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) have shown great promise for the use in personalized cancer diagnostics, treatment monitoring, and research, especially among metastatic patients. The power of ctDNA analyses in early detection of acquired resistance against e.g. EGFR-targeted therapies in non-small cell lung cancer (NSCLC) have been demonstrated in several studies. However, NSCLC patients display lower ctDNA and CTC concentrations than expected and in particular brain metastases pose a challenge, probably due to the blood-brain barrier. In this study, we used a novel highly sensitive assay to detect clinically relevant mutations in ctDNA and detected CTCs in blood from metastatic NSCLC patients. 56 metastatic NSCLC cases were enrolled in this study. 37 patients presented with brain metastases and 20 of them had brain as the only site of metastasis (oligo-metastatic disease). Cell free (cf)DNA was isolated from 1.5-2ml of plasma using the Circulating Nucleic Acid kit (Qiagen). The ctDNA quantity was measured by Qubit Fluorometer (Thermo Fisher) and the quality was assessed by Tapestation (Agilent technologies). The UltraSEEK™ Lung Panel on the MassARRAY® System (Agena Bioscience) was used for the detection of mutations in five genes EGFR, KRAS, BRAF, ERBB2 and PIK3CA covering 67 different hot spot mutation loci. CTCs were detected by the CellSearch system. The total cfDNA concentration yields were low in most cases (median; 21ng/30µl, mean; 39ng/30µl, ± SD 2.6 ng/µl isolated from 1.5-2ml of plasma). Mutations could be detected in 26 samples (46.4%), with the variant allele frequency ranging between 0.1% and 5%. EGFR mutations were detected in 23.2% (13/56), KRAS in 21.4%, ERBB2 in 7.1%, PIK3CA in 3.6% and BRAF in 1.8% of the samples. Mutations were detected in 57.9% of patients with peripheral (non-brain) metastases, whereas 40.5% of brain metastatic patients had mutations detected in blood. Among oligo-metastatic patients, 30.5% had detectable mutations in plasma. In 42 samples, the EGFR mutation status in the primary tumor tissue was also determined. In 90.5% of samples, concordant results between plasma and tissue were found. In two patients, an EGFR mutation was only detected in the plasma, whereas in two other samples the mutation was only detected in the tumor tissue. In 46 patients, CTCs were measured. In 11 patients (23.9%), ≥ 1 CTC was detected. In 64.7% of patients, an informative result was obtained when results from both liquid biopsy approaches were combined. The UltraSEEK technology was able to detect mutations in cfDNA in plasma from NSCLC patients with limited, oligo-metastatic tumor burden, low amounts of total cfDNA, and very low fractions of ctDNA. Parallel assessment of CTCs in patients without detectable ctDNA enlarged the population of patients amenable to successful liquid biopsy analysis.

#5582

Use of circulating tumor DNA (ctDNA) to predict superiority of dosing schedules in early clinical development.

Juliann Chmielecki,1 Zhongwu Lai,1 Kristy Potts,2 Kavita Garg,2 Mark Li,2 J Carl Barrett1. 1 _AstraZeneca, Waltham, MA;_ 2 _Resolution Bioscience, WA_.

Changes in ctDNA can be used as a surrogate molecular marker of response to determine clinical efficacy and often precede imaging changes. This analysis is valuable to early clinical trials for dose optimization as response data may not be available for patients because of the short period of time on drug. Unlike other ctDNA applications where tumor-specific single gene mutations are followed over time, inclusion of "all comers" populations in phase I trials necessitates a comprehensive assay design optimized to detect multiple ctDNA changes from diverse tumors. Therefore, we developed a highly sensitive bespoke ctDNA panel (Resolution Bioscience) suitable for monitoring applications in "all comers" solid tumor populations. The panel uses next-generation sequencing technology to interrogate mutational hotspots from 8 oncogenes as well as the complete coding sequences of 3 tumor suppressor genes. Validation studies established the mutant allele frequency (MAF) limit of detection for short variants and small insertions/deletions as 0.1%; additionally, mutant alleles detected at or above this frequency can be followed below this threshold in serial samples. The assay requires 2mL plasma and results are reported within 14 days. In silico analysis predicted detection of at least one monitorable somatic alteration from ~63% of solid tumors that shed ctDNA. To demonstrate applicability, we used this assay to analyze serial ctDNA samples from patients enrolled in dose-escalation cohorts for the Wee1 inhibitor, AZD1775 (NCT02610075). Dosing schedules were compared for the degree of ctDNA clearance after completion of the first cycle, and correlated with subsequent RECIST response data, where available. To date, 15 patients were evaluable, with more anticipated as additional cohorts accrue. Greater ctDNA decreases were observed with higher drug dose, and longer drug exposure. Together with safety and exposure data, our studies demonstrate that ctDNA dynamics can be used to inform decisions around optimal dosing schedule. This enables rapid decision making before imaging studies are available, and complements traditional measurements of target engagement.

#5583

Plasma levels of circulating MACC1 transcripts co-segregate with other prognostic markers and treatment response in glioblastoma patients.

Carsten Hagemann,1 Nikolas Neuhaus,1 Mathias Dahlmann,2 Almut F. Kessler,1 Dennis Kobelt,2 Pia Herrmann,2 Matthias Eyrich,1 Benjamin Freitag,1 Thomas Linsenmann,1 Camelia Monoranu,1 Ralf-Ingo Ernestus,1 Mario Löhr,1 Ulrike S. Stein2. 1 _University of Würzburg, Würzburg, Germany;_ 2 _Charité Berlin and Max Delbrück Ctr. for Molecular Medicine, Berlin, Germany_.

IDH1-mutation and MGMT-promoter methylation are established prognostic markers in Glioblastoma multiforme (GBM); however, invasive procedures are needed to obtain the required tumor tissue. Metastasis-associated in colon cancer-1 (MACC1) can easily be measured in peripheral blood and has been established as a prognostic plasma marker for several solid cancer entities. Further, MACC1 has been identified by us and others as a promising biomarker for prognostication for GBM, when detected in the tumor tissue. Here, we evaluated whether circulating MACC1 transcripts could be useful to predict clinical outcome and response to therapy in GBM.

Plasma samples were collected from GBM patients (IDH1 wildtype (wt), IDH1R132H, and unspecified) before surgery. Samples from healthy volunteers served as controls. MACC1-transcript levels, determined by qRT-PCR, were correlated with patient overall survival (OS) and clinical data of known prognostic significance. Cut-off values for Kaplan-Meier analyses were determined by ROC calculations. Cluster analyses were performed.

MACC1 transcripts were higher in plasma of GBM patients compared to healthy controls (P<0.0001), yet differentially distributed within the cohort. Low MACC1 levels clustered together with all other favorable markers, i.e. small tumor volume, young age, IDH1R132H and MGMT promoter methylation. MACC1 levels correlated with patient prognosis in conjunction with the IDH mutation status: patients with IDH1R132H mutation and low MACC1 levels showed the most favorable outcome (mean OS 54.0 months (m), SD=9.8; 95% CI 34.7-73.3), those with IDH1wt and high MACC1 had the worst prognosis (mean OS 10.5 m), while those with IDH1wt and low MACC1 were intermediate (mean OS 20.3 m). No patients in this cohort displayed IDH1R132H and high MACC1 expression. Interestingly, patients with low MACC1 levels receiving standard therapy (operation and radiochemotherapy according to Stupp) survived longest (mean OS 36.9 m, SD=6.5; 95% CI 24.1-49.7). Patients with high MACC1 showed shorter OS (mean OS 13.0 m). The worst prognosis had patients not receiving the standard regimen, independently of their MACC1 levels (MACC1 low: 5.2 m; MACC1 high: 5.5 m).

The inclusion of circulating MACC1 transcript levels into the diagnostic workup might improve the accuracy of outcome prediction and thus help to define more precise risk categories of GBM patients. Whether pharmaceutical inhibition of MACC1 expression might be of general benefit for the treatment response and survival of GBM patients remains to be determined.

#5584

Circulating tumor cells in the peripheral blood and leukapheresis product of non-small cell lung cancer patients.

Kiki C. Andree,1 Menno Tamminga,2 Anouk Mentink,1 T Jeroen Hilterman,2 Harry J. Groen,2 Leon W. Terstappen1. 1 _University of Twente, Enschede, Netherlands;_ 2 _University Medical Center Groningen, Groningen, Netherlands_.

Introduction The number of circulating tumor cells (CTC) isolated by the FDA approved CellSearch (CS) system in 7.5 mL of blood of metastatic and non-metastatic non-small cell lung cancer (NSCLC) patients is low or absent. Processing a larger blood volume could yield a greater amount of CTC from these patients. An approach to probe larger volumes is using diagnostic leukapheresis (DLA) where the blood can be split into its components after which selective harvesting of mononuclear cells (MNC) including CTC can take place.

Methods Patients with histologically proven NSCLC who either have been recently diagnosed or are eligible for new therapy can participate in this ongoing study. Patients had DLA for MNC collection derived from one total body blood volume. Before and after DLA, whole blood was drawn in a CellSave blood collection tube and analyzed by CS. The obtained DLA product was divided for analysis. 2x108 cells (on average 2mL of DLA product, representing ~100mL of blood) was diluted in 7.5 mL buffer, and analyzed by CS. 9 mL of the DLA product (on average 1x109 cells) was fixed and depleted of leukocytes using the RosetteSep CD45 depletion kit (Stemcell Technologies), allowing processing of a larger volume, followed by CS analysis. Results of molecular characterization of the detected CTC is pending.

Results So far, a total of 8 DLA's were performed in 7 stage 4 NSCLC patients. Sample collection took place before (t=0) and/or after (t=1) treatment. Patients underwent DLA in about 110 minutes, at this time an average of 4860 mL of blood was processed. No complications occurred, no post-procedure complaints were recorded. Similar DLA products with an average of 95 mL were obtained. CTC counts for these patient samples are shown in the table below.

CTC counts blood and DLA

---

Patient | t= | #CTC pre blood | #CTC post blood | #CTC DLA (~2mL, 200x106 cells) | #CTC depleted DLA (~9mL, 900x106 cells)

1 | 0 | 1 | n/a | 11 | 4

1 | 1 | 3 | 0 | 2 | 0

2 | 0 | 35 | 14 | 60 | 20

3 | 1 | 0 | 0 | 0 | 0

4 | 0 | n/a | 0 | 4 | 49

5 | 0 | 0 | 0 | 1 | 6

6 | 0 | 0 | 0 | 0 | 2

9 | 0 | 2 | 0 | 11 | 4

Conclusion DLA is a novel method to collect CTC from the blood. DLA can result in the detection of CTC where the use of 7.5mL of blood is not sufficient. Additionally, DLA shows an increase of CTC yield in patients with CTC detected in 7.5 mL of blood.

#5585

Extracellular vesicle poly(A)+ RNA analysis for cancer biomarker screening.

Taku Murakami,1 Cindy Yamamoto,1 Mieko Ogura,1 Melanie Oakes,1 Hiroshi Harada2. 1 _Hitachi Chemical Co. America, Ltd., Irvine, CA;_ 2 _Sapporo City General Hospital, Sapporo, Japan_.

Extracellular vesicles (EV) are a promising biomarker source and present in several biological fluids such as plasma (8.0 x 1011 particles/mL, 121 ± 2.3 nm in diameter) and urine (1.1 x 1011 particles/mL, 152 ± 11.2 nm in diameter). In order to streamline marker screening and validation in translational studies, we developed and optimized high throughput filter-based EV capture, poly(A)+ RNA isolation and gene expression assay protocols for RT-qPCR and RNA-seq. First, we confirmed that plasma and urinary EV mRNA profiles (12 to 15 genes by RT-qPCR) are stable up to 1 week at 4°C, and up to 5 repeats of freeze-thaw cycles, and urinary EV was stable up to 10 months at -80°C, indicating EV mRNA could be a useful biomarker source. Although EV mRNA is less abundant (550 pg mRNA from 1 mL plasma) compared to tissue mRNA or EV non-coding RNA, the optimized protocol allows quantification of about 9,000 genes in average by RNA-seq from 2 mL frozen plasma (N=3) with high reproducibility among the replicates (Spearman correlation: 0.9086 to 0.9326) and between the commercial EV kits (Spearman correlation: 0.9207 to 0.9683). About 77% of the 1000 most abundant genes in plasma EV are expressed ubiquitously throughout various organs but some are expressed specifically in blood (12%), spleen (4.4%), esophagus mucosa (1.2%), etc. according to the Genotype-Tissue Expression (GTEx) database. In urinary EV, other than ubiquitously expressed genes (88%), the genes specific to liver (1.5%), bladder (1%), brain (1%), etc. are present. These data suggest EV mRNA are released from various organs and circulating in biological fluids. The protocol was further applied to biomarker discovery for bladder cancer, and differential gene expression analysis of EV mRNA was conducted using bladder cancer (N=4), cancer remission (N=2), and healthy/disease control urine samples (N=4). Ingenuity pathway analysis indicated that cancer-related molecular and cellular functions are activated in bladder cancer urinary EV but not in remission, while immune-related functions are activated both in cancer and remission. These data indicate that EV mRNA is a promising biomarker source for cancer biomarker screening, and could be used in translational studies.

#5586

Circulating tumor cells as the prognostic marker for non-remission and disease-specific mortality of patients with thyroid cancer.

Ching-Ping Tseng,1 Jen-Der Lin,2 Miaw-Jene Liou,2 Wei-Shan Hung,1 Hsueh-Ling Hsu,1 Kong-Kit Leong,1 Yu-Ting Chen,1 Ju-Chien Cheng3. 1 _Chang Gung University, Taoyuan, Taiwan;_ 2 _Chang Gung Memorial Hospital, Taoyuan, Taiwan;_ 3 _China Medical University, Taichung, Taiwan_.

Patients with thyroid cancer usually have good prognosis. However, 15-20% of patients ultimately develop recurrence and disease-related death. In this study, we investigate whether the number of circulating tumor cells (CTCs) expressing either epithelial cell adhesion molecule (EpCAM), podoplanin (PDPN, a marker related to poor prognosis in patients with thyroid cancer), or thyroid-stimulating hormone receptor (TSHR, a marker for thyroid cells) is a prognostic marker for non-remission and disease-specific mortality (DSM) of patients with thyroid cancer. Blood samples were collected from patients (n = 128) after thyroidectomy or radioactive iodide therapy. After enrichment of CTCs by a negative selection PowerMag system, enumeration and subtyping of the CD45-depleted cells were performed by immunofluorescence staining using the antibodies aginst EpCAM, TSHR, and PDPN, respectively. Our data revealed that the number of EpCAM+-CTCs (p < 0.001) and PDPN+-CTCs (p < 0.001), and TSHR+-CTCs (p < 0.001) for patients in the non-remission group (n = 43) was significantly higher when compared to the remission group (n = 85). At the cutoff of 40, 14, and 47 cells/mL for EpCAM+-CTCs, TSHR+-CTCs, and PDPN+-CTCs, the accuracy of the assay was equivalent to 80.4%, 76.6%, and 77.3%, respectively. On the other hand, the number of EpCAM+-CTCs (p < 0.001), PDPN+-CTCs (p = 0.013), and TSHR+-CTCs (p < 0.001) for patients in the DSM group (n = 17) was significantly higher when compared to the patients who survived (n = 111). At the cutoff of 27, 25, and 9 cells/mL for EpCAM+-CTCs, TSHR+-CTCs, and PDPN+-CTCs, the accuracy of the assay was equivalent to 69.5%, 67.2%, and 68.5%, respectively. These data together indicate that CTC testing is worthy to be considered as a routine clinical test to benefit clinical care of patients with thyroid cancer.

#5587

Sample quality control of cell-free DNA.

Melissa H. Liu,1 Eva Graf2. 1 _Agilent Technologies, La Jolla, CA;_ 2 _Agilent Technologies, Germany_.

Sequencing of cell-free DNA (cfDNA) extracted from blood specimens or other body fluids is possible due to the establishment of low input library protocols for next-generation sequencing workflows. Accurate quantification of cfDNA samples is essential to determine suitable input amounts for cfDNA library preparation prior to sequencing. The main component of cfDNA samples is the mononucleosome with a size around 170 bp, sometimes with additional species representing nucleosome multimers. Further, cfDNA samples may contain larger DNA fragments dependent on the donor's health status, preanalytical sample treatment, or extraction method. High molecular weight material can negatively influence library preparation and subsequently result in lower sequencing depth. Therefore, reliable quantification of cfDNA requires a method that separates DNA fragments by size, such as electrophoresis. This poster shows the use of a new cfDNA assay for the 2100 Bioanalyzer system as an add-on for the High Sensitivity DNA assay. The assay enables automated cfDNA quantification with pre-defined regions. Moreover, the results include sample purity as a score to qualify cfDNA samples according to their contamination level with high molecular weight material. The features of the assay are described with examples of typical sample patterns.

#5588

Uneven distribution of CTCs in magnetic fields following binding with iron nanoparticles.

Chengsen Xue, Christina D. Swenson, Thomas W. Mc Closkey. _ICON Laboratory Services, Farmingdale, NY_.

Circulating tumor cell (CTC) detection and characterization have recently become an integral part of precision medicine. Several large, multicenter clinical studies confirmed that the prevalence of CTC is a useful predictor of treatment efficacy in metastatic breast cancer. The number of CTCs is strongly correlated with clinical progression. Oncology clinics widely use this biomarker to provide prognostic information. There are several platforms to isolate and measure CTCs. The only FDA-approved method is the CellSearch system developed by Veridex. The CellSearch System uses anti-EpCAM antibodies and polymer-coated iron nanoparticles in order to isolate CTC with the use of a magnetic field. The enriched CTC fraction is then placed in a MagNest chamber and the transparent slide is read by the automatic fluorescent CellTracks Analyzer. By using prelabeled breast cancer cells as quality control (QC) samples, we semiquantitated the distribution of CTC cells on slides in eleven QC samples. We found that CTC cells distribute unevenly on the slides. In order to count all CTCs, the whole area of the slide is divided into 175 small regions by the manufacturer. To better quantitate the CTC number, it is important to have all cells evenly spread over the slide. Our results showed that 41 out of 175 regions contained higher QC CTC counts. The heat map indicates that the magnetic field lines may have an effect on CTC localization. The CTC platforms are susceptible for underestimating actual CTC counts. Our findings indicate that cell concentration in certain regions caused by a magnetic field effect could be one of several contributing factors.

#5589

Detection of epidermal growth factor receptor mutations from circulating tumor DNA versus archival tissue of lung cancer.

In-Jae Oh,1 Hyeong-Won Seo,1 Hyun-Ju Cho,1 Ha-Young Park,1 Bo-Gun Kho,1 Jin-Sun Chang,1 Tae-Ok Kim,1 Hong-Jun Shin,1 Cheol Kyu Park,1 Jung-Hwan Lim,1 Yong-Soo Kwon,1 Yu-Il Kim,1 Sung-Chul Lim,1 Young-Chul Kim,1 Yoo-Duk Choi2. 1 _Chonnam National Univ. Hwasun Hospital, Jeonnam, Republic of Korea;_ 2 _Chonnam National Univ. Hospital, Gwangju, Republic of Korea_.

Background: Epidermal growth factor receptor (EGFR) mutations are well known predictive marker of targeted therapy. We compared the sensitivity of EGFR mutation detection techniques between matched achival lung cancer tissue and peripheral blood sample.

Methods: We collected the paired samples from plasma and paraffin-embedded tumor tissue obtained before EGFR-tyrosine kinase inhibitor (EGFR-TKI). DNA extraction was performed using the QIAamp MinElute virus spin kit and EGFR mutation analysis was done by two detection methods. The current standard test is the PNAClampTM (Clamp) which is the PNA-based PCR clamping that selectively amplifies only the mutated target DNA sequence. The new technique is the PANAMutyperTM EGFR test (Mutyper), which use PNA clamping-assisted fluorescence melting curve analysis.

Results: A total of 295 (188 male and 107 female) patients were analyzed in this study. The histologic types were composed of 258 adenocarcinoma, 22 squamous cell carcinoma and 15 others. Most were clinical stage IV (137, 46.4%) and the EGFR mutation rate of tissue sample by standard Clamp test was 25.7%. Plasma sensitivity was significantly higher in Mutyper than Clamp (71.2% vs. 30.0%, p<0.001) with tissue as reference. The overall concordance and degree of agreement between two samples were better in Mutyper (91.9%, k=0.762, p<0.001) than Clamp (81.7%, k=0.344, p<0.001). In tissue sample, the median progression-free survival (PFS) of EGFR sensitive group was significantly longer than negative group regardless of the two methods. In plasma sample, the median PFS was significantly longer only by Mutyper (9.9 vs. 2.2 months, p=0.001), not by Clamp (9.8 vs. 7.6 months, p=0.968).

Conclusions: Mutyper was useful liquid biopsy technique with better sensitivity than standard Clamp test.

#5590

MiSelect R: A novel single-cell retrieval system for CTC from whole blood for biomarker analysis in cancer targeted and immunotherapy.

Ju-Yu Tseng,1 Yen-Ru Chen,1 Chia-Ying Lee,1 Li-Fan Wu,1 Cheng-Hsu Wang,2 Shin-Hang Wang,1 Hui-Min Yu,1 Wei-Feng Fang,1 Mu-Yi Chen,1 Chwen-Cheng Chen3. 1 _MiCareo, Taipei, Taiwan;_ 2 _Chang Gung Memorial Hospital, Keelung, Taiwan;_ 3 _JN Biopharma Consulting, Taipei, Taiwan_.

Background: Circulating tumor cells' (CTCs) presence in patient's peripheral circulation has been recognized as a useful tool of liquid biopsy for cancer treatment. In addition to enumeration, analysis of associated protein and molecular alterations offers great potential for precision medicine. MiSelect R was invented for purification and characterization of rare cells including CTC. This study evaluates MiSelect R analytic performances and validates with clinical samples.

Methods: Lung cancer cell line H2228 was spiked into 8 mL of whole blood (16 cells/ 8mL) for single-cell retrieval, subsequent RT-PCR and sequencing analysis on ALK-translocation. Breast cancer cell lines, SKBr3 (high EpCAM expressing) and MDA-MB-231 (low EpCAM expressing), were spiked into 8mL whole blood from healthy subjects at various concentrations (4-1024 cells/ 8mL) for recovery rate and linearity. Experiments with low cell spikes (32 cells/ 8mL) vs. high cell spikes (1024 cells/ 8mL) were carried out to assess the within-device precision. The assay specificity was examined in blood of healthy subjects. Further evaluation was performed in metastatic breast cancer (mBC) patients.

Results: Spiked H2228 cells were retrieved with greater than 90% recovery rate and 70% purity from whole blood. Each retrieved single H2228 cell was confirmed to have ALK-EML4 translocation by RT-PCR and sequencing analysis. The detection ranges of 4 to 1024 SkBr3 and MDA-MB-231 cells per 8mL were confirmed to be linear with average recovery rate of 91% (R2 = 0.9949) and 70% (R2 = 0.9983), respectively. The within-device precision showed 9.23% and 7.82% CV for low cell spikes vs. high cell spikes. Great assay specificity was achieved with no CTC detected in healthy subjects (N = 42). 100% detection rate of CTC was achieved in untreated mBC patients and 47% for patients regardless of treatment history. Also, the HER2 score analyzed by MiSelect R in isolated CTCs was in agreement with that by IHC analysis. Nonetheless, the HER2 expression level was heterogeneous in the CTCs isolated from HER2-positive patients. Furthermore, the PD-L1 expression in CTCs was shown to be heterogeneous within and between cancer patients.

Conclusion: The MiSelect R System showed excellent capability for single-cell retrieval of rare cells from whole blood. It also demonstrated outstanding analytic performances and clinical sensitivity for CTC detection as well as versatile capability for multiple-biomarker characterization. It potentially provides important cellular and molecular information at single-cell level from liquid biopsy for prognosis.

#5591

Analytical and clinical validation of blood-based NGS panel for detection of genomic alterations in DNA repair pathways.

Zhixin Zhao. _Predicine, Inc, CA_.

Circulating cell-free DNA (cfDNA) and cell-free RNA (cfRNA) in cancer patients are valuable resource of biomarkers for early detection, diagnosis, treatment and monitoring of disease progression. Compared to cfDNA, cfRNA is more labile to degrade and fluctuate, and thus more challenging to be detected in blood. In the present study, we reported PrediSeq, a validated NGS-based liquid biopsy assay to detect fusions, splicing and mutation events using cfRNA. By using Predicine proprietary ultra-sensitive DeepSeaTM (Deterministic, Efficient, Precise, Sensitive Algorithm) variant detection algorithm, pre-analytical study demonstrated RNA-based detection of fusion, RNA splicing, and mutations using reference materials. In a LOD study, PrediSeq NGS assay is more sensitive than the digital PCR based assay in terms of mutation detection. In clinical validation study, tissue and plasma samples from various types of cancer patients (lung, breast, prostate etc) were used, where concordant fusion events with tissue were observed and RNA mutations confirmed variants detected at DNA level. In conclusion, a highly sensitive and accurate RNA-based liquid biopsy test has been developed to complement the use of cfDNA for detection of fusion, splicing and mutations.

#5592

HER-2 copy number measurement in tissue and cell-free plasma DNA of gastric cancer patients using droplet digital PCR method.

Boram Kim,1 Soo Kyung Nam,2 Soo Hyun Seo,2 Sang-Hoon Ahn,2 Do Joong Park,2 Hyung-Ho Kim,2 Kyoung Un Park,2 Hye Seoung Lee2. 1 _Seoul National University Hospital, Seoul, Republic of Korea;_ 2 _Seoul National University Bundang Hospital, Seongnam, Republic of Korea_.

In around 10% of gastric cancer, amplification and overexpression of the human epidermal growth factor receptor 2 (HER2) is observed and trastuzumab is adopted as a target therapy agent for HER2-positive gastric cancer. Because of efficiency and accuracy, droplet digital PCR (ddPCR) is one of the best methods for measuring HER2 amplification, especially in cell-free plasma samples. In this study, we aimed to investigate the correlation between tissue and plasma HER2 status by using ddPCR method in gastric cancer patients. We collected formalin-fixed and paraffin-embedded tissue (FFPE) and cell-free plasma samples of 81 gastric cancer patients, including 30 HER2 immunohistochemistry (IHC)-negative (0 or 1+) cases and 51 HER2 IHC-positive cases (IHC 2+, n=20; IHC 3+, n=31), with informed consents. We performed ddPCR of a target gene HER2 and a reference gene EIF2C1 in both tissue and cell-free plasma samples, and gene copy number (GCN) of HER2 was calculated. The median HER2 GCN of tissue DNA ddPCR was 2.61 (1.47-49.00) and the median GCN of plasma ddPCR was 1.83 (0.97-7.67). HER2 IHC results showed moderate correlation with HER2 GCN of tissue ddPCR (r=0.455, p<0.001) and weak correlation with plasma ddPCR (r=0.321, p=0.003). A ratio >2.405, which showed the best discriminating level from a receiver operating characteristic curve, was defined as a cut-off for tissue HER2 amplification using ddPCR method. Comparing the HER2 amplification results of tissue DNA ddPCR to HER2 IHC results, the concordance rate was 86.4% with Cohen's kappa of 0.715. Sensitivity and specificity of tissue ddPCR were 86.3% and 86.7%, respectively. HER2 amplification by tissue ddPCR was significantly associated with advanced gastric cancer and lymphatic invasion (p<0.05). HER2 GCN of tissue ddPCR was weakly correlated with plasma ddPCR (r=0.250, p=0.025). Sensitivity and specificity of plasma ddPCR for HER2 IHC positivity were 35.3% and 93.3%, respectively. However, sensitivity and specificity of plasma ddPCR were 66.7% and 60.0% with another cut-off of 1.715. In 51 HER2 IHC-positive cases, intratumoral heterogeneity was observed in 100% (5/5) of both tissue and plasma ddPCR-negative cases, 100% (2/2) of tissue-negative and plasma positive cases, 64.3% (18/28) of tissue-positive and plasma-negative cases, and 50.0% (8/16) of both positive cases (p=0.026). In conclusion, HER2 GCN of plasma ddPCR showed a weak correlation with tissue ddPCR, and the sensitivity of plasma ddPCR was not high. Intratumoral heterogeneity is suggested to be one of the possible causes, and these findings may contribute to developing plasma HER2 testing useful in daily practice.

#5593

The fragment size and levels of cell-free DNA provide prognostic information in patients with advanced pancreatic cancer.

Morten Lapin,1 Satu Oltedal,1 Kjersti Tjensvoll,1 Tove Buhl,1 Rune Smaaland,1 Nils Glenjen,2 Bjørnar Gilje,1 Oddmund Nordgård1. 1 _Stavanger University Hospital, Stavanger, Norway;_ 2 _Haukeland University Hospital, Bergen, Norway_.

Purpose: To investigate the prognostic value of cell-free DNA (cfDNA) fragment size and cfDNA levels in patients with advanced pancreatic cancer. Methods: Blood samples were acquired from patients with locally advanced or metastatic pancreatic cancer at baseline (n = 61) and one month (n = 48) after initiation of chemotherapy. All samples were processed by Lymphoprep™ (Axis Shield) density centrifugation followed by DNA isolation from the plasma fraction using the QIAamp Circulating Nucleic Acid kit (Qiagen). The cfDNA fragment size and cfDNA levels were then determined using the High Sensitivity DNA kit on an Agilent 2100 Bioanalyzer. A cohort of healthy age-matched volunteers (n = 28) constituted the control group. Results: Both the cfDNA fragment size (median 167 vs 176.5 bp; p < 0.001) and the cfDNA levels (median 4.48 vs 0.33 ng/mL plasma; p < 0.001) were significantly different between patient and healthy control samples. A pre-treatment cfDNA fragment size of ≤167 bp (median) was associated with both shorter progression-free survival (PFS) (4 vs. 7.7 months; log-rank p = 0.002) and overall survival (OS) (4.6 vs. 10.5 months; log-rank p = 0.001). Similarly, cfDNA levels above the median value were associated with both shorter PFS (3.3 vs. 7.7 months; log-rank p < 0.001) and OS (5.4 vs. 8.8 months; log-rank p = 0.001) for samples obtained before initiation of chemotherapy. Survival analysis on the combination of fragment size and cfDNA levels showed a significantly longer PFS (8.1 vs 4.6 vs 2.6 months; log-rank p < 0.001) and OS (10.6 vs 7.9 vs 3.8 months; log-rank p = 0.001) for patients negative for both parameters compared to patients with 1 or 2 positive parameters, respectively. Analyses of plasma samples obtained one month after initiation of chemotherapy indicated a trend towards an association with PFS (p = 0.066 and p = 0.025), but not OS (p = 0.504 and p = 0.213) for cfDNA fragment size and cfDNA levels, respectively. Univariate cox regression using both the continuous and the categorical variable for cfDNA fragment size and cfDNA levels, and the categorical variable for the combination test, confirmed the results from the Kaplan-Meier estimates. Conclusion: Determination of cfDNA fragment size and cfDNA levels is a non-invasive and simple method for predicting prognosis in patients with advanced pancreatic cancer. Further investigations in larger patient cohorts are needed to elaborate the consistency and clinical impact of this finding.

#5594

Circulating tumor cells measured in the pulmonary vein and radial artery during surgery of non-small cell lung cancer.

Menno Tamminga,1 S de Wit,1 Joost F. Swennenhuis,2 Ellen Heitzer,3 Michael Speicher,4 T. Jeroen N. Hiltermann,1 Ed M. Schuuring,1 Leon W.M. Terstappen,5 Harry J. Groen1. 1 _University Medical Centre Groningen, Groningen, Netherlands;_ 2 _University Twente, Groningen, Netherlands;_ 3 _Medical University Graz, Graz, Austria;_ 4 _Medical University Graz, Graz, Netherlands;_ 5 _University Twente, Enschede, Netherlands_.

Background: The number of circulating tumor cells (CTCs) detected by the CellSearch system is usually low or absent in NSCLC, possibly by elimination of CTCs in the capillary system. Our hypothesis is that the number of CTCs will be higher in the pulmonary vein draining the primary tumor compared to peripheral blood vessels and is influenced by the manipulation of the tumor during surgery.

Methods: In an ongoing study, patients with NSCLC undergoing open thoracotomy or video assisted surgery (VATS) lobectomy are included. During surgery, blood was drawn from the draining pulmonary vein (dv) of the lobe containing the tumor (T1,T2) and from the radial artery (ra) (T0,T1,T2). T0 was at start of surgery, T1 upon identification of the draining pulmonary vein and T2 right before the pulmonary vein was dissected. Dissection of the pulmonary artery occurred in open surgeries before, and in VATS after T2. EpCAM+ CTCs were detected by CellSearch and EpCAM- CTCs after filtration of the EpCAM depleted samples. EpCAM+ CTCs were isolated by FACS and analyzed for copy number variations (CNV) by single-cell whole genome sequencing and genomic alterations of EpCAM- CTC by FISH on the microsieves.

Results: So far, 17 patients have been included, one with a benign inflammatory tumor (pt 8). CTC counts were higher in the pulmonary vein than in the distal artery, with CTC clumps observed in some patients (* in table). No consistent effect of manipulation could be determined. In patient 4 and 5, EpCAM+ CTCs from the vein showed CNVs aberrations, whereas in patient 6 CTCs in the radial artery were of normal epithelial origin. EpCAM- CTCs of patient 1 and 2 were of both tumor and normal origin.

Conclusion: In NSCLC patients high amounts of single and clumps of EpCAM+ and EpCAM-CTCs were observed in the draining pulmonary vein during surgery. In contrast, the amount of CTCs measured in the radial artery is low (even after correction of blood flow from non-tumor vessels), suggesting loss of CTCs in the cardiac system.

EpCAM+ and EpCAM- CTCs in the draining vein and the radial artery during surgery

---

|

Draining vein T1 (EpCAM+) | Draining vein T1 (EpCAM-) | Draining vein T2 (EpCAM+) | Draining vein T2 (EpCAM-) | radial artery T0 (EpCAM+) | radial artery T0 (EpCAM-) | radial artery T1 (EpCAM+) | radial artery T1 (EpCAM-) | Radial artery T2 (EpCAM+)

Patient 1 | 85* | 35 | 1 | 5 | ND | ND | 1 | 2 | 2

Patient 2 | 10* | 4 | 19 | 11 | ND | ND | 0 | 5 | 0

Patient 3 | 50* | 130 | 101* | 10 | 3* | 2 | 0 | 0 | 0

Patient 4 | 2 | 11 | 7 | 1 | 1 | 0 | 0 | 0 | 0

Patient 5 | 1 | 2 | 7 | 3 | 1 | 2 | 2 | 3 | 1

Patient 6 | 0 | 0 | 0 | 5 | 0 | 0 | 27 | 21 | 0

Patient 7 | >3000* | >26000 | 1 | 320 | 0 | 0 | 0 | 0 | 0

Patient 8# | 0 | 17 | 596* | 10 | 0 | 0 | 0 | 5 | 0

Patient 9 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0

Patient 10 Lower lobe | 73* | 255 | 204* | 173 | ND | ND | 0 | 0 | 0

Patient 10 Middle lobe | 1 | 4 | 260 | 16 | ND | ND | 0 | 0 | 0

Patient 11 | ND | ND | 0 | 5 | ND | ND | ND | ND | 0

Patient 12 | 1 | 0 | 1 | 3 | ND | ND | 0 | 1 | ND

Patient 13 | 4 | 4 | 0 | 4 | 0 | 0 | 0 | 0 | 0

Patient 101 | 2 | 1 | 1 | 0 | 2 | 0 | 1 | 0 | ND

Patient 102 | 3 | 2 | 1 | 0 | 0 | 0 | 0 | 0 | 0

Patient 103 | 0 | 5 | >1000* | >1000 | 0 | 1 | ND | ND | 0

Patient 104 | 2 | 8 | 20* | 7 | ND | ND | 1 | 0 | 0

|  | |  | |  | |  | |

#5595

Predictive impact of sequential evaluation of PD-L1-expressing circulating tumor cells in NSCLC patients treated with nivolumab.

Yasuhiro Koh,1 Hiroaki Akamatsu,1 Keita Mori,2 Kuninobu Kanai,1 Atsushi Hayata,1 Nahomi Tokudome,1 Masayuki Higuchi,3 Keiichiro Akamatsu,1 Masanori Nakanishi,1 Hiroki Ueda,1 Nobuyuki Yamamoto1. 1 _Wakayama Medical University, Wakayama, Japan;_ 2 _EORTC, Brussels, Belgium;_ 3 _Hitachi Chemical Co. Ltd., Chikusei, Japan_.

Background: Anti-PD-1 antibody nivolumab has become a new standard treatment for pretreated, advanced non-small cell lung cancer (NSCLC). Although PD-L1 expression on tumor tissue has a predictive value, significance of its expression on circulating tumor cells (CTCs) is unknown. Here, we conducted a sequential evaluation of PD-1-expressing CTCs in NSCLC patients treated with nivolumab.

Methods: Advanced NSCLC patients who received nivolumab at Wakayama Medical University Hospital were enrolled in the study (UMIN000024414). Nivolumab was administered 3 mg/kg bi-weekly until progressive disease (PD) or unacceptable toxicity. Peripheral whole blood (3 mL) was collected in an EDTA collection tube (BD vacutainer) and processed within 3 hours for CTC evaluation at baseline, week 4 and week 8. CTCs were detected using automated microcavity array system (Hitachi Chemical Co.). PD-L1 expression was immunohistochemically examined on both tumor tissues and CTCs using anti-PD-L1 antibody, clone 28-8 (Abcam).

Results: Thirty-eight patients were registered in the study between January 2016 and September 2016. Clinical characteristics of the patients were as follows: median age 68 (range, 49 to 86); male 73 %; stage IV 100 %; squamous/non-squamous, 30/65 %. Regarding nivolumab treatment, overall response rate (ORR) was 22% (95% confidence interval [CI]: 10-38%), and median progression-free survival (PFS) was 62 days (95%CI: 40-235 days). At baseline, CTCs were detected in all patients (median, 15; range, 1-90) and PD-L1-expressing CTCs were detected in 87% of patients. Tumor proportion score (TPS) of PD-L1 expression on CTCs varied from 6% to 100%. Matched tumor tissues were available from 14 patients and 7 showed the PD-L1 TPS ≥50%. PD-L1 status on CTCs was not correlated with that on tumor tissues both using proportional score and H score (Spearman's correlation: r = 0.0007 and 0.08, respectively). On CTCs, patients with PD-L1 ≥50 % have significantly higher disease control rate than those with below 50% (83.3% versus 36.4%, p<0.01). Similarly, patients with PD-L1 ≥50 % on CTC had significantly longer PFS compared with those with below 50% (293 days versus 49 days, hazard ratio 2.41 (95% CI: 1.05-5.54), p = 0.03). During nivolumab treatment, number of CTCs was decreased (median number was 10 at 4 weeks, and 5 at 8 weeks). Among those with PD-L1 ≥50 % on CTC at baseline, changes in number and PD-L1 status on CTCs at 4 weeks did not correlate with PFS.

Conclusions: Sequential monitoring of PD-L1 expression on CTCs during nivolumab treatment was successfully conducted. PD-L1 expression on CTCs at baseline was a strong predictor in efficacy. Predictive significance of PD-L1-positive CTCs should be evaluated in a larger validation cohort.

#5596

Impact of blood collection tubes on CTC-, ctDNA- and miRNA recoveries in malignant melanoma patients.

Svenja Schneegans,1 Lelia Lück,1 Leonie Bluhm,2 Janina Staub,3 Rüdiger Greinert,2 Beate Volkmer,2 Alexander Sartori,4 Darryl Irwin,4 Taija af Hallstrom,5 Melanie Hussong,6 Jonathan Shaffer,6 Markus Sprenger-Haussels,7 Stefan W. Schneider,3 Peter Mohr,2 Klaus Pantel,1 Harriet Wikman1. 1 _University Medical Center Hamburg-Eppendorf, Department of Tumor Biology, Hamburg, Germany;_ 2 _Elbe Clinics, Centre of Dermatology, Buxtehude, Germany;_ 3 _University Medical Center Hamburg-Eppendorf, Department of Dermatology and Venereology, Hamburg, Germany;_ 4 _Agena Bioscience GmbH, Hamburg, Germany;_ 5 _Orion Pharma, Orion Corporation, Espoo, Finland;_ 6 _QIAGEN Inc, Frederick, MD;_ 7 _QIAGEN GmbH, Hilden, Germany_.

Liquid biopsy represents a powerful clinical tool that makes use of the detection of circulating tumor cells (CTCs), circulating tumor DNA (ctDNA) and exosomes in peripheral blood. Each different analysis is complementary to each other and can give additional valuable clinical information. These biomarkers are susceptible to quick degradation, presenting a challenge in a clinical environment and may require different blood collection tubes. The aim of this CANCER-ID study was to analyze the influence of different blood tubes on quantity and quality of CTCs, ctDNA and total and exosomal miRNA isolated from the same blood tube.

After confirming that plasma removal does not influence CTC recovery rates, peripheral blood from 20 cases of metastatic melanoma patients was collected in EDTA, Streck and Transfix tubes. CTC isolation from the PBMC fraction was performed by the ClearCell device (Clearbridge BioMedics). CTCs were identified by immunofluorescence staining. CtDNA was extracted from Streck and EDTA plasma samples, quantity (Qubit) and quality (Tapestation) measurements were performed before analyzing 86 hot-spot mutations in 13 genes by the UltraSEEK chemistry (Agena Bioscience). Ten of the 20 metastatic melanoma patients and 5 healthy donors were chosen for miRNA analysis. MiRNAs were extracted with miRNeasy Serum/Plasma Advanced Kit and miRNeasy Serum/Plasma Kit (QIAGEN) from total plasma and from extracellular vesicles (EVs), respectively. EVs were isolated by ultracentrifugation. QIAseq miRNA libraries were produced and sequenced (Illumina NextSeq 550). The reads were mapped to miRBase and normalized (geNorm). Volcano plots of fold change versus p-value were used to display the miRNAs that are significantly regulated.

The CTC enrichment results showed that 3/20 of the EDTA blood samples were positive, whereas samples from Streck and Transfix tubes were negative. The quantity or quality of the ctDNA did not significantly differ between the EDTA and Streck tubes. Mutation analysis of ctDNA performed so far showed similar detection sensitivities between the two tubes. The morphology, particle concentration and size distribution of EVs did not either differ between the two tubes. MiRNA-NGS analyses from plasma revealed that seven (EDTA) and four (Streck) miRNAs are significantly differentially expressed in patients compared with healthy donors. No overlap of these miRNAs was found between the two tubes. In the EV fraction 24 and 31 miRNAs were found significantly differentially expressed in the EDTA and Streck tubes, respectively. Here, one EV miRNA is up-regulated and six are down-regulated in both tubes.

In conclusion, the CTC recovery rates and minimal overlap of circulating miRNAs indicate that the different tubes affect CTC, ctDNA and miRNA results. Thus, the outcome of liquid biopsy analyses strongly depends on the choice of blood collection tubes as important pre-analytical variable.

#5597

TargetSelector™ CTC technology demonstrates clinical utility in monitoring treatment response in germ cell tumors (testicular cancer).

Shilpa Gupta,1 Benjamin L. Maughan,2 Cecile Rose T. Vibat,3 Veena M. Singh3. 1 _University of Minnesota, Minneapolis, MN;_ 2 _University of Utah, Salt Lake City, UT;_ 3 _Biocept, San Diego, CA_.

Background: Liquid biopsy has recently emerged as a minimally invasive and cost effective means to assess cancer biomarkers without the risk of surgical biopsy complications. Using a simple blood draw, circulating tumor cell (CTC) analysis is capable of providing information with implications for diagnostics, prognosis, and treatment decisions of various carcinomas. Serial CTC measurements can aid monitoring disease progression or response to therapy. Biocept's proprietary TargetSelector™ technology captures CTCs and provides assessment of enumeration, protein expression by immunofluorescence, and gene amplifications or translocations via FISH. There is limited data on clinical utility of CTCs in germ cell tumors (GCT). We aim to evaluate the prognostic and predictive significance of CTCs in refractory GCT in an ongoing clinical trial of brentuximab vedontin and bevacizumab in refractory CD 30+ GCT (NCT02988843). CTC enumeration will be performed at baseline, prior to cycle 3 of treatment, and at progression to monitor treatment response in blood from patients with refractory testicular cancer. Methods: Peripheral whole blood samples from refractory testicular cancer patients are collected into Biocept CEE-Sure™ blood collection tubes that are validated to preserve CTCs for up to 96 hours. Biocept's TargetSelector™ platform utilizes a proprietary antibody capture cocktail and microchannel enabling enrichment, enumeration, and CTC analyses which were performed at Biocept's CLIA-certified and CAP accredited laboratory. Results: From a patient with heavily pretreated refractory testicular cancer, serial blood collections were obtained at baseline and prior to cycle 3 (approximately six week interval). Two CTCs were detected at baseline. Prior to cycle 3, there was 1 CTC (corresponding to tumor marker response and CT scan showing stable disease). At progression after cycle 4, the CTC count increased to 4, corresponding with disease progression in tumor markers and CT scan. In this patient, the CTC capture utilizing the TargetSelector™ CTC technology was very predictive of clinical response and progression. Conclusions: Clinical application of Biocept's TargetSelector™ CTC technology enables the sensitive detection of CTCs in testicular cancer. Longitudinal CTC assessment implemented in the clinical setting can be used to assess drug responsiveness and follow disease progression in this rare disease, as well as in more common solid tumor cancers. The TargetSelector™ liquid biopsy platform provides an economical, non-invasive, and reliable means to arm physicians with valuable information for disease management and patient care. Ongoing collection of blood samples and data analysis from patients being enrolled on the study will provide us a better understanding of the potential clinical use of this novel technology in GCT patients. 

### Liquid Biopsy 6

#5598

Development and optimization of a comprehensive high-sensitivity NGS cancer assay and bioinformatics pipeline for plasma cfDNA profiling.

Juber Patel,1 Maysun Hasan,1 Fanli Meng,1 Xiaohong Jing,1 Dilmi Perera,1 Jonathan Reichel,1 Erika Gedvilaite,1 Julie Yang,1 Maha Shady,1 Sandeep Raj,1 Preethi Srinivasan,1 Ian Johnson,1 Jiashi Wang,2 Mirna Jarosz,2 Aliaksandra Samoila,1 Agnes Viale,1 Bob Li,1 Pedram Razavi,1 Dana Tsui,1 Michael Berger1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Integrated DNA Technologies, IA_.

The accessibility of tumor-derived cell-free DNA (cfDNA) in blood plasma provides a means to non-invasively profile somatic mutations in solid tumor patients. Clinical applications include longitudinal monitoring of disease burden and acquired drug resistance, identification of clinically relevant alterations and mutation signatures, and detection of minimal residual disease. However, the low fraction of tumor-derived cfDNA in plasma in many patients requires assays and bioinformatics methods that are much more sensitive than have been used for traditional tissue-based analysis.

The design of our cfDNA NGS panel is based on prospectively-collected clinical sequencing data obtained from more than 20,000 patients at Memorial Sloan Kettering Cancer Center using MSK-IMPACT, a custom 468-gene sequencing test authorized by the FDA for somatic mutation profiling. Exons harboring hotspot mutations, clinically actionable mutations, and elevated somatic mutation rates were selected for inclusion in the cfDNA panel. Additional non-coding content was included to enable optimal detection of selected copy number alterations, regions of loss of heterozygosity, rearrangement breakpoints, and microsatellite instability. Altogether the panel contains 208 kilobases of sequence from 129 cancer genes. Ultra-deep sequencing and unique molecular indexing enable PCR-generated replicate sequences to be collapsed into error-free consensus sequences, thereby facilitating the high-confidence detection of mutations present at low allele fractions.

We developed an open source bioinformatics tool, Marianas, for collapsing PCR replicates into consensus sequences and computing associated quality and performance metrics. Marianas incorporates many empirically derived features that lead to significant noise reduction. It efficiently processes a bam file with 20,000X coverage in 20 minutes on a single processor. We benchmarked the performance of Marianas against other available tools for collapsing and consensus base calling. The relative contributions of sources of error such as barcode contamination and sample

cross-talk during PCR and sequencing were also quantified. We found that using unique dual sample indexes in multiplexed sequencing runs was essential to suppress these sources of noise.

Applying these aggregated methods to analyze plasma cfDNA samples obtained from patients across a range of solid tumor types and disease stages, we were able to reliably detect clinically relevant mutations with variant allele fractions below 0.003, including subclonal mutations associated with acquired drug resistance. This approach, when applied prospectively on clinical specimens, has the potential to facilitate diagnosis, prognosis, and treatment selection in an era of precision oncology.

#5599

Noninvasive genomic profiling of cerebral spinal fluid in breast cancer patient with leptomeningeal disease.

Masahiro Oikawa, Naveen Ramesh, Emi Sei, Shanshan Bai, Min Hu, de Groot F. John, Murthy Rshmi, Barbara O'Brien, Nicholas Navin. _UT MD Anderson Cancer Center, Houston, TX_.

Leptomeningeal disease (LMD) involves the dissemination of tumor cells from the primary breast tumors into the membranes surrounding the central nervous system and spinal cords. LMD occurs in about 5% of breast cancer patients and is associated with very poor survival. The genomic evolution of LMD in breast cancer patients has remained difficult to study, in part due to technical challenges in collecting longitudinal biopsy samples from the central nervous system. Liquid biopsies of the cerebral spinal fluid (CSF) may provide a unique opportunity to profile circulating tumor DNA (ctDNA) and has not been compared directly to non-invasive monitoring of ctDNA in blood samples. Furthermore, most non-invasive ctDNA profiling methods are limited to a targeted set of genes and have not allowed unbiased whole-genome profiling. To address these limitations, we developed an unbiased method for ctDNA analysis called PEGASUS (Plasma Exome and Genome Analysis by Size-selection and Unbiased Sequencing) that enables whole-genome sequencing of copy number aberrations and somatic mutations from ctDNA. We applied PEGASUS to sequence matched blood samples and CSF from 10 breast cancer patients with LMD. Quantitation of ctDNA levels showed that ctDNA was detected in CSF samples from 9/10 patients, while blood ctDNA was detected in only 1 LMD patient with extensive metastatic disease. Whole-genome copy number profiling at 220kb resolution and mutational profiling of a 2000 cancer gene panel of the matched CSF and blood samples was performed in 10 LMD patients using PEGASUS. Our data identified aneuploid copy number aberrations and somatic mutations (range: 11-25) in the CSF of 7 patients, including mutations in known driver genes such as BRAF, RB1, TP53 and amplifications of MYC and ERBB2. In contrast the matched blood samples showed only diploid copy number profiles and no detectable somatic mutations in 9/10 LMD patients. In one patient with extensive cranial metastatic disease with matched CSF and blood ctDNA, we found a high concordance in copy number profiles (R= 0.75) and modest concordance of somatic mutations (59.5%), but also additional CNAs and mutations that were specific to the CSF. Collectively, these data show that genomic profiling of ctDNA in CSF is technically feasible for patients with LMD, and provides a major advantage over blood ctDNA which cannot be detected in mostcases.

#5600

Establishment and characterization of a unique circulating tumor cells-derived xenograft (CDX) in prostate cancer.

Vincent Faugeroux,1 Emma Pailler,1 Olivier Deas,2 Virginie Marty,1 Kamélia Alexandrova,1 Kiki Andree,3 Jean-Yves Scoazec,1 Nikolas Stoecklein,4 Nicolo Manaresi,5 Dominique Tramalloni,1 Maud Ngo-Camus,1 Claudio Nicotra,1 Leon Terstappen,3 Valérie Lapierre,1 Karim Fizazi,1 Yohann Loriot,1 Jean-Gabriel Judde,2 Françoise Farace1. 1 _Institut Gustave Roussy, Villejuif, France;_ 2 _XenTech, Evry, France;_ 3 _University of Twente, Enschede, Netherlands;_ 4 _University Hospital of the Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany;_ 5 _Menarini Silicon Biosystems S.p.A., Bologna, Italy_.

Background: The rarity of in vivo and in vitro human prostate cancer (PCa) models has hampered progress in understanding disease pathogenesis, metastatic progression and drug resistance mechanisms. Using CTCs from a leukapheresis product of a patient with advanced PCa, we report the establishment of a CDX and an in vitro cell line derived from this CDX. The phenotypic and molecular characterization of patient tumor-biopsies, CTCs, CDX and CDX-derived cell-line are presented.

Methods: Leukapheresis was performed in seven patients with advanced castration-resistant prostate cancer (CRPC). CTCs from the seven leukapheresis products were enriched by RosetteSep and implanted in Nod/Scid-IL2Rγ-/-mice. The CDX tumor was propagated in successive generations of mice. All samples, including eight tumor-biopsies performed at diagnosis two years prior leukapheresis and CTCs isolated at the single cell level during leukapheresis were characterized by immunofluorescence, immunohistochemistry, and whole-exome sequencing (WES).

Results: Based on CellSearch® counts in leukapheresis products, the estimated median number of engrafted CTCs was 697 (range: 10-19988). A mouse engrafted with 19988 CTCs developed a tumor within 193 days. Immunohistochemistry performed on the CDX and two tumor-biopsies indicated that the CDX and biopsies were positive for EpCAM, CK5/6/8/18, negative for CK7 and vimentin, and weakly positive for synaptophysin. While biopsies expressed PSA and the androgen receptor, the CDX was negative for both indicating tumor evolution. In contrast to tumor biopsies, the CDX strongly expressed Ki67, NSE and chromogranin, evidencing emergence of a neuroendocrine phenotype. The in vitro cell line established by culturing dissociated CDX cells for five months, grew in microspheres and expressed epithelial and ALDH and CD133 cancer stem-cell markers. By WES, a high degree of intra-tumor heterogeneity was observed in the eight tumor biopsies and CTCs as already reported in this tumor type. Only 2.8% (58/2087) and 2.3% (49/2087) of the mutations present in the tumor biopsies were identified in CTCs and the CDX respectively, indicating that a very few number of mutations have the potential to support the dissemination and tumorigenic activity of CTC. Trunk mutations in TP53, NF1 and LRP1B genes were identified in all samples including the CDX while PTEN gene loss was acquired lately and detected only in CTCs and the CDX. Mutational similarity of the CDX and the in vitro cell line was 91%. The analysis of copy number variations is ongoing in all samples and will be presented.

Conclusion: We report the first PCa CDX model, demonstrating the tumorigenicity of CTCs from CRPC. This CDX model represents a unique tool to identify clonal mutations associated with the tumor-initiating capacity of CTCs and explore the genetic and phenotypic basis of metastasis and drug resistance in advanced CRPC.

#5601

Single-cell molecular profiling of circulating tumor cells (CTCs) within the TRACERx study reveals heterogeneous patterns in early non-small cell lung cancer (NSCLC).

Francesca Chemi,1 Sakshi Gulati,1 Dominic G. Rothwell,1 Debbie Burt,1 Daniel Slan-Tan,1 Barbara Mesquita,1 Chris Wirth,1 Gareth Wilson,2 Jackie Pierce,1 Ged Brady,1 Charles Swanton,3 Caroline Dive1. 1 _CRUK Manchester Institute, Manchester, United Kingdom;_ 2 _University College London Cancer Institute, The Francis Crick Institute, London, United Kingdom;_ 3 _University College London Cancer Institute, The Francis Crick Institute, On behalf of TRACERx consortium and Cancer Research UK Manchester/UCL Lung Cancer Centre of Excellence, London, United Kingdom_.

Introduction: For early stage NSCLC, surgery with curative intent is the most common therapeutic option; however, tumour recurrence occurs in approximately 50% of cases and most commonly at distant sites. Analysis of CTCs represents a potential means of tracking cancer cells from the primary tumour and has the potential to provide insights into the metastatic process and mechanisms of resistance to therapy linking cell phenotype and genotype. Within the TRACERx study, we are carrying out whole genome analysis of single CTCs obtained from NSCLC patients undergoing surgery in order to examine the relationship between CTCs, the primary tumour and metastatic relapse.

Methods: Blood from 163 patients was collected at surgery from the tumour draining pulmonary vein and from peripheral vein in longitudinal samples. CTCs were enriched based on their EpCAM expression using CellSearch® and then enumerated according to their positivity for epithelial markers (Cytokeratins) and lack of detectable blood markers (CD45). Samples containing at least five CellSearch® CTCs (CS-CTCs) were processed through DEPArray™ for single cell isolation followed by whole genome amplification (WGA) using the Ampli1™ WGA kit. Genome wide copy number alterations (CNA) were detected through low-depth whole genome sequencing (WGS) and mutational analysis of individual CTCs was carried out using whole exome sequencing (WES). CTC genomic profiles were compared to the corresponding spatially separated tumour sections obtained at surgery.

Results: In a pilot study of 30 patients undergoing tumour resection, the presence of pulmonary vein CS-CTCs has been linked to poor clinical outcome (Crosbie et al, J Thorac Oncol, 2016). Here we extended the evaluation of pulmonary vein CS-CTCs to a further 122 patients and confirmed the initial observation that the presence of pulmonary vein CS-CTCs is a poor prognostic indicator. CNA analysis of 100 single CTCs from the first 12 patients revealed heterogeneous patterns between patients and within individual patients. Three types of pulmonary CS-CTC candidates were observed: Type 1, which share clear copy number changes with both tumour and other CS-CTCs; Type 2, where copy number changes were detected but were not shared with the tumour; Type 3, where no copy number changes were detected. Using WES of both CTCs and matching excised tumour regions, we identified common genetic alterations shared by CTCs and the tumour, as well as private genetic changes detected only in CTCs. We are currently evaluating metastatic biopsies from relapsing patients and comparing their molecular profiles to those seen in CS-CTCs obtained at surgery.

Conclusion: The combined tumour and CTC data show that single CTC analysis provides an additional layer of complexity with a valuable new perspective on tumour heterogeneity and early dissemination in NSCLC.

#5602

Detection of circulating cell-free tumor DNA (ctDNA) in patients with small cell lung cancer (SCLC).

Sumitra Mohan,1 Victoria Foy,1 Hui Sun Leong,1 Pietà G. Schofield,1 Mahmood Ayub,1 Nigel K. Smith,1 Sudhakar Sahoo,1 Chang Sik-Kim,1 Lynsey Priest,1 Mathew Carter,2 Hedley T. Carr,3 Crispin Miller,1 Corinne Finn-Faivre,2 Fiona Blackhall,2 Dominic G. Rothwell,1 Caroline Dive,1 Gerard Brady1. 1 _Cancer Research UK-Manchester Institute, Manchester, United Kingdom;_ 2 _The Christie NHS Foundation Trust, Manchester, United Kingdom;_ 3 _Astrazeneca, Cambridge, United Kingdom_.

Introduction: Tumor genomes can be reconstructed from the molecular information obtained from circulating cell-free DNA (cfDNA) and circulating tumor cells (CTCs) obtained from the peripheral blood of patients with cancer. The analysis of cfDNA and CTCs is a minimally invasive approach and represents a powerful research tool, with potential as a companion diagnostic for both patient stratification and monitoring. Here, we use cfDNA next-generation sequencing (NGS) analysis to assess and compare ctDNA profiles in patients with SCLC.

Methods: Whole genome sequencing (WGS) libraries were prepared from cfDNA isolated from the pre-treatment peripheral blood samples from 69 patients and 32 cancer-free controls. Libraries were subjected to WGS to establish genome wide copy number aberrations (CNA) as well as targeted mutation analysis of 110 SCLC associated genes. Quantitative metrics were calculated from CNA such as Percent Genome Altered (PGA; percentage of genomic regions altered), Z-score (measure of standard deviation) and Moran's I (measure of spatial autocorrelation). In addition CellSearch®, an epitope dependent enrichment platform was used to enumerate CTCs from a parallel blood sample.

Results: An examination of CNA patterns revealed SCLC associated changes such as losses on chromosomes 3p, 5q and 17p and gains on chromosome 3q and 5p as well as amplification of MYC in 21/69 (30%) and SOX2 in 36/69 (52%) as well losses on FHIT in 40/69 (58%), RASSFI in 38/69(55%) and RB1 in 24/69 (35%) patients. A combination of three CNA metrics enabled detection of tumor associated changes in 58/64 (84%) patients, with Moran's I emerging as the most sensitive CNA metric for detecting ctDNA. Targeted NGS detected tumor associated mutations in 60/64 (94%) with TP53 mutations detected in 50 patients (83% of patients with any detectable ctDNA). Somatic mutations and CNA were detected in both limited stage SCLC (LS-SCLC, confined to 1 hemithorax) and extensive stage (ES-SCLC, with distant metastases) with statistically significant differences seen for CNA metrics and variant allele frequencies of mutations consistent with higher levels of ctDNA in ES-SCLC. However, no significant differences between ES and LS were observed in the mutation patterns with respect to DNA damage repair, RAS and PI3K and transcriptional regulation pathways between the two stages of SCLC. There was considerable overlap between the detection of ctDNA and CTC counts, with cfDNA NGS readouts detecting tumor related changes in 96% of patients and CTCs were detected only in 76% of patients.

Conclusion: We have established sensitive methods for detecting ctDNA in cfDNA and combined with CTC enumeration we have an effective liquid biopsy for 98% of patients (96% in LS and 100% in ES) in this cohort. Future work will involve utilising this optimised NGS approach in an independent cohort of patients to correlate cfDNA metrics with clinical outcome.

#5603

Analytical validation of a comprehensive 500-gene ctDNA panel designed for immuno-oncology and DNA damage research.

Elena Helman, Carlo Artieri, James V. Vowles, Jennifer Yen, Tracy Nance, Marcin Sikora, Joshua Gourneau, Mohit Goel, Stefanie Mortimer, Darya Chudova, Justin Odegaard, Richard B. Lanman, AmirAli Talasaz. _Guardant Health, Redwood City, CA_.

Background: Translational research and enrollment in clinical trials are limited by the rarity of individual mutations and lack of sufficient tissue for comprehensive testing. To address these limitations, we developed GuardantOMNI (OMNI), a highly sensitive 500-gene cfDNA sequencing test requiring as little as 2 mL of plasma and designed for broad genomic detection of somatic single-nucleotide variants (SNVs) and small indels in 497 genes, copy number amplifications (CNAs) in 106 genes, and fusions in 21 genes. Additionally, the OMNI panel enables assessment of tumor mutational burden (TMB), and DNA damage and mismatch repair, with coverage of over 30 genes associated with the DDR pathway. Here we present the first analytical validation study of OMNI.

Methods: Analytical performance was assessed as per Nex-StoCT Working Group guidelines using precharacterized cell lines and healthy normal donor-derived samples. Qualitative and quantitative orthogonal confirmation was provided by exome sequencing, microarrays, and data from published compendia.

Results: Seventy-three validation and 150 development plasma samples were processed for this study, using both 5ng and 30ng cfDNA input levels, and all samples passed sequencing QC metrics established prior to testing. Reportable ranges for SNVs were ≥0.04% variant allele fraction (VAF), ≥0.02% for indels, ≥2 supporting molecules for fusions, and ≥2.18 copies for CNAs. Cell line-based dilution studies demonstrated 95% limits of detection (LoD) of 0.24-0.6% VAF for SNVs (depending on known cancer association), 0.4-0.8% for non-homopolymeric indels (depending on clinical relevance), 0.1-0.2% for fusions, and 2.2-2.9 copies for 90% of CNA genes targeted. Comparison of diluted cell line and healthy donor samples to orthogonal sequencing and published genotype data demonstrated accuracies of 98.7% for SNVs, 97.2% for indels, and 100% for CNAs and fusions across the reportable range. The analytical false-positive rate per sample measured across 24 healthy donors was 0.25 for SNVs, 0.04 for indels, and 0 for CNAs and fusions, with positive predictive values (PPVs) of 97.5% for SNVs, 98% for indels, and 100% for CNAs and fusions. Quantitative correlation of allele fraction with confirmatory methods was high (r2 > 0.99).

Conclusions: To our knowledge, OMNI is the largest comprehensive ctDNA cancer gene panel available. It detects alterations in genes under study in over 98% of current clinical trials with sensitivity, specificity, and accuracy similar to currently available targeted ctDNA sequencing tests. OMNI has the potential to accelerate clinical trial enrollment, research and discovery with a single, noninvasive blood sample.

#5604

Novel DNA methylation biomarkers show high sensitivity and specificity for blood-based detection of colorectal cancer - A clinical biomarker discovery and validation study.

Sarah Østrup Jensen,1 Mai-Britt Worm Ørntoft,1 Nadia Øgaard,1 Helle Kristensen,2 Mads Heilskov Rasmussen,1 Jesper Bertram Bramsen,1 Peter Mouritzen,2 Mogens Rørbæk,3 Anne-Sofie Kannerup,4 Søren Laurberg,5 Hans Jørgen Nielsen,6 Claus Lindbjerg Andersen1. 1 _Aarhus Univ. Hospital, Aarhus N, Denmark;_ 2 _Exiqon, Vedbæk, Denmark;_ 3 _Regional Hospital West,, Herning, Denmark;_ 4 _Randers Regional Hospital, Randers, Denmark;_ 5 _Aarhus Univ. Hospital, Aarhus C, Denmark;_ 6 _University of Copenhagen, Hvidovre, Denmark_.

Background: Screening for colorectal cancer (CRC) using fecal occult blood tests (FOBT) reduces CRC mortality, and many CRC screening programs therefore use FOBT. However, FOBT is not the best approach; first, because population acceptance is low due to unpleasantness of fecal sampling; second, because bowel tumors bleed only intermittently, which limits FOBT sensitivity. Development of a novel blood-based screening approach may alleviate these problems.

Objective: This study aims to develop and validate novel blood-based biomarker assays to achieve high patient compliance, sensitivity and specificity.

Methods and materials: CRC-specific DNA methylation biomarker candidates were identified using a genome-wide discovery strategy based on >4,000 Illumina 450K DNA methylation arrays. We designed digital droplet PCR assays to detect top biomarker candidates in circulating cell-free DNA (cfDNA) isolated from plasma. Initially, sensitivity and specificity of biomarkers were evaluated in validation cohort 1 consisting of plasma collected from 114 symptomatic CRC patients and 86 colonoscopy-confirmed healthy controls. To ensure comparable technical sensitivities for all assays in all samples, we used a fixed cfDNA input of 4,500 copies per ddPCR reaction. Next, we tested markers in validation cohort 2, a selected cohort of 8 mL plasma collected from participants in the Danish national screening program. This cohort comprised 131 asymptomatic CRCs and 869 controls enriched for comorbidities like adenomas, other cancers, diabetes, arthritis, hypertension, inflammatory bowel disease and arteriosclerosis.

Results: Our discovery identified 12 DNA methylation biomarkers. Their performance was evaluated in validation cohort 1, and the three best performing markers showed a sensitivity of 89% at a specificity of 99%, which is superior to FOBT. Sensitivity increased with stage, reaching 65%, 85%, 78% and 83% for stage I-IV, respectively. When evaluated in validation cohort 2, the sensitivity was 51% for CRC samples with an input of at least 4,500 copies of cfDNA, and sensitivity was reduced equally for all disease stages compared with results from validation cohort 1. Sensitivity correlated positively with cfDNA input, and it is therefore critical to collect sufficient plasma volumes to achieve high sensitivities in clinical practice. Finally, specificity was 92.4 % even though we enriched for comorbidities in this cohort, and specificity was 95.7% in controls with a clean colon and no comorbidities.

Conclusion: Our systematic biomarker discovery and validation study identified a three-gene DNA methylation panel with superior performance in plasma compared to FOBT.

### Therapeutic Antibodies, Including Engineered Antibodies 4

#5606

Eradication of triple-negative breast cancer cells by targeting glycosylated PD-L1.

Seung-Oe Lim,1 Chia-Wei Li,2 Mien-Chie Hung2. 1 _Purdue University, West Lafayette, IN;_ 2 _UT MD Anderson Cancer Center, Houston, TX_.

EGF signaling inhibits GSK3β-β-TrCP-mediated degradation of N-linked glycosylated programmed death ligand-1 (PD-L1), resulting in PD-L1 protein destabilization and enhanced immune checkpoint blockade efficacy. Here we show that EGF also mediates PD-L1 and receptor programmed cell death protein-1 (PD-1) interaction, requiring β-1,3-N-acetylglucosaminyl transferase (B3GNT3) expression in triple-negative breast cancer cells. Downregulation of B3GNT3 enhances cytotoxic T cell-mediated anti-tumor immunity. A monoclonal antibody targeting glycosylated PD-L1 (gPD-L1) blocks PD-L1/PD-1 interaction and promotes PD-L1 internalization and degradation. In addition to immune reactivation, drug-conjugated gPD-L1 antibody induces potent cell-killing effect as well as bystander-killing effect on adjacent cancer cells without PD-L1 expression with virtually no detectable toxicities. Our work suggests targeting protein glycosylation as a potential strategy to enhance immune checkpoint therapy.

#5607

Tissue factor is a novel oncotarget for immunotherapy of triple-negative breast cancer using a second generation ICON in orthotopic mouse models of human and murine TNBC cell line- and patient-derived xenografts.

Zhiwei Hu,1 Rulong Shen,1 Amanda Campbell,1 Elizabeth McMichael,1 Lianbo Yu,1 Bhuvaneswari Ramaswamy,1 Cheryl A. London,1 Tian Xu,2 William E. Carson1. 1 _The Ohio State University, Columbus, OH;_ 2 _Yale University, New Haven, CT_.

Triple-negative breast cancer (TNBC) is often associated with BRCA1 and BRCA2 mutation and is a leading cause of breast cancer death. Due to the lack of validated target molecules, there is currently no approved targeted therapy for TNBC. Tissue factor (TF) is a common yet specific surface target receptor for cancer cells, tumor vascular endothelial cells and cancer stem cells in several types of solid cancers. Here we report evidence supporting the idea that TF is a novel surface target for TNBC patients and cancer lines with BRCA1 and BRCA2 mutations. We showed that TF is over-expressed on TNBC cells and tumor neovasculature in greater than 85% of TNBC patients (n=14) when using standard paraffin-embedded tumor tissues and in nearly 60% of TNBC patients (n=157) when employing tissue microarray slides, but was not detected in adjacent normal breast tissue. We also describe the development of a second-generation TF-targeting immunoconjugate with improved efficacy and higher safety threshold compared to the original immunoconjugate. We showed that L-ICON1 is effective in killing TNBC cells via antibody-dependent cell-mediated cytotoxicity in vitro and in treating human and murine TNBC cell line- and patient-derived xenografts in vivo in preclinical orthotopic mouse models. These results suggest that TF is a novel oncotarget in TNBC and targeting TF may constitute a novel targeted immunotherapy for TNBC patients. The findings in this preclinical study warrant further clinical investigation.

#5608

Evaluation of retargeted T cell cytotoxicity of a bispecific antibody targeting CDH17 and CD3 in 2D- and 3D-colon cancer models.

Macarena Irigoyen,1 Eric Dai,2 John Luk,2 Gonzalo Castillo,1 Don Staunton2. 1 _BIOENSIS, Bothell, WA;_ 2 _Arbele Corporation, Redmond, WA_.

Cadherin-17 (CDH17) is an adhesion molecule that binds to self and integrin a2b1, its expression in the adult is limited to gastrointestinal tissue and is involved in cancer progression. CDH17 is expressed de novo or overexpressed in several gastro-intestinal cancers including colorectal, gastric and pancreatic, making it an ideal target for immunotherapies. A humanized bifunctional antibody was developed, ARB-201, that binds to both CDH17 and the CD3/TCR complex (CD3) with high affinity. ARB-201 induced retargeted T cell cytotoxicity was determined in a 2D and 3D model. Relative to 2D, cytotoxicity of 3D tumors may be considered more predictive of efficacy for solid tumors. DLD-1, a colorectal adenocarcinoma cell line, was targeted in the 2D and 3D T cell cytotoxicity assays. Pre-labeled DLD-1 were grown in standard 384-well microplate (2D) or Kuraray's 384-well Elplasia microplates (3D). Effector PBMCs, freshly isolated from healthy donors were added to the target DLD-1 cells containing a concentration response curve of ARB-201. Samples were evaluated for the specific cellular cytotoxicity using cell imaging. The results show at 48 hours an EC50 of 12 pM for 2D and 47 pM for the 3D model. ARB-201 induced potent T cell cytotoxicity of DLD-1. Although cytotoxicity in the 3D tumor model was relatively less, we observed an increase over time, demonstrating that ARB-201 progressively kills 3D tumor cells and possesses cytotoxic activity that may translate into clinical efficacy for solid tumors.

#5609

ERY974, a novel T cell-redirecting bispecific antibody targeting glypican-3, shows antitumor activity in gastric cancer patient-derived xenograft models with varying glypican-3 expression.

Azuma Yumiko, Yuji Sano, Toshiaki Tsunenari, Yasuko Kinoshita, Yoko Miyazaki, Junko Shinozuka, Etsuko Fujii, Atsuhiko Kato, Takahiro Ishiguro, Shohei Kishishita, Junichi Nezu, Yoshiki Kawabe, Mika Endo. _Chugai pharmaceutical CO., LTD., Japan_.

Background: ERY974 is a humanized IgG4 bispecific T cell-redirecting antibody (TRAB) currently in a Phase 1 clinical trial (NCT02748837) in patients with solid tumors that are glypican-3 (GPC3)-positive. ERY974 consists of a common light chain but has two different heavy chains that each recognize a different protein, GPC3 or CD3. ERY974 simultaneously binds to GPC3 on the cancer cell surface and to CD3 on the T cell surface to induce cellular cytotoxicity mediated by the potent effector function of T cells. The Fc portion of ERY974 is modified to eliminate FcγR binding and prevent GPC3-independent Fc-mediated effector function. However, binding to FcRn, an important factor in the PK profile of IgG, is maintained. ERY974 shows strong antitumor activity against gastric, lung, ovarian, head & neck, hepatic, and esophageal cancer-derived tumors in a non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mouse model injected with human T cells. In a cohort expansion of the Phase 1 trial, the activity of ERY974 will be examined in gastric cancer; however, individual clinical samples do not show uniform expression levels of GPC3 in tumor lesions, reflecting the heterogenous and complex structure of patients' tumors. To predict the potential efficacy of ERY974 in gastric cancer patients, we examined whether ERY974 alone or in combination with chemotherapy, could show substantial activity against heterogenous tumors with different GPC3 expression profiles using patient-derived xenografts (PDX) of gastric cancer.

Method & results: We evaluated the antitumor effect of ERY974 in PDX gastric cancer tumors that have high, moderate, or low expression levels of GPC3 in a NOD-SCID mouse model injected with human T cells that were expanded in vitro using CD3/CD28 beads. The expression of GPC3 was evaluated by immunohistochemistry and quantitative RT-PCR. The efficacy of ERY974 monotherapy seemed to correlate with the GPC3 expression levels in each PDX tumor. The combination of ERY974 and chemotherapy, such as paclitaxcel, cisplatin or capecitabine, showed increased antitumor activity compared with ERY974 or chemotherapy alone.

Conclusion: These preclinical data support the possibility that ERY974 alone or in combination with chemotherapy will demonstrate activity in patients with gastric cancer, and the GPC3 expression profile might be a useful predictive biomarker of ERY974 efficacy for patient selection.

#5610

CD125xCNE bispecific antibody development to treat bladder cancer.

Judit Hunyadkurti,1 Jeffrey Leyton,1 Laurent Fafard-Couture,1 Vincent Lacasse,1 Marc-Andre Bonin,1 Angel Lopez2. 1 _University of Sherbrooke, Sherbrooke, Quebec, Canada;_ 2 _University of South Australia, Australia_.

Background: Bladder cancer is one of the most prevalent cancers impacting adults worldwide.Although,successfully treated in its early stages, there are no effective therapies when the cancer has progressed into the surrounding muscle layer, clinically classified as muscle invasive bladder cancer (MIBC). CD125 was recently shown to be involved in MIBC progression and uniquely overexpressed in MIBC tumors relative to healthy urothelium or superficial tumors. Cyclin E (CNE) is a critical cell cycle protein and regulates progression of normal cells to replicate their DNA. There is a strong link between CNE dysregulation and tumorigenesis and was recently found to be overexpressed in MIBC. We characterized the monoclonal antibodies (mAbs) A14 specific for CD125, and HE-12 and HE-172 specific for CNE with a long-term purpose to develop a CD125xCNE therapeutic bispecific antibody. In order, for the antibody to efficiently target CNEour group developed a natural composite compound (termed Accum)that conjugates to surface lysines and enables mAbs to escape endosomal entrapment followed by active routing to and efficient accumulation in the nucleus. Methods: For Accum conjugation, maleimide groups were introduced into A14 by reaction with 10-to-100-fold molar excess of a PEGylated SMCC crosslinker at RT for 1 h. Purified and concentrated maleimide-derivatized A14 was reacted with 100-fold molar excess Accum for 18 h at 4 °C. Excess Accum-A14 was purified and concentrated. Conjugation was characterized by SDS-PAGE. CD125-positive MIBC cells were treated with Accum-A14 and nuclear localization efficiency determined by confocal microscopy.HE-12 and HE-172 mAbs were evaluated for binding nuclear CNE by flow cytometry using the whole cell and fractionated cell lysate along with saponin to enable the mAbs to diffuse into the cell. In addition, HE-12 and HE-172 mAb binding was evaluated by co-immunoprecipitation. PCR amplification of A14, HE-12, and HE-172 was performed. Results: A14 is readily loaded with Accum with minimal aggregation.Accum-A14 undergoesCD125-specific internalization and has 25-60-fold increased nuclear localization relative to A14 in MIBC cells.The binding of the intracellular CNE by mAb HE-12 and HE-172 was specific. Conclusion: This preliminary data demonstrates that Accum modification of A14 retains CD125 specificity and efficiently localizes to the nucleus of MIBC cells. HE-12 and HE172 recognized CNE. On going work will construct the CD125xCNE bispecific antibody. Future studies will determine the efficiency to target CNE and its impact to MIBC progression.

#5611

Inhibition of IL-6 trans-signaling in HNSCC.

Andrean L. Simons,1 Rachel A. Dahl,1 Madelyn Espinosa-Cotton,1 Samuel N. Rodman,1 Stefan Rose-John2. 1 _University of Iowa, Iowa City, IA;_ 2 _University of Kiel, Kiel, Germany_.

Various types of cancers including head and neck squamous cell carcinomas (HNSCCs) are highly inflammatory, and the cytokine interleukin-6 (IL-6) is believed to be associated with this inflammatory phenotype. Unfortunately, global inhibition of IL-6 signaling has demonstrated little to no clinical efficacy in various types of cancers. Previous studies in the field have reported major differences between IL-6 classical signaling through the membrane bound IL-6R (mIL-6R) and IL-6 trans-signaling through the soluble IL-6R (sIL-6R), and the idea that these 2 pathways may have vastly different functions. Recent studies in models of inflammation have proposed that IL-6 classical signaling serves an anti-inflammatory/homeostatic role while IL-6 trans-signaling stimulates proinflammatory events. Therefore, IL-6 trans-signaling (and not classical signaling) may be associated with the proinflammatory phenotype observed in HNSCCs and may also be responsible for the increased tumor progression and treatment resistance frequently encountered with this disease. The goal of this work is to investigate if selective inhibition of IL-6 trans-signaling will demonstrate superior antitumor efficacy compared to global inhibition of IL-6 signaling in HNSCC cells and if selective inhibition of IL-6 trans-signaling will enhance the antitumor efficacy of chemo/radiotherapy in HNSCC cells. We observed that the HNSCC cell lines SQ20B, SCC-25, FaDu and Cal-27 all express mIL-6R and secrete IL-6, sIL-6R, and the natural inhibitor of sIL-6R signaling--soluble glycoprotein 130 (sgp130)--at varying levels. Simultaneous inhibition of IL-6 classical and trans-signaling using the IL-6R antagonist tocilizumab, and selective inhibition of IL-6 trans-signaling using the extracellular portion of gp130 fused to the Fc portion of human IgG1 (sgp130-Fc), showed no significant effects on cell survival in vitro. However, sgp130-Fc significantly suppressed SQ20B tumor growth in athymic nude mice while tocilizumab had no effect. Significant antitumor effects of sgp130-Fc were also observed in a murine SCCVII/C3H syngeneic mouse model. Cell lines treated with cisplatin and/or X-ray radiation (0-4 Gy) increased IL-6, sIL-6R and sgp130 in a dose-dependent manner and sgp130-Fc significantly increased SQ20B tumor response to chemo/radiotherapy in vivo. Altogether, this work suggests that selective inhibition of IL-6 trans-signaling using sgp130-Fc may be more effective than global IL-6 inhibition with respect to suppressing HNSCC tumor growth and warrants further study as an adjuvant to standard HNSCC therapy.

#5612

Evaluating the cytotoxic effectiveness of a novel TCR-like bispecific T cell engager targeting the PR1/HLA-A2 leukemia antigen.

Amanda Cernosek Herrmann, Jin Seon Im, Hong He, Sergueeva Anna, Sijie Lu, Jeffrey Molldrem. _UT MD Anderson Cancer Ctr., Houston, TX_.

Overall 5-year survival rates of myeloid leukemia patients remain low at <30%, and new therapies for these patients are desperately needed. PR1 (VLQELNVTV) is a human leukocyte antigen (HLA)-A2 restricted peptide derived from serine proteases proteinase-3 and neutrophil elastase, which are aberrantly expressed in myeloid leukemia blasts, including acute myelogenous leukemia (AML). PR1 was shown to be immunogenic in myeloid malignancies, and we have conducted preclinical development and humanization of a T cell receptor-like monoclonal antibody (h8F4) that targets the leukemia-associated PR1/HLA-A2 complex and eliminates AML xenografts by antibody-dependent cellular cytotoxicity (ADCC) following repeat dosing. To improve the potency of h8F4, we have developed a bispecific T cell-engaging antibody that targets PR1/HLA-A2 on leukemia and CD3 on neighboring T cells. Here we demonstrate successful production and purification of the h8F4 bispecific antibody. Utilizing flow cytometry, we confirm PR1/HLA-A2 and CD3-specific binding characteristics, T cell activation in the presence of PR1/HLA-A2, and importantly AML target cell cytotoxicity after h8F4 bispecific antibody engagement with healthy donor effector T cells. Cytotoxicity assays were performed with both AML cell lines and primary patient AML blasts serving as target cells and health donor PBMC as a source of effector T cells at an E:T ratio of 2:1. Results indicate up to 60% leukemia-specific lysis with 2nM h8F4 bispecific antibody after only 18 hours of incubation. In vivo data also confirm significant elimination of the leukemia cell line U937 (as evidenced by % leukemia cells detected in peripheral blood) in an NSG-U937 AML xenograft mouse model when compared to control groups treated with effector cells alone. Mice were infused with 5,000 leukemia cells followed by 3 million normal healthy donor PBMC. These infusions were followed by a week of daily h8F4 bispecific antibody injections (20ng/injection). Both bioluminescence and flow cytometry analysis two weeks following the final injection show a marked difference between the h8F4 bispecific antibody treatment group and controls, with a 1.5-fold reduction in circulating leukemia cells and an overall increase in survival. In conclusion, these studies demonstrate the therapeutic potential of a novel bispecific antibody targeting the PR1/HLA-A2 leukemia-associated antigen. This bispecific antibody appears to increase the potency of h8F4 with rapid elimination of AML compared to h8F4, and our studies justify potential development as a treatment option for patients with high-risk AML.

#5613

Generation of a therapeutic monoclonal antibody for acute myeloid leukemia by employing the U937 cell line as a surrogate antigen.

Bethany Chen,1 Evelyn Kandov,2 Berhane Ghebrehiwet2. 1 _Winston Churchill High School, Potomac, MD;_ 2 _Stony Brook University, Stony Brook, NY_.

The prognosis of acute myeloid leukemia (AML) has improved for younger patients, but it still remains grim for patients over 65 years old with a likelihood of death within a year of diagnosis. Here, we evaluated the effects of novel anti-gC1qR monoclonal antibodies (mAbs) that were generated against intact U937 cells, which are used here as a model for AML. The U937 cell line is known to overexpress gC1qR, which in turn serves not only as a proliferative molecular signal but also provides a platform for the generation of angiogenic and metastatic factors. Cell viability results reveal that all of the mAbs significantly decrease (p<0.05) the percentage of viable AML cells although the mixture of those mAbs was not significant when compared to the individual mAbs themselves. The anti-U937 antibodies, monoclonal and polyclonal, showed a significant difference (p<0.05) against the anti-purified recombinant gC1qR antibodies. The anti-U937 mAb methodology of immunizing with the mammalian cell instead of the purified protein allowed the mAbs to better target the native endogenous protein present on human cells. Our study not only provided novel and potent mAbs against AML, but also implicated a potentially more efficient technique of creating therapeutic mAbs against cancer cells.

#5614

Targeting the radiation-inducible neoantigen GRP78 as a novel strategy for non-small cell lung carcinoma treatment.

Abhay Singh, Vaishali Kapoor, David Dadey, Dinesh Thotala, Dennis Hallahan. _Washington University in St. Louis School of Medicine, St. Louis, MO_.

Non-small cell lung cancer (NSCLC) ranks among the highest cancer-related mortalities worldwide. NSCLC immunotherapy has not been successful in the past. Although lung cancer is not typically believed to be an immunogenic malignancy, more and more evidence suggests that immune responses to lung cancer may be important. The objective of this research was to address a critical barrier and develop a new paradigm in the treatment of NSCLC. Development of anticancer antibodies is limited by the scarcity of antigens that are specifically overexpressed in lung cancer, resulting in too few molecular targets and small percentages of patients who can be treated with the therapeutic antibody. We established a platform technology to develop antibodies to radiation-inducible neoantigens. Several radiation-inducible antigens have been identified by our group. Glucose-regulated protein 78 (GRP78), a multifunctional protein folding chaperone and co-receptor that is highly expressed on the surface of NSCLC, holds significant promise as a cancer-specific target. Although the potential of targeting GRP78 has been demonstrated in prostate cancer, melanoma, and breast cancer, there are limited data on the impact of targeting cell surface-associated GRP78 in NSCLC. We show that GRP78 undergoes radiation-induced translocation to the surface of NSCLC cells. We developed several GRP78-targeting monoclonal antibodies. NSCLC-specific antibody binding is achieved for several days by anti-GRP78 monoclonal antibodies after irradiation. Optical imaging and immunohistochemical staining indicated that the GRP78-specific antibodies achieve specific binding to irradiated NSCLC in mouse models. The anti-GRP78 antibodies also have a direct cytotoxic effect on NSCLC cells. The combination of anti-GRP78 antibodies and radiation showed an additive effect. Overall, this study suggests that GRP78 is a promising therapeutic target for the treatment of lung cancer. We propose to harness the physiologic response of cancer to radiotherapy and evolve it for immunotherapy of lung cancer using specific targeting antibodies.

#5615

Antibodies targeting TIP1 enhance the efficacy of radiotherapy in lung cancer and glioblastoma.

Vaishali Kapoor, Abhay K. Singh, David Dadey, Kelly Hoye, Andrea Collins, Dinesh Thotala, Dennis Hallahan. _Washington University in St. Louis School of Medicine, St. Louis, MO_.

Antigens that are over-expressed in cancer in response to radiation are being used as novel targets. We showed tax interacting protein 1 (TIP-1) to be radiation-inducible that translocated to the surface of the cancer cell following irradiation. TIP-1, which consists of a single PDZ domain plays an important role in cell signaling, cancer development, and progression. TIP-1's involvement in various survival pathways makes it an attractive target for anticancer therapeutics. We used antibodies specific to this PDZ domain to determine its role in cancer cell survival. We monitored the proliferation of lung cancer (A549 and H460) and glioblastoma (D54 and U251) cells after 24, 48, 72 and 96h treatment with the anti-PDZ antibody. We observed a time-dependent proliferation arrest with anti-PDZ antibody treatment which was associated with increased apoptosis. The anti-PDZ antibody, when combined with radiation (3Gy), led to reduced proliferation and colony formation. Anti-PDZ antibody had no effect on the proliferation of normal lung (MRC-5) and endothelial (HUVEC) cells. Cells treated with anti-PDZ antibody showed decreased levels of the phosphorylated forms of AKT, mTOR, and a downstream substrate of mTOR, 4EBP1. Anti-PDZ antibody treatment also led to an overall reduction in basal levels of AKT, mTOR, and 4EBP1. Further, we evaluated the effect of the anti-PDZ antibody on tumor growth in heterotopic mouse models of lung cancer (A549) and glioma (U251). We observed significant growth delay in mice treated with anti-PDZ antibody treatment when compared to mice treated with the isotype control. The combination of the anti-PDZ antibody with radiation showed an additive effect. Immunoblot analysis of tumor tissues also showed downregulation of phosphorylated and total levels of AKT, mTOR and 4EBP1 in the tumors treated with anti-PDZ antibody. Overall, our results suggest that TIP-1 is a promising therapeutic target for treatment of lung cancer and glioblastoma. Antibodies specific to the PDZ domain of TIP-1 enhance the efficacy of radiotherapy. The anti-PDZ antibodies need to be optimized further before translating it into the clinic.

#5616

Rituximab chemotherapy induces a partial recovery from T-cell exhaustion in follicular lymphoma patients.

Benoit Milcent,1 Pauline Brice,2 Catherine Thieblemont,2 Jean-Luc Teillaud,1 Sophie Siberil1. 1 _Cordeliers Research Center-Inserm UMR-S 1138; Sorbonne Universités, UPMC Univ Paris 06; Paris Descartes-Paris 5 University, Paris, France;_ 2 _APHP, Saint-Louis Hospital, Hemato-oncology, Paris, France_.

For many years, the clinical responses observed in cancer patients treated with antitumor antibodies have been viewed only as a consequence of the recruitment and activation of innate immune cells. Several preclinical studies have indicated that anti-tumor antibodies can also induce a potent anti-tumor adaptive T-cell response capable of protecting animals from tumor challenge. Our studies have shown that CD4+ Th1 cells play a central role in the response to anti-CD20 tumor therapy by opposing Treg expansion and that CD20-specific memory T cells are responsible for anti-tumor protection observed in surviving animals subjected to tumor rechallenge. Whether an adaptive T-cell response is elicited in patients with Follicular Lymphoma (FL) receiving anti-CD20-based treatment is not known. In the present work, we analyzed peripheral T cells before and during anti-CD20-chemotherapy treatment of FL patients (R-CHOP) with high tumor burden. We first examined in ELISPOT assays the IFN-γ production by PBMC from 20 patients against human CD20-derived peptides previously selected by combining in silico and in vivo screening using transgenic human HLA-DR mice. Both FL patients before treatment and patients undergoing R-CHOP treatment exhibited peripheral T-cell responses to the selected CD20-derived peptides. However, these responses were markedly lower than those observed in healthy donors. Similar results were also obtained when T cell responses against viruses commonly infecting large numbers of individuals (CMV, EBV, influenza) were assessed, suggesting that high tumor burden FL patients exhibit an immune exhaustion of T cells. Multiparametric flow cytometry analyses suggested that their poor ability to respond to viral and CD20-derived peptides is related to the presence of an immunocompromised and exhausted blood T-cell peripheral compartment with higher percentages of TIGIT- and PD-1-expressing T cells, of peripheral Treg, and terminally differentiated CD8+ TEMRA. R-CHOP therapy induced a shift of CD4+ and CD8+ T cells toward a central memory phenotype (TCM), an additional shift of CD8+ T cells to a naive phenotype, and a decrease in the number of PD-1+ and TIGIT+ T cells, making these cells evolving toward a lesser degree of exhaustion. However, it did not restore a level of specific responses similar to those observed in healthy donors. These results support the use of anti-PD-1 and anti-TIGIT antibodies in combination with anti-CD20 in FL patients with high-tumor burden.

#5617

A core fucose-deficient anti-podocalyxin antibody exhibits antitumor activity via augmented antibody-dependent cellular cytotoxicity in oral squamous cell carcinoma.

Shunsuke Itai, Mika K. Kaneko, Shinji Yamada, Yukinari Kato. _Tohoku University, Sendai, Japan_.

Background: Podocalyxin is a mucin-like type I transmembrane protein that is highly glycosylated with O- and N-glycans. It was originally found in renal podocytes and is also expressed in many kinds of cancers such as colorectal, breast and oral cancers. In addition, it was reported that podocalyxin overexpression is associated with progression, metastasis and poor outcomes; consequently, it is known as a diagnostic marker. However, the function of podocalyxin in oral cancers, including oral squamous cell carcinomas (OSCCs), has not been fully elucidated because there are few monoclonal antibodies (mAbs) suitable for analyzing podocalyxin expression in OSCCs. We recently produced sensitive and specific anti-podocalyxin mAb PcMab-47, which reacts with endogenous podocalyxin-expressing cancer cell lines. Furthermore, we have produced a mouse-human chimeric PcMab-47 (chPcMab-47) and demonstrated its antitumor activity against podocalyxin-expressing xenograft models using HCT-15 colon cancer cells and Chinese hamster ovary (CHO)/podocalyxin cells.Methods: We first engineered PcMab-47 into a mouse IgG2a-type mAb (47-mG2a) to add antibody-dependent cellular cytotoxicity (ADCC) activity and further developed a core fucose-deficient 47-mG2a (47-mG2a-f) to augment its ADCC activity using a fucosyltransferase 8-knockout CHO-S cell line. The binding affinity was determined using flow cytometric analysis. Using PcMab-47, 47-mG2a, and 47-mG2a-f, immunohistochemical analysis was performed against 201 cases of oral cancer tissues, and the sensitivity was compared. ADCC and complement-dependent cytotoxicity (CDC) activities were investigated using the 51Cr release assay. Finally, antitumor activity in OSCC xenograft models was compared using 100 or 500 μg /mouse, three times injection of 47-mG2a and 47-mG2a-f. Results: Both 47-mG2a and 47-mG2a-f exhibited higher binding affinity than PcMab-47 for podocalyxin-expressing OSCC cells such as SAS and HSC-2 cells. In immunohistochemical analysis of OSCCs using PcMab-47 and 47-mG2a, it was revealed that 47-mG2a showed high-sensitivity for staining OSCC cells in a cytoplasmic pattern at a much lower concentration. PcMab-47 detected podocalyxin in 163 cases (81.1%); in contrast, 47-mG2a did in 197 cases (98.0%). 47-mG2a-f exhibited the similar sensitivity as 47-mG2a, indicating that 47-mG2a and 47-mG2a-f are useful for immunohistochemical analyses of podocalyxin expression in OSCCs. In vitro analysis revealed that 47-mG2a-f exhibited much stronger ADCC than 47-mG2a, whereas these mAbs did not display CDC. Furthermore, 47-mG2a-f exerted antitumor activity in SAS and HSC-2 xenograft models, whereas 47-mG2a did not. Conclusions: A core fucose-deficient anti-podocalyxin mAb could be useful for antibody-based therapy against podocalyxin-expressing OSCCs.

#5618

Anti-FGFR4 antibody drug conjugate for immune therapy of rhabdomyosarcoma and hepatocellular carcinoma.

Adam Cheuk,1 Nitya Shivaprasad,2 Martin Skarzynski,2 Sivasubramanian Baskar,2 Peter Azorsa,2 Javed Khan2. 1 _Leidos Biomedical Research, Inc., Bethesda, MD;_ 2 _National Cancer Institute, Bethesda, MD_.

Rhabdomyosarcoma (RMS), an aggressive soft tissue sarcoma originating from skeletal muscle in children and adolescent young adults. It is divided in two main histological subtypes including embryonal RMS driven by RAS pathway mutations and alveolar RMS driven by a chimeric fusion gene involving PAX3 or PAX7 with FOXO1. FGFR4 is a cell surface tyrosine kinase receptor that is a critical molecule in RMS biology. In RMS it is a direct target and strongly induced by PAX3-FOXO1 as well as PAX3, and PAX7. By immunohistochemistry we found high expression of FGFR4 in RMS cancers but low expression in normal human organs. Of note FGFR4 has been reported overexpressed in hepatocellular carcinoma (HCC). We therefore hypothesized that FGFR4 will provide a rational target for immune therapy in cancers with high expression. We generated fifteen binders against FGFR4 and characterized them further as candidates for immunotherapy. We found that 3A11, a mouse IgG antibody, bound to FGFR4 positive cell lines with an observed avidity of 1.9nM. By ELISA using the extra cellular domain of human FGFR1, FGFR2, FGFR3 or FGFR4, 3A11 showed dose dependent binding to FGFR4 only. 3A11 was also found internalized upon binding to FGFR4 positive cell lines. The VL and VH domain of 3A11 was cloned and chimeric 3A11 scFvFc (mouse Fv and Human IgG1 Fc) construct was made. The chimeric form of 3A11 antibody was successfully produced in vitro and retained its specificity. To investigate FGFR4-mediated cytotoxicity in RMS cell lines, we cultured RH30 (a RMS cell line expresses FGFR4) in the presence of the chimeric 3A11 and polyclonal anti-mouse 2°ADC conjugated to Duocarmycin DM for 72 hours. Dose-dependent cytotoxicity was observed in RH30, suggesting that chimeric form of 3A11 could deliver a cytotoxic payload to FGFR4 positive cells. We are currently developing antibody drug conjugate using the cytotoxic drug, pyrrolobenzodiazepine which will be tested in vitro and in vivo models of RMS. We have thus developed a chimeric antibody that may provide effective immune therapy for cancers with high FGFR4 expression including RMS and HCC

#5619

Additional mechanisms of action of SGN-CD48A in multiple myeloma and improved antitumor activity in combination with daratumumab.

Devra J. Olson, Bernard A. Liu, Margo Zaval, Anthony Cao, Jesse Gurgel, Julia Cochran, Nicole Stevens, Martha Anderson, Timothy S. Lewis. _Seattle Genetics, Inc., Bothell, WA_.

SGN-CD48A is a novel multiple myeloma (MM) antibody-drug conjugate (ADC) composed of a CD48-directed antibody conjugated to 8 molecules of a cytotoxic antimitotic drug, monomethyl auristatin E (MMAE), via a next generation β-glucuronidase-cleavable linker. Previously, we showed that SGN-CD48A has potent single agent antitumor activity against MM cells, and produces durable complete remissions in mouse xenograft models. Here, we describe the bystander effect activity and immunogenic cell death (ICD) potential of SGN-CD48A in MM models. In addition, combination studies with approved MM therapeutic antibodies were explored. ADC bystander effect is the additional cytotoxic activity on antigen-negative cells in the presence of antigen-positive tumor cells. To measure bystander effect, we generated CD48 knock-out (KO) myeloma cell lines, which also express luciferase (Luc+). SGN-CD48A demonstrated bystander effect cell killing of the CD48KO-Luc+ cells when co-cultured with parental CD48+ MM cell lines in vitro. Next, a novel imaging-based method for measuring in vivo bystander effect within the mouse bone marrow compartment was developed using surgical intratibial implantation of admixed parental CD48+ and engineered CD48KO-Luc+ MM cells. SGN-CD48A in vivo bystander effect activity was evaluated over time using bioluminescence imaging and ex vivo flow cytometry of bone marrow aspirates. We then measured SGN-CD48A activation of ICD and endoplasmic reticulum (ER) stress signaling markers. SGN-CD48A treatment increased exposure of intracellular ER membrane markers calreticulin and heat-shock protein 70 (HSP70) on the MM cell surface, hallmarks of ICD. Myeloma cells already have high basal levels of ER stress markers due to constitutive production of monoclonal immunoglobulin. However, we observed increased phospho-JNK and a minor elevation of ATF-4 expression following SGN-CD48A treatment. Finally, we tested SGN-CD48A antitumor activity in combination with MM therapeutic antibodies, daratumumab and elotuzumab, in a MOLP8-Luc+ disseminated xenograft mouse model. SGN-CD48A, daratumumab, and elotuzumab showed delay of tumor progression as single agents in this system. However, the combination of SGN-CD48A with daratumumab produced durable complete remissions in all animals. In contrast, combination of SGN-CD48A with elotuzumab did not produce additional antitumor activity. In summary, SGN-CD48A has potential antitumor mechanisms of action beyond direct delivery of cytotoxic MMAE to CD48+ myeloma cells, and preclinical data has identified daratumumab as a plausible combination partner.

#5620

Multispecific antibodies targeting CD38 and PD-L1 show potent tumor cytotoxicity.

Wim van Schooten. _Teneobio, Inc., Menlo Park, CA_.

Multivalent antibodies targeting either CD38 alone or CD38 in conjunction with PD-L1 may yield therapeutics with superior biologic activities and provide benefit for treating malignancies expressing low levels of CD38 (DLCBL, daratumumab refractory MM, and NSCLC). Multivalent, multispecific antibodies kill CD38low cells through a variety of mechanisms including stronger and more specific engagement of CD38. Potent and directed immune checkpoint inhibition is realized by adding an anti-PD-L1 binding domain. Teneobio's discovery platform utilizes VH domains (UniDabs) of fully human heavy chain antibodies (UniAbs) to develop bi-, tri-, and tetravalent antibodies. Individual UniDabs targeting CD38 and PDL1 were identified using our unique sequence-based discovery platform and high-throughput lead evaluation pipeline (TeneoSeek). This robust screening workflow enables evaluation of a large diversity of natural fully human antibodies, targeting multiple epitopes on a single antigen and uncovering important sequence activity relationships. We have identified UniDabs that bind five different functional epitopes on human, cynomolgus and mouse CD38 including potent inhibitors of hydrolase and cyclase enzymatic functions. In addition, we generated a large panel of PD-L1 inhibitors that bind with high affinities human and cynomolgus PD-L1. Using different combinations and arrangements of UniDabs, a variety of multivalent antibodies were constructed and evaluated in in vitro models. Data from a range of assay types show that multivalent UniAbs targeting CD38 can be engineered to display superior tumor cell cytotoxicity through multiple mechanisms of action. Summary: Teneobio's discovery platform utilizes VH domains (UniDabs) of fully human heavy chain antibodies (UniAbs) to develop bi-, tri-, and tetravalent antibodies. Multivalent UniAbs targeting CD38 and PD-L1 can be engineered to display superior tumor cell cytotoxicity through multiple mechanisms of action.

#5621

FAP-4-1BBL: A novel versatile tumor-stroma targeted 4-1BB agonist for combination immunotherapy with checkpoint inhibitors, T-cell bispecific antibodies, and ADCC-mediating antibodies.

Johannes Sam, Christina Claus, Claudia Ferrara, Sabine Lang, Valeria Nicolini, Sara Colombetti, Volker Teichgräber, Stefan Evers, Marina Bacac, Pablo Umana, Christian Klein. _Roche Pharma Research & Early Development, Schlieren, Switzerland_.

For tumor specific co-stimulation via 4-1BB (CD137) a novel tumor-stroma targeted FAP-4-1BB ligand (FAP-4-1BBL) was designed composed of a trimeric split 4-1BBL, a tumor-stroma targeted 4B9 Fab moiety recognizing fibroblast activation protein (FAP) and a heterodimeric Fc-region devoid of FcgR binding but maintained FcRn binding. In presence of a TCR signal 1 either from a T cell receptor MHCI/peptide interaction or through TCR engagement by a T-cell bispecific antibody, FAP-4-1BBL provides co-stimulation to T cells strictly dependent on cross-linking by FAP-expressing fibroblasts. Similarly, FAP-4-1BBL can provide co-stimulation to NK cells with activated FcgRIIIa signaling. FAP-4-1BBL was tested as monotherapy and combined with CEA-TCB, an anti-CEA T-cell bispecific antibody, in the s.c. gastric MKN45 xenograft model co-grafted with NIH-3T3 fibroblasts in human stem cell engrafted (HSC) NSG mice in comparison to DP47-4-1BBL, an analogous untargeted 4-1BBL fusion protein. For use in syngeneic mouse models in immunocompetent mice muFAP-4-1BB, a murine surrogate made of a mu4-1BB agonistic surrogate antibody fused to the variable region of the FAP antibody 4B9, was used. muFAP-4-1BB was tested as monotherapy and combined with a murine specific CEA-TCB surrogate (muCEA-TCB) and the muPD-L1 specific surrogate antibody 6E11 in the s.c. colorectal MC38-CEA model in CEA transgenic (Tg) C57BL/6 mice. For combination with ADCC-mediating antibodies FAP-mu4-1BBL, a hybrid FAP-mu4-1BBL fusion protein, was generated. FAP-mu4-1BBL was tested as monotherapy and combined with the ADCC-mediating anti-HER2 antibody trastuzumab in the s.c. gastric N87 xenograft model in human CD16 Tg Scid mice. In the s.c. gastric MKN45 xenograft model co-grafted with NIH-3T3 fibroblasts in HSC-NSG mice FAP-4-1BBL resulted in combined anti-tumoral efficacy in combination with CEA-TCB, whereas the respective monotherapies as well as the combination with the untargeted DP47-4-1BBL did not improve anti-tumor efficacy. In the syngeneic s.c. MC38-CEA model in CEA Tg C56BL/6 mice, a model with natural FAP expression due to fibroblast infiltration, the muFAP-4-1BB surrogate antibody resulted in combined anti-tumor efficacy, both combined with muCEA-TCB and muPD-L1 antibodies including the induction of tumor remission. Finally, the hybrid FAP-mu4-1BBL surrogate resulted in improved anti-tumor efficacy combined with the ADCC-mediating antibody trastuzumab in the s.c. gastric N87 xenograft model in human CD16 Tg Scid mice. These data show that FAP-4-1BBL is a versatile combination partner for cancer immunotherapy mediating FAP-dependent co-stimulation to T and NK cells in combination with PD-L1 checkpoint inhibition, TCBs and ADCC-mediating antibodies. Clinical evaluation of this novel therapeutic approach is planned early 2018.

#5622

A novel immunocytokine fusion protein combining tumor-targeting anti-CD47 antibody with GM-CSF cytokine for enhanced antitumor efficacy.

Zhengyi Wang, Wei Cao, Taylor Guo, Jingwu Zang. _I-Mab Biopharma, Shanghai, China_.

Introduction

CD47 blockade has emerged as a promising cancer immunotherapy by promoting phagocytosis of tumor cells. However, this treatment strategy may be limited by the scarcity of macrophages with phagocytic potential at the tumor site. The key myeloid cytokine GM-CSF has been approved as an adjunctive agent in the treatment of cancer by virtue of its ability to stimulate hematopoiesis and particularly induce the differentiation of pro-inflammatory M1 macrophages. Here we report the development of a novel immunocytokine composed of a newly discovered CD47 IgG1 fused with GM-CSF at the C-terminus that exhibited enhanced phagocytosis and M1 macrophage activation, leading to superior antitumor efficacy.

Methods

A lead anti-CD47 mAb was identified from panning of a naïve human library and a series of functional screening assays. The human GM-CSF sequence was then fused via various flexible linkers to the C-terminus of 1F8's heavy chain to generate different formats of anti-CD47-GMCSF fusions. The resultant fusion proteins were tested for retention of anti-CD47- and GM-CSF-mediated biological activities. A lead molecule (1F8-GMCSF) was then tested for its ability to reduce tumor growth in immunodeficient NSG mice. In addition, phenotype of tumor associated macrophages (TAM) was investigated in tumor sections by immunohistochemistry (IHC).

Results

In macrophage-tumor cell co-culture, treatment with 1F8-GMCSF fusion molecule led to significantly higher levels of tumor cell phagocytosis than that with anti-CD47 alone. Moreover, higher levels of pro-inflammatory cytokines were detected in co-culture supernatant after treatment with 1F8-GMCSF than that with GM-CSF alone. These data suggest functional cooperation between phagocytosis pathways and GM-CSF-mediated growth and differentiation pathways, leading to synergism for enhanced phagocytosis and increased M1 polarization. Consequently, treatment of 1F8-GMCSF in a tumor xenograft model demonstrated superior efficacy in suppression of tumor growth compared to 1F8 or GM-CSF treatment alone or in combination. IHC analysis of tumor sections confirmed a shift from M2 to M1 phenotype in infiltrating macrophages after 1F8-GMCSF treatment.

Conclusion

We have successfully generated a novel anti-CD47-GM-CSF immunocytokine fusion protein that not only retains key properties of its components but also demonstrates enhanced anti-tumor efficacy and M1 macrophage polarization. This fusion protein represents a second generation anti-CD47 molecule that may be used for treatment of solid tumors and is undergoing preclinical development with an aim to enter clinical studies in 2018.

#5623

Development of a CD47-blocking antibody as a cancer therapy.

Joshua Oaks, Ming Wang, Hui Zou. _Phanes Therapeutics, San Diego, CA_.

Immune system avoidance by cancer cells is controlled through a variety of mechanisms such as CTLA-4 and PD-1/L1, which inhibit the T-cell mediated adaptive immune response to cancer. Similar mechanisms exist for evasion from innate immunity, for example, through CD47 overexpression. On healthy cells, CD47 serves as a "don't eat me" signal by the binding to the transmembrane SIRPα protein on phagocytic cells, and prevents the engulfment of "self" by macrophages. In several cancer types, tumor cells overexpress CD47 to elude the immune system. This property of CD47 makes it an attractive target for cancer treatment. Here we describe a humanized monoclonal antibody that is a potent disruptor of the CD47/SIRPα interaction and therefore presents itself as a potential therapeutic anti-CD47 antibody. This antibody binds robustly to recombinant extracellular domains (ECD) of human and cynomolgus CD47, and to full-length human and cynomolgus CD47 expressed on HEK293 cells. Functionally, it blocks the CD47/SIRPα interaction in an ELISA-based assay with an IC50 of single-digit nM. In a cell-based assay, effect on phagocytosis by this antibody was assessed. In addition, disruption of the TSP-1/CD47 interaction has been postulated to have positive outcomes in cancer therapy and the efficacy of this antibody on inhibiting this interaction is also investigated. Finally, the effect of the antibody on Raji human Burkitt's lymphoma xenograft growth in mice will be presented. A concern with anti-CD47 therapy is the undesired targeting of red blood cells (RBCs). This antibody does not induce marked hemagglutination, suggesting a limited impact on RBCs. Taken together, our data suggest that we have developed a novel, humanized anti-CD47 antibody that can restore immune system targeting of cancer cells without inducing significant RBC agglutination.

#5624

Development of novel bispecific immune modulating antibodies.

Laura A. Vitale,1 Lawrence J. Thomas,2 Li-Zhen He,1 Thomas O'Neill,1 Jenifer Widger,1 Andrea Crocker,1 James Testa,1 Karuna Sundarapandiyan,1 Eric Forsberg,2 James Boyer,2 James Storey,2 Joel Goldstein,1 Henry C. Marsh,1 Tibor Keler1. 1 _Celldex Therapeutics, Inc., Hampton, NJ;_ 2 _Celldex Therapeutics, Inc., Needham, MA_.

The initial success with checkpoint blocking antibodies has evolved into a tremendous effort in combination therapy to further improve outcome in responsive cancers and to overcome resistance. There is strong rationale for this approach, as the cancer-immunity cycle has many points for intervention, and the immune system has developed multiple pathways to the same end. The use of bispecific antibodies provides opportunities to engage two pathways simultaneously that can result in greater efficacy, improved safety profile, and simplified development compared to separately administered antibodies. To generate bispecific antibodies that blocked the PD-1 signaling pathway, we developed and characterized fully human PD-L1 specific antibodies generated using human Ig transgenic mice. A lead antibody was chosen for its potent activity in functional assays and good manufacturing properties. Our initial bispecific construct couples our CD27 agonist antibody with the PD-L1 antibody (αCD27xαPD-L1) as we have previously shown that this combination led to improved therapeutic activity in the BCL1 disseminated lymphoma model. We found that the αCD27xαPD-L1 was more potent in CD27 costimulation assays such as NFkB activation, and T cell cytokine production compared to the parental antibodies. Similarly, αCD27xαPD-L1 was more efficacious in vivo as measured by Ag-specific T cell IFN-γ production and antitumor activity. Importantly, the bispecific antibody was more effective than the combination of anti-CD27 and anti-PD-L1 antibodies in the BCL1 lymphoma model. The enhanced efficacy was attributed to more efficient cross-linking of the bispecific antibody/CD27 receptor, resulting in stronger T cell activation. In addition, we observed that CD27 signaling increased PD-L1 expression on both tumor and immune cells, which may further enhance the activity of the bispecific antibody. The use of bispecific antibodies can improve the efficacy and profile of immunotherapeutic monoclonal antibodies by combining complementary functionalities and enhancing target cross-linking in the relevant tissues. We are currently exploring this approach with multiple antibodies from our pipeline.

#5625

Myeloid derived suppressor cells (MDSCs) express Sialyl Tn (STn) and are a therapeutic target for anti-STn antibody drug conjugates.

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

Introduction: Myeloid derived suppressor cells (MDSCs) are functionally defined by their capacity to suppress T cell immunity; therefore inhibiting these cells is of great interest for immuno-oncology applications. STn, the sialylated version of the carbohydrate Tn antigen, is broadly expressed in human cancers such as breast, ovarian, bladder, cervical, colon, and lung and is rarely expressed in normal adult human tissues. The presence of STn in tumors is associated with metastatic disease, poor prognosis, and reduced overall survival. Tumor STn expression is well established and can be leveraged for targeted cancer therapy, however its expression on immuno-suppressive tumor infiltrating lymphocytes such as MDSCs has not been established. Methods: We generated humanized anti-STn mAbs having no cross-reactivity to the asialylated form of STn (Tn) or other glycan antigens. The epitope targeted by these mAbs is the STn glycan itself, not a particular glycopeptide or carrier protein, which offers the broadest potential to bind to multiple STn-glycosylated proteins on cell surfaces. We used these mAbs to quantify specifically the expression of tumor and MDSC STn expression across a set of patient tumor samples. We further treated mice with a humanized anti-STn-MMAE antibody-drug conjugate to evaluate targeted depletion of STn+ MDSCs. In order to evaluate the therapeutic potential of targeting this MDSC phenotype, we further evaluated the immunosuppressive environment of tumors containing STn+ and STn- MDSCs. Results: For the first time, we report the detection of STn on the surface of MDSCs in a growing body of patient tumor samples. Our data in patients as well as xenograft models additionally links STn expression on MDSCs to tumor cell STn expression. We have also demonstrated depletion of STn+ MDSCs after treatment with an anti-STn antibody-drug conjugate in an ovarian carcinoma xenograft model. Conclusions: Our data demonstrated that STn provides a uniquely glycan-specific and potent target for treatment of solid tumors. Anti-STn therapeutics offer the potential to go beyond tumor targeting to also directly target and deplete immune-suppressive MDSCs, thus fostering immune re-engagement and increasing the potential for better patient outcomes.

#5626

Preclinical characterization of Sym022, a novel anti-LAG3 antibody.

Michael M. Grandal, Maria C. Melander, Vikram K. Bhatia, Torben Gjetting, Trine Lindsted, Camilla Fröhlich, Johan Lantto, Ivan D. Horak, Michael Kragh, Klaus Kofoed, Mikkel W. Pedersen. _Symphogen A/S, Ballerup, Denmark_.

Immunotherapy has become a major focus of research in oncology and blockade of immune checkpoints such as cytotoxic T-Lymphocyte associated protein 4 (CTLA4) and programmed cell death protein 1 (PD1) has been some of the most successful immunotherapies. Lymphocyte-activation gene 3 (LAG3) belongs to the second-generation immune modulatory targets. LAG3 is expressed by activated T-cells and tumor infiltrating lymphocytes (TILs) and negatively regulates T-cell activity upon ligand engagement. LAG3 binds major histocompatibility complex class II (MHCII) molecules found on the surface of antigen presenting cells and tumor cells. Sym022 is a Fc-inert human monoclonal antibody targeting LAG3. Sym022 binds to human and cynomolgus monkey LAG3 with high affinity and blocks the interaction between LAG3 and MHCII molecules. Functionally, Sym022 increases cytokine production by T-cells in vitro and tumor growth inhibition in vivo. Mechanistically, Sym022 not only blocks ligand binding, but also decreases total LAG3 surface levels through internalization and/or shedding.

#5627

Anti-TIGIT biomarker study: Inhibition of TIGIT induces loss of Tregs from tumors and requires effector function for tumor growth inhibition.

Gretchen M. Argast, Belinda Cancilla, Fiore Cattaruzza, Pete Yeung, Erwan le Scolan, Rose Harris, Reyhaneh Lahmy, Alayne Brunner, Min Wang, Gilbert O'Young, Earth Light Lowe, Fumiko Axelrod, Jorge Monteon, Jennifer Elechko, Andrew Lam, MingHong Xie, Austin Gurney, John Lewicki, Ann Kapoun. _OncoMed Pharmaceuticals, Inc, Redwood City, CA_.

The immune checkpoint co-inhibitory receptor TIGIT (T cell immunoreceptor with Ig ITIM domain) is expressed on regulatory T cells (Tregs) and on activated CD4+ T, CD8+ T, and NK cells. We have reported that by blocking TIGIT activity with an IgG2a anti-TIGIT antibody (313R12), CD8+ and CD4+ Tcells and NK cells were activated, resulting in dose-dependent tumor growth inhibition (TGI) in multiple syngeneic mouse models. To explore the pharmacodynamics (PD) and mechanism of action of tumor growth inhibition (TGI) by anti-TIGIT antibodies, we examined the kinetics of immune cell frequency and activation in tumor by flow cytometry, qPCR and immunohistochemistry (IHC). We performed in vivo time course studies in the CT26.WT colon carcinoma model using weekly dosing at 0.1, 0.5 and 12.5 mg/kg anti-TGIT. Mice were sacrificed at 24 hours, 7 days and 14 days after the first dose for biomarker analysis. After 24 hours of treatment, Tregs in the tumor decreased and this reduction of Tregs was sustained at 7 and 14 days. Markers of immune cell activation and exhaustion such as CD69, PD1 and intracellular cytokines were modulated during the course of the study, suggesting a more cytotoxic intratumoral environment after 313R12 treatment. In addition, CD226, a binding partner of TIGIT, was significantly upregulated in T cells, Tregs and NK cells throughout the study, reflecting a feedback loop activated by inhibiting TIGIT activity. The anti-TIGIT antibody used in these studies, 313R12, is effector function competent and is able to induce cell-mediated cytotoxic effector functions upon binding. In order to determine whether effector function is necessary for anti-TIGIT antibody activity, we compared 313R12 with an effector function-deficient molecule, 313R13, in CT26.WT tumors. After 7 days, only 313R12 showed significant TGI compared to control-treated animals, suggesting that effector function is required for efficacy. While the effector function-deficient molecule 313R13 was able to similarly induce some changes in PD biomarkers, including immune cell activation, it required a higher dose than 313R12 to do so. To develop biomarkers for anti-TIGIT, we used microarray analyses to identify anti-TIGIT gene signatures in tumors and blood from multiple syngeneic models. In addition, we developed multiplexed IHC panels (e.g., TIGIT+CD8, TIGIT+FOXP3) to quantify expression of TIGIT and TIGIT ligand-positive immune cells in the tumor and surrounding stroma, and we profiled a panel of 80 human tumors with these panels. In summary, we examined the effects of anti-TIGIT antibodies on preclinical mouse models. Biomarker analysis demonstrated loss of Tregs and activation of T cells and NK cells, as well as effector function, as part of the mechanism of action of the molecule. We have also identified biomarkers that can be used for PD and potential predictive analysis in clinical trial samples.

#5628

**Measurement of Fc-mediated ADCC and CDC activity of anti-TNFΑ and anti-VEGF therapeutic antibodies using reporter-based bioassays and engineered TNFΑ** + **and VEGF** + **target cells.**

Denise Garvin, Jamison Grailer, Rich Moravec, Jim Hartnett, Vanessa Ott, Frank Fan, Mei Cong, Zhi-jie Jey Cheng. _Promega, Madison, WI_.

Fc-mediated effector functions are critical to the efficacy and safety of therapeutic antibodies. Measurement of Fc-mediated antibody-mediated cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) during antibody drug discovery and development is not only important for antibodies that harness ADCC and/or CDC as their primary mechanism of action (e.g. rituximab, trastuzumab), but also for antibodies designed to target and block soluble ligands such as TNFΑ and VEGF. We previously reported the development of a cell-based reporter bioassay platform which has been used to measure ADCC and ADCP mediated through FcΓRI, FcΓRIIa and FcΓRIIIa. These reporter bioassays exhibit the specificity, accuracy, precision, and robustness necessary for qualification according to ICH guidelines and have been used extensively to characterize and measure the potency of antibody-based biologics drugs that target cell surface immune receptors. In the current study, we sought to evaluate Fc-mediated ADCC and CDC activities of therapeutic antibodies designed to target and block soluble ligands including TNFΑ and VEGF. To measure ADCC activity of anti-TNFΑ and anti-VEGF blocking antibodies, we developed engineered target cells that express either membrane-bound TNFΑ or VEGF. When used as target cells with reporter-based effector cells expressing a relevant FcΓR, ADCC activity of adalimumab (anti-TNFΑ) and bevacizumab (anti-VEGF) was detected in a specific and dose-dependent manner. Similarly, when used in a luminescence-based CDC assay, the engineered target cells elicited an appropriate FcΓR-mediated response. The assay signals demonstrated IgG isotype specificity as IgG4 variants showed minimal activity in both ADCC and CDC assays. Our results demonstrate that the combined use of cell-based reporter bioassays with target cells engineered to express membrane-bound soluble ligands can provide a simple, specific, and quantitative platform to measure Fc-mediated effector functions of therapeutic antibodies targeting soluble ligands.

#5629

Preclinical characterization of Sym023 a human anti-TIM3 antibody with a novel mechanism of action.

Trine Lindsted, Monika Gad, Michael V. Grandal, Camilla Frölich, Vikram K. Bhatia, Torben Gjetting, Johan Lantto, Ivan D. Horak, Michael Kragh, Klaus Koefoed, Mikkel W. Pedersen. _Symphogen, Ballerup, Denmark_.

Immunotherapy has become a major focus of research in oncology and blockade of immune checkpoints such as cytotoxic T-Lymphocyte Associated Protein 4 (CTLA4) and programmed cell death protein 1 (PD1) has been some of the most successful immunotherapies. The next wave of immunomodulatory targets that are being explored for cancer therapy include T cell immunoglobulin and mucin domain protein 3 (TIM3). TIM3 is constitutively expressed on cells of myeloid origin whereas the TIM3 expression is induced on T-cells upon activation. The exact function of TIM3 on the different immune cells is not clear and may be context dependent suggesting that TIM3 is not a classical immune check-point.

Sym023 is a human anti-TIM3 antibody, which binds human TIM3 and cross-reacts with cynomolgus monkey TIM3. Sym023 blocks binding of phosphatidyl serine but not galectin 9 and stimulates T-cell proliferation in mixed lymphocyte reactions and tumor growth inhibition in vivo. Here, we present data demonstrating that ligation of TIM3 by Sym023 increase cytokine production and T cell proliferation in vitro through a novel mechanism of action.

### Vaccines 2

#5630

Immune profile of tumor microenvironment helps predict response in patients treated with an investigational immunotherapeutic consisting of a retroviral replicating vector (Toca 511) and an extended-release formulation of 5-fluorocytosine (Toca FC).

Derek Ostertag, William Accomando, Leah Mitchell, Maria Rodriguez-Aguirre, Daniel Hogan, Oscar Diago, Dawn Gammon, Ali Haghighi, Harry Gruber, Asha Das, Douglas Jolly. _Tocagen, Inc., San Diego, CA_.

Toca 511 (vocimagene amiretrorepvec) is an investigational, conditionally lytic, retroviral replicating vector (RRV). RRVs selectively infect cancer cells due to defects in innate and adaptive immune responses found in cancers that support virus replication, and cell division requirements for virus integration into the genome. Toca 511 spreads through cancer cells, stably delivering an optimized cytosine deaminase (CD) gene that converts the prodrug Toca FC (investigational, extended-release 5-fluorocytosine) into 5-fluorouracil (5-FU), a canonical chemotherapeutic. In preclinical tumor models, as infected cancer cells are killed, diffusible 5-FU also kills nearby susceptible cells, including uninfected cancer cells, and myeloid derived suppressor cells (MDSC) that contribute to immune-suppression in the tumor microenvironment. This action by Toca 511 and Toca FC has been shown in animal models to generate a durable anti-tumor immune response that can be transferred to naïve, untreated animals. The Toca 511 and Toca FC immunotherapeutic are proposed to remodel the tumor microenvironment to break tumor tolerance resulting in induction of antitumor activity by the patient's immune system and durable complete responses. In a phase 1 clinical study for recurrent high grade glioma (NCT01470794), Toca 511 was injected into resection cavity walls at time of resection followed by multiple courses of oral Toca FC. We observed multi-year durable and objective responses; including 5 ongoing complete responses in a group of 23 patients in the higher dose single agent treatment cohorts given approximately the same Toca 511 doses and having the same entry criteria as an ongoing Phase 3 study in recurrent high grade glioma (NCT02414165). Patient tumors at time of resection were analyzed by exome sequencing, RNA sequencing, IHC, and TCR sequencing, before Toca 511 treatment. In this study, we report higher levels of tumor infiltrating T cells by TCR sequencing, before the start of treatment, were significantly associated with responding patients compared to patients whose disease progressed. The significance of this data is supported by the preclinical mechanism of action reported previously. Additionally, we plan to report on T cell, B cell, and myeloid populations in the tumor as measured by IHC and RNA sequencing and their relationship to clinical response. Data reported here will provide mechanistic context to the immunotherapeutic mode of action proposed to account for durable responses seen in treatment of brain tumors with Toca 511 and Toca FC.

#5631

Personal neoantigen-targeting vaccination generates neoepitope-specific T cell responses in tumors of patients with glioblastoma.

Derin B. Keskin,1 Itay Tirosh,2 Annabelle Anandappa,1 Jing Sun,1 Nathan D. Mathewson,1 Sachet A. Shukla,1 Evisa Gjini,1 Shuqiang Li,1 Letitia Li,1 Anita Giobbie-Hurder,1 Phuong M. Le,1 Zhuting Hu,1 Wandi Zhang,1 Oriol Olive,1 Christine McCluskey,1 Heather Daley,1 Patrick Y. Wen,1 Antonio E. Chiocca,1 Maegan Harden,2 Niall Lennon,2 Stacey Gabriel,2 Gad Getz,2 Donna Neuberg,1 Jerome Ritz,1 Eric S. Lander,2 Aviv Regev,2 Kai Wucherpfennig,1 Mario Suva,3 Edward F. Fritsch,4 Rodig Scott,5 Keith L. Ligon,5 Kenneth J. Livak,1 Hacohen Nir,3 Patrick A. Ott,1 Catherine J. Wu,1 David A. Reardon1. 1 _Dana Farber Cancer Institute. Harvard Medical School, Boston, MA;_ 2 _Broad Institute, Cambridge, MA;_ 3 _Massachusetts General Hospital, Boston, MA;_ 4 _Neon Therapeutics, Cambridge, MA;_ 5 _Brigham And Women's Hospital. Harvard Medical School, Boston, MA_.

Personal vaccines directed at neoantigens arising from tumor mutations can induce neoepitope-specific T cell responses in patients with highly mutated tumors such as melanoma. It remains unknown if this approach can be successfully applied in tumors with low mutation frequency. We conducted a phase 1/1b study to determine the safety and feasibility of patient-specific neoantigen-targeting vaccination in patients with newly diagnosed, methylguanine methyltransferase (MGMT) unmethylated glioblastoma, administered following maximum surgical resection and conventional radiotherapy. Tumor-specific mutations were identified by whole-exome sequencing. Each vaccine, composed of up to 20 synthetic long peptides containing predicted tumor neoepitopes admixed with poly-ICLC, was administered subcutaneously on a prime-boost schedule. Among 8 treated patients, adverse events were limited to mild injection site reactions. Seven patients (88%) received a vaccine with ≥10 neoepitope peptides (median 12, range, 7-20), with median time to vaccine initiation of 18.6 weeks from surgery. All patients died from progressive disease. Median progression-free and overall survival was 7.5 months (95% CI: 6.2, 9.7) and 16.8 months (90% CI: 9.6, 21.3), respectively. 3 patients dropped out of the study due to disease progression. We analyzed vaccine responses on 5 patients that got at least one booster immunization. All 3 patients who required dexamethasone during vaccine priming failed to generate interferon-ɣ responses in peripheral blood mononuclear cells. In contrast, 2 patients who did not receive dexamethasone during vaccine priming, generated robust de novo immune responses against multiple personal neoantigens. Circulating vaccine-induced polyfunctional neoantigen-specific CD4+ and CD8+ T cell responses were enriched for memory and activated phenotypes, and increased numbers of tumor-infiltrating CD4+ and CD8+ T cells were detected in these 2 patients. T cell receptor analysis identified identical clonotypes isolated from post-vaccination glioblastoma tissue and peripheral blood including a clonotype specific for ARHGAP35, a neoantigen targeted by vaccination. To our knowledge we provide the first evidence that tumor specific T cells can traffic from the periphery into glioblastoma tumors and that neoantigen-targeting vaccines can favorably alter the tumor immune milieu of glioblastoma. In conclusion, individualized, multi-neoepitope vaccination is feasible, safe, and generates neoantigen-specific T cell responses in the periphery and intracranial tumors of patients with glioblastoma.

#5632

Targeted immunotherapy with SV-BR-1-GM: Mechanism of action and companion diagnostic development.

Markus D. Lacher,1 Sanne Graeve,1 Charles L. Wiseman,1 Ying Y. Kong,2 William W. Kwok,2 William V. Williams1. 1 _BriaCell Therapeutics Corp., Berkeley, CA;_ 2 _Benaroya Research Institute at Virginia Mason, Seattle, WA_.

SV-BR-1-GM is a GM-CSF-engineered whole-cell targeted immunotherapy derived from a breast cancer cell line (SV-BR-1). We are currently assessing SV-BR-1-GM in a phase I/IIa clinical trial in metastatic and locally recurrent breast cancer (ClinicalTrials.gov identifier NCT03066947). Subjects who fail to respond with tumor regression are offered participation in a "roll-over" study in which an immune checkpoint inhibitor (pembrolizumab or ipilimumab) is added to the SV-BR-1-GM regimen (ClinicalTrials.gov identifier NCT03328026). To identify patients likely to respond to SV-BR-1-GM we are co-developing a companion diagnostic.

In a pilot phase I study with four evaluable patients, one subject (A002), with metastatic breast cancer, experienced systemic tumor regression including in soft tissue, lung, and brain lesions. In contrast to the three nonresponders, A002 matched with SV-BR-1-GM at both a class I (HLA-A) and a class II (HLA-DRB3) HLA allele. Strikingly, SV-BR-1-GM cells also express HLA class II "accessory" factors such as HLA-DMA and -DMB, HLA-DRA, and CD74 (encoding invariant chain and CLIP), and immunostimulators such as IL6, Il18, and KITLG, and the damage-associated molecular pattern (DAMP) protein HMGB1. SV-BR-1-GM cells do not express the T cell co-stimulatory ligands CD80 or CD86. These findings are consistent with a potential to activate previously primed but not naïve CD4+ T cells. In agreement, peptide-pulsed SV-BR-1-GM cells selectively activated peptide-HLA specific, pre-primed T cells.

Given this functional data and the expression of the "immune signature", comprised of factors including those mentioned above, we hypothesized that SV-BR-1-GM cells can act as antigen-presenting cells (APCs) and present tumor-associated antigens (TAAs) directly to HLA-matched T cells, which in turn would induce an immune response directed to the patients' tumors. To evaluate this hypothesis, we have begun developing bioassays for assessing antibody and T cell responses to SV-BR-1-GM.

Here, we present data on SV-BR-1-GM's molecular makeup and development of anti-SV-BR-1-GM humoral and cellular immune responses. Our findings support SV-BR-1-GM acting as antigen-presenting cells for HLA class I and class II restricted immune responses.

#5633

Development of glycoconjugate-based anticancer vaccines.

Xuefei Huang. _Michigan State University, East Lansing, MI_.

The development of an effective vaccine construct against tumor associated carbohydrate antigens (TACAs) is an attractive approach towards cancer treatment and prevention. However, a significant challenge is that TACAs are only weakly immunogenic and direct administration of TACAs cannot elicit a powerful antibody response to protect the host from cancer development. In this presentation, we will present our strategies to significantly enhance the immunogenicity of TACAs as potential anti-cancer vaccines using mucin-1 (MUC1) as a representative antigen. Mucin-1 (MUC1) is one of the top ranked tumor associated antigens. MUC1 peptides and glycopeptides have been covalently conjugated to an immunogenic carrier. Immunization of mice with these constructs led to super strong antibody responses with IgG titers over one million, which are one of the highest IgG titers reported to date. Furthermore, the constructs elicited MUC1 specific cytotoxic T cells, which can selectively kill MUC1 positive tumor cells. The abilities of MUC1 conjugates to induce both antibody and cytotoxic T cell immunity targeting tumor cells highlight the potential of these constructs as anti-tumor vaccines.

#5634

Eradication of breast carcinoma with bone metastasis by autologous formalin-fixed tumor vaccine (AFTV) combined with radiation and chemotherapy.

Fumito Kuranishi,1 Yuki Imaoka,2 Hiroshi Iwako,1 Yoji Uemae,3 Takeshi Ishihara,3 Tsubasa Miyazaki,3 Tadao Ohno3. 1 _Innoshima Ishikai Hospital, Onomichi, Japan;_ 2 _Hiroshima university school of medicine, Hiroshima, Japan;_ 3 _Cell-medicine Inc., Tsukuba, Japan_.

No standardized treatment has been established for skeletal metastasis of breast carcinoma which is refractory to intensive chemo-radiation therapy and therefore is assumed impossible to cure. Here we show, however, the 3rd successful case concomitantly treated with the tumor vaccine, AFTV, and chemo-radiation therapy (the 1st and 2nd cases have been presented in AACR2013 and AACR2015, respectively). A 50-year old woman presented 2.5-cm mass of tubular carcinoma in her left breast in a MRI image. The tumor accompanied with no lymph node metastasis was resected in March 2009. We found the tumor was ER(+), PgR(-), and HER2 (-). Four months after the resection, she selected one course of AFTV as the first-line therapy. Response to delayed-type hypersensitivity (DTH) test became strongly positive. We then treated her with the aromatase inhibitor, anastrozole. Six years later, in March 2015, a PET image revealed bone-metastasis to sternum which was confirmed by needle-biopsy and ABC immunostaining as class V metastatic ductal carcinoma. We treated her again with second course of AFTV and palliative radiation therapy (36Gy/12fractions/19days) together with letrozole, bisphosphonate, additional 2 shots of nivolmab (40mg/48kg body) in October and November 2015. She was diagnosed to be complete response by PET imaging in February 2016, August 2016, and August 2017. Alteration of her blood CEA level suggests that the combined treatments eradicated the metastatic breast carcinoma. In conclusion, the present case suggests that AFTV in combination with palliative X-ray irradiation and adjuvant aromatase inhibitors eradicates skeletal metastasis of breast carcinoma with enhancement of a specific anti-tumor immune reactivity.

#5635

The development to peptide vaccine targeting heat shock protein 90 to overcome resistance to HER-2 target drugs in breast cancer.

Jinho Kang. _Korea University, Seoul, Republic of Korea_.

HER-2 overexpression is observed in approximately 25% of all breast cancers and is associated with poorer overall survival and decreased time to relapse. Target therapies directed against HER-2 have been developed and used clinically, but many patients continue to develop resistance. Breast cancer is immunogenic and well suited to treatment via immunomodulation. HSP90 is a chaperon for most of the important signal transduction system in HER-2 type breast cancer; HER-2, ER / PR and Akt, the downstream protein of PI3K. Therefore, HSP90 is considered as a potential therapeutic target in order to improved treatment of HER-2 positive resistant breast cancer. Immunotherapy targeting HSP90 has been tried before, however, no clinically available vaccine due to limitation of autologous cell-based approach. We questioned whether HSP90 was an immune target in HER-2 positive breast cancer. First, HSP90-specific IgG antibody immunity was detected in the sera from HER-2 positive breast cancer patients. Next, potential MHC class II epitopes derived from HSP90 with high binding affinity across multiple human HLA genotypes were identified using in silico algorithms. Top 11 peptides were synthesized and Th1 vs Th2 immunity were assessed in human PBMCs. After IFN-γ and IL-10 ELISPOT assays, epitopes inducing Th1-directed immunity were selected for in vivo experiments. Immunogenecity of HSP90 peptides vaccine was evaluated in FVB mice model and showed strong antigen-specific T cell responses in IFN-γ ELISPOT assay. Then, we assessed the anti-tumor effect of immunization with HSP90 peptides vaccine in MMTV-neu-transgenic mouse model after autologous cancer cell implantation. Peptides vaccines derived from HSP90 significantly inhibited tumor growth compared with control group (mean ± SD; 1896 ± 1211 mm3 vs. mean ± SD; 774 ± 136 mm3, respectively; p < 0.05). The tumor-inhibitory effect was associated with HSP90 peptide-specific IFN-γ-secreting T cell responses in immunization group. Also, immunohistochemical staining of tumors derived from immunized mice was performed to examine immune microenvironment and the protein expression of HER-2 pathways. Taken together, multi-epitope peptides vaccine derived from HSP90 is immunogenic in breast cancer patients and is a promising tumor antigen. Based on efficacies in tumor-rejection in vivo, further research in combination with immune modulating antibodies will be performed in the lapatinib-resistant tumor model.

#5636

Identification of bladder cancer-associated cancer-testis antigens-derived long peptides encompassing both CTL and promiscuous HLA class II-restricted Th cell epitopes.

Yasuharu Nishimura,1 Miki Tsuruta,2 Shohei Ueda,3 Poh Yin Yew,4 Isao Fukuda,4 Sachiko Yoshimura,4 Hiroyuki Kishi,5 Hiroshi Hamana,6 Masatoshi Hirayama,2 Junji Yatsuda,7 Atsushi Irie,8 Satoru Senju,8 Eiji Yuba,9 Tomomi Kamba,7 Masatoshi Eto,10 Hideki Nakayama11. 1 _Nishimura Project Laboratory, Institute of Resource Development and Analysis, and Department of Immunogenetics, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan;_ 2 _Department of Immunogenetics and Department of Oral and Maxillofacial Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan;_ 3 _Department of Urology, Graduate School of Medical Sciences, Kyushu University, and Department of Immunogenetics, Graduate School of Medical Sciences, Kumamoto University, Fukuoka and Kumamoto, Japan;_ 4 _Tumor Immunoanalysis Department, OncoTherapy Science, Inc., Kawasaki, Japan;_ 5 _Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences (Medicine), University of Toyama, Toyama, Japan;_ 6 _Department of Innovative Cancer Immunotherapy, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan;_ 7 _Department of Urology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan;_ 8 _Department of Immunogenetics, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan;_ 9 _Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University, Sakai, Japan;_ 10 _Department of Urology, Graduate School of Medical Sciences, Kyushu University and Kumamoto University, Fukuoka and Kumamoto, Japan;_ 11 _Department of Oral and Maxillofacial Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan_.

DEP domain containing 1 (DEPDC1) and M-phase phosphoprotein 1 (MPHOSPH1) are human cancer testis antigens that are frequently overexpressed in urinary bladder cancer. The growth of bladder cancer cells was significantly inhibited by knockdown of DEPDC1 or MPHOSPH1 expression using small interfering RNAs; thus, DEPDC1 and MPHOSPH1 are essential for tumor growth and survival, indicating the clinical significance of these antigens as targets for antigen-specific cancer immunotherapy. Subsequent reports further identified highly immunogenic DEPDC1- and MPHOSPH1-derived short peptides (SPs) that can induce HLA-A24-restricted cytotoxic T lymphocytes (CTLs) from peripheral blood mononuclear cells (PBMCs) isolated from patients with bladder cancer. These DEPDC1- and MPHOSPH1-derived SPs vaccine administered to patients with progressive bladder cancer was well tolerated and effectively activated SPs-specific CTLs in vivo, and the better CTL induction was associated with longer survival of patients in a recent phase I and II clinical trial (Obara W et al. Ann. Oncol. 28: 798, 2017). Here, we aimed to identify long peptides (LPs) derived from DEPDC1 and MPHOSPH1 that induced both T-helper (Th) cells and tumor-reactive CTLs. Stimulation of PBMCs isolated from healthy donors with the synthetic DEPDC1- and MPHOSPH1-LPs predicted to bind to promiscuous human HLA class II molecules by a computer algorithm induced specific CD4-positive T cells as revealed by interferon-gamma enzyme-linked immunospot assays. Three of six LPs encompassed SPs recognized by either HLA-A2- or -A24-restricted CTLs, and all six LPs stimulated DEPDC1- or MPHOSPH1-specific Th cells restricted by promiscuous and frequently observed HLA class II molecules in the Japanese population. Some LPs are naturally processed from the proteins in DCs, and the capacity of these LPs to cross-prime SP-specific CTLs was confirmed in vivo using HLA-A2 or -A24 transgenic mice. The LP-specific and HLA class II-restricted T-cell responses were also observed in PBMCs isolated from patients with bladder cancer. Repeated stimulation of PBMCs with DEPDC1-LPs and MPHOSPH1-LPs yielded clonal Th cells expressing specific T-cell receptor (TCR)-alpha and beta genes. These DEPDC1- or MPHOSPH1-derived LPs may have applications in immunotherapy in patients with bladder cancer, and the TCR genes identified may be useful for monitoring of Th cells specific to LPs in vivo. [ This research was supported by a MEXT Grant-in-Aid for Scientific Research on Innovative Areas 22133005; JSPS KAKENHI 23650609, 24300334, 15H04311 and 16H06498 for Scientific Research on Innovative Area "Neo-self"; the Project for Cancer Research And Therapeutic Evolution (P-CREATE) from the Japan Agency for Medical Research and Development, AMED; and OncoTherapy Science, Inc.. ]

#5637

Combination of oncolytic vaccinia virus and immune checkpoint blockade overcomes resistance to immunotherapy in renal cell carcinoma.

Hongjae Chon, Chan Kim. _CHA Bundang Hospital, Seongnam-si, Republic of Korea_.

Renal cell carcinoma (RCC) is a hypervascular tumor with variable immunogenic responses, and VEGF-targeted anti-angiogenic agent has become a standard treatment during the past decade. Recently, immune checkpoint inhibitor (ICI) has merged as another standard of care in RCC, but response is limited as a monotherapy. To determine the optimal combination strategy for immunotherapy in RCC, we employed JX594 (Pexa-Vec), an oncolytic vaccinia virus currently in phase 3 clinical trial, as a combination partner for ICI in RCC. Intratumoral injection of JX594 remodeled the tumor microenvironment of Renca tumor model through dynamic changes in immune system, leading to the conversion of an immunuosuppressive, non-inflamed tumor into an inflamed tumor. The JX594 virotherapy increased the intratumoral infiltration of CD8+ T cell by 16-fold, resulting in systemic and tumor-specific anti-cancer immune response against RCC. Moreover, the expression of immune checkpoint molecules such as PD-1 (4.0-fold), CTLA-4 (2.3-fold), and LAG-3 (3.1-fold) were increased. Depletion experiment showed that the therapeutic effect of JX594 is largely dependent on the cooperation of CD8+ and CD4+ T cells. In addition, GM-CSF has been shown to potentiate the immunotherapeutic efficacy of JX594. Combination of JX594 and ICI targeting PD-1 or CTLA-4 further increased the intratumoral infiltration of activated CD8+ T cells and effectively suppressed the growth of RCC. Furthermore, the optimal schedule was rationalized by

combination treatment of various administration methods. Finally, based on these combination results, triple combination of JX594, anti-PD-1, and anti-CTLA-4 maximized the anti-cancer immune responses and induced durable responses with improved overall survival. Collectively, we demonstrate that intratumoral injection of JX594 sensitizes non-inflamed RCC to ICI treatment through CD8+ T cell infiltration in tumor and induces anti-cancer immune responses that can overcome immunotherapy resistance.

#5638

A tetanus-way of improving synthetic long peptide tumor vaccination.

Jan Wouter Drijfhout,1 Erika Fletcher,2 Justyna Leja-Jarblad,3 Iliana Kerzeli,2 Robert Cordfunke,1 Gunilla Tornqvist,3 Frida Lindqvist,3 Greta Hultqvist,2 Rob Valentijn,4 Sam Ladjervardi,2 Michael Haggman,2 Gustav Ullenhag,2 Sara Mangsbo2. 1 _Leiden University Medical Center, Leiden, Netherlands;_ 2 _Uppsala Univ., Uppsala, Sweden;_ 3 _Immuneed AB, Uppsala, Sweden;_ 4 _Leiden University Medical Center, Uppsala, Netherlands_.

We have previously identified a natural B cell epitope derived from tetanus toxin named Minimal Tetanus Toxin Epitope (MTTE)[1]. Most healthy individuals have IgG1 antibodies to this peptide sequence but no IgM antibodies. We have assessed the possibility to apply this MTTE sequence in drug development with the intention to create immune complexes and use them as vehicles to carry antigen-material into dendritic cells. Thru a conjugation method three MTTE sequences and a longer synthetic peptide stretch harboring T cell epitopes are linked together. As immune complexes are efficient carriers of antigen material and can promote cross-presentation the aim is to use these conjugates for therapeutic purposes in diseases where a strong cellular immune response should be induced.

To further evaluate the MTTE peptide conjugates in drug development we have investigated how a diphtheria, tetanus, pertussis (DTP) vaccination induces antibody titers against the linear tetanus sequence. Herein we have studied the MTTE location in the tertiary structure of the protein, how cancer patients respond with antibody titers against MTTE along with a subsequent T cell response and in vivo anti-tumor responses (mice). In addition we have studied safety in terms of cytokine release assessment pre and post a DTP vaccination. We found that the MTTE sequence is part of an alpha-helix that is externally oriented and thereby accessible for peptide binding. We found that not helper epitope is part of the MTTE sequence and that healthy individuals as well as cancer patients can mount IgG titers against the epitope, this also result in more efficient induction of recall responses post a DTP booster than pre boost. Mice seropositive to the MTTE sequence can mount anti-tumor responses, while this is not seen in seronegative animals. In addition, a cytokine release assessment using a modified chandler loop model with intact complement does not display a broad immediate cytokine release pre or post a DTP booster. We conclude that it is efficacious and safe to make use of a peptide conjugate in a drug development project with the aim to use it as a vaccine strategy.

[1] Mangsbo et al. In press. Molecular Immunology 2017

#5639

Changes in local and peripheral T cell diversity after HPV E7 antigen-expressing Listeria-based immunotherapy (ADXS11-001) prior to robotic surgery for HPV-positive oropharyngeal cancer.

Rosemarie Krupar,1 Ravi R. Pathak,2 Naoko Imai,3 Eric Genden,4 Krzys Misiukiewicz,3 Elizabeth G. Demicco,5 Jigneshkumar Patel,2 Falguni Parikh,2 Michael Donovan,5 Seunghee Kim-Schulze,3 Sven Perner,1 Marshall Posner,3 Brett Miles,4 Sacha Gnjatic,3 Andrew G. Sikora6. 1 _Pathology of the University Medical Center Schleswig-Holstein, Campus Luebeck and Research Center Borstel, Luebeck, Germany;_ 2 _Bobby R. Alford Department of Otolaryngology-Head and Neck Surgery, Baylor College of Medicine, Houston, TX;_ 3 _Division of Hematology and Medical Oncology, Department of Medicine, Mount Sinai School of Medicine, New York, NY;_ 4 _Department of Otolaryngology, Mount Sinai School of Medicine, New York, NY;_ 5 _Division of Pathology, Mount Sinai School of Medicine, New York, NY;_ 6 _Bobby R. Alford Department of Otolaryngology-Head and Neck Surgery, Baylor College of Medicine, New York, TX_.

Human papilloma virus associated oropharyngeal cancers (HPVOPC) account for about one third of OPC and express foreign viral antigens such as the E6 and E7 oncogenes, which are suitable for immune targeting. We performed a "window of opportunity" trial of patients undergoing standard-of-care transoral surgery for HPVOPC to assess the effect of the HPV16-E7 targeting vaccine ADXS11-001 on the intratumoral and systemic immune response. We reported, in a prior abstract, that 5 of 8 ADXS11-treated patients showed increased E6 or E7-specific IFN-γ responses post-treatment and 4/8 patients demonstrated increased CD8 and CD4 tumor infiltrating lymphocytes (TILs) after vaccination. In the present study, we profiled peripheral blood immunocyte populations (PBMCs) pre-vaccination, post-vaccination and post-surgery via multicolor flow cytometry in 8 patients. Clonal expansion and diversification of PBMCs and tumor TILs post-treatment were determined via T cell receptor (TCR) DNA sequencing and correlated to HPV antigen specific immune responses determined by ELISPOT as well as tumor immune profile determined by quantitative immunofluorescence. While peripheral CD8 cytotoxic T cell (CTL) levels remained constant over the course of the study, we observed trends towards their increased expression of LAG-3 (1.6 fold, p=0.0821) and PD-1 (1.6 fold, p=0.0748) 6 weeks post-surgery, consistent with activation. We did not observe statistically significant changes in levels of CD4 T helper cells, MDSC or Tregs, although a modest trend towards decreased CD8/MDSC ratio was observed post-surgery (0.5 fold, p=0.0864). Overall TCR diversity/clonality of PBMCs and TILs did not change. However the comparison of similarity between T cell repertoires of the tumor and PBMCs (Morisita's index) demonstrated an increase of expanded clones post-vaccination limited to TILs in 5/6 patients, indicating clonal expansion in ADXS11-treated patients. When we analyzed T cell repertoire changes in individual patients, 4 of 8 patients demonstrated expansion of specific TCR clones post-vaccination. The two patients with the greatest increase in number of expanded TCR clones among both, PBMCs and TILs, also had the strongest increase of E6 or E7-specific IFN-γ responses post-treatment, and also had strongly increased CD8 TILs. Our results demonstrate that immunomodulatory effects of ADXS11-001 on the local and peripheral immune response vary among patients, with 2/8 treated patients demonstrating evidence of an integrated adaptive immune response in blood and tumor, consistent with selective clonal expansion in the context of a vaccine-induced anti-tumor T cell response. Profiling of additional patients will permit more detailed analysis of ADXS11-induced immune modulation and impact on TCR diversity.

#5640

Development of a personalized pancreatic cancer vaccine.

Darren R. Cullinan, Michael McLellan, Xiuli Zhang, Timothy M. Nywening, Chris A. Miller, S. Peter Goedegebuure, William G. Hawkins, William E. Gillanders. _Washington University in St. Louis, St. Louis, MO_.

Introduction: Cancer neoantigens result from mutations accumulated in an individual tumor. These mutations are usually private and yield high-affinity T-cells as they are not limited by mechanisms of self-tolerance. Neoantigens presented in the context of MHC class I can be recognized by anti-tumor CD8+ T-cells.

Methods: Three consecutive patients with confirmed pancreatic ductal adenocarcinoma (PDAC) had DNA and RNA isolated from areas of high tumor cellularity. Comparative normal DNA was isolated from peripheral blood mononuclear cells (PBMC). Nonsynonymous mutations were identified by tumor/normal whole exome sequencing and expression in the tumor was verified by cDNA capture. HLA typing was inferred from exome sequencing. Candidate neoantigens were identified using multiple epitope prediction algorithms for MHC class I. Matched patient PBMC were cultured in serum-free media for 48 hours with synthetic mutated short peptides to determine the baseline immune response by interferon-gamma (IFNγ) enzyme-linked immunospot assay.

Results: A KRAS driver mutation was identified in all three patients. Using KRAS variant allele frequency (VAF) as a surrogate for tumor purity, we achieved greater than 40% tumor purity for all three samples. This provides an adequate level of purity to proceed with neoantigen identification. In these samples, the mean number of nonsynonymous mutations identified was 27 (range: 18-32; DNA VAF cutoff 10%). Of these nonsynonymous mutations, a mean of 8.6 mutations (range: 7-10) were expressed (RNA VAF cutoff >10% and FPKM >1). Following confirmation of mutation expression, a mean of 5 mutations (range: 4-6) were predicted to bind to MHC class I using multiple prediction algorithms (binding affinity <500nM). IFNγ immunospot assay revealed a baseline immune response to one neoantigen in each patient (Patient A - SDHA: 75 spot forming units per millions cells (SFU/M) vs Negative: 12.5 SFU/M, p=0.01; Patient B - ZRANB1: 588 SFU/M vs Negative: 17.5 SFU/M, p<0.001; Patient C - ATAD3C: 340 SFU/M vs Negative: 75 SFU/M, p=0.002).

Conclusion: Utilizing advanced genomic and bioinformatics tools we have successfully characterized the cancer neoantigens in a series of three consecutive patients with PDAC. From this data, we plan to open a phase I clinical trial to assess the safety and immunogenicity of a neoantigen DNA vaccine.

#5641

Synergy of immunomodulators delivered by an HSV-1 oncolytic virus can enhance its therapeutic effects in vivo.

Yanal M. Murad,1 Jun Ding,1 Dmitry Chouljenko ,1 Erica Lee,1 Luke Bu,2 Guoyu Liu,2 Ronghua Zhao,1 Zahid Delwar,2 Will Liu,1 William Jia2. 1 _Virogin Biotech Canada, Vancouver, British Columbia, Canada;_ 2 _University of British Columbia, Vancouver, British Columbia, Canada_.

Oncolytic HSV-1 (oHSV-1) are among the most promising therapies for cancer. Besides the oncolytic activity delivered by the virus against tumor cells, this treatment can also induce a potent immune response against tumor antigens (whether classic TAA, or neoantigens specific to the tumor being targeted). We have constructed an oncolytic HSV-1 virus (VG161) to deliver 2 immunomodulator cytokines, IL12 and IL15, to the tumor micro-environment. These two cytokines can work synergistically to trigger an efficient anti-tumor immune response. A PD-L1 mimic peptide capable of blocking PD-1/PD-L1 interaction is also delivered as a Fc fusion peptide (TF-Fc) to help in maintaining an effective immune response against the tumor. For the construction of the recombinant virus, the exogenous components were cloned under CMV promoter control. The ICP34.5 gene was deleted as a safety measure for the recombinant virus. The expression of Il12, IL15 and TF-Fc were confirmed, in vitro and in vivo, by qRT-PCR, ELISA and Western blot. Upon intra-tumoral injection of VG161, expressed IL12, IL15 and TF-Fc were detected only in the injected tumor mass, but not in the animals' blood or other organs. The bio-distribution of the virus was tested by qPCR after injecting VG161 intratumorally, and most of the injected virus were concentrated in the tumor mass. Minimal amounts of the virus were detected in the liver and spleen for short periods after virus injection. We have demonstrated the superior activity of the VG161 virus, compared to the back-bone virus (with no payloads) using immune-competent mouse models (CT26) and nude mice implanted with human tumor cells lines (LNCaP and U87). VG161 induced tumor oncolysis resulting in complete tumor regression in nude mice. In the CT26 model, the tumor regressed to undetectable limits upon intra-tumoral injection with VG161. When the treated mice were challenged with the same tumor, the tumor cells did not grow. The added efficacy in CT26 tumor model can be attributed to the immune response generated by the modified virus, as demonstrated by a higher number of tumor-infiltrating CD8 T cells. The specific activity of T cells against the tumor cells was also demonstrated by ELISpot assay. Finally, memory T-cells were evident in the treated animals demonstrated by multiple assays. Conclusions This work has demonstrated the safety and efficacy of VG161, a novel oncolytic virus which can induce a strong anti-tumor immunity and oncolytic activity. We have also demonstrated that the Intra-tumoral expression of multiple immune regulatory factors may significantly change the tumor immune microenvironment to enhance the efficacy of the oncolytic virus.

#5642

Electroporation as a feasible method for antigen delivering into vectors loaded with NKT cell ligand.

Akira Arimoto,1 Masayasu Nishi,2 Kimihiro Yamashita,1 Yutaka Sugita,1 Eiji Fukuoka,1 Tomoko Tanaka,1 Takashi Kamigaki,3 Rishu Takimoto,4 Yoshihiro Kakeji1. 1 _Kobe University Graduate School of Medicine, Kobe, Japan;_ 2 _Hyogo Cancer Center, Japan;_ 3 _Juntendo University Faculty of Medicine, Japan;_ 4 _SETA Clinic Group, Japan_.

Background: Invariant natural killer T (iNKT) cells have been shown as an ideal immunetherapeutic target of which the activation via α-Galactosylceramide (α-GalCer) conjugated with the MHC class I-like CD1d molecule induces both the innate and adaptive immune system. With transfection of antigen-coding mRNA, α-GalCer loaded CD1d-expressing cells can act as a vector to enhance the antigen-specific immune responses. However, the method using the transfection of antigen-coding mRNA is sometimes too complicated. Electroporation (EP) has been reported as a way to induce substances easily into cells. We applied EP as a simple method to deliver the antigen into the vector for iNKT activation. In this study, we aimed to show the feasibility of EP as a method to deliver the antigen into α-GalCer loaded DCs, a naturally CD1d-expressing vector, in terms of inducing antigen-specific antitumor responses.

Methods: DCs were generated from bone marrow of C57BL/6 mice. Ovalbumin (OVA) was electroporated to DCs as a tumor model antigen. To load α-GalCer, DCs were cultured with α-GalCer for 48 hours. We compared the antitumor responses of α-GalCer loaded DCs with or without electroporated OVA in C57BL/6 mice. Mice were prophylactically administered each type of DCs intravenously followed by subcutaneous injection of EG7, which is OVA induced EL4, thymoma (n = 5, respectively). We evaluated the tumor size and the survival rate.

Results: With electroporated OVA, tumors did not grow in mice administered α-GalCer loaded DCs whereas tumors in mice administered α-GalCer loaded DCs without electroporated OVA aggressively progressed (tumor size on day 28 after tumor inoculation; 3 ± 3 vs 4176 ± 1571 mm3, P < 0.05). In mice administered α-GalCer loaded DCs with electroporated OVA, no death was observed during the whole observation period. Survival rate was significantly better for mice administered α-GalCer loaded DCs with electroporated OVA than those administered α-GalCer loaded DCs without electroporated OVA (P < 0.05).

Conclusion: α-GalCer loaded DCs with electroporated OVA showed significantly higher rate of tumor rejection and longer survival in the mice model inoculated OVA-induced tumor compared to α-GalCer loaded DCs without electroporated OVA. EP was feasible as a way to deliver the antigen into the vector, which induced antigen-specific antitumor responses. We are trying to show the activity of antigen-specific cytotoxic T lymphocytes following the administration of α-GalCer loaded DCs with electroporated antigen.

#5643

TCRβ repertoire analysis from a prophylactic MUC1 cancer vaccine trial.

Michelle L. Miller, Jason Lohmueller, John R. McKolanis, Robert Schoen, Olivera J. Finn. _University of Pittsburgh, Pittsburgh, PA_.

Mucin 1 (MUC1) is a large transmembrane glycoprotein that is overexpressed and hypoglycosylated in many adenocarcinomas, including colon cancer. In the hypoglycosylated state, the peptide core of the 20aa variable number of tandem repeats (VNTR) region is exposed, allowing it to function as an altered-self ligand for antibodies and T cells. VNTR peptides have been previously used as antigens in therapeutic cancer vaccines. A clinical trial was conducted to test immunogenicity and safety of a MUC1 100mer VNTR peptide+polyICLC adjuvant vaccine in a prophylactic setting in patients with premalignant colon adenomas who were at high risk for later developing colon cancer. In this premalignant setting, where patients were expected to have fewer immunosuppressive countermeasures usually present in patients with cancer, nearly half of the individuals generated IgG antibodies with no noted adverse effects. Because the antibody response was isotype-switched, it suggested simultaneous T cell activation. To determine whether changes in T cell clonal abundance following immunization could be detected, we sequenced and analyzed TCRβ repertoires from PBMCs of 21 antibody-responders collected before and after both primary and one-year booster vaccinations. Of the patients analyzed, we identified 8 TCRβ sequences from three patients that were significantly elevated after both priming and boosting immunizations, potentially identifying T cell clones that expanded in response to the vaccine. 16 TCRβ sequences from four individuals were significantly decreased at both time points, potentially identifying T cell clones that left circulation to participate in immune responses in secondary lymphoid organs or tissues. The timing of blood collection at two weeks post immunization may have been too late to allow us to detect all of the vaccine-expanded clones before they left circulation. In summary, we found 24 TCRβ CDR3 sequences from 7/21 vaccine responders that significantly changed in frequency after both primary and booster vaccinations. TCR sequencing studies from patients' T cells expanded in vitro with MUC1 peptide will be performed to determine if they overlap with those sequenced ex vivo.

#5644

Generation of clinical grade autologous TLR 7,8-polarized fast dendritic cell vaccines for active immunotherapy of patients with AML.

Iris Bigalke,1 Lisbeth Johanne Skoge,1 Kirsti Hønnåshagen,1 Christiane Geiger,2 Gunnar Kvalheim,1 Dolores J. Schendel2. 1 _Oslo University Hospital, Oslo, Norway;_ 2 _Medigene AG, Planegg-Martinsried, Germany_.

Although progress has been made in the treatment of AML in recent years, relapse rates following chemotherapy are still high resulting in only low long-term patient survival rates. Treatment options are often limited following relapse, emphasizing the continued need for new and more effective treatments options.We have developed an active immunotherapy using TLR7/8-polarized fast dendritic cells (DCs), which is currently under evaluation for treatment of patients with AML in morphological remission in a non-randomized phase I/II clinical trial (NCT02405338). The trial subjects receive repeated immunotherapy with autologous vaccine cells, transfected with RNA encoding two leukemia-associated antigens WT1 and PRAME. Patients are vaccinated for two years or until disease progression. Here we present data on the feasibility of clinical grade production of our TLR7/8-polarized fast DC vaccine for long-term vaccination of chemotherapeutically pretreated AML patients. A total of 20 patients with a median age of 59 years (range 24 - 73 years) were recruited to this ongoing trial. For vaccine production, autologous apheresis material was collected from each patient and fast DC generation was performed following isolation of DC precursor cells using a TLR7/8-agonist containing maturation cocktail. Final vaccine cells were cryopreserved in multiple aliquots prepared to deliver 5x106 \- 10x106 cells per vaccine dose. Vaccine cells could be generated from all 20 AML patients. A second apheresis was performed during the trial for a second production in order to generate sufficient vaccine doses for the intended treatment period for 4 of 20 patients. The majority of production runs (17 out of 24) yielded 20 or more vaccine doses (8 batches: 20-29 doses, 6 batches: 30-39 doses, 2 batches: 40-49 doses and more than 60 doses from one batch). For seven patients less than 20 vaccine doses were cryopreserved from one production run. To evaluate vaccine cell purity, presence of autologous non-DC cells was determined and revealed a purity of more than 70% for 22 vaccine batches (17 batches 80% - 99%, 5 batches 71% - 77%). Only two productions yielded lower purities (range 64% - 69%). The average post-thawing vaccine cell viability was 83% (range 68%-95%) with the majority of cells showing a viability of >70%. Additionally, DCs generated from all patients showed a mature surface phenotype as demonstrated by high expression of typical DC surface markers such as CD80, CD86, CD83, CD40 and HLA-DR. Furthermore, high expression of the lymphocyte homing receptor CCR7 could be detected for all cell preparations. Taken together, these results clearly demonstrate the feasibility and robustness of our protocol for production of mature clinical grade TLR7/8-polarized fast DCs in high numbers from heavily pretreated post-remission AML patients allowing for long-term vaccination of trial subjects.

## CANCER CHEMISTRY:

### Emerging Proteomic Technologies for Cancer Research

#5645

Critical variables to reduce Western blot variability.

Russ Yukhananov, Alex Margulis. _Precision Biosystems, Mansfield, MA_.

Reliable immunodetection of proteins is a critical step in the discovery of new biomarkers and diagnostic. The immunodetection protocol consists of multiple steps including blocking of nonspecific binding sites, incubation using primary and secondary antibodies and extensive washing between steps that easily introduce bias and errors. The quality and reproducibility of results depends on multiple subjective and objective factors such as the qualification and technical skills of the personnel performing the assay and the accuracy of temporal and temperature control, especially during the immunodetection step. BlotCycler™, automated western blot processor, use fluidic control system that allows to eliminate the variability associated with immunodetection and to achieve higher sensitivity by optimized washing procedure. We used BlotCycler to analyze critical source of errors during immunodetection. Relatively small changes in duration and temperature of incubation significantly affect not only the intensity of signal but also the specificity of antibody interaction with antigen. Automated immunodetection at 4ºC significantly improve the specificity of detection and sensitivity that allows reproducible detection of low expressing proteins. Automated processing using BlotCycler allows optimization and control of critical variables leading to improved reproducibility and specificity of immunodetection and should be routinely used for new biomarker discovery and diagnostics, reliable protein quantification, primary antibody specificity testing, and optimization of primary and secondary antibodies concentrations.

#5646

Serum functional biomarker discovery with PEP technology.

Xing Wang, Michael Davies. _Array Bridge Inc, St. Louis, MO_.

Proteins play essential roles in numerous biological processes and being able to pinpoint functional differences between cell or tissue samples can greatly aid in understanding disease processes and metabolic changes. The PEP technology allows systematic analysis of protein functions within a proteome. Hundreds of functional proteins can be separated and functionally assayed to generate a comprehensive three-dimensional landscape of protein families such as protein kinases, phosphatases, proteinases and oxido-reductases. This information can then be integrated into other genomic and proteomic knowledge bases to provide further insight of important biological processes such as cancer development and aging. Using the PEP technology, functional biomarker candidates have been identified from liver, lung and breast cancer patient serum. Both qualitative and quantitative differences of metabolic enzymes and proteases were observed when comparing the cancer patient serum and normal serum. Some of the active enzymes were identified by mass spectrometry and validated in selected bioassays. Deep mass spectrometry analysis revealed that some of the functional biomarkers are protein variants of important biological function. It is believed that this functional proteomics technology will provide a unique approach in the discovery of potential cancer biomarkers for diagnostic and prognostic applications. Further efforts are ongoing to develop a prototype of diagnosis kit for cancer detection based on the current discoveries.

#5647

Multivariant analysis to decipher the human pancreatic cancer proteome to identify novel biomarkers and therapeutic targets.

Henry Chun Hin Law, Dragana Lagundzin, Zachary S. Wagner, Nicholas Woods. _University of Nebraska Medical Center, Omaha, NE_.

The microenvironment of a tumor plays a vital role in its development and progression. We hypothesized that the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment could elucidate proteins associated with patient survival. This project utilized a quantitative proteomics approach designed to: 1) Identify differentially expressed proteins in the tumor microenvironment of PDAC liver metastases that exhibited different levels of cancer cell differentiation; 2) Distinguish proteins associated with PDAC patient survival to build a predictive model for prognosis; and 3) Identify potential therapeutic targets to promote patient survival. A cohort of PDAC liver metastases from 60 patients was collected from the Pancreatic SPORE Rapid Autopsy Program at the University of Nebraska Medical Center. The tissue samples were preserved within 1-3 hours to avoid post-mortem protein degradation. The tissues were divided into 7 batches and were lysed, digested and differentially labeled with isobaric Tandem Mass Tags. The tagged peptides were mixed, fractionated with C18 spin columns and analyzed by an Orbitrap Fusion Lumos LC-MS/MS system. 30811 peptides and 3960 proteins were identified from the analysis. 1842 of them were quantified and 917 proteins were submitted for principal component analysis. Tissue samples with similar protein expressions were grouped into 4 major clusters. Samples exhibit a tendency to cluster depending on similar histological features and tumor cell differentiation. Gene ontology enrichment analysis of the corresponding protein clusters suggests samples group according to tumor cellularity, the degree of differentiation and stroma involvement. The proteome data was then correlated to the survival days post diagnosis with partial least square analysis to identify PDAC prognostic markers. Thymidine phosphorylase (TYMP) was one of the highest scoring proteins in the model. This protein converts 5'-deoxy-5-fluorouridine into 5-fluorouracil and is a known predictive marker for capecitabine treatment. Current data suggests patients with higher TYMP expressions tend to have longer survival after the diagnosis. One of the high scoring proteins identified in the model is endoplasmic reticulum aminopeptidase 1 (ERAP1). ERAP1 is an enzyme which trims precursor peptides in the endoplasmic reticulum. Antigenic peptides from the cancer cells might be altered in the process. With further validation, the biomarkers identified with this methodology could provide insight into the design of personalized cancer treatment for PDAC patients.

#5648

Proteomics analysis of oral cancer cell-derived extracellular vesicles.

Ana K. Oliveira, Ariane F. Busso-Lopes, Jamile Oliveira Sá, César Rivera, Adriana F. Paes Leme. _CNPEM/LNBio, Campinas, Brazil_.

More than 300,000 new cases of head and neck cancer occur each year around the world, and although it is not one of the most common tumors in the world, has an elevated morbidity rate (37.8%) after five years of diagnosis. The development and progression of the Oral Squamous Cell Carcinoma (OSCC) involve complex cellular mechanisms which contribute to a tumor progression. Recently extracellular vesicles (EVs) have taken a significant physiological and pathological role in cell-cell communication through interactions mediated by receptors or by transfer of bioactive molecules such as receptors, proteins, lipids, mRNA, microRNA, and DNA, by changing the phenotype of the recipient cell. The tumor cells can transfer information to different cell types using EVs as a vehicle, modifying the characteristics of the tumor microenvironment, and contributing to cancer sustaining. The detection of alterations in the EVs proteome may, therefore, contribute to the understanding the cellular mechanisms modulated in OSCC and help to improve the clinical prognosis and potential therapeutic targets in oral cancer. Thus, this study analyzed the cargo proteins of different EVs from less and high aggressive oral cancer cells to understand their possible role in microenvironment modulation and oral cancer progression. Our results indicate that EVs of aggressive cells have exclusive proteins that may contribute to lymph node metastasis, which is the worse prognostic factor in OSCC and can be considered potential targets in OSCC.FAPESP: 2016/19337-3

#5649

Multi-omics data indicate that primary and lymph node oral cancer cells-derived extracellular vesicles carry cargo molecules with a specific aggressive pattern.

Ariane F. Busso-Lopes,1 Carolina M. Carnielli,1 Flávia Winck,2 Diego M. Riaño-Pachón,2 Ana K. Oliveira,1 Rute A. Costa,1 César Rivera,1 Camila Caldana,1 Jay W. Fox,3 Adriana F. Paes Leme1. 1 _Brazilian Center for Research in Energy and Materials - CNPEM, Campinas, Brazil;_ 2 _University of Sao Paulo, Sao Paulo, Brazil;_ 3 _University of Virginia, Charlottesville, VA_.

Oral squamous cell carcinoma (OSCC) exhibits high incidence and morbidity, representing 300,000 new cases and 145,000 deaths per year. Moreover, the 5-year survival rate is 50% and this is directly associated with regional lymph node metastases. To explore how the extracellular vesicles (EVs) play a significant role on OSCC lymph node metastasis, we characterized the EVs released by tumor primary site cells and their paired lymph node metastasis cells using nanoparticle tracking analysis, high-resolution microscopy (Cryo-EM), EVs uptake by HUVEC, BJ-5ta and gingival primary fibroblasts recipient cells and the proteins, lipids, metabolites and miRNAs contents. Both cells-derived EVs showed a similar size, morphology, and the presence of annexin-2 and flotillin-1 surface markers. Interesting, only gingival primary recipient cells were more likely to uptake primary OSCC-derived EVs compared with lymph node-derived EVs. This effect is probably associated with the differential abundance of 490 proteins, 316 miRNAs, 63 lipids and 17 metabolites between the oral cancer cells-derived EVs. Markedly, multilevel omic data integration indicates that metabolic processes were over-represented mainly in primary OSCC-derived EVs, suggesting that these EVs carry specific cargo molecules associated with a more aggressive pattern compared with lymph node OSCC-derived EVs.

Financial support: FAPESP (Process number 2016/50005-7).

#5650

Functional proteomics for precise cancer diagnosis and therapy using the MSK Quantitative Cancer Proteomics Atlas.

Paolo Cifani, Ahmet Dogan, David B. Solit, Alex Kentsis. _Memorial Sloan Kettering Cancer Center, New York, NY_.

The advent of molecular biology and molecular profiling in clinical medicine has transformed our understanding of the molecular basis of human cancer. As a result, we are increasingly improving the classification of human tumors based on their specific genetic and molecular mechanisms of pathogenesis. However, currently only a small number of mutant alleles guide treatment decisions, while most observed mutations remain of unknown pathologic and clinical significance. In addition, even for recently approved drugs, such as those targeting activated kinase signaling, clinical efficacy is highly varied, with no currently satisfactory means to identify molecular markers of response and resistance. To address this need, and broadly enable transformative future advances in precision oncology and patient outcomes, we have now developed the Quantitative Cancer Proteomics Atlas (QCPA). This technology permits highly multiplexed, quantitative analysis of the expression and biochemical activity of hundreds of proteins, covering most recurrently mutated and known pathogenic pathways in cancer cells, and designed to be applied to clinically accessible patient specimens. QCPA enables concomitant measurement of cellular concentration and stoichiometry of regulatory post-translational modification of key proteins mediating aberrant survival and proliferation. The activation status of these proteins is then used as a proxy to infer regulatory status of relevant biochemical pathways. These sentinel proteins include 467 known effectors of kinase regulatory signaling, apoptosis, stress response, and proliferation. QCPA measurements can be made using conventional parallel reaction monitoring mass spectrometry, or using an improved method termed accumulated ion monitoring (Cifani & Kentsis 2017). We established analytical metrics of QCPA assays in cancer cell lines, and demonstrated sensitivity and quantification accuracy amenable to measure endogenous chemically modified proteins from microgram specimens. We then demonstrated the ability of QCPA profiling to detect differential biochemical activities in response to defined stimuli in vitro. Next, we deployed QCPA profiling to reveal functional differences between primary human acute myeloid leukemia specimens and CD34+ myeloid progenitors, measuring specific activation of biochemical pathways in cancer cells. Finally, we tested the clinical utility of functional proteomic profiling by determining molecular markers of response and resistance to ERK pathway inhibitors in patient specimens. Thus, QCPA functional proteomic profiling provides a tool to achieve precise diagnosis and rational targeted cancer therapies.

#5651

Achieving simultaneous improvements in workflow and multiplexing in whole-slide tissue imaging.

Michael Natan. _Ultivue, Cambridge, MA_.

With very high spatial and biological complexity, tumor micro-environments present a significant

characterization challenge, which is only heightened by the need to image entire sections of cancer

tissue on many different samples. A variety of techniques offer very high levels of multiplexing, but

always fail on either speed requirements (incredibly long times required to image a whole slide) or on

compatibility issues (expensive, hard-to-operate CapEx not suitable for high-throughput CLIA operation).

Ultivue has developed a platform reagent technology (InSituPlex™) that simultaneously enables whole-

slide higher multiplexing by immunofluorescence AND complete compatibility with existing

instrumentation.

The InSituPlex technology comprises five key foundational elements. First, short unique DNA barcodes

are covalently attached to each primary antibody (Ab). These conjugates are purified and functionally

characterized in tissue for optimal staining specificity. Importantly, all of this work is done in advance by

Ultivue. Second, tissue samples are stained in a single step. Third, all the DNA sequences attached to

targets (via primary Ab binding) are simultaneously amplified, allowing for detection of less abundant

markers e.g. PD-1, PDL-1 etc. Fourth, complementary DNA sequences each with different, covalently-

attached fluorescent labels ("imager strands") are simultaneously hybridized to the amplified DNA on

the targets. All these wet chemistry steps are carried out in walkaway mode using commercially

available auto-stainers. The samples are then imaged using a standard, whole-slide multi-color

fluorescence imaging system. Finally, in an optional fifth step, the imager strands are gently removed

via an enzymatic dehybridzation- with complete retention of antigenicity- to enable higher multiplexing

via additional rounds of hybridization/imaging.

The InSituPlex reagent technology offers many advantages for cancer research over current approaches:

• the ability to detect 5, 10, or more targets simultaneously via whole-slide imaging on an each

and every sample, with no loss in antigenicity;

• the ability to leverage existing auto-stainers and existing fluorescent microscopes with

Ultivue's reagent kits to carry out high-throughput analyses of multiple cancer

biomarkers; and

• the ability to design custom biomarker panels that leverage Ultivue's unique barcoding

capability and infrastructure.

This poster will elaborate on each step of the technology, and provide multiple examples of data

acquired using different biomarker panels and different cancer tissue types.

#5652

Validation of novel InSituPlex™ technology utilizing standard chromogenic IHC and multiplexed tyramide-based immunofluorescence.

Katir Patel, Courtney Hebert, Jamie Buell, Sean Downing. _Ultivue, Cambridge, MA_.

Introduction: The demands of today's immuno-oncology research has driven the development of

more robust tools to interrogate the tumor microenvironment, specifically using multiplex

immunofluorescence (mIF). Multiplex immunofluorescence allows investigators to visualize

multiple biomarkers in tissue sections while preserving spatial context. The current gold standard

of immunohistochemistry is "brown" staining using DAB with a hematoxylin counterstain. In

recent years, multiplex immunofluorescence has been accepted in the field using tyramide

molecules (TSA) that permanently fix fluorescent dyes to the tissue. Consequently, tissue integrity

is compromised and cannot be utilized in additional forms of analysis. In addition, high-level

plexing using TSA requires the use of highly specialized and costly imaging equipment. Here we

validate a novel technology, InSituPlex™, utilizing DNA barcoding of antibodies and the detection

of markers using simple DNA hybridization techniques. InSituPlex allows for high-leveling of

multiplexing, preservation of tissue integrity for additional analyses, and implementation on a

variety of commonly used imaging platforms.

Methods: Performance of the InSituPlex technology was assessed using 15 immuno-oncology

relevant markers (CD3, CD4, CD8, CD20, CD25, CD68, CD163, FoxP3, Granzyme B, Ki67, PD1,

PD-L1, pan-cytokeratin, Sox10, and TTF1). Each marker was benchmarked by performing a

single-plex DAB chromogenic IHC and an immunofluorescence assay. Antibodies to be used for

InSituPlex testing were first conjugated to unique DNA barcodes and the resulting conjugates were

assessed utilizing the previously described assays to ensure equivalent performance of the

InSituPlex conjugate to unconjugated antibodies. The performance of the complete InSituPlex

assay was evaluated using InSituPlex HRP labelled imaging strands and the newly barcoded

antibodies. InSituPlex vs. DAB/TSA performance was compared across platforms. All staining

was performed manually and on the Leica BOND RX autostainer. Images were acquired across

multiple imaging platforms, including the Leica VERSA whole slide scanner, and image analysis

was performed using HALO from Indica Labs.

Results: All antibodies performed equally well across all the staining methodologies as assessed

qualitatively. Quantitatively, cell counts were performed on serial sections. Statistical analysis

comparing InSituPlex to standard chromogenic and immunofluorescent assays across all 15

antibodies did not yield any significance (p-value > 0.1).

Conclusions: Detection of relevant immune and cancer related markers utilizing InSituPlex

technology is equivalent to standard chromogenic IHC and immunofluorescent assays.

#5653

VMax™: A new platform for recombinant protein expression.

Eric Hesek, Chris Wilson, Lindsey Wolf, Sanjay Vasu, Michael Sturges, Daniel Gibson, Matthew Weinstock. _Synthetic Genomics, La Jolla, CA_.

Here we describe Vmax™, a next-generation protein expression platform based on the organism V. natriegens, an extremely rapidly growing gram-negative prokaryote capable of reaching high cell density. Vmax™ contains an IPTG inducible T7 expression system, enabling high levels of recombinant protein expression. By combining a robust expression system with a highly productive organism, Vmax allows researchers to perform experiments faster, shorten workflow times, and generate large amounts of recombinant protein.

#5654

Fully automated tissue classification by imaging mass spectrometry, moving towards a hundred samples a day.

Emrys A. Jones,1 Richard Chapman,1 Emmy Hoyes,1 Lukasz Migas,2 Fiona Henderson,2 Steven Pringle,1 James Langridge,1 Adam McMahon2. 1 _Waters, Wilmslow, United Kingdom;_ 2 _University of Manchester, Manchester, United Kingdom_.

Desorption electrospray ionisation mass spectrometry is a surface analysis technique that can rapidly analyse the lipid and metabolite composition of a tissue section. By moving the tissue under the analysis probe, a chemical map of the sample can be generated in a matter of minutes. As has been shown in the literature 1,2, each tissue type or disease state has its own characteristic molecular fingerprint, therefore, using histologically annotated databases of these molecular profiles these maps can be classified using machine learning approaches. With the increased rate of analysis made possible by recent technological advances, the timeframes of a single sample makes automation an attractive proposition. With a slide loading system and a range of software solutions, up to two hundred slides can be queued for analysis. Each will be loaded in turn onto the analysis stage, the location of the tissue section determined with optically directed object detection algorithms, and the analysis started. During the analysis the information from each pixel is projected into the classification model and colour coded by designated tissue type. Applications of this approach that have been demonstrated from a research perspective are the analysis of serial sections of drug dosed spheroids and tumour xenografts for studying the chemical heterogeneity of these systems in three dimensions, in the latter case allowing comparison with other imaging modalities such as MRI and PET. Initial studies will also be presented which will investigate the use of the system for the classification of large cohorts of unknown tissues with subsequent comparison to histological classification by conventional methodologies.l 1. Calligaris, D.Caragacianu, D.Liu, et al; PNAS, 2014, 111, 15185-15189 2. Guenther, S., Muirhead, L.J., et al, Cancer Res, 2015, 75, 1828-1837

#5655

Biophysics in the cell—CETSA to study drug binding and cellular processes in cancer therapy.

Ying Yu Liang, Anderson Ramos, Henriette Laursen, Olga Surova, Johan Lengqvist, Sara Lööf, Anette Langebäck, Smaranda Bacanu, Jonas Bergh, Pär Nordlund. _Karolinska Institute, Solna, Sweden_.

A key step of the action of most drugs is their binding (engagement) of the target protein(s). However, limitations in the available methods for directly accessing this critical step have added uncertainties in many stages of drug development. We have developed a generic method for evaluating drug binding to target proteins in cells and tissues (Martinez Molina et al. Science, 341:84). The technique is based on the physical phenomenon of ligand-induced thermal stabilization of target proteins; the method is therefore called the cellular thermal shift assay (CETSA). The technique allows for the first time to directly measure the biophysical interactions between a drug and protein target in non- engineered cells and tissues. We show that using CETSA a range of critical factors for drug action can be addressed at the target engagement level, including drug transport and activation, off-target effects, drug resistance as well as drug distribution in cells, patient and animal tissues.

Although CETSA was first developed for evaluating drug binding to target proteins in cells it is also useful for characterizing physiological interactions, including protein-protein, protein-metabolite and protein-nucleic acid interactions (Martinez Molina & Nordlund, Ann Rev Pharm Toxic 2016 56:141). In the proteome-wide CETSA experiment, interactions with more than 7000 proteins can be measured in parallel (Savitski, Science 2014 346;6205). This strategy therefore constitutes a novel mean for discovering key interactions determining the fate of cancer cells during therapy and resistance – interactions that can serve as biomarkers or candidates for novel drug targets in cancer therapy.

#5656

Developing metal-labeled antibody panels for multiplex proteomic characterization of rare circulating tumor cells by imaging mass cytometry.

Milind Pore,1 Erik Gerdtsson,1 Christopher Boldt,1 Sophia Wix,1 Preeya Katti,1 Mohan Singh,2 Akil Merchant,2 James Hicks,1 Peter Kuhn1. 1 _University of Southern California David and Dana Dornsife College of Letters, Arts, and Sciences, Los Angeles, CA;_ 2 _University of Southern California Keck School of Medicine, Los Angeles, CA_.

Imaging mass cytometry (IMC) is a novel technology with the potential of multiplex proteomic profiling of rare circulating and disseminated tumor cells (CTCs and DTCs) and immune cells from liquid biopsies. The high-definition single-cell analysis (HD-SCA) identifies CTCs and DTCs by the virtue of morphometric and fluorescent staining with exact locations of all the cells, which could be further characterized using downstream genomics and proteomics platform. This study aims to develop and employ metal-labeled antibody panels for multiplex proteomic characterization of CTCs, DTCs and immune cells obtained from longitudinal liquid biopsies from cancer patients on the basis of their epithelial-to-mesenchymal transition (EMT) status, cancer stem cell (CSC), dormancy, proliferation and drug resistance markers. For every antibody panel, 6-10 biomarkers were chosen, and metal-labeled antibodies were processed in a three-stage validation program using known positive and negative control cell lines. In the first stage, basal level marker expression was evaluated in positive control cells seeded on a glass slide. In the second stage, cell lines were spiked in blood and slides were prepared as per the HD-SCA protocol, stored at -800 C, and expression of biomarkers was evaluated in IMC staining. In the third stage, cell line-spiked slides were first stained in three colors immunofluorescent staining in HD-SCA, mimicking the clinical setup followed by IMC staining with metal-labeled antibodies. Antibodies showing expressions in all three stages were included to build a panel targeting EMT, CSC, vasculature mimicry, dormancy, proliferation and drug resistance biomarkers. Out of 69 metal-labeled antibodies, 24 antibodies targeting cancer and immune cell-specific biomarkers have been validated for multiplex proteomic profiling in HD-SCA platform. This includes epithelial markers EpCAM and E-cadherin; mesenchymal markers vimentin and fibronectin; CSC markers CD44, CD24, ALDH and beta-catenin; prostate-specific markers PSA, PSMA, AR-C terminal, AR-N terminal; and immune cell markers CD45, CD66a, CD3, HLA-DR, CD45RA, CD8a, CD4, CD38, CD14 and CD31. Building and validating metal-labeled antibody panels for EMT, CSC, dormancy and drug resistance biomarkers could achieve multiplex proteomic characterization of the rare cell IMC. It would be further used in monitoring treatment efficacy, mutational switchovers, and immune surveillance from longitudinal liquid biopsies collected from cancer patients to better understand the biology of a disease.

#5657

Use of a novel immunofluorescence multiplexing technology, InSituPlex™, for the simultaneous detection of immune cells in multiple cancer types.

Sean Downing, Katir Patel, Jamie Buell, Courtney Hebert. _Ultivue, Cambridge, MA_.

Introduction: The field of immuno-oncology has enthusiastically adopted multiplexed

immunohistochemistry (IHC) techniques to establish the spatial relationships between various

immune cells in tumor biology. Multiplexing enables researchers to gain a deeper understanding

and insight into the tumor microenvironment. Unfortunately, many of the multiplexing

technologies currently utilized in the immuno-oncology field face a number of challenges. These

challenges include complicated and difficult assay optimizations for high-level multiplexing and

the need of specialized imaging instrumentation. In addition, harsh stripping methods and

irreversible staining techniques compromise the integrity of precious tissue samples that ultimately

can no longer be used for additional forms of testing. InSituPlex™ is a new method of multiplexed

IHC that relies on the detection of a unique DNA barcode conjugated to primary antibodies. This

technology removes the need to perform multiple, harsh stripping methods to perform multiplexed

IHC and allows for higher order of multiplexing in less time than currently available multiplexing

techniques, all the while maintaining tissue integrity for use in downstream analyses.

Methods: InSituPlex was used to perform a multiplexed IHC assay on 5 samples from 4 tumor

types (breast, lung, colon, and melanoma). The assay consisted of the following markers: CD4,

CD8, CD20, CD68, PD1, Ki67, FoxP3, and a tumor marker; pan-cytokeratin (CK) for breast,

colon, and lung carcinomas and Sox10 for melanoma. Staining was performed using the Leica

BOND RX autostainer, imaging was performed on various platforms including the Leica VERSA

whole slide scanner, and image analysis was performed using HALO from Indica Labs.

Data: All markers were detected utilizing the InSituPlex technology across all tumor types. Data

indicates a range of phenotypes for infiltrating immune cells in tumor types from highly inflamed

to "immune deserts". The panel provided for analysis of many phenotypic subtypes, (e.g.,

CD4+/PD1+, CD8+/Ki67+, and Ki67+/CK+), cell densities, proliferative indexes, and spatial

analysis (e.g., CD8+ to tumor cells).

Conclusion: InSituPlex technology is proficient in maintaining tissue integrity and detecting a

range of immune cell phenotypes in multiple tumor types. InSituPlex can be implemented by any

lab possessing a standard fluorescent microscope and does not rely on the purchase of, nor access

to, more expensive platforms.

#5658

From staining to analysis: End-to-end application of InSituPlexTM technology for multiplexed immuno-profiling in FFPE tumor samples.

Mael Manesse, Amanda Bares, Heike Boisvert, Stephanie Walter. _Ultivue, Cambridge, MA_.

Introduction: The development of new and improved cancer diagnostic and therapeutic methods

relies on a deep understanding of the protein expression profile in the tumor microenvironment.

Conventional immunohistochemistry (IHC) methods only provide insights into a limited number

of markers of interest, prompting the need for the development of multiplexed methods to assess

and characterize multiple cell phenotypes and their spatial context in a single sample. We have

developed an approach to enable high-multiplexing detection of protein markers in FFPE tumor

samples using an integrated workflow with the Leica Bond RX autostainer and Leica Versa whole

slide scanner.

Methods: A cocktail of primary antibodies of interest, modified with unique, addressable DNA

barcodes, is applied to tissue samples in a single staining step. The barcodes are then amplified

simultaneously to improve sensitivity. Finally, a cocktail of fluorescently labeled oligonucleotides,

complementary to the barcodes on the antibodies, is used to tag the different targets of interest for

multiplexed imaging. Formalin-fixed, paraffin-embedded samples from both human tonsil and

primary tumor biopsies were stained manually and automatically on the BondRX, using a panel of

antibodies specific to cancer immuno-profiling markers. Images were acquired using the Leica

Versa whole slide scanner, and analyzed with Leica Aperio Image Analysis and Indica Labs Halo

software.

Results: Compared to typical multiplex IHC methods, the InSituPlex single staining step offers

multiple advantages. First, the workflow preserves the integrity and antigenicity of the samples by

avoiding any antibody stripping steps and streamlining the assay development process by

eliminating the need to optimize staining order. In addition, the total assay time stays constant

regardless of the multiplexing level. Adding targets to the panel does not increase staining time,

facilitating the optimization of higher multiplex assays, as well as the development of streamlined

automated assays. A fully automated staining protocol was developed on the BondRX by

modifying assay parameters such as reagent concentrations and volumes, optimizing the timing of

each step, and evaluating the impact on the resulting images in terms of specificity, sensitivity and

overall signal to background ratio. After optimization, the automated staining protocol delivered

similar or better results compared to the manual assay protocol, while decreasing the total assay

time by approximately 40%. The optimized protocol was subsequently used to carry out a

multiplexed analysis of immune cell phenotypes in tumor samples.

Conclusions: the InSituPlex technology enables multiplexed immune-profiling in tumor FFPE

sections, and provides a streamlined, high-throughput workflow with existing automated staining

and imaging instrumentation.

#5659

Multiplex, single-cell proteomic analysis using imaging mass cytometry for circulating tumor cells.

Sophia Wix, Erik Gerdtsson, Milind Pore, James Hicks, Peter Kuhn. _Bridge@USC, Los Angeles, CA_.

Current methods of solid tumor biopsy are invasive and potentially risky for metastatic cancer patients. The high-definition single-cell assay (HD-SCA) is a well-documented, minimally invasive alternative to tissue biopsy that harnesses morphometric and proteogenomic analysis of circulating tumor cells (CTCs) in blood to identify the cellular and molecular characteristics underlying the disease. These "liquid biopsies," utilizing the HD-SCA, are a promising prognostic and predictive platform for assessing the state of cancer during treatment for metastatic patients. Immunofluorescent methods for biomarker analysis are generally limited to four channels for signal detection. Imaging mass cytometry (IMC) achieves multiplexed, highly targeted proteomic analysis with subcellular resolution for up to 37 MaxPar metal-conjugated antibodies quantified by time of flight (TOF) mass spectrometry. We have developed an application of IMC technology for the HD-SCA workflow using the Fluidigm Hyperion Imaging System (HIS), and have employed the combined platform in a validation study across cell lines and patient samples. The HIS platform was tested and effectively integrated into the HD-SCA pipeline with validation of 35 proteomic markers. Metal-tagged antibodies to protein biomarkers for hormone receptor status, epithelial versus endothelial marker expression, epithelial-to-mesenchymal transition, stemness, along with standard leukocyte markers, were validated in appropriate cell lines and used to evaluate patient CTCs. We hope to detect a predictive biomarker using IMC proteomics, single-cell genomics, and high-resolution morphologic data. Coupling IMC-based proteomics with single-cell genomics and morphometrics in liquid biopsies has the potential to significantly increase the potential clinical utility of the liquid biopsy for both prognostic and predictive applications.

#5660

Proteomic comparative assessment of flash-frozen and OCT embedded breast cancer tissues for utilization in precision oncology discovery studies.

Kiki Panagopoulos,1 Punit Shah,1 Albert Kovatich,2 Michael A. Kiebish,1 Jeffrey Hooke,3 Leigh Campbell,3 Mary Lou Cutler,4 Audrey Kovatich,5 Hai Hu,6 Brenda Deyarmin,6 Lori Sturtz,6 Praveen Kumar Raj Kumar,6 Stella Somiari,6 Emily Y. Chen,1 Elder Granger,1 Viatcheslav Akmaev,1 Rangaprasad Sarangarajan,1 Niven Narain,1 Craig D. Shriver1. 1 _BERG, Framingham, MA;_ 2 _Henry M. Jackson Foundation, Bethesda, MA;_ 3 _Henry M. Jackson Foundation, Bethesda, MD;_ 4 _Uniformed Services University, Bethesda, MD;_ 5 _MDR Global Systems, Windber, PA;_ 6 _Chan Soon-Shiong Institute of Molecular Medicine at Windber, Windber, PA_.

Proteomic analysis is an important tool in classifying molecular architecture of tissue samples for subtyping and precision oncology applications. Tissues are commonly either flash frozen (FF) or embedded in optimal cutting temperature (OCT) compound for long-term storage. OCT provides a convenient specimen matrix for cryostat sectioning but contains glycols and resins which are incompatible with mass spectrometric analysis of proteins. In contrast, flash frozen preservation of tissue is historically considered to be more suitable and compatible with mass spectrometry applications. As a pilot study, we evaluated different tissue preservation methods, sample processing methods, and protein extraction methods, and their impact on quantitation and coverage of the proteome. In this pilot study, 30 breast cancer cases with matching flash frozen and OCT-embedded samples were used for proteomic analysis. Tissues were procured from the Clinical Breast Care Project (CBCP) in partnership with the Chan Soon-Shiong Institute of Molecular Medicine at Windber, and the Walter Reed National Military Medical Center (WRNMMC). Breast cancer subtypes were determined by clinical and expanded immunohistochemical panels. These subtypes were Her2, Luminal A (LA), Luminal B (LB) and Triple Negative (TN). Flash frozen tissues were cryosectioned to isolate 60-100 mg total tissue per case, and proteins were extracted using an 8M urea lysis buffer. The OCT specimens were laser microdissected to isolate primarily tumor cells from the samples. Proteins were extracted using the illustra TriplePrep kit. Digested proteins were then multiplexed using Tandem-Mass-Tag (TMT) 10plex isobaric labeling reagents, and chromatographically resolved using a Waters 2D nanoAcquity liquid chromatographer (LC) coupled to a Thermo Q Exactive Plus mass spectrometer (MS). Data was then analyzed using Proteome Discoverer. In total, 6130 proteins were identified using a 1% peptide FDR confidence filter in the 30 samples analyzed. Unsupervised hierarchical clustering of proteins across the samples resulted in two primary clusters, OCT and FF. Within the primary clustering, there were sub-clusters of Luminal, Her2, and Triple Negatives. At 2-fold threshold, 246 proteins were differentially expressed between OCT and FF tissues. However, the differential proteins between ER+ (LA and LB) and ER- (Her2 and TN) tumors correlate irrespective of the cryopreservation and processing methods. In conclusion, proteome analysis of samples stored under two different conditions provide subtyping information; however, there is differential expression of proteins between the two types of sample handling techniques which can impact biological interpretation of breast cancer precision oncology studies.

#5661

A multi modal mass spectrometry imaging strategy to profile the metabolic hallmarks of colorectal cancer.

Josephine Bunch,1 Rory T. Steven,1 Adam J. Taylor,1 Spencer A. Thomas,1 Alan M. Race,1 Alex Dexter,1 Gregory Hamm,2 Nicole Strittmatter,2 Rasmus Havelund,1 Renata F. Soares,3 Andrew D. Campbell,4 Owen J. Sansom,4 Richard J. Goodwin,2 Zoltan Takats3. 1 _National Physical Laboratory, Teddington, London, United Kingdom;_ 2 _Astra Zeneca, Cambridge, United Kingdom;_ 3 _Imperial College London, London, United Kingdom;_ 4 _Beatson Institute for Cancer Research, Glasgow, United Kingdom_.

New methods are required to image the metabolic hallmarks of cancerous tissues and correlate this information with patient tumour pathology, cancer phenotype and treatment outcome. Mass spectrometry imaging (MSI) offers a powerful suite of techniques for imaging metabolites, drugs and proteins in cells and tissues. Here we present a multi-modal MSI strategy and use it to profile PDX, GEMM and biopsy samples.

Human and murine colon and small intestine samples were sectioned onto glass slides and stored at -80°C until use. MALDI-MSI (Waters and Bruker) was carried out with pixel sizes between 10 and 50 microns, DESI (Waters) at 20-50 microns and SIMS (IONTOF) at approximately 200 nm per pixel. Data were converted from proprietary format to imzML and analysed in MatLab (2015b, MathWorks) via the SpectralAnalysis software package using non-negative matrix factorisation (NMF) and principal component analysis (PCA). Further reduction and segmentation was achieved using stacked autoencoders and t-distributed stochastic neighbour embedding (t-SNE). To assign the peaks detected to molecular identities, peaks picked from mean spectra were matched to the human metabolome database (HMDB) using custom Matlab scripts. Possible adducts were queried against the database masses, and filtered based on comparison of expected and observed mass, and correlation between the images of the monoisotopic mass and 13C isotope image. Preliminary data show the successful preparation and analysis of multiple GEMM and human tissue samples across several sites by MALDI-MSI, DESI-MSI and SIMS imaging. Converting data to imzML and combining into large, single multi-file images enabled multivariate analysis (MVA) to be performed simultaneously within the same comparison. Of these MVA methods, mass spectrometry imaging (MSI) in combination with t-SNE has been shown to differentiate tumour subpopulations linked with patient outcomes. Current t-SNE methods suffer from three main limitations as compared to other MVA methods; poor computational scaling to large datasets, inability to add new data into the embedding, and inability to return to spectral contributions. By combining t-SNE performed on a subset of data and learning the embedding via deep learning, almost limitless size datasets can be analysed by t-SNE, new data can be incorporated into the embedding, and the spectral contributions towards the three dimensional space can be determined.

Database matching on the data from these GEMM samples to the HMDB reveals over 1000 possible molecular assignments, primarily structural lipids, within 10 ppm mass accuracy and with isotope image correlations above 0.6. From these methods, comparisons can be made between the unique molecules detected in different tissues, as well as the relative changes in intensity of those detected between different tissues.

#5662

N-linked glycosylation site mapping in prostate cancer and matched normal tissue: Defining glycan microheterogeneity.

Sarah Michelle Totten,1 Cheylene Tanimoto,1 Abel Bermudez,1 Amy Hembree,1 James D. Brooks,2 Sharon J. Pitteri1. 1 _Stanford Univ. School of Medicine, Palo Alto, CA;_ 2 _Stanford Univ. School of Medicine, Stanford, CA_.

The primary goal of this study is to map specific glycan structures occupying glycosylation sites on proteins from prostate cancer tissue in men treated with radical prostatectomies, and to compare detailed glycosylation patterns in malignant tissue to that of matched normal tissue. Glycosylation is a common, yet complex, post-translational modification known to play an integral role in a number of biologic processes. It has become well recognized that glycosylation is aberrantly expressed in a variety of cancers, including prostate cancer, making glycosylation an attractive source for potential biomarkers and therapeutics targets. Although altered expression of glycosylation in prostate cancer is well reported in literature, detailed characterization of the microheterogeneity of glycan structures occupying specific sites on glycoproteins derived from cancer tissue is largely unknown. Defining glycosylation in a site-specific manner is key to understanding glycosylation's involvement in cellular functions that promote tumorigenesis, invasion, and metastasis. Elucidating alterations among specific glycoforms may reveal novel biomarkers with increased specificity and clinical utility for prostate cancer diagnosis. Using state-of-the-art mass spectrometry, this study represents the most comprehensive, site-specific glycosylation analysis of primary prostate tissues comparing malignant and normal states, and builds on our previously described global glycoproteomic analysis. In this study, intact glycopeptide analysis was performed on prostate cancer and matched normal prostate tissue from eleven men with high-grade prostate cancer post prostatectomy. Proteins were extracted from tissue lysates, reduced, alkylated, and digested with trypsin. The resulting digest was enriched for glycopeptides using strong anion exchange in electrostatic repulsion hydrophilic interaction liquid chromatography mode. Using this technique, releasing the glycan is not needed for elution and glycopeptides remain intact for site-specific analysis. Enriched fractions were analyzed by LC-MS/MS, using higher-energy collision-induced dissociation for glycan and peptide backbone fragmentation. Protein Metrics Byonic software was used to make protein and glycopeptide identifications, which were subsequently confirmed by manual expertise. Preliminary results reveal an average of 125 glycopeptides identifications per patient across 70 unique glycoproteins. Glycopeptide identifications provide peptide sequence, glycosylation site localization, and glycan composition. Among annotated proteins with mapped glycosylation sites were prostatic acid phosphatase, nidogen, zinc-alpha-2-glycoprotein, integrin alpha-1, hemopexin, and hypoxia upregulated protein 1. Commonly identified glycan structures include multiply sialylated and fucosylated species.

#5663

Proteomic analysis of exosomes isolated from ccRCC cell lines.

Kosuke Mizutani,1 Kyojiro Kawakami,2 Yasunori Fujita,2 Kengo Horie,1 Masafumi Ito,2 Takashi Deguchi1. 1 _Gifu Univ. Graduate School of Medicine, Gifu, Japan;_ 2 _Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan_.

Purposes of the study: Clear renal cell carcinoma (ccRCC) is one of the most lethal malignant tumors. Molecular targeted therapies have been used to improve outcomes for metastatic or advanced ccRCC. However, currently approved therapies have limited success and a cure remains elusive. Since ccRCC could be curable if diagnosed at the early stage, novel diagnostic approach is needed to identify circulating markers that may help diagnose early and evaluate prognosis. Exosomes are membrane vesicles with a diameter of 40-150 nm that are secreted by various types of cells including cancer cells. Exosomes contain a variety of molecular constituents of their origin such as proteins and nucleic acids. Over the years, exosome has been suggested to be a novel diagnostic marker. The purpose of this study is to find candidates of ccRCC markers in exosomes. Experimental procedures: Exosomes were collected from cell culture supernatant of RPTEC human renal proximal tubule epithelial cells and ccRCC cell lines (Caki-1, KMRC-1, and OS-RC-2) by ultracentrifugation. Isolation of exosome was confirmed by western blot analysis for CD9. Exosome fraction was analyzed by iTRAQ-based quantitative proteomics. Results: A total of 52 proteins were detected and 3 proteins were found to be upregulated by more than 1.2 fold in exosomes isolated from ccRCC cells compared with those from RPTEC cells. Twelve proteins were found to be downregulated by less than 0.8 fold in exosomes from ccRCC cells compared with those from RPTEC cells. The expression profiles of several proteins were verified by western blot analysis. Conclusions: While major challenges still remain, exosome studies in ccRCC could provide a wealth of information to find novel diagnostic and prognostic markers.

#5664

Chemical proteomic platforms to probe metabolic heterogeneity in the tumor microenvironment.

Raymond E. Moellering. _University of Chicago, Chicago, IL_.

Proteomic platforms provide quantitative snapshots of the proteins present in a biological sample. Despite widespread use, these methods typically require homogenization of samples, signal-averaging over thousands-to-millions of cells and provide no information on protein function. This talk will focus on several novel technologies to circumvent these limitations and to interrogate protein activity within the complex architecture of biological samples, and on spatial scales ranging from single cells to whole organisms. At the single cell level we have developed a chemical proteomic technology, activity-dependent proximity ligation, which enables multiplexed quantification of enzyme activity with high spatial resolution in cellular co-culture and in patient tissue samples. Application of this platform has enabled detection of several metastasis-associated biomarker activities in primary patient samples, as well as simultaneous detection of activity and mRNA expression profiles. These data provide insight into the progression lineage of metastatic ovarian cancers. On the other end of the spectrum, we have recently developed a first-in-class activity-dependent PET imaging probe that specifically and irreversibly labels one of these enzyme biomarkers in vivo. This provides an opportunity to detect aggressive tumor populations, both at sites of primary occurrence and metastasis, with this novel PET radiotracer. Together, these approaches offer new paradigms for studying protein function in basic and translational biology and medicine, ideally providing new tools and biological hypotheses to understand and target tumor malignancy.

## IMMUNOLOGY:

### Emerging Tools and Models in Immuno-oncology Research

#5665

**NOD-** scid IL2rgnull **(NSG) mice deficient in murine MHC Class I and Class II expression support engraftment of functional human T cells in the absence of acute xenogeneic GVHD following injection of PBMC.**

Michael A. Brehm,1 Michael Wiles,2 Laurie Kenney,1 Ben Low,2 Roland M. Tisch,3 Lisa Burzenski,2 James G. Keck,4 Dale L. Greiner,1 Leonard D. Shultz2. 1 _University of Massachusetts Medical School, Worcester, MA;_ 2 _The Jackson Laboratory, Bar Harbor, ME;_ 3 _University of North Carolina at Chapel Hill, School of Medical, Chapel Hill, NC;_ 4 _The Jackson Laboratory, Sacramento, CA_.

The study of human immune system-tumor interactions has been limited historically by a paucity of models that fully reproduce human immune system function. Humanized mice are emerging as an exciting platform to study human immuno-oncology and provide preclinical tools to test cutting edge immunotherapies. An effective and simplistic approach to engraft immunodeficient mice with functional human T cells is the injection of human peripheral blood mononuclear cells (PBMC). This PBMC engraftment model (termed Hu-PBL-SCID mice) has supported studies of human infectious agents, allograft rejection, and human T cell immune function. The Hu-PBL-SCID model has also supported studies of human immune system-tumor interactions following injection of human PBMC into immunodeficient mice implanted with patient-derived (PDX) tumors. However, a major limitation of this model is the development of acute xenogeneic graft-versus-host disease (GVHD) due to human T cell recognition of murine MHC class I and class II. To address this limitation, we created two strains of NSG mice that lack murine MHC antigens, one by crossing NSG-B2Mnull with NSG-(IA IEnull) mice, the second by knocking out the MHC class II IAb gene in NSG-(Kb Db)null mice using TALEN technology. We observed that human IgG clearance was rapid in NSG-B2Mnull (IA IEnull) mice that lack functional FcRn activity whereas clearance in NSG-(Kb Db)null (IAnull) mice was comparable to that observed in NSG mice. Injection of human PBMC into both strains led to engraftment of human T cells that exhibited an approximate 3:1 CD4:CD8 ratio with long term engraftment and without development of acute GVHD. The engrafted human T cells were functional as documented by their ability to reject human islet allografts. Administration of human recombinant IL2 using AAV-IL2 to NSG-(Kb Db)null (IAnull) mice resulted in increased human CD45 cell engraftment, including heightened levels of human Tregs. However, high IL2 levels led to the development of acute GVHD. Moreover, NSG-(Kb Db)null (IAnull) mice injected with a PDX tumor and PBMC from an allogeneic donor supported significant tumor growth in the absence of acute GVHD. These data document that NSG mice deficient in murine MHC class I and class II can support studies of human immune system-tumor interactions in the absence of acute GVHD and provide a model for evaluation of human antibody therapeutics.

#5666

Octaplex spectral imaging of non-small cell lung cancer (NSCLC): In-depth insights into lung cancer immune micro-environment.

Houssein Abdul Sater,1 Scott Allen,2 Chetan Pundkar,2 Kimya Jones,2 Nikhil Patel,2 Ravi Kolhe,2 Roni Bollag,2 Carsten Schroder2. 1 _National Institutes of Health/Leidos, Bethesda, MD;_ 2 _Augusta University, Augusta, GA_.

Introduction: Lung cancer continues to be the lead cause of death among all cancer types. Checkpoint Inhibitors (CPI) showed great promise in curing or prolonging survival in various cancer types including advanced NSCLC, and others (HNSCC, RCC, Melanoma). The key limiting steps to such success is to find the right target population, minimize toxicities and better understand resistance mechanisms. Despite recent discoveries (1), there continues to be a huge need for development of better predictive biomarkers. Combining immunology and pathology knowledge will help guide the field of immune-oncology. Our group is interested in more in-depth anatomic dissection of immune TME (iTME).

Methods: To better understand the iTME in NSCLC and get deeper insight into the interaction of immune infiltrate with tumor cells, we successfully performed 8-color multispectral imaging (MSI) on 7 available cases of early stage NSCLC at Georgia Cancer Center. All 7 cases were successfully stained for CD4, CD8, FOXP3, PDL1, Ki67, CD68, CK, and DAPI. The regions of interest were identified by a research pathologist and images were captured using the Vectra 3 system (Perkinelmer).

Results: Octaplex staining was successful in this pilot study. All cases had significant immune infiltrate consistent of T and B lymphocytes as well as Tregs and CD68+ macrophages. The distribution of most infiltrates took a network like picture highlighting the piecemeal approach of immune infiltrate to attack cancer cells. In many instances, the infiltrate traces the tumoral capillary network. None of the cases followed the patterns described previously (2), but ather a mixture of these morphologies. Areas highly expressing PD-L1 tended to be devoid of Tcells but highly expressed on the edges of highly infiltrated areas. Tumor cells cruise through iTME by forming small agglomerates of cells or elongated shaped individual cells. Surprisingly CD4:CD8 ratio was inversely related to tumor differentiation with low ratio in poorly differentiated, solid patterns. In addition, immune infiltrates seem to be locked within areas where PD-L1 is highly expressed by tumor or its vascular network.

Conclusion: This pilot study provides insights from guided pathology. Under sampling is a major problem in studies and might lead to false conclusions. New insights into immune resistance through "Egress Lock" is a potential resistance mechanism to immune therapies. Type of infiltrate and its behavior still need further in-depth examination.

References:

1. Patel, S. P., & Kurzrock, R. (2015). PD-L1 expression as a predictive biomarker in cancer immunotherapy. Molecular cancer therapeutics, 14(4), 847-856.

2. Fridman, W. H., Galon, J., Pagès, F., Tartour, E., Sautès-Fridman, C., & Kroemer, G. (2011). Prognostic and predictive impact of intra-and peritumoral immune infiltrates. Cancer research, 71(17), 5601-5605.

#5667

High-throughput analysis of MAPK and JAK-STAT signaling in CT26 tumors using a combination of immunophenotyping and phospho-flow cytometry.

David Draper, Alden Wong, Philip Lapinski, Sheri R. Barnes, Maryland Rosenfeld Franklin, Scott C. Wise. _MI Bioresearch, Ann Arbor, MI_.

The development of robust platforms that enable the analysis of cell signaling proteins is crucial for small molecule drug development. Analysis of phospho-proteins in solid tumors is one area that remains a challenge due to the complex nature of the tissue. The most common approaches used to measure the effects of in vivo therapy are immunoblot and ELISA-based assays. However, these bulk analysis methods have drawbacks. This is primarily due to their inability to distinguish between phospho-protein levels in different cell subsets that exist in the tumor. Using the CT26 syngeneic model for colorectal cancer, we describe a novel high-throughput phospho-flow platform that can simultaneously analyze the phosphorylation status of up to 6 signaling proteins in both tumor cells and immune cells. To first validate the phospho-flow assay, splenocytes from naïve mice were treated in vitro with various stimuli that preferentially target distinct signaling proteins. We then tested the platform in vivo. To this end, CT26 tumor-bearing mice were dosed with the MEK inhibitor trametinib or vehicle. At the appropriate time point post-dosing, tumors were harvested and dissociated into single cell suspensions. Live tumor cells and CD8+ T cells were first identified by flow cytometry using antibodies against CD45, CD3, and CD8. Following fixation, MAPK and JAK-STAT signaling activity was analyzed by measuring the levels of phosphorylated MEK, ERK1/2, STAT1, STAT2, STAT5, and STAT6 proteins using phospho-flow. Our results demonstrated that trametinib triggered a reduction in both pMEK and pERK1/2 levels in tumor cells that was evident 24 hours after the final trametinib dose. We also found phosphorylated STAT1, STAT3, and STAT5 was readily detectable in tumor cells, while STAT6 phosphorylation was not obvious. In contrast, we found that the CD8+ T cell levels of pMEK and pERK1/2 were not affected by trametinib. Furthermore, when JAK-STAT signaling was analyzed, only phosphorylation of STAT3 was detected in CD8+ T cells, which was also unaffected by trametinib. These results demonstrate successful combination of immunophenotyping and phospho-protein analysis in solid tumor-derived cells using flow cytometry. This platform is a tool that can be used to provide valuable insight into the effects that small molecule inhibitors may have on distinct cell subsets within a heterogenous tumor microenvironment.

#5668

The rational design and evaluation of a peptide inhibitor of the PD-1/PD-L1 interaction.

Rebecca J. Boohaker, Vijaya Sambandam, Mark Suto, Bo Xu. _Southern Research Institute, Birmingham, AL_.

Tumor cells exploit immune checkpoints by expressing checkpoint ligands such as PD-L1, effectively masking them from immune destruction. Programmed cell death- 1 (PD-1), plays a major role in tumor immune escape. The interaction of PD-1/PD-L1 inhibits cytotoxic T lymphocyte (CTL) proliferation, induces apoptosis of tumor-specific T cells, and promotes the resistance of tumor cells to CTL attack. Monoclonal antibodies to these proteins have been developed and approved for use in the clinic. Theoretically, peptide mimics, which have the benefit of lower half-life and significantly smaller size, are able to penetrate into solid tumors and tissues better than antibodies, and can function as inhibitors for the PD1/PD-L1 interaction. Here we report for the first time the rational design and validation of a peptide inhibitor to the PD-1/PD-L1 immune checkpoint as a viable alternative to current inhibitory antibodies. We demonstrated, by biolayer interferometry and in silico docking simulations, that a PD-L1 peptide mimetic (PL120131) can interfere with the PD-1/PD-L1 interaction by binding to PD-1. We established the binding constant for the interaction and PL120131 reduces association of PD-L1 in a dose dependent manner. We show that PL120131 is capable of inhibiting PD-1 mediated apoptotic signaling pathway and rescuing Jurkat cells and primary lymphocytes from apoptosis. Additionally, we show that PL120131 treatment allows for CTL anti-tumor activity. Furthermore, PL120131 can maintain co-culture survivability and activity of T Cells in a 3D co-culture model. Together, the characterization of this PD-1/PD-L1 inhibiting peptide provides insight regarding the ability to inhibit PD-L1 binding while maintaining CTL viability and activity. Understanding the mechanism(s) of action of

this peptide has yielded information on the pathways that are drivers of immune evasion in a tumor microenvironment, which may improve and synergize with other forms of immunotherapy, thus increasing efficacy of cancer treatment. Outcomes from this study have provided a path forward to further development of small molecule inhibitors to the PD-1/PD-L1 interaction, and outlines a strategy for the design and development of small molecules to additional cell-surface checkpoint proteins.

#5669

Establishment of a human CD3ε transgenic mouse model to assess anti-tumor efficacy of human T-cell-redirecting bispecific antibodies.

Mengmeng Yang,1 Mingkun Zhang,2 Sandra Verploegen,3 Patrick Engelberts,3 Yuxi Zhang,2 Lei Zheng,1 Keyi Zhu,1 Annie Xiaoyu An,1 Cunxiang Ju,2 Jing Zhao,2 Xiang Gao,2 Qian Shi,1 Davy Xuesong Ouyang1. 1 _Crown Bioscience, Inc., Taicang, Jiangsu, China;_ 2 _Nanjing Biomedical Research Institute, Nanjing University, Nanjing, Jiangsu, China;_ 3 _Genmab B.V., Utrecht, Netherlands_.

T-cell redirecting therapy has taken a prominent area in immuno-oncology. T cells driven by a tumor-specific antigen, traffic into the tumor and initiate tumor killing. However, this is often hampered by inhibitory factors in the tumor microenvironment. In recent years, researchers have been actively developing T-cell redirecting bispecific antibodies, which binds to a specific tumor associated antigen (TAA) on tumor cells and CD3 (usually epsilon chain, CD3E) on T cells. This physically links the tumor cell to the T-cell which leads to MHC-independent recognition and killing of cells carrying the TAA. Several CD3 recruiting bispecific antibodies that have been approved are now in clinical trials and demonstrate promising efficacy. However, research on new therapeutic T-cell redirecting antibodies is often hampered by a lack of proper in vivo models, due to the absence of cross-reactivity with mouse CD3ε. We have now built a human CD3E BAC transgenic model in BALB/c background to address this issue. These transgenic mice express both human- and mouse CD3ε in more than 80% of the T cells. Unlike previously developed transgenic lines, where early T lymphocyte and natural killer cell development were blocked in mice with high copy numbers of the human CD3ε gene, our model is phenotypically normal with levels of T , B , and NK cells comparable to those in wild-type BALB/c mice. In an ex vivo T-cell stimulation assay, spleen-derived T cells could be activated by either anti-human CD3 antibody (OKT3) or anti-mouse CD3 antibody (145-2C11), indicated by significantly elevated CD25+/CD69+ population, as well as IL-2 and IFNγ release. In an efficacy assay, we inoculated syngeneic mouse CD20-expressing A20 lymphoma cells into the transgenic mice and treated with mCD20xmCD3 or mCD20xhCD3 bispecific antibodies, containing a human- or mouse CD3ε-specific CD3 arm, respectively. We found complete depletion of peripheral B cells 48 hours after dosing of either bispecific antibody. Treatment also induced 33% and 39% tumor growth inhibition at day 10, following 3 doses of 1 mg/kg mCD20xhCD3 and mCD20xmCD3 bispecific antibody, respectively. Taken together, our human CD3ε transgenic mice offer a novel model to assess the preclinical in vivo efficacy of human CD3-T-cell redirecting therapeutics.

#5670

Systemic lymphoid architecture response assessment (SYLARAS): Application to system-wide immunophenotyping of glioblastoma.

Gregory J. Baker,1 Sucheendra K. Palaniappan,2 Jodene K. Moore,1 Stephanie H. Davis,1 Peter K. Sorger1. 1 _Harvard Medical School, Boston, MA;_ 2 _Systems Biology Institute, Tokyo, Japan_.

Interactive flow cytometry analysis software allows users to make binary decisions on immunomarker status of single cells through arbitrary placement of one- and two- dimensional gates. However, manual gating through a point-and-click graphical user interface (GUI) is impractical for large datasets. Here we describe SYLARAS, a suite of experimental and computational tools for the systematic and chronological evaluation of lymphoid tissue architecture. SYLARAS takes multiplex single-cell data as input, bins cells in n-dimensional orthant space, and returns schematized summary statistics on immunophenotypes of interest. We programmatically curated data on 33 immunophenotypes constituting primary and secondary lymphoid tissue of immunocompetent mice bearing syngeneic, orthotopic glioblastoma (GBM) at 3 stages of tumor growth. SYLARAS permitted the discovery of tumor-induced immune signatures whose divergence from mock engrafted, age-matched controls was quantified and statistically analyzed. By integrating information on peripheral immune composition with the levels of 111 blood cytokines of the same mice, we uncovered novel cell types, cytokines, and correlation networks perturbed by the mouse GL261 glioma model. SYLARAS facilitates the discovery of immune regulatory mechanisms and their response to therapy and is broadly applicable to the fields of immuno-oncology, autoimmunity, and infectious disease.

#5671

Humanized mouse models for evaluation of cancer therapies.

Jean-Francois Mirjolet, Josselin Caradec, Olivier Duchamp, Francis Bichat, Caroline Mignard. _Oncodesign S.A., Dijon Cedex, France_.

Mice with a humanized immune system, so called "humanized" mouse models, can be used to study the complex interactions between the human immune system and tumor cells. In order to assess compounds efficiency in immune-oncology, the in vivo model should recapitulate the biological characteristics of the human tumor and the related immune microenvironment. The choice of mouse strain is the first critical factor as genetically engineered mouse strains differentially express cytokines and growth factors. Then the choice of human antigen/target to be assessed drives the choice of the tumor models and the immune population to be transferred into mice. We developed on different immunodeficient mouse strains multiple humanization strategies using either human PBMCs, Hematopoietic Stem Cells (HSCs), or specific human immune cells such as Dendritic Cells (DCs), T cells, subset of T cells (e.g. gamma9 delta2 T cells) and NK cells. We also developed mouse humanization models using combinations of immune subpopulations such as co-transfer of autologous T cells and DCs.Humanized models were then used for in vivo proof of concept studies with mice xenografted with cell lines (human disseminated lymphoma, orthotopic/subcutaneous solid tumors) or patient-derived xenografts. Tumor-bearing humanized mice were treated with cancer therapeutics such as bispecific antibodies, ADCC-inducing antibodies, Treg targeting antibodies, TLR agonists, vaccines and adoptive T cells transfer therapeutic antibodies. Therapeutics efficiency was assessed by following up mice survival and tumor growth. The impact of therapeutics on tumors and immune cells was also assessed by flow cytometry and immunohistochemistry analyses. In case of disseminated lymphoma and ovarian human tumor in mice reconstituted with human PBMCs, significant antitumor activity of bispecific antibodies were evidenced by an increase in survival, decrease in specific biomarkers (CA125 for ovary) and decrease of residual disease.

#5672

Facile generation of single-cell transcriptome and immune repertoire freshly isolated from clinical tumor specimens.

Junjie Zhu,1 Ameen A. Salahudeen,1 Valeria Giangarra,2 Luz Montesclaros,2 Jerald Sapida,2 Osman Sharifi,2 Josephine Lee,2 Grace X. Zheng,2 Dhananjay Wagh,3 John Coller,3 Chiara Sabatti,1 Calvin J. Kuo1. 1 _Stanford University, Stanford, CA;_ 2 _10x Genomics Inc, Pleasanton, CA;_ 3 _Stanford Functional Genomics Facility, Stanford, CA_.

Immunotherapies including cell-based therapies generate deep and durable responses in patients with chemotherapy-refractory cancers. However, in solid tumors, particularly those with stereotypic driver mutations and resultant neoantigens, the clonality and cell states of tumor-infiltrating lymphocytes (TILs) remain poorly understood. Here, we use a droplet-based 5' single-cell RNA-sequencing (scRNA-seq) to simultaneously profile transcriptome and immune repertoire of the same cells, enabling the phenotypic characterization (including cytotoxic and helper T, B, T regulatory and exhausted T cells) of each clonotype. We performed scRNA-seq on unsorted and CD45+-FACS sorted cells from fresh clinical samples of multiple tissue types, including lung, liver and kidney, and obtained on average tens of thousands of cells per sample. We demonstrated that high-quality single-cell suspension can be rapidly and reliably generated from clinical samples. We devised different diversity metrics to appropriately classify the immune repertoire within samples, and cross samples of different tissues. We observed distinct cell type compositions and, more importantly, context-specific T-cell clonal expansion patterns, suggesting the activation of different molecular programs in these tumors. In addition, comparison of non-small cell lung cancer (NSCLC) tumors varying by histology type and patient smoking status showed differences among these phenotypes, suggesting a potential link between immune microenvironments and cancer etiology. In summary, we provide a proof of concept for rapid generation of large number of single-cell transcriptomes of TILs paired with their corresponding TCR cDNA sequence in fresh tumor samples across different tissue types. Further analysis with this methodology on larger clinical cohorts will provide robust correlative prognostic markers of clinical phenotypes of immunotherapy responses. Our analysis strategy of TCR sequences among large clinical cohorts across multiple tumor types will facilitate cell-based therapeutic efforts, including CAR T cell or autologous T cell therapies.

#5673

Complex, patient-derived, multi-cell type, 3D models of breast cancer for personalized prediction of therapeutic response.

Qi Guo,1 Melissa Millard,1 Christine Wilhelm,1 Ashley Elrod,1 Nick Erdman,1 Lacey E. Dobrolecki,2 Brian McKinley,3 Mary Rippon,3 Wendy Cornett,3 John Rinkliff,3 Amanda Scopteuolo,3 Linda Gray,3 James Epling,3 Barbara Garner,3 Jeff Hanna,3 Eric McGill,3 C. David Williams,3 David Schammel,4 David L. Kaplan,5 Christopher Corless,6 Jeff Edenfield,3 Michael T. Lewis,2 Howland E. Crosswell,1 Teresa M. DesRochers1. 1 _KIYATEC, Inc., Greenville, SC;_ 2 _Baylor College of Medicine, Houston, TX;_ 3 _Greenville Health System, Greenville, SC;_ 4 _Pathology Associates, Greenville, SC;_ 5 _Tufts University, Medford, MA;_ 6 _Knight Diagnostics Laboratories, Portland, OR_.

Breast cancer survival has drastically improved over the past decades; however, drug resistance and subsequent disease progression is responsible for the incurability of advanced disease. While the focus of many drug response studies is the transformed tumor cells, there is increasing evidence suggesting a role for stromal cells in tumorigenesis and drug resistance. Microenvironmental components, including extracellular matrix, fibroblasts, leukocytes, and adipocytes, all contribute to physiological mammary gland biogenesis. Accordingly, these stromal elements contribute to disease progression and resistance. However, many in vitro drug response studies still utilize 2D monolayer cultures with purified breast tumor cells. In vivo studies remain the gold standard for drug development, even though they are performed with immune-compromised mice that may not reflect the physiological tumor microenvironment and have been repeatedly shown to be a poor representation of clinical outcomes. Thus, there is a need for more complex in vitro models to test drug response effectively and accurately. We have previously demonstrated the benefits of using a patient-derived, tri-culture (3x), 3D perfusion microtumor (3DpMT) system. To further replicate the complex tumor microenvironment, we have expanded to a penta-culture (5x) model by incorporating macrophages and lymphocytes alongside the tumor cells, fibroblasts, and adipocytes of the 3x model. We have accrued over 207 primary tumor samples, including both resected tumor and core biopsies, from which we have generated 12 stable PDX models (~50% ER+) and >20 3x, 4x, and 5x 3DpMT with a focus on triple negative (TNBC). The 5x patient-derived 3DpMT tissues represent our most complex breast cancer in vitro model and have been cultured successfully for up to 5 weeks allowing for high-throughput, long term drug response testing with different dosing strategies. They have been characterized by flow cytometry, IHC, RNA expression, NGS, DNA methylation patterns, and cytokine/chemokine secretion. When possible, marker expression has been compared to the primary tumor. Furthermore, the accuracy of our models to replicate clinical tissue is evident in the similar toxicities of chemotherapies observed in clinical use. With these models we can replicate physiological processes including cell migration, polarization of macrophages, activation of lymphocytes, and changes in molecular profiles throughout the duration of our 5x culture assays. Our model has the potential to test a myriad of drugs, from conventional chemotherapies to novel immunotherapies over extended time periods with different dosing strategies in order to provide a more accurate prediction of patient-specific clinical response.

#5674

**Transgenic expression of human IL15 in NOD-** scid IL2rgnull **(NSG) mice enhances the development and survival of functional human NK cells.**

Ken-Edwin Aryee,1 Lisa Burzenski,2 Dale L. Greiner,1 Raymond M. Welsh,1 Leonard D. Shultz,2 James G. Keck,3 Michael A. Brehm1. 1 _University of Massachusetts Medical School, Worcester, MA;_ 2 _The Jackson Laboratory, Bar Harbor, ME;_ 3 _The Jackson Laboratory, Sacramento, CA_.

The human innate immune system plays critical roles in tumor surveillance and in immunoregulation within the tumor microenvironment. Natural killer (NK) cells are innate lymphoid cells that mediate tumor cell killing by antibody-dependent cell mediated cytotoxicity (ADCC), through direct recognition, and by the expression of chimeric antigen receptors that directly target tumors. However, NK cell subsets with regulatory functionality also contribute to the tumor immune suppressive environment that enables tumor growth. The balance between effector and regulatory NK cell subsets has been studied extensively in murine models of cancer, but there is a paucity of models to study human NK cell function in tumorigenesis, which is restricted primarily to in vitro experiments. Humanized mice are a powerful alternative to syngeneic mouse tumor models for the study of human immuno-oncology and have proven effective tools to test immunotherapies targeting T cells. However human NK cell development and survival in humanized mice are severely limited. Previous studies have demonstrated that injection or transient expression of human IL15 enables efficient development of functional human NK cells within immunodeficient mice that were engrafted with CD34+ HSC. Based on these results we established NSG mice that constitutively expresses human IL15. The NSG-Tg(Hu-IL15) mice were generated using a BAC containing the human IL15 gene, and express a physiological level of human IL15 (7.1 ± 0.3 pg/ml). To evaluate human NK cell development, 8 to 12-week-old NSG and NSG-Tg(Hu-IL15) mice received 200 cGy irradiation and were then injected intravenously with 1x105 CD34+ HSC derived from umbilical cord blood. No difference in overall survival of HSC-engrafted NSG-Tg(Hu-IL15) mice compared to NSG mice were observed during the experiment, indicating that expression of IL15 did not increase mortality. Levels of circulating human CD45+ cells, T cells and B cells were similar between the HSC-engrafted NSG-Tg(Hu-IL15) and NSG mice. Significantly higher levels of human CD56+ NK cells were found in NSG-Tg(Hu-IL15) mice as compared to NSG mice at all time points tested in the peripheral blood and within the spleen and bone marrow. A higher proportion of human CD56+ NK cells recovered from the blood and spleen of NSG-Tg(Hu-IL15) mice expressed granzyme A, granzyme B and perforin as compared to NK cells from NSG mice, suggesting that the NK cells were functional. Moreover, human NK cells enriched from the NSG-Tg(Hu-IL15) mice lysed K562 cells in an in vitro cytotoxicity assay. These data demonstrate that HSC-engrafted NSG mice expressing human IL15 support enhanced development of functional human NK cells and suggest that HSC-engrafted NSG-Tg(Hu-IL15) mice are a powerful model to study the role of NK cells in tumor-immune system interactions and to test immunotherapies targeting NK cells.

#5675

Single-cell mass cytometry of classical Hodgkin lymphoma defines an exhausted and immunosuppressive microenvironment.

Fathima Z. Cader,1 Ron C. Schackmann,2 Xihao Hu,1 Kirsty Wienand,1 Robert A. Redd,1 Bjoern Chapuy,1 Jing Ouyang,1 Nicole E. Paul,1 Evisa Gjini,3 Mikel Lipschitz,3 Laura M. Selfors,2 Philippe Armand,1 David Wu,4 Jonathan R. Fromm,4 Donna Neuberg,1 Xiaole S. Liu,1 Scott J. Rodig,3 Margaret A. Shipp1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Harvard Medical School, Boston, MA;_ 3 _Brigham and Women's Hospital, Boston, MA;_ 4 _University of Washington, Seattle, WA_.

Background: In classical Hodgkin lymphoma (cHL), the rare malignant Hodgkin Reed-Sternberg (HRS) cells are surrounded by an inflammatory infiltrate. Yet, the host anti-tumor immune response is ineffective. HRS cells have multifaceted mechanisms to evade the immune system including 9p24.1/PD-L1/PD-L2 genetic alterations leading to overexpression of PD-1 ligands and subsequent T cell exhaustion, aberrant antigen presentation and modulation of the tumor microenvironment (TME).

The clinical success of PD-1 blockade in cHL suggests the TME contains reversibly exhausted T-effectors (Teff). Paradoxically, durable responses are observed in patients with β2M/MHC class I loss on HRS cells, raising the possibility of non-CD8+ mediated mechanisms of efficacy of PD-1 blockade. For this reason, we sought to characterize HRS cells and the surrounding TME.

Methods: Using CyTOF technology, we evaluated 7 primary cHL suspensions and 10 reactive lymphoid tissue (RLT) samples at the single-cell protein level. We designed a custom panel of 39 isotope-conjugated antibodies. A combination of surface and intracellular markers distinguish T cell subsets according to lineage, differentiation, polarization, activation and exhaustion. Additional markers were incorporated to identify B cells, NK cells and macrophages. HRS cells were defined by CD15/CD30/Pax5 positivity. Inclusion of β2M and MHC class I allowed assessment of antigen presentation on HRS cells.

The data was acquired on a Helios CyTOF and analyzed using a fast k-weighted nearest neighbor algorithm, X-shift. X-shift clustered cells with phenotypic similarities together. Then, samples were separated into cHL and RLT and the contribution of a sample to a given cluster was quantified.

Results: Comparison of viable cell suspensions from RLT and cHL revealed loss of naïve T-cells and skewing towards differentiation of Teff in both CD4+ and CD8+ subsets in cHLs. This prompted a second X-shift analysis focused on CD3+ cells, which highlighted salient differences between cHL and RLT within the CD4+ subset. In cHL, we found expansion of Teff and regulatory T cells (Treg) with a reduction of follicular helper T cells. Furthermore, both Treg and Teff populations were largely Th1 (T-bet+/CCR5+) polarized. Evaluation of PD-1 expression showed Tregs had little/no PD-1 while Teff had intermediate/high expression. Hence, Tregs retain functionality in contrast to Teff, which are exhausted, providing two mechanisms of immunosuppression.

Manual gating identified HRS cells with a characteristic phenotype: CD15, CD30, Pax5, rosetted by CD4+ T cells. Importantly, we found loss or decrease of β2M and MHC class I in 5/7 cases.

Conclusions: The TME in cHL is CD4+ T cell rich with frequent loss of MHC class I on HRS cells. Differential PD-1 expression results in functional CD4+ Tregs and exhausted Teff, a synergistic bases for the observed immunosuppression in cHL.

#5676

Patient-derived tumor xenografts in humanized NSG-SGM3 mice: An improved immuno-oncology platform.

Li-Chin Yao,1 Mingshan Cheng,1 Ken-Edwin Aryee,2 Pooja Kumar,3 Nicole Walsh,2 Dale Greiner,2 Leonard Shultz,4 Edison T. Liu,4 Michael Brehm,2 James G. Keck1. 1 _The Jackson Laboratory, Sacramento, CA;_ 2 _University of Massachusetts Medical School, Worcester, MA;_ 3 _The Jackson Laboratory, Farmington, CT;_ 4 _The Jackson Laboratory, Bar Harbor, ME_.

The JAX® Onco-Hu® platform utilizes humanized mice engrafted with tumors to enable in vivo investigation of the interactions between the human immune system and human cancer. We have recently shown that humanized NOD-scid IL2Rγnull (NSG™) mice bearing patient-derived xenografts (PDX) allow efficacy studies of checkpoint inhibitors. A major avenue of our investigation is to generate murine humanized models containing a more complete human hematopoietic system and robust innate immune cell population. Next-generation NSG strains include triple transgenic NSG mice (NSG-SGM3) expressing myelosupportive human cytokines KITLG, CSF2, and IL-3. When engrafted with CD34\+ human hematopoietic progenitor cells (HPCs) from CD3-depleted umbilical cord blood, NSG-SGM3 mice produce higher myeloid and Treg populations in the circulation as compared to NSG mice over 18 weeks post engraftment. We implanted an array of PDX tumors into humanized NSG-SGM3 mice at 2-3 months post engraftment. Tumors were dissociated and single-cell infiltrates were analyzed by multicolor flow cytometry with a focus on examining overall immune cell infiltration and the levels of hCD33+ myeloid cells. In the PS4050 melanoma PDX model, we found that hCD45+ cell infiltration was significantly increased in hu-NSG-SGM3 mice as compared to hu-NSG mice engrafted with the same HPC donor (3.7% vs. 1% of viable cells). The majority of tumor-infiltrating cells in hu-NSG-SGM3 mice expressed hCD33 (55% of hCD45+) and the percentage was significantly higher than that in hu-NSG mice (13%). hCD3+T cell infiltration level was similar between these two strains (~20% of hCD45+). PS4050-bearing hu-NSG-SGM3 mice treated with the anti-PD-1 antibody pembrolizumab (Keytruda) showed a significant reduction in tumor growth and the PD-1 levels in tumor-infiltrating T cells were greatly reduced by flow cytometry analysis. The overall hCD45+ cell infiltration and the frequencies of hCD4+T, hCD8+T, and hCD33+myeloid cells in tumors remained similar after treatment. Lastly, we observed that the effect of Keytruda on tumor growth reduction in hu-NSG-SGM3 mice is PD-L1-dependent using the human lung carcinoma cell line NCI-H460 depleted of PD-L1 expression by CRISPR. Keytruda treatment significantly reduced mock-transfected NCI-H460 cell growth. By comparison, PD-L1 KO NCI-H460 cells grew more slowly than the mock cells and lost the response to Keytruda. Together, these results indicate that PDX tumor-implanted hu-NSG-SGM3 mice serve as an important platform for understanding human immune system and tumor microenvironment interactions and for preclinical immuno-oncology efficacy studies.

#5677

Establishment and application of a panel of PBMC-humanized mouse tumor models in immune-oncology and targeted cancer immunotherapy.

Lan Zhang,1 Haochen Wu,1 Fei Chen,1 Lianqi Zhao,1 Xiaoyu An,1 Weibin Tan,2 Xiaoyan Fu,2 Meng Qiao,1 Qian Shi,1 Wenqing Yang1. 1 _CrownBio, Taicang, Jiangsu Province, China;_ 2 _Taicang Blood Center, China_.

Background: Monoclonal antibodies and checkpoint blocking approaches have achieved remarkable success in cancer immunotherapy in clinical practices. Besides the success of anti-PD-1 and anti-PD-L1 antibodies (such as Keytrude and Tecentriq), two bispecific antibodies, Catumaxomab and Blinatumomab have been approved to treat cancer patients, and many more bispecific antibodies are currently in pre-clinical or clinical development. To meet the increasing market needs for fast, reliable and cost effective mouse tumor model systems, we developed a panel of humanized tumor models, designated as MiXenoTM platform. MiXenoTM could be used in a broad spectrum of applications in immuno-oncology drug discovery, including targeted cancer immune-therapy.

Method: CrownBio has a collection of about 200 xenograft models, including subcutaneous, orthotropic and systemic models, which covers majority of cancer type. Gene expression and mutation status are often profiled in these models. To engage both host immune system and tumor antigens, we have developed some specific MixenoTM tumor models by inoculating tumor cells over-expressing specific anti-tumor antigens (e.g. EGFR, CD47, Braf or PD-L1) into PBMC-humanized immunocompromised mice. Reconstitution of human immune component with human PBMCs in these tumor-bearing mice provides a useful tool to evaluate the targeted immune-therapeutics including bispecific T cell engagers.

Results: To validate the MiXenoTM models for targeted cancer immunotherapy, several xenograft models have been profiled and selected based on their tumor antigens or gene expression. Models with over-expression of a variety of tumor antigens (e.g. EGFR, CD47, Braf, PD-L1, etc.) were used to develop specific MixenoTM tumor models. The immune and tumor cells were engrafted either simultaneously or sequentially. Graft versus host disease (GvHD) can be managed by optimizing the immune cell constitution and tumor cell engraftment. Some models were also validated using stand of care I/O drugs and characterized by immune-phenotyping.

Conclusions: MiXenoTM tumor models with specific tumor antigen targeting strategy are valid model systems to evaluate the human immuno-modulatory drugs including bispecific antibodies. Further studies are needed to expand model collections and to extend their applications in I/O space.

#5678

Developing a preclinical immunotherapy platform using syngeneic mouse models of human melanoma.

Corinne Rauck,1 Eva Perez-Guijarro,1 Zoe W. Ohler,2 Rajaa El E. Meskini,2 Howard Yang,1 Suman Vodnala,1 Cari Graff-Cherry,2 Sung Chin,2 Anyen Fon,1 Helen Michael,1 Maxwell Lee,1 Terry Van Dyke,1 Shyam Sharan,1 Glenn Merlino,1 Chi-Ping Day1. 1 _National Cancer Institute, NIH, Bethesda, MD;_ 2 _Frederick National Laboratory for Cancer Research, Frederick, MD_.

Melanoma is an aggressive and lethal disease with no efficacious therapies for a broad subset of late stage patients. Current immunotherapies, including immune checkpoint blockade (ICB), may prolong survival in certain patients, but generate responses in less than 40% of the treated cohort. This demands a better understanding of molecular mechanisms underlying the lack of response and acquired resistance to ICB. However, functional studies are limited in patients, and current preclinical studies are handicapped by the absence of appropriate mouse models that recapitulate the pathological and immunological diversity of human melanomas. Here we develop four syngeneic melanoma mouse models with human-relevant genetic modifications and carcinogenic agents, which we hypothesize will mirror the spectrum of responses to ICB and offer a platform for future mechanistic studies in melanoma. The models are: 1) neonatal ultraviolet radiation (UV)-induced melanoma in a HGF-transgenic mouse, in which melanocyte localization at the epidermal-dermal junction mimics human distribution (HU); 2) 7,12-Dimethylbenz(a)anthracene (DMBA)-induced melanoma in a HGF-tg and Cdk4R24C mouse (HC4D); 3) UV-induced melanoma in a BrafCA/+; HGF-tg; Cdkn2aflox/+; Tyr-CreERT2-t mouse (BHCU); and 4) UV-induced melanoma in a BrafCA/+; Ptenflox/+; Cdkn2aflox/+; Tyr-CreERT2-tg mouse (BPCU). Exome sequencing of the four models reveals a high correlation with mutational subtypes previously described in human melanoma. BPCU and BHCU represent different Braf mutant patient populations and HU and HC4D represent triple wildtype melanoma (non-BRAF, -NRAS, -NF1). The four mouse models demonstrate distinct responses to ICB with anti-CTLA-4 treatment. While HU and HC4D melanomas show high or partial sensitivity to anti-CTLA-4, respectively, BPCU and BHCU do not respond to treatment. In vivo vaccination assays demonstrate that the anti-CTLA-4 response in our models is linked to increased tumor immunogenicity. However, the number of non-synonymous mutations and antigen presentation functionality do not correlate with ICB efficacy. Tumor infiltration by T cells was assessed by CD3 and FoxP3 immunostaining and gene expression analysis. Although clear differential gene expression profiles are noted among the four models and in those tumors responding to the treatment, we unexpectedly found that "hot" melanomas (e.g., showing upregulation of inflammatory pathways and high T-cell infiltration) do not necessarily predict ICB efficacy. These results suggest that additional mechanisms could help determine the response or intrinsic resistance to anti-CTLA-4 and open new avenues for future research and treatment. Overall, our study offers four genetically and phenotypically distinct mouse models representing diverse human melanoma subtypes as powerful tools for the mechanistic study of the response to immunotherapies in melanoma.

#5679

Predicting prognosis in papillary thyroid carcinoma: Clues in the tumor microenvironment.

Donna C. Ferguson, William D. Dupont, Thomas Stricker, Vivian L. Weiss. _Vanderbilt Univ. Medical Ctr., Nashville, TN_.

The incidence of thyroid carcinoma is dramatically increasing in the U.S. Papillary thyroid carcinoma (PTC) represents 85% of thyroid cancer and has a ~15% recurrence rate and a 5-10% metastasis rate. Current molecular assays fail to predict the biologic behavior of these tumors. Here we perform a computational immunogenomic evaluation of 568 PTCs and correlate immune infiltrate with tumor behavior. Computational immunogenomic analysis was performed on RNA sequencing data from 568 PTCs in The Cancer Genome Atlas (TCGA) using the CIBERSORT deconvolution analytic tool for characterizing cellular composition (Newman et al. Nature Methods 2015). Immunologic cell fractions from tumors with global p values <0.05 were characterized as having "high confidence" and those with p values ≥0.05 were characterized as having "low confidence." The immune cell fractions were correlated with tumor pathologic T stage and lymph node (LN) stage. Immune cell fractions for activated dendritic cells (DCs) were categorized as low (<0.5%), medium (0.5-3.0%) and high (>3.0%); and for follicular helper T cells (TFH) were low (<1%), medium (1-5%), and high (>5%). A high CD8/T regulatory cell (CD8/Treg) ratio was defined as ≥4. For the logistic regression, pathologic tumor T stage was divided into low stage (T1 and T2) and high stage (T3 and T4), and LN stage was classified as no metastasis (N0) and LN metastasis (>N0). Among tumors with high CIBERSORT confidence, there is a significant negative correlation between high TFH fraction and advanced pathologic tumor stage (OR=0.12, 95%CI=0.02-0.62, p = 0.01) and a significant positive correlation between high activated DC fraction and advanced T stage (OR= 8.75, 95%CI= 1.7-45, p=0.009). The observed odds ratio for cases with a medium infiltrate of activated DCs is 2.31 (CI: 0.93-5.7, p=0.07). Finally, among tumors with high CIBERSORT confidence, a high CD8/Treg ratio is negatively associated with the presence of LN metastases (OR=0.39, 95%CI=0.19-0.81, p=0.01). Here we present a robust computational analysis of immune cell infiltrates and their correlation with tumor and lymph node stage. We demonstrate that increased DCs and decreased TFH cells correlate with increased local disease. We also show that decreased CD8/Treg ratios correlate with LN metastases. Additional studies are needed to confirm these computational findings in PTCs, evaluate for predictors of tumor recurrence, and expand this analysis to include other thyroid neoplasms. In conclusion, immunologic evaluation of thyroid tumors may provide more information on the biologic behavior of these lesions and inform both diagnosis and treatment.

#5680

Quantitative cellular and molecular imaging of the intact tumor microenvironment.

Jan Martinek, Hannah M. Brookes, Te-chia Wu, Lili Sun, Philipp Henrich, Kyung In Kim, Joshy George, Paul Robson, Jacques Banchereau, Karolina Palucka. _Jackson Laboratory, Farmington, CT_.

The causes underlying the extent and character of tumor-associated immune responses in cancer are not well defined and are likely multifactorial, including cancer cell heterogeneity, host genotype, and the immune status of individual patients. Tumors are organized tissues with numerous reciprocal local and systemic connections with immune cell populations of both the myeloid and lymphoid lineages. Understanding the cellular and molecular composition of tumors could help uncover the variation in immune response to cancer in patients and to delineate interpatient patterns that could help elucidate causes of disease progression. Multiparameter flow cytometry and single-cell transcriptomics technologies have provided an unprecedented level of cell typing and annotation of cells present in tumors. However, these methods require tissue dissociation into single-cell suspensions. This is routinely linked with cell loss and artifacts due to enzymatic treatment, and cell sorting en route to transcriptomic analysis as well as cell contamination. Furthermore, tissue dissociation and single-cell analysis do not enable the determination of cellular context in which the cells find themselves at the time of analysis. Given this, we have established a comprehensive approach to cellular and molecular analysis of intact non-dissociated tissues. To this end, we have developed polychromatic (10+) immunofluorescence staining protocols for frozen tissue sections that are analyzed by confocal microscopy and whole-tissue scanning. Post-acquisition quantitation is based on adapted and improved histocytometry method based on Gerner et al. (Immunity 2012 Aug 24;37(2):364-76) with dynamic cell segmentation using Imaris software and a flow cytometry-like analysis conducted with Flowjo, allowing us to gate on different immune populations and subpopulations based on their phenotype while preserving each cell's original location within the tissue. In the next step, specific cells of interest are harvested based on their location with laser capture microdissection and profiled with RNA-seq. Transcripts derived by this computational pipeline are further analyzed using in situ hybridization with ViewRNA to confirm cell expression and establish the cellular context. We are also implementing light-sheet microscopy for 3D assessment of tissue architecture and organization by a given molecule. Thus, we are generating extensive quantitative and qualitative "maps" of cancer, stromal and immune infiltrate, allowing us to study its dynamic within the tissue.

#5681

Prediction of immunotherapy outcome by multimodal assessment of minimal residual disease and persistence of allogeneic anti-CD123 CAR T-cells (UCART123) in pre-clinical models of acute myeloid leukemia.

Mayumi Sugita,1 Nuria Mencia-Trinchant,1 Nathan Ewing-Crystal,1 Gabrielle Suppa,1 Roman Galetto,2 Agnès Gouble,2 Julianne Smith,3 Gail J. Roboz,1 Duane C. Hassane,1 Monica L. Guzman1. 1 _Weill Cornell Medicine, New York, NY;_ 2 _Cellectis SA, Paris, France;_ 3 _Cellectis Inc, New York, NY_.

Acute myeloid leukemia (AML) is a fatal disease. The promise of autologous T-cells expressing chimeric antigen receptors (CARs) in targeting B-cell malignancies has encouraged extension of this approach to AML. However, clinical guidance regarding infusion and re-infusion regimens for CAR-T technology is unclear for any cancer including AML. Given that studies demonstrate clear benefit for minimal residual disease (MRD) assessment in predicting relapse for AML, we thus sought to ascertain whether simultaneous molecular assessment of MRD markers and CAR-T-specific markers could inform decisions around CAR-T dosing and re-infusion. We tested this approach using patient-derived xenograft (PDX) models with allogeneic anti-CD123 CAR-T cells (UCART123). UCART123 are genetically modified allogeneic T-cells expressing an anti-CD123 CAR. These cells lack expression of the T-cell receptor (TCRabKO), in order to minimize graft vs. host disease (GvHD).

PDX were established using prognostically adverse AML (FLT3-ITD+NPM1+) and treated with 1x106 or 2.5x106 UCART123. The median overall survival (OS) of control mice injected with saline or CAR-T negative (TCRabKO) T-cells succumbed to disease was 124.5 and 126 days, respectively. In contrast, UCART123 groups survived >180 days (hazard ratio 0.08, P=0.003). Clonal dynamics between disease and CAR-T were simultaneously monitored post-infusion by quantifying mutated NPM1 and CAR-T genetic markers, respectively, using digital droplet PCR (ddPCR). We found that ddPCR monitoring was more sensitive than multiparameter flow cytometry (MFC) at detecting MRD and persistence of UCART123. Using ddPCR, leukemia and UCART123 cells were detected when human cells were not evaluable using MFC in peripheral blood (PB). Mice with persistent UCART123 remained disease-free. Importantly, when mutated NPM1 levels became elevated with simultaneous loss of UCART123, relapse was evident by MFC in PB in subsequent time-points (2 out 20 mice, all at 1x106 dose, ~180 days) re-infusion of UCART123 cells resulted in effective elimination of AML.

Taken together, we have demonstrated that simultaneous monitoring of disease and UCART123 cells provides valuable insight into the kinetics and effectiveness of UCART123 cells. Currently, we have implemented the ddPCR assay in the phase I clinical trial of UCART123 in AML allowing to simultaneously detect UCART123 cells and blasts in peripheral blood of NPM1 mutant AML patients.

#5682

Histomorphological and molecular immune classification of head and neck cancer and its relation to prognosis.

Jan Budczies, Mohamed Badr, Ingeborg Tinhofer, Carsten Denkert, Korinna Jöhrens. _Charité - Universitätsmedizin Berlin, Germany_.

Recently, nivolumab and pembrolizumab have been approved as second-line treatment for recurrent and/or metastatic squamous cell carcinomas of the head and neck (SCCHN). However, while nivolumab improved response rate and overall survival in the CheckMate-141 trail and led to long-lasting responses, the overall response rate of 13% was limited. Therefore, biomarkers to select patients for immune therapy and to analyze and overcome mechanisms of resistance are urgently needed. To gain inside into the immunological status of SCCHN, we evaluated digital slides (HE stain) and molecular data of 528 primary tumors from TCGA. Tumor infiltrating lymphocytes (TILs) were evaluated separately intra-epithelial and in tumor stroma using a semi-quantitative method. The content of T cells, CD8+ T cells, cytotoxic lymphocytes, B lineage cells, NK cells, monocytic lineage cells, myeloid dendritic cells, neutrophils, fibroblasts and endothelial cells was estimated from RNA-seq data using the MCP-counter method. The T cell level was classified as high and low using a median cutoff point. As both intra-epithelial TILs and stromal TILs followed bimodal distributions, cutoff points were introduced leading to the flowing classification: Intra-epithelial TILs were present in 61% and absent in the remaining 39% of tumors. Levels of stromal TILs were high in 52% and low in the remaining 48% of tumors. Intra-epithelial and stromal TILs correlated highly significant (p=1.1E-06), but there were considerable proportions of tumors with absent intra-epithelial and high stromal TILs (13%) as well was with present intra-epithelial and low stromal TILs (21%). Intra-epithelial TILs correlated significantly with HPV status, but not with PD-L1 mRNA. Stromal TILs correlated significantly with PD-L1 mRNA, but not with HPV status. In univariate Kaplan-Meier analysis, intra-epithelial TILs correlated significantly with better DFS (p=0.009), but not with OS, while high stromal TILs correlated borderline significantly with better OS (p=0.071), but not with DFS. Tumors with many T cells had both significantly better DFS (p=0.018) and better OS (p<0.0001). PD-L1 mRNA expression correlated significantly with T cell content (R=0.45), but did not correlate significantly with DFS and OS. However, in a bivariate analysis of DFS including PD-L1 mRNA and T cells, PD-L1 was a significant negative prognostic maker, (HR per doubling=1.13, 1.01-1.25), while T cells were a strong positive prognostic maker (HR per doubling=0.74, 0.64-0.68). These results remained significant in bivariate analysis of OS and in a multivariate analysis including correction for clinical-pathological patients and tumors characteristics. The histopathological and molecular features describing the immune status of SCCHN investigated here should be further analyzed for their relevance in immune therapy patients.

#5683

Value of new spontaneous and carcinogen-induced mouse-derived isograft (MDI) tumor models for drug development of novel immune therapeutic approaches.

Janette Beshay, Peter Jantscheff, Cynthia Obodozie, Christoph Schaechtele, Holger Weber. _ProQinase GmbH, Freiburg, Germany_.

Checkpoint inhibitor treatment has already become a common therapy of various cancer types. However, there is still a growing need for well-characterized preclinical mouse models, as clinical data indicate that patients only partially respond to this regiment. Currently, cell lines cultured from the 1970s are used frequently to evaluate novel therapies. In a research development program, new mouse-derived isografts (MDI) were established from spontaneously occurring and carcinogen-induced tumors. These tumors are transplanted as tissue pieces in a PDX-like manner and tested for their solid growth. Furthermore, the efficacy of immune checkpoint inhibitor treatment is evaluated and the presence of different immune cell populations in the tumor is characterized by FACS analysis. In addition, RNA-seq data complete the first characterization of these models and will give insights into the expression level and genetic modification of genes in these models. Here, the spontaneous MDI model JA-0009 and two carcinogen-induced MDI models JA-2042 and JA-2011 are presented. Whereas JA-0009 and JA-2011 tumors are only partially affected by immune checkpoint inhibitor treatment, JA-2042 shows a significant response in a combined treatment of anti-PD-1/anti-CTLA-4. Characterization of the infiltrated immune populations revealed a high number of T cells, especially Treg cells. In contrast, the JA-0009 tumor is infiltrated mainly by macrophages of M2 type with a low number of CD8+ T cells and the JA-2011 tumor by neutrophils. Taken together, the new MDI models complement the existing GEMs and cell-line derived syngeneic tumor models by their new properties (low passage number, no tissue culture, conserved original tumor properties and intra-tumoral immune cell populations) to characterize novel immune-modulatory agents in drug development.

#5684

A methodology for designing and validating computational pathology scores for immune cell clustering in tumor biopsies.

Logan Cerkovnik, Karen Ryall, G David Young, Kristen Wilson, Joseph Krueger. _Flagship Biosciences, Westminster, CO_.

Abstract: Introduction: Immune cell clustering is commonly observed in histopathology images. As the frequency and nature of immune cell clustering may represent biological phenomenon critical to response to immunotherapy, it is an import feature to measure for differentiating immune phenotypes which may predict patient response to immune modulating therapy. Computational pathology data sets and unsupervised machine learning approaches are capable of describing immune cell clustering using a variety of methods. However, it has not been clear how such measurements might be applied to generate a validated computational pathology score that truly captures the immune phenotype sought to score. This work explores a methodology for the development and analytical validation of digital pathology scores for immune cell clustering derived from the application of unsupervised learning to digital pathology data sets.

Methods and Experimental Design: Computational pathology data derived with Flagship's cTA™ (Computational Tissue Analysis) platform from 12 NSCLC biopsy samples stained with a validated CD8 -Ki67 duplex IHC assay was used to create a virtual library of scores that described the clustering of CD8+ staining cells. The library was created by using different combinations of clustering methods, parameters, and scoring schemes. Scores with high distinguishability measured through a two-way intraclass correlation coefficient, inter-run precision measured through the coefficient of variation, and dynamic range were considered analytically validated. Principal component analysis and hierarchical clustering of the analytically validated scores were used to further optimize selection of the most informative subset of clustering scores from the library.

Results and Conclusions: 17 clustering scores passed the validation criteria. 4 of these 17 scores appear to be relatively uncorrelated with each other and capture unique information about the spatial relationships of CD8 positive cells. A further analysis of these scores demonstrates the ability to distinguish different immune cell clustering profiles in samples that contain similar biomarker expression levels using the common scoring methods of both overall percentage of positive cells and percentage of positive cells per tissue area. This process for screening and analytically validating a virtual library of computational biomarker scores appears to hold much promise for bringing cluster-derived computational pathology scores into future clinical applications in a way that is analogous to analytical validation of traditional IHC assays in support of oncology drug development.

#5685

Nanocage-therapeutics prevailing phagocytosis and immunogenic cell death awakens immunity against cancer.

Gihoon Nam, Gi Beom Kim, In-San Kim. _Korea Inst. of Science & Technology, Seoul, Republic of Korea_.

A growing appreciation of the relationship between the immune system and tumorigenesis has led to the development of strategies aimed at 're-editing' the immune system to kill tumors. Here, we report a novel tactic for overcoming the activation-energy threshold of the immunosuppressive tumor microenvironment and mediating the delivery and presentation of tumor neoantigens to the host's immune system. This nature-derived nanocage not only efficiently presents ligands that enhance cancer cell phagocytosis, it also delivers drugs that induce immunogenic cancer cell death. The designed nanocage-therapeutics induces the release of neoantigens and danger signals in dying tumor cells, and leads to enhancement of tumor cell phagocytosis and cross-priming of tumor specific T cells by neoantigen peptide-loaded antigen-presenting cells. We observed potent inhibition of tumor growth and complete eradication of tumor through systemic tumor-specific T-cell responses in tumor draining lymph node and spleen and further, infiltration of CD8+ T cells into the tumor site. Remarkably, after removal of the primary tumor, all mice treated with this nanocage-therapeutics are protected against subsequent challenge with the same tumor cells, suggesting development of lasting, tumor-specific responses. This designed nanocage-therapeutics 'awakens' the host's immune system and provokes a durable systemic immune response against cancer.

#5686

Utilization of fluorescent multiplex IHC and digital image analysis for studying LAG-3, CD3 and CD8 positive TIL subsets in NSCLC tissue.

Malik Khenkhar,1 Daniel Biljes,1 Nickels Winkler,1 Philipp C. Uhlig,1 Hartmut Juhl,1 Alison L. Bigley,2 Lorcan Sherry2. 1 _Indivumed GmbH, Hamburg, Germany;_ 2 _OracleBio, Biocity Scotland, United Kingdom_.

Targeting checkpoint molecules expressed on immune cells has shown promising results in the treatment of non-small cell lung cancer (NSCLC). One interesting molecule that is frequently expressed on exhausted tumor-infiltrating lymphocytes (TIL) is lymphocyte activation gene-3 (LAG-3). We recently implemented chromogenic anti-LAG-3/CD3 dual immunohistochemistry (IHC) and digital image analysis to quantify LAG-3 positive T cells in NSCLC tissue. Here, we extended this work by establishing an anti-LAG-3/CD3/CD8/pan-Cytokeratin (pan-CK) fluorescent multiplex IHC (mIHC) assay followed by digital image analysis to examine the composition of T cells infiltrating NSCLC tissue in more detail. Tyramide signal amplification (TSA) based fluorescent 5-color mIHC (LAG-3/CD3/CD8/pan-CK + DAPI) was developed by Indivumed on the Leica BOND RX automated staining platform and applied to formalin-fixed paraffin-embedded (FFPE) NSCLC tissue samples. Image analysis was performed by OracleBio. Tumor and stroma regions of interest (ROI) were classified according to the pan-CK and DAPI signals. LAG-3, CD3 and CD8 single positive cells, as well as dual and triple positive cells, were then quantified in the tumor and stroma ROIs. Fluorescent mIHC allowed for a specific quantification of LAG-3, CD3 and CD8 single, LAG-3/CD3, LAG-3/CD8, CD3/CD8 dual and LAG-3/CD3/CD8 triple labeled cells in the tumor and stroma ROIs of the analyzed NSCLC samples. As part of the assay validation, similar ratios of the differently labeled cells, in particular LAG-3 positive T cells (LAG-3/CD3 dual positive), were observed with fluorescent mIHC and chromogenic IHC, demonstrating a good concordance between the two approaches. Among the analyzed NSCLC samples, different ratios of LAG-3 positive TILs in adeno- and non-adenocarcinoma samples were detected. Determining the ratios of dual or triple positive TIL subsets by mIHC in combination with digital image analysis allows for a clearer understanding of the lymphocyte composition within NSCLC tissue than a simple quantification of LAG-3, CD3 and CD8 single positive cells. Furthermore, such information will contribute to a deeper understanding of the role of LAG-3 positive immune cell sub-populations in the progression and treatment of NSCLC.

#5687

Integrative -omics analysis to identify drug targets for ccRCC immunotherapy.

Anna Reustle,1 Moreno Di Marco,2 Florian Büttner,1 Stefan Winter,1 Siarhei Kandabarau,1 Daniel Kowalewski,2 Linus Backert,2 Steffen Rausch,3 Joerg Hennenlotter,3 Marcus Scharpf,4 Falko Fend,4 Arnulf Stenzl,3 Jens Bedke,3 Stefan Stevanovic,2 Matthias Schwab,1 Elke Schaeffeler1. 1 _Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology Stuttgart and University of Tuebingen, Stuttgart, Germany;_ 2 _Department of Immunology, University of Tuebingen, Tuebingen, Germany;_ 3 _Department of Urology, University Hospital Tuebingen, Tuebingen, Germany;_ 4 _Institute of Pathology and Neuropathology, University Hospital Tuebingen, Tuebingen, Germany_.

Background

Clear cell renal cell carcinoma (ccRCC) is the dominant subtype of renal cancer and frequently diagnosed at advanced disease stages. Cure of advanced and metastatic ccRCC is achieved only in rare cases with currently available therapies, consisting of tyrosine kinase and mTOR inhibitors, due to intrinsic or acquired resistance mechanisms. We developed a workflow to identify potential drug targets involved in ccRCC pathogenesis for immunotherapeutic approaches and combinatorial treatment strategies.

Methods

We analyzed frequent ccRCC-specific peptides by HLA ligandomics of 55 ccRCC tumors, paired non-tumor tissues and 158 other benign tissues from various organs. To extract cellular pathways with enrichment in ccRCC compared to the S3 region of kidney proximal tubules, which represents the region of ccRCC origin, we performed transcriptome and gene set enrichment analyses in 51 of the 55 tumor tissues of our study cohort and in a microdissected sample of the S3 region (S3-transcriptome data taken from Cheval et al., PloS One 2012). ccRCC-specific pathway genes were intersected with the tumor-exclusive peptide source genes from the HLA ligandome analysis to retrieve a list of candidate target genes. By integrating gene expression data of an independent ccRCC cohort from the Cancer Genome Atlas (TCGA KIRC, n=452), the candidates were validated on the level of individual gene expression and pathway enrichment. Integration of DNA methylation (TCGA KIRC, n=273) and somatic mutation data (TCGA KIRC, n=392) was used to refine the candidate list.

Results

Of the frequent ccRCC-specific HLA class I and II peptide source genes, 203 were involved in ccRCC-enriched pathways in both cohorts. 136 of the genes passed the selection criteria of minimal expression in tumors (> 7.2 log2 FPKM-UQ) and induction compared to paired non-tumor tissues. 89 of the candidate targets were affected by somatic point mutations in one up to eight patients, potentially giving rise to patient-specific neo-epitopes. 110 of the candidate genes displayed altered DNA methylation patterns in tumors compared to non-tumor tissues, offering the possibility of epigenetic targeting.

Conclusion

The presented workflow, integrating HLA ligandomics, transcriptomics and genomics, yields a list of genes with frequent ccRCC-specific HLA-presented peptides that could be targeted in immunotherapeutic approaches, such as ccRCC-directed peptide vaccines. Moreover, the selected genes are involved in ccRCC-enriched cellular pathways and might therefore be important factors of ccRCC pathogenesis, potentially restraining rapid resistance development when they are used as drug targets. The list represents a pre-selection of potential drug targets for novel immunotherapeutic and combinatorial therapy approaches, which need to be further evaluated.

#5688

Humanized double knock-in mouse models for evaluating in vivo efficacy of the combination therapy of immune-oncology drugs.

Tian Gan,1 James Jin,2 Chaoshe Guo,1 Yuelei Shen,1 Yanan Guo1. 1 _Beijing Biocytogen Co., Ltd, Beijing, China;_ 2 _Biocytogen LLC., Worcester, MA_.

Over the past years, monoclonal antibodies have been successfully utilized in clinical trials to block or activate key mediators of immune checkpoint pathways, including CTLA-4, PD-1, PD-L1 and OX40 et.al,. Although significant benefits including complete regression and long-term survival were reported among the majority of participants, not all patients respond. In order to optimize the efficacy and minimize toxicities of anticancer immunotherapies, immune checkpoint antibodies are being tested in combination with other conventional therapeutics (radiation, chemotherapy and targeted therapies) and newer immunotherapies (cancer vaccines, oncolytic viruses). However, along the IO drug development process, in vivo efficacy models have always been a rate-limiting step, especially for the combination therapy of two IO antibodies. Therefore, we generated humanized double knock-in mouse to evaluate the in vivo efficacy of combination therapy. For example, humanized double Knock-in B-hPD-1/hOX40 mice pairing with mouse cancer cells, demonstrated a synergistic effect of using hPD-1 and hOX40 antibodies. Additionally, more double knock-in mice targeting immune checkpoint molecules were developed, such as B-hPD-1/hPD-L1, B-hPD-1/hCTLA-4, B-hPD-1/hLAG3, B-hPD-1/hTIM3, B-hPD-1/hTIGIT, B-hPD-1/hBTLA, et al,. Humanized double knock-in mice, represent useful in vivo efficacy models for evaluation of the combination effects of various IO drugs.

#5689

Characterization of immune infiltrate and checkpoint protein expression patterns in murine syngeneic tumors via multiplex immunohistochemistry.

Jennifer Ziello, Sarah Klein, Katherine Crosby. _Cell Signaling Technology, Inc, Danvers, MA_.

Murine syngeneic tumor models are increasingly utilized for preclinical immuno-oncology studies as immunotherapeutic strategies continue to make clinical strides. However, the immunologic features of the tumor microenvironment (TME) in these models remain largely undefined. In this study, we applied a 7-color multiplex immunohistochemistry panel to visualize and quantify the immune infiltrate within formalin-fixed, paraffin-embedded RENCA, CT26.WT, and LL/2 tumor tissues derived from subcutaneous mouse models of renal cell carcinoma, colon carcinoma, and lung carcinoma, respectively. Additionally, we applied this panel to a 4T1 orthotropic primary mammary tumor and lung metastasis formed via tail vein injection. The multiplex panel included antibodies detecting CD3 and CD8 as T cell markers, F4/80 as a myeloid cell marker, the immunosuppressive receptor PD-1 as well as its ligand PD-L1, pan-keratin as a tumor mask, and DAPI as a nuclear counterstain. We characterized the tissue localization of tumor-infiltrating immune cells and analyzed the trends in co-expression and frequency patterns of immunosuppressive proteins. This study strives to better understand the underlying differences in the immunologic landscapes of these tumors, which in turn has implications for researchers studying responses to immunotherapeutic approaches and combination strategies.

#5690

Characterization of tumor growth and immune microenvironment in humanized NOG-EXL mice implanted with A549, MDA-MB-436 and A375 cells.

Srimoyee Ghosh,1 Anne Fiore,2 Christoph Eberle,2 Sarah Wang,1 Keith Mikule,1 Kristen McEachern,1 Sujatha Kumar,1 David Jenkins,1 Geeta Sharma2. 1 _TESARO Inc., Waltham, MA;_ 2 _Charles River Laboratories (Agilux), Worcester, MA_.

Syngeneic mouse models are the most extensively explored mouse models for evaluation of immune-oncology therapeutic modalities in preclinical settings. One of the major limitations with syngeneic models is that both the tumor and the immune system is murine and thus do not express any human targets. Also, this precludes the evaluation of human clinical product (antibody or protein) mandating the synthesis and use of murine surrogates. Therefore, development of models of human tumors in mice with competent human immunity became an urgent need. Tumor-bearing, humanized NOG-EXL mice are a new and valuable preclinical testing platform for immuno-oncology, to simulate trials, evaluate multiple drugs alone or in combination, and produce predictive data both in cell line derived (CDX) and patient derived (PDX) models. With the objective of understanding the tumor growth and basal immune microenvironment in Hu-NOG-EXL mice (NOG mice humanized using CD34+ cells isolated from cord blood), we implanted three different human tumor cell lines (A549: Human Lung Carcinoma; MDA-MB-436: Triple Negative Breast Cancer; A375: Human Melanoma) in Hu-NOG EXL mice procured from Taconic, NY. The tumors were collected at a tumor volume of 1000-1500mm3, and profiled for the lymphoid and myeloid compartments using flow cytometry. The corresponding splenic compartment was also profiled to monitor the correlation or lack thereof between the tumor and splenic compartments. Finally the tumors were compared and categorized based on their basal infiltrates. The results demonstrated that Hu-NOG-EXL mice could be an useful tool to dissect the immune response in human tumors. It was possible to identify and quantitate the myeloid cells which are absent in a model that has been implanted with human PBMNC's (Peripheral Blood Mono Nuclear Cells) instead of human CD34 stem cells.

#5691

Evaluation of immunomodulatory agents in classically immunologically 'cold' cancers using syngeneic mouse models of breast and ovarian cancer.

Dylan Daniel, Sumithra Urs, Kevin Guley, Sarah Krueger, David Draper, Alden Wong, Hillary Evens, Claire Higginbottom, Dan Saims, Scott Wise, Maryland Rosenfeld Franklin. _MI Bioresearch Inc., Ann Arbor, MI_.

The clinical successes of immunotherapy in immunologically "hot" cancers such as kidney, bladder and lung cancers has lead researchers to pursue strategies to improve the initiation of immune responses in immunologically more "cold" cancers such as breast and ovarian cancer. We have characterized two syngeneic mouse models of breast cancer, 4T1 and E0771, and an ovarian cancer model, ID8, for their baseline immune profile and responsiveness to various immunotherapy approaches in an effort to enable rational combination therapy.

The 4T1 model has useful traits for immuno-oncology research including a highly metastatic phenotype. However, our data illustrate that the tumors have a paucity of CD8 T cells and a highly immunosuppressed microenvironment with Tregs and ~80% G-MDSCs of CD11b+ cells leading to primary resistance to checkpoint blockade. Radiation can induce changes in an immunosuppressive microenvironment and radiotherapy remains an important clinical modality for the treatment of breast cancer. Treatment of 4T1 tumors with radiation resulted in a ~15% reduction of total MDSCs in the mice receiving 8Gy daily for three days. For the purpose of guiding future immunotherapy combinations, we established a focal beam radiation dose response and found single dose 5Gy had no activity while 10 or 20Gy resulted in increasing anti-tumor effects.

The baseline immune cell profiling for E0771, a triple negative breast cancer, revealed notable differences to 4T1. While both models have a similar proportion of CD4 T cells and Tregs, 4T1 has ~55% G-MDSCs of CD11b+ cells while E0771 has ~0.4%. The content of M-MDSCs is nearly reciprocal with E0771 having ~49% M-MDSCs of CD11b+ cells and 4T1 having ~7%. The E0771 model also has a higher proportion of CD8 T cells than 4T1. Based on its immune profile, E0771could be a better candidate for responding to checkpoint inhibition. An efficacy study in the E0771 model demonstrated high sensitivity to inhibitors of PD-1, PD-L1, and CTLA-4, although the activity of anti-PD-1 on established tumors was more modest. Costimulatory agonistic antibodies to OX40, CD137 and GITR were also highly active. These data suggest that E0771 may represent a more immunologically "warm" breast cancer.

We have developed the ID8-Luc-mCh-Puro syngeneic mouse cell line as an orthotopic (intraperitoneal) model of ovarian cancer wherein tumor burden is monitored by bioluminescence imaging. Immune profiling on ascites from ID8-Luc-mCh-Puro bearing mice shows heterogeneity in the profiles between mice; although, all mice have robust CD8+ T cell infiltration with favorable CD8+ T cell/Treg ratios. The activity of checkpoint inhibitors against orthotopic ID8-Luc-mCh-Puro is currently being evaluated. Together, these models of breast and ovarian cancer can be used to evaluate anti-tumor immune responses in immunologically more quiescent indications.

#5692

Low frequency electromagnetic field suppresses macrophage phagocytosis.

Weston Scot Burrup, Evita G. Weagel, Richard A. Robison, Kim L. O'Neill. _Brigham Young Univ., Provo, UT_.

The purpose of this study is to determine the role of low frequency electromagnetic fields (EMF) on human macrophages and their inflammatory response. The effects of EMF on humans are highly dependent on frequencies. Studies have shown that low EMF (25-30 Hz) frequencies can help lower inflammation and suppress pain, as well as induce healing in benign prostatic hyperplasia. Macrophages are known for their pro-inflammatory responses against foreign antigens and their anti-inflammatory and angiogenic responses in cancer. We wanted to determine the effects of 27Hz EMF on macrophage phagocytosis and gene expression to determine whether low frequency EMF can decrease inflammation. This can help us understand the role of low frequency EMF in macrophage function. This may provide further insight as to how macrophages may be affected when fighting disease, especially cancer. We incubated the monocytic cell line U937 with 200-400 ng of phorbol-12 myristate 13-acetate (PMA) for 24 hours to allow for differentiation into macrophages. Once cells had differentiated, we stained the cells with the eFluor 670 dye for 30 minutes. The cells were then washed and plated in 12-well plates at 1X106 cells/mL and allowed to attach for 15 minutes. Cells were exposed to 27 Hz EMF treatment for 30 or 60 minutes or no exposure at all (control). After EMF treatment, a solution of PE fluorescent beads was added to the cells. U937 cells were allowed to phagocytose for 60 minutes. Engulfment levels were then measured with Flow Cytometry and results were analyzed with FlowJo Software. The percentage of macrophages involved in engulfment of 1 bead, 2 beads, and 3+ beads were determined. With 30 minutes of 27Hz EMF treatment the macrophages showed a significant reduction in total engulfment compared to the control. When looking at the percentages of macrophages that engulfed 1 bead or 2 beads, a higher percentage of treated macrophages engulfed 1 bead and a similar percentage engulfed two beads as compared to the control. Interestingly, the percentage of macrophages that engulfed 3+ beads was reduced significantly. This suggests 30 minutes of exposure to EMF treatment decreased the aggressiveness of the macrophages. In contrast, after 60 minutes of exposure to EMF treatment, the total engulfment percentage increased by more than 5%. This was seen by a higher percentage of cells participating in engulfment of 3+ beads as compared to the control. In conclusion, we observed that with 30 minutes of exposure, macrophages tend to be less aggressive but after 60 minutes a shift in aggressiveness is observed. Our research implies that macrophages can be induced to a more aggressive state with 60 minutes of electromagnetic field treatment. Further research is ongoing to understand the effects of different time intervals of EMF treatment on macrophages.

#5693

Tumor whole-transcriptome sequencing and multiplex immunohistochemistry of immune cell populations in 158 Asian colorectal cancers.

Clarinda Wei Chua,1 Engin Cukuroglu,2 Elisa Fontana,3 Si Lin Koo,1 Joe Poh Yeong,4 Andy Nguyen,5 J. Zachary Sanborn,5 Steve Benz,5 Emile John Tan,4 Ronnie Mathew,4 Ee-Lin Toh,4 Sarah Boon Ng,2 Tony Kiat Lim,4 Anders Jacobsen Skanderup,2 Shahrooz Rabizadeh,5 Anguraj Sadanandam,3 Jonathan Göke,2 Iain Bee Tan1. 1 _National Cancer Centre Singapore, Singapore, Singapore;_ 2 _Genome institute of Singapore, Singapore, Singapore;_ 3 _Institute of Cancer Research, London, United Kingdom;_ 4 _Singapore General Hospital, Singapore, Singapore;_ 5 _NantOmics LLC, CA_.

Background: Colorectal cancer (CRC) is a cancer largely refractory to immune checkpoint inhibition. There is substantial interpatient molecular heterogeneity in colorectal cancer reported from studies on tumor RNA and studies on immunohistochemical analyses of fixed tumor tissue. Several major transcriptomic analyses from microarrays and RNA-seq data have identified major transcriptomic subtypes and major activated pathways and deconvoluted cell-type enrichments. Immunohistochemical (IHC) analyses of formalin-fixed, paraffin-embedded (FFPE) tissue microarrays have identified several different histomorphologic/spatial patterns of immune infiltrates in the tumor microenvironment. Here, we perform large-scale -omic analyses on tumor RNA and multiplex IHC simultaneously on 158 Asian colorectal cancers.

Methods: We performed whole-genome sequencing (WGS) (60x tumor, 30x normal) and deep whole-transcriptomic sequencing (RNA-seq) (∼200x106 reads per tumor) on 158 colorectal cancers. To evaluate the spatial patterns of the tumor microenvironment, we constructed a tissue microarray comprising the tumor core, tumor edge and normal adjacent tissue of these 158 CRCs. We performed H&E analyses of the TMA and multiplex immunohistochemistry to simultaneously evaluate 7 markers, i.e., cytokeratin (CK), CD3, CD8, FOXP3, CD68, PD-L1, DAPI, using the an Opal Multiplex fIHC kit. Images were acquired using a Vectra 3 pathology imaging system microscope (PerkinElmer, Waltham, MA, USA).

Results: 32 are microsatellite instability high (MSI-H) tumors and 126 are microsatellite stable. (MSS). The major transcriptomic subtypes (Consensus molecular subtypes (CMS 1-4) and CRC assigner (Goblet-like, Enterocyte, Stem-like, Inflammatory, Transit-amplifying subtypes) were identified with good concordance between both classification systems. CMS1 and Inflammatory subtypes were enriched amongst MSI-H tumors. Major oncogenic pathway activations (RAS, Wnt), cell-cycle and inflammatory signatures (interferon-rich) were also identified across the populations. We deconvoluted cell-type enrichment scores from the transcriptomic data to identify different cell-type enrichment patterns across the cohort. On the TMAs, we identified cell type populations and immune infiltrate patterns in the tumor core and invasive edge across the cohort. Correlations across these analyses will be presented at the meeting.

Conclusions: There is substantial interindividual variability in the transcriptomic landscape and spatial patterns of immune cell infiltrates in CRCs.

#5694

Evaluation of the in vivo efficacy of immune-oncology drugs targeting stimulatory immune checkpoint molecules using humanized knock-in mice.

Tongmei Xia, Chaoshe Guo, Yuelei Shen, Yanan Guo. _Beijing Biocytogen Co., Ltd, Beijing, China_.

Cancer immunotherapy is one of the most promising research areas in the field of cancer therapy. Many pharmaceutical and biotech companies in the world are devoting great effort to develop cancer immunity-related treatment antibodies. However, along the IO drug development process, in vivo efficacy models have always been a rate-limiting step. In most cases, a human monoclonal antibody does not have mouse cross-reactivity. Mouse surrogate antibodies were often used in immune-competent syngeneic mouse models to evaluate in vivo efficacy of IO drugs. However, the efficacy of a surrogate antibody cannot fully represent the human drug in the clinical scenario. Therefore, we generated humanized knock-in mice to evaluate the in vivo efficacy of human IO antibodies. For example, human OX40 knock-in (B-hOX40) mice were generated with a chimeric OX40 receptor, which is recognizable by stimulatory human OX40 antibodies. Additionally, more knock-in mice targeting stimulatory immune checkpoint molecules were developed and validated, such as B-hCD137, B-hGITR, B-hCD27, B-hCD40, B-hCD28, B-hCD3 et al,. All these mouse models response well to the corresponding human IO antibodies, proving that they are powerful tools for in vivo efficacy evaluation of human stimulatory immune checkpoint antibodies.

### Immune Monitoring / Clinical Correlates

#5695

Use of certain antihistamines among breast cancer patients confers survival benefit.

Hakan L. Olsson. _Univ. of Lund, Lund, Sweden_.

H1-antihistamines have not been widely studied in relation to cancer therapy, despite being among the most common medications used globally, and having become increasingly sophisticated, cheap and safe.

In a nation-wide study of the entire Swedish population using the Drug Prescription Registry, Cancer Registry and Cause of Death Registry- the first of its kind - we investigated the effects of the six major antihistamines (cetirizine, clemastine, desloratadine, ebastine, fexofenadine and loratadine) on breast cancer-related (as well as all-cause) mortality. Our study includes 61,627 Swedish women with breast cancer diagnosed between July 1st 2005 and December 31st 2013. 14,711 used at least one of the six major antihistamines. Both peri- and post-diagnostic antihistamine use was analyzed, as well as the use among different subgroups of patients, divided according to age, stage and estrogen receptor status, was analyzed.

Here, we show a consistently beneficial effect of desloratadine and ebastine use on breast cancer survival (in the peri-diagnostic analysis, for desloratadine, HR=0.79 [95% CI 0.63-0.99], P=0.044; and for ebastine users, HR=0.57 [95% CI 0.32-1.03], P=0.064; relative to non-users) and a consistently negative effect on breast cancer survival of clemastine use (in the peri-diagnostic analysis, HR=1.24 [95% CI 1.05-1.47], P=0.013; relative to non-users). Loratadine use is associated with better survival in the peri-diagnostic analysis (HR=0.78 [95% CI 0.63-0.97], P=0.022) relative to non-use. No significant effects were seen for cetirizine use or fexofenadine use. Post diagnostic use. A woman with breast cancer using desloratadine will lower her relative risk of breast cancer-related death with 15% after one

year's use and 17% after two years' use (both risks relative to a woman of the same age and the same tumor status).

Conclusion. Use of desloratadine and ebastine may may significantly improve survival in breast cancer with few side effects.

#5696

Discovery of putative breast cancer antigens using genomics-driven immunoproteomics.

David K. Han. _University of Connecticut School of Medicine, Farmington, CT_.

Recent advances in cancer immunotherapeutics such as checkpoint inhibitors, chimeric antigen-receptor T cells, and tumor-infiltrating T cells (TIL) are now significantly impacting cancer patients in a positive manner. Although very promising, reports indicate no more than 25% of cases result in complete remission. One of the limitations of these treatments is the identity of cancer antigens in each patient as it is technically challenging to identify cancer antigens in a rapid fashion. Thus, identification of cancer antigens for each patient followed by targeted treatment will likely increase the efficacy of cancer immunotherapies. To achieve this goal, a combined technologies of deep genomic sequencing and personalized immune assessment platform was devised, termed Genomics-Driven Immunoproteomics (GDI). Using this technological platform, we report the discovery of 149 putative tumor antigens from human breast cancer patients. Significant numbers of these cancer antigens arise from single nucleotide variants (SNVs), as well as insertions and deletions that results into frame-shift mutations. We propose a general model of anticancer immunity and suggest that GDI platform may help identify patient-specific tumor antigens in a timely fashion for precision immunotherapies.

#5697

Simultaneous assessment of PD-L1 and IFR1 expression on breast cancer circulating tumor cells.

Lance U'Ren,1 Nolan Ericson,1 Ryan Huston,1 Elisabeth Mahen,2 VK Gadi,2 C. Anthony Blau,2 Eric Kaldjian1. 1 _RareCyte, Inc., Seattle, WA;_ 2 _University of Washington, Seattle, WA_.

Background: There is a need for noninvasive predictive biomarkers of response to anti-PD1/PDL1 therapies. Assessment of circulating tumor cells (CTCs) is a rational approach to noninvasive sampling of tumors to understand the potential response or nonresponse to anti-PD1/PD-L1 therapies. IFN-gamma signals through the JAK/STAT cascade to induce PD-L1 via the Interferon Regulatory Factor-1 (IRF1) transcription factor, and is a potent inducer of PD-L1 expression in tumor cells. Recent studies have shown that low or absent IRF1 expression can identify melanomas that lack IFN-gamma responsiveness, and that IRF1 can have a higher predictive value of response to anti-PD1/PD-L1 therapy than PD-L1 itself. Using CTC models and the RareCyte platform, we developed a multiparameter assay that allows simultaneous PD-L1 and IRF1 assessment after CTC identification. We used this assay to better understand the relationship between IRF1 and PD-L1 expression in CTC model systems and applied this assay to blood samples from breast cancer patients.

Materials and Methods: Peripheral blood from normal donors or cancer patients under an IRB-approved protocol was collected into RareCyte blood collection tubes. PD-L1(+) and PD-L1(-) CTC models were created by culturing A549 overnight with or without 10ng/mL INF-gamma. The A549 cells were spiked into normal donor blood and buffy coats isolated from 7.5mL of blood by AccuCyte® separation and spread onto slides. MDA-MB-231 cells were used as a model to represent breast cancer cells that express PD-L1 in the absence of IFN-gamma signaling. Slides were stained with a 6-marker panel that included antibodies to pan-cytokeratin (CK), EpCAM, CD45, PDL1, IRF1, and a nuclear dye on the Leica Bond Rx auto-stainer. Slides were scanned with CyteFinder® and CTCs identified by CK and/or EPCAM positivity and negative CD45 staining. Confirmed CTCs were then assessed for expression of PD-L1 and IRF1; cellular compartment was recorded for IRF1 staining.

Results: Nuclear expression of IRF1 correlated with PD-L1 expression in IFN-gamma stimulated A549 cells. Unstimulated MDA-MB-231 cells expressed high levels of PD-L1, but this did not correlate with nuclear IRF1 expression. Breast cancer patient samples were identified having CTCs that expressed PD-L1. A PDL1 mean fluorescence intensity (MFI) threshold was set at the upper 95% CI of unstimulated A549 cells to define "high" and "low" PD-L1 expression in the patient samples. The population of high-PD-L1 CTCs had higher nuclear IRF1 MFI than low-PD-L1 CTCs, with mean fold-increase of 2.7 (range from 1.4 to 3.3).

Conclusions: Nuclear IRF1 expression correlates with PD-L1 expression in breast cancer CTCs. Using CTCs to evaluate expression of IRF1 and PD-L1 together with assessment of CTC mutational load could be a powerful noninvasive assessment of response to anti-PD1/PD-L1 therapies.

#5698

Infiltrating immune cells in breast cancer subtypes.

Jaime L. Matta,1 Jarline Encarnacion,1 Carmen Ortiz,1 Julie Dutil,1 Amy Weber,2 Shari Pilon-Thomas2. 1 _Ponce Health Sciences University, Ponce, PR;_ 2 _H. Lee Moffitt Cancer Center, Tampa, FL_.

In 2017, breast cancer (BC) affected around 29% of Hispanic women in the US according to the American Cancer Society. Among Hispanics, Puerto Rican women are one of the most diverse groups in terms of ancestry and culture. This diversity adds complexity to the BC etiology in this population. Several population studies have suggested ancestry as a BC risk factor which needs to be considered in clinical trial design aiming at selecting better therapies. In contrast to chemotherapeutic treatments known to have significant toxicities, some immunotherapies are recognized by having modest toxicity grades with a high capacity for individualized medicine. Monoclonal antibodies such as trastuzumab are used to treat BC by stratifying patients according to their molecular subtypes. Depending on whether BC tumors are (+) or (-) for the estrogen (ER), progesterone (PR), and HER2, four molecular subtypes have been identified: luminal A (ER+, PR+/−, HER2-), luminal B (ER+, PR+/−, HER2+), HER2+ (ER−, PR−, HER2+), and basal-like (ER−, PR−, HER2−). The objective of this study is to elucidate the distribution of molecular BC subtypes and ancestry markers in Puerto Rican women and to establish the relationship between these distributions with their tumor infiltrating lymphocytes (TIL). Our overall hypothesis is that Hispanic women with BC will have distinct molecular subtypes and ancestry distributions that will be associated with their TIL signature. By combining samples collected through the Puerto Rico Bio-Bank and from our BC cohort, 129 blood and tumor tissue samples were selected. Molecular BC subtypes were classified using PAM50. A panel of ancestry informative markers was genotyped on iPLEX technologies and global ancestry proportions were estimated using ADMIXTURE. PanCancer Immune Panel was used to characterize the TIL based on RNA signatures. The initial results obtained through PAM50, showed that 45% of the tumors were luminal A, 27% luminal B, 18% basal-like, and 7% HER2+. Global ancestry proportions for each patient grouped by PAM50 subtype were performed. Overall, the ancestry proportions were: 67% European (SD ± 12%), 19% African (SD ± 11%), and 14% Native American (SD ± 7%). To evaluate the TIL RNA signature among subtypes, a principal component analysis (PCA) was performed followed by an immune score using the PCA2. The PCA2 score was able to distinguish among the basal-like, luminal A and luminal B subtypes; however, the HER2+ samples could not be clearly distinguished from the luminal A or B subtypes. These results show a qualitative tendency intrinsic of each molecular BC subtype and the TIL signature. Long-term, this approach of studying BC subtypes, ancestry, and immune infiltrates while increasing sample size is expected to provide data needed to improve prognostic tools to support the design of immunotherapies for Hispanic women with BC. This study was supported by: #S06GM008239-20, 9SC1CA182846-04, #U54CA163068, 2U54CA163071-06, and R003050/MD007579.

#5699

A new method for evaluating T cell response after stimulation on gastrointestinal cancer patients.

Tomoko Tanaka, Kimihiro Yamashita, Yutaka Sugita, Eiji Fukuoka, Akira Arimoto, Yoshihiro Kakeji. _Kobe University Graduate School of Medicine, Kobe, Japan_.

Background: In anti-tumor immunity, T cells play a central role to eliminate cancer cells. Immune responses of T cells are initiated by communication with antigen presenting cells (APCs). It became clear that an immunological synapse (IS) is formed at the T cell-APC interface and is made up of a supramolecular activation cluster (SMAC) containing the T cell receptors (TCR) microclusters (MCs) and CD28 MCs. TCR MCs consist of TCR, kinases, and adaptor molecules, and are the minimal unit of T cell signal transduction. CD28 MCs also function as the signalosomes of CD28 activation. By using total internal reflection fluorescence microscopy to observe the interaction of T cell and planar bilayer, it was elucidated that TCR activation is regulated by the amount of MCs aggregation, which shows the extent of IS formation. Based on this finding, we hypothesized that ex vivo T cells could be examined IS formation and that exhausted T cells of advanced cancer patients should be low in response to a stimulus.

Method: To simplify IS formation of T cells, we used human monoclonal T cells by αCD3 and/or αCD28 antibody and replaced to evaluate the aggregation of MCs by detecting the CD3 or CD28 molecules on the surface of T cells. This phenomenon was defined as IS-like formation. In this method, IS-like formation was detected by confirming an image of each cell with imaging cytometry. After these conditions was established, it was verified whether it is possible to measure IS-like formation rate of T cells. Additionally, the correlation between IS-like formation and interferon-γ cytokine production after this activation was examined. In the same manner as above, IS - like formation rates of T cells derived from PBMCs of healthy donors or esophageal and gastric cancer patients were measured.

Result: We have succeeded in observing that CD3 or CD28 molecules on a T cell aggregated in one area after stimulation as IS-like formation by imaging cytometry. In dot plot study, positive area of CD3 or CD28 decreased after the stimulation, which showed the aggregation of these molecules. Accordingly, we could measure IS-like formation rate of a population of T cell clones. In addition, the rate of IS-like formation showed a positive correlation with interferon-γ production. Similar results were obtained T cell from PBMC in healthy donors. Moreover, IS-like formation rate of T cell from cancer patients decreased compared with healthy donors.

Conclusion: IS-like formation of T cell succeeded to observe by CD3 and/or CD28 antibody stimulation. We established the evaluation method of IS-like formation degree by imaging cytometry. Moreover, IS-like formation rate correlated with cytokine production of IFN-g. We established new method to evaluate the strength of T cell after activation.

#5700

IDO1 high expression is associated with a poor prognosis in patients with esophageal cancer.

Yuki Kiyozumi. _Kumamoto University, Graduate School of Medical Sciences, Kumamoto, Japan_.

[Background] Esophageal cancer is the sixth most common cause of death from cancer worldwide. Although the development of multidisciplinary treatment for esophageal cancer, the prognosis of those patients remains poor. Recently, immunotherapy is expected as innovative therapy for several types of cancer. Indoleamine 2, 3- Dioxygenase (IDO1) is an enzyme responsible for the oxidation of tryptophan into kynurenine and implicated in immune modulation through its ability to limit T cell function and engage mechanisms of immune tolerance. IDO1 is focused as one of the immune subversion strategy and the therapy of targeting IDO1 is in clinical development for many types of cancer. IDO1 activation induces the downregulation of activating natural killer (NK) cell receptors on NK cells and inhibition of cytotoxic T cells by promotion of cell cycle arrest and apoptosis. However, the significance of IDO1 expression in esophageal cancer remains still unknown. The aim of this study is to examine the relationship between IDO1 expression and tumoral immune status, and its prognostic impact in esophageal cancer.

[Methods] IDO1 expression, cluster of differentiation (CD8) expression as a marker of cytotoxic T cells, forkhead Box P3 (FOXP3) as a maker of regulatory T cells were evaluated by immunostaining in 305 clinical samples of curative resected esophageal cancer.

[Result] In 305 clinical samples, high IDO1 high expression (n=71) was significantly associated with poor prognosis in overall survival(OS)(p=0.0034). In uni-and multivariate analysis, IDO1 high expression was an independent prognostic factor for OS. In those patients, CD8 low expression was also associated with poor prognosis in OS (p = 0.025). Furthermore, double immunostaning with IDO1 and CD8 revealed that CD8 expression levels was inverse correlated with IDO1 expression levels in those patients. In combination analysis of IDO1 and CD8 expression, IDO1 high expression and CD8 low expression group experienced worse overall survival (p=0.0024). Conversely, IDO1 low expression and CD8 high expression group was shown the best overall survival. In addition, IDO1 high expression group is correlate with the high counts of FOXP3 positive cells (p = 0.02).

[Conclusion] IDO1 expression was associated with an unfavorable clinical outcomes by induced immune tolerance and the classification of IDO1 and CD8 expression levels might serve as a predictive biomarker in esophageal cancer. The correlation between IDO1 and FOXP3 expression suggested that IDO1 involved in immune tolerance in esophageal cancer. These findings indicate that IDO1 would be a regulator of, and candidate therapeutic target in esophageal cancer.

#5701

Verification of the methodology for evaluating tumor-infiltrating lymphocytes in colorectal cancer.

Shinji Matsutani, Masatsune Shibutani, Kiyoshi Maeda, Hisashi Nagahara, Tatsunari Fukuoka, Tatsuro Tamura, Go Ohira, Sadaaki Yamazoe, Kenjiro Kimura, Takahiro Toyokawa, Ryosuke Amano, Hiroaki Tanaka, Kazuya Muguruma, Kosei Hirakawa, Masaichi Ohira. _Osaka City University Graduate School of Medicine, Osaka city, Japan_.

Background: The density of tumor-infiltrating lymphocytes (TILs) have been reported to reflect antitumor immune response and correlate with prognosis in malignancy. However, the methodology for evaluating the density of TILs by an immunohistochemical analysis differs among reports. The aim of this study was to verify the methodology for evaluating the density of TILs by immunohistochemical analysis and thereby identify the optimum methodology in clinical setting.

Methods: Three-hundred-thirteen patients who underwent curative operation for stage II/III colorectal cancer were enrolled. We retrospectively examined the density of TILs using immunohistochemical staining according to each method as follows: 1) subset of lymphocytes (i.e. CD4+/CD8+), 2) selected fields (i.e. at random or focusing on hot spots), 3) location in low-power field (i.e. the invasive margin [TILsIM] or the center of the tumor [TILsCT] or the surface of the tumor [TILsST]), and 4) location in high-power field (i.e. in tumor stroma [sTILs] or intra-tumor cells [iTILs] or total TILs [tTILs: sTILs+iTILs]). We then assessed the prognostic value of the density of TILsIM evaluated as described above. We also evaluated the correlation between the density of TILsIM and that of TILsCT/TILsST.

Results: In randomly selected fields, the densities of CD8+sTILsIM and CD8+tTILsIM were significantly associated with the survival. However, the densities of TILs evaluated via other methods were not associated with the survival. Furthermore, the density of CD8+TILsIM was significantly associated with that of CD8+TILsCT and CD8+TILsST.

Conclusions: We concluded that best and easiest way to evaluate the density of TILs in the clinical setting may be to assess the density of total CD8+TILs at the invasive margin in randomly selected fields.

#5702

Characterization of circulating tumor cells during immune checkpoint inhibition in metastatic renal cell carcinoma and malignant melanoma.

Tian Zhang,1 SinJung Park,2 Daniella Runyambo,1 Michael J. Poellmann,2 Marco Reyes-Martinez,1 Zahra Mahbooba,3 Rebecca Green,3 Carrie Lee,3 Daniel J. George,1 Andrew J. Armstrong,1 Seungpyo Hong,2 Andrew Z. Wang3. 1 _Duke Cancer Institute, Durham, NC;_ 2 _University of Wisconsin-Madison, Madison, WI;_ 3 _University of North Carolina-Chapel Hill, Chapel Hill, NC_.

Background: Although immune checkpoint inhibitors (ICIs) have shown efficacy in a range of solid tumors, there are no established circulating biomarkers able to identify early responders to treatment. Circulating tumor cells (CTCs) allow a non-invasive peripheral assessment of disease burden over time. We evaluated the prevalence of CTCs before and during treatment with ICIs.

Methods: Patients were enrolled prospectively at Duke Cancer Center and UNC Lineberger Cancer Center and selected for metastatic renal cell carcinoma (mRCC) and metastatic malignant melanoma (mMM) receiving standard of care immunotherapy. Using E-selectin for slowing cell rolling and a nanoparticle dendrimer based technology for cell capture with EpCAM, HER2, and EGFR, the CapioCyte platform was used for CTC capture at baseline, during treatment, and upon disease progression.

Results: We enrolled 5 subjects with mRCC and 4 subjects with mMM. Prior to treatment, subjects had a median of 293 CTCs/mL (all healthy volunteers (HVs, n=10) had <8 CTCs/mL). All subjects had more CTCs at baseline than HVs. One subject had CTCs rise within the first 4 weeks on ICI treatment, which corresponded to clinical disease progression. Several other subjects showed a rapid decrease in CTCs within 4 weeks but increased at the time of disease progression. In most cases of disease control, CTCs decreased by at least 50% at the 4-week timepoint and increased at time of disease progression.

Conclusions: CapioCyte is a sensitive platform to detect CTCs in mRCC and mMM. Based on CTC detection, many patients had early response to ICI treatment, but rapid progression corresponding to clinical progression. This pilot data suggest that CTCs can be a valuable biomarker for monitoring the clinical benefit of cancer immunotherapies.

#5703

Immunoscore as prognostic biomarkers in patients with epithelial ovarian cancer.

Shin-Wha Lee,1 Min-Je Kim,2 Ju-hyun Kim,1 Young-Jae Lee,1 Yong-Man Kim,1 Yong-Man Kim1. 1 _University of Ulsan, ASAN Medical Center, Seoul, Republic of Korea;_ 2 _ASAN Institute for Life Science, Seoul, Republic of Korea_.

Background: The analysis of single parameters alone may not provide sufficient insights about complex immune system-tumor interactions. This study is to validate the immune contexture as prognostic biomarkers in high-grade serous ovarian cancer (HGS-OC) and to find new era of immunoscore in HGS-OC.

Methods: We collected FFPE samples from 187 patients with HSOC and produced TMA samples. We accomplished the OPAL multiplex IHC assay for the quantitative analysis of immune markers, including CD4, CD8, CD20, FoxP3, PD-L1, and CK. Multiplex Biomarker Imaging and inForm® Image Analysis Software was used.

Results: FIGO stage III and IV patients were 84.5% (158/187). The optimal debulking surgery was done in 66.8% (125/187). The 3-year disease-free survival and 5-year overall survival were 35.1% and 50.0%, respectively. Any single marker was not related to the survival, including CD8, FoxP3, and PD-L1. However, high CD8:FoxP3 and CD8:PD-L1 ratios were correlated with good survival. In Cox regression model, the risk factors for HGS-OC survival were FIGO stage (HR 1.784, 95% CI: 1.295-2.457, p<0.001) and platinum resistance (HR 4.257, 95% CI: 2.753-6.582, p<0.001). Additionally, CD8:PD-L1 ratio was a favorable prognostic factor (HR 0.621, 95% CI: 0.042-0.917, p=0.017).

Conclusion: These findings indicate that although any single immune marker is not related to the survival, CD8:FoxP3 and CD8:PD-L1 ratios provide the positive correlation with the prognosis in HGS-OC; especially, CD8:PD-L1 ratio is prognostic biomarker that is comparable to clinical biomarkers. The next study for immunoscore is necessary to define immunoscore in ovarian cancer.

#5704

Mass cytometry identifies the expansion, persistence, and immune checkpoints of adoptively transferred memory-like NK cells in patients with leukemia.

Melissa M. Berrien-Elliott, Julia A. Wagner, Rizwan Romee, Michelle Becker-Hapak, Timothy Schappe, Carly Neal, Ethan McClain, John DiPersio, Peter Westervelt, Amanda F. Cashen, Todd A. Fehniger. _Washington University School of Medicine, Saint Louis, MO_.

NK cells are an emerging cell therapy for cancer, however, the optimal approaches to maximize NK cell anti-tumor attack are unclear. NK cells exhibit memory-like (ML) properties following combined cytokine (IL-12/15/18) pre-activation, evidenced by enhanced responses to cancer cells upon re-stimulation weeks later. A first-in-human clinical trial for acute myeloid leukemia (AML) (Romee R et al., Sci Transl Med, 2016) revealed that 7 of 11 (54%) evaluable patients responded to ML NK cell therapy. To inform key aspects of response, we used mass cytometry to track ML NK cell diversity, checkpoints, and effector functions in AML patients treated with ML NK cells. Multidimensional analyses (viSNE) of patient samples collected 7 days after NK transfer accurately identified in vivo-differentiated ML NK cells that were distinct from conventional NK (cNK) cells: CD56hiCD11bloCD62L+ NKG2AhiNKp30hi Ki-67+ (cNK: 3%±0.5% vs. ML: 87%±5%, mean±SEM within ML gate, P<0.05, N=10). In a second clinical trial of MHC-haploidentical hematopoietic transplantation (HCT), augmented with same-donor IL-12/15/18 activated NK cells (NCT02782546), mass cytometry identified marked ML NK cell expansion in vivo in this immune-compatible environment. In the first two patients treated, ML NK cells expanded (>1000-fold expansion in vivo, peak >2000 cells/uL blood), persisted >= 60 days, and were distinct from immature CD56brightKIR-CD16- NK cells developing from the graft. These ML NK cells exhibited potent anti-leukemic functional responses at day +28. Utilizing the phase 1 study cohort, Citrus analysis identified increased NKG2A expression as significantly correlated with treatment failure [median NKG2A = 89±25 (treatment failure); 8±3 (clinical response); p=0.007, FDR<0.1]. NKG2A is an inhibitory receptor that binds to non-classical MHC HLA-E expressed on AML. We hypothesized that NKG2A/HLA-E interactions in vivo represent a key checkpoint on ML NK cell responses. Consistent with this idea, HLA-Ehi AML blasts resulted in reduced ML NK cells responses (P<0.05) in vitro. ML NK cells also triggered with HLA-E+ K562-AML in the presence of control or anti-NKG2A blocking antibodies. Increased functional responses including IFN-γ (p=0.02) and TNF (p=0.05) production by NKG2A-blocked ML NK cells were detected, compared to isotype-treated ML NK cells. Similar results were observed with HLA-E+ primary AML blasts as targets, showing that ML NK cells treated with NKG2A blockade produced significantly more IFN-γ (p=0.001). Thus, mass cytometry identified that in vivo-differentiated ML NK cells are distinct from cNK cells, and exhibit marked expansion and persistence in an immune-compatible environment. NKG2A was identified as a key ML NK cell checkpoint in vivo, and blockade of NKG2A signals may enhance the clinical efficacy of ML NK cell therapy for AML patients.

#5705

The key role of kynurenine in anti-PD-1 failure.

Andrea Botticelli,1 Bruna Cerbelli,2 Luana Lionetto,1 Ilaria Zizzari,2 Annalina Pisano,2 Michela Roberto,1 Elisa Onesti,1 Francesca Romana DI Pietro,1 Chiara Napoletano,2 Laura Pizzuti,3 Patrizia Vici,3 Giulia D'Amati,2 Federica Mazzuca,1 Maurizio Simmaco,1 Marianna Nuti,2 Paolo Marchetti1. 1 _Sant'Andrea Hospital, Sapienza University of Rome, Rome, Italy;_ 2 _Sapienza University of Rome, Rome, Italy;_ 3 _Division of Medical Oncology 2, Regina Elena National Cancer Institute, Rome, Italy, Rome, Italy_.

Background: Immune checkpoint inhibitors have revolutionized treatment and outcome of severe and often fatal disease, as metastatic lung cancer, demonstrating long-term tumor control and extended patient survival. Unfortunately, only 25-30% of patients have a long-term benefit from immunotherapy, while the remaining 70-75% demonstrate primary or acquired resistance. Recently, indoleamine 2,3-dioxygenase (IDO) has been proposed as a possible mechanism of resistance to anti-PD1 treatment. Indeed, IDO catalyzes the degradation of tryptophan (Trp) into kynurenine (Kyn), which seems to enhance the activity of Treg, leading to an immunosuppressive microenvironment.

Methods: The serum concentrations of Trp and Kyn were measured by high-performance liquid chromatography tandem mass spectrometry in 26 patients affected by non-small cell lung cancer (NCLSC) before the start of the second-line therapy with nivolumab. The IDO activity was expressed with Kyn/Trp ratio. The associations between Kyn/Trip ratio and early progression, PS, age, sex, brain metastases and pleural effusion were analyzed using Spearman test and Mann Whitney test.

Results: 14 out of 26 patients (54%) presented early progression (defined as progression of the disease within 6 months from the beginning of nivolumab treatment). The median value of Kyn/Trp ratio was 0.073 (0.024-0.18). We found a significant association between Kyn/Trp ratio and early progression (p= 0.009), while no statistical associations were found between Kyn/Trp ratio and PS, age, sex, brain metastases and pleural effusion. Indeed, patients with early progressive disease presented a median value of Kyn/Trp ratio significantly higher than other patients (0.094 vs 0.052; p= 0.01).

Conclusion: The pretreatment evaluation of IDO activity, expressed as Kyn/Trp ratio, seems to be associated with response to immunotherapy. In particular, higher Kyn/Trp ratio could predict resistance to anti-PD-1 treatment. These preliminary results suggest the possibility of using anti-PD-1 plus IDO inhibitor in patients with high level of Kyn/Trp ratio.

#5706

A blood-based next-generation sequencing assay to determine tumor mutational burden (bTMB) is associated with benefit to an anti-PD-L1 inhibitor, atezolizumab.

David Fabrizio,1 Daniel Lieber,1 Christine Malboeuf,1 Jacob Silterra,1 Emily White,1 Michael Coyne,1 Tina Brennan,1 Jie Ma,1 Mark Kennedy,1 Erica Schleifman,2 Sarah Paul,2 Yan Li,2 David Shames,2 Craig Cummings,2 Eric Peters,2 Marcin Kowanetz,2 Doron Lipson,1 Geoff Otto1. 1 _Foundation Medicine, Cambridge, MA;_ 2 _Genentech, South San Franscisco, CA_.

Background: Tumor mutational burden (TMB) has emerged as a new biomarker for response to checkpoint inhibitor immunotherapy. Recently, we described a novel assay to calculate TMB from the circulating tumor DNA (ctDNA) in the blood (bTMB) and demonstrated validation to support its application in a prospective phase III trial in 1L NSCLC comparing the anti-PD-L1 agent, atezolizumab, against standard of care chemotherapy. Herein, we demonstrate clinical utility of the bTMB assay across a retrospective study of 794 NSCLC patients from two randomized clinical trials evaluating progression free survival (PFS) and overall survival (OS) between atezolizumab and chemotherapy. Furthermore, we compare TMB between tissue and blood, and evaluate variant-level concordance between the bTMB assay and the CLIA-validated ctDNA assay, FoundationACT (FACT).

Methods: Cell free DNA (cfDNA) from plasma was isolated from retrospective clinical samples. The bTMB assay delivered a count of somatic base substitutions in the ctDNA down to 0.5% allele frequency across 394 genes from as little as 1% tumor content, yielding a bTMB score. Tissue TMB was evaluated from formalin-fixed, paraffin embedded specimens using the FoundationOne assay.

Results: Patients with bTMB ≥14 mut/Mb were considered high, and demonstrated significant enrichment for both PFS and OS in the phase II POPLAR study evaluating atezolizumab vs. chemotherapy (PFS HR = 0.57, OS HR = 0.56, n=211). Survival results were validated in the larger phase III study, OAK (PFS HR = 0.65, OS HR = 0.64, n=583). Significant enrichment in PFS was observed between bTMB-high and non-high patients receiving atezolizumab, however this effect was not observed for patients receiving chemotherapy. Amongst 259 patients with blood and tissue TMB, overall and positive percent agreement (PPA) in the TMB categorical result were 81.5% and 63.6%, respectively. When bTMB was restricted to the 62 gene FACT assay, the PPA dropped to 17.0%, suggesting a sufficiently sized panel is required to sensitively identify patients with high TMB. When samples were TMB-high in blood, the median percent overlap of TMB variants between blood and tissue was 70%. Blood samples tested in both the bTMB and FACT assays revealed that 93% of variants were detected in both assays across overlapping regions, with ≥99% of variants detected in both platforms with variant allele frequency ≥1.0%.

Conclusions: The bTMB assay is analytically validated for high accuracy and precision, and demonstrates a high degree of variant level concordance with the FACT assay. High bTMB is significantly associated with improved survival to atezolizumab vs. chemotherapy in 2L NSCLC, and the survival benefit is specific to atezolizumab and not prognostic. Finally, TMB between blood and tissue is correlated and largely explained by overlapping variants present in both sample types.

#5707

**On-treatment changes in CRP as a surrogate for overall survival for atezolizumab benefit in 2** nd **line NSCLC.**

Namrata Patil, Wei Zou, Simonetta Mocci, Vinita Gupta, Luciana Molinero, Alan Sandler, Marcus Ballinger, Susan Flynn, Marcin Kowanetz, Priti Hegde. _Genentech, Inc., South San Francisco, CA_.

Background: Overall survival (OS) is an optimal endpoint for evaluation of checkpoint inhibitors (CPI). Identification of early surrogates of OS is important to inform early clinical signals and treatment decisions. We evaluated C-reactive protein (CRP) in clinical trials with atezolizumab (anti-PD-L1 antibody). CRP is elevated in different inflammatory and malignant conditions and is often considered to be a marker of poor prognosis.

Materials and Methods: Serum from patients enrolled in the 2L+ NSCLC studies POPLAR and OAK (randomized phase 2 and phase 3 controlled trials of patients with non-small cell lung cancer (NSCLC) who progressed on post-platinum chemotherapy) was analyzed for CRP levels over time. Patients were binned by changes in CRP at 6 weeks post-treatment (C3D1; Cycle 3 Day 1) as compared to pre-treatment levels, by either greater than 1.5 fold, less than 1.5 fold or unchanged (within +1.5 fold).

Results: C3D1 decreases in serum CRP were associated with RECIST1.1 based responses to atezolizumab but not with docetaxel in the POPLAR study, and these findings were further validated in the OAK study. Patients with RECIST based disease progression (PD) demonstrated an increase in serum CRP as early as Cycle 2 (3 weeks post atezolizumab therapy). Notably, decrease in CRP levels was strongly associated with improved PFS and OS. Results from OAK are shown below (table). Furthermore, in patients with RECIST PD or stable disease (SD), decrease in on-treatment CRP also predicted long overall survival.

Conclusion: CRP is a soluble biomarker that is modulated post-atezolizumab treatment. This simple lab test may provide utility in informing OS benefit for CPIs and combinations.

C3D1 CRP fold change relative to pre-treatment | HR | CI | p value | HR | CI | p value

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

ITT | 0.75 | 0.62-0.91 | 0.004 | 0.74 | 0.6-0.91 | 0.0053

CRP <1.5 FC | 0.48 | 0.33-0.69 | 0.000095 | 0.49 | 0.32-0.75 | 0.0011

CRP +1.5 FC | 0.85 | 0.62-1.15 | 0.28 | 0.61 | 0.43-0.86 | 0.0049

CRP>=1.5 FC | 0.96 | 0.69-1.34 | 0.81 | 1.25 | 0.86-1.81 | 0.25

#5708

Associations between soluble immune checkpoint molecules and overall survival in advanced non-small cell lung cancer (NSCLC) patients treated with either anti-PD-1/-L1 monoclonal antibodies or single-agent chemotherapy.

Imad A. Tarhoni,1 Ibtihaj Fughhi,1 David Gerard,1 Sanjib Basu,1 Cristina Fhied,1 Wen-rong Lie,2 Donna Russell,2 Marta Batus,1 Nisha Thakar,1 Philip Bonomi,1 Mary Fidler,1 Jeffrey Borgia1. 1 _Rush University Medical Center, Chicago, IL;_ 2 _Millipore-sigma, Burlington, MA_.

Background: While several of the KEYNOTE trials showed that NSCLC patients whose tumors expressed high levels of the programmed death ligand-1 (PD-L1) were more likely to have superior survival with pembrolizumab compared to chemotherapy, other trials failed to confirm the predictive value of PD-L1 tissue expression. And with response rates for PD-1/-L1 directed immunotherapy approximating 20%, development of improved molecular diagnostics to accurately identify patients likely to have long-term disease control is needed. The goal of the current study is to evaluate potential relationships between a panel of soluble immune checkpoint molecules and disease control in NSCLC patients treated with PD-1/-L1 monoclonal antibodies. Method: Pretreatment sera from 128 cases of advanced NSCLC that failed frontline chemotherapy were evaluated for 16 soluble checkpoint molecules and immune regulators using the Human Immuno-Oncology Checkpoint Protein Panel (MilliporeSigma). This panel consists of the following targets: BTLA, CD27, CD28, TIM-3, HVEM, CD40, GITR, GITRL, LAG-3, TLR-2, PD-1, PD-L1, CTLA-4, CD80/B7-1, CD86/B7-2, and ICOS. All patients tested received either PD-1/-L1 targeting checkpoint inhibitors (nivolumab, atelizumab, or pembrolizumab; n=79) or single-agent chemotherapy (n=49). All kits were processed according to manufacturer-defined protocols and read using a Luminex® FLEXMAP 3D®. Finally, statistical relationships were determined using the Log-Rank test in relation to overall survival, defined as the interval from diagnosis to last follow up or death. Results: Pretreatment sera from 128 cases of NSCLC were evaluated for 16 soluble checkpoint molecules and immune regulators using the Luminex immunobead platform. In analysis of the cohort, lower levels of CD80/B7-1 were found to have significant association (p=0.0372) with superior overall survival. Patients who received PD-1/L1-directed immunotherapy, however, demonstrated low circulating levels of the T-cell associated molecules CD28 and CD80/B7-1, which were associated with superior overall survival (p=0.0178 and 0.036, respectively), whereas low circulating levels of the tumor-associated molecules LAG-3 and CD86/B7-2 were associated with an inferior overall survival (p=0.009 and 0.0278, respectively). There were no significant associations (all p>0.05) identified in the cohort that received chemotherapy alone. Conclusion: These findings suggest that soluble immune-checkpoint molecules may identify advanced NSCLC patients most likely to benefit from anti-PD-1/-L1 immunotherapy. Prospective study of these biomarkers is planned to determine if they have predictive value for anti-PD-1/-L1 therapy and to explore implications for developing combination immunotherapy regimens.

#5709

Effect of IL-18 on the expansion and phenotype of human natural killer cells.

Hiroaki Senju,1 Asuka Kumagai,2 Yoichi Nakamura,3 Shinnosuke Takemoto,4 Masaaki Fukuda,5 Hiroshi Gyotoku,1 Katsumi Nakatomi,1 Minoru Fukuda,1 Hiroshi Mukae,1 Yoshimasa Tanaka2. 1 _Nagasaki University Hospital, Nagasaki, Japan;_ 2 _Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan;_ 3 _Tochigi Cancer Center, Tochigi, Japan;_ 4 _Nagasaki Medical Center, Nagasaki, Japan;_ 5 _Nagasaki Genbaku Hospital, Nagasaki, Japan_.

When pathogenic stresses are recognized by innate immune cells, inflammasomes are assembled and caspase-1 is activated, resulting in the conversion of pro-IL-18 into mature IL-18. Because natural killer (NK) cells express IL-18 receptors, IL-18 may play roles in the immune functions of NK cells. In the present study, we examined the effect of IL-18 on NK cells derived from lung cancer patients and healthy adult volunteers. When peripheral blood NK cells were stimulated with IL-2, the cells formed clusters beginning on day 5-6 and proliferated thereafter, in which the number of NK cells increased by 10-fold in 10 days. When IL-18 was added, cell clusters were observed as early as on day 4 and NK cells proliferated vigorously. On day 10, the expansion rate was 56-fold on average, showing that IL-18 promoted the expansion of NK cells. It was also notable that IL-18 enhanced the expression of CD80, CD86, HLA-DR and HLA-DQ on NK cells, suggesting that IL-18 conferred NK cells an APC-like phenotype. When cellular cytotoxicity was determined, APC-like NK cells efficiently killed tumor cells and the anti-tumor activity was augmented by the addition of tumor antigen-specific mAbs. In addition, IFN-γ was produced by APC-like NK cells in response to tumor cells, and the cytokine production was further enhanced by mAbs. Taken together, IL-18 not only promoted the expansion of NK cells, but also changed the phenotype of NK cells, and the IL-2/IL-18-induced NK cells might serve as a bridge between innate immunity and adaptive immunity and be useful for cancer immunotherapy.

#5710

Molecular profiling of anti-PD-1 treated melanoma patients reveals importance of assessing neoantigen burden and tumor escape mechanisms for clinical treatment.

Jie Wang,1 Sean Michael Boyle,1 Christina Lee,2 Eric Levy,1 Zeid Rusan,1 Sekwon Jang,3 Richard Chen1. 1 _Personalis, Menlo Park, CA;_ 2 _Georgetown University, DC;_ 3 _Inova Schar Cancer Institute, VA_.

Despite the remarkable response of some melanoma patients to checkpoint inhibitor therapy, significant numbers of patients do not achieve complete response. To understand this differential response, there is an increasing interest in identifying biomarkers and mechanisms that influence immunotherapy effectiveness. In this study, we characterize the immuno-genomics of tumors from a series of melanoma patients that have received anti-PD-1 checkpoint inhibitors to assess potential factors influencing response.

To better understand mechanisms of anti-PD-1 response, we sequenced and genomically profiled tumors from 19 stage III and IV melanoma patients where response was evaluated using RECIST criteria. Of the 19 patients, there were 5 complete responders (CR), 8 partial responders (PR), and 6 progressive disease (PD) patients. Immuno-genomic profiling was performed using Personalis' ACE ImmunoID platform, an augmented exome/transcriptome platform and analysis pipeline that allows for assessment of tumor mutations, neoantigens, HLA typing, gene expression quantification, tumor micro-environment, and tumor escape mechanisms. The molecular information for each of the 19 melanoma patient samples was then analyzed together with the corresponding clinical response to anti-PD-1 therapy.

We identified 3 outlier patients, which, while having very high neoantigen burden, did not achieve complete response (2 PR & 1 PD). One of these patients had extremely high expression of IDO1, which may facilitate immune escape in a PD-1 independent manner. Two independent HLA mutations in HLA-A and HLA-B (stop-gain mutation and splice site mutation, respectively) were found in the second patient, leading to the likely loss of surface expression of two classes of HLA-A and HLA-B proteins. If these three high neoantigen burden individuals with proposed tumor escape mechanisms are removed from consideration, we found a highly significant association between neoantigen burden and response to anti-PD-1 therapy (PD + PR vs CR, P = 0.00046). We also observed that, in our cohort, response to anti-PD-1 therapy was more accurately predicted by neoantigen burden than mutational burden.

In conclusion, we observed a strong correlation between response to anti-PD-1 therapy in melanoma patients and neoantigen burden when tumor escape mechanisms are considered. In our patients, we saw highly suggestive resistance mechanisms that involve perturbations to elements of the antigen presenting machinery and checkpoint blockade. This highlights the potential importance of broad immuno-genomic profiling of patients that are candidates for receiving immunotherapy. We are continuing to increase our cohort size to observe both how well the neoantigen burden holds to anti-PD-1 response and to identify additional mechanisms for immune evasion.

#5711

The impact of combination oral azacitidine (CC-486) + pembrolizumab (PEMBRO) on the immune infiltrate in metastatic melanoma (MM).

Emily Z. Keung, Isabella C. Glitza, Elizabeth Burton, Rodabe N. Amaria, Sapna P. Patel, Adi Diab, Cassian Yee, Michael K. Wong, Wen-Jen Hwu, Patrick Hwu, Scott E. Woodman, Michael T. Tetzlaff, Nallely Trujillo-Conley, Denai R. Milton, Michael A. Davies, Kunal Rai, Irina Fernandez, Jorge M. Blando, Luis M. Vence, Padmanee Sharma, James P. Allison, Jennifer A. Wargo, Hussein Tawbi. _MD Anderson Cancer Center, Houston, TX_.

Introduction: Although immune checkpoint inhibitors have improved survival for many with MM, most pts do not respond and median PFS is only 6 months for single agent therapy. Cancers subvert the cellular epigenetic machinery to facilitate immune escape. Epigenetic mechanisms of resistance are potentially reversible by DNA hypomethylating agents (HMA). Based on preclinical models, we hypothesized that CC-486, an oral HMA, will enhance response to PEMBRO in PD-1 naïve pts and reverse resistance in anti-PD-1 refractory pts. The aim of this study is to determine the safety, efficacy, and characterize the pharmacodynamic impact of CC-486 + PEMBRO on immune infiltrates in pts with MM.

Experimental: NCT02816021 is an ongoing phase II study of CC-486 + PEMBRO in MM pts who are PD-1 naïve (Arm A) or who have progressed on prior PD-1 directed therapy (Arm B). Pts receive 300mg PO of CC-486 on days 1-14 and 200mg IV of PEMBRO q3 weeks. Serum and tumor biopsies are obtained at baseline, prior to cycles 3 and 5. Immune monitoring studies were performed by the Immunotherapy Platform at MD Anderson. Immune cell phenotyping by CyTOF was performed using 36 metal-conjugated antibodies targeting myeloid and T cell surface markers. FCS files were exported and manually gated for lymphocytes using FlowJo (version 10.1) and subjected to multidimensional phenographic analysis.

Results: Thirteen pts (Arm A, n=6; Arm B, n=7) have been enrolled. Two of the 3 pts remaining on study are PD-1 naïve and have received 13 and 8 cycles of CC-496 + PEMBRO with partial responses by RECIST1.1 at 6 months, respectively. One pt on Arm B remains on study with stable disease after 4 cycles. The combination was considered safe after a run-in phase (6 pts/arm treated without DLTs) and the study is open to full accrual. The most common grade 3/4 AEs were leukopenia , neutropenia, vomiting, and diarrhea (2 each) with 1 grade 5 AE unrelated to treatment (hepatic rupture/bleeding due to progressive disease). Serum and tumor biopsies from 6 pts (3 per Arm) were available for interim analysis, with additional samples in process. Of these 6 pts, 1 pt (PD-1 naïve) responded to therapy by RECIST1.1. We evaluated the blood and tumor samples by CyTOF. In the tumor samples, we observed an increase in frequency of T cells in 2 PD-1 naïve patients but did not observe similar changes in PD-1 refractory patients in this small cohort. Similar data was found with immunohistochemistry. These changes were not observed in the blood samples.

Conclusion: The regimen CC-486 + PEMBRO is not marrow suppressive and is well tolerated. Changes in the peripheral lymphocyte cell populations upon treatment are not necessarily concordant with changes occurring in the tumor. Analysis of collected samples is ongoing and will be presented at the meeting, and will help corroborate initial findings and yield further insight into the effect of this combination on the immune response.

#5712

Identification of a highly suppressive Treg subset associated with immunotherapy response.

Teresa Troiani,1 Simona Romano,2 Paolo D'Arrigo,2 Anna Rea,2 Martina Tufano,2 Emilio F. Giunta,1 Giuseppe Matarrese,2 Claudio Procaccini,2 Nunzia Novizio,2 Vincenza Vigorito,2 Deriggio Faicchia,2 Giuseppe Argenziano,1 Fortunato Ciardiello,1 Maria F. Romano2. 1 _Università degli studi della Campania Luigi Vanvitelli, Napoli, Italy;_ 2 _Università di Napoli Federico II, Napoli, Italy_.

Melanoma often exploits Treg to avoid immune attack. Treg is a heterogeneous population with respect to immunosuppressive capability. Lymphocytes are particularly rich in FKBP51 (encoded by FKBP5 gene), known as the receptor for FK506. Melanoma aberrantly expresses this protein, which sustains resistance and invasion. Melanoma/immune cell interaction, through PD-L1/PD1, bidirectionally generates FKBP5 splicing inducing a lower molecular weight form termed FKBP51s. In 64 advanced melanoma patient PBMCs, we found that FKBP51s marked a Treg subset that correlated to anti-CTLA4 response. More precisely, a Treg FKBP51s+ count <1% was associated with unresponsiveness (normal donor-range 0.1-0.7%). Aim of the present study was to assess the role of Treg FKBP51s+ as potential biomarker of response in a different cohort of patients treated with anti-PD1. In addition, the suppressive potential of Treg FKBP51s+ in comparison with that of Treg FKBP51s- is investigated. To date, we have outcomes of 11 patients. For each patient, we have collected up to 10 blood samples, at T0 and before each treatment, to monitor Tregs. In 5 responder patients, Treg FKBP51s+ was >1.2 and <4.8; in 5 non responder patients, the count was >0.04 and <0.8 After a transient increase following the first administration, the count decreased to 0.3+0.2% in responder patients. Interestingly, a patient with count =0.7% developed autoimmune side effects that led to therapy discontinuation. Resolution of side effects was accompanied by a value increase to 9.9%; anti-PD1 re-administration was then successful. In vitro iTreg generation showed that FKBP51s was upregulated in Treg CD25high, Ki67high and p70S6khigh, corresponding to a highly metabolically active profile with strong suppressive capability. In conclusion, melanoma patients that benefit from immune checkpoint targeted therapy are recognizable by an expansion of a Treg subset which is marked by FKBP51s, a splicing protein isoform generated by triggering of surface antigens (PD-L1, PD1), abundantly expressed on highly suppressive Tregs.

#5713

Promising predictive biomarkers for immunotherapy in metastatic melanoma.

Marta Nyakas, Elin Aamdal, Tormod Guren, Steinar Aamdal, Kari Dolven Jacobsen, Paal Brunsvig, Kristin Austlid Tasken, Gunhild Mælandsmo, Arne Yndestad, Bente Halvorsen, Paal Aukrust, Thor Ueland. _Oslo University Hospital, Oslo, Norway_.

Aim: to explore biomarkers in order to predict outcome in patients with metastatic melanoma who have had immunotherapy with ipilimumab in a real-world setting. Metastatic melanoma is a very aggressive, incurable cancer with historically few therapeutic options and poor survival. Immunotherapy represents a revolution for metastatic melanoma treatment but there is a lack of biomarkers to predict treatment response. Material and methods: In the Norwegian National Phase 4 Multicenter Study, IPI4, 150 patients were included to receive ipilimumab (anti-CTLA3). A subgroup of 73 patients (4 screening failure) was included at Oslo University Hospital -The Norwegian Radium Hospital. Serum was available from 56 patients of this subgroup and were examined before and during ipilimumab treatment concerning possible predictive biomarkers. Expression of a panel of 17 inflammatory markers reflecting different inflammatory pathways including extra cellular matrix remodeling and fibrosis, vascular inflammation, notch signaling, inflammation in general and monocyte/macrophage activation were measured at baseline and at the 2nd and/or 3rd treatment with ipilimumab. Results: During an average 33.7 months follow-up, 33 (59%) patients died. Six promising candidates (endostatin, osteoprotegerin, C-reactive protein, pulmonary and activation-regulated chemokine and galectin-3 binding-protein) were higher in non-survivors. In particular, high endostatin and galectin-3 binding protein levels were independently associated with poor long time survival also in adjusted analysis (age, gender, lactate dehydrogenase). A 1 standard deviation (SD) increase in Gal3BP gave a 1.8 x times higher risk of death (95% CI 1.10-2.95, p=0.019) while a 1 SD increase in endostatin was associated with a 2x higher risk of death (95% CI 1.12-3.64, p=0.020) in the final model. Conclusion: Endostatin and galectin-3 binding protein may represent biomarkers for prognosis during immunotherapy with ipilimumab and should be further evaluated.

#5714

SPADE identification of novel MDSC subsets.

Kathryn Cole, Holly Britton, Phyllis Warkentin, James E. Talmadge. _Univ. of Nebraska Medical Ctr., Omaha, NE_.

Myeloid derived suppressor cells (MDSC's) are a heterogeneous population of myeloid cells at varying stages of differentiation that occur in increased numbers in the blood of cancer patients. The phenotypes of MDSC's, are controversial but are generally subdivided into three phenotypes: macrophage (M-), granulocytic (G-) and immature (i-) MDSC's. Our studies used a single staining tube with antibodies to linage markers (CD3, CD19 and CD56), HLA-DR, CD11b, CD14, CD15, CD16, CD33, CD34, CD45, PD-L1 and lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1). Mobilized apharesis products from lymphoma patients provided a product with a high frequency of MDSCs compared to normal donor blood samples. A total MDSC population (Lin-HLA-DR-CD11b+CD14+ and Lin-HLA-DR-CD11b+CD14-CD33+) was exported from FlowJo to Spanning tree progression analysis of density normalized events (Spade) in Matlab. This analysis identified nine MDSC sub-populations as nodes. Impressively, cells from the non-MDSC populations, including monocytes and granulocytes did not occur within these nodes. The SPADE analysis identified four M-MDSC nodes, four G-MDSC nodes and one iMDSC node. LOX-1 expression was limited to two G-MDSC and one M-MDSC subset. The nodes were differentiated based on CD14, CD15, CD33, CD16, LOX-1, and CD34 expression. The total populations were SPADE analyzed by overlaying non-MDSCs populations onto MDSCs and monocytes and granulocytes subset to nodes independent of MDSC nodes. The assessment of MDSC function used intracellular arginase-1 and nitric oxide synthetase-2 expression with the expected differential expression consistent with the M and G-MDSC subsets. The proportions of the different MDSC nodes varied between cancer patients and were generally at least a log higher than in the blood of normal donors. In summary, a SPADE analysis can cleanly identify MDSCs from monocytes and granulocytes and provide insight into novel MDSC subsets.

#5715

Monitoring immune cells-mediated cytolysis through impedance technology allows kinetic analysis of reagent efficacy.

Fabio Cerignoli, Biao Xi, Garret Guenther, Lincoln Muir, Brandon Lamarche, Jason Shields, Yama Abassi. _ACEA Biosciences, Inc., San Diego, CA_.

In vitro characterization of immunotherapy reagents efficacy and combination synergies is necessary before moving to expensive animal models and clinical studies. Current standard assays like Chromium-51 release, ATP-based luminescence cell monitoring or flow cytometry are difficult to implement in a high throughput environment and are based on end point methodologies that are unable to capture the full dynamic of the immune response. Here we validate an impedance-based platform for monitoring cytotoxic activity of immune cells in the context of cancer immunotherapy assays. The technology detects cell death and proliferation of adherent cells by measuring changes in conductance of microelectrodes embedded in 96 and 384-wells cell culture plates, without the use of labeling or cell modification. We present a large set of validation including CD19 Car T models, EpCAM/CD3 BiTE antibodies and checkpoint combination therapies with anti PD-1/PD-L1 and CTLA-4 antibodies. Data comparison with Annexin V staining/Flow Cytometry shows perfect correlation between the drop in impedance signal and % of apoptotic cells. Long time monitoring (> 7 days) in combination with low effector:target ratios reveal the occurrence of resistance mechanisms in target cells and exhaustion in effector cells. Such dynamic response has been observed in animal models and patient clinical trials, but it is difficult to monitor in short time assays. Overall, our results demonstrate the value of such label-free approach in measuring the cytotoxic response across the temporal scale, an aspect that is otherwise very difficult to assess with more canonical end point assays but that is highly relevant when testing combinations. Thanks to the availability of 384-wells format and minimal sample handling, the technology is ideally suited for applications in large reagent validation screening or therapeutic protocol validation directly on patient samples.

#5716

Functional evidence for an immunosuppressive role of kynurenine in cancer patients.

Alban Bessede,1 Antoine Italiano,2 Assia Chaïbi,1 Christophe Rey,1 Imane Nafia,1 Sylvestre le Moulec,2 Sophie Cousin,2 Maud Toulmonde,2 Céline Auzanneau,2 Marina Pulido2. 1 _ImmuSmol, Pessac, France;_ 2 _Institut Bergonié, Bordeaux, France_.

Background: While being neglected for decades, tumor immune escape is now considered as a new paradigm that promotes tumor growth and evasion. Besides the prototypical PD1 / PDL1 axis, several mechanisms has been identified as important actor in immune editing process. Indoleamine 2,3 dioxygenase (IDO1), a first rate-limiting enzyme of the Kynurenine pathway, has been described to favor tolerance and immune escape through the production of a series of metabolites including L-Kynurenine. However, functional evidences have not yet been clearly demonstrated.

Methods: We reconstituted in vitro a human immune - tumor cell synapse where ability of primary immune cells to promote tumor cell death has been kinetically followed through fluorescent-based live cell imaging in the presence and absence of L-Kynurenine. Two different donors as source of Peripheral Blood Mononuclear Cells (PBMCs) and three different tumor cell lines - namely IB115 and IB136 as sarcoma models & H1299 as reference lung adenocarcinoma model - have been used in this study. Finally, in order to determine cancer subtypes that might be highly exposed to Kynurenine pathway dysregulation and associated immune dysfunction, a comprehensive analysis of Kynurenine and Tryptophan levels was done in a series of 691 patients with advanced solid tumors

Results: L-Kynurenine demonstrated a clear propensity to limit the immune cell-mediated tumor apoptosis elicited by TCR ligation - an event that was consistent within the two different donors and the three different cell lines. Interestingly, this immune suppressive feature of L-Kynurenine was associated with a limited amount of gamma-Interferon (IFNγ) in the cell culture supernatant while no effect was observed on Tumor Necrosis Factor alpha (TNFα). It's worth to note that L-Kynurenine had no effect on tumor cells without immune cells thus suggesting that Kynurenine has direct action on immune cells through which it limits their anti-tumor function -The Kyn/Trp ratio in cancer patients was significantly higher than that reported in healthy subjects (median: 71.2, range: 5.8-2825.2). Median value was the highest in breast cancer (126) and the lowest in colorectal (54) and pancreatic cancer (51) patients. No correlation with IDO1 tumor expression neither with systemic markers of inflammation (C-reactive protein) was observed.

In conclusion, these results represent the first large description of the Kynurenine/Tryptophan ratio levels in cancer patients and the first functional evidence of a direct inhibition by Kynurenine of lymphocytes's effector functions.

### Neoantigens in Cancer

#5717

Mass spectrometry as a tool for MHC class I and II neoantigen discovery.

Michael J. Ford,1 Richard Jones,1 David Allen,1 Ravi Amunugama,1 Michael Pisano,2 James Mobley,2 Paul Domanski,2 Bill Ho,2 Daniel Bochar,2 Melissa Holt2. 1 _MS Bioworks, Ann Arbor, MI;_ 2 _Cayman Chemical, Ann Arbor, MI_.

A neoantigen is a patient-specific tumor antigen resulting from mutations during oncogenesis (1). Advances in genome sequencing of cancer tumor tissues combined with bioinformatics have enabled the identification of tumor specific mutations and the prediction of the peptides that will be presented by the MHC complex. The presented peptides are recognized as foreign by the immune system and can be used to discriminate cancerous from normal cells. Neoantigen prediction by gene sequencing and in silico approaches can be strengthened and further supported by mass spectrometry. The molecular level characterization of peptides associated with molecules of the major histocompatibility complex requires a targeted protein complex enrichment, an unbiased peptide elution, and finally a peptide analysis method. We use immunoaffinity to isolate the target complex, elute the peptides under conditions minimizing protein contamination and finally analyze the peptides by mass spectrometry. Here we present a case studies of our recent work applying workflows for the analysis of peptides associated with both Class I and Class II MHC molecules. Combining state-of-the-art mass spectrometry and bioinformatics, we demonstrate the utility of this approach for neoantigen identification and quantitation.

Reference: 1. Sun et al. Cancer Lett 2017;392:17-25.

#5718

Ex vivo **ATLAS-identification of neoantigens for personalized cancer immunotherapy in mouse melanoma.**

Hanna Starobinets,1 Catarina Nogueira,1 Kyle Ferber,1 Huilei Xu,1 Abha Dhaneshwar,2 Jason R. Dobson,1 James Loizeaux,1 James Foti,1 Michael O'Keefe,1 Erick Donis,1 Wendy Broom,1 Pamela Carroll,1 Paul Kirschmeier,2 Jessica B. Flechtner,1 Hubert Lam1. 1 _Genocea Biosciences, Cambridge, MA;_ 2 _Belfer Center for Applied Cancer Science, Dana-Farber Cancer Institute, Boston, MA_.

Neoantigens are emerging as attractive vaccine targets for personalized cancer immunotherapy. As opposed to tumor-associated antigens, neoantigens contain non-synonymous mutations that enable their identification as foreign targets not subject to central tolerance in the thymus. Personalized cancer vaccines leverage neoantigens to specifically direct the immune system to recognize cancer cells for the coordinated attack and destruction of tumors. While in silico methods are commonly used to predict immunogenic neoantigens primarily via putative binding to major histocompatibility complexes (MHC), the positive predictive value of these approaches is low as they cannot account for the complexity of antigen processing, the diversity of MHC class I and class II alleles, and the additional steps of T cell activation. Ex vivo technologies have the potential to overcome the limitations of neoantigen identification by utilizing biologically-relevant testing. ATLAS™ is an unbiased immune response profiling platform that enables comprehensive screening of a tumor mutanome by using a patient's own autologous immune cells, specifically monocyte-derived dendritic cells (MDDC) as antigen presenting cells (APCs) and sorted CD8+ and CD4+ T cells. By utilizing autologous APCs and T cells, ATLAS is agnostic to MHC diversity and assesses preexisting T cell responses to any given mutation. Patient MDDC are pulsed with an ordered array of Escherichia coli expressing patient-specific mutations as short polypeptides. CD8+ and CD4+ T cell response screening is performed using APCs and E. coli with and without pore-forming lysteriolysin O (cLLO) facilitating MHC class I or class II presentation, respectively. Thus, preexisting patient T cell responses to cancer antigens can be characterized by inflammatory cytokine secretion. We utilized a mouse melanoma model to demonstrate the capability of the ATLAS platform for identification of vaccine neoantigens. Whole exome sequencing was performed on B16F10 melanomas resected from C57BL/6 mice, identifying >1600 non-somatic, non-silent mutations. E. coli libraries individually expressing all mutations were constructed and used to screen APCs and T cells from the spleens of B16F10 tumor-bearing mice. Biologically relevant neoantigens were identified by their ability to modulate the secretion of inflammatory cytokines by CD4+ and CD8+ T cells. The significance of the identified neoantigens in comparison to predicted and previously reported B16F10 antigens is described. Top neoantigen candidates were selected and manufactured as synthetic long peptides. Therapeutic vaccination with ATLAS-identified neoantigens in tumor challenge studies is planned and progress will be reported. These studies demonstrate a biologically-relevant approach to improve neoantigen selection for personalized cancer vaccine design enabling improved therapeutic efficacy.

#5719

RNA-based tumor neoantigens from intron retention.

Claire A. Margolis,1 Alicia C. Smart,1 Dennis Adeegbe,2 Diana Miao,1 Meng Xiao He,1 Harold Pimentel,3 Tim Fugmann,4 Kwok-Kin Wong,2 Eliezer M. Van Allen1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _NYU Langone Medical Center, New York, NY;_ 3 _Stanford University, Stanford, CA;_ 4 _Philochem AG, Otelfingen, Switzerland_.

Background: Tumor neoantigens contribute to cancer immunity and may influence selective immune checkpoint inhibitor response. Translation of nonsynonymous somatic mutations in tumor cells generates mutant peptides that can be recognized as foreign by the host immune system. Alterations in tumor RNA, rather than DNA, may also contribute to the tumor neoantigen landscape. Intron retention, a widespread feature of cancer transcriptomes, represents a novel source of tumor neoantigens.

Methods: We developed an in silico pipeline leveraging existing tools and novel methods to computationally identify retained-intron neoantigens (RI-neoantigens) from transcriptome sequencing data. We applied our computational pipeline to three cohorts of melanoma patients (n = 89) treated with immune checkpoint blockade.

Results: This approach identified a mean RI-neoantigen burden of 3,643 across cohorts, which augments the mean DNA-derived somatic neoantigen burden by roughly 4.5-fold. 3,253 RI-neoantigens were exclusively present in two or more patients who responded to immunotherapy. A subset of responder-exclusive RI-neoantigens were validated by peptide restimulation assay. A candidate RI-neoantigen from a cancer cell line was identified in complex with MHC I by mass spectrometry, supporting the hypothesis that aberrant splicing results in intron retention, which generates abnormal transcripts that are translated into immunogenic peptides and presented to the immune system.

Conclusions: Our results demonstrate the contribution of transcriptional dysregulation to the overall burden of tumor neoantigens. Further study of RI-neoantigens may expand our understanding of tumor immunity and identify immunogenic peptide sequences for personalized cancer vaccines.

#5720

Functional characterization of neoantigens determining immune checkpoint blockade response in mouse models of human melanoma.

Eva Perez Guijarro,1 Chi-Ping Day,1 Zoe W. Ohler,2 Rajaa El Meskini,2 Howard Yang,1 Suman Vodnala,1 Cari Graff-Cherry,2 Sung Chin,2 Anyen Fon,1 Helen Michael,1 Maxwell Lee,1 Terry Van Dyke,2 Shyam Sharan,2 Glenn Merlino1. 1 _National Cancer Institute, National Institutes of Health, Bethesda, MD;_ 2 _Frederick National Laboratory for Cancer Research, Frederick, MD_.

Melanoma is the deadliest form of skin cancer due to the lack of widely effective therapies for advanced disease. Recently FDA-approved immunotherapies, such as immune checkpoint blockade (ICB) by CTLA-4 and PD-1/PD-L1 antibodies, provide unprecedent durable responses but in less than 40% of late stage melanoma patients. While high mutational loads characteristic of responsive tumors has not shown predictive value of patient outcome, accumulating evidences suggest a key role for neoantigens in the response to ICB. Moreover, new T-cell transfer- and vaccine-based therapeutic strategies highlighted the relevance of an efficient identification and prioritization of highly immunogenic neoantigens to improve immunotherapies. However, mechanistic studies are not possible in humans, and the development of adequate predictive methods is still the center of intense debate in the field. Here we use two genetically engineered melanoma mouse models exhibiting distinct response to ICB. Melanomas induced by neonatal ultraviolet radiation (UV) in a HGF-transgenic mouse (HGF-tg) showed high sensitivity to anti-CTLA-4, whereas UV-induced melanomas in HGF-tg; BrafV600E; Cdkn2a+/- mouse (Braf/HGF) did not respond. We hypothesized these models will allow us to identify the neoantigen features required for ICB response including type, expression levels and allele frequency patterns. High tumor immunogenicity, assessed by in vivo vaccination assays, as well as increased T-cell infiltration upon anti-CTLA-4 treatment were correlated to greater response. Moreover, exome and RNA sequencing analyses revealed similar mutational and neoantigen load in both models, albeit with no common expressed mutations. Notably, both models expressed similar levels of antigen presentation related genes (e.g. B2m, H2-Kd, Tap1) suggesting that specific neoantigens in HGF-tg melanoma cells may contribute to their sensitivity to anti-CTLA-4. To test this, we predicted MHC-I/-II binding of HGF-tg melanoma mutated epitopes in silico and generated a "neo-epitope" library. Importantly, the expression and allele frequency of most of selected mutations were decreased in anti-CTLA-4 responder HGF melanomas. The "neo-epitope" library was transduced into Braf/HGF non-responder cells and future studies will identify the neo-epitopes lost upon ICB, representing determinants of therapeutic success. Additionally, in vivo vaccination assays using synthetic mutant peptides will be performed to validate each neoantigen candidate. We anticipate that our studies will provide insight into the role that neoantigens play in melanoma immunotherapy responses.

(EPG and CPD contribute to this abstract equally).

#5721

Neoantigen load and HLA-class I expression characterize a subset of HR-proficient high-grade serous ovarian carcinomas with favorable prognosis and T cell-inflamed phenotype.

Hirokazu Matsushita,1 Kosei Hasegawa,2 Katsutoshi Oda,3 Shogo Yamamoto,3 Kayo Asada,3 Akira Yabuno,2 Akira Nishijima,3 Takahiro Karasaki,1 Yuji Ikeda,2 Keiichi Fujiwara,2 Hiroyuki Aburatani,3 Kakimi Kazuhiro1. 1 _The University of Tokyo Hospital, Tokyo, Japan;_ 2 _Saitama Medical University International Medical Center, Saitama, Japan;_ 3 _The University of Tokyo, Tokyo, Japan_.

Aim: Around 50% of high-grade serous ovarian carcinomas (HGSC) have deficiency in homologous recombination (HR) pathways. There has been increased evidence of benefit for the use of poly ADP ribose polymerase (PARP) inhibitors in a group of HGSC patients who especially have HR-deficient tumors. Therefore, it is needed to develop a new treatment strategy for the rest, an HR-proficient tumor. The aim of this study is to investigate immunological background of HR-proficient HGSC by integrated molecular analysis to explore the subset that would become candidates for immunotherapy such as immune checkpoint inhibitors.

Methods: In total, 80 cases of HGSC were analyzed in this study. Exome and RNA sequencing were performed to identify germline and somatic mutations. Methylation arrays were also carried out to evaluate BRCA1 and RAD51C promoter methylation status. Predicted neoantigens derived from mutations were identified based on the MHC class I binding prediction algorithm NetMHCpan 2.8. Immune profile in the tumor was assessed by differential gene expression analysis and gene set enrichment analysis (GSEA) using RNA sequencing data. Neoantigen load, antigen presentation, immune profile and their relevance to clinical outcomes were also investigated.

Results: Either BRCA1/2, RAD51C/D mutations, or BRCA1, RAD51C promoter methylation was defined as HR-deficient. A total of 34 (42.5%) and 46 (57.5%) patients were classified as having HR-deficient and HR-proficient tumors, respectively. As expected, the numbers of predicted neoantigens were lower in HR-proficient than HR-deficient tumors (p<0.01). However, 40% of the patients with HR-proficient tumors still had high numbers of neoantigens and displayed better survival trend than those who had lower numbers of neoantigens. Incorporation of HLA-class I expression status in the analysis revealed that the patients who had both high neoantigen number and high HLA-class I expression showed improved survival in HR-proficient HGSC (p=0.02). GSEA demonstrated that the gene sets for effector memory CD8, TH1, and type I and type II interferon (IFN) responses were enriched in those patients.

Conclusions: The number of neoantigens and HLA-class I expression characterized a subset of HR-proficient HGSC with improved prognosis and immunologically hot phenotype. This subset might be a candidate target for immune checkpoint inhibitors rather than PARP inhibitors.

#5722

Antigen identification for cancer immunotherapy by deep learning on tumor HLA peptides.

Brendan Bulik-Sullivan,1 Jennifer Busby,1 Matthew Davis,1 Lauren Young,1 Tyler Murphy,1 Andrew Clark,1 Fujiko Duke,1 Michele Busby,1 Adnan Derti,1 Mojca Skoberne,1 Karin Jooss,1 Corinne E. Gustafson,2 Assunta De Rienzo,2 William G. Richards,2 Nhien T. Dao,2 Hyeong R. Kim,3 Jamie E. Anderson,2 Chang-Min Choi,3 Vincent De Montpreville,4 Se Jin Jang,3 Olaf Mercier,4 Raphael Bueno,2 Elie Fadel,4 Joshua Francis,1 Roman Yelensky1. 1 _Gritstone Oncology, Cambridge, MA;_ 2 _The Thoracic Surgery Oncology laboratory, Division of Thoracic Surgery and the Lung Center, Brigham & Women's Hospital, Boston, MA; _3 _Asan Medical Center, Seoul, Republic of Korea;_ 4 _Centre Chirurgical Marie Lannelongue, Le Plessis-Robinson, France_.

A key goal in immuno-oncology is the identification of tumor antigens recognized by CD8 T cells. Immune modulators such as PD-1 inhibitors indirectly promote T-cell attack against tumor antigens, and may be augmented by antigen-directed therapeutic immunization or adoptive cell therapy to increase clinical benefit. Such personalized therapeutics require accurate antigen identification from patient samples, which remains elusive today.

Methods: We generated the largest reported dataset of human tumor transcriptomes and HLA class I peptidomes (N=111) from specimens of multiple tumor types. We used these data to train a deep learning model of HLA peptide presentation for antigen prediction. Our model architecture addressed two key challenges: (1) learning HLA-allele-specific models from tumor data where each sample expressed up to 6 unique HLA class I alleles and (2) incorporating information about all aspects of HLA presentation, including gene expression, proteasomal processing and stable binding of peptides to HLA. We evaluated the performance of the model on two independent test datasets. First, we tested the model on HLA presented peptides from five held-out tumor mass spectrometry samples. Then, to establish that accurate prediction of HLA presentation translates to prediction of antigens recognized by T cells in vivo, we compiled from three published studies a dataset of >2,000 mutations in 16 patients, with 23 mutations (i.e. neoantigens) recognized by PD1+ PBMC or TIL CD8 T cells. Recognition of a peptide by TIL or activated peripheral T-cells implies not only tumor presentation of the peptide, but also its ability to prime T cell responses, and thus represents the most stringent test of tumor antigen prediction.

Results: The model demonstrated a breakthrough in prediction accuracy. On the mass spectrometry test data, it achieved a >10-fold improvement in positive predictive value (PPV) vs standard HLA binding affinity (~50% PPV at 40% recall for the MS-based model vs ~5% PPV for binding affinity prediction). On the T-cell test dataset, the model ranked T-cell recognized neoantigens on average >4-fold higher than standard prediction (median rank ~7 for the MS-based model vs ~30 for binding affinity). When selecting candidate neoantigens for hypothetical 10-neoantigen personalized immunotherapy, the model prioritized at least 1 T-cell recognized neoantigen in the top 10 for 9/11 patients with neoantigen responses vs 3/11 for binding affinity. For a hypothetical 20-neoantigen immunotherapy, the model correctly selected the majority (16/23, 70%) of recognized neoantigens.

Conclusion: We used the largest dataset of tumor transcriptomes and HLA peptidomes reported to-date to train a deep learning model of HLA epitope presentation. The new model significantly outperforms state of the art methods, and has sufficient predictive accuracy for in silico antigen selection for personalized cancer immunotherapy.

#5723

Inactivation of DNA repair triggers neoantigen generation and impairs tumor growth.

Giovanni Germano,1 Simona Lamba,1 Giuseppe Rospo,1 Ludovic Barault,1 Alessandro Magri',1 Federica Maione,1 Mariangela Russo,1 Giovanni Crisafulli,1 Alice Bartolini,1 Giulia Lerda,1 Giulia Siravegna,1 Benedetta Mussolin,1 Roberta Frapolli,2 Monica Montone,1 Federica Morano,3 Filippo de Braud,3 Nabil Amirouchene-Angelozzi,1 Silvia Marsoni,1 Maurizio D'Incalci,2 Armando Orlandi,4 Enrico Giraudo,1 Andrea Sartore-Bianchi,5 Salvatore Siena,5 Filippo Pietrantonio,3 Federica DiNicolantonio,1 Alberto Bardelli1. 1 _Candiolo Cancer Institute, Candiolo (TO), Italy;_ 2 _IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy;_ 3 _Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy;_ 4 _Policlinico Universitario A. Gemelli, Roma, Italy;_ 5 _Niguarda Cancer Center, Grande Ospedale Metropolitano Niguarda, Milan, Milan, Italy_.

Colorectal, ovarian, endometrial and other tumors carrying defects in DNA mismatch repair often show favorable prognosis and indolent progression. The genomes of these tumors bear hundreds of thousands of somatic mutations, a feature which fosters cancer progression and might lead to rapid evolution of resistance to targeted therapies. Recent evidences that a subset of MSI (microsatellite instable) tumors respond prominently to immune checkpoint blockade led to the hypothesis that the presence of high number of somatic mutations may be responsible for effective immune-surveillance. However, several reports indicate that a relevant fraction of hyper-mutated tumors have unfavorable prognosis and do not respond to immune-modulators. To understand the molecular and functional bases of response to immune checkpoint inhibitors, we genetically inactivated MutL homolog 1 (MLH1) in colorectal, breast and pancreatic mouse cancer cells. The growth of MMR deficient cells was comparable to their proficient counterparts in vitro and upon transplantation in immune-compromised mice. However, isogenic MMR deficient cancer cells, acquiring alterations over time, were unable to form tumors when injected subcutaneously or orthotopically in syngeneic mouse models according to cell-passage number. Moreover, we found that MMR-driven dynamic generation of neoantigens, when restricted to a clonal population, further increases immune detection. Mechanistically, MLH1 inactivation increased the mutational burden and led to dynamic mutational profiles, resulting in persistent renewal of neoantigens in vitro and in vivo, while control cells exhibited stable mutational loads and neoantigen profiles over time. These results led us hypothesize that enforced increase of the number of mutations in cancer cells could restrict cancer growth and might be beneficial for therapeutic purposes. We therefore performed a pharmacological screen to identify agents capable of permanent inactivation of MMR in colorectal, breast and PDAC cancer cells. We found that temozolomide triggers MLH1 inactivation in cancer cells that -as a result- are unable to form tumors in syngeneic animals. Genomic analysis of temozolomide resistant cells revealed that fluctuating levels of neoantigens, rather than the absolute number of mutations, might be critical to provoke immune surveillance. Overall, these results provide the rationale for developing innovative anticancer therapies based on inhibition of DNA repair mechanisms.

#5724

Homologous recombination repair gene mutations for DNA repair and immune oncology drug combinations.

Dry R. Jonathan, Zhongwu Lai, Brian Dougherty, Carl Barrett. _AstraZeneca, Waltham, MA_.

Exciting clinical benefit has been seen from both immune checkpoint inhibitors and PARP inhibitors in several cancer types. Initial reports have suggested the tumor mutational burden (TMB) may be a tumor agnostic marker for predicting immunotherapy (IO) response. Deficiencies in DNA damage repair (DDR) resulting from BRCA1 and BRCA2 mutations can lead to an accumulation of tumor mutations, and sensitivity to PARP inhibitors. We hypothesized that patients with germline or somatic mutations in BRCA1/2 or other homologous recombination repair (HRR) genes, termed "HRRm", will have higher TMB, higher immune infiltration and activity, and greater benefit from the combination of DDR and IO therapies. We investigated the relationship between DDR deficiencies, TMB, PDL1, transcriptomic immunoscores and drug response across The Cancer Genome Atlas (TCGA) disease cohorts, data from AstraZeneca's clinical studies and a linked NSCLC genomic and EHR dataset from Foundation Medicine and FlatIron Health. HRRm cancer samples show higher TMB in several cancer types. This does trend with higher PDL1 but does not associate with transcriptomic immunoscores. HRRm could be a practical clinical biomarker to select patients with DDR defects likely to benefit from combination of DDR and IO therapies.

#5725

Systematic identification of tumour-specific neoantigens(by whole-genome sequencing) and correlation between tumour neoantigen burden, PD-L1 expression and immune infiltrates in 158Asian colorectal cancers.

Si-Lin Koo,1 Joe Poh Sheng Yeong,2 Andy Nguyen,3 Clarinda Wei Ling Chua,1 J Zachary Sanborn,3 Steve Benz,3 Wah Siew Tan,2 Choong Leong Tang,2 Su Yan,4 Min Hoe Chew,5 Brian Goh,2 Chung Yip Chan,2 Xiao Qing Koh,6 Alexander Lezhava,4 Tony Kiat Hon Lim,2 Shahrooz Rabizadeh,3 Anders Skanderup,4 Iain Beehuat Tan1. 1 _National Cancer Centre Singapore, Singapore;_ 2 _Singapore General Hospital, Singapore;_ 3 _NantOmics, LLC, Culver City, CA;_ 4 _Genome Institute of Singapore, A*STAR, Singapore;_ 5 _Sengkang General Hospital, Singapore;_ 6 _Nanyang Technological University, Singapore_.

Background: Somatic mutations are attractive therapeutic targets for "individualized neoantigen vaccines" because of lack of host central tolerance and reduced risk of autoimmunity. Here, we perform large-scale-omic analyses to assess the neoantigen landscape of colorectal cancer (CRC), a cancer largely refractory to immune-checkpoint inhibition.

Methods: We performed whole genome sequencing (WGS) (60x tumor, 30x normal) and deep whole transcriptomic sequencing (RNA-Seq) (∼200x106 reads per tumor) on 158 colorectal cancers of which 32 are microsatellite instability high (MSI-H) tumours and 126 are microsatellite stable (MSS). Whole exome sequencing (200x tumor, 100x normal) was also performed on 120 tumours. HLA typing, somatic mutations, gene expression and neoepitope predictions were computationally evaluated. Inferred HLA-A alleles were orthogonally validated with Pacbio long-read sequencing. Tissue microarrays (TMAs) with tumour core, tumour edge and normal adjacent tissue of these 158 CRCs were constructed. Histopathological analyses using multiplex immunohistochemistry (mIHC) to simultaneously evaluate 7 markers, i.e. cytokeratin (CK), CD3, CD8, FOXP3, CD68, PD-L1, DAPI, have been performed.

Results: The most common HLAs were, by allele count: A*11:01: 56; A*33:03: 38; B*58:01: 33; B*46:01: 29; B*40:01: 26; C*01:02: 41; C*07:02: 33. Inferred HLA-A alleles from WGS data was largely concordant (>90%) with Pacbio long-read sequencing. There were a median of 2,850 (1229-6909) [MSI] & 213 (27-13,835) [MSS] coding variants, from which 10,487 (4,307-27,365) [MSI] & 726.5 (50-59,096) [MSS] possible neoepitopes were derived, after accounting for epitope processing, the normal proteome and general population variome based on dbSNP, Of these, 5,707 (2,608-15,218) [MSI] & 320 (14-25,243) [MSS] neoepitopes are expressed (based on RNA-Seq). Epitope prediction algorithms revealed a median of 423 (17-1,056) [MSI] & 26 (0-1,102) [MSS] bound & expressed neoepitopes. 5 MSS tumors did not have any predicted bound nor expressed neoepitopes, 112 of 126 (89%) of MSS tumors had at least 5 predicted bound, expressed neoepitopes. Histopathological correlations between extent of immune infiltrates in fixed tissues, tumour PD-L1 expression and neoantigen burden is ongoing.

Conclusions: There is substantial variability in the neoantigen landscape amongst MSI & MSS CRCs. MSI contains multiple-fold higher neo-antigens. Amongst MSS tumours, 89% of patients have at least 5 predicted bound and expressed neo-epitopes that could be targeted in neoantigen-based vaccines for personalized immunotherapy.

#5726

Rearrangement-related peptides with neoantigenic potential in malignant pleural mesothelioma.

Aaron S. Mansfield,1 Tobias Peikert,1 James B. Smadbeck,1 Julia B. Udell,2 Farhad Kosari,1 Stephen J. Murphy,1 Hongzheng Ren,1 Vishnu V. Serla,1 Janet L. Schaefer Klein,1 Giannoula Karagouga,1 Faye R. Harris,1 Carlos Sosa,1 Sarah H. Johnson,1 Wendy Nevala,1 Svetomir N. Markovic,1 Aaron O. Bungum,1 Eric S. Edell,1 Haidong Dong,1 John C. Cheville,1 Marie Christine Aubry,1 Jin Jen,1 George Vasmatzis1. 1 _Mayo Clinic, Rochester, MN;_ 2 _Center for International Blood and Marrow Transplant Research, Minneapolis, MN_.

Malignant pleural mesothelioma is a disease primarily associated with exposure to the carcinogen asbestos. Consistent with this carcinogenic exposure, cytogenetic analyses have identified multiple recurrent structural chromosomal abnormalities in this malignancy, but more recent high-throughput sequencing evaluations of point mutations suggest that there is a low mutational burden in mesothelioma. Since tumor mutational burden has been correlated with responses to treatment with immune checkpoint inhibitors such as nivolumab, it was not consistent that patients with mesothelioma and low mutation burdens would have similar response rates in clinical trials with immune checkpoint inhibitors as patients with non-small cell lung cancer which is associated with a high mutation burden. In order to reconcile these differences, and given the potential for an improved understanding of the molecular pathogenesis of mesothelioma to improve therapeutic options, we used mate-pair sequencing (MPseq) and RNA sequencing (RNAseq) to understand how structural variants affect the transcriptome. MPseq differs from standard next generation sequencing approaches by tiling the whole genome with larger fragments (2-5kb) to reliably detect structural variants such as insertions, deletions and rearrangements. Amongst 22 mesothelioma specimens there were 1535 chromosomal rearrangements (median 41, range 3-298 per specimen), that resulted in junctions or novel fusions of non-coding DNA or genes. Six-hundred thirty-seven of these rearrangements (median 22, range 5-103 range per specimen) resulted in novel fusions of genes. Many of these inter- or intra-chromosomal rearrangements were consistent with a pattern of chromoanagesis such as chromoplexy or chromothripsis. Chromosomal rearrangements detected by MPseq were used to guide analysis of RNAseq data and revealed that these chromosomal junctions resulted in the expression of 179 novel amino acid sequences (median 5, 0-51 range per specimen). To determine whether transcription of chromosomal rearrangement-related junctions have neoantigenic potential, we used in silico tools to determine whether any of the expressed junctions contained peptides that could be presented by patient-specific HLA molecules. The top candidate rearrangement-related peptides with neoantigenic potential bound patient-specific HLA molecules nearly as well or as well as a positive control in competitive binding assays. Our findings represent the discovery of potential neoantigen expression driven by structural chromosomal rearrangements. These results may have implications for the development of novel therapeutic strategies, the selection of patients to receive immunotherapy, and blood-based treatment monitoring strategies.

#5727

MHC II complements MHC I in shaping the mutational landscape of tumors.

Rachel Marty, Wesley Thompson, Trey Ideker, Maurizio Zanetti, Hannah Carter. _University of California, San Diego, La Jolla, CA_.

The major histocompatibility complex (MHC) class I and class II molecules present peptide antigens to T cells to enable immune detection of foreign or mutated peptides. Mutant peptides presented by MHC-I on the cell surface have the potential to activate a cytotoxic CD8+ T-cell response, imposing selective pressure on tumor cells harboring neoantigens. Recently, we demonstrated that patient MHC-I genotype results in individual differences in which cancer-causing mutations can be effectively presented on the cell surface to stimulate CD8+ T cells and is a determinant of the probability of observing a particular mutation (Marty et al., Cell 2017). However, since cytotoxic CD8+ T cell responses require CD4+ T cells that are activated by MHC-II-based antigen presentation, we hypothesized that individual variation in MHC-II genotype must also be an important determinant of immune selection of tumor cells. To investigate the role of MHC-II restriction in shaping the mutational landscape of tumors, we first developed a score that represents the ability of an individual MHC-II genotype to present a mutation on the cell surface. The score maps affinities of mutated peptides for different MHC-II molecules into a single value and was able to distinguish peptides found in complex with MHC-II in vitro by mass spectrometry from random peptides with an AUC of 0.69. Next, we assessed the relationship between MHC-II presentation of 1,018 cancer mutations and their occurence in 7,137 tumors across 30 tissues types from The Cancer Genome Atlas (TCGA). Across all MHC-II genotypes in TCGA, we observed an unexpected bias for recurrent cancer mutations to be less well presented than random mutations (p < 2.2e-16), while foreign pathogenic sequences were more likely to be presented (p < 2.2e-16). Moreover, we found that higher mutation frequency among tumors correlated with overall poorer presentation across cancer patients. Comparing mutation presentation by MHC-II with our previous results for MHC-I, we found only a modest correlation in presentation scores across mutations (rho=0.28). Combining MHC-I and MHC-II genotype-derived presentation scores into a single model increased overall performance for predicting occurrence of mutation. Notably, MHC-II genotype had an even stronger influence over the probability for specific oncogenic mutations to arise than MHC-I genotype (OR 2.12 vs. OR 1.43). We also observed differences in the extent to which MHC-based presentation could predict driver mutation status in different tumor types, with the best performance observed in thyroid cancer (AUC=0.91). These results reveal that immunoediting during tumor development is dependent on MHC-II as well as MHC-I genotype, further implicating the inherited immune system as a key risk factor for cancer. 

### Oncogenes, Inflammation, and Cancer

#5728

DNA demethylating agents and interferons as modulators of Wnt/β-catenin signaling in colorectal cancer.

Ilias Ettayebi,1 Parinaz Mehdipour,2 Rajat Singhania,2 Ankur Chakravarthy,2 Tiago Medina,2 Shu Yi Shen,2 Charles Ishak,2 David Roulois,3 Daniel De Carvalho2. 1 _University of Toronto, Toronto, Ontario, Canada;_ 2 _University Health Network, Toronto, Ontario, Canada;_ 3 _Université de Rennes 1, France_.

p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 12.0px 'Times New Roman'} Colorectal cancer (CRC) is the second leading cause of death from cancer in men and the third in women in Canada. There is accumulating evidence suggesting that CRC is organized in a hierarchical manner, at the apex of which are the Cancer-Initiating Cells (CIC). Furthermore, results from xenograft models and human clinical trials indicate a selective enrichment of CICs in tumours that are resistant to therapy, suggesting that targeting CICs may represent a new paradigm in cancer treatment. We have previously reported the treatment with low dose of the DNA demethylating agent, 5-Aza-2'-deoxycytidine (5-AZA-CdR), can target CICs through the activation of the RIG1-MDA5 viral sensing sensing pathway, leading to an anti-viral response in cancer cells. Although it is now known that 5-AZA-CdR induces a state of "viral mimicry" in these cancer cells, the mechanism by which 5-AZA-CdR can specifically target CICs is not well understood. In this study, we propose a novel intersection between RIG1-MDA5-IRF7 and Wnt/β-catenin pathways. We found that treatment of CIC enriched cancer cells with both 5-AZA-CdR and type I and III interferons reduced canonical Wnt/β-catenin signalling. Furthermore, treatment with type I and III interferons showed a reduced CIC frequency in colorectal cancer cells in vitro. Mechanistically, we found that IRF7 and β-catenin interact in the nucleus, giving rise to a novel, non-canonical pathway that can modulate WNT signalling. These findings may explain a novel pathway by which 5-AZA-CdR can specifically target CIC enriched populations in colorectal cancer, and highlights the importance of innate immune pathways in epigenetic therapy.

#5729

**Differential activation of PI3K signaling predicts for immunogenicity in** BRCA1/2 **-deficient ovarian cancer.**

Adam Kraya, Kara N. Maxwell, Bradley Wubbenhorst, Brandon Wenz, John Pluta, Nicole Lunceford, Amanda Barrett, Jennifer J.D. Morrissette, Anupma Nayak, Michael Feldman, Susan Domchek, Robert H. Vonderheide, Katherine L. Nathanson. _University of Pennsylvania, Philadelphia, PA_.

Ovarian cancers with mutations in BRCA1/2 exhibit defects in homologous recombination-based DNA repair and are sensitive to DNA damaging agents, such as platinum chemotherapy. However, most develop resistance, necessitating orthogonal treatment strategies. Checkpoint blockade is one potential strategy, as BRCA1/2 ovarian cancers have higher neoantigen load relative to HR-proficient tumors and increased T cell infiltrates. However, phase I/II trials in ovarian cancer using CTLA4 or PD1/PDL1 inhibitors have shown response rates of 10-15% and have failed to establish selective benefit in patients with BRCA1/2 mutations, suggesting immunosuppressive mechanisms may impede clinical benefit. We investigated the determinants of immunogenicity through genomic analysis and immunohistochemistry (IHC) in 86 ovarian cancers with germline or somatic alterations in BRCA1/2 from TCGA (n=68) and Penn (n=18). Using TCGA data, we found that copy number (CN) loss in the tumor suppressor PTEN was associated with lower cytolytic index (p=0.04) and immune ESTIMATE scores (p=0.02), both RNA-based measures of immune activity, despite similar neoantigen loads (p=0.36). Further, BRCA1/2 mutant cancers with PTEN loss had decreased expression of costimulatory immune molecules CD28, CD86, TNFSF13B, TNFRS13B, TNFRSF17, and TNFSF18 and counter-regulatory immune inhibitors DOK3, HAVCR2, PDCD1LG2, and TGFB1 versus PTEN (wild type) WT, suggesting lower immune activity with PTEN loss. Gene set analysis found that BRCA1/2 mutant tumors with PTEN loss had heightened inositol phosphate metabolism (p=0.03) and decreased cytokine production (p=0.03). As BRCA1 hypermethylated ovarian tumors often amplify PIK3CA to activate PI3K signaling, we evaluated PIK3CA CN in PTEN WT tumors and found that both BRCA1 promoter hypermethylated (n=11) and BRCA1/2 mutant (n=45, germline or somatic) tumors exhibited PIK3CA amplification (CN=5.3). However, among PTEN WT tumors, BRCA1/2 mutant tumors also exhibited amplified AKT2 (CN=4.1), increased NFKB1 expression (p=0.025), and increased cytolytic index (p=0.028) and immune ESTIMATE scores (p=0.009) as compared to BRCA1 hypermethylated tumors, whose immunogenicity resembled PTEN loss tumors. We confirmed the effects of PTEN loss by IHC in tumors associated with germline mutations in BRCA1/2 from Penn and found lower intratumoral levels of CD3+ (p=0.05), CD8+ (p=0.01), and FoxP3+ (p=0.009) T cells with PTEN loss. Our study illustrates that while activation of PI3K signaling by PTEN loss or PIK3CA amplification can lead to immune suppression in BRCA1/2 ovarian cancer, as seen in melanoma and uterine leiomyosarcoma, selective activation of the AKT2-NFκB signaling arm of the PI3K pathway may drive immune responses in these tumors. These findings have important implications for clinical use of immune checkpoint therapies in BRCA1/2 ovarian cancers.

#5730

Co-targeting toll-like receptor and PI3K survival signaling pathways in stem-like castration resistant prostate cancer cells.

Saheed Oluwasina Oseni,1 Rolando Branly,2 Mirjana Pavlovic,3 James Kumi-Diaka1. 1 _Florida Atlantic University, Davie, FL;_ 2 _Broward College, Davie, FL;_ 3 _Florida Atlantic University, Boca-raton, FL_.

The development of castration-resistant prostate cancer (CRPCs) has been linked to the ability of prostate cancer stem cells (PCSCs) to either resist or survive the inhibitory effect of androgen deprivation and radiation therapies. Dysregulation of the PI3K/Akt/mTOR pathway is a common phenomenon in CRPCs, resulting in survival, growth enhancement, resistance to apoptosis and altered metabolism. Toll-like receptors (TLRs) have been shown to be surprisingly expressed in prostate cancer cells, which when activated could lead to activation of innate immune response and activation of other cell signaling pathways. Since, recent studies have identified links between TLR signaling and prostate cancer, TLR polymorphisms and prostate cancer risk, as well as possible cross-talks between TLR signaling and the PI3K pathway in immune cells, we hypothesized that there is a link between PI3K signaling pathway and TLR signaling in CRPCs. Our aim is to determine the synergism between targeting PI3K and TLR signaling in a combination regimen. Methods: PTEN-/- (PC3) and PTEN-/+ (DU145) castration-resistant prostate cancer cell lines (CRPCs) were used for this experiment. CRPCs cells were cultured and enriched for CD44+ spheroid cells either by low dose radiation or in serum-free media. CRPCs and stem-like spheroid cells were then treated with different concentration of PI3K, AKT, and mTOR inhibitors, as well as TLR agonists /antagonists as monotherapy. Combination regimens were also done as assess the synergistic effects of PI3K/AKT/mTOR inhibitors and/or TLR ligands (agonists)/antagonists in CRPCs and/or spheroid cells. CRPCs or spheroid cells were assessed for treatment-induced effects such as cell viability (MTT assay), cell cytotoxicity (LDH), mode of cell death (fluorescent staining), protein expression profile (Western blot analysis), and redox activity (NBT assay). We also tested to assess treatment-induced inflammatory response via ELISA assay. Results: Our result shows that TLR 3, 4 and 9, were significantly differentially overexpressed in PTEN-/- CRPCs compared to PTEN-/+ CRPCs. The activation of these pathway results in PI3K signaling downstream. The co-targeting of PI3K signaling pathway with both PI3K inhibitors and TLR signaling modulators appears to be beneficial in preventing proliferation and stemness in PTEN-/- CRPCs compared to PTEN-/+ CRPCs. Co-administration of PI3K and TLR 4/9 agonist appears to be antagonistic but become synergistic when TLRs 4 and 9 signaling were inhibited using siRNAs. Conclusion: One of the advantages of this combination treatment method is that it proffers a solution to the often-encountered feedback loop observed when PI3K signaling inhibitors are used as monotherapy. This study has provided more insights on how targeting can be beneficial in the eradication of CRPCs when used along with PI3K signaling inhibitors.

#5731

Synergistic effect of cigarette smoke and bacterial-induced chronic obstructive pulmonary disease type airway inflammation on promotion of K-ras mutant lung cancer.

Marco A. Ramos, Misha Umer, Susana Castro, Berenice Adriana Gutierrez, Nassim Khosravi, Seyed Javad Moghaddam. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Lung cancer, particularly K-ras mutant lung cancer, is the leading cause of cancer death worldwide, and cigarette smoking (CS) is its main cause. Epidemiologic studies had consistently revealed a strong association (3 to 9 fold increase) between lung cancer and COPD (chronic obstructive pulmonary disease), after controlling for CS exposure. COPD is an inflammatory disease of the airways with smoking being the main cause of it. Importantly, lung inflammation persists and lung function continues to deteriorate as does the increased risk of lung cancer even after cessation of cigarette smoking among former smokers with COPD. These facts suggest a strong link between COPD-related airway inflammation and lung cancer promotion independent of smoking. We have previously shown that weekly exposure to an aerosolized bacterial lysate of nontypeable Haemophilus influenzae (NTHi), induces lung inflammation with a profile of mediators and inflammatory cells similar to that observed in COPD patients excluding mucous metaplasia. We further showed that NTHi-induced COPD type airway inflammation promotes lung cancer 3.2-fold in a K-ras mutant mouse model remarkably similar to that in the epidemiologic literature. NTHi is the most common colonizing bacteria in the lower respiratory tract of patients with COPD and could be a potential cause of perpetuating and promoting persistent airway inflammation after CS exposure in these patients. Therefore, we further studied the effect of combined CS and NTHi exposure in the induction of COPD phenotype and promotion of lung cancer. Briefly, 6-week old K-ras mutant mice were exposed to NTHi lysate once a week for 8 weeks and to cigarette smoke (CS) daily for 2 hours/day, 5 days/week for 8 weeks and studied at the age of 14 weeks. The CS exposure was conducted by burning 3R4F reference cigarettes (University of Kentucky, Tobacco Research Institute), using an InExpose System (SCIREQ Scientific Respiratory Equipment Inc). We found that CS exposure alone caused a mild macrophage dominant airway inflammation, induced airway epithelial mucous metaplasia, and led to a 2.3 fold increase in lung tumor burden. Notably, combined NTHi and CS exposure resulted in a robust neutrophilic lung inflammation and mucous metaplasia and promoted K-ras mutant lung cancer by 4.3 folds (2 times more than CS alone). Our experimental results suggest that CS exposure and colonization of smoke-injured airways with NTHi induce an inflammatory and structural COPD phenotype fully recapitulating human COPD and provide a microenvironment that has a significant promoting effect on K-ras mutant lung cancer.

#5732

Zinc transporter, ZIP14, as a mediator of systemic muscle wasting in metastatic cancers.

Anup Biswas,1 Gang Wang,2 Wanchao Ma,1 Courtney Coker,1 Kurenai Tanji,1 Manoj Kandpal,3 Ramana Davuluri,3 Swarnali Acharyya1. 1 _Columbia University, New York, NY;_ 2 _Cornell Medical Center, New York, NY;_ 3 _Northwestern University Feinberg School of Medicine, New York, NY_.

Metastasis contributes to the vast majority of cancer-related mortalities. Metastatic tumors secrete factors that systemically affect various organs leading to dysfunction and accelerated death. More than 80% of metastatic cancer patients experience a progressive and debilitating loss of muscle mass and function by a process known as cachexia. Cachectic patients suffer deterioration of diaphragm and cardiac muscles and often die prematurely due to respiratory and cardiac failure. The prognosis for these patients is further diminished by the fact that they are often too weak to tolerate standard doses of anti-cancer treatments. Cachexia is therefore an important determinant of therapeutic response, outcome and patient survival in advanced cancer patients. Although systemic metabolic derangements and chronic inflammation predominate in cachexia, the underlying molecular mechanisms driving its development are not well understood. Therefore, insights into the specific interventions that could either reverse or prevent cachexia are expected to improve treatment outcome, survival and quality of life in cancer patients. In this study, we identified a metal ion transporter, ZIP14 that was upregulated in cachectic muscles from five independent metastatic models, as well as in metastatic cancer patients. We find that TNF-alpha and TGF-beta cytokines upregulate ZIP14 in muscles, which in turn results in the accumulation of intracellular zinc. Increased zinc influx in muscle cells is associated with the degradation of myofibrillar proteins that contributes to muscle atrophy and muscle mass loss. Importantly, germline ablation or muscle-specific depletion of Zip14 markedly inhibits cancer-induced muscle wasting. Our study demonstrates a novel function of ZIP14 in muscles as a mediator of cachexia in advanced cancers. Insights from this study can be used to develop therapeutic strategies to prevent cachexia, and improve the survival and quality of life in metastatic cancer patients.

#5733

IL-11 as a therapeutic target to treat colorectal cancer.

Jennifer Huynh, Ashwini Chand, Matthias Ernst. _Olivia Newton-John Cancer Research Institute, Melbourne, Victoria, Australia_.

Introduction Colorectal cancer is a pressing health concern and there is a demand to develop novel therapeutics and improve current therapies to treat this disease. Immune checkpoint inhibitors (i.e. anti-PD-1 treatment) have been highly successful for the treatment of solid malignancies however, the majority of colorectal cancers do not typically respond to immunotherapy. Accumulating evidence alludes to a role for IL-11 signaling in tumor development although the mechanisms underlying IL-11 biology in colorectal cancer remain largely unknown. We postulate that IL-11 could be a mode of immunosuppression which can be targeted to overcome the poor responses of colorectal cancer to immunotherapy. Here we aim to investigate IL-11 signaling as a therapeutic strategy which can also be targeted in combination with anti-PD-1 treatment. Moreover, we present data that indicates an immunological function for IL-11 in modulating T cell anti-tumor activity.

Method To assess the therapeutic potential for targeting IL-11 signaling in colon cancer in vivo, wild type and Il11r-/- C57BL/6 mice were injected subcutaneously in the flank on Day 0 with 1 x 106 MC38 colon cancer cells. The mice were then treated with an anti-PD-1 antibody i.p. dose once every 3 days. Mice were euthanised at Day 18. Harvested tumors were analysed by FACs and immunohistochemistry. To ascertain a role for IL-11 signaling in T cell activity, CD8+ and CD4+ T cells were FACs-sorted from wild type and il11r-/- mice, and activated with PMA/ionomycin for 24h. T cell activation was assessed by measuring mRNA levels of granzyme B and perforin by qPCR, and IFNγ production by ELISA.

Results MC38 growth was attenuated in il11r-/- mice compared to wild type hosts. Lower tumor burden corresponded with a modest increase in tumor-infiltrating CD8+ T cells however we observed no differences in other immune cell subtypes (i.e. CD4+ T cells, MDSCs and macrophages). In addition, lower tumor burden corresponded with a marked reduction in Ki67 as assessed by immunohistochemistry. In addition, T cells harvested from il11r-/- hosts expressed lower levels of granzyme B, perforin and IFNγ under both basal conditions and following PMA/ionomycin stimulation.

Conclusion Here we report that IL-11 signaling supports tumorigenesis using the MC38 colon cancer mouse model. For the first time, we have characterized a functional role for IL-11 in modulating T cell function. Overall, the findings from this study indicate IL-11R as a potential therapeutic target to improve the efficacy of immune checkpoint inhibitors for the treatment of colorectal cancer.

#5734

Interleukin-1 signaling is required for HNSCC tumor response to cetuximab.

Madelyn M. Espinosa-Cotton,1 Rachel A. Dahl,1 Elana Fertig,2 Isaac Jensen,1 Ayana J. McLaren,3 Kenley Miller,4 Samuel N. Rodman,1 Sandra Schmitz,5 Andrean L. Simons1. 1 _University of Iowa, Iowa City, IA;_ 2 _Johns Hopkins University, Baltimore, MD;_ 3 _Lincoln University, Lincoln University, PA;_ 4 _San Jacinto College, Pasadena, TX;_ 5 _Universite Catholique de Louvain, Louvain-la-Neuve, Belgium_.

Despite the incorporation of the epidermal growth factor receptor (EGFR) inhibitor cetuximab into the clinical management of head and neck squamous cell carcinoma (HNSCC), limited to no long-term changes in overall survival are observed in HNSCC patients even though EGFR is expressed at high levels in these tumors. Therefore, the identification of novel therapeutic approaches to enhance the clinical efficacy of cetuximab could lead to improved long-term survival for HNSCC patients. Our previous work suggests that cetuximab activates the interleukin-1 (IL-1) pathway via tumor release of IL-1 alpha (IL-1α), although the implications of activating this pathway are unclear. The IL-1 pathway plays a central role in immune response and displays both pro-tumor and anti-tumor activities. IL-1 may promote tumor growth by upregulating the secretion of pro-inflammatory mediators involved in angiogenesis and metastasis. On the other hand, IL-1 signaling may promote antitumor immunity specifically via natural killer (NK)-cell mediated antibody-dependent cell-mediated cytotoxicity (ADCC), which is also an important mechanism of action of cetuximab. The goal of our work is to determine how modulation of the IL-1 pathway affects HNSCC tumor response to cetuximab. We found that blockade of IL-1 signaling using an IL-1-receptor antagonist (IL-1RA, anakinra), neutralizing IL-1α/IL-1β antibodies, and genetic knockdown of the IL-1R all suppressed the anti-tumor efficacy of cetuximab, while IL-1α overexpression and treatment with recombinant IL-1α enhanced HNSCC tumor response to cetuximab in immunodeficient and immunocompetent HNSCC mouse models. Mechanistically, these results appear to be due to modulation of ADCC, as we found that IL-1 blockade significantly reduced cetuximab-mediated ADCC in vitro. Additionally, we found that HNSCC patients with high baseline circulating levels of IL-1 ligands (IL-1α, IL-1β) were significantly more likely to respond favorably to cetuximab monotherapy compared to patients with low or no baseline circulating IL-1 ligands. Altogether, these results suggest that IL-1 signaling is necessary for HNSCC tumor response to cetuximab. Therefore, IL-1α warrants further study as a novel therapeutic to enhance response to cetuximab and as an immunologic biomarker for cetuximab response.

#5735

Amino acid transporter SLC6A14: A novel drug target for colorectal cancer and colitis and its transcriptional regulation by TCF4/beta-catenin pathway.

Mohd Omar Sikder, Sathish Sivaprakasam, Ganapathy Vadivel. _TTUHSC, Lubbock, TX_.

Amino Acid Transporter SLC6A14: A Novel Drug Target for Colorectal Cancer & Colitis and Its Transcriptional Regulation by TCF4/β-catenin Pathway

Mohd Omar Faruk Sikder, Sathish Sivaprakasam, Vadivel Ganapathy

Texas Tech University Health Sciences Center

SLC6A14 is a Na+/Cl− -dependent amino acid transporter capable of transporting 18 of the 20 proteinogenic amino acids, including leucine (mTOR activator), glutamine (necessary for nucleotide biosynthesis), and arginine (substrate for iNOS). This transporter is expressed at basal levels in normal colon but significantly upregulated in colorectal cancer (CRC) and colitis. However, the relevance of this upregulation to disease progression and the mechanisms involved in the upregulation remain unknown. We postulated that deletion of SLC6A14 or pharmacological blockade of its function may suppress CRC by depleting amino acids and interfering with mTOR signaling selectively in tumor cells. It may also ameliorate the severity of colitis by diminishing NO synthesis by iNOS in colonic epithelial cells. Since TCF4/β-catenin/Wnt signaling is activated in CRC and colitis, we postulated that TCF4/β-catenin might control the expression of SLC6A14. We tested these hypotheses in the present study. CRC cells treated in vitro with α-methyl tryptophan, a selective blocker of SLC6A14, showed evidence of amino acid deprivation, decreased mTOR activity, and increased autophagy and apoptosis. In nude mouse xenografts with LS174T cells (a CRC cell line), silencing of SLC6A14 by shRNA markedly reduced tumor growth. Accordingly, Slc6a14-/- mice showed reduced tumor incident and tumor size compared to the wild type mice in AOM/DSS-induced colitis-associated colon cancer mouse model. Furthermore, Slc6a14-null mice showed decreased disease progression in an experimental colitis model (Dextran sulfate sodium-induced colitis) compared to wild type mice. In vitro treatment of LS174T cells with calphostin-C, a potent inhibitor of TCF4/β-catenin, dramatically reduced the expression of SLC6A14 mRNA and protein levels, whereas treatment of KM12L4 and KM12C cells with Wnt agonist AMBMP showed the opposite trend. Finally, ChIP assay demonstrated that TCF4/β-catenin complex directly regulates the expression of SLC6A14 in human CRC cells by binding with its promoter. We conclude that the increased β-catenin/Wnt signaling in CRC and colitis is responsible for the upregulation of SLC6A14 under these pathological conditions and that deletion of the transporter or its pharmacological blockade

protects against colitis and CRC. These studies identify SLC6A14 as a novel drug target for the treatment of colonic inflammation and CRC.

#5736

In silico analysis shows that PTEN loss and AR overexpression are associated with increased CD8+ T-cell and Treg density and earlier disease recurrence in prostate cancer.

Thiago Vidotto,1 Madhuri Koti,2 Jeremy A. Squire2. 1 _University of São Paulo, Ribeirão Preto, Brazil;_ 2 _Queen's University, Kingston, Ontario, Canada_.

PTEN loss occurs in 20-30% prostate cancers (PCa), is associated with worse outcome, and regulates the type I interferon response through the interferon regulatory factor 3 (IRF3). Both PTEN loss and the overexpression of the androgen receptor (AR) promote castrate-resistant PCa leading to a more aggressive disease. To characterize the associations between PTEN loss and AR expression in immune cell infiltration in the tumor microenvironment (TME), we conducted an in silico analysis in two PCa cohorts. RNAseq and gene expression array data were imputed in CIBERSORT to evaluate the relative frequency of 22 immune infiltrating cells in the TME. PTEN was lost in 19.7% (97/491) and 19.8% (26/131) in the TCGA and MSKCC cohorts, respectively. AR was overexpressed in 56% (279/491) and 54% (71/131) of the TCGA and MSKCC cohorts, respectively. Remarkably, AR overexpression was associated with decreased CD8+ T-cells infiltration in both the TCGA (p<0.0001) and MSKCC (p=0.001) cohorts. In addition we found that PTEN loss was associated with increased Treg infiltration in both the TCGA (p=0.002) and MSKCC (p<0.0001) cohorts. PTEN loss was also associated with low plasma cell infiltration in both the TCGA and MSKCC cohorts (p=0.001 and p=0.002, respectively). More detailed analysis of the MSKCC cohort showed that PTEN loss correlated with increased CD8+ T-cell density in the TME (p<0.0001). When we compared the immune cell infiltration profile from the primary and metastatic tumors in the MSKCC cohort, we found an increased infiltration of memory B-cell, Treg, and active dendritic cells (p<0.0001, p=0.005, and p=0.002, respectively) in the metastatic tumors. We then investigated the association between PTEN and AR expression with the most common immunotherapeutic targets. We found that PTEN loss was associated with increased PD-1 expression in MSKCC cohort (p<0.0001) and with reduced PD-L1 expression in the TCGA cohort (p=0.006). Increased AR expression correlated with reduced PD-1 expression in both TCGA and MSKCC cohorts (p<0.0001) and with reduced CTLA-4 expression in TCGA cohort (p=0.009). There was a positive association between PD-1 and CTLA-4 expression with CD8+ T-cell and Treg infiltration in both cohorts. Log-rank analysis showed that PTEN loss and high Treg infiltration predicted earlier disease recurrence in both the TCGA and MSKCC cohorts (p=0.021 and p=0.027, respectively). In addition, combined PTEN loss and increased CD8+ T-cell infiltration promotes earlier disease recurrence in both the TCGA and MSKCC cohorts (p=0.05 and p=0.029, respectively). These findings imply that the immune cell profile in the TME can be influenced by changes in the expression of PTEN and AR, suggesting that novel immunotherapies may promote better responses in castrate-resistant PCa.

#5737

Genomic drivers of cancer are enriched and mutually exclusive within non-T cell-inflamed tumors.

Brian Olson, Riyue Bao, Jessica Fessler, Jason Luke, Thomas F. Gajewski, Akash Patnaik. _University of Chicago Comprehensive Cancer Center, Chicago, IL_.

While immunotherapy has seen dramatic successes in a variety of malignancies, the majority of patients fail to respond. In particular, less than 10% of patients with metastatic, castrate-resistant prostate cancer (mCRPC) respond to immunotherapy. Based on previous work demonstrating robust clinical activity of immunotherapies in patients with a T cell-inflamed tumor microenvironment, we investigated whether non-T cell-inflamed cancers show evidence for distinct genomic alterations that might mediate immune escape. RNA-seq gene expression data were analyzed from two prostate cancer cohorts, The Cancer Genome Atlas (TCGA) of primary tumors (n=437) and a Stand Up to Cancer (SU2C) mCRPC cohort (n=43). These datasets were interrogated for common molecular alterations and their association with non-T cell-inflamed and T cell-inflamed cancers, focusing on copy number deletions of PTEN, RB1/CDKN2A, and activating mutations in CTNNB1 (β-catenin). Similar approaches were used to analyze 30 solid tumor types from TCGA (n=9555). Our analysis identified that 94% of primary prostate cancers harbor a non-T cell-inflamed tumor microenvironment, with only 6% of patients having a T cell-inflamed gene signature. Similar results were found in mCRPC patients from the SU2C cohort. Higher frequencies of PTEN loss and CTNNB1 activation, as well as a significant enrichment of RB1/CDKN2A deletion, were found in non-T cell-inflamed primary and metastatic prostate tumors. Moreover, we observed a direct correlation between the expression of PTEN/RB1 and CD8A expression within the tumor microenvironment. Strikingly, analysis of 30 human cancers revealed that loss of PTEN and/or RB1/CDKN2A correlates with T-cell exclusion across multiple malignancies. When combined with β-catenin pathway activation, we found that these alterations are mutually exclusive, with only 1% of non-T cell-inflamed cancers sharing all three mechanisms, 24% sharing two out of the three, and 75% of the samples harboring only one mechanism. Taken together, these data reveal that deregulated PTEN/PI3K, Rb and Wnt-β-catenin signaling networks are enriched in a mutually exclusive manner within non-T cell-inflamed cancers, suggesting that such cancers evolutionarily co-opt nonredundant signaling pathways to achieve a common goal of immune evasion. Targeting these pathways pharmacologically may provide an opportunity to enhance responsiveness to immunotherapy across multiple malignancies.

#5738

Hypoxia-inducible factor-1 in myeloid cells is required to protect the irradiated lung from metastasis.

Hoibin Jeong, Beom-Ju Hong, Hyung-Seok Choi, Jung-Min Oh, G-One Ahn. _Pohang Univ. of Science and Technology, Pohang, Republic of Korea_.

Recent evidence suggests that metastasis is proceeded by premetastatic niche formation where bone marrow-derived cells (BMDC), particularly those of myeloid cells (monocytes and macrophages), form clusters preparing for the arrival of cancer cells in the metastasis-prone organs such as the lung. Many molecules involved in the premetastatic niche formation has been identified, some of which include vascular endothelial growth factor (VEGF), lysyl oxidase (LOX), and S100A8. Since VEGF, LOX, and S100A8 are downstream targets of hypoxia-inducible factor (HIF), a major transcription factor stabilized under hypoxic conditions, we investigated whether HIF-1 in myeloid cells would affect the premetastatic niche formation hence metastasis to the lung. To do this, we used our novel strain of myeloid specific Hif-1α knockout (KO) mice using human S100A8, an intracellular calcium-binding protein as the myeloid promoter. We first performed bone marrow (BM) transplantation from GFP-expressing myeloid specific Hif-1α KO (hereafter denoted as Hif-1α KO) mice or GFP-expressing littermate control mice to lethally irradiated wild-type (WT) mice. Two weeks later, these mice were subcutaneously implanted for Lewis lung carcinoma (LLC) primary tumors followed by 20 Gy chest irradiation to promote the premetastatic niche formation. Primary tumors were then removed and luciferase-expressing LLC cells were intravenously injected to induce the lung metastasis. We observed to our surprise that mice receiving Hif-1α KO BM had 10-fold increase in the lung metastasis, indicating that HIF-1α in myeloid cells is required to protect the lung from the metastasis. While the total number of GFP-expressing BMDC was similar, there were significantly decreased alveolar macrophages (GFP+F4/80+CD11c+CD11b-) in the irradiated lung of mice receiving BM of Hif-1α KO BM than the littermate control mice. Because alveolar macrophages are specialized immune cells involved in phagocytosis of numerous airborne antigens, we hypothesized that HIF-1α-deficient alveolar macrophages are deficient in phagocytosis and this may be responsible for the increase in the lung metastasis. Indeed, we observed that alveolar macrophages isolated from bronchioalveolar lavage fluid of Hif-1α KO mice demonstrated a decreased phagocytic activity. We are currently investigating whether we could pharmacologically modulate phagocytic activities of alveolar macrophages to regulate the lung metastasis. In conclusion, we demonstrate that HIF-1 in alveolar macrophages is required to protect the irradiated lung from the lung metastasis, possibly in a mechanism involving phagocytosis.

#5739

Targeting EZH2 histone methyltransferase activity alleviates inflammatory bowel disease.

Haixia Long, Jie Zhou, Zhongyu Wang, Shuo Huang, Bo Zhu. _Institute of Cancer, Xinqiao Hospital, Chongqing, China_.

Objective: Inflammation plays crucial roles in the pathogenesis of inflammatory bowel disease (IBD). Enhancer of zeste homolog 2 (EZH2)-mediated trimethylation of histone 3 lysine 27 (H3K27m3) is critical to immune regulation. However, evidence is lacking to address the effect of EZH2 enzyme's activity on intestinal immune responses during IBD. This study aimed to explore the effects of targeting EZH2 methyltransferase activity on IBD progression in mice.

Design: EZH2's enzymatic activity inhibitor GSK343 or GSK126 were delivered by intravenous injection to C57BL/6 mice at the onset, or during the ongoing of IBD, induced by dextran sodium sulfate (DSS). Azoxymethane (AOM)/DSS protocol was applied to trigger colitis-associated colorectal cancer (CAC). Immune cells in the colon of colitis mice were analyzed by flow cytometry, immunofluorescence staining and myeloid-derived suppressor cells (MDSCs) deletion experiments. We differentiated mouse hematopoietic progenitor cells (HPCs) into MDSCs in vitro to investigate the effect of GSK343 on MDSCs generation.

Results: Inhibition of EZH2 activity significantly attenuated IBD-related symptoms and inflammation, substantially delaying the onset of CAC. Injection of GSK343 increased the number of functional MDSCs in the colons during IBD. Deletion of MDSCs abrogated the protective effects of GSK343 on IBD. Moreover, inhibition of EZH2 activity promoted the generation of MDSCs from HPCs.

Conclusion: Inhibition of EZH2 activity alleviates DSS-induced colitis by promoting MDSC development, and might serve as a new therapeutic strategy to treat IBD. Additionally, EZH2 inhibition enhances MDSC generation, therefore, EZH2 enzymatic inhibitors now in clinical trials for cancer treatment may hinder beneficial anticancer immunity responses.

#5740

**PD-L1 overexpression induced by oncogenic** KRAS **and** BRAF **mutations through MAPK pathway activation in non-small cell lung cancer.**

Yosuke Miura,1 Noriaki Sunaga,1 Pinjie Bao,1 Luc Girard,2 Yusuke Tsukagoshi,1 Tomomi Masuda,1 Norimitsu Kasahara,1 Reiko Sakurai,1 Kyoichi Kaira,1 Takeshi Hisada,1 John Minna,2 Masanobu Yamada1. 1 _Gunma University Graduate School of Medcine, Maebashi, Japan;_ 2 _University of Texas Southwestern Medical Center at Dallas, Dallas, TX_.

Mutant KRAS and BRAF are important oncogenic drivers in non-small cell lung cancer (NSCLC), however, effective treatment strategies for most of these mutants have not been established. Immuno-oncology clinical trials have shown promising results of anti-programmed death 1 (PD-1) antibodies for a subset of NSCLC patients and tumor expression of programmed-death ligand 1 (PD-L1) is regarded as a predictive marker for anti-PD-1 therapy. We found that CD274 mRNA and PD-L1 protein expression were reduced in NSCLC lines by small interfering RNAs (siRNAs) targeting mutant KRAS, but not wild-type KRAS, and also by pharmacologic inhibitors of MEK and ERK. In addition, there was a positive correlation between CD274 expression and KRAS expression in NSCLC lines, and analyses of The Cancer Genome Atlas (TCGA) database revealed that NSCLCs with high KRAS expression exhibited higher levels of CD274 compared to the tumors with low KRAS expression among KRAS-mutant NSCLCs. Similarly, siRNA-mediated BRAF knockdown and inhibitors of BRAF and MEK reduced PD-L1 expression in BRAF-mutant NSCLC cells, suggesting that PD-L1 expression was also upregulated by oncogenic BRAF. Mechanistically, these results suggest that oncogenic KRAS and BRAF induce PD-L1 overexpression in NSCLCs by activation of the MEK-ERK pathway. For clinical translation, our results suggest that: 1. in NSCLCs the combination of KRAS and PD-L1 overexpression could be a surrogate predictive marker for anti-PD-1 treatment; and 2. that since the goal of checkpoint inhibitor immuno-oncology therapy is to inhibit the PD-L1 axis, that a combination of anti-PD-1 and targeted therapy to inhibit the MEK-ERK pathway could be beneficial.

#5741

**Dexamethasone suppresses cytokine-induced dual oxidase 2 and VEGF-A expression in human pancreatic cancer cells in vitro** **and pancreatic cancer growth in xenografts.**

Yongzhong Wu, James H. Doroshow. _National Cancer Inst., Bethesda, MD_.

Chronic pancreatic inflammation is strongly associated with pancreatic cancer. We previously demonstrated that inflammatory cytokines interact to produce a DUOX2 -dependent, reactive oxygen species (ROS)-related, pro-oxidant milieu that could increase the pathologic potential of pancreatic inflammation, enhancing malignant transformation. Furthermore, we have shown that DUOXuox2 expression is upregulated in patients with chronic pancreatitis as well as pancreatic ductal adenocarcinoma (PDAC). Dexamethasone (Dex) has been reported to inhibit PDAC invasiveness, the formation of pancreatic intraepithelial neoplasia in genetically-engineered mouse models of pancreatic cancer, as well as epithelial to mesenchymal transition in PDACs, and local tumor recurrence and metastasis in vivo. Using cultured human pancreatic cancer cell lines, we found that in a dose- and time-dependent fashion Dex inhibited cytokine (IFN-γ/LPS, IL-4, and IL-17)-mediated up-regulation of DUOXuox2 and VEGF-A expression in several human pancreatic cancer cell lines, including BxPC-3, ASPC-1, and CFPAC-1. Dex pretreatment also blocked DUOX2-related DNA double strand breaks (detected as activated γH2AX) and the expression of HIF-1α in PDAC cells treated with pro-inflammatory cytokines. The effects of Dex were abolished by pre-treatment with the Dex antagonist RU-486. As expected, we did not observe anti-proliferative effects of Dex on PDAC cells in vitro. However, Dex strongly repressed DUOXuox2 mRNA and protein expression as well as the growth of xenografts initiated from BxPC-3 cells; in contrast, for the MIA-Paca line that is unresponsive to cytokines in culture, Dex produced no effect on DUOXuox2 expression and tumor growth when these cells were grown as xenografts. Examination of the human DUOXuox2 promotor in silico revealed a putative negative glucocorticoid receptor (GR) binding element. Western analysis, using nuclear extracts from pancreatic cancer cells treated with Dex, revealed that both activated glucocorticoid receptor and certain co-repressors, such as NCOR-1/2 and histone deacetylases (HDAC1, 2, and 3) exist in human pancreatic cancer cell nuclei. Our ongoing experiments are focused on understanding the molecular mechanism of Dex-mediated DUOXuox2 repression both in vitro and in vivo. In summary, these studies suggest that cytokine-related oxidant stress, generated by Duox2, could play a role in the progression of pancreatic cancer.

#5742

p38 MAPK functions as a tumor suppressor in skin epithelial cells, but as a tumor promoter in myeloid cells.

Min-Kyung Choo, Jin Mo Park. _MGH/Harvard Medical School, Charlestown, MA_.

The p38 MAP kinase plays a pivotal role in cellular responses to stress, mitogenic stimuli and immune signaling. Evidence from clinical observations and cell culture experiments reveals a role for p38 in many aspects of cancer and immunity. Meanwhile, attempts to determine the physiological role of p38 in mice have been hampered due to the early embryonic lethality of mice null for p38alpha, the most abundant and ubiquitously expressed p38 isoform. To overcome this challenge, we generated conditional knockout mice in which p38alpha expression was specifically ablated in epithelial cells and immune cells, and have discovered that p38alpha plays a crucial role in inflammation and immune response in a cell type-dependent manner. Here, we investigated the role of p38alpha in keratinocytes and myeloid cells in a chemically induced skin carcinogenesis model. Our study using conditional knockout animals showed that keratinocyte- and myeloid-specific p38α ablation resulted in an increase and decrease, respectively, in tumor incidence. Furthermore, tumors from the two groups of mutant mice displayed contrasting histological features in the intratumoral and tumor-stromal areas. Interestingly, tumors derived from p38alpha-deficient keratinocytes highly expressed p63 positive stem cell populations. High p63 expression was also detected in p38alpha-deficient epidermis in adult skin. p38alpha-ablated epidermis harbored greater numbers of colony-forming cells and side population cells. Mechanistically, we discovered that p38alpha directly phosphorylated and thereby destabilized p63. We identified the amino acid residues on p63 that were directly phosphorylated by p38alpha in vivo and in vitro. p63 phosphorylation by p38alpha contributed to shaping keratinocyte gene expression related to stem cell homeostasis, inflammation, and tumorigenesis. These findings illustrate a novel link between stress-induced protein kinase signaling and p63-dependent epidermal homeostasis. On the other hand, p38 ablation in macrophages upregulated chemokine expression to recruit cytotoxic T cells to tumor microenvironments and resulted in tumor regression. LPS-induced ATF3 expression was reduced in p38alpha-deficient mouse and human macrophages, and p38alpha-overexpression increased ATF3 level. According to the publicly available datasets, T cell-recruiting chemokine expression is upregulated in ATF3 KO macrophages. Therefore, ATF3 may link to p38alpha to chemokine expression in macrophages. These data suggest that p38alpha in epithelial cells and myeloid cells plays a distinct role in skin tumorigenesis and cell type-specific targeting should be considered to increase drug efficacy and reduce adverse effects.

#5743

Aberrant microRNA expression dysregulates the head and neck squamous cell carcinoma immune landscape.

Yuanhao Qu,1 Wei Tse Li,1 Christine O. Honda,1 Omar Saad,2 Aswini R. Krishnan,1 Sunil J. Advani,1 Weg M. Ongkeko1. 1 _University of California, San Diego, CA;_ 2 _University of California, Santa Babara, CA_.

Immunotherapy has emerged as one of the most promising treatments for several cancers, including head and neck squamous cell carcinoma (HNSCC). However, the failure of a large percentage of patients to respond to immunotherapy remains the ultimate obstacle to successful treatment. Etiology-associated dysregulation of genes involved in the immune system may be central to the development of differential clinical response to immunotherapy. Smoking, one of the main causes of HNSCC, may have a dual effect on the efficacy of immunotherapy. On one hand, mutations caused by tobacco are very effective in promoting antigenicity, thus enhancing immunogenicity. On the other hand, tobacco may alter cellular processes in a way that causes immunotherapy to fail. For example, microRNA-induced mRNA degradation or inhibition of translation could be critical in the dysregulation of immune associated (IA) genes. Using next generation sequencing data from The Cancer Genome Atlas (TCGA), we identified a comprehensive panel of dysregulated IA genes, including tobacco-associated dysregulated IA genes, in HNSCC samples compared to normal controls. To validate our findings, normal and cancer epithelial cell lines (HaCaT and UMSCC22B) were exposed to physiologically relevant doses of cigarette smoke condensate, and qPCR was performed to determine tobacco regulation of IA gene expression. Several IA genes, including FOXA2, SEMA3G, PLAU, OLR1, and DKK1, were significantly dysregulated and corroborated with the direction of dysregulation from our analysis of clinical data. The expressions of these genes also showed significant correlation with patient survival in Kaplan-Meier survival analysis (univariate cox regression, p<0.05). Several of these genes correlate significantly with clinical variables, including neoplasm histological grade and pathological stage, as assessed using the Kruskal-Wallis test (p<0.05). In order to understand the molecular mechanism by which these IA genes are dysregulated, we also identified a panel of tobacco-dysregulated microRNAs in HNSCC and distinguished those that can potentially target dysregulated IA genes using the online target prediction service provided by TargetScan, which utilizes a computational model that takes into account miRNA-mRNA complementarity as well as conservation of binding sites. We found a large number of microRNAs to be negatively enriched in relation to IA gene expression using gene set enrichment analysis (GSEA). These findings will be very important in understanding not only how tobacco use is critical to the pathogenesis and progression of HNSCC, but also the molecular mechanisms underlying the failure of immunotherapy for certain patients. Furthermore, these IA genes and their regulatory microRNAs in HNSCC can potentially be therapeutically targeted as a novel treatment strategy or as a complement to existing immunotherapy treatments.

#5744

Chronic inflammation activates IL6 signaling to upregulate DNMT1 and DNMT3b to promote colon tumorigenesis.

Mohammed L. Ibrahim, John D. Klement, Daneila Payne, Chunwan Lu, Priscilla S. Redd, Kebin Liu. _Augusta University. Georgia Cancer Center, Augusta, GA_.

A strong correlation has been well documented between chronic inflammation and tumorigenesis. Colitis Associated Cancer (CAC) stands as a solid example for the inflammation-mediated tumorigenesis and is considered as the most serious complication of inflammatory bowel diseases. Pro-inflammatory cytokines are believed to regulate the neoplastic transformation of colonic epithelial cells. However, the underlying mechanism of inflammation-driven tumorigenesis is still a matter of debate. As a key player in the tumor inflammatory microenvironment, the pro-inflammatory cytokine IL6 is one of the most extensively studied cytokine in tumor biology research. IL6 is a pleiotropic cytokine secreted by different cell types including macrophages, fibroblast, epithelial cells and myeloid cells. IL6 and its downstream oncogenic transcription factor STAT3 have been shown to act as a tumor promoter through stimulating the proliferation and growth of malignant cells, inhibiting cell death and maintaining the inflammatory tumor-promoting milieu. Moreover IL6 protein and mRNA levels were found to be elevated in serum and tumor specimen from human and mice with different types of cancers including colon cancer; suggesting that IL6 may play a pro-tumorigenic function during the chronic inflammation-mediated tumorigenesis. However, the molecular mechanisms underlying IL6 tumor-promoting functions in CAC is still elusive. In this study, we aimed at determining the expression profiles and underlying mechanism of function of IL6 in the colon. Our data indicate that IL6 is significantly elevated in the colon in DSS-induced colitis mouse model and in AOM-DSS induced-CAC. Interestingly, myeloid derived suppressor cells (MDSCs) were shown to be the major source of IL6 in the lamina propria in vivo and in vitro .Given the role of IL6/STAT3/DNMTs axis in tumor promotion, we further hypothesized that IL6 may directly contribute to the inflammation-mediated colon tumorigenesis. To test our hypothesis, we generated IL6-overexpressing colon cancer cell lines and observed that IL6 overexpression is associated with STAT3 phosphorylation and resultant DNMT1 and DNMT3b upregulation in colon cancer cells. Similarly, treatment of colon cancer cells with exogenous IL6 induces STAT3 phosphorylation and up-regulation of DNMT1 and DNMT3b. Moreover, IL6-overexpression alters cell proliferation and survival. Collectively, our data highlighted a new crucial role of IL6 in the inflammation-mediated tumorigenesis through epigenetic alteration of colonic epithelial cells; suggesting that prolonged autocrine and paracrine IL6 signaling creates an inflammatory microenvironment favors tumor initiation and growth.

#5745

Effect of activator of G-protein signaling 3 (AGS3) on prostate tumorigenesis.

Timothy O. Adekoya, Nikia Smith, Tonelia Mowatt, Temilade Aladeniyi, Ricardo M. Richardson. _North Carolina Central University, Durham, NC_.

Prostate cancer (PCa) is a leading cause of cancer death among men, with greater prevalence of the disease among the African American population in the US. Activator of G-protein Signaling 3 (AGS3/GPSM1), a receptor-independent activator of G-protein signaling has been shown to affect different cellular processes and cell cycle activity as well as tumor growth and development. AGS3 contains seven tetratricopeptide (TPR) repeats in its N-terminal half and four G-protein regulatory (GPR) motifs in its C-terminal half. The aim of this study is to assess the role of AGS3 in prostate cancer development and metastasis as well as to understand the molecular dynamics involved. To that end, the metastatic PCa cell lines LNCaP, PC3, MDA PCa 2b and DU-145 were analysed for AGS3 expression relative to RWPE-1, a non-metastatic Pca cell line. Quantitative RT-PCR and western blot analysis have shown that AGS3 expression varies in the PCa cell lines relative to control RWPE-1. Overexpression of AGS3 in PC3 and LNCaP cells significantly enhanced tumor progression in nude mice xenografts. Interestingly, expression of the TPR, not the GPR, repeats in LNCaP promoted tumor growth as well as the full length AGS3. Preliminary studies with a Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice deficient in AGS3 expression (TRAMP+/AGS3-/-) displayed decrease prostate tumorigenesis. Taken together, these results indicate that AGS3 expression promotes prostate tumor growth. The data also suggest that the effect of AGS3 in prostate tumorigenesis is mediated via its TPR, not GPR motif.

#5746

**CRISPR9-mediated FGL2-KO in tumor cells impairs tumor progression in brain and triggers antitumor immune memory systematically via NF-** κ **B dependent Batf3 expression in DCs.**

Jun Yan, Qingnan Zhao, Konrad Gabrusiewicz, Ling-Yuan Kong, Longfei Huo, Xueqing Xia, Jun Wei, Jingda Xu, Eric Davis, Amy B. Heimberger, Shulin Li. _MD Anderson Cancer Center, Houston, TX_.

Brain cancer is the 10th leading cause of death in United States with increasing primary tumor morbidity and secondary brain metastasis morbidity. Glioblastoma (GBM) is most lethal brain tumor with less than 5% 5 years survival rate marked by significant resistance to majority of therapies targeting tumor cells or tumor microenvironment. Here we shown one approach to convert aggressive GBM cells to immunogenic tumor cells by knockout FGL2 in GBM cells to evoke efficient anti-tumor immunosurvilliance. FGL2 is an immunosuppressive molecule and its expression level is strongly correlated with glioma grade and poor diagnosis. FGL2 from GBM cells, but not from host cells, is essential for immune evasion dependent GBM progression using both preclinical GBM and brain metastatic tumor models. Knockout FGL2 in GBM cells completely abolish tumor progression of GBM models challenged with low or high number of tumor cells in immunocompetent mice and protect mice from tumor rechallenge in periphery. This eradication effect is impaired in immune deficient mice and CD8+ T cells depleted mice, but not in FGL2KO mice, showing the necessarily of immune system to eliminate FGL2KO tumor cells and the requirement of FGL2 in tumor cells to evade immunosurvilliance. To understand how the systemic CD8+ T cell-dependent anti-tumor response and memory was induced by FGL2KO GBM cells, we studied the impact of FGL2KO tumor cells on dendritic cells (DCs) which control immune response via its critical role in the generation of effective cytotoxic T lymphocyte (CTL) responses. Knockout FGL2 in GBM cells induced CD8a+/CD103+ DCs accumulation in brain in vivo; DCs proliferation and CD103+ DCs induction in vitro. In agreement with this discovery, knockout Batf3, a key transcription factor regulating CD8a+/CD103+ DCs, reverse the impact of FGL2KO in GBM cell in vivo. By molecule analysis, a novel signaling pathway was revealed that knockout FGL2 in GBM cells induce NF-κ B activation, and this activation induced Batf3 and associated CD103 expression in DCs. Therefore, target specific molecule in GBM cells, such as knockout FGL2, can convert aggressive GBM cells to immunogenic cells, pointing to new approach for immune therapy.

#5747

Lack of FGF21 accelerates the Th17-IL-17 axis-mediated transition from nonalcoholic steatohepatitis to hepatocellular carcinoma.

Qianqian Zheng,1 Harshul Pandit,2 Xingkai Liu,3 Youxi Yu,3 Suping Li,2 Shelby Turner,2 Xin Meng,1 Robert Martin,2 Yan Li2. 1 _China Medical University, China;_ 2 _Univ. of Louisville, Louisville, KY;_ 3 _The First Hospital of Jilin University, China_.

Background: Hepatocellular carcinoma (HCC) has been increasingly recognized in nonalcoholic steatohepatitis (NASH) before cirrhosis stage and the estimated annual HCC incidence in NASH patients is about 0.3%. Accumulating evidence shows that T helper (Th) 17-Interleukin (IL)-17 axis mediates the transition from NASH to HCC, while fibroblast growth factor 21 (FGF21) exerts pharmacologic efficacy in downregulation of Th17- IL-17 axis. In fact, FGF21 has recently emerged as a promising therapeutic candidate for the treatment of obesity and diabetes. However, the NASH-related carcinogenesis and role of FGF21 played during NASH-HCC transition are largely unknown.

Aim: To investigate the effect of FGF21 on Th17-IL-17 axis during the NASH-HCC carcinogenetic transformation and to reveal the potential NASH-HCC mechanism.

Methods: NASH-HCC was induced in FGF21 knockout (KO) mice treated with DEN at age 2 weeks at 100 mg/kg body weight (i.p.), and followed feeding with high-fat diet (HFD,60% kcal% fat) or methionine/choline-deficient diet (MCD). In an orthotopic HCC model with inoculation of 106 hepal-6 cells into 10 week-old male C57L/J mice, fenofibrate (50 mg/kg) was administered to induce FGF21. In vitro studies were performed using shRNA-FGF21 knockdown hepatocyte and HCC cell lines. The cellular and molecular events, in turn, white adipose tissue (WAT) lipolysis, insulin resistance (IR), release of free fatty acids (FFAs), Th17 cell infiltration and IL-17A production, and NASH-HCC initiation, were determined both in vivo and in vitro.

Results: Early HCC detection and increased HCC incidence were found in the FGF21 knockout (KO) mice (P<0.05 versus wild-type controls). Increased WAT lipolysis, hepatic FFAs uptake, Th17 cells/IL-17A, and aberrant NF-κB and p53 signaling were found in NASH and HCC tissues (adipose, spleen and liver) from in FGF21-KO mice by flow cytometry, immunohistochemistry (IHC) and Western blot. IL-17A significantly induced FFAs uptake in the shRNA-FGF21 knockdown hepal-6 cells and FL83B cells. IL-17A promoted the shRNA-FGF21 knockdown hepal-6 cells spheroid formation through Wnt/β-catenin pathway. Fenofibrate treatment significantly increased PPAR-α and native FGF21 protein levels in the liver tissues, and decreased the tumor volumes.

Conclusions: 1) Lack of FGF21 played a key role in hepatic lipid accumulation and subsequent upregulated Th17-IL-17 signaling in liver; 2) lack of FGF21 accelerated the Th17-IL-17 axis promoted NASH-HCC transition; 3) upregulation of native FGF21 not only attenuates NASH, but also prevents HCC carcinogenetic transformation via downregulation of Th17-IL-17 axis.

This work was supported by an Institutional Development Award (IDeA) from the NIGMS of the National Institutes of Health under grant number P20GM113226.

#5748

Downregulation of glycine decarboxylase renders ovarian cancer cells less proliferative and more chemoresistant.

So-Jin Shin, Hyewon Chung, Jin Young Kim, Hyera Kim, Chi-Heum Cho, Eunyoung Ha. _Keimyung Univ. School of Medicine, Daegu, Republic of Korea_.

Glycine decarboxylase (GLDC) is a very recently identified metabolic oncogene that links glycine metabolism with tumorigenesis. We designed this study to determine the role of GLDC in chemoresistance in ovarian cancer cells. First, we found GLDC is downregulated in paclitaxel-resistant ovarian cancer cells (OVCAR3/PTX). Prompted by decreased glucose transport in OVCAR3/PTX cells, we transplanted OVCAR3 and OVCAR3/PTX onto the back of the NOD-scid IL2Rgammanullmice and found that the OVCAR3/PTX tumor grew less rapidly than paclitaxel-sensitive ovarian cancer cells (OVCAR3). We also found that GLDC is downregulated in OVCAR3/PTX tumor. In vitro study revealed that the inhibitory effect of siGLDC on proliferation was significantly greater in OVCAR3 than in OVCAR3/PTX. We also found that knockdown of GLDC in OVCAR3 cells rendered ovarian cancer cells PTX-resistant. Finally, we determined GLDC expressions in patients with chemosensitive and chemoresistant serous-type ovarian cancer and found that GLDC expression was markedly downregulated in chemoresistant cancer patients. In summary, these results suggest that GLDC is associated with ovarian cancer chemoresistance.

#5749

Interleukin-1 (IL-1) induces a treatment-resistant gene expression pattern in prostate cancer (PCa).

Shayna Elizabeth Thomas-Jardin,1 Mohammed Kanchwala,2 Joan Jacob,1 Rachel Meade,1 Nagham Gahnim,1 Nikki Delk1. 1 _UT Dallas, Richardson, TX;_ 2 _UT Southwestern, Dallas, TX_.

Inflammation promotes the development and progression of cancer, where inflammatory cytokines secreted by infiltrating immune cells and tumor cell autocrine signaling are usurped to promote tumor cell survival and disease progression. The interleukin-1 (IL-1) inflammatory cytokine is elevated in prostate cancer (PCa) patient tissue and serum, where it promotes PCa metastasis and bone colonization. Performing in vitro analysis on PCa cell lines and RNA sequencing, we found that IL-1 concomitantly upregulates many pro-survival and lineage proteins, and represses the hormone receptor, androgen receptor (AR). Despite AR loss, a subpopulation of PCa cells remain viable. Our observation could reflect a role for IL-1 in the de novo accumulation of AR- PCa subpopulations. Indeed, AR- PCa subpopulations, such as stem and neuroendocrine cells, are implicated in PCa resistance to AR-targeted therapies. Thus, we hypothesize that IL-1 may reprogram AR+ PCa cells into treatment-resistant AR- PCa cells that survive hormone receptor repression through the upregulation of pro-survival pathways. To begin to address this hypothesis, we performed RNA sequencing and pathway analysis to identify an IL-1-conferred expression pattern in the LNCaP AR+ PCa cell line that mimics the PC3 AR- PCa cell line. We identified canonical inflammatory and immune response pathways, including NFκB, IL-8, and iNOS signaling cascades, likely facilitating PCa cell survival in an inflammatory tumor microenvironment. We also identified novel IL-1-regulated proteins that are known to promote PCa cell survival, such as SQSTM1/p62, SOX9, and ELF3. Experiments are underway to determine the functional significance of our IL-1-conferred gene expression profile in cell line and PDX models. Our investigation will provide insight into the mechanistic function of IL-1 in PCa resistance to AR-targeted therapies

#5750

Underlying inflammatory processes in non-neoplastic female thyroid serve as an immunological basis for the sex disparity in papillary thyroid cancer.

Anvita Gupta,1 Karnika Singh,2 Timmy J. O'Connell,1 Melanie Jones,3 JK Rasamny,1 Monica Schwarcz,1 Augustine Moscatello,1 Edward Shin,4 Raj Tiwari,1 Jan Geliebter1. 1 _New York Medical College, Valhalla, NY;_ 2 _Weill Cornell Medicine, New York, NY;_ 3 _United States Military Academy Preparatory School, West Point, NY;_ 4 _New York Eye and Ear Infirmary, New York, NY_.

Background: The American Cancer Society (ACS) estimated 56,870 new cases of thyroid cancer in the United States in 2017, with roughly 2,010 deaths attributed to the disease. The incidence of thyroid cancer has been increasing steadily over the past three decades, with Papillary Thyroid Cancer (PTC) being the most prevalent form of this endocrine malignancy, comprising of about 90% of all cases. Appreciably, Surveillance, Epidemiology, and End Results (SEER) and the ACS indicate that the incidence of PTC in the age group of 20-49 years is three to four times more prevalent in women than in men. This epidemiologic finding suggests that the female thyroid is at a higher risk of developing PTC.

Methods: The Cancer Genome Atlas (TCGA) database provides the means to address the differences between male and female thyroids on the molecular level. Using the normalized Level 3 RNA Seq Data and patient pathology reports available on the TCGA Data Portal, differential gene expression of 56 non-neoplastic/normal thyroid samples (40 women; 16 men) was investigated using the biological analysis software, Subio platform, version v1.20.5031. (Subio Inc., Tokyo, Japan).

Results: Immune/inflammatory genes exhibited a higher expression in uninflamed, normal thyroid tissue of women than in men (>1.5 fold; p<0.05). The expression levels of these genes were increased in male PTC samples, compared to their matched normal tissue. The level of expression of immune/inflammatory genes in women approximated their expression levels in matched PTC tissue of women and men, indicating that normal thyroid tissue in women exhibits a neoplastic level of inflammation.

Conclusions: Our data suggest that the female thyroid may be "at-risk" for developing PTC due to underlying and on-going immune/inflammatory processes.

#5751

Dietary cholesterol promotes steatohepatitis-related hepatocellular carcinoma by inducing aberrant gene expression in metabolism and mutations in calcium signaling.

Qiaoyi Jessie Liang,1 Narcissus Teoh,2 Lixia Xu,1 Geoffrey Farrell,2 Jun Yu1. 1 _The Chinese University of Hong Kong, Shatin, Hong Kong;_ 2 _Australian National University, Garran, Australia_.

Background and Aims: Dietary cholesterol and nonalcoholic steatohepatitis (NASH) are risk factors for hepatocellular carcinoma (HCC), but their molecular mechanisms are undefined.

Methods: We investigated the effects of cholesterol on NASH and HCC in diethylnitrosamine-injected mice fed high-fat diets with or without high cholesterol. mRNA microarray and whole-exome sequencing analyses were applied for expressional and genetic aberrations. Identified molecular changes were validated in 37 human NASH-HCCs.

Results: Whereas non-cholesterol-fed mice developed simple steatosis, high-cholesterol-fed mice developed NASH, with inflammatory, metabolic and oncogenic genes upregulated in NASH versus steatosis. In cholesterol-induced NASH, HCCs were larger and more numerous than in non-cholesterol-induced steatosis. Although similar numbers of differentially expressed genes were identified between NASH- and steatosis-HCCs, more pathways were found to be uniquely affected in NASH-HCCs, including calcium signaling, insulin signaling, cell adhesion, and axon guidance. In addition, significantly more nonsynonymous somatic mutations occurred in NASH-HCCs (335±84/sample) than steatosis-HCCs (43±13/sample) (P<0.05). Integrated analysis demonstrated that genetic and expressional alterations in NASH-HCCs uniquely affected distinct genes pertinent to five pathways of calcium signaling, insulin signaling, cell adhesion, axon guidance and metabolism. Twelve aberrantly expressed genes in murine NASH-HCC were verified in human NASH-HCC, including metabolism-related genes ALDH18A1, CAD, CHKA, POLD4, PSPH, SQLE and CFD. Among the 80 recurrent mutations particular in murine NASH-HCCs, 21 were also recurrently mutated in human NASH-HCCs, including potential mutational cancer drivers MTOR and SDK1, and three calcium signaling-related genes (RYR1, CACNA1H and RYR2). With calcium signaling affected most significantly by mutations in murine NASH-HCCs, we further verified that calcium signaling was significantly dysregulated by 19 recurrently mutated genes in human NASH-HCCs (n=37), 11 of which encode calcium channel proteins (RYR1, RYR2, CACNA1B, CACNA1E, CACNA1H, CACNA1I, GRIN2C, ATP2A2, ATP2B4, SLC8A1 and ITPR3). RYR1 was mutated most frequently in both murine (4/5) and human (13.5%) NASH-HCCs, with loss-of-function stop-gain/truncating mutations; silencing of RYR1 promoted hepatocyte growth.

Conclusions: Dietary cholesterol causes NASH by dysregulating genes involved in inflammation and metabolism, and promotes NASH-HCC by inducing oncogenic expression and mutations. We identified novel aberrant gene expression, mutations and core oncogenic pathways that contribute to cholesterol-associated NASH-HCC in mice that are relevant to human NASH-HCC.

#5752

Protumoral and pro-metastatic effects of TLR7 in lung cancer.

Marion Dajon,1 Kristina Iribarren,1 Marco Alifano,2 Diane Damotte,3 Isabelle Cremer1. 1 _INSERM UMRS1138, Paris, France;_ 2 _Hopital Cochin, Paris, France;_ 3 _Hopital Cochin, Université Paris Descartes, INSERM UMRS1138, Paris, France_.

TLR7 agonists are currently under investigations for their ability to enhance anti-tumor immune responses. However, in some tumor models, these agonists also stimulate tumor cells, which can express high levels of TLR7, a receptor for single-stranded RNA [1]. We have demonstrated that stimulation of lung tumor cell lines expressing TLR7 with synthetic TLR7 agonists led to upregulation of the antiapoptotic protein Bcl-2, tumor cell survival and chemoresistance [2]. In Non-Small-Cell Lung Carcinoma (NSCLC) cohorts of patients, we have observed high expression of TLR7 on tumor cells of 70% of patients, which conferred poor clinical outcome and was strongly associated with resistance to chemotherapy [3]. This pro-tumoral effect of TLR7 has been validated in murine models in which the injection of TLR7 agonists in NOD/SCID mice, in C57BL/6 wild-type or in TLR7-deficient mice grafted with lung adenocarcinoma tumor cells led to increased tumor progression, increased lung metastasis, and resistance to chemotherapy. On the contrary, we demonstrated that TLR7 antagonist injection led to antitumoral effect. Additionally, TLR7 stimulation resulted in a significant increase of Myeloid-Derived Suppressor Cells (MDSC) in the tumor microenvironment. Depletion experiments of MDSC indicated that these cells are involved in the pro-tumoral effect induced upon TLR7 stimulation. Finally, we have demonstrated that the pro-tumoral effect of TLR7 stimulation, mediated by the MDSC recruitment, was independent of TLR7 stimulation on immune cells. Our results reveal the mechanism by which TLR7 stimulation induce the pro-tumoral effect in mice, and open the way to the development of novel cancer therapeutics including TLR7 inhibitors, for NSCLC patients. This results demonstrate the important role of TLR7 in lung tumor progression. Knowing that natural ligands of TLR7 are ssRNA, we suppose that this effect could be linked to viral infections or RNA released in the tumor microenvironment. [1] Dajon, et al. Immunobiologie. 2016 [2] Cherfils-Vicini J, et al. J Clin Invest. 2010 [3] Chatterjee J, et al. Cancer Res. 2014

#5753

Cancer stem cell markers allow for immune evasion in triple-negative breast cancer.

Andrew D. Hoffmann. _Northwestern University, Chicago, IL_.

Triple-negative breast cancer tumor-initiating cells, or cancer stem cells (CSCs) are identified as a population that is CD44+ and CD24low. These cells are important drivers of distant metastasis and recurrence. An important component of both the recurrence and metastasis functions of CSCs is the capacity to evade immune detection. Our work has identified key members of the antigen presentation machinery required for proper immunosurveillance as being differentially expressed in tumor cells expressing CD44 compared to cells lacking CD44, including a differential response to important immune system cytokines. We are currently working to identify functional differences related to the altered protein levels in these cells.

#5754

Hypoxia-induced tumor plasticity and immune resistance involves an alteration of target recognition by a mechanism involving TGF-beta signaling.

Stephane Terry, Stephanie Buart, Jean-Paul Thiery, Fathia Mami-Chouaib, Salem Chouaib. _Institut Gustave Roussy, Villejuif, France_.

Backgound: Hypoxia is known to shape the tumor microenvironment of developing tumors and contributes to both tumor progression and escape to immune surveillance. Previous work using non-small cell lung carcinoma cells (NSCLC) showed that hypoxia promotes epithelial-mesenchymal transition (EMT) in a fraction of carcinoma cells and that acquisition of a more mesenchymal (Mes) phenotype by certain cancer subclones correlates with an increased propensity to resist cytotoxic T lymphocytes (CTL) attacks as compared to more Epithelial (Epi) cancer subclones. Moreover, numerous studies identified alterations or loss of major histocompatibility complex (MHC) class I as a potential mechanism of tumor escape in cancer. Here, we asked whether hypoxia-induced EMT could regulate MHC class I expression and whether that could impact on resistance to CTL-mediated killing.

Methods and Results : By comparing CMH class I in various NSCLC cancer subclones that have experienced hypoxic stress, we found that cancer clones with enhanced Mes features have a reduced expression of CMH class I molecules, as compared to those with a more Epi phenotype. Transient siRNA knockdown of hypoxia effectors HIF-1α or HIF-2α did not affect the expression of CMH class I molecules suggesting that EMT effectors, rather that HIFs, directly regulate CMH class I expression. Among EMT effectors, TGF-β expression was found to be highly upregulated in Mes cancer clones. Moreover, treatment of these cells with TGF-βR inhibitors markedly upregulated CMH class I expression. and as target cells, increases their susceptibility to CTL-mediated killing. However, blocking of TGF-β using Ab to TGF-β during the coculture time had no significant effects on CTL-mediated killing. This suggests that the observed changes in target cell susceptibility occurs through TGF-β-mediated repression of MHC class I rather than by cancer cell production of TGF-β affecting CTL effector functions. Conclusions: These results suggest that the increase in TGF-β signaling, especially found in EMTed Mes carcinoma cells in response to hypoxic stress, contributes to downregulate CMH class I expression in these cells while coinciding with a reduced susceptibilty to CTL attacks. The development of agents targeting hypoxia or EMT components should provide new therapeutic opportunities in combination with current approaches.

#5755

Cancer immunity contributes to genome stability by suppressing L1 retrotransposition in gastrointestinal cancers.

Hyunchul Jung,1 Jung Kyoon Choi,1 Eunjung Alice Lee2. 1 _KAIST, Republic of Korea;_ 2 _Boston Childrens Hospital & Harvard Medical School, Boston, MA_.

Long interspersed nuclear element-1 (L1) retrotransposons are normally suppressed in somatic tissues, mainly by DNA methylation and antiviral defense. However, L1s can be de-suppressed in cancers to act as insertional mutagens and cause genomic instability by creating DNA double-strand breaks and chromosomal rearrangements. Whereas the frequency of somatic L1 insertions varies greatly among individual tumors, much remains to be learned about underlying genetic, cellular, or environmental factors. Here, our pan-gastrointestinal cancer genome analyses for stomach, colorectal, and esophageal tumors identified multiple correlates of L1 activity. Clinical indicators of tumor progression, such as tumor grade and patient age, showed positive association. Potential L1 expression suppressors such as TP53 and DNMT1, a DNA methyltransferase, were inactivated in tumors with frequent L1 insertions. Importantly, tumors with high immune activity, for example, due to viral infection or high tumor-antigen load, tended to carry a low number of L1 insertions in their genomes with high expression levels of L1 suppressors such as APOBEC3s and SAMHD1. Our analysis of the transcriptional effects of intragenic retrotransposon insertions demonstrated an increased risk of gene disruption in retrotransposition-prone cancers. In particular, we found a splicing-disrupting L1 insertion in an exon of MOV10, a key L1 suppressor, which caused exon skipping with evidence of nonsense-mediated decay in a tumor with a high L1 insertion load. Our results indicate that cancer immunity may contribute to genome stability by suppressing L1 retrotransposition, particularly in gastrointestinal cancers.

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

### Antibodies, Fusion Proteins, and Related Biologics

#5756

Apelin-blocking antibodies as potent therapeutics for oncology.

Arvin Tam, Ming Wang, Hui Zou. _Phanes Therapeutics, San Diego, CA_.

Apelin (APLN) is the endogenous ligand of the G-protein-coupled receptor APJ (APLNR) and triggers a wide range of downstream signaling events, including downregulation of the production of forskolin-induced cAMP and phosphorylation of ERKs, Akt, and p70 S6 kinase. Several lines of evidence suggest that the apelin/APJ axis promotes tumor growth. First, apelin is a potent inducer of angiogenesis and cell proliferation, and its expression is upregulated in hypoxic conditions. Apelin is upregulated in several cancer types and can directly promote tumor growth by inducing cell proliferation through ERK and/or Akt activation. Second, Berta et al. (2010) reported that elevated apelin level correlated with increased microvessel densities and poor survival. Third, blocking APJ signaling reduced proliferation and inhibited tumor growth in human colon adenocarcinoma cell lines expressing high levels of apelin (Picault et al., 2014) and in patient-derived hepatocellular carcinoma cells (Muto et al., 2014). Fourth, although anti-VEGF therapies were developed to block tumor angiogenesis, tumors can acquire drug resistance by bypassing the VEGF pathway and initiating an alternative angiogenic pathway such as the apelin/APJ axis to support growth. Therefore, blocking apelin signaling may be an effective therapeutic strategy for various types of cancer. To block the apelin pathway in cancer therapy, we developed several monoclonal antibodies (mAbs) that bind to the apelin peptide and block apelin-APJ signaling. The antibodies bind to all the physiologically active forms of apelin, including apelin-13, -17, -36, and pyro-apelin-13. These antibodies bind to unique epitopes with an affinity in the picomolar range. The antibodies potently neutralized apelin activity in a cell-based assay with IC50 values in the low nanomolar range. Two antibodies were tested in a mouse oxygen-induced retinopathy (OIR) model to assess their inhibitory effect on angiogenesis. Humanization of the antibodies identified several potent leads that are being characterized for their effect in suppressing the proliferation of tumor cells overexpressing APJ. A search of PDX models found that apelin is overexpressed in specific tumor types including colorectal cancer, kidney cancer, and sarcomas. Remarkably, in PDX models of brain cancer, both apelin and APJ are overexpressed. Indeed, a recent report by Harford-Wright et al. (2017) identified the apelin-APJ system as a central regulator of expansion of patient-derived glioblastoma cells. For recurrent glioblastoma, current anti-VEGF therapies have little to no effect, making the apelin-APJ system an important and exciting target for treating this devastating disease. We will show the efficacy of our lead antibody in PDX models of brain cancer as well as data in support of using apelin-neutralizing mAbs as cancer therapies.

#5757

Preclinical development and testing of a therapeutic antibody against LRG1.

Stephen Moss, David Kallenberg, Vineeta Tripathi, Sterenn Davis, Jestin George, Marie O'Connor, Laura Dowsett, John Greenwood. _University College London, London, United Kingdom_.

Introduction: The purpose of this study was to generate a therapeutic function-blocking antibody against leucine-rich alpha-2-glycoprotein 1 (LRG1), a novel regulator of pathological angiogenesis that is up-regulated in many cancers, and demonstrate efficacy in proof of principle experiments in mouse models.

Experimental Procedures: Mice were immunized with recombinant human LRG1 and hybridomas generated. More than 100 monoclonal antibodies (mAbs) were first screened by surface plasmon resonance (SPR) to measure affinity for target, and then in a co-culture angiogenesis assay to evaluate functional activity. The most promising candidates were then tested in vivo in the mouse model of laser-induced choroidal neovascularization, and from this study a lead was humanized and deimmunized. The resulting antibody is a hinge-stabilized IgG4 that is now undergoing manufacturing and pre-clinical safety and toxicology testing.

Results: Of the original set of mouse mAbs, an initial screen to eliminate low affinity (KD greater than 1nM) binders resulted in 25 progressing to functional testing in angiogenesis assays. The seven most effective blocking mAbs were then tested in vivo in the mouse model of laser-induced choroidal neovascularization. Intraocular injections of mAb yielded two that exhibited equivalent inhibition of neovascularization to a therapeutic VEGF-blocker, and of these, the higher affinity mAb (15C4) was selected for humanization. Epitope mapping identified an amino-acid sequence within human LRG1, that in the form of a synthetic peptide was able to compete the binding of 15C4 to LRG1. Multiple species sequence alignment revealed the epitope to be partially conserved with the corresponding mouse sequence, and SPR analysis showed that 15C4 binding to recombinant mouse LRG1 was several orders of magnitude lower than to human LRG1. Nevertheless, administration of 15C4 to tumor-bearing mice significantly inhibited tumor growth in a range of subcutaneous and genetic models. Furthermore, due to the vessel normalizing effects of 15C4, combinations of mAb with both immunotherapy and cytotoxics were significantly more effective at restricting tumor growth than either monotherapy.

Conclusions: We have generated and characterized a novel vessel-normalizing, fully humanized function-blocking mAb against LRG1 that shows efficacy in mouse tumor models. GMP manufacture is on-going, and early stage clinical trials are planned in 2019.

Financial Support: The work was supported by the Medical Research Council, the Rosetrees Trust and UCL Business.

#5758

Generation and characterization of novel monoclonal antibodies against overexpressed CD109 on pancreatic cancer cells for use in diagnosis and therapy.

Gustavo A. Arias-Pinilla,1 Angus Dalgleish,2 Satvinder Mudan,3 Izhar Bagwan,4 Tony Walker,1 Helmout Modjtahedi1. 1 _Kingston University London, Kingston-upon-Thames, United Kingdom;_ 2 _St George's University of London, London, United Kingdom;_ 3 _The Royal Marsden NHS Trust, London, United Kingdom;_ 4 _Royal Surrey County Hospital, Guildford, United Kingdom_.

Pancreatic cancer is one of the most aggressive and lethal types of cancer, and more effective therapeutic agents are urgently needed. Overexpressed cell surface antigens are ideal targets for therapy with monoclonal antibody (mAb)-based drugs, and several have been approved for the treatment of human cancers; however, none have approval for pancreatic cancer. Our aim was to discover novel overexpressed cell surface antigens in human pancreatic cancer cells using mAb technology. We have generated two novel mouse mAbs, KU42.33C and KU43.13A, against the human pancreatic cancer cell line BxPC-3. Using ELISA, flow cytometry, competitive assay and mass spectrometry, we discover that these two mAbs target two distinct epitopes on the external domain of CD109 that are overexpressed by varying amounts in human pancreatic cancer cell lines. Treatment with these two naked antibodies alone did not affect tumor cell growth in vitro or cell migration. Of the two mAbs, only KU42.33C was useful in determining the expression of CD109 in tumor cells by Western blot and immunohistochemistry. Immunohistochemistry of human pancreatic carcinoma arrays with mAb KU42.33C showed that 94% of the 65 human pancreatic adenocarcinoma cases were CD109 positive, with no expression in normal pancreatic tissues. Our results suggest that these two novel mAbs are excellent tools for determining the expression level of CD109 in the tumor specimens and sera of patients with a wide range of cancers, in particular pancreatic cancer, and for investigating its diagnostic, prognostic and predictive value. Further research should unravel the therapeutic potential of the humanized forms or the conjugated versions of such antibodies in patients whose tumors overexpress CD109 antigen.

#5759

Discovery of a novel claudin 6 (CLDN6) specific monoclonal antibody.

Lewis J. Stafford, Brad Screnci, Chidananda Sulli, Erin Rosenberg, Nicholas Molino, David Tucker, Jonathan Sullivan, Trevor Barnes, Jennifer Pfaff, Tanmayee Hazarika, Thomas Charpentier, Samantha Gilman, Rebecca Rimkunas, Rona Wilf, Sharon Willis, Benjamin Doranz, Joseph Rucker, Ross Chambers. _Integral Molecular, Philadelphia, PA_.

Claudin 6 (CLDN6) is a tight junction molecule that is involved in cell to cell adhesion of epithelial and endothelial cell sheets. CLDN6 is considered an oncofetal protein which is not expressed in normal human tissue but is expressed in some cancers such as endometrial, ovarian and testis cancer. Expression of CLDN6 in endometrial and urothelial cancer leads to a poor prognosis. The problem of developing antibodies against CLDN6 is that the family member claudin 9 (CLDN9) is highly homologous, only varying by 2 amino acids in the extracellular domain. To address this need, Integral Molecular has developed the MPS Discovery Engine® to enable the isolation, characterization, and engineering of monoclonal antibodies for tight junction proteins, GPCRs, ion channels, and transporters. MPS utilizes a collection of technologies to address each of the barriers to monoclonal antibody development against the native extracellular epitopes of multispan membrane proteins. These include, antigen engineering to attain high levels of surface expression, DNA and Lipoparticle immunization to present native epitopes to the immune system, diverse immunization host species to deal with highly conserved proteins, Lipoparticles (high concentration native membrane proteins) to enable phage display, microfludic B-cell isolation to isolate rare MAbs, and shotgun mutagenesis (comprehensive alanine scanning) for epitope mapping. Using the MPS Discovery Engine® we were able to successfully screen a large panel of clones for claudin 6 specificity. From these clones there were 72 potential antibodies that reacted with either claudin 6 or 6/9. A subset of these antibodies reacted only to claudin 6 and not to claudin 9 which has led to our lead drug candidate. With our MPS Discovery Engine® platform, we have the ability to target intact, conformationally specific, and functional antibodies to multipass membrane proteins.

#5760

An IgG1 version of the anti-TfR1 antibody ch128.1 shows significant antitumor activity against different xenograft models of multiple myeloma.

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

The transferrin receptor 1 (TfR1), also identified as CD71, is a type II transmembrane homodimeric protein involved in the cellular uptake of iron and in the regulation of cell growth. TfR1 is a meaningful target for antibody-based cancer immunotherapy due to its high expression levels on the surface of cancer cells and its central role in cancer pathology. We previously developed a mouse/human chimeric IgG3 specific for human TfR1 (ch128.1), which exhibits antitumor activity in SCID-Beige mice bearing disseminated multiple myeloma (MM) KMS-11 cells. Since human IgG1 has been the isotype of choice for therapeutic antibodies targeting malignant cells and has several advantages compared to IgG3, including a more established developability, we constructed an IgG1 version of ch128.1. This novel antibody, ch128.1/IgG1, expressed in murine cells (NS0/1), is properly assembled and secreted and binds antigen (TfR1) similar to its IgG3 counterpart, as demonstrated by ELISA and flow cytometry. Administration of a single dose of the IgG1 completely blocks subcutaneous KMS-11 tumor formation in SCID-Beige mice. Importantly, a single dose of the antibody also shows significant protection, including long-term survival, in this same strain of mice bearing disseminated human MM cells KMS-11 (Asian origin) or MM.1S (African American origin), at different stages of the disease. As expected, the protection was stronger when the mice were treated at an earlier disease stage. Studies using MM.1S cells are of particular relevance given the higher incidence and mortality of MM in African Americans compared to other racial groups. Importantly, we also found that ch128.1/IgG1 confers in vivo protection against MM.1R cells, the MM.1S variant that is dexamethasone resistant. We also developed a ch128.1/IgG1 triple mutant (L234A/L235A/P329S) antibody that does not bind to FcγRs and C1q (the initial protein of the complement pathway), but retains TfR1 binding, as well as binding to FcRn, the neonatal Fc receptor (also known as the Brambell receptor), which is critical in retaining antibody bioavailability in the blood. The triple mutant failed to confer protection in vivo against KMS-11 and MM.1S cells, suggesting a critical role of the antibody Fc fragment in ch128.1/IgG1-mediated antitumor activity, consistent with a potential role of functional effector cells present in this mouse model, such as macrophages. In fact, we found that ch128.1/IgG1 elicits antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) in the presence of murine bone marrow derived macrophages as effectors and KMS-11 cells as targets, similar to its IgG3 counterpart. Taken together, our results suggest that ch128.1/IgG1 has great potential as an effective therapy for incurable human B-cell malignancies such as MM.

#5761

Identification of Fc mutations that optimize agonistic activity of human anti-CD40 monoclonal antibodies by enhancing appropriate Fcg receptor interactions.

Shiming Ye, Diane Cohen, Donghee Choi, Siu sze Tan, Joanna Xiong, Yoshiko Akamatsu, Debra Chao, Diane Hollenbaugh, Fiona Harding. _AbbVie Biotherapeutics Inc., Redwood City, CA_.

Several agonistic antibodies against human CD40 are currently being explored as immune therapy for cancer patients. Previously, anti-CD40 antibody therapy showed some clinical efficacy, but significant toxicity at the efficacious dose limited further development. To develop an anti-CD40 agonistic antibody with an improved clinical therapeutic index, we focused on modifications to the heavy chain isotype that would impact interactions with specific Fc receptors.

In mouse models, anti-CD40 murine IgG1 demonstrated stronger immune stimulatory activities than murine IgG2a. The CD40 murine IgG2a antibody induced acute cytokine release within hours of dosing and led to mortality weeks later. To generate human antibodies with an Fc structure that functions similarly to murine IgG1, human IgG1 Fc mutations with reduced binding on human CD16, enhanced binding on CD32A/B, and maintained binding on CD64 were identified through a proprietary mammalian cell surface display technology. A panel of anti-human CD40 antibodies carrying different Fc regions were created. In a CD40 transfected HEK293 NFκB reporter cell assay, where agonistic activity was not related to Fcγ receptor interactions, anti-CD40 human IgG2 demonstrated stronger agonistic activity than human IgG1 antibodies with either wild type or mutated Fc. This result confirmed the reported role of human IgG2 hinge in impacting the agonistic activity of anti-CD40. In assays using primary B cells or monocyte-derived dendritic cells, which constitutively express Fcγ receptors, a subset of anti-CD40 human IgG1 antibodies showed stronger potency than the corresponding human IgG2, due to the better Fcγ receptor crosslinking for IgG1 antibodies. Swapping the human IgG2 hinge into IgG1 Fc did not further enhance the agonistic activity of anti-CD40 antibodies in the primary cell assays, suggesting that the Fc-crosslinking plays a dominant role in mediating the agonistic activity of these anti-CD40 antibodies in biological systems with cells expressing Fcγ receptor. Engineering the proprietary human IgG1 Fc mutations to these anti-CD40 antibodies further enhanced agonistic activity compared to wild type IgG1 in primary cell assays. In addition to the potent agonistic activity, anti-CD40 with the IgG1 Fc mutations demonstrated lower ADCC and reduced cytokine production driven by Fc receptor activation in a co-culture system. Finally, an Fc modified anti-CD40 agonistic antibody demonstrated an acceptable safety profile in cynomolgous monkey toxicity studies. These data suggest anti-CD40 with the proprietary IgG1 Fc mutations represents a newer generation of CD40 agonists for oncology immune therapy.

#5762

Fast screening of CD40 agonistic antibodies for cancer immunotherapy.

James Jin,1 Yanan Guo,2 Yuelei Shen,2 Benny Yang3. 1 _Biocytogen LLC., Worcester, MA;_ 2 _Beijing Biocytogen Co., Ltd, Beijing, China;_ 3 _Eucure (Beijing) Biopharma Co., Ltd, Beijing, China_.

CD40, Cluster of Differentiation 40, is a member of the TNF-receptor superfamily. CD40 is a co-stimulatory protein found primarily on antigen presenting cells, including B cells, macrophages and dendritic cells. Its ligand, CD40L (CD154) is predominantly expressed on T helper cells, whose engagement activates antigen presenting cells and induces a variety of downstream effects. Whereas most immune checkpoint receptors are expressed on the surface of T cells, CD40 stands out as it is expressed by antigen presenting cells and controls the proper activation of these cells. In turn, CD40 activation exerts a significant impact on T cell-mediated activity against cancer cells. Several pharmaceutical companies are pursuinganti-CD40 therapiesfor treating hematological and solid cancers. Having an access to Biocytogen's antibody discovery and in vivo efficacy screening platform for immune checkpoints, we asked whether we could find suitable anti-CD40 reagents which can successfullycombat developed tumors in preclinical settings. We designed a powerful screening strategy using Biocytogen's humanized CD40 mouse (B-hCD40). Candidate antibodies were generated by immunizing wild-type mice with recombinant hCD40 protein. Then the hybridomas were screened by high-throughput flow cytometry. Instead of examining the immune modulating activity in cell culture, we applied these antibodies to B-hCD40 mice bearing MC38 tumors. Through this straightforwardscreening, several clones stood out by their unparallelingbenefits. These clones were further selected for humanization. Again, the cohort of recombinant humanized CD40 antibodies was screened in B-hCD40 mice. In the end, we discovered the leads with the most potent anti-tumor activity. In conclusion, our work successfully identified several CD40 antibodies that are highly effective for cancer treatment in the preclinical setting.

#5763

Large-scale use of knockout validation to confirm antibody specificity to key oncology targets.

Sam Heaton, Monica Sassi, Alejandra Solache, Bruce Hamilton, Sambhav Dave. _Abcam, Cambridge, United Kingdom_.

Antibodies are the most common tools in basic science and clinical research. Over the past several years there has been an ongoing discussion around antibody validation in light of poor experimental reproducibility between laboratories. This is in part due to the cross-reactivity between antibodies and off-target proteins and the variability between different antibody batches. As a result, experimental irreproducibility leads to wasted resources and compromises the advancement of science. In response to this, Abcam has introduced knockout (KO) validation to help produce target-specific antibodies. This initiative uses KO cells lines made possible through a partnership with Horizon Discovery. Target genes are mutated via CRISPR-Cas9 within a haploid cell line. This results in a frameshift and a complete loss of gene expression. These KO cell lines provide a true negative control for antibody validation as a lack of signal confirms that the antibody binds to its intended target. As a result, both antibody specificity and reproducibility on a large scale can be addressed. We have KO-validated over a thousand antibodies, including several that are relevant to immuno-oncology research. Examples of such targets are the ERK1/2 pathway, which regulates cell survival, proliferation and cell death. Inhibition of this pathway is an attractive target for the treatment of malignant tumors with increased ERK1/2 activity. Similarly, Ki67 expression is strongly associated with tumor cell proliferation and can be used as a marker for tumor aggressiveness. Finally, overexpression of vimentin in tumors correlates with increased growth and invasion, leading to poor prognosis. It is imperative that reproducible data can be produced on these targets to further our understanding of tumor biology and patient prognosis. Here we present the data for antibodies to these key targets that have been validated in both flow cytometry and immunocytochemistry. The antibodies have been tested in KO and wild-type cells to confirm their specificity. The antibodies shown gave signal in wild-type cells but showed complete loss of signal when tested in knockout cell lines. By providing researchers with reliable and specific antibodies that work first time we hope to minimize wasted resources and improve reproducibility.

#5764

Discovery of therapeutic human monoclonal antibody combinations using microfluidics and molecular genomics.

David S. Johnson, Adam S. Adler, Rena Mizrahi, Carter Keller. _GigaGen, Inc., South San Francisco, CA_.

Monoclonal antibodies (mAbs) have proven to be extremely effective immuno-oncology therapeutics that lead to previously unseen durable responses in melanoma, bladder cancer, RCC, lung cancer, and other malignancies. Most mAb therapeutics, including current anti-PD-1 drugs, were discovered by mouse immunization followed by hybridoma isolation. Unfortunately, mouse hybridomas are highly inefficient, capturing only about 0.1% of the total B-cell diversity in immunized mice. As a result, discovery programs that use mouse hybridomas often struggle to find high-affinity, developable antibodies against certain challenging targets, even when using humanized mice. We have developed a novel method for mouse mAb discovery that leverages microfluidics, multiplex PCR, yeast single chain variable fragment (scFv) display, and fluorescence-activated cell sorting (FACS). Our technology is significantly faster and more comprehensive than conventional hybridoma screening. We recently embarked on a discovery campaign for 17 immuno-oncology targets, including PD-1, CTLA-4, GITR, and OX40. We injected chimeric humanized mice (Trianni) with antigen twice weekly for three weeks. Lymph nodes, splenocytes, and bone marrow were removed from the mice and made into single-cell suspensions. We then isolated B cells using negative selection magnetic beads, and used microfluidics to process >4 million cells per mouse, generating DNA libraries that keep single cell heavy and light chain immunoglobulin (Ig) pairing intact. We built yeast scFv libraries for each of the 17 antigen-specific B cell repertoires with a C-terminal c-myc tag. To select scFv-expressing cells that bind to antigen, biotinylated antigen was added to the yeast cells at 7-250nM and then stained with fluorescent streptavidin. We then flow sorted for the top double-positive cells (c-myc+/antigen+). Finally, we deep sequenced the libraries of enriched high-affinity scFvs, which yielded the sequences for >2,000 candidate scFv binders. Hundreds of these candidate scFvs were engineered into full-length mAb constructs and expressed transiently in Chinese hamster ovary (CHO) cells. To develop candidates for clinical use, we performed assays for binding kinetics, cell surface antigen binding, in vitro T-cell activation, and epitope binning. Candidates that are high affinity, bind cell surface antigen, and induce immune cell activation are now being tested in combinations for in vitro and in vivo efficacy. Combinations that have significantly better in vitro and in vivo efficacy than benchmark checkpoint inhibitors currently on the market will be moved forward into clinical studies.

#5765

Immunobiomarkers: Structural and functional characterization of single chain fragment variable (scFv) to ERG from a mouse monoclonal antibody.

Shyh-Han Tan,1 Anshu Rastogi,1 Sreedatta Banerjee,1 Annie Bagga,1 Charles Xavier,1 Ahmed Mohamed,1 Denise Young,1 Gyorgy Petrovics,1 Albert Dobi,1 Isabell A. Sesterhenn,2 Jacob Kagan,3 Sudhir Srivastava,3 David G. David,1 Inger L. Rosner,1 Shiv Srivastava,1 Alagarsamy Srinivasan1. 1 _Center for Prostate Disease Research, Uniformed Services University and the Walter Reed National Military Medical Center, Rockville, MD;_ 2 _Joint Pathology Center, Silver Spring, MD;_ 3 _National Cancer Institute, Bethesda, MD_.

Introduction: Biomarkers for early detection, disease stratification as well as treatment strategies are a major area of focus in prostate cancer (CaP) research. The gene fusion involving ERG and the TMPRSS2 gene promoter contributes to expression of ERG in CaP, serving as a biomarker for CaP. This led to the development of monoclonal or polyclonal antibodies against ERG protein for diagnostic or therapeutic purposes. As the use of intact monoclonal antibody is limited due to large size and low cell penetration, we hypothesized that single chain fragment variable (scFv) and single domain antibody (sdAb) known as minibodies will be valuable as therapeutic agents targeting ERG protein. Towards this goal, the present study addresses the following: i) Characterization of variable heavy (VH) and variable light (VL) chain sequences and generation of expression vectors; ii) Assessing the specificity of binding of scFv and sdAb to ERG protein; iii) Functional evaluation of scFv and sdAb in cellsMethods: We generated VH and VL encoding sequences using RNA derived from hybridoma cells secreting anti-ERG monoclonal antibody designated 9FY. scFv-9FY and sdAb-9FY fragments represent scFv and sdAb , respectively were cloned in an eukaryotic and prokaryotic expression vectors. The proteins (scFv and sdAb) were analyzed by western blot and ELISA. The binding of scFv and sdAb to ERG protein in live cells was evaluated by bimolecular fluorescence complementation (BiFC) assay. Biological effects of scFv and sdAb were assessed in HEK293, VCaP and LNCaP cells. Results: Analysis was carried out to determine the primary DNA sequence of scFv fragments. Using the predicted amino acid sequence, we identified CDR1, CDR2 and CDR3 regions. scFv expressed in prokaryotic vector was purified and used for the evaluation of immunoreactivity to ERG by ELISA. The affinity of scFv and sdAb to ERG was demonstrated through their binding to ERG protein in HEK293 cells by using BiFC assay. This involved the generation of chimeric proteins in which scFv or sdAb coding sequences were fused in-frame to the Venus N- or C-terminal segment separated by a flexible linker of 10 amino acids. Minibodies scFv and sdAb, upon introduction into VCaP cells through transfection, showed cytotoxic effects similar to the observations noted with siRNA against ERG.Conclusions: Here we showed that scFv and sdAb minibodies exhibit high affinity for ERG protein. This property enables them to disrupt the functions mediated by ERG in cells. Given the prevalence of ERG overexpression in prostate cancer, the novel agents based on scFv and sdAb have the potential to interfere with the effect or functions of ERG and are likely to be beneficial in the treatment of CaP.

Source of Funding: Center for Prostate Disease Research, USU Grant HU0001-10-2-0002, NCI/EDRN Grant ACN12011-001-0, and the NCI Grant R01CA162383.

#5766

**Multifunctional biologics for targeted T-cell therapy based on** in vivo **matured fully human VH domains.**

Brian Mcguinness, James Legg, Phil Bland-Ward, Peter Pack. _Crescendo Biologics Ltd, Cambridge, United Kingdom_.

Crescendo Biologics has created a proprietary transgenic mouse devoid of any antibody light chains from which it generates highly diverse fully human VH domain ('Humabody') building blocks. In vivo maturation optimizes Humabody potency and develops superior biophysical properties. Their small size (13kDa) and high stability permits Humabody assembly into an almost limitless array of multifunctional formats optimally configured for therapeutic efficacy. These small versatile single VH domain building blocks permit the rapid exploration of a vast range of 3D format space to identify optimal therapeutic solutions. This fully modular plug & play approach lacks the constraints of traditional mAbs and enables a radical rethink of how a multifunctional molecule can be designed and assembled to deliver enhanced therapeutic benefit. Such molecules are capable of target engagement that is unachievable using regular mAbs. Using Humabody VH addressing key targets in the immune-oncology space, Crescendo has exemplified the potential for optimally configured molecules to deliver enhanced efficacy both in vitro and in vivo. Examples include: CB307, a unique T-cell engaging format with a novel mode of action. CB201, a biparatopic antagonist which 'handcuffs' PD1: one domain binding and blocking PD1 from engaging with its ligands, the other binding a nonblocking epitope. With a binding affinity of 175pM and a unique ability to localise rapidly to PD-1 positive T-cells, CB201 demonstrates significantly enhanced in vivo efficacy over IgG-based PD-1 antagonists in a PDX tumour model in NSG mice. CB108, a half-life extended paratopic Humabody binding PSMA has demonstrated superior tumour targeting with enhanced internalisation and highly efficient accumulation in target-positive tumours with up to 18 percent ID per g recorded in a 24hr imaging study. These data support the progression of highly differentiated therapeutics into the clinic.

#5767

Vascular endothelial growth factor receptor 2 targeted photoimmunotherapy.

Makoto Mitsunaga, Takashi Nishimura, Kimihiro Ito. _Jikei University School of Medicine, Tokyo, Japan_.

Background

Photoimmunotherapy (PIT) is a new class of molecular targeted cancer theranostics, which employs monoclonal antibody (mAb) conjugated to a photosensitizer, IR700, that is activated by focal near-infrared (NIR) light irradiation leading to necrotic cell death by cell membrane rapture where mAb-IR700 conjugates binds to target membrane proteins specifically. We have previously demonstrated HER2-targeted PIT employing trastuzumab-IR700 conjugates for HER2 expressing cancers. However, some cancer cells were survived partly because of tumor heterogeneity and inhomogeneous micro-distribution of mAb-IR700 conjugates. In this study, we developed a new type of PIT agents targeting VEGFR-2 expressed on vascular endothelium in a tumor and evaluated the feasibility by comparing conventional PIT in vitro and in vivo.

Methods

HER2-positive human gastric cancer cells, NCI-N87, were used for the experiments. HER2-targeting trastuzumab and VEGFR-2-targeting ramucirumab were conjugated to IR700. Cells were treated with mAb-IR700 conjugates followed by NIR light irradiation after washing the cells. LIVE/DEAD assay was performed to assess short-term phototoxicity and trypan blue dye exclusion assay to assess long-term phototoxicity. Next, mouse tumor xenograft models were created for in vivo PIT. Tumor-bearing mice were randomized and treated with mAb-IR700 conjugates followed by NIR light irradiation under anesthesia. Antitumor effects were monitored by measuring tumor diameters with a caliper.

Results

PIT utilizing ramucirumab-IR700 conjugates did not induce phototoxic effect in vitro because of the absence of membranous expression of VEGFR-2 in NCI-N87 cells, while PIT utilizing trastuzumab-IR700 conjugates induced rapid phototoxic effect because of the strong membranous expression of HER2 in NCI-N87 cells. By contrast, antitumor effects were observed in NCI-N87 xenograft tumors in vivo utilizing both ramucirumab- and trastuzumab-IR700 conjugates followed by NIR light irradiation. Microscopic analysis of the harvested tumors showed decreasing tumor micro-vessel densities only when tumors were treated with PIT utilizing ramucirumab-IR700 conjugates but not trastuzumab-IR700, which represents a different mechanism than that of conventional PIT targeting antigens expressed on the tumor cell membrane.

Conclusion

We demonstrated a new mechanism of PIT utilizing ramucirumab-IR700 conjugates. As VEGFR-2 is upregulated in many types of solid cancers, this method may be considered as being applicable to various types of cancers in future clinical settings.

#5768

MG1122, a whole IgG-like bispecific antibody targeting mesothelin and CD3, induces T cell-mediated killing of MSLN-expressing tumor cells.

Yun-Jung Lee, Okjae Lim, Munkyung Kim, Jeewon Lee, Kisu Kim, Junhong Jung, SuA Lee, Sung Keun Kim, Haenaem Kwon, Yangmi Lim, Yong Yea Park, Jonghwa Won. _Mogam Institute, Yongin-si, Republic of Korea_.

Mesothelin (MSLN) is an antigen overexpressed in several malignancies, including mesothelioma and ovarian and pancreatic adenocarcinoma. It has been studied as a marker for diagnosis and a target for immunotherapy. Here, we adopted a bispecific antibody format for recruiting cytotoxic T cells to kill tumor cells. MG1122 is a novel, whole IgG-like bispecific antibody which recognizes CD3 on T cells and MSLN on tumor cells. MG1122 induced effective killing of MSLN-expressing tumor cells. This response was associated with robust activation of T cells as shown by nuclear factor of activated T cells (NFAT) activation, CD25 and CD69 upregulation and increased cytokine release. In addition, using a live cell analysis system, we found that levels of activated caspase 3/7 were amplified in the tumor cells by MG1122 treatment. Although MSLN is a surface antigen, it also exists as a secreted isoform, which can lead to tumor evasion against MSLN targeting antibodies. However, despite the high concentration of soluble MSLN, MG1122 still effectively bound and showed strong killing effects against MSLN-expressing tumor cells. Taken together, our MSLN/CD3 bispecific antibody, MG1122 offers a promising opportunity to redirect T cells to kill MSLN-expressing cancer cells.

#5769

Targeted Engineered Toxin Bodies provide a novel mechanism of action against HER2 positive cancers.

Brigitte Brieschke, Garrett L. Robinson, Sangeetha Rajagopalan, Hilario J. Ramos, Jensing Liu, Jack P. Higgins, Erin K. Willert. _Molecular Templates Inc., Austin, TX_.

Molecular Templates develops highly potent, specific, next-generation immunotoxins that are proprietarily de-immunized to avoid both innate and adaptive immune recognition. These Engineered Toxin Bodies (ETBs) destroy cancer cells by enzymatic destruction of ribosomes, a mechanism of action (MOA) distinct from that of other therapeutics. Molecular Templates' lead compound, MT-3724, has seen clinical activity in heavily pre-treated lymphoma patients, underscoring the potential for this class of agents to work in the refractory/relapsed setting.

Current modalities for the targeted treatment of HER2 positive breast cancer include monoclonal antibodies, antibody-drug conjugates, and tyrosine kinase inhibitors. Although all these modalities have demonstrated clinical benefit, the majority of patients will unfortunately relapse due to a variety of resistance mechanisms. Upon relapse, most of these patients will still express HER2, allowing for the development of new HER2-targeted modalities.

MT-5111, a HER2-targeted ETB with picomolar potency against HER2 expressing cells, was designed to overcome mechanisms of resistance to current HER2 modalities such as: escape from antibody dependent cell-mediated cytotoxicity (ADCC), alterations in signal transduction, epitope masking, and enhanced small molecule efflux. Additionally, MT-5111 was genetically engineered to reduce the anti-drug antibody response and signalling through innate receptors, allowing for repeat dosing.

MT-5111 binds to an epitope on HER2 distinct from trastuzumab and ado-trastuzumab emtansine (T-DM1), and exhibits effective cytotoxicity on both T-DM1 sensitive and T-DM1 resistant HER2 positive cell lines. The targeting of MT-5111 to a distinct epitope allows for activity of the ETB on cell lines where the trastuzumab epitope is masked, and allows for combination with targeted agents trastuzumab or T-DM1 without competition for binding to HER2. Previous reports have shown that resistance to T-DM1 may be due to increased drug efflux; however, the MT-5111 cytotoxic payload is a large molecule not subject to this mechanism of resistance. Additionally, the novel MOA for MT-5111 should allow for cell kill independent of changes to the tumor microenvironment or alterations in HER2-mediated signal transduction.

Pre-clinical data demonstrate the potent activity of MT-5111, including in T-DM1-resistant cell lines, as well as the potential for co-administering MT-5111 and trastuzumab or T-DM1, allowing for concurrent treatment of two HER2-targeted agents with distinct and non-interfering mechanisms of action. In vitro and in vivo data will be presented, highlighting the potential for MT-5111 as a novel agent under development for treatment of breast carcinomas, and other malignancies overexpressing the HER2 receptor. Molecular Templates intends to initiate clinical studies with MT-5111 in 2018.

#5770

Engineering and characterization of anti-PSMA humabody-deBouganin fusion proteins.

Jeannick Cizeau,1 Shilpa Chooniedass,1 Rachelle L. Dillon,1 Arjune Premsukh,1 Gregory P. Adams,1 Glen C. MacDonald,1 Normann Goodwin,2 Lorraine Thompson,2 Bethan Archer2. 1 _Eleven Biotherapeutics, Cambridge, MA;_ 2 _Crescendo Biologics, Cambridge, United Kingdom_.

Targeted protein therapeutics or TPTs are single-protein therapeutics composed of targeting moieties genetically fused via peptide linker to cytotoxic protein payloads. TPTs are designed to overcome efficacy and safety challenges inherent within ADCs. For tumor indications only reachable by systemic delivery, we have designed a de-immunized cytotoxic protein payload, deBouganin. DeBouganin is a T-cell epitope-depleted variant of bouganin, a type I Ribosome Inactivating Protein (RIP) that blocks protein synthesis by the deadenylation of ribosome RNA resulting in programmed cell death. DeBouganin's biophysical and biochemical properties, in combination with its unique mechanism of action, make it an attractive cytotoxic payload for targeted drug delivery. Using different protein scaffolds and in vivo matured human VH domains, we have shown that deBouganin exhibits certain advantages over more conventional small molecule cytotoxins, with respect to cell killing power, including the ability to kill cancer stem cells, circumvent multi-drug resistance, and avoidance of cross-resistance mechanisms.

Crescendo has created a proprietary transgenic mouse devoid of any antibody light chains from which it generates highly diverse fully human VH domain ('Humabody®') building blocks. In vivo maturation optimizes Humabody® potency and develops superior biophysical properties. Their small size and high stability permits Humabody® assembly into multifunctional formats optimally configured for therapeutic effcacy. Such molecules are capable of target engagement that is unachievable using regular mAbs and may include a half-life extending serum albumin-binding binding domain or a cytotoxic payload. Using monovalent and biparatopic PSMA targeting Humabody® formats, we describe the molecular engineering and biological testing of novel anti-PSMA Humabody-deBouganin fusion constructs with or without a VH half-life extender. The data show that Humabody-deBouganin fusion proteins are expressible as a soluble protein in E. coli supernatant. Moreover, biological testing demonstrated the preference of a non-cleavable linker composed of a single G4S motif over a furin linker so as to ensure serum stability. IC50 value of all fusion proteins was approximately 0.2 nM against PSMA-positive LnCAP tumor cells. In vitro data support the potential of Humabody-deBouganin fusion constructs as anti-cancer therapeutics.<!--EndFragment-->

#5771

Targeting EphA2 in bladder cancer using a novel antibody-directed nanotherapeutic.

Walid Kamoun,1 Elden Swindell,1 Christine Pien,1 Lia Luus,1 Jason Cain,1 Irawati Kandela,2 Richard Huang,1 Suresh Tipparaju,1 Dmitri Kirpotin,1 Wiam Bshara,3 Vasileios Askoxylakis,1 Carl Morrison,3 Daryl Drummond1. 1 _Merrimack Pharmaceuticals, Cambridge, MA;_ 2 _Northwestern University, Evanston, IL;_ 3 _Roswell Park Cancer Institute, Buffalo, NY_.

Ephrin receptor A2 (EphA2) is a member of the Ephrin-Ephrin receptors cell to cell signaling family of molecules and plays a key role in proliferation, differentiation, and migration. EphA2 is overexpressed in a broad range of cancers, including bladder cancer, and is associated with increased metastasis and poor prognosis. Several potential EphA2-targeted therapies were developed and showed promising preclinical activity which failed to translate clinically due to narrow therapeutic window. we propose to use a novel antibody-directed nanotherapeutic (ADN) approach to target EphA2 for the treatment of bladder cancer. This work characterizes the expression of EphA2 in bladder cancer patients, evaluates its prognostic power, and tests an EphA2-targeted ADN, MM-310, in patient-derived xenograft (PDX) models. EphA2 expression in tumors was investigated using a validated immunohistochemistry assay performed in 147 bladder cancer samples, enabling analysis of prevalence and prognostic power. Whole sections of primary and metastatic tumor resections were used to assess EphA2 expression in tumor-associated vessels and tumor cells. Four EphA2+ PDX models were used to evaluate the activity of MM-310 compared to free docetaxel, alone or in combination with gemcitabine.

EphA2 was expressed in tumor cells and tumor-associated blood vessels in primary and metastatic lesions. EphA2 overexpression was seen in 80%-100% of tumors and correlated with shorter survival. In the PDX models, MM-310 controlled tumor growth, mediated greater regression, and was more active than free docetaxel at equitoxic dosing (Table). Additionally, the combination of MM-310 and gemcitabine controlled tumor growth better than each drug alone, and outperformed the combination of free docetaxel and gemcitabine in the single PDX model where it was compared.

Thus, in bladder cancer models, a docetaxel-based ADN as a monotherapy and in combination with gemcitabine targeted EphA2 and led to significant tumor regression.

Maximum tumor regression (%) & Time to regrowth (days)

---

Model Name | Control | Docetaxel | MM-310 | p value

BL0293 | -2% & 3 days | -58% & 55 days | -100% & 120 days | <.05 & <.001

BL0382 | 0% & 3 days | -16% & 34 days | -100% & 106 days | <.001 & <.001

BL0440 | 0% & 7 days | -43% & 41 days | -84% & 67 days | <.01 & <.01

BL417362 | 0% & 5 days | -47% & 69 days | -100% & 121 days | <.05 & <.05

#5772

Engineered lectins to treat cancer.

Thomas Murarasu, Ludger Johannes. _Curie Institute, Paris, France_.

Targeted delivery of cytotoxic compounds to tumors has become one of the most dynamic areas of research and development in the cancer therapy field. In this context, glycosphingolipids (GSLs) represent a promising class of tumor markers. Indeed, cancer patients have altered GSL expression patterns on their tumoral cells compared to healthy tissue (Krengel et al., 2014). In a recent prioritization of cancer antigens by the National Cancer Institute at Rockville (USA), 4 out of 75 were GSLs (GD2, GD3, fucosyl-GM1, and GM3), with GD2 arriving already at the 12th position (Cheever et al., 2009). However, GSLs are only rarely addressed in the context of tumor targeting, obtaining GSL specific antibodies being a notorious difficult task. An exception is the anti-GD2 antibody, dinutuximab, which entered the market for the treatment of high-risk neuroblastoma patients, which is unfortunately associated to dose-limiting acute toxicity (Yu et al., 2010). This project aims at developing a novel class of therapeutic products for the delivery of therapeutic compounds by targeting tumor-specific GSLs. Our products are based on a naturally occurring GSL-binding lectin with intrinsic tumor targeting capacity. We will rely on a proprietary technology to derive products which are tuned to transport cytotoxic compounds specifically into cancer cells. As a proof of concept, we apply our technology to a tumor-specific GSL, GD2, to generate a therapeutic formulation against neuroblastoma, one of the most frequent cancer in children under 5 years old. Our technology has truly groundbreaking potential as, in principle, it can apply to any GSL to specifically target several common tumors.

#5773

**Sequencing and cloning IDH1 R132H-targeted monoclonal T cell receptors from CD4** + **T cells facilitated by opto-electric-positioning technology.**

Duane Smith,1 Payal Watchmaker,2 Guido Stadler,1 Natalie Marks,1 Yelena Bronevetsky,1 Keviin Chapman,1 Hideho Okada2. 1 _Berkeley Lights Inc, Emeryville, CA;_ 2 _University of California, San Francisco, San Francisco, CA_.

Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are enzymes that catalyze the conversion of isocitrate to α-ketoglutarate, while reducing NADH to NADPH. More than 80% of low-grade gliomas and secondary glioblastomas harbor point mutations in IDH. The R132H mutation is the most common mutation in IDH1, and vaccination with the mutant peptide elicited an MHC class II-specific antitumor response, suggesting mutant IDH1 as a potential target for immunotherapy (1). Discovering monoclonal T cell receptors (TCRs) that specifically recognize the R132H mutation is a key but challenging step towards viable immunotherapy for glioma/glioblastomas. Using opto-electric-positioning (OEP) technology in a chip-based, NanoPen culture environment, we have successfully identified and isolated a monoclonal T cell that specifically recognizes the R132H mutant IDH1 peptide. We cultured and identified monoclonal CD4+ T cells on chip, stimulated by monocyte-derived dendritic cells, pulsed with R132H IDH1 peptide. Activated, CD4+ T cells were selectively exported from the chip and their T-cell receptors (TCRs) were subsequently deep sequenced. The most likely pairs of alpha and beta TCR chains were cloned into mammalian expression vectors for functional validation in vitro, producing a TCR that specifically binds mutant IDH1 tetramer. Taken together, we show that the combined platform of the OEP and nanofluidic device provides a powerful solution to help identify cell type-specific, monoclonal T cells that can be exported and sequenced to reveal distinct TCRs, which in turn can be cloned, expressed and validated for future immunotherapies.

Reference: 1. Schumacher et al., Nature 2014;512:324-27.

#5774

Novel cell-based high-throughput hybridoma screening method using the Celigo image cytometer for antibody discovery.

Leo L. Chan,1 Haohai Zhang,2 William Rice,1 Nasim Kassam,3 Maria S. Longhi,2 Haitao Zhao,4 Simon C. Robson,2 Wenda Gao,5 Yan Wu2. 1 _Nexcelom Bioscience LLC, Lawrence, MA;_ 2 _Beth Israel Deaconess Medical Center, Boston, MA;_ 3 _Brigham Woman's Hospital, Boston, MA;_ 4 _Chinese Academy of Medical Sciences, China;_ 5 _Antagen Pharmaceuticals, Inc, Boston, MA_.

Hybridoma screening is a highly important process for antibody discovery, which can identify potential clones from hundreds to thousands of hybridoma cultures against the desired target antigen. Traditional screening methods using ELISA and flow cytometry presented technical issues such as limited accuracy, reduced high-throughput capability, increased time and cost. Conventional ELISA screening with coated antigen can also be impractical for difficult to express hydrophobic membrane antigens or multi-chain protein complexes. Here, we demonstrate novel cell-based high-throughput screening methodology using plate-based Celigo Image Cytometer, which avoids nonspecific signals by contrasting antibody binding signals directly on living cells, with and without recombinant antigen expression. The image cytometry-based high-throughput screening method was optimized by detecting the binding of hybridoma supernatants to the recombinant antigen CD39 expressed on Chinese hamster ovary (CHO) cells. Both target CHO and CFSE-stained wild type were co-cultured to simultaneously detect target antibodies (Alexa Fluor 594) and nonspecific binding, which can eliminate the need to produce a separate control sample for each hybridoma supernatant. Next, the image cytometer was used to screen 672 unique hybridoma supernatants to develop the high-throughput screening workflow. The Celigo was used to quickly image and analyze antibody binding of 672 samples, using Hoechst, Alexa Fluor 594 and CFSE fluorescence to identify high, medium, and low binding hits. Finally, the sensitivity of the image cytometer was demonstrated by serial dilution of purified CD39 antibody. Celigo was used to measure antibody affinities of commercial and in-house antibodies to membrane-bound CD39. This cell-based screening procedure can be completely accomplished within one day, significantly improving throughput and efficiency of hybridoma screening. Furthermore, measuring direct antibody binding to living cells eliminated both false positive and false negative hits. The image cytometry method was highly sensitive and versatile, and could detect positive antibody in supernatants at concentrations as low as ~5 ng/mL, with concurrent Kd binding affinity coefficient determination. We propose that this screening method will greatly improve screening technologies and facilitate more efficient antibody discovery. 

### Canonical Targets 2

#5775

Identification of SAR439859, an orally bioavailable selective estrogen receptor degrader (SERD) that has strong anti-tumor activity in wild-type and mutant ER+ breast cancer models.

Maysoun Shomali,1 Youssef El-Ahmad,2 Frank Halley,2 Jane Cheng,1 Michael Weinstein,1 Muchun Wang,1 Fangxian Sun,1 Natalia Malkova,1 Mikhail Levit,1 Malvika Koundinya,1 Zhuyan Gou,1 Andrew Hebert,1 Jessica McManus,1 Dietmar Hoffman,1 Hui Cao,1 Joonil Jung,1 Jack Pollard,1 Sylvie Vincent,1 Timothy Ackerson,1 Francisco Adrian,1 Chris Winter,1 Victoria Richon,1 Hong Chen,1 Karl Hsu,1 Joanne Lager,1 Albane Courjaud,2 Rosalia Arrebola,2 Laurent Besret,2 Pierre-Yves Abecassis,2 Laurent Schio,2 Gary McCort,2 Michel Tabart,2 Victor Certal,2 Fabienne Thompson,2 Bruno Filoche-Rommé,2 Laurent Debussche,2 Patrick Cohen,2 Carlos Garcia-Echeverria,2 Monsif Bouaboula1. 1 _Sanofi, Cambridge, MA;_ 2 _Sanofi, Vitry-sur-Seine, France_.

Nearly 70% or more of newly diagnosed cases of breast cancer (BC) are estrogen receptor positive (ER+) where endocrine therapy is a primary treatment. However, substantial evidence describes a continued role of ER signaling in tumor progression, where approximately 40% of patients on endocrine therapy develop resistance that include mutations in the ER that drive a constitutively active receptor. Fulvestrant, an estrogen receptor degrader, is effective at shutting down ER signaling. However, fulvestrant efficacy studies report insufficient blockade of ER signaling in patients that may be a consequence of poor pharmaceutical properties.

Here we describe the discovery of SAR439859, a novel, orally bioavailable SERD with potent antagonist and degradative properties against ER both in vitro and in vivo. SAR439859 has robust inhibition of ER signaling activity in multiple ER+ breast cancer cell lines including tamoxifen resistant lines harboring ER mutations. Across a large panel of ER+ cells, SAR439859 demonstrated broad and superior ER degradation activity as compared to other SERDs including improved inhibition of ER signaling and inhibition of cell growth. Similarly, in vivo treatment with SAR439859 demonstrated significant tumor regression in ER+ BC models including MCF7-ESR1 mutant-Y537S model and endocrine therapy resistant patient-derived xenograft tumor transplantation.

Collectively, these results showed that SAR439859 is an oral, nonsteroidal, selective estrogen receptor antagonist and degrader that could provide therapeutic benefit to ER+ breast cancer patients. SAR439859 is currently being evaluated in a phase I clinical trial.

#5776

Pharmacologic and PK/PD study of D-0502: An orally bioavailable SERD with potent antitumor activity in ER-positive breast cancer cell lines and xenograft models.

Yaolin Wang, Zhe Shi, Yueheng Jiang, Xing Dai. _InventisBio Inc., Shanghai, China_.

Breast cancer is the most common form of cancer in women in the developed world, with estrogen receptor-positive (ER+) breast cancer accounting for 70-80% of all breast cancers. Endocrine therapy such as selective estrogen receptor degrader (SERD) fulvestrant has been used effectively to extend the life of ER+ breast cancer patients, either alone or in combination with CDK4/6 inhibitor (palbociclib or abemaciclib). However, due to poor pharmacokinetic property, fulvestrant has to be given via intramuscular injection. In addition, patients relapsed after prolonged endocrine therapy with aromatase inhibitors such as letrozole and anastrozole due to emergence of ER mutations. Here, we report the discovery of D-0502, an orally bioavailable SERD with potent activity in various ER+ breast cancer cell lines and xenograft models. In addition, combination of our SERD candidates and palbociclib in both MCF-7 xenograft model and ESR-1 mutated (Y537S) patient-derived breast cancer xenograft model resulted in further tumor growth inhibition or regression. Comparison of various SERD molecules showed our candidate has more potent antitumor activity in MCF-7 xenograft model than GDC-0810, AZD9496 and fulvestrant. More importantly, drug metabolism and pharmacokinetic (PK) studies both in vitro and in vivo demonstrated that our SERD molecule D-0502 exhibits superior PK profiles suitable for clinical development.

#5777

**Ormeloxifene augments the therapeutic response of enzalutamide** via **targeting androgen receptor splice variant 7.**

Bilal B. Hafeez, Andrew E. Massey, Vivek K. Kashyap, Mohammed Sikander, Advit Shetty, Mehdi Chaib, Hassan Mandil, Shabnam Malik, Mohan M. Yallapu, Meena Jaggi, Subhash C. Chauhan. _Univ. of Tennessee Health Science Ctr., Memphis, TN_.

Enzalutamide (ENZ) is a second generation anti-androgen drug in clinical use for the treatment of advanced prostate cancer (PrCa). Use of ENZ has some limitations because of its toxic side effect and developing chemoresistance. It has been shown that expression of androgen receptor splice variant 7 (ARV7) in advanced PrCa is involved in ENZ therapy resistance. It is noteworthy that ENZ does not target AR splice variants because these lack the ligand binding domain of AR. Thus, identification of non-toxic agents which can target ARV7 can improve the enzalutamide therapy response and could be used for the management of advanced stage PrCa. Herein, we identified a clinically approved selective estrogen receptor modulator ormeloxifene (ORM), which augments ENZ therapy response via suppression of ARV7 expression in PrCa cells. We used androgen-independent human prostate cancer cells (22Rv1), which express ARV7 as a model system to study the effect of ORM alone or in combination with ENZ. ORM treatment showed a more potent effect in 22Rv1 cells as compared to ENZ alone. ORM treatment also sensitized the effects of ENZ in 22Rv1 cells. ORM treatment effectively inhibited colony formation ability of 22Rv1 cells; this effect was additive in combination with ENZ. We next performed Western blot analysis of ARV7 in control and ORM treated 22Rv1 cells. ORM effectively inhibited the protein level of AR V7 at 12 hrs post-treatment in 22Rv1 cells; however, no effect was observed with the treatment of ENZ. ORM treatment also inhibited the promoter activity of the AR target gene PSA as determined by luciferase assay. To translate these finding to an in vivo model system, we investigated the chemo-sensitization potential of ORM in a 22Rv1 cell-derived xenograft mouse model. ORM treatment (200 µg/mouse three times a week for 8 consecutive weeks) significantly (P<0.01) inhibited prostate tumor growth. However, we did not observe any significant tumor growth inhibition with oral administration of ENZ (10 mg/kg body weight). ORM treatment improved the therapeutic response of ENZ in 22Rv1 cells derived xenograft mouse model as monitored by significant (P<0.01) decreases in both tumor volume and tumor weight as compared to ENZ treatment alone. Our findings indicate that ORM alone or in combination with ENZ could be an effective therapeutic approach for the treatment of advanced PrCa.

#5778

Preclinical evaluation of TQB3616, a highly potent and selective small-molecule CDK4/6 inhibitor.

Xu Zhaobing,1 Hu Lihong,1 Liu Yingchun,1 Charles Z Ding,2 Xiaoyu Zhu,2 Chen Chen,2 Yusong Zhu,2 Jiahu Wu,2 Dongdong Wu,2 Shuhui Chen,2 Xiquan Zhang,3 Ling Yang,3 Xin Tian3. 1 _WuXi Apptec, Wuhan, China;_ 2 _WuXi Apptec, Shanghai, China;_ 3 _Chia Tai Tianqing Pharmaceutical Group Co. Ltd., Nanjing, China_.

Objective: The G1 restriction point is critical for regulating the cell cycle and is controlled by the retinoblastoma protein (Rb) pathway (CDK4/6-cyclin D1-Rb-p16/ink4a). Selective CDK4/6 inhibitors, such as palbociclib and ribociclib have been approved by US FDA in combination with letrozole to treat patients with HR+/Her2- breast cancer, and selective CDK4/6 inhibitor LY2835219 is in Phase 3 trials for the same and related indications. Here, we disclose TQB3616 for the first time, a novel selective, potent, oral inhibitor of CDK4/6.

Method: The anti-proliferative activity of TQB3616 was evaluated in a panel of 18 Rb-proficient cell lines in a 72-hour viability assay. Daily oral administration was used to evaluate in vivo antitumor activity of TQB3616 in the MCF-7 cell-derived xenograft (CDX) breast cancer model. Effect on CDK4/6-cyclin D1-Rb signaling was assessed by western blot analysis of downstream effector protein p-Rb. In vivo antitumor activity of TQB3616 was also tested in a patient-derived xenograft (PDX) lung cancer model LU-01-0393.

Result: TQB3616 displayed potent anti-proliferative activity in 8 of 18 Rb-proficient cancer cell lines (>50% inhibition at 0.3 µM). Inhibition of p-Rb in the tumor samples was verified by western blot analysis. TQB3616 showed in vivo antitumor activity in the MCF-7 CDX model with TGI of 60% @7.5 mpk and 93% @15 mpk, better than palbociclib (TGI = 52% @20 mpk, 80% @40 mpk) and LY2835219 (TGI = 45% @7.5 mpk, 76% @15 mpk). TQB3616 also showed good in vivo antitumor activity in the LU-01-0393 lung cancer PDX model with 65% of TGI @35 mpk.

Conclusion: TQB3616 is a potent and selective CDK4/6 inhibitor and shows excellent antitumor activity in preclinical tumor models. These results warrant TQB3616 going to the clinic for exploration of its potential for treating HR+/Her2-/Rb+ breast cancer as well as other Rb+ cancer types.

#5779

CDK4/6 inhibitors and anaplastic thyroid cancer: An investigation into the retinoblastoma cancer pathway and potential therapeutic targets.

Kristen A. Wong, Antonio Di Cristofano. _Einstein College of Medicine/Montefiore, Bronx, NY_.

Introduction/Background: Recently, Cyclin-dependent kinase (CDK) 4/6 inhibitors have been shown to increase progression-free survival in estrogen receptor-positive, stage IV breast cancer patients. We aim to assess whether Palbociclib, a selective CDK4/6 inhibitor, prevents tumor cell growth in vitro and in vivo in anaplastic thyroid cancer (ATC). ATC carries an automatic stage IV classification and a 6-month survival prognosis; there has been very little advancement in chemotherapeutic options, making it a good disease target for this new class of drug. Retinoblastoma (Rb) is a tumor suppressor protein that is rarely mutated in ATC (<3%). It blocks cell entry into S phase, preventing DNA synthesis and cell proliferation. Hyper-phosphorylation of Rb by CDK4/6 causes the release of transcription factors and induces cell division. By blocking CDK4/6 with the inhibitor, the tumor suppressor function of Rb stays activated. Methods: We characterized our large library of thyroid cancer cell lines by mRNA and proteins involved in the Rb pathway using qPCR and western blot. We then performed 72-hour dose-dependent cell growth assays using viable cell counts with differing concentrations of Palbociclib to determine EC50 values. The same cell counting technique was used to test Palbociclib in combination with other targeted chemotherapy agents and again in resistance/adaptation experiments with long term exposure. Cell cycle analysis was performed using BD FACSCanto II fluidics system. Results: Our human lines show more susceptibility compared to murine lines. Interestingly, the PI3K and RAS pathway-activating mutated lines required the highest relative doses of Palbociclib followed by p53 and BRAF mutated cells. The Rb negative cells showed no growth inhibition when treated. Using flow cytometry, it was confirmed that Palbociclib's cystostatic effect is due to a G1 arrest. The presence of synergistic effects between Palbociclib and Omipalisib, a PI3K inhibitor, occurs in the majority of the cell lines and preliminary experiments show this combination treatment slows the development of resistance to Palbociclib. Conclusions: In vitro testing has shown that Palbociclib causes dose-dependent growth inhibition on Rb positive ATC cells, and genetic mutations may predict response to the drug. Rb protein is necessary for the drug to have effect. Current work is focused on designing a novel mouse model for in vivo study and to further investigate the related parallel pathways for combined targeted therapies, namely those whose over-activation is associated with the least sensitivity to Palbociclib - PI3K/AKT and RAS pathways.

#5780

E6201, a novel MEK1 inhibitor, suppresses the metastatic capability of triple-negative breast cancer cells.

Jangsoon Lee,1 Bora LIM,1 Kuicheon Choi,1 Troy Pearson,1 Linda Paradiso,2 Thomas Myers,2 Debu Tripathy,1 Naoto T. Ueno1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Strategia Therapeutics, Inc, Spring, TX_.

Background: Triple-negative breast cancer (TNBC) lacks the receptor targets ER, PR, and HER2 and thus it does not respond to receptor-targeted treatments such as hormonal therapy and trastuzumab, leaving chemotherapy as the mainstay of treatment. TNBC also has higher recurrence, metastasis, and mortality rates than other receptor subtypes. The MAPK (RAS/MEK/ERK) pathway plays a crucial role in cancer cell survival, invasion, and metastasis and critically contributes to aggressive features of TNBC, as demonstrated by our novel therapeutic synergy identification screening of RNA interference. Two BRAF inhibitors (vemurafenib and dabrafenib) and two MEK inhibitors (trametinib and cobimetinib) have received U.S. Food and Drug Administration approval for treatment of melanoma, clinically validating the potential of MAPK pathway inhibition to meaningfully benefit patients with TNBC. However, the rapidly emerging resistance of TNBC to current standard treatments and disease progression remain clinical challenges for these pathway-targeted agents. E6201 is a novel MEK1 inhibitor being evaluated in phase 1 clinical trials in patients with advanced solid tumors (NCT00794781) and with melanoma metastasized to the central nervous system (NCT03332589). In the preclinical study described herein, we determined the in vitro anti-tumor efficacy and in vivo anti-metastatic efficacy of E6201 in TNBC to prepare for further development of E6201 in the clinic.

Methods: The anti-tumor effects of E6201 were examined using cell proliferation and anchorage-independent colony-formation assays. To evaluate the anti-metastatic activity of E6201 in vitro and in vivo, a migration/invasion assay and an experimental/spontaneous metastasis assay were performed, respectively.

Results: In vitro cell proliferation data demonstrated that E6201 inhibited growth more effectively (half maximal inhibitory concentration [IC50] range: 0.05-5 µM) than did other MEK inhibitors (selumetinib, pimasertib, and trametinib). E6201 inhibited TNBC cell-colony formation in a dose-dependent manner (P = 0.0001). Also, E6201 suppressed the migration and invasion of MDA-MB-231 (IC50: 1 µM) and SUM149 (IC50: 0.25 µM) cells in a dose-dependent manner (P = 0.001). In vivo experimental and spontaneous metastasis assays demonstrated that E6201 inhibited lung metastases of the SUM149 and MDA-MB-231-LM2 cell lines (P = 0.0001). Immunohistochemical staining demonstrated that E6201 decreased the metastatic burden in the lung (P = 0.011) and decreased phosphorylated ERK expression in a dose-dependent manner.

Conclusion: Taken together, these data demonstrate that E6201 is an effective inhibitor targeting the MAPK pathway in TNBC, showing anti-tumor efficacy in vitro and anti-metastatic efficacy in vivo. Our data provide a rationale for further clinical development of E6201 as MEK-targeted therapy for treatment of TNBC.

#5781

A novel ERK1/2 inhibitor has potent activity in KRAS-mutant non-small cell lung cancer models.

Aurelie Courtin, Luke Bevan, Tom Heightman, Birikiti Kidane, Justyna Kucia-Tran, John Lyons, Sandra Muench, Alpesh Shah, Lukas Stanczuk, Neil Thompson, Nicola Wallis, Nicola Wilsher, Joanne Munck. _Astex Pharmaceuticals, Cambridge, United Kingdom_.

Non-small cell lung cancer (NSCLC) molecular profiling is a key factor in treatment selection. Although, patients with NSCLC tumors harboring EGFR or ALK mutations can benefit from personalized therapies, there are currently no approved targeted therapies for KRAS mutant tumors which occur in 25% to 30% of patients with NSCLC. The constitutive activation of the MAPK pathway in these tumors provides a rationale for targeting effectors such as MEK1/2 (MEK) or ERK1/2 (ERK). Inhibitor of MEK kinase have been tested clinically in KRAS-mutant NSCLC but results have been disappointing, possibly because compensatory signaling such as the reactivation of ERK is triggered following the inhibition of MEK, leading to cancer cell survival. Therefore, targeting ERK directly represents an attractive therapeutic approach. As previously described, we have developed a novel, potent and selective ERK inhibitor identified by fragment-based drug discovery which has potent activity in vitro and in vivo. Here, we demonstrate the activity of this lead compound in KRAS-mutant NSCLC models. Our novel ERK inhibitor was tested in a panel of 440 human cancer cell lines of which the KRAS NSCLC population was identified as particularly sensitive. 62% of the KRAS-mutant NSCLC cell lines tested, exhibited antiproliferative IC50s ranging from 1 nM to 500 nM. This lead compound also inhibited ERK downstream signaling in KRAS NSCLC models both in vitro and in vivo. Indeed, the phosphorylation level of the ERK substrate, RSK, was strongly decreased in HCC-44 and Calu-6 xenograft tumors 2h after oral administration of the lead compound at 50 mg/kg. Levels of pRSK remained below those of untreated tumors for up to 16h in HCC-44 tumors and 24h in Calu-6 tumors. We also confirmed that, the ERK inhibitor conferred a decrease in phosphorylation of ERK itself in both models. The inhibition of ERK signaling corresponded to significant anti-tumor activity in these models with a daily oral administration of 50 mg/kg compound leading to significant tumor regression in subcutaneous models of HCC-44 (18.3% T/C) and Calu-6 (8.9% T/C) xenograft tumors. This work demonstrates the in vitro and in vivo activity of a novel, highly potent, selective ERK inhibitor in models of KRAS-mutant NSCLC. These data support the further optimisation of this series of compounds for clinical development.

#5782

Selumetinib-based therapy in uveal melanoma patient-derived xenografts.

Didier Decaudin,1 Rania EL Botty,1 Béré Diallo,1 Gerald Massonnet,1 Justine Fleury,1 Adnan Naguez,1 Marine Dubois,1 Emma J. Davies,2 Aaron Smith,2 Joanne Wilson,2 Colin Howes,2 Paul Smith,2 Nathalie Cassoux,1 Pascale Mariani,1 Sophie Piperno-Neumann,1 Sergio Roman Roman,1 Fariba Nemati1. 1 _Inst. Curie, Paris, France;_ 2 _AstraZeneca, Macclesfield, United Kingdom_.

Purpose: The prognosis of metastatic uveal melanoma (UM) patients remains one of the worst observed in human cancers. The identification of frequently mutated GNAQ/GNA11 and the activation of MAPK signaling in UM raise the possibility of using efficient targeted therapies such as MEK inhibition. The aim of our study was to evaluate drug combinations that might enhance the efficacy of the MEK inhibitor, selumetinib (AZD6244, ARRY-142886), using patient-derived xenografts.

Materials: Five UM PDXs were used in this study, i.e., MP34, MP46, MP55, MP77, and MM26, all mutated for GNAQ or GNA11. Selumetinib was administered alone or combined with chemotherapies (dacarbazine and docetaxel) or targeted therapies: the ERK inhibitor, AZ6197 and the mTORC1/2 inhibitor, vistusertib (AZD2014). All targeted therapies were administered orally five days/week at the following doses: selumetinib at 25 mg/kg/day BID, AZ6197 at 50 mg/kg QD, AZD2014 at 15mg/kg QD. Dacarbazine was administered at 40 mg/kg for 5 consecutive days every 28 days and docetaxel at 15 mg/kg weekly. Both were administered intraperitoneally.

Results: The combination of selumetinib and dacarbazine did not show improvement of antitumor efficacy in MP34, MM26 and MP55.The results showed a slight increase of overall response rate when selumetinib was combined with docetaxel, in comparison to the treatment with selumetinib or docetaxel alone. Importantly, we observed a significant antitumor effect when selumetinib was used in combination with AZ6197 or AZD2014 with respect to the monotherapies. Preliminary pharmacodynamics studies showed variation of the expression of p-ERK and also p-AKT and/or p-S6 when UM PDX were treated with AZ6197 or AZD2014 alone or in combination with selumetinib.

Conclusion: Our data suggest that ERK and mTORC1/2 targeting could represent promising therapeutic approaches for the treatment of metastatic UM patients.

#5783

Strong antitumor activity of ASN007, an oral ERK1/2 inhibitor, in PDX tumor models with MAP kinase pathway alterations including KRAS mutations.

Sanjeeva P. Reddy, Niranjan S. Rao, Roger A. Smith, Scott K. Thompson, Sarper Toker. _Asana Biosciences, Lawrenceville, NJ_.

The RAS/RAF/MEK pathway is frequently dysregulated in a broad range of cancers through mutations in several targets such as KRAS, NRAS, HRAS, BRAF, NF1 and receptor tyrosine kinases. Although MEK and BRAF inhibitors improve the outcome for patients with BRAF mutant melanoma and lung cancers, the duration of response is limited due to reactivation of the pathway. The extracellular-signal-regulated kinases, ERK1 and ERK2 (ERK1/2), which are downstream of MEK and BRAF kinases, serve as a key node in this crucial signaling pathway. Targeting ERK1/2 offers a promising therapeutic strategy for a broad range of cancers, particularly the ones driven by RAS mutations, and in overcoming resistance to MEK/BRAF inhibitors. Here, we present discovery and characterization of a novel orally bioavailable compound, ASN007, that showed inhibition of ERK1/2 kinases in a biochemical assay with IC50 = 2 nM for both targets. Interestingly, ASN007 displayed a much slower dissociation rate (longer target residence time) as compared to other ERK inhibitors. ASN007 inhibited the phosphorylation of ERK1/2 substrates such as RSK1, FRA1 and Elk1 in various cell lines with low nM IC50 values. ASN007 showed potent anti-proliferative activity that was selective for MAPK-pathway dependent cancer cell lines. Indeed, ASN007 showed much more robust activity against RAS mutant cell lines as compared to other ERK1/2 inhibitors. In a daily oral dosing regimen, ASN007 demonstrated strong tumor growth inhibition in BRAF, KRAS and NRAS mutant xenograft models in mice and was well tolerated at efficacious doses. Of note, ASN007 showed strong efficacy using various intermittent dosing regimens. It also showed a strong anti-tumor activity in colorectal PDX models with various KRAS mutations. Furthermore, ASN007 showed regression of tumor growth in a melanoma PDX model that was resistant to both MEK and BRAF inhibitors. In addition to the single agent activity, greater efficacy was observed when ASN007 was combined with immunotherapeutic agents such as an anti-PD-1 antibody or an IDO-1 inhibitor. ASN007 does not exhibit significant inhibition of CYP enzymes or the hERG channel. A Phase 1 trial with ASN007 in patients with BRAF and K-, H- and N-RAS mutant melanoma, colorectal, non-small lung and pancreatic cancers is being planned.

#5784

A novel ERK inhibitor in the treatment of uveal melanoma.

Elgilda Musi,1 Grazia Ambrosini,1 Joanne Munck,2 Gary K. Schwartz1. 1 _Columbia University Medical Center, New York, NY;_ 2 _Astex Pharmaceuticals, Cambridge, United Kingdom_.

Uveal melanoma is one of most common intraocular malignancies of the adult eye. GNAQ/ GNA11 genetic alterations are present in 85% of diagnosed cases resulting in activation of MAPK pathway. Recently, MEK inhibitors, such as selumetinib have been used to treat GNAQ/GNA11-mutant uveal melanoma patients. However, selumetinib has shown modest effect compared to chemotherapy. As ERK is the primary downstream effector of the MAPK pathway, it is hypothesized that ERK inhibitors have the potential to improve clinical activity in these patient populations. We examined the effects of a selective and potent ERK inhibitor (Compound A), on several uveal melanoma cell lines including 4 GNAQ cell lines (92.1, Mel202, Omm1.3, MP46), 2 GNA11 cell lines (MP41, Omm1), 2 BRAF cell lines (OCM1A, OCM1) and a Wild Type (Mel290) cell line. The uveal melanoma cell lines were sensitive to ERK inhibition by Compound A, with IC50s concentrations determined to be between 50 to 250 nM. Compound A was more effective at inhibiting growth than the MEK inhibitor selumetinib at nanomolar concentrations. Compound A at 250 nM inhibited pERK1/2 within at least 2 hours of drug exposure. By 24 hours inhibition of pRB and suppression of cyclin D1 was detected in all the cell lines tested except the Wild type cell line Mel290. Cell cycle analysis of Compound A dosed at 250 nM for 24 hours showed significant G1 arrest (85-90% G1). These in vitro results support further evaluation of ERK inhibition in the treatment of uveal melanoma.

#5785

EML4-ALK variant E6a/b;A20 positive NSCLC cell lines are associated with growth upon blocking MEK-ERK pathway.

Bengt Hallberg,1 Ruth H. Palmer,1 Ganesh Umapathy,1 Dan E. Gustafsson,1 Joachim T. Siaw,1 Wasi Alam,1 Jikui Guan,1 Robert Doebele,2 Anh T. Le,2 Andrea Doak2. 1 _Inst. of Biomedicine, Gothenburg, Sweden;_ 2 _Division of Medical Oncology, Denver, CO_.

Background:

The protein kinase B (PKB/AKT) and RAF/MEK/ERK signaling pathways are activated in a wide range of human cancer types. In many cases, concomitant inhibition of both pathways is necessary to block proliferation and induce cell death and tumor shrinkage. Several feedback mechanisms have been described in which inhibition of one pathway leads to activation of a parallel signaling pathway, thereby decreasing the effectiveness of targeted monotherapies.

Materials and Methods:

In order to determine which signalling core should be blocked for combinatorial treatment, we treated a panel of EML4-ALK positive lung cancer cell lines using MEK-ERK inhibitors. Resazurin assay was performed to evaluate cell viability. Protein levels were determined using western blotting.

Results:

In this study, we describe a feedback mechanism in which MEK-ERK inhibition leads to increased activation of AKT signaling in EML4-ALK variant (E6a/b;A20) positive NSCLC cell lines. Interestingly, EML4-ALK variant (E13;A20) NSCLC cell lines responds to MEK-ERK inhibitors and shows synergism when combined with ALK tyrosine kinase inhibitors. We found that feedback response in EML4-ALK variant (E6a/b;A20) positive NSCLC cells was mediated by the mammalian target of rapamycin complex 2-associated protein SIN1, resulting in increased survival and proliferation that depended on AKT signaling.

Conclusions:

Taken together, these results elucidate an important feedback network and contraindicate the use of MEK inhibitors as effective therapeutic strategy in EML4-ALK variant (E6a/b;A20) positive NSCLC.

#5786

Grabbing GRB2: The use of liposome-incorporated Grb2 antisense oligonucleotides as a novel therapy in gynecologic malignancies.

Olivia D. Lara,1 Cristian Rodriquez-Aguayo,1 Emine Bayrakter,1 Paola Amero,1 Selanere Mangala,1 Chris LaFargue,1 Ana Tari-Ashizawa,2 Gabriel Lopez-Berestein,1 Anil Sood1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Biopath Holdings, Houston, TX_.

Prexigebersen (liposome-incorporated nuclease resistant antisense oligonucleotides specific for GRB2, L-GRB2) has proven highly effective in the treatment of leukemia, however its application in solid malignancies including gynecologic cancer has yet to be investigated. Given the biological significance of GRB2, we aim to assess the effect of prexigebersen in pre-clinical models. We tested the clinical significance of GRB2 using publicly available high-throughput data. We investigated the expression of GRB2 in a series of in vitro (Western Blotting) experiments in high-grade serous (HGSC) and uterine (UC) carcinoma models. We also tested in vivo (orthotopic mouse model) the biological effects of prexigebersen in HGSC models (OVCAR 5), first in a dose-defining experiment then in combination with standard dose paclitaxel. We first examined GRB2 using TCGA and GISTIC data and found alterations (amplifications, deletions and mutations) in 6% (33 of 594) of HGSC cases and 5% (25 of 547) of UC cases. Amplifications of GRB2 are associated with a decreased patient survival (p<0.01) in UC, suggesting it to be an attractive therapeutic target. GRB2 was expressed in most ovarian (HEYA8, OVCAR5, SKOV3, and OVCAR3) and uterine (MFE 319, Ishikawa and Hec1a) cancer cell lines tested. We performed a dose-defining in vivo experiment on an orthotopic mouse model of ovarian cancer (OVCAR 5). We found a maximum decrease in GRB2 expression at a dose of 15 mg/kg prexigebersen and a dose dependent decrease in tumor burden (p<0.05). We then performed an additional in vivo model in which prexigebersen was combined with standard dose paclitaxel. There was an eighty-six percent decrease in tumor burden (p<0.05), and multinodular burden (p<0.01) in the combination prexigebersen/paclitaxel group compared to control. Prexigebersen shows promising preclinical efficacy and may be a novel therapeutic strategy in gynecologic malignancies.

#5787

Ipatasertib impairs tumor growth in androgen sensitive and castrate resistant prostate cancer: Evidence in preclinical models.

Remi M. Adelaiye-Ogala, Yen T. Nguyen, Jose A. Rodriguez-Nieves, Aian Neil Alilin, Supreet Agarwal, David Vanderweele. _National Cancer Institute, NIH, Bethesda, MD_.

Introduction: Therapeutic targeting the PI3K/AKT signaling pathway is an attractive strategy to combat solid tumors that exhibit hyper-AKT activity. Ipatasertib, a potent pan-AKT inhibitor, has exhibited significant inhibitory effects on tumor growth in preclinical studies, including models of Androgen Receptor (AR)-independent prostate cancer (PCa) that lack PTEN, a negative regulator of the PI3K/AKT pathway. It is currently being investigated in several ongoing clinical trials, including evaluating its efficacy in combination with abiraterone in castrate resistant PCa (CRPC) (NCT01485861). There is cross-talk between androgen signaling and PI3K/AKT pathway activation, however the optimal setting for AKT inhibition in PCa is not clear. Here we seek to determine the benefit of ipatasertib for androgen sensitive (AS) PCa alone or in combination with androgen deprivation (AD), as well as the benefit of ipatasertib alone or in combination with the antiandrogen enzalutamide in CRPC. Methods: In vitro studies utilized human androgen sensitive PCa cell lines LAPC, LAPC4shPTEN, and LNCAP, as well as two LNCaP-derived, castrate resistant cell lines. Cell viability was determined using CellTitre Glo assay and apoptosis by flow cytometry. Inhibitory effects of AKT signaling pathway was determined by western blot assays against AKT phosphorylation sites and downstream targets as well as detection for hormone receptor expression. In vivo studies utilized LuCaP 136 and LuCaP 136-derived castrate resistant (LuCaP 136CR) patient derived xenograft (PDX) models. Cells were injected into athymic mice and grouped into 4 arms. Intact mice bearing AS tumors were treated with vehicle, ipatasertib (100mg/Kg), AD, or ipatasertib + AD. Castrated mice bearing castrate-resistant tumors were treated with vehicle, ipatasertib, enzalutamide (10mg/Kg), or ipatasertib + enzalutamide. Tumor growth was determined twice/week via blinded caliper measurements. Results: Ipatasertib effectively inhibited AKT signaling, as indicated by significant decrease in phosphorylation of the downstream targets P70S6K and 4E-BP1. Our in vitro data indicate significant anti-proliferation effects of ipatasertib across all PCa models, both androgen sensitive and castrate resistant. Interestingly, this was irrespective of PTEN status. In addition, we observed a decrease in glucocorticoid receptor (GR) expression in enzalutamide resistant lines, although AR expression was not significantly altered. Preclinical animal studies showed significant response to tumor growth inhibition with ipatasertib monotherapy, which was enhanced with combination treatment. Conclusion: Ipatasertib is beneficial in models of androgen sensitive and castrate resistant PCa and can be effective in the treatment of PCa alone or in combination with current standard therapies targeting the androgen receptor.

#5788

Preclinical evaluation of HZB0071, a small molecule inhibitor of AKT.

Gang Li,1 Lihong Hu,1 Weifeng Mao,2 Dongfang Li,2 Charles Z. Ding,2 Shuhui Chen2. 1 _WuXi AppTec (wuhan), Wuhan, China;_ 2 _WuXi AppTec (shanghai), Shanghai, China_.

Objective: AKT is a component of the PI3K/AKT/mTOR (PAM) pathway, which plays an important part in cell proliferation, growth and survival, activated in many cancers and has been validated as a therapeutic target in the clinic. Inhibition of AKT activity can block the PAM pathway and proliferation of various types of tumor cells with loss of tumor suppressor PTEN, mutational activation of p110a catalytic subunit of PI3K and amplification of the gene encoding either AKT or PI3K. The purpose of this study is to investigate the in vitro and in vivo antitumor activity of HZB0071, a small molecule AKT kinase inhibitor, in preclinical models of solid cancer with AKT overexpression.

Method: Kinase inhibiting activity of HZB0071 was determined with AKT1, 2, 3 kinase assays. Cellular anti-proliferative activity was evaluated with AKT overexpressed LNcap cell line and DU145 cell line without AKT overexpression. The antitumor activity of HZB0071 was evaluate in vivo and the synergistic effect combined with Paclitaxel in AKT highly expressed patient-derived mouse xenograft (PDX) models of Gastric Cancer ST-02-0013 and cell-derived mouse xenograft (CDX) models of Breast Cancer BT474.

Result: HZB0071 displayed potent kinase inhibiting activity for AKT1, 2, 3 with IC50 1.3±0.41 nM, 50.3±13.4 nM and 90.4±5.84 nM, respectively. HZB0071 inhibited cell proliferation in AKT overexpressed LNcap cells with IC50 67±7 nM. In contrast, HZB0071 showed much weaker anti-proliferative activity in DU145 cells with IC50 >10,000 nM. HZB0071 showed antitumor efficacy in AKT highly expressed gastric cancer PDX model ST-02-0013 (TGI = 62% @30 mpk, QD as single agent; TGI = 61% @15 mpk + Paclitaxel @10 mpk, QD, in combination with Paclitaxel), as well as in the breast cancer BT474 CDX model (TGI = 88% @30 mpk + Paclitaxel @15 mpk, QD).

Conclusion: We have identified a novel potent AKT inhibitor HZB0071. Preclinical studies shows antitumor efficacy of HZB0071 in AKT overexpressed solid cancer models. HZB0071 represents a promising clinical candidate for treating solid cancers with high AKT expression.

#5789

Discovery of clinical candidate BMS-986158, an oral BET inhibitor, for the treatment of cancer.

Ashvinikumar V. Gavai, Derek Norris, David Tortolani, Daniel O'Malley, Yufen Zhao, Claude Quesnelle, Patrice Gill, Wayne Vaccaro, Tram Huynh, Vijay Ahuja, Dharmpal Dodd, Christopher Mussari, Lalgudi Harikrishnan, Muthoni Kamau, John S. Tokarski, Steven Sheriff, Richard Rampulla, Dauh-Rurng Wu, Jianqing Li, Huiping Zhang, Peng Li, Dawn Sun, Henry Yip, Yingru Zhang, Arvind Mathur, Haiying Zhang, Christine Huang, Zheng Yang, Asoka Ranasinghe, Celia D'Arienzo, Ching Su, Gerry Everlof, Lisa Zhang, Nirmala Raghavan, John T. Hunt, Michael Poss, Gregory D. Vite, Richard A. Westhouse, Susan Wee. _Bristol-Myers Squibb, Princeton, NJ_.

Background: The bromodomains and extra-terminal domain (BET) proteins are a family of 4 adapter proteins, BRD2, BRD3, BRD4, and BRDT, that bind to specific acetylated lysine residues on the histone tails of chromatin and recruit additional proteins to regulate gene transcription. The c-MYC oncogene, which is amplified and deregulated in 40% to 70% of all cancers, is directly regulated by BET proteins. Preclinical studies provide a strong rationale for pursuing transcriptional regulation via BET inhibition in cancer treatment (Lenhart, et al. Mol Cancer Ther. 2015;14:2167-2174; Filippakopoulos, et al. Nature. 2010;468:1067-1073). Here, we present results of crystal structure-guided structure-activity relationship (SAR) studies that resulted in the identification of BMS-986158, a highly potent BET inhibitor.

Methods: Using fluorescence resonance energy transfer (FRET), we screened a library of compounds and identified a carbazole series of BET inhibitors. Alkylation of the carbazole nitrogen resulted in a 10-fold boost in potency against BET. We then created a differently oriented carbazole series and, subsequently, a carboline series of compounds to improve potency and pharmaceutical properties. A thermal shift assay was used to evaluate selectivity for binding to the BET family of bromodomains.

Results: Crystal structure and subsequent SAR studies demonstrated that the isoxazole moiety formed critical interactions with the BET bromodomains. Lead compounds demonstrated potent binding to BRD4 and reduction in c-MYC expression and proliferation in cell lines such as KMS-11. Accessing a second lipophilic pocket in the BRD4 binding site increased potency significantly. Modification of the lead series from a carbazole carboxamide to a carboline resulted in significant improvement in pharmaceutical properties and led to the identification of BMS-986158, which demonstrated in vitro and in vivo potency against a variety of tumor types. In c-MYC-driven cancer cell lines, BMS-986158 caused dose-dependent downregulation of c-MYC expression and induced cancer cell death. BMS-986158 demonstrated > 70% tumor growth inhibition at tolerated doses in patient-derived xenograft models (lung, colorectal, and triple-negative breast cancers). Antitumor activity in mice and pharmacokinetic properties in animal studies support oral dosing in humans.

Conclusions: Structure-based drug design led to the discovery of BMS-986158, a highly potent BET inhibitor. With promising antitumor activity in preclinical studies, BMS-986158 is currently being evaluated in a phase 1/2a clinical trial in patients with advanced cancers.

#5790

TG-1601 is a novel BET inhibitor with strong binding affinity and long-lasting effect in pre-clinical models.

Emmanuel Normant,1 Leonid Gorelik,2 Rama Shmeis,1 Henry Le,1 Robert Nisch,1 Karen TenHuisen,1 Teja Turpuseema,1 James Oliviero,3 Hari P. Miskin,1 Peter Sportelli,1 Michael S. Weiss1. 1 _TG THERAPEUTICS, New York, NY;_ 2 _Fortress Biotech, New York, NY;_ 3 _Checkpoint Therapeutics, New York, NY_.

BET (bromodomain and extra-terminal) proteins bind to acetylated lysine residues on chromatin and participate in the regulation of gene transcription. Inhibition of BET protein binding to chromatin with small molecules selectively suppresses the transcription of a set of oncogenes, including MYC and BCL-2. TG-1601 is a novel, selective and potent small molecule inhibitor of BET bromodomains. TG-1601 binds to the first and second bromodomains (BD1, BD2) of the BET protein family, BRD2, BRD3, BRD4, and BRDT, with Kd values ranging from 0.5 nM to 9.1 nM. MYC protein expression is strongly inhibited in the MV4-11 cancer cell line with an EC50 of 5 nM, with GI50 comprised between 15 nM and 85 nM in a variety of leukemia and myeloma cancer cell lines, indicating potent inhibition of cell proliferation. Time course and dose-response studies conducted in mice bearing subcutaneous MV4-11 xenografts showed that MYC protein was undetectable 3 hours following a single 25 mg/kg oral dose, with a TG-1601 tumor concentration of 6uM achieved. Interestingly, at 24h post-dose, while TG-1601 is cleared from the tumor, MYC protein level remains below 40% of its initial level, indicating a long-lasting effect pharmacodynamic of TG-1601, potentially attributable to enhanced binding affinity compared to earlier generation molecules. In agreement with this long-lasting effect, efficacy studies in MV4-11 tumor-bearing mice, dosed with a 20 mg/kg/day PO regimen interrupted by increasing drug holiday periods, showed that drug holidays of 2, 3 and 4 days per week only modestly affected efficacy (3%, 15% and 12% TGI respectively), suggesting discontinuous dosing of TG-1601 in clinic may not significantly impact efficacy. Anti-cancer agents have been shown - in vitro or in vivo - to synergize with BET inhibitors. Here we show that TG-1601 and anti PD-1 antibody demonstrated synergistic anti-tumor activity when combined in the B16F10 model, an aggressive syngeneic model of melanoma. TG-1601 inhibition of MYC, CCR-2 and IL1RN gene expression was tested in a whole blood ex-vivo experiment, and the genes were validated as pharmacodynamic markers to monitor TG-1601 activity in clinic. In conclusion, TG-1601 is a novel BET inhibitor with remarkably strong affinity and a potent ability to inhibit MYC expression and cell growth, with favorable pharmacokinetic properties supporting clinical development. Its properties in vivo provide an opportunity for the rational development of TG-1601 as an anti-cancer agent, taken alone or in combination with other small molecules or antibodies. IND enabling studies are underway, with clinical evaluation expected to commence in the first half of 2018.

#5791

Bromodomain inhibitors for the treatment of invasive lobular carcinoma.

Darran P. O'connor,1 Louise Walsh,1 Yue Fan,2 Finbarr Tarrant,2 Suet-Feung Chin,3 Philip Schouten,4 Carlos Caldas,3 Rene Bernards,4 Triona ni Chonghaile,1 William Gallagher2. 1 _Royal College of Surgeons in Ireland, Dublin, Ireland;_ 2 _University College Dublin, Dublin, Ireland;_ 3 _University of Cambridge, Cambridge, United Kingdom;_ 4 _Netherlands Cancer Institute, Amsterdam, Netherlands_.

Invasive lobular carcinoma (ILC) is the second most common type of breast cancer after invasive ductal carcinoma (IDC), accounting for approximately 10-15% of all breast tumors. ILC is characterized by inactivation of E-Cadherin and neoplastic cells that invade the stroma in a "single-file" pattern. Women with ILC are usually older, have used hormone replacement therapy and are more likely to have hormone receptor-positive disease. ILCs have similar survival to IDCs at both five and 10 years, but despite this, the clinical course is distinct: ILCs are three times more likely to metastasize to the peritoneum, gastrointestinal tract, and ovaries and are more frequently bilateral. Therefore, tailored therapeutic options for this distinct, hard-to-treat subtype of breast cancer are required.

As part of the RATHER FP7 HEALTH consortium (www.ratherproject.com), we carried out RNA-Seq analysis of 61 primary ILC samples and identified that high expression of the BET family protein Brd3 (uniquely among BRD family members) was associated with poor recurrence free survival (p=0.03, HR 8.63, CI 1.22-60.85). This observation was further validated in the independent METABRIC cohort (n=99), where again, high Brd3 expression (and not other BRD members) was associated with poor recurrence-free survival (p<0.01, HR=3.16, CI 1.24-8.03). Using a two ILC cell lines (SUM44PE and MDA-MB134VI) we found that ILC cells were relatively resistant to the anti-estrogen therapies tamoxifen and fulvestrant compared to those derived from IDC. Next, we tested whether the ILC cell lines were sensitive to BET protein inhibition using the pan-BET family inhibitor JQ1. Interestingly, while JQ1 inhibited cell growth in both ILC cell lines tested, apoptosis was only induced in SUM44PE cells, while MDA-MB134VI cells exhibited G1 arrest. Dynamic BH3 profiling was used to dissect the underlying anti-apoptotic dependencies in each ILC cell type and showed that in the JQ1-resistant MDA-MB134VI cells, survival was predominantly Bcl2-dependent. Combination of JQ1 and the Bcl2-inhibitor venetoclax (ABT-199) synergistically killed MDA-MB134V1 cells compared to treatment with JQ1 alone, while combination with the Bcl2/Bcl-Xl/Bcl-W inhibitor navitoclax (ABT-263) added further synergy.

With a number of BET inhibitors now entering clinical trials, the data described here suggest that BET inhibition is a rational therapeutic option for some ILC cases, and for those that do not respond, combination with venetoclax may be a suitable therapeutic strategy. In our cell line models, baseline Bcl-2 expression was sufficient to predict induction of apoptosis in response to JQ1 and could be used to guide therapeutic choice.

#5792

Preclinical antitumor activity of BMS-986158, an oral BET inhibitor, for the treatment of cancer.

Susan Wee, Donald Jackson, Heshani Desilva, Maya Dajee, Julie Carman, Petra Ross-MacDonald, Jinping Gan, Richard A. Westhouse, Christine Huang, Zheng Yang, Michael Poss, John T. Hunt, Gregory D. Vite, Ashvinikumar V. Gavai. _Bristol-Myers Squibb, Princeton, NJ_.

Background: Regulation and recognition of covalent chromatin modifications by cellular proteins are key determinants of gene expression. Bromodomain and extra-terminal (BET) proteins, including BRD2, BRD3, BRD4, and BRDT, bind directly to acetylated lysine on histone tails to promote gene transcription. Oncogenes such as c-MYC, BCL2, and ASCL1, are directly regulated by BET proteins. Here, we report the preclinical evaluation of BMS-986158, an orally bioavailable, potent, and selective BET inhibitor.

Methods: BMS-986158 activity was assessed in tumor growth assays across solid and hematologic cancer cell lines in vitro and in vivo in patient-derived xenografts (PDX). Dose-response gene expression profiling of ex vivo blood from healthy donors was used to identify transcripts exhibiting a pharmacodynamic effect with BMS-986158 treatment.

Results: Tumor growth inhibition (TGI) with BMS-986158 was observed across different solid and hematologic cancer cell lines. Mutations in chromatin regulators were identified among BMS-986158‒sensitive cell lines. BMS-986158 also demonstrated antitumor activity (TGI ≥ 70%) in 24 of 82 PDX models tested (Table). Genetic alterations that correlate with BMS-986158 responsiveness will be presented. Finally, an ex vivo dose-response profiling study in healthy human blood identified 124 genes responsive to BMS-986158, including chemokines and chemokine receptors.

Conclusions: BMS-986158, a potent and selective BET inhibitor, demonstrated robust anti-proliferative activity in vitro across a broad range of cancer cell lines. Consistent with the in vitro data, preclinical antitumor activity was observed in various PDX tumor models. Furthermore, gene expression profiling studies in human blood samples identified select target genes responsive to BMS-986158 treatment that may be useful as clinical pharmacodynamic markers.

TGI assay in PDX models

---

Tumor Type | Total sample, n | Responders, na

Adenocarcinoma | 31 | 9

Lung squamous cell carcinoma | 21 | 7

Breast cancer | 8 | 2

Brain cancer | 1 | 0

Colorectal cancer | 6 | 3

Esophageal cancer | 3 | 1

Gallbladder cancer | 2 | 1

Head and neck cancer | 3 | 1

Mixedb | 7 | 0

Total | 82 | 24

a Responders were defined as having > 70% TGI

b Mixed tumor types include large cell neuroendocrine carcinoma of the lung, liver, liver metastases from rectum, mixed adenocarcinoma, neuroendocrine tumor, and non-small cell lung cancer

#5793

Anti-tumor efficacy of INCB057643, a novel BET bromodomain inhibitor, in castration-resistant prostate cancer as single agent and in combination therapy.

Ramiro Vázquez,1 Gianluca Civenni,1 Giada Zoppi,1 Dheeraj Shinde,1 Aleksandra Kokanovic,1 Phillip Liu,2 Bruce Ruggeri,2 Giuseppina M. Carbone,1 Carlo V. Catapano1. 1 _Institute of Oncology Research, Bellinzona, Switzerland;_ 2 _Incyte Corporation, DE_.

Castration-resistant prostate cancer (CRPC) is an advanced stage of the disease for which there are limited treatment options. Multiple genetic and epigenetic events contribute to the emergence of CRPC. Bromodomain and extra-terminal (BET) proteins are attracting considerable attention as targets for prostate cancer therapy due to their regulatory role and impact on multiple genes involved in tumor progression and treatment resistance. Several BET bromodomain inhibitors are currently in clinical trials for cancer treatment. In this study, we evaluated the efficacy of the BET inhibitor INCB057643, which is currently in phase 2 clinical trials, as single agent and in combination with enzalutamide or docetaxel in prostate cancer models. The anti-proliferative activity and the effects of INCB057643 on colony and tumor-sphere forming capacity were evaluated in vitro in androgen-dependent (LNCaP and VCaP) and androgen-independent (DU145, PC3, 22Rv1) cells. The effect of the combination of INCB057643 with enzalutamide or docetaxel on cell growth was evaluated with MTT or SRB methods. The in vivo efficacy of INCB057643 as single agent and in combination was assessed in 22Rv1 mouse xenografts. INCB057643 showed significant anti-proliferative activity in all the prostate cancer cell lines. Interestingly, INCB057643 exhibited substantially higher activity in colony and tumor-sphere forming assays in all cell lines. This was particularly evident in 22Rv1 cells, suggesting a strong impact on tumorigenic stem-like cell subpopulation in this CRPC cell model. The combination of INCB057643 with docexatel was additive or synergistic in DU145, 22Rv1 and LNCaP cells (CI of 0.46, 1.04 and 0.66, respectively). Also, concomitant and sequential treatment with INCB057643 and enzalutamide resulted in potentiation of the antiproliferative effect in 22Rv1 and LNCaP cells. These results were mirrored in 22Rv1 tumor xenografts, where the INCB057643/docetaxel and INCB057643/enzalutamide combinations resulted in potentiation and significant reduction of tumor growth compared to control and/or single agent-treated mice. In summary, INCB057643 has significant activity both in vitro and in vivo and enhances the antitumor effect of both docetaxel and enzalutamide in 22Rv1 cells, a model of CRPC. These results point to INCB057643 as promising agent for treatment of CRPC and development of novel drug combination strategies.

#5794

Targeting the BD2 BET bromodomain to inhibit proliferation of pancreatic ductal adenocarcinoma cells.

Aubrey Lynn Miller,1 Peter J. Slavish,2 Brandon M. Young,2 Philip M. Potter,2 Anang A. Shelat,2 Karina J. Yoon1. 1 _Univ. of Alabama at Birmingham, Birmingham, AL;_ 2 _St. Jude Children's Research Hospital, Memphis, TN_.

Pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic cancer, is a highly aggressive tumor with a 5-year survival of <6%. While surgery is the only curative treatment for PDAC patients, the majority of patients present with advanced disease and are ineligible for resection. These patients usually receive gemcitabine, and have a median survival of ~6 months. Identification of novel targets and development of effective therapies are imperative to improve outcome for patients with this tumor type.

We and others document that inhibiting the function of BET bromodomain proteins suppresses PDAC tumor growth in preclinical models. The BET family of proteins is characterized by two N-terminal bromodomains, BD1 and BD2, which bind to acetylated lysine residues on specific histones. The binding of BET proteins to acetylated lysines promotes the formation of transcriptional complexes, and impacts gene transcription and chromatin structure. The literature suggests the potential specific roles for BD1 vs BD2 using pharmacological inhibitors and dominant negative mutational analyses. However, the relative importance of each bromodomain with respect to transcriptional regulation compared to chromatin structure is not well understood. Further, there is suggestion in the literature that each bromodomain function may differ in specific tumor cell types. Functional differences between BD1 and BD2 in PDAC cells have not been reported.

The goal of the current study was to compare the efficacy of the BD2-specific BET inhibitor (BETi) SJ018 with the efficacy of the pan-BET inhibitor I-BET762 in PDAC cells. We exposed four PDAC cell lines (Panc1, BxPC3, MiaPaCa2, and CFPAC) to a range of concentrations of SJ018 or I-BET762 for 96 hours, and assessed cell viability using alamarBlue. Data, analyzed using GraphPad Prism 7, demonstrated that SJ018 exerted up to ~60-fold lower IC50 values across all cell lines tested compared to the pan-BET inhibitor I-BET762.

Further, we recently made a novel observation that the pan-BETi JQ1 induced DNA damage and inhibits expression of the DNA repair protein Rad51 in PDAC cells. Immunoblot analysis for phosphorylated Ser-139 H2AX (γH2AX), a marker of DNA damage, demonstrated that SJ018 and I-BET762 also increased DNA damage, and that the BD2-specific SJ018 induces DNA damage to a greater extent and at a lower dose than the BD1/BD2 inhibitor I-BET762 in BxPC3 cells. The data also showed that both SJ018 and I-BET762 inhibited expression of the homologous recombination protein Rad51 in vitro. We conclude that SJ018 is more potent than I-BET762 in PDAC cells as a single agent, and that degree of potency is reflected by higher levels of DNA damage.

#5795

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

Steven Kregel,1 Rohit Malik,1 Irfan A. Asangani,2 Ester Fernandez-Salas,1 Kari Wilder-Romans,1 Xia Jiang,1 Thekkelnaycke Rajendiran,1 Xuhong Cao,1 Corey Speers,1 Shaomeng Wang,1 Arul M. Chinnaiyan1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _University of Pennsylvania, Philadelphia, PA_.

Metastatic castration-resistant prostate cancer (mCRPC), a lethal disease primarily driven by androgen receptor (AR) signaling, results in approximately 30,000 annual deaths in the U.S. While the AR is necessary for the function, survival, and differentiation of normal prostatic tissue, AR function and signaling shift from tumor suppressive to tumor promoting during prostate carcinogenesis. Surgical or chemical castration targeting the AR signaling axis has been the mainstay of prostate cancer (PCa) treatment. Unfortunately, despite castration, PCa will inevitably progress into castration resistant prostate cancer (CRPC) in which AR remains an important oncogene. Current CRPC therapeutics, such as second-generation AR antagonists, elicit only temporary responses and patients eventually succumb to the disease. Therefore, new therapeutics against the AR axis in CRPC are urgently needed. One such novel strategy for targeting the AR-pathway and inhibiting the growth of CRPC has been the use of bromodomain and extraterminal (BET) protein inhibitors such a JQ1, which disrupt AR and oncogenic c-MYC transcriptional activity. While BET inhibitors may seem to be attractive candidate drugs for clinical translation, their off-target effects, such as binding to other proteins, toxicity, and the rapid development of treatment resistance, limit their translation. However, a new class of molecules that target BET proteins through proteasomal degradation can improve efficacy and specificity over standard inhibitors. Based on our preliminary findings, we have hypothesized that pharmacologic BET degradation represents an important advance in CRPC treatment and may provide a novel therapeutic strategy that can enhance efficacy and disrupt resistance to AR-targeted therapy. Utilizing prostate cancer cell lines, organoids, and patient derived xenografts, we performed RNA-seq, ChIP-seq, and proteomic studies to assay the effects of BET inhibitors and degrader compounds in preclinical models. These inhibitors affected AR-positive prostate cancer cells preferentially over AR-negative cells, and proteomic and genomic mechanistic studies confirmed the disruption of oncogenic AR and MYC signaling both in vivo and in vitro. In conclusion, we are developing highly potent, small molecules that lead to the proteasomal-degradation of BET proteins. With optimized in vivo properties, BET degraders promise to be a novel potential therapeutic strategy for patients with mCRPC.

#5796

Modulation of transcription factor landscape through BET family protein inhibition is dependent on specific driver mutations in pancreatic cancer.

Mikele G. Weldon, Albert R. Wang, Hope A. Beyer, Shannon G. Stiles, Gopal Iyer. _University of Wisconsin-Madison, Madison, WI_.

Targeting of bromodomain and extra-terminal domain (BET) family protein activity has the potential to reduce proliferation in many cancers. However, changes in transcription factor (TF) landscape can be influenced by different mutational profiles of specific cancers. Within this mutational background, BET proteins can target these TFs and create an oncogenic phenotype. To exploit these altered TF readouts, we tested the hypothesis that inhibition of BET activity in different genetic mutations of pancreatic cancer will lead to antiproliferative effects. Towards this, we treated pancreatic cancer cell lines that had mutations in KRAS, CDKN1A, SMAD4 and TP53 with a bromodomain inhibitor. We showed that cell lines with CDKN1A deletion and wild-type SMAD4 were highly resistant to BET inhibition and had a high nanomolar IC50 concentration. Through targeted transcription factor gene expression profiling of over 400 TFs, we discovered that LMO2, GTF2B, ID1, and CREB1 were upregulated, while SALL4, TCF4, and TP63 were downregulated in these cell lines. This suggests that these TFs may be partly responsible for resistance to BET inhibition. Furthermore, we showed dramatic changes in TF landscapes in pancreatic cancer cells sensitive to BET treatment compared to the resistant cells. This indicates that for antiproliferative effects in pancreatic cancer cells, modulating BET activity may be necessary to switch on or off specific TFs. Together, these results suggest that BET inhibitors may be useful in pancreatic cancer treatment and that mutation-specific differences in TF targets of BETs may dictate the progression of pancreatic cancer.

#5797

BET inhibitor synthetic lethality in malignant peripheral nerve sheath tumors.

Jonathan M. Cooper, Amish J. Patel, Zhiguo Chen, Chung-Ping Liao, Kun Chen, Yong Wang, Lu Q. Le. _UT Southwestern Medical Center, Dallas, TX_.

BET bromodomain inhibitors have emerged as a promising therapy for numerous cancer types in pre-clinical studies, but molecular indicators of tumor response are lacking. Through modeling tumor evolution by studying genetic lesions underlying the development of Neurofibromatosis Type 1 (NF1)-associated Malignant Peripheral Nerve Sheath Tumors (MPNST), a lethal sarcoma with no effective medical treatment, we identified a BRD4 dependency that serves as a controlled model system to delineate mechanisms of sensitivity or resistance to BET bromodomain inhibitors in this disease. In this study, we show that loss of NF1 and p53 is associated with increased BRD4 protein levels and partial sensitivity to BET inhibition in MPNST. Strikingly, genetic depletion of BRD4 profoundly sensitizes MPNST cells to diverse BET inhibitors in culture and in vivo, while wild-type cells are spared. Collectively, genetic and pharmacological inhibition of BRD4 reveals that BET inhibitor resistance in MPNST requires wild-type BRD4 to support growth in a bromodomain-independent manner. Our findings provide a framework for understanding BET inhibitor sensitivity and resistance within distinct molecular subsets of MPNST. In addition, discovery that a synthetic lethality exists between BET inhibition and reduced BRD4 levels nominates a BRD4-low patient subpopulation as that which might best respond to a BET inhibitor therapeutic strategy for MPNST.

#5798

Elucidating novel role of bezafibrate, a PPARα agonist as potential anticancer agent in the treatment of renal cell carcinoma.

Srikanth Iyer, Jeevan Ghosalkar, Geena Malhotra, Kalpana Joshi. _Cipla Ltd., Mumbai, India_.

Renal Cell Carcinoma (RCC) accounts for 2 - 3% of all the malignant diseases worldwide, leading to a mortality of around 1.7 lakh every year. Incidence of RCC in eight major markets has been predicted to increase by 63% over the period from 2013 to 2023. The current treatment modules are dominated by anti-angiogenic agents and tyrosine kinase inhibitors (TKIs). In the coming years, Programmed Cell Death - 1 inhibitors are set to replace TKIs as the preferred standard of care (SOC) drug. However, owing to the long waiting periods associated with these agents to reach the market, there is a significant unmet medical need for development of new drugs. Discovery and development of new drugs is an expensive process associated with long incubation periods. Hence repurposing of an already existing drug is an attractive and cost effective substitute. We have identified Bezafibrate, a FDA approved drug for the treatment of hyperlipidaemia, as a potential anti-cancer agent against RCC. It acts as a PPARα agonist, thereby promoting the uptake and catabolism of fatty acids. Several studies have shown its role in inhibition of cyclin-dependent kinases, thereby arresting the cells in G0/G1 phase of cell cycle. It has been reported to be successfully used in combination with other respective SOC drugs in the treatment of leukaemia and colorectal cancer. However, the efficacy of bezafibrate as a single agent or in combination with SOC for the treatment of renal cancer has not yet been reported. In this study, we have evaluated Bezafibrate against a panel of 10 RCC cell lines including patient derived cells (PDX). It displayed an in vitro dose dependent cytotoxicity with a mean IC50 of 1222 μM and 985 μM in a 2D and 3D setup respectively. Importantly, the combination of sunitinib, a SOC and bezafibrate showed an overall additive effect in suppressing colony formation by 1183L (PDX) and SN12C cell lines seeded on soft agar coated plates. Further, in vivo efficacy studies were performed in sub cutaneously implanted A498 cells in athymic nude mice xenograft model treated with drug alone and in combination. Sunitinib and bezafibrate alone exhibited a mean tumor growth inhibition of 50.78% and 31.59% respectively on day 20 as compared to the control group. Interestingly, the combination was able to inhibit the growth of tumor by 66.76% on the same day (n = 7). Moreover, the cell cycle analysis, differential gene and protein expression studies involving markers for cell cycle regulation, proliferation and apoptosis show promising results. Thus, our results indicate bezafibrate as a promising candidate against RCC. Furthermore, the novel combination involving Bezafibrate and Sunitinib synergistically ameliorates the proliferation and migration potential of the cancer cells. Hence, this combination may be significantly helpful in reducing the dosage of Sunitinib without compromising on its therapeutic potential.

#5799

Characterization and profiling of CB-103, a novel small-molecule protein-protein interaction inhibitor targeting the NOTCH transcription complex.

Rajwinder Lehal,1 Charlotte Urech,1 Michele Vigolo,2 Maximilien Murone,1 Freddy Radtke2. 1 _Cellestia Biotech AG, Basel, Switzerland;_ 2 _Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland_.

NOTCH signalling is a key development pathway whose aberrant activation is known to play a role in multiple human cancers. In human tumors the NOTCH pathway can be activated by various genetic lesions such over expression of ligands/receptors, GOF mutations in NOTCH receptors, including protein stabilizing mutations in the PEST domain of NOTCH, chromosomal translocations, or loss-of-function mutations in the E3 ubiquitin ligase FBXW7 and other negative regulators of the pathway (SPEN, NUMB). Activation of signalling due to above mentioned mechanisms can be addressed in part using blocking antibodies against NOTCH ligands/receptors or small molecule inhibitors of the gamma secretase enzyme (GSIs). However, in human tumors where NOTCH signalling is constitutively activated due to chromosomal translocations in the NOTCH receptors (NOTCH1 and 2), none of the above-mentioned strategies will be effective. Moreover, due to on-target and off-target toxicities associated with blocking antibodies and GSIs, these anti-NOTCH agents failed to advance in clinical trials, although some of them showed signs of clinical efficacy. Given the role of NOTCH signalling in human tumors, there is a need to identify novel targets in the NOTCH pathway and develop new and more selective anti-NOTCH agents. To inhibit pan-NOTCH signalling in human tumors independently of the mechanisms of NOTCH activation, and in the most downstream part of the pathway, we have previously reported the discovery of a new class of small molecules able to target the NOTCH transcription complex enabling the specific inhibition of NOTCH target gene expression (e.g. cMYC, HES1, DTX1, CCND1). These small molecules act as protein-protein interaction inhibitors, and thereby compromise the assembly of functional NOTCH transcription complex.

Here we present further in vitro and in vivo characterization of the lead molecule CB-103. The anti-cancer activity of CB-103 was extensively profiled in several human cancer cell lines representing NOTCH positive solid tumors, leukemias and lymphomas. Moreover, CB-103 responsiveness of these cell lines correlates with a downregulation of the NOTCH signature following treatment with CB-103. Specifically, we will present data outlining the in vivo pharmacokinetic and pharmacodynamic properties of CB-103.

#5800

Vorinostat and indole-3-carbinol modulate microRNAs and histone deacetylase activity in subtypes of triple-negative breast cancer.

Beverly Lyn-Cook,1 Fatemeh NouriEmamzaden,1 Anfernee Hawkins,2 George Hammons,1 Beverly Word1. 1 _NCTR, Jefferson, AR;_ 2 _University of Arkansas at Pine Bluff, Pine Bluff, AR_.

Triple-negative breast cancer is a highly heterogeneous and aggressive subtype of breast cancer. Epigenetic drugs are continuing to emerge as promising therapies for triple-negative breast cancer (TNBC). These drugs, including the one used in this study, vorinostat, a histone deacetylase inhibitor (HDAC), are capable of multiple actions and are considered multifunctional drugs. Epigenetic mechanisms have been shown to play a role in the etiology of this cancer, through modulation of DNA methylation, histone modification or microRNAs. We have shown in this study that vorinostat and indole-3-carbinol (I3C) modulated the expression of microRNAs 221 and 222 differently in basal-like-2 (BL2), MSL, basal-like1 (BL1) and M subtypes of TNBC cells. Additional studies demonstrated that combination of tamoxifen and vorinostat significantly decreased expression of miRNA 221 in TNBC cell lines. MiRNAs are short noncoding RNAs that regulate the function of target genes post-transcriptionally. In addition, breast cancer microarrays further showed modulation of a number of other miRNAs that play a role in cell proliferation, drug resistance, cell invasion and metastasis. MicroRNAs are epigenetic targets for drug development. Using a global HDAC activity assay, it was also shown that subtypes of TNBC cells respond differently to vorinostat and I3C treatment, depending on their HDAC activity. HCC70, an African American cell line and a BL2 subtype, had the highest HDAC activity and its activity was significantly decreased by vorinostat treatment compared to BT549 (M), HCC 1806 (BL2), MCF7 (ER positive) and a basal nonmalignant cell line, MCF10A. Both vorinostat and I3C show great promise alone and in combination as effective agents in triple-negative breast cancer. Histone deacetylase inhibitors and dietary agents are emerging as therapy for triple-negative breast cancer and are currently in clinical trials.

#5801

Unexpected selectivity of histone deacetylase inhibitors in live MDA-MD-231 triple-negative breast cancer cells.

Shaimaa M. Aboukhatwa,1 Thomas W. Hanigan,1 Jayaprakash Neerasa,1 Taha Y. Taha,1 Nathan D. Brown,1 Eman E. El-Bastawissy,2 Mohamed A. Elkersh,2 Tarek F. El-Moselhy,2 Jonna Frasor,1 Pavel A. Petukhov1. 1 _University of Illinois at Chicago, Chicago, IL;_ 2 _Tanta University, Tanta, Egypt_.

While inhibition of histone deacetylases (HDAC) is a promising strategy for the treatment of cancer, HDACs do play essential roles in normal cellular function, limiting their use as therapeutic targets. Increasing evidence shows that each of the HDAC isoforms carry out unique biological functions; therefore, by inhibiting a single isoform, one could regulate a focused subset of genes and achieve the desired therapeutic effect and lower toxicity. To investigate this aspect, many isoform-selective inhibitors have been synthesized, characterized for selectivity against recombinant HDACs in biochemical assays, and tested for anti-cancer activity. Despite significant progress, the actual engagement of HDAC isoforms is poorly characterized since methods to determine selectivity of HDAC inhibitors in live cells with unperturbed epigenetic machinery and in vivo are missing. Recently, using a novel photoreactive HDAC probe (PRP), we demonstrated that its isoform selectivity in live breast cancer cells is cell-type dependent, differs from that determined in the biochemical assays, and, in the case of HDAC3, is regulated by phosphorylation by c-Jun N-terminal kinase (JNK). Similar cell-type dependent variability in HDAC isoform selectivity was also observed for other HDAC isoforms. In this report, we extended our photolabeling approach to characterize HDAC isoform selectivity of the commonly used in pre-clinical and clinical research non-selective and selective inhibitors: SAHA, panobinostat, pimelic diphenylamide 106, PCI-34051, and TMP-269. Seven novel PRPs based on the chemical scaffolds of these HDAC inhibitors were synthesized and tested in the biochemical assay and the cell-based photolabeling assay in live triple negative breast cancer cells MDA-MB-231. We found that the cell-based profiles of these PRPs were skewed toward inhibition of HDAC3 irrespective of their biochemical selectivity, chemotypes, or isoform abundance, suggesting that the actual HDAC isoform selectivity and target engagement in live cells is different from that perceived based on the results of the biochemical assay. These studies suggest that a substantial correction in our understanding of the role HDAC isoforms and their inhibitors play in live cells in culture and in vivo may be necessary. Further expansion of these studies to other types of cells and animal tissues ex vivo and in vivo as well as identification of the mechanisms responsible for modulation of HDAC isoform selectivity and target engagement are in progress.

#5802

Efficacy of PI3Kβ inhibitor AZD8186 in PTEN-deficient triple-negative breast cancer.

Nicci Owusu-Brackett, Ming Zhao, Argun Akcakanat, Kurt W. Evans, Erkan Yuca, Funda Meric-Bernstam. _University of Texas MD Anderson Cancer Center, Houston, TX_.

Background: Phosphatase and tensin homologue (PTEN) loss is a frequently disrupted tumor suppressor in cancer. PTEN serves as a negative regulator of the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathway to promote balanced cell proliferation, survival and differentiation. Consequently, loss of PTEN function increases cell proliferation, decreases cell death and contributes to tumor initiation. PTEN loss occurs across a variety of cancer subtypes, and these PTEN deficient tumors are dependent on PI3Kβ activity. As a result, therapeutic approaches that focus on the inhibition of PI3Kβ isoform in PTEN-null cells are of interest. We evaluate the efficacy of PI3Kβ inhibitors focused on triple negative breast cancers with loss of PTEN function. Methods: We studied the effects of p110β inhibitor AZD8186 in a panel of ten TNBC cell lines, which included four PTEN-mutant cell lines. Three of four cell lines were confirmed to have PTEN loss on western blot. In vitro, cell proliferation assays were performed to determine the half maximal inhibitory concentration (IC50) after 3 days of treatment and to test the effects in combination with chemotherapy. We confirmed the sensitivity of the PTEN-null cell lines to AZD8186 with colony formation assays. Western blot analysis was performed to assess PTEN expression and PI3K pathway activation in MDA-MB-436 and MDA-MB-468 cell lines, and to evaluate effects of AZD8186 on PI3K signaling. Results: Cell lines with PTEN loss were significantly more sensitive to AZD8186 in vitro (p= 0.008). AZD8186 inhibited PI3K signaling. Western blot revealed decreased activation of pAKT, pGSK3β, pPRAS40 and pS6. Activation of other pathways were evaluated with no activation of the MAPK/ERK or MEK pathways appreciated. AZD8186 treatment resulted in increased apoptosis but did not have a significant effect on cell-cycle progression in PTEN-deficient cell lines. AZD8186 was not synergistic with eribulin, paclitaxel or carboplatin; combinations with novel targeted therapies in vitro and in vivo are ongoing. Conclusion: AZD8186 had single agent efficacy in PTEN-deficient triple negative breast cancer cell lines in vitro. Further study is needed to identify rationale combinations and in vivo efficacy.

#5803

**Homoharringtonine, a natural protein synthesis inhibitor, inhibits growth of triple negative breast cancer** in vitro **and** in vivo **.**

Mohamad Yakhni,1 Arnaud Briat,2 Elisabeth Miot-Noirault,2 Florent Cachin,1 Frederique Penault-Llorca,1 Nina Radosevic-Robin1. 1 _INSERM U1240/University Clermont Auvergne, Centre Jean Perrin, Clermont-Ferrand, France;_ 2 _INSERM U1240/University Clermont Auvergne, Clermont-Ferrand, France_.

Introduction: Triple negative breast cancer (TNBC) is a heterogeneous disease for which new therapeutic approaches are urgently needed. Homoharringtonine (HHT) is a protein synthesis inhibitor clinically approved in chronic myeloid leukemia. Its multi-factor mechanism of action includes downregulation of short-lived cellular proteins like Mcl-1. As increased protein synthesis lies behind several hallmarks of cancer present in TNBC, we investigated the effect of HHT on TNBC in vitro and in vivo.

Methods: TNBC cell lines CAL-51, MDA-MB-231 and MDA-MB-468, representative of aggressive, BRCA1 non-mutated TNBC (Lehmann, J Clin Invest 2011) were used. HHT was provided by LeukePharma, Houston, TX, USA. Cell viability was assessed by sulphorhodamine B assay. Number of cells in different cell cycle phases or in apoptosis was determined by flow cytometry. Protein expression was evaluated by western blot. For in vivo experiments, xenografts of MDA-MB-231 were established in Swiss nu/nu mice, treated for 7 days by subcutaneous HHT (1 mg/kg, bi-daily), and analyzed on day 10. Evaluation of HHT on MDA-MB-468 and CAL-51 xenografts is ongoing.

Results: Already after 24h of exposure to 100 ng/ml HHT (mean plasma HHT concentration after 5 mg/m2/day in humans (Sentenac S et al, Blood 2003)), viability of CAL-51 and MDA-MB-468 cells was strongly reduced (10.4±1.0% and 19.2±2.2% of control, respectively). This inhibition level was obtained in MDA-MB-231 cells only after 72h (18.7±4.8%). After 48h in 100 ng/ml HHT, MDA-MB-231 and -468 cells accumulated in the S phase of the cell cycle, whereas the CAL-51 accumulated in the G1/0 phase. This was associated with an increase of apoptosis in CAL-51 (42.0±2.4% vs 9.2±1.3%, untreated vs treated cells, respectively) and in MDA-MB-468 (24.2±2.8% vs 12.2±3.6%) but not in MDA-MB-231 (9.0±2.2% vs 3.6±1.7%). Concordantly, 100 ng/ml of HHT induced, after only 2h, a marked decrease of the level of anti-apoptotic proteins Mcl-1, Bcl-2, survivin and XIAP in CAL-51 and MDA-MB-468 cells, which preceded by 2h the reduction of caspase-3 level. No change in expression of those proteins was observed in MDA-MB-231 during all 48h of exposure to HHT. On the other side, the quantity of phosphorylated S6 increased in MDA-MB-231 but decreased in CAL-51 and MDA-MB-468 already after 24h of incubation with 100 ng/ml HHT. In vivo, HHT reduced growth of MDA-MB-231 xenografts for 36.5% (tumor volume on day 10: 230.4±27.7% mm3 vs 146.2±15.5% mm3, untreated vs treated, respectively).

Conclusion: HHT exerts cytotoxic action on TNBC cell lines CAL-51 and MDA-MB-468 whereas its effect on MDA-MB-231 is dominantly cytostatic. Because of its capacity to rapidly reduce the levels of anti-apoptotic proteins, HHT is worth further studies as potential sensitizer of TNBC to chemo- and/or targeted therapy.

#5804

A novel copper(II) complex of tolfenamic acid exhibits anticancer activity in pancreatic cancer cells via downregulation of transcription factors Sp1 and Sp3 and an anti-apoptotic protein, survivin.

Myrna L. Hurtado,1 Umesh T. Sankpal,1 Shahela Mahammad,1 Jaya Chhabra,2 Deondra K. Brown,2 Raj K. Gurung,2 Alvin Holder,2 Riyaz Basha1. 1 _University of North Texas Health Science Center, Fort Worth, TX;_ 2 _Old Dominion University, Norfolk, VA_.

Despite advances in research, pancreatic cancer (PC) remains one of the most lethal malignancies. The five-year survival rate is 8% and standard treatment options often cause high toxicity. Thus, there is a pressing need for identifying less toxic yet more effective agents for the treatment of this malignancy. Tolfenamic acid (TA) is a NSAID used as migraine medicine but has recently been demonstrated in pre-clinical studies to have anti-cancer properties. It is known to downregulate the transcription factor Specificity protein 1 and 3 (Sp1, Sp3). Sp1 and Sp3 regulate several genes involved with apoptosis and cell growth, including survivin, an inhibitor of apoptosis. Recently, it has been suggested that a copper(II) complex of TA (Cu-TA) can produce a higher therapeutic response; however, its efficacy has yet to be tested in gastro-intestinal cancers. Previously we presented that both TA and Cu-TA caused a dose-dependent response in inhibition of cell growth in PC cells, however, Cu-TA had an enhanced effect. Here, we used human PC cell lines (MIA PaCa-2 and Panc1) to evaluate the therapeutic efficacy of Cu-TA. Physical characterization of Cu-TA was performed using Fourier-transform infrared spectroscopy (FTIR) to analyze purity. To demonstrate the compound's stability, dose curve with Cu-TA was carried out using a stock solution that is one-year old as well as a 6-month old stock solution. Cells were also treated with the components used in the formation of Cu-TA (CuCl2 and BPY) to ensure the anti-cancer activity is due to the Cu-TA compound as a whole and cell viability was measured at 24 and 48 h. Western blot and quantitative PCR (qPCR) was done to assess Cu-TA's effect on Sp1, Sp3 and survivin protein and mRNA levels at 24 and 48 h post-treatment. To evaluate potential cardiotoxicity, cardiomyocytes (H9C2) were treated with increasing dosages of Cu-TA or TA and cell viability was measured at 24 and 48 h. Cu-TA was highly effective in suppressing Sp1, Sp3 and survivin protein expression. qPCR results showed that survivin mRNA expression was significantly lower following both Cu-TA and TA treatment; however, the mRNA expression of Sp1 and Sp3 remained unaltered. This suggests that both Cu-TA and TA could be working through a similar mechanism by effecting Sp1 and Sp3 post-translationally, possibly by proteasome-dependent degradation. FTIR results confirmed Cu-TA's purity (>99%) while the dose curves demonstrated the compound's stability. Both Cu-TA and TA did not cause any significant cytotoxicity to H9C2 cells. These results show that Cu-TA is more effective than TA and potentially useful for PC treatment. Studies to precisely understand the underlying mechanisms of Cu-TA are under investigation through molecular profiling analysis.

### Combination Chemotherapy 2

#5805

Palbociclib synergistically enhances the anticancer activity of cisplatin in P53 mutant model of upper gastrointestinal cancers.

Dhvanir Kansara,1 Amruta Samant,1 Priya Pancholi,1 Tanvi Visal,1 Shraddha Patel,1 Vikas Sehdev2. 1 _Arnold & Marie Schwartz College of Pharmacy and Health Sciences, Long Island University, Brooklyn, NY; _2 _Presbyterian College School of Pharmacy, Presbyterian College, Clinton, NY_.

Background: Upper Gastrointestinal Cancers (UGCs) respond poorly to conventional chemotherapy due to overactive intrinsic mechanisms that mediate cellular proliferation and drug resistance. Dysregulated cell division due to increased activity of cyclin-dependent kinases 4/6 (CDK4/6) is one such mechanism that drives progression of UGCs, making them an important therapeutic target. Palbociclib is a second generation cdk4/6 specific inhibitor approved for treatment of ER+ breast cancers. Since Cisplatin (CDDP) is a first line therapeutic agent frequently used for treatment of UGCs we hypothesized that Palbociclib would complement the DNA-damaging activity of CDDP. To test this hypothesis, we investigated the potential therapeutic benefit of Palbociclib alone and in combination with CDDP in P53 mutant model of UGCs. Methods: In this study, we evaluated the effect of Palbociclib treatment alone and/or in combination with CDDP on FLO-1 (P53 mutant) UGC cell viability, survival, and cell cycle progression, respectviely. The MTT cell viability assay and Compusyn mediated median effect plot analysis (MEPA)(Chou and Talaly) were used to determine synergistic drug combinations of Palbociclib and CDDP in FLO-1 UGC cells. Results: The cell viability data and MEPA indicated that Palbociclib and CDDP show significant synergistic anticancer activity in FLO-1 UGC cells at a ratio of 1:2 and 1:3, respectively. The clonogenic cell survival assay data showed that in comparison to treatment with Palbociclib (0.5µM) or CDDP (1.0 and 1.5µM) alone, the combination treatments (Palbociclib:CDDP) at a ratio of 1:2 and 1:3 exhibited significantly elevated (p<0.05) inhibition of cellular survival in FLO-1 UGC cell lines. In addition, the Combination Index (CI) values of 0.66 and 0.71 for the aforementioned combination ratios suggests a strong synergism between the two drugs. Furthermore, the cell cycle data exhibited a marked increase (p<0.05) in the percentage of FLO-1 cells in the sub-G1-phase (cell death) after treatment with Palbocicb and CDDP combinations for 72 hrs., respectively . Conclusions: Our in vitro data indicate that Palbociclib, as a single agent, is an effective therapeutic strategy for inhibiting UGC cells. Additionally, Palbociclib in combination with CDDP synergistically enhances the anti-tumor activity of CDDP against P53 mutant UGC cells[MM1] . The observed synergism can potentially translate into reduced individual drug dosages and toxicity for Palbociclib and/or CDDP when used in combination.

#5806

Combination of the miniature drug conjugate PEN-866 with PARP inhibitors as a rational approach to overcoming limitations of PARP inhibitor monotherapy.

Samantha Perino, James M. Quinn, Marrissa Callahan, Gitanjali Sharma, Brian White, Mark Bilodeau, Leila Alland, Kerry Whalen, Richard Wooster. _Tarveda Therapeutics, Watertown, MA_.

PEN-866 is a novel miniature drug conjugate comprised of a Heat Shock Protein 90 (HSP90) targeting ligand attached through a cleavable linker to SN-38, the active metabolite of the approved topoisomerase I inhibitor, irinotecan. The conjugate accumulates and is retained in tumor cells through the HSP90 targeting ligand. PEN-866 linker cleavage provides sustained release of SN-38 at a high local tumor concentration leading to DNA damage and apoptosis of tumor cells resulting in broad preclinical antitumor activity. Reducing the DNA repair activity in tumor cells is an opportunity to significantly potentiate the efficacy of PEN-866.

Poly ADP ribose polymerase (PARP) has an important role in the repair of DNA. PARP inhibitors have shown activity in the clinical setting, both in patients whose cancers harbor mutations in BRCA1 and BRCA2, and in those that are BRCA1/2 wildtype, and are approved as monotherapy for use in treatment of patients with advanced ovarian cancers. Combining PARP inhibitors with other therapeutics has been challenging due to dose limiting toxicities. Combining PARP inhibitors with PEN-866 is mechanistically appealing with the potential for greater efficacy as compared to these agents used alone and the potential to mitigate the toxicity of the combination of a PARP inhibitor with conventional cytotoxic therapy due to the high levels of accumulation and retention of PEN-866 in xenograft tumors.

To evaluate these hypotheses, we have combined PEN-866 and PARP inhibitors in preclinical models of human cancer. Efficacy studies were carried out in both BRCA mutant and BRCA wildtype tumor xenografts combining PEN-866 and PARP inhibitors, and the combinations resulted in greater efficacy than that of the single agent in tumors with both genetic backgrounds. In addition, a pharmacodynamic assessment of DNA damage was performed in tumors responsive to the combination treatment. These data demonstrate that combination of PEN-866 and PARP inhibitors provide greater efficacy than single agent activity alone, including BRCA wildtype tumors, and support the evaluation of such combinations in the clinical setting. The authors acknowledge David Proia, Weiwen Ying and others from Synta Pharmaceuticals who contributed to this work.

#5807

The dual c-Met/VEGFR2 inhibitor foretinib augments chemotherapy response in preclinical models of gastric cancer.

Niranjan Awasthi, Meghan Grojean, Sheena Monahan, Sazzad Hassan, Urs von Holzen, Margaret A. Schwarz, Roderich E. Schwarz. _Indiana Univ. School of Medicine, South Bend, IN_.

BACKGROUND: Gastric adenocarcinoma (GAC) is the fourth most common malignant tumor in the world. Several growth factors and their receptors including c-Met and VEGFR, are overexpressed in GAC and thus provide a potentially effective therapeutic target. Foretinib is a novel small molecule inhibitor of the c-Met and VEGFR pathways. We evaluated the therapeutic efficacy of foretinib to enhance the antitumor response of nab-paclitaxel (NPT), a water-soluble albumin-bound formulation of paclitaxel, or oxaliplatin in preclinical models of GAC.

METHODS: Tumor growth experiments were performed in subcutaneous xenografts in NOD/SCID mice using 5x106 MKN-45 cells. Animal survival study was performed as peritoneal dissemination model in NOD/SCID mice using 10x106 MKN-45 cells. The mechanistic evaluation involved immunohistochemical and Immunoblot analyses.

RESULTS: In subcutaneous GAC xenografts, NPT and foretinib monotherapies demonstrated inhibition in tumor growth, while NPT plus foretinib combined showed additive effects. Net tumor growth in different therapy groups was 581.7 mm3 in controls, 397.9 mm3 after oxaliplatin, 229.9 mm3 after NPT, -82.6 mm3 (tumor regression) after foretinib, -74.1 mm3 after oxaliplatin+foretinib and -96.3 mm3 after NPT+foretinib. No significant change in body weight was observed for those mice treated with nab-paclitaxel, oxaliplatin or foretinib. In the GAC survival model, median animal survival compared to controls (23 days) remained unchanged after oxaliplatin therapy (24 days, p=ns) but increased after monotherapy with NPT (42 days, an 83% increase, p=0.0014) or foretinib (46 days, a 100% increase, p=0.0006). Importantly, a further increase in animal survival was observed in combination therapy groups: oxaliplatin+foretinib (55 days, a 139% increase, p=0.0006) and NPT+foretinib (76 days, 230% increase, p=0.0006). Effects of therapy on intratumoral proliferation and microvessel density corresponded with tumor growth inhibition data. In vitro studies demonstrated inhibition in the proliferation of GAC cells by both NPT and foretinib, with additive effects in combination. Immunoblot analysis revealed that foretinib pre-exposure blocked HGF-induced expression of phospho-c-Met. Furthermore, the foretinib treatment caused a decrease in phosphorylation of AKT, ERK, and PLC-γ in GAC cells, either alone or in combination with nab-paclitaxel.

CONCLUSION: These findings suggest that the antitumor effect of chemotherapy can be significantly enhanced by the c-Met/VEGFR pathway inhibitor foretinib, which might lead to clinically relevant therapeutic combinations to increase survival of GAC patients.

#5808

Hedgehog pathway inhibitors itraconazole and cyclopamine produce synergistic suppression of Pten deficient prostate cancer.

Vaqar M. Adhami, Imtiaz A. Siddiqui, Mohammad Imran Khan, Islam Rady, Leanna Sako, Hasan Mukhtar. _Univ. of Wisconsin-Madison, Madison, WI_.

Hedgehog (Hh) signaling plays an important role in prostate development and is also a characteristic feature of prostate cancer (PCa). The ability of Hh pathway activation to promote cell invasiveness, epithelial to mesenchymal transition and metastasis suggest that targeting Hh pathway may provide an important clinical avenue for the treatment of advanced PCa. Loss of PTEN function is considered a characteristic feature of advanced PCa. To gain insight into the status of Hh signaling in PCa in relation to PTEN status we selected three cell lines based on their PTEN expression. Gli1 mRNA was upregulated 2000 fold in PC3 (PTEN-/- null) cells, 800 fold in DU145 (PTEN+/-) cells when compared with normal prostate epithelial cells suggesting that PTEN loss is associated with increased Hh signaling. We also observed significant increase in the mRNA levels of Hh ligand (Shh), Hh receptors (PTCH2 and SMO) and effectors of Hh signaling (Gli1 and Gli2) in the prostate tissues of PTEN knockout mice. A 400 fold increase in the mRNA levels of Gli1 and 15 fold increase in the levels of Gli2 mRNA levels was also observed. We also observed significant decrease in the mRNA levels of the negative regulators of Hh signaling, HHIP and SuFu in the prostate tissues of PTEN knockout mice with similar inhibition in the transcript levels of GAS1. We next investigated the effect on Hh signaling in PCa cells treated with itraconazole and cyclopamine alone and in combination. PCa 22Rν1 and PC-3 cells transfected with Gli-dependent luciferase reporter construct were treated with itraconazole (1 and 2 μM), cyclopamine (5 and 10 nM) and combination of itraconazole (1 μM) and cyclopamine (5 nM) for 40 hours. Gli-dependent luciferase reporter activity was inhibited by itraconazole and cyclopamine treatments; however the inhibition was significantly greater in combination. Treatment of PCa cells with the combination of itraconazole and cyclopamine led to increased cell growth inhibition that was greater than the sum of the individual agents alone suggesting synergism. We next analyzed the effect of the combination on the growth of CWR22Rν1 tumors under in vivo conditions using athymic nude mice. In control animals, targeted tumor volume of 1200 mm3 was reached at 6 weeks post cell inoculation and significantly later time in animals treated with itraconazole alone (9 weeks) and cyclopamine alone (8 weeks). Notably, tumor volumes stayed under 165 mm3 in mice that received both itraconazole and cyclopamine. In the prostate specific Pten knockout mice combination of itraconazole and cyclopamine produced 57% inhibition of tumor growth compared to 30% in cyclopamine alone and 45% in itraconazole alone treated mice. These data show that a combination of itraconazole and cyclopamine is considerably more efficient in inhibiting tumor growth highlighting the synergistic efficacy of this combination for the treatment of PCa.

#5809

Synergistic effect of alectinib and everolimus on anaplastic large cell lymphoma cells via ALK-mTOR pathway inhibition.

Dongchan Kim,1 Hyejoo Park,1 Youngil Koh,2 Sung-Soo Yoon2. 1 _Seoul National Univ. College of Medicine, Seoul, Republic of Korea;_ 2 _Seoul National University Hospital, Seoul, Republic of Korea_.

Purpose Anaplastic lymphoma kinase (ALK) fusion is associated with various kinds of cancer, including anaplastic large cell lymphoma (ALCL). We investigated the efficacy of combination between ALK inhibitor and mammalian target of rapamycin (mTOR) inhibitor in ALK-positive ALCL cells.

Methods & Results ALK-positive cancer cell lines were treated with ALK and/or mTOR inhibitors and assessed for cell viability. Treatment of ALK-positive ALCL cell lines with the combination resulted in a synergistic effect in Karpas299 cells but not in SU-DHL-1 cells. Viability of Karpas299 cell was reduced to 26.4% (p<0.001) by alectinib-everolimus combination treatment compared to the single treatment groups (alectinib: 59.5%, everolimus: 51.6%); the Combination Index (CI) value of 0.316 indicated a synergistic effect of the combination treatment. Moreover, changes in intracellular signaling were analyzed by western blot. Combination treatment augmented increase of ALK-mTOR de-phosphorylation, and cleavage of poly ADP ribose polymerase (PARP) molecules. Combination treatment significantly increased the numbers of Karpas299 cells in subG1 (p = 0.0096 everolimus), and G0/G1 (p = 0.0018 alectinib; 0.0013 everolimus) cell cycle arrest compared to single treatment groups. We investigated the biological differences between Karpas299 and SU-DHL-1 cell lines using RNA sequencing data obtained from Sequence Read Archive (SRA). The RNA expression pattern of the ALK isoforms ALK-201 and 203 was different between the two cell lines. These data indicated that the relatively high expression of ALK-203 in SU-DHL1 cells could explain the lack of synergism in response to the combination treatment observed.

Conclusions Combination treatment with alectinib and everolimus synergistically reduced cell survival, augmented inhibition of cellular signaling, and increased cell cycle arrest in Karpas299 but not in SU-DHL1 cells. These differences might be attributable to differential expression of ALK isoforms between the two cell lines.

#5810

Crizotinib and PARP inhibitors act synergistically by triggering apoptosis in high-grade serous ovarian cancer (HGSOC).

Irem Durmaz, Ingrid Hedenfalk. _Division of Oncology and Pathology, Department of Clinical Sciences, Lund University, Lund, Sweden_.

Introduction: Epithelial ovarian cancer is the seventh most common malignancy diagnosed in women, but the fifth most common cause of cancer-related deaths with a relative five-year survival less than 50%. High-grade serous ovarian cancer (HGSOC) is the predominant histological subtype. PARP inhibitors such Olaparib and Niraparib have been approved by the FDA for the maintenance treatment of platinum-sensitive, recurrent HGSOC. However, similar to many other targeted agents, the efficacy of PARP inhibitors is limited by the development of drug resistance. The aim of this study was to investigate new combinatorial treatment strategies to prolong anti-cancer activities of PARP inhibitors using HGSOC cell lines, which might also translate to overcoming resistance in clinical tumors.

Methods: The cytotoxic activities of various drugs (Carboplatin, the PI3K inhibitor LY294002 and the c-met inhibitor Crizotinib) alone or in sequential combination with the PARP inhibitors Niraparib and Olaparib were analyzed using the NCI-SRB assay and xCeLLigence platform and combination index (CI) values were calculated using the Chou-Talalay method. Phospho-H2AX staining was used to address the accumulation of DNA double strand breaks and FACS analysis using propidium iodide was conducted to investigate cell cycle phase distribution. Western blot experiments were performed to reveal the effects of the compounds alone or in combination on the protein level. All experiments were conducted using the OVSAHO (BRCA2 homozygous deletion), KURAMOCHI (BRCA2 mutant) and CAOV3 (BRCA1/2 wild-type) HGSOC cell lines. The clear cell ovarian cancer (CCOC) cell line JHOC5 was used as a reference. Patient-derived primary cells collected from ascites were used for some experiments.

Results: All drugs showed high cytotoxic effects on HGSOC cell lines and on primary cells, however, very low effects were observed in the CCOC cell line. PARP inhibitors and Crizotinib significantly induced accumulation of DNA double strand breaks, which was also observed to a limited extent with LY294002. PARP inhibitors and Carboplatin alone and in combination resulted in G2/M cell cycle arrest. In contrast, Crizotinib alone or in combination with PARP inhibitors induced a SubG1 arrest. Among all sequential combinations, Crizotinib with both of the PARP inhibitors was found to be more strongly synergistic compared to Carboplatin and PARP inhibitor or PARP inhibitor and LY294002 combinations. Treatment of HGSOC cells with a sequential combination of Crizotinib and PARP inhibitors resulted in deregulation of various proteins including ERK, Akt, and p53, all contributing to caspase-3 induced apoptosis.

Conclusion: Our results indicate the possible benefit of combining the c-met inhibitor Crizotinib with PARP inhibitors in HGSOC to enhance the activity of PARP inhibitors as a new therapeutic strategy.

#5811

**Evaluation of antitumor efficacy of CA102N, H-Nim and hyaluronic acid (HA) conjugate, alone or in combination with 5-fluorouracil (5-FU) in** in vivo **human colorectal cancer models.**

Eskouhie Tchaparian,1 Louis Lin2. 1 _Holy Stone Healthcare Co., Ltd., Redwood City, CA;_ 2 _Holy Stone Healthcare Co., Ltd., Taipei, Taiwan_.

CA102N is a conjugate of HA and H-Nim (N-(4-Amino-2-phenoxyphenyl, methanesulfonamide), a derivative of cyclooxygenase-2 inhibitor Nimesulide, and a CD44 receptor targeted system currently in preclinical development for metastatic colorectal cancer. The in vivo antitumor activities of CA102N was evaluated singly or combined with 5-FU in CR5038 (Patient Derived Xenograft (PDX) and HT29 Xenograft models. Inhibitory activity was assessed according to tumor-volume changes. CA102N (i.v. 200mg/kg, BIW) monotherapy did not show significant tumor growth inhibition at either the end of the treatment (21 days) or prolonged observational (45 days) period, in CR5038 PDX-SCID metastatic model in comparison to vehicle control or 5-FU (i.v. 50mg/kg, QW). Notably, the combination therapy displayed a marked reduction in tumor growth (>70%) compared to 5-FU alone (p=0.0003) or vehicle control (p<0.0001) in addition to prolonged median survival (p<0.05). Alternatively, HT29 xenografts nude mice were found to have significantly reduced tumor burden treated with either single agents CA102N (400 mg/kg, QW) or 5-FU (50 mg/kg, QW), (p<0.012) compared to control vehicle. The drug combination further displayed a synergistic antitumoral effect and greater median survival (p <0.05) in HT29 model. CA102N was well tolerated alone or in the combination therapy. Based on the results of this study, combined treatment of CA102N and 5-FU resulted in a complete response at the regimen given. The combination of CA102N and 5-FU represents a promising potential therapeutic strategy and a new approach to reverse drug resistant in the treatment of colorectal cancer. Studies presently are ongoing to further evaluate its efficacy in CT26 murine colorectal models.

#5812

Synergistic induction of urinary bladder cell death and reduction of drug resistance in vitro and effective control of xenograft tumor growth in vivo by a triple combination of gemcitabine, cisplatin, and romidepsin.

Hwa-Chain R. Wang, Pawat Pattarawat. _Univ. of Tennessee, College of Veterinary Medicine, Knoxville, TN_.

Human urinary bladder cancer is the fifth most commonly diagnosed cancer in the United States. Long-term survival of patients is still suboptimal with current therapeutic regimens failing to impede drug resistance and recurrence. Thus, it is urgent to develop therapeutic regimens that effectively control tumor growth, drug resistance, and recurrence to reduce the morbidity and mortality of this disease. We investigated the efficacy of combination regimens to control various human urinary bladder cancer cells in vitro and cell-derived xenografts in vivo. Our studies revealed, for the first time, a combination of gemcitabine plus cisplatin and romidepsin effective in synergistically inducing cell death and reducing drug resistance in vitro. The enhanced Ras-ERK-Nox pathway played an essential role in mediating signals to elevate reactive oxygen species, leading to enhanced caspase activation, enhanced DNA damage and oxidation, and reduced glutathione, which synergistically increased cell death and reduced drug resistance in cells treated with combined gemcitabine, cisplatin, and romidepsin. The unfolded protein response also played a role in the modulation of cyto-protection and cell death through the combined regimen. In vivo studies verified the efficacy of gemcitabine plus cisplatin and romidepsin in control of xenograft tumor growth in immune-deficient mice. Hence, our results lead us to suggest that a combination of gemcitabine, cisplatin, and romidepsin should be seriously considered as an advanced regimen over the conventional gemcitabine plus cisplatin regimen for controlling the development and recurrence of human urinary bladder cancer.

#5813

Thymoquinone influences the anticancer properties of paclitaxel and gemcitabine against breast cancer cells.

Hanan A. Bashmil,1 Aliaa A. Alamoudi,1 Abdulwahab Noorwali,2 Gehan A. Hegazy,3 Ghada Ajabnoor,1 Ahmed M. Al-Abd3. 1 _Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia;_ 2 _King Abdulaziz University, Jeddah, Saudi Arabia;_ 3 _National Research Ctr. of Egypt, Cairo, Egypt_.

The use of anti-cancer adjuvant therapy is rationalized by potentiating the efficacy, and/or protecting from major side effects of chemotherapeutics. Thymoquinone is naturally occurring compound with cumulative evidences of anticancer properties. In this study, we assessed the role of thymoquinone as a potential adjuvant therapeutic in human breast cancer cell lines. Sulfa-Rhodamine-B assay was used to evaluate the effect of thymoquinone on the cytotoxic profile of paclitaxel and gemcitabine in breast cancer cell lines (MCF-7 and T47D). After 72 h of exposure, thymoquinone showed cytotoxic effects against MCF-7 and T47D with IC50's of 64.93±14 µM and 165±2 µM respectively. Combining thymoquinone with paclitaxel increased its IC50 from 0.28±0.1 to 0.3±0.004 µM in MCF-7 cells, with combination index indicative of antagonism (CI-value = 1.53). Similarly, the combination of thymoquinone with paclitaxel in T47D resulted in increasing its IC50 by approximately 1.6 folds (0.09±8 to 0.14±0.02 µM); and the CI-value was 1.66 indicating an antagonistic effect. On the other hand, the IC50 of gemcitabine was significantly reduced after the combination with thymoquinone in both cell lines; MCF-7, T47D, by 15 folds (0.9±0.1 to 0.058±12 µM) and 6 folds (14.3±2 to 2.3±0.2 µM), respectively. In this combination the CI- values were indicative of strong synergism in both cell lines; MCF-7, T47D (0.15 and 0.30 respectively). Further investigation showed that thymoquinone did not affect the cell cycle distribution, however, paclitaxel showed a significant accumulation of cells at G2/M phase in both cell lines after 24, 48h of exposure. On the other hand, gemcitabine caused significant anti-proliferative effect reflected by increasing cell population in S-phase with reciprocal decrease in G2/M cells in both cell lines. The combination of thymoquinone with paclitaxel in T47D didn't enhance paclitaxel-induced G2/M arrest, however it caused significant increase in cell death attributed to this arrest from 38.1% to 69.5%; and from 44.5% to 60.4% after 24 and 48 h of exposure, respectively. Moreover, the combination of thymoquinone with gemcitabine increased S-phase arrest induced by gemcitabine from 44.3% to 49.9%. Accordingly, thymoquinone increased total cell death induced by gemcitabine against T47D from 23.1% to 49.3% and from 15.1 to 64.5 after 24 h and 48 h of exposure, respectively. Using annexin-V/FITC differential staining, thymoquinone significantly increased the percent of apoptotic/necrotic cell death after combination with paclitaxel or gemcitabine. In conclusion, thymoquinone possesses potential chemomodulatory effects to paclitaxel and gemcitabine against breast cancer cells. Despite the promising anti-proliferative activity of thymoquinone against breast cancer, better understanding of these effects are needed to provide a novel approach for the treatment of breast cancer as a disease.

#5814

Synthetic lethal therapy to overcome resistance to AURKA inhibition in HPV-negative head and neck cancer.

Alexander Y. Deneka,1 Margret B. Einarson,1 Jong Woo Lee,2 Anna S. Nikonova,1 Barbara Burtness,2 Erica A. Golemis1. 1 _Fox Chase Cancer Ctr., Philadelphia, PA;_ 2 _Yale University School of Medicine Cancer Center, New Haven, CT_.

Head and neck squamous cell carcinomas (HNSCC) affect more than a half million people annually worldwide. Most currently available treatments, which include cisplatin and irradiation, are associated with significant morbidity, while many targeted inhibitors show limited single agent efficacy: novel therapeutic approaches which combine efficacy and limited toxicity are needed. The most common mutational event in human papillomavirus (HPV)-negative HNSCC is mutation of TP53. As control of the G1/S transition is entirely p53-dependent, cells with TP53 mutations lose the G1/S checkpoint, and evade radiation- and cisplatin-induced cellular senescence. Targeting the serine/threonine mitotic kinase Aurora-A (AURKA) emerged as a promising synthetic lethal approach in this setting. AURKA is negatively regulated by p53, which causes upregulation of AURKA in the majority of cases of HNSCC, correlating with poor prognosis and cisplatin resistance. The single agent AURKA inhibitor MLN8237 (alisertib) had a 9% monotherapy response rate in treatment-refractory HNSCC, with all responses occurring in HPV-negative or -unknown disease. In our study, we used initial high throughput screening in multiple genomically characterized HNSCC cell lines to identify synergistic combinations involving AURKA inhibitors and 20 other inhibitors of cell cycle, and cell cycle checkpoint controls. This work has identified has several effective combinations, particularly in between alisertib (targeting AURKA), danusertib (AURKA and AURKB), LY2606368/prexasertib (CHK1), and reversine (MPS1). Multiplexing analysis with measures of cell metabolism, direct visualization of mitotic nuclei, and additional assays has allowed us to dissect distinct execution points for different drug combinations. We will describe in detail mechanistic interactions between specific combinations of value for HNSCC. These drug combinations are likely to represent a new and effective modality for treating TP53-mutated cancers.

#5815

Aldose reductase inhibition prevents doxorubicin-induced inflammatory response in endothelial cells and macrophages.

Himangshu Sonowal, Ashish Saxena, Kota V. Ramana, Satish K. Srivastava. _University of Texas Medical Branch, Galveston, Galveston, TX_.

Anthracycline class of chemotherapeutic drugs such as doxorubicin and daunorubicin has been known to be effective in the therapy of various types of cancer. However, in certain cancers such as colon cancer, the use of anthracyclines is restricted due to acquired drug resistance, which requires high amounts of drugs that cause severe toxic side effects. We have recently demonstrated that aldose reductase (AR) inhibitor, fidarestat, increases the sensitivity of doxorubicin towards colon cancer cells death in vitro and in vivo and decreases cardiotoxicity. However, the mechanisms through which AR inhibitor prevents doxorubicin-induced toxicity is not understood. We have examined how AR inhibition prevents doxorubicin-induced endothelial and cardiac toxicity along with inflammatory response in in vitro and in vivo models. Our results demonstrate that fidarestat prevents doxorubicin-induced oxidative stress, endothelial cell death, and monocyte adhesion. Further, fidarestat prevents the activation of NF-κB-dependent expression of endothelial cell markers ICAM-1 and VCAM-1 and expression of various inflammatory cytokines and chemokines in human vascular endothelial cells. In addition, fidarestat prevented the doxorubicin-induced decrease in eNOS and increase in iNOS expression. Similarly, AR inhibition prevented doxorubicin-induced endothelial dysfunction, cardiac hypertrophy, and cardiomyocyte injury in mice. Fidarestat also prevented doxorubicin-induced activation of monocytes and macrophages to proinflammatory phenotype in culture as well as in mouse models. Thus, our results suggest that by preventing doxorubicin-induced inflammatory as well as oxidative stress signals, fidarestat decreases cardiotoxic effects of doxorubicin. This is a novel adjuvant therapy of cancer using aldose reductase inhibitors with anthracyclines that would increase cancer therapeutic efficacy and decrease cardiotoxic side effects.

#5816

Inhibition of Hsp90 sensitizes pancreatic cancer in vitro and in vivo to chemo-radiotherapy.

Ganji Purnachandra Nagaraju,1 Bassel F. El-Rayes2. 1 _Emory Univ., Marrietta, GA;_ 2 _Emory Univ., Atlanta, GA_.

Purpose: Important signaling pathways contributing to proliferation, development, metastasis and resistance to chemo-radiotherapy in pancreatic adenocarcinoma (PDAC) includes the PI3K/AKT/HIF-1α/STAT-3 pathway. The molecular chaperone heat shock protein 90 (HSP90) controls the activity, stability, turnover, and trafficking of many molecules involved in these major signaling pathways. Functional inhibition of HSP90 may inhibit these pathways simultaneously. We evaluated the efficacy of HSP90 functional inhibitor ganetespib in combination with chemo-radiotherapy against PDAC in vitro and in vivo.

Methods and Materials: Cell proliferation (Br-dU), colony formation (clonogenic assay), migration (spheroid), and Western blot analyses were carried out in HPAC and PANC-1 PDAC cell lines to determine the effect of ganetespib alone or in combination with 5-FU+ 2Gy XRT. Tumor regression study of PDAC with ganetespib alone and in combination with chemo-radiation was analyzed in xenograft mouse models.

Results: At the clinically achievable dose of 50 nM, ganetespib inhibited PDAC cells proliferation (p<0.001), potentiated 5-FU chemotherapy and 2Gy of ionizing radiation (p<0.001), and decreased the levels of survival molecules such as PI3K, p-AKT, and p-ERK in vitro. Additionally, ganetespib indeed potentiates the effects of chemo-radiation in both cell lines: significant decrease in colony formation as well as decrease in migration. Combinations of ganetespib and chemo-radiation therapy demonstrated synergistic ability in human xenograft mouse models (p<0.001).

Conclusion: Ganetespib potentiates the effects of chemo-radiation in preclinical studies. Evaluating HSP90 inhibitors in PDAC patients is a rational approach in assessing the fatal PDAC.

#5817

Oxaliplatin and ATR inhibitors show strong synergy in Ewing sarcoma cells.

Kathleen Sorensen, Jennifer Jess, Susan Goosen, Guillermo Flores, Zach Madaj, Elissa Bogulaslawski, Patrick J. Grohar. _Van Andel Institute, Grand Rapids, MI_.

Background: Ewing sarcoma (ES) is the second most common childhood bone tumor that is in need of new molecularly targeted agents. This tumor is driven by the aberrant activity of the EWS-FLI1 transcription factor that both drives proliferation and blocks differentiation. We, and others, have focused on developing methods to directly target EWS-FLI1. A complementary approach, employed in this study, is to target inherent sensitivities to specific agents created by the many gene expression changes induced by the fusion protein.

Experimental Design: It is known that EWS-FLI1 alters the expression of multiple gene involved in the DNA damage response. Therefore, an initial unbiased drug matrix screen was conducted to determine synergistic combinations between DNA damage response inhibitors and twelve different chemotherapeutic agents. The most synergistic combinations were analyzed by GI50 shifts to determine the sensitivity of ES cells to the chemotherapeutic agent and DNA damage response inhibitor alone and in combination and linked to mechanism via western blot analysis. Finally, gene transfer studies are used to link the effects to EWS-FLI1.

Results: Ewing sarcoma cells show increased sensitivity to an ATR inhibitor (ATRi) relative to control cell lines. In addition, we found marked synergy with platinum agents that was superior to synergy the other 11 chemotherapeutic agents evaluated. This sensitivity was observed with three different platinum agents and shifted the IC50 by over an order of magnitude in five different ES cell lines. In addition, although oxaliplatin showed a lower magnitude of shift in IC50, the synergy could be linked to EWS-FLI1. Oxaliplatin was antagonistic to ATRi in HT1080 cells that lack EWS-FLI1 but showed the opposite effect when these cells were forced to express EWS-FLI1 and the two agents showed synergy. Work continues to understand the mechanism of this heightened sensitivity of oxaliplatin in combination with ATRi as it relates to EWS-FLI1 and to evaluate both combinations in vivo in animal models of the disease.

Conclusion. In conclusion, ES cells are sensitive to ATRi with a striking hypersensitivity when used in combination with platinum agents. Specifically, ES cells are more sensitive to a combination therapy with cisplatin and ATRi. Yet, experiments with oxaliplatin indicate that this platinum agent in combination with ATRi affects cells transfected with EWS/FLI1 more than cells lacking this key transcription factor. Therefore, it is likely that the combination of oxaliplatin and ATRi would show a broad therapeutic window as the two agents may be antagonistic in normal cells that do not express EWS-FLI1 while synergize in Ewing sarcoma cells.

#5818

Globally optimizing therapeutic combinations against bortezomib-resistant multiple myeloma using a quantitative parabolic optimization platform.

Masturah Rashid,1 Tan Boon Toh,1 Lissa Hooi,1 Aleidy Silva,2 Yanzhou Zhang,1 Neerja Karnani,3 Sudhakar Jha,1 Chih-Ming Ho,2 Wee Joo Chng,1 Dean Ho,2 Edward Kai-Hua Chow1. 1 _National University of Singapore (NUS), SINGAPORE, Singapore;_ 2 _University of California, Los Angeles (UCLA), Los Angeles, CA;_ 3 _Singapore Institute for Clinical Sciences (SICS), A*STAR, SINGAPORE, Singapore_.

Multiple myeloma is an incurable hematological malignancy that relies on drug combinations as first and secondary lines of treatment. The inclusion of proteasome inhibitors, such as bortezomib, into these drug combination regimens has improved median survival. Resistance to bortezomib, however, is a common occurrence that ultimately contributes to treatment failure. Thus, there remains a need to identify improved drug combinations that may serve as later lines of treatment for improved treatment against bortezomib-resistant multiple myeloma. We have developed the quantitative parabolic optimization platform (QPOP) to optimize drug combinations against bortezomib-resistant multiple myeloma. By mapping phenotypic output data to parabolic response surfaces, QPOP is able to deterministically optimize drug combinations as well as drug dosages. By continuously optimizing in multiple systems of interest, from in vitro to in vivo, drug combinations can be globally optimized for greater efficacy in increasingly complex biological systems. While QPOP does not rely on molecular mechanism modeling or prediction, identified optimal drug combinations can reverse DNA hypermethylation and silencing of tumor suppressors that occurs following acquired bortezomib-resistance in multiple myeloma. Furthermore, this drug combination is broadly effective across a range of primary multiple myeloma patient samples. Beyond bortezomib-resistant multiple myeloma, global optimization of drug combinations by QPOP can serve to improve drug combination design across a range of other cancers and diseases through a continuous optimization process across the entire drug development pipeline.

#5819

Colossolactone-G enhances the anticancer properties of 5-fluorouracil and gemcitabine against colorectal cancer cells.

Rinad A. Aljohani,1 Gehan A. Hegazy,1 Aliaa A. Alamoudi,1 Ali M. El-Halawany,2 Riham S. Eldine,2 Ghada Agabnoor,1 Ahmed M. Al-Abd3. 1 _King Abdulaziz University, Jeddah, Saudi Arabia;_ 2 _Cairo University, Cairo, Egypt;_ 3 _National Research Ctr. of Egypt, Cairo, Egypt_.

The use of anti-cancer adjuvant therapy is rationalized by potentiating the efficacy, and/or protecting from major side effects of chemotherapeutics. Gemcitabine and 5-fluorouracil are important chemotherapeutic agents which are frequently used to treat cancers such as colorectal cancer. Colossolactone-G is a naturally occurring triterpenoid metabolite isolated from the fruiting body of Ganoderma colossum. Herein, we assessed the potential chemomodulatory effects of Colossolactone-G to 5-fluorouracil and gemcitabine against human colorectal cancer cells. After 72 h of exposure, colossolactone-G per se possesses cytotoxic effects against HCT116 and HT29 cells with IC50's of 15.9±3.6 µM and 90.5±1.7 µM, respectively. In addition, colossolactone-G improved the cytotoxic profile of gemcitabine against HCT116 cells reducing its IC50 from 0.28±0.1 µM to 19.41±3.1 nM with combination index indicative of synergism (CI-value = 0.154). Similarly, colossolactone-G enhanced the cytotoxicity of gemcitabine against HT29 cells reducing its IC50 from 6.2±1.1 µM to 0.6±0.3 µM with combination index indicative of synergism (CI-value = 0.219). Besides, colossolactone-G synergized the cytotoxic profile of 5-fluorouracil against HCT116 and HT29 cells decreasing its IC50's from 10.8±4.6 to 3.1±0.5 µM (CI-value = 0.302), and from 12.9±3.7 to 3.2±0.6 µM (CI-value = 0.426), respectively. By assessing cell cycle distribution after 24 h using DNA content flowcytometry, both gemcitabine and 5-fluorouracil induced moderate cell cycle arrest at S-phase which was increased by combination with colossolactone-G and was further extended to induce accumulation of cells at G0/G1-phase. Longer exposure (48 h) of cells to colossolactone-G alone induced antiproliferative effect and accumulation of cells at G0/G1-phase. Besides, the S-phase arrest induced by gemcitabine and 5-fluorouracil was further increased by combination with colossolactone-G for 48 h and was accompanied by reciprocal decrease in cells in G2/M-phase. Cell cycle arrest attributed to gemcitabine and 5-fluorouracil treatment resulted in increasing cells undergoing apoptosis as shown by annexin-V/FITC staining. Similarly, combination with colossolactone increased the percentage of cells undergoing apoptosis in both cell lines. In conclusion, colossolactone-G represents promising chemomodulatory compound of natural origin which improves the anti-colorectal cancer activity of gemcitabine and 5-fluorouracil in a cell cycle dependent manner. Detailed molecular assessment for the underlying mechanisms of colossolactone-G chemomodulatory activity is currently under investigation.

#5820

Farnesyl transferase inhibitors in combination with agents targeting factors downstream of RAS demonstrate strong drug synergism in malignant peripheral nerve sheath tumors.

Elliot J. Kahen, Darcy Welch, Damon R. Reed. _Moffitt Cancer Center, Tampa, FL_.

Background: Dysfunction of NF1, which codes for the neurofibromin protein, is responsible for development of neurofibromatosis type 1 and is strongly associated with an increased risk of Malignant Peripheral Neural Sheath Tumors (MPNST). Though the role of neurofibromin as a negative regulator of the RAS oncogene has been characterized, effective chemotherapy treatments for MPNST have still proven elusive, and the prognosis for unresectable or metastatic tumors remains poor. Therapeutics that target RAS are an attractive avenue for discovery not only for MPNST but also for many other RAS-associated diseases. Members of the RAS family of proteins undergo posttranslational modification with farnesyl, which is essential for its subcellular localization and function. Attempts have been made to employ Farnesyl transferase inhibitors (FTIs) that block the enzyme responsible for this modification RAS-associated diseases, but K-RAS and N-RAS are able to overcome this inhibition via geranylgeranylation. Nonetheless, FTIs may still prove to be a useful tool in H-RAS dependent cancers. Interestingly, recent molecular characterization has suggested that H-RAS may be the predominant RAS member active in MPNSTs. Though FTIs have been assessed in NF1-associated diseases in the past, there has been little work looking at combination of FTIs with other relevant small molecule inhibitors.

Methods: We used a combination of molecular biology, proteomics, high-throughput drug screening analysis to elucidate to relative importance of H, K, and N RAS in MPNSTs and to explore the efficacy of FTIs in combination with other drugs that target the RAS pathway. Across 5 cell line models, we used our high-throughput screening platform to assess the efficacy of an array of drug combinations containing FTIs and other agents of interest at clinically relevant doses, guided by published pharmacokinetics.

Results: We found that opposed to many other RAS-associated diseases, K-RAS carried a diminished role in MPNSTs, with very little expression detected at the protein level. H-RAS and N-RAS were observed, with H-RAS demonstrating prominence among the RAS members. In line with previous studies, single agent treatment with FTIs was not efficacious at physiologically relevant concentrations. However, combination of FTIs with inhibitors of MEK and mTOR resulted in remarkably strong synergism and potent effects across 5 MPNST cell lines. These findings suggest promising opportunities for developing combination clinical trials that contain FTIs along with other relevant therapeutics in the treatment of MPNSTs.

#5821

Atorvastatin antagonizes carboplatin, synergizes with the glycolytic inhibitor PFK-158, and induces lipid laden multilamellar bodies in ovarian cancer.

Eleftheria Kalogera, Debarshi Roy, Jamie N. Bakkum-Gamez, William A. Cliby, Sean C. Dowdy, Viji Shridhar. _Mayo Clinic, Rochester, MN_.

Objectives: To investigate the antitumor potential of the widely used anticholesterol drug Atorvastatin (ATV) alone, in combination with Carboplatin (CBPT) and with the glycolytic inhibitor PFK158 (PFKi) in ovarian cancer (OC).

Methods: MTT assay and the Chou-Talalay methodology were used to test anticancer activity in vitro; half maximal inhibitory concentration (IC50) and combination index (CI) were calculated. Cellular changes were studied with transmission electron microscopy (TEM). ATV was combined with PFKi as we previously showed that PFKi, in addition to glycolysis, inhibits lipid metabolism by targeting PLA2G3. PLA2G3 expression was studied by western blot given that downregulation in OVCAR8 using two shRNAs (sh33, sh35) induced autophagy, a prodeath condition in OC, and was associated with chemosensitization compared to nontargeted controls (NTC) (unpublished data). A random sample of 10 patient-derived OC xenografts (PDX) was studied by TEM.

Results: OVCAR8 exhibited a dose dependent proliferation inhibition when treated with ATV (IC50 8μM). PFKi and CBPT IC50 were 4μM and 78μM. PFKi treatment combined with 4μM and 8μM of ATV resulted in ATV sensitizing cells to PFKi by shifting PFKi IC50 down to 2.7μM and 0.4μM, respectively. When ATV was combined in constant ratio with CBPT, CIs were >1.1 indicating antagonism. Combination of ATV 4μM with PFKi 2μM resulted in greater decrease in PLA2G3 and p62 and greater increase in cleaved PARP compared to PFKi alone suggesting higher levels of autophagy and apoptosis. ATV and combination treatment resulted in increased caveolin-1 levels, previously shown to be associated with apoptotic cell death. TEM analysis of ATV and PFKi treated cells showed greater number of lipid rich multilamellar bodies (MLB) compared to controls. PLA2G3 knockdowns exhibited higher MBL formation, lower p62, and when treated with statin and with PFKi, cleaved PARP was significantly higher compared to NTCs. TEM showed that PDXs clustered in low and high MLB groups suggesting a distinct novel phenotype.

Conclusions: ATV could enhance PFKi antitumor activity by jointly targeting PLA2G3 leading to metabolic reprogramming of OC. Importantly, ATV antagonized CBPT. Additional studies are in progress to explore the mechanistic pathway of ATV and characterize the significance of MLB in OC as a prodeath phenotype and biomarker of response.

#5822

Secretory acetylated APE1/Ref-1 requirement for suppression of tumor growth in triple-negative breast cancer in vivo: Extracellular apoptotic initiator.

Yu Ran Lee,1 Myoung Soo Park,2 Hee Kyoung Joo,1 Ki Mo Kim,3 Jeryong Kim,1 Byeong Hwa Jeon,1 Sung Choi1. 1 _School of Medicine, Chungnam National University, Daejeon, Republic of Korea;_ 2 _Chungnam National University Hospital, Daejeon, Republic of Korea;_ 3 _Korean Medicine Convergence Research Division, Korea Institute of Oriental Medicine, Daejeon, Republic of Korea_.

Triple-negative breast cancer (TNBC) represents a relatively small proportion of all BCs but a relatively large proportion of BC-related death. Thus more effective therapeutic strategies are needed for the management of TNBC. We previously demonstrated that the stimulation of apoptosis by the binding of secreted acetylated-apurinic apyrimidinic endonuclease 1/redox factor-1 (Ac-APE1/Ref-1) to the receptor for advanced glycation end products (RAGE) was essential for the TNBC cell death in response to hyperacetylation. The aim of the present study was to assess the potential therapeutic efficacy of secretory Ac-APE1/Ref-1 in orthotopic TNBC xenografts in vivo. We found that hyperacetylation in xenografts caused secretion of Ac-APE1/Ref-1 into the blood, where the factor bound directly to RAGE in hyperacetylated tumor tissues. Hyperacetylation in the TNBC xenografts induced strong inhibition of tumor growth and development. Hyperacetylation also caused cell death in tumors, accompanied by increased RAGE expression and generation of reactive oxygen species, which induced apoptosis. Tissues exhibited markedly higher counts of apoptotic bodies, a reduced proliferation index, and reduced neovascularization compared with control tumors. Ac-APE1/Ref-1-stimulated apoptosis was markedly reduced in RAGE-knockdown tumors compared with RAGE-overexpressing tumors, even in the presence of hyperacetylation. The function of secreted Ac-APE1/Ref-1 was confirmed in other hyperacetylated TNBCs xenografts using BT-459 and MDA-MB-468 cells, demonstrating its relevance as an anticancer molecule. Our findings suggest that the Ac-APE1/Ref-1 possesses potent chemotherapeutic efficacy against hyperacetylated TNBCs based on stimulation of cell death by RAGE-dependent triggering of Ac-APE1/Ref-1.

#5823

Palmatine as a potential pancreatic cancer therapeutic agent.

Amanda R. Muñoz,1 Roble Bedolla,2 Shih-Bo Huang,2 Xiaou Yang,2 Paul Rivas,2 Robert Reddick,2 Martha Hanes,2 Glenn Halff,2 Rita Ghosh,2 Addanki P. Kumar1. 1 _South Texas Veterans Health Care System, San Antonio, TX;_ 2 _UT Health San Antonio, San Antonio, TX_.

Over the last 30 years, little improvement has been made to the 5 year survival rate of pancreatic cancer (PanCA) patients. While survival has increase by 2% over the last several years, the current rate is still less than 8%. This depressing fact demonstrates the importance of developing or improving therapies to more effectively manage this disease. Along these lines, published studies from our laboratory demonstrated the anti-tumorigenic potential of the cork tree bark extract, Nexrutine® (Nx). Nx suppressed growth of pancreatic cancer cells through downregulation of STAT3/NF-κB activation. Subsequent biochemical and molecular investigations revealed palmatine (PMT) (i) as an active constituent of Nx able to suppress the growth of pancreatic cancer cells; (ii) synergizes with gemcitabine (GEM); and (iii) downregulates GLI1, COL1A1 and Survivin. Despite such promising in vitro observations however, the in vivo relevance of PMT is undefined. Furthermore, it is unclear if PMT can recapitulate the biological activities of Nx in vivo. In this investigation, we tested the hypothesis that PMT recapitulates the biological activities of Nx and enhances GEM activity. This hypothesis was tested by comparing the efficacy of Nx and PMT using (i) athymic mice implanted with Capan-2 cells; (ii) a syngenic mouse model using C57BL/6 mice implanted with KPC-GFP-Luc cells; and (iii) a short term ex vivo model utilizing cells isolated from primary pancreatic tumors following surgical resection. Additionally, in vitro experiments were also done to assess the underlying molecular mechanism. Analysis of these data show that both Nx and PMT are well tolerated in vivo and a significant reduction in the levels of serum inflammatory cytokines including IL-6, granulocyte-colony stimulating factor (G-CSF), and CXCL1. Interestingly, animals receiving PMT, but not Nx, showed a trend towards decreased pancreatic tumor weight that was associated with histopathological changes. Investigation into the potential mechanism revealed that Nx and PMT mediated inhibition of STAT3, EP4, Src, TrkA, and RPS6 activities may contribute to the observed growth inhibitory and anti-inflammatory effects. Incredibly, our ex vivo analysis of patient derived PanCA cells demonstrated that both Nx and PMT could inhibit the growth of these cells. Collectively, our data demonstrates PMT recapitulates biological activities of Nx and that there is potential for developing PMT as an agent for clinical management of PanCA. Supported by NCCIH (R01 AT007448; APK) and VA-MERIT Award (I01 BX 000766; APK).

#5824

The BET inhibitor JQ1 sensitizes cholangiocarcinoma cells to PARP inhibitors.

Samuel C. Fehling,1 Aubrey L. Miller,1 James E. Bradner,2 Karina J. Yoon1. 1 _University of Alabama at Birmingham, Birmingham, AL;_ 2 _Novartis Institutes for Biomedical Research, Cambridge, MA_.

Cholangiocarcinoma (CCA) is an aggressive bile duct neoplasm, and the second most common primary hepatic malignancy. CCA is usually diagnosed at a late stage where the current standard of care, resection followed by gemcitabine with or without cisplatin, is not effective. Up to 90% of CCA patients are ineligible for resection and, of those eligible, postoperative chemotherapy does not significantly prolong overall survival. The limited number of CCA models hinders drug development and discovery of new therapeutic targets. Mutations have been identified in KRAS (17% of CCA tumors), p53 (44%) and SMAD4 (17%), but are not recognized as essential to CCA progression. c-Myc protein is highly expressed in 94% of CCA cases while low to undetectable in normal adult liver, suggesting c-Myc overexpression may contribute to CCA tumor progression but this hypothesis has not been tested. Toward developing novel effective therapies for CCA, we evaluated the effect of the bromodomain and extraterminal domain (BET) inhibitor JQ1 on c-Myc expression and on the proliferation of CCA cells. JQ1 inhibits c-Myc transcription by binding to the acetylated lysine (K-Ac) binding pockets of BET protein family members (BRD2, BRD3, BRD4 and BRDT), preventing their interaction with K-Ac residues on nuclear proteins, disrupting the formation of functional transcriptional complexes and inhibiting c-Myc transcription. We have shown that JQ1 inhibited tumor growth in a patient-derived xenograft (PDX) model of CCA and downregulated expression of c-Myc and its transcriptional targets BRCA2 and Chk1, both involved in DNA damage response. Our current data show that JQ1 decreased c-Myc protein expression, CCA cell viability, and clonogenic potential in KKU-055 CCA cell line. Of note, the literature suggests that tumors deficient in BRCA2 or Chk1 are relatively sensitive to PARP inhibitors (PARPi). Therefore, we hypothesize that JQ1-induced downregulation of c-Myc and its transcriptional targets BRCA2 and Chk1 would potentiate the efficacy of PARPi. We exposed CCA cell line KKU-055 with a range of JQ1 and PARPi (olaparib and veliparib) concentrations as single agent or combination therapy. Each combination of JQ1 with either PARPi was more effective than any of the three drugs as single agents. Combination indices (CI), calculated using CompuSyn 1.0, ranged from 0.05 to 0.09 which indicated strong synergy for JQ1 with olaparib or veliparib. Further, synergy with JQ1 was not dependent on a single PARPi. JQ1 with olaparib (1:1) also decreased the clonogenic potential of KKU-055 cells, compared to each drug as a single agent. These data suggest that inhibition of BET protein function sensitizes CCA cells to PARPi, coincident with downregulation of c-Myc and its transcriptional targets BRCA2 and Chk1. Research funded by NIH/NCI R21 CA205501

#5825

Structure-based drug design to overcome temozolomide resistance in glioblastoma (GBM) through a dual inhibition of MGMT and base excision repair.

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

The incidence of pediatric and adult brain tumors has continued to rise and temozolomide (TMZ) has remained the sole choice for chemotherapy in these cancers. TMZ alkylates the DNA to generate N7-methylguanine (~70%) and O6-methylguanine (~6-9%) lesions, with the latter being considered the cytotoxic residue. However, the N7-methylations can also contribute to cell killing by producing abasic sites, strand-breaks, and mutagenicity. Therefore, inhibiting the two major mechanisms of resistance to TMZ in brain cancers, namely, the MGMT DNA repair, which removes the O6-methyl groups, and the APE1 endonuclease, which cleaves the apurinic/apyrimidinic sites, is a logical choice for enhancing the alkylator therapy; incidentally, both these proteins are highly expressed in brain tumors. Pseudosubstrates for MGMT such as O6-BG + TMZ therapies suffer from a high level of bone marrow suppression and there is no clinically approved inhibitor for APE1. Recently, we discovered that nitroaspirin (NCX-4016) is a powerful and clinically relevant inhibitor of human MGMT for increasing the efficacy of alkylating agents (Proc of AACR 54, 2013;1094). Further extensive structure-activity relationship studies revealed that the 3-hydroxybenzyl nitrate moiety of NCX-4016 is essential for its MGMT inhibition activity. To develop a dual inhibitor of APE1 and MGMT, we designed a hybrid compound called MGAP-9 by combining 3-hydroxybenzyl nitrate with 7-nitroindole-2-carboxylic acid, a known potent, nontoxic, and cell-permeable APE1 inhibitor through an ester bond. MGAP-9 exhibited significant inhibition of MGMT activity and a corresponding decrease in protein levels in both concentrations- and time-dependent manner. Similarly, we observed potent inhibition of APE1 nuclease activity both in vitro and in tumor cells using a biotin-labeled oligonucleotide/polyacrylamide gel-electrophoresis assay. Inhibition of both targets occurred at pharmacologically achievable concentrations (5-25 µM). MGAP-9 exhibited more than 100-fold sensitization of various GBM cell lines towards the methylating agents such as TMZ and dacarbazine, but surprisingly not to BCNU or CCNU. A substantial increase in γ-H2AX further confirmed the DNA damage induced by MGAP-9 through a dual inhibition. In addition, the hybrid drug displayed potent antiangiogenic activity against human umbilical vein cells (HUVECs). Finally, MGAP-9 showed significant regression of tumor growth in mouse orthotopic glioblastoma xenograft models and did not elicit systemic toxicity at the therapeutic doses used. The findings suggest the clinical relevance of MGAP-9 and its usefulness in reducing the TMZ doses to curtail the marrow suppression in brain tumor therapy.

Supported by CPRIT grants RP130266 and RP170207 to KSS.

#5826

Enhanced anti-tumor effects of selinexor and niraparib in preclinical models of ovarian cancer.

Hua Chang,1 Trinayan Kashyap,1 Sophie Debler,1 Thaddeus J. Unger,1 Sarah Wang,2 Keith Mikule,2 Jing Wang,2 Mansoor R. Mirza,1 Sharon Shacham,1 Yosef Landesman1. 1 _Karyopharm Therapeutics, Inc., Newton, MA;_ 2 _TESARO Inc., Waltham, MA_.

Introduction: Selinexor (KPT-330) is a first-in-class oral exportin-1 (XPO1/ CRM1) inhibitor that induces cell cycle arrest and apoptosis in cancer cells through reactivation of tumor suppressor proteins and inhibition of DNA damage repair genes. Here, we studied selinexor in combination with niraparib, an inhibitor of the DNA damage repair proteins PARP1 and 2, in preclinical models of ovarian cancer. Given that both compounds can inhibit DNA damage repair responses, we hypothesized the combination of selinexor and niraparib would enhance cancer cell death by accumulation of DNA damage that cannot be resolved in ovarian cancer.

Methods: Selinexor and niraparib alone and in combination were tested in vitro on the BRCA wildtype ovarian cancer cell line A2780. Total RNA and protein were extracted from cell lysates and analyzed by qPCR and immunoblots. In vivo, a subcutaneous A2780 xenograft mouse model was treated with selinexor [10 mg/kg, once per week (M) for three weeks] or niraparib [37.5 mg/kg, once-daily for five days per week (M-F) for three weeks] as single agents or in combination. Tumor growth and body weights were measured for 21 days. Tumors were harvested at the end of the study and analyzed by immunohistochemistry (IHC).

Results: Selinexor and niraparib as single agents inhibited A2780 cell proliferation (selinexor IC50: 200nM; niraparib IC50: 800nM). The combination of selinexor and niraparib showed synergistic cytotoxicity in A2780 cells. Increased expression of phospho (S139) H2A.X with the combination confirmed induction of DNA damage. In vivo, the combination enhanced tumor inhibition (51.0% in combination versus 24.3% and 29.6% in selinexor and niraparib respectively) and improved median survival compared to vehicle or each agent alone (18 days in combination group versus 11, 14 and 16 days in vehicle, selinexor, and niraparib groups, respectively). IHC analysis showed enhanced nuclear p53 and p21 staining in selinexor treated tumors as well as in tumors treated with the combination. Increased apoptosis was observed in tumor samples treated with both selinexor and niraparib as compared to vehicle control or each agent alone.

Conclusions: Selinexor plus niraparib demonstrated enhanced anti-tumor activity in preclinical models of human ovarian cancer. Since both drugs could inhibit DNA damage repair pathway, the drug combination efficiently accumulated DNA damage, which was associated with reduced cell proliferation, and induced apoptosis. This combination therapy warrants further investigation as a treatment option for patients with ovarian cancer.

#5827

Enzymatic combination investigation in cancer therapy.

Karine Aguera, Fanny Gallix, Fabien Gay, Karine Senechal, Severine Cire, Françoise Horand, Alexander Scheer, Vanessa Bourgeaux. _ERYTECH PHARMA, LYON, France_.

L-asparaginase (ASNase), an enzyme currently used in combination with chemotherapy for Acute Lymphoblastic Leukemia (ALL) treatment, hydrolyzes L-asparagine (Asn) - a non-essential amino acid - into aspartic acid (Asp) and ammonia, leading to Asn removal from the circulation. Similarly, restriction to exogenous L-methionine (Met) - an essential, sulfur-containing proteinogenic amino acid - blocks cell cycle in the late S or G2 phase in Met-dependent cancer (Hoffman and Jacobsen 1980; Guo et al., 1993; Kokkinakis et al., 1997, 2004, 2005 and 2006; Pavillard et al., 2006; Yano et al., 2014). ERYTECH Pharma's strategy was to combine Met depletion, by using Methionine-γ-lyase (MGL) (a pyridoxal-dependent enzyme) with Asn depletion (via ASNase activity), and investigate its in vitro and in vivo therapeutic potential.

In vitro sensitivity to MGL combined with ASNase was assessed on 7 cell lines representative of different cancers (glioblastoma, AML, gastric and pancreatic adenocarcinoma or carcinoma). Fixed enzyme concentrations (IC50), determined previously in dose-response studies using each enzyme alone, were used in a sequential therapeutic scheme adding MGL first and ASNase 3 or 4 days later. Tumor cells sensitivity was evaluated by measuring cell viability. In vivo combination study, in a human gastric xenograft mouse model, was performed using ASNase and MGL encapsulated into red blood cells, respectively named erymethionase and eryaspase. The scheme of administration consisted in erymethionase treatment on Day 7 and 15 followed by eryaspase treatment on Day 21. Control arms received vehicle or single therapy with 3 repeated administrations.

In vitro study results showed enhanced mortality with MGL-ASNase sequential therapy vs single therapies (ASNase and MGL tested alone) in 72% (5/7) of the tested cellular models (AML, glioblastoma, gastric and pancreatic cancers models). Those data were confirmed in vivo with an enhanced tumor inhibition when erymethionase was administered twice before eryaspase, compared to vehicle or erymethionase-alone treatments (respectively 57% and 36% on Day 37). The result significance is supported by a p-value inferior to 0.0001.

To conclude, our in vitro results showed promising potential for enzymatic bi-therapy treatment on several cancer models and that intrinsic tumor cells properties determined its efficiency. In vivo data confirmed, in a gastric cancer model, the therapeutic efficacy of this sequential erymethionase-eryaspase treatment. However, this enzymatic bi-therapy still requires further investigation. The identification of involved mechanisms of action should allow to optimize the therapeutic design and select best responders. A preclinical program is currently ongoing on specific biomarkers, investigation on tumor cell synchronization with ASNase therapy and optimal therapeutic enzyme sequence for treatment.

#5828

Withaferin A and tumor treating fields synergistically inhibit glioma proliferation.

Edwin Chang,1 Chirag Patel,1 Christoph Pohling,1 Nooshin Beygui,2 Dong Ho Ha,1 Sanjiiv S. Gambhir1. 1 _Stanford University, Canary Center, Palo Alto, CA;_ 2 _Scripps College, Claremont, CA_.

Glioblastoma multiforme (GBM) is the most lethal primary brain cancer (median survival: 15-17 months, 5-year survival: 5-10%). Current interventions consist of equally aggressive surgical resection, radiotherapy, and chemotherapy. However, this cancer is heterogeneous and not easily accessible so standardized, non-specific therapies are often ineffective. Novel, unorthodox approaches against GBM need consideration. Withaferin A is a steroidal lactone that originates from the roots and leaves of the winter cherry plant (Withania somnifera). Within Ayurvedic medicine, Withania somnifera extracts are applied to many disorders including several cancers. Extracts such as AshwaMAX can contain up to 4.3% (w/w) Withaferin A, a biologically active compound from Withania somnifera. Tumor Treating Fields (TTFields) are thought to disrupt mitotic spindle formation and stymie proliferation of actively dividing cancer cells. We report on the anti-cancer properties of both Withaferin A and TTFields. We also examined the hypothesis that combining TTFields with Withaferin A would synergistically inhibit GBM growth. We used GBM cells (U87-MG, GBM2, and GBM39) and breast adenocarcinoma cells (MDA-MB-231) isolated from human primary tumors. Each GBM cell line was modified to express firefly luciferase. Proliferative potential was assessed by bioluminescent imaging (BLI), cell counting via hemocytometer, or through the CellTiter-Blue® viability assay. Intracranial orthotopic U87-MG GBM xenografts were grown in the right frontal lobe of female nude mice (n=5/experiment). TTFields were imposed on cell cultures with the inovitroTM system from Novocure Ltd. Neurosphere cultures (U87-MG, GBM2, and GBM39) were inhibited by AshwaMAX at IC50 of 1.4, 0.19 and 0.22 µM (Withaferin A equivalent), respectively, and by Withaferin A with IC50 of 0.31, 0.28, and 0.25 µM, respectively. Oral gavage, every other day, of AshwaMAX (40mg/kg per dose) significantly reduced BLI signal (n=5 mice/group, p<0.02, four-parameter non-linear regression analysis) in preclinical models. Comsol modeling showed that the inovitroTM device imposed optimal TTFields in the center of the cell culture dishes. TTFields at 4 V/cm significantly inhibited (n=4 measurements per time point, p<0.001, 2-way ANOVA) growth of MDA-MB-231 cancer lines and all three human GBM lines. The effects were due to the TTFields alone and not to the slight temperature rises caused by the TTFields. Combination of Withaferin A (10-100 nM) with TTFields (4 V/cm) significantly inhibited GBM growth, to a degree beyond that of Withaferin A or TTFields alone. Poisson-based analysis of significance for regression indicated that the interaction of the combined treatments on GBM cells was synergistic (p<0.01, n=3 experiments). The findings suggest a novel approach to treat GBM in a manner that is likely better than either treatment (Withaferin A or TTFields) alone and that is synergistic.

#5829

CDK8 inhibitor SEL120-34A targets CD34 positive AML cells by regulation of various transcriptional programs involved in maintenance of leukemia stem cells.

Eliza Majewska,1 Milena Mazan,1 Michal Mikula,2 Agnieszka Dreas,1 Katarzyna Wiklik,1 Aniela Golas,1 Katarzyna Wojcik,1 Magdalena Masiejczyk,1 Malgorzata Statkiewicz,2 Urszula Kuklinska,2 Krzysztof Goryca,2 Aleksandra Grochowska,3 Aleksandra Cabaj,2 Michal Combik,1 Jerzy Ostrowski,2 Krzysztof Brzozka,1 Tomasz Rzymski1. 1 _Selvita S.A., Kraków, Poland;_ 2 _Maria Sklodowska-Curie Memorial Cancer Center, Warsaw, Poland;_ 3 _Medical Center for Postgraduate Education, Warsaw, Poland_.

Despite heterogeneity deregulated transcription is one of the universal features of leukemia cells. This has paved the way for the development of active compounds targeting epigenetic and transcriptional factors. A substituted tricyclic benzimidazole, SEL120-34A, is a novel inhibitor of Cyclin-dependent kinase 8 (CDK8), which regulates transcription by associating with the Mediator complex. In many cancer cells SEL120-34A regulates expression of STAT1- dependent genes as a major transcriptional effect. In AML cells regulation of NUP98-HOXA9 - dependent transcription and other transcriptional programs involved in maintenance of leukemia stem cells have been observed as a dominant mechanism of action. Treatment with the compound resulted in a differential efficacy on CD34 positive AML cells with elevated STAT5 S726 levels and stem cell characteristics. In contrast, resistant cells were negative for activated STAT5 and revealed lineage commitment. Prolonged exposure of AML cells lines to chemotherapy led to the enrichment in population of resistant CD34 positive cells, which proved to be oversensitive to SEL120-34A treatment, providing rationale for rational combinations with approved drugs. Further clinical development of SEL120-34A is warranted as a novel therapeutic approach in relapsed and refractory AML.

#5830

Combination of volasertib (BI6727) and abemaciclib (LY2835219) for melanoma management: Concomitant targeting of multiple cell cycle phases.

Sanghamitra Nethramurthy, Mary A. Ndiaye, Chandra K. Singh, Nihal Ahmad. _University of Wisconsin-Madison, Madison, WI_.

Melanoma is one of the most lethal forms of skin cancer that kills an average of around one person every hour in the United States alone. Melanoma can be surgically removed if diagnosed early, however, undiagnosed or untreated melanomas can rapidly metastasize to become lethal. Significant progress has been made in the treatment of metastatic melanoma over the past decade, especially with novel targeted therapeutic approaches (such as BRAF- and MEK- inhibitors or immunotherapies). However, even with these newer therapies, melanoma can acquire resistance and become untreatable, with fatal consequences. Therefore, novel therapeutic approaches are required for the management of this neoplasm. It is commonly accepted that mechanism-based combination therapeutics could be better for an efficient and complete elimination of cancer cells, thereby limiting the chances of recurrence and resistance. Based on this rationale, we reasoned that a simultaneous targeting of G2/M and G1 phases of the cell cycle could provide a more efficacious response against melanoma. Studies in our laboratory have suggested that polo-like kinase 1 (PLK1), which is a mitotic regulator acting in the G2/M phase of the cell cycle, is overexpressed in melanoma and is a potentially druggable target for melanoma management. We demonstrated that BI6727, a small-molecule PLK1 inhibitor, resulted in delay and regression of melanoma xenografts. Similarly, dysregulation of the p16-cyclin D1-CDK4/6-Rb pathway has been shown to frequently occur in melanoma, and CDK4/6 inhibition is being extensively investigated in the management of several cancers, including melanoma. In this study, we evaluated the anti-proliferative efficacy of a combination of G2/M-phase targeting by BI6727 and G1-phase targeting by the CDK4/6 inhibitor LY2835219 in human melanoma cells. We determined the effect of BI6727 and/or LY2835219 on growth and viability of human melanoma cells using the RealTime-Glo Cell Viability- and Trypan Blue exclusion- assays. We found that the combination of BI6727 and LY2835219 resulted in a better anti-proliferative response, when compared to either of the agents alone. Further, the combination treatment was found to result in a marked decrease in the clonogenic survival of melanoma cells, as assessed by colony formation assay. Cell cycle analysis showed a G2/M-phase arrest in BI6727 treated cells and G1-phase arrest in LY2835219 treated cells. Interestingly, the combination was found to cause G2/M as well as G1- phase arrest of melanoma cells. Overall, our data suggests that a concomitant inhibition of PLK1 with CDK4/6 provides a superior anti-proliferative response, suggesting that dual inhibition should be further evaluated in detailed in vitro and in vivo experiments. Additional studies to determine the mechanisms of this combination are currently underway in our laboratory.

#5831

Biopolymer β-brachytherapy delivered concomitantly with systemic paclitaxel outperforms traditional x-ray radiation to induce complete regression in multiple pancreatic tumor xenograft models.

Jeffrey L. Schaal, Jayanta Bhattacharyya, Xinghai Li, Samagya Banskota, Jeremy Brownstein, David Kirsch, Michael Zalutsky, Ashutosh Chilkoti. _Duke Univ., Durham, NC_.

Pancreatic tumors remain one of the deadliest manifestations of cancer in clinical oncology. Even when tumors are diagnosed early, remain loco-regionalized, and receive cutting-edge cancer treatment, the 5-year survival rate remains less than 27%. This high mortality rate arises from the significant biologic barriers that inhibit drug delivery effectiveness in pancreatic tumors, as well as their underlying genetic mutations that confer resistance to traditional radiation and chemotherapeutic standards of care. In this work, we hypothesized that a new form of radiation brachytherapy might prove highly synergistic for overcoming the resistance native to pancreatic tumors. Utilizing the thermally stabilized biopolymer ELP, 131iodine radionuclides can be delivered intratumorally to provide continuous, focalized, high-dose β-radiation to treat pancreatic cancer from the inside out. Dose-escalation studies revealed that the two important variables for improving outcomes were the 131I-ELP radioactivity dose and the duration of concurrent paclitaxel therapy. 100% tumor regression was achieved across multiple genotypic models of pancreatic cancer models: MIA PaCa-2, AsPc-1, and BxPc3 (both subcutaneous and orthotopic). The potent synergistic results of 131I-ELP brachytherapy were replicated using both a novel micelle formulation of paclitaxel and the clinical standard Abraxane. Multigroup analysis revealed true molecular synergy as assessed by the Bliss Independence framework (p < 0.01). External beam irradiation, when similarly employed as comparison, merely induced growth inhibition when combined with paclitaxel utilizing a 5Gy hypofractionation regimen. Immunohistochemical analysis of treated tumors revealed differential modulation of biomolecular components that regulate the effectiveness of drug delivery and radiotoxicity. Levels of PECAM, VE-cadherin, claudin-4, HIF-1α, and stromal collagen were all specifically investigated, while macroscopic analysis revealed significantly enhanced accumulation and retention of fluorescently tagged paclitaxel when tumors were treated with biopolymer brachytherapy. Standard x-ray therapy indicated little to no improvement. No signs of any toxic side effects were observed in serologic enzyme analysis and multiorgan biodistribution studies.

#5832

Sphingolipid metabolism determines the efficacy of nanoliposomal ceramide in acute myeloid leukemia.

Charyguly Annageldiyev,1 Arati Sharma,1 Brian M. Barth,2 Todd E. Fox,3 Tye Deering,4 Viola Devine,1 Nicole R. Keasey,1 Stephan T. Stern,5 Megan M. Young,1 Hong-Gang Wan Wang,1 Jason Liao,6 Junjia Zhu,6 Aaron D. Viny,7 Ross L. Levine,7 Thomas P. Loughran,8 Mark Kester,4 David F. Claxton1. 1 _Penn State Univ. College of Medicine, Hershey, PA;_ 2 _University of New Hampshire, Durham, NH;_ 3 _University of Virginia , School of Medicine, Charlottesville,, VA;_ 4 _nanoSTAR Institute, University of Virginia, Charlottesville,, VA;_ 5 _National Cancer Institute, Frederick, MD;_ 6 _Penn State University, Hershey, PA;_ 7 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 8 _University of Virginia School of Medicine, Charlottesville,, VA_.

Therapeutic advances for the treatment of acute myeloid leukemia (AML) have been limited in part due to the heterogeneity and complexity of the disease and a poor understanding of its underlying biology. The leukemia stem cell (LSC) arguably resists current therapy resulting in relapses for most initially treatment sensitive patients. AML with myelodysplastic syndrome related changes (AML-MRC) highlights this challenge, representing a very poor outlook subset. The present study sought to understand the underlying sphingolipid biology in and AML, and to evaluate the efficacy of nanoliposomal ceramide (Lip-C6). Sphingolipids play essential roles in cell survival and proliferation, as well as stress and death. Lip-C6, which delivers a short-chain analog of the pro-apoptotic sphingolipid ceramide, has been in development as an anticancer therapeutic. The efficacy of Lip-C6 therapy was evaluated in both in vitro and in vivo models using primary AML cells and AML cell lines. Evaluation and characterization of the effect of treatment with Lip-C6 was done through lipidomic, short term assays such as apoptosis, autophagy and colony formation assays. Efficacy of Lip-C6 and vinblastine was tested in patient derived xenograft models and mouse - human cell line xenograft MV-411. NOD SCID gamma (NSG) mice were injected with luciferase/YFP labeled cells and monitored by bioluminescence imaging (BLI) for the leukemia progression and efficacy. Sphingolipid metabolism was observed to be elevated in patient samples with De Novo AML but not those with AML-MRC. Apoptosis induced by Lip-C6 in CD34+ve/CD38-ve "LSCs" was robust in AML-MRC, but limited in De Novo AMLs. Similarly, AML colonies forming cells were more sensitive to Lip-C6 in AML- MRC than in De Novo cases. It was hypothesized that elevated sphingolipid metabolism and the upregulation of pro-survival pathways such as autophagy contributed to Lip-C6 resistance in De Novo AML. Vinblastine, when combined with Lip-C6, focused sphingolipid metabolism towards pro-apoptotic metabolites and blocked autophagy. In-vivo combination of Lip-C6 and vinblastine uniquely yielded long term control of leukemia progression without systemic toxicity, translating in to prolonged overall leukemia free survival compared to single agents. Altogether, this study shows fundamental biological differences in sphingolipid metabolism between De Novo AML and AML-MRC. The combination of Vinblastine and Lip-C6 targets the LSC and yields apparent cure of lethal human xenograft AML.

### Epigenetic and Metabolic Pathways in Mediating Therapeutic Resistance

#5833

Escape form adaptive drug tolerance through OGT and TET1 mediated H3K4me3 remodeling in MAPKi resistant melanoma.

Dinoop Ravindran Menon,1 Heinz Hammerlindl,1 Abdullah Al Emran,1 Joachim Torrano,1 Sabrina Hammerlindl,1 Gao Zhang,2 Rajasekharan Somasundaram,2 Richard A. Sturm,1 Nikolas K. Haass,1 Keith Flaherty,3 Meenhard Herlyn,2 Helmut Schaider1. 1 _The University of Queensland, Brisbane, Australia;_ 2 _The Wistar Institute, Philadelphia, PA;_ 3 _Harvard Medical School, Boston, MA_.

Background Acquired drug resistance in BRAF mutant melanoma is the main cause for disease relapse. We previously described a slow cycling induced drug tolerant state upon continuous BRAF/MEK treatment preceding permanent resistance with generic changes in histone methylation. Rationale Genetic alterations linked to acquired BRAF inhibitor resistance are absent in about 40% of relapsed melanoma patients suggesting the involvement of epigenetic alterations in the development of acquired drug resistance. We investigated epigenetic remodeling in BRAF mutant melanoma upon BRAF/MEK inhibition. Methods An in-vitro model of time lapse dependent transition to acquired drug resistance using mutant BRAF melanoma was used to investigate epigenetic changes following chronic drug exposure. Histone methylation patters were investigated using ChIP-seq, followed by target gene promoter ChIP-PCR and functional verification. Findings were confirmed by gene silencing, combined treatment regimes in vivo, in PDX tumors and clinical data sets. Results A state dependent response to chronic drug treatment was observed. Long term treatment of more than 45 days enables the cells to escape the slow cycling state which results in proliferating cellular clusters (drug-tolerant persister colonies) with stem-like characteristics that regain global H3K4me3. Persister colonies are then giving rise to fast proliferating BRAF/MEK inhibitor resistant cells. H3K4me3 ChIP-seq of colonies compared to parental cells revealed differential marking at promotor regions of several target genes involved in MAPKi resistance, including ARAF, BRAF, and CRAF. H3K4me3 remodeling corresponded to increased gene expression and susceptibility to pan-RAF inhibitors, suggesting an H3K4me3 mediated increase of ARAF and CRAF as a mechanism of BRAF/MEK inhibitor resistance. Two enzymes, OGT and TET1 that are both linked to H3K4me3 regulation in embryonic stem cells are highly upregulated in persister colonies and tumor tissue of PDXs from BRAF mutant melanoma patients under MEK1/2 inhibition. A shift in OGT nuclear localization and O-linked glycosylation patterns was observed in colonies compared to parental cells suggestive of altered transcriptional and protein activity. OGT ChIP-PCR of colonies compared to parental cells confirmed a set of genes with exclusively H3K4me3 marking in colonies. shRNA mediated knockdown of OGT and TET1 blocked H3K4me3 increase in IDTC colonies, prevented colony formation and delayed tumor relapse in a BRAF mutant xenograft mouse model. High TET1 mRNA expression is linked to significantly shorter survival in TGCA data. Conclusion OGT and TET1 mediated epigenetic remodeling through H3K4me3 with upregulation of MAPKi resistant genes is responsible for the emergence of permanent resistance. Both enzymes are promising targets to combat treatment failure and prolong overall survival.

#5834

Epigenetic re-programming sensitizes prostate cancer cells to anti-androgen therapy.

Lanbo Xiao, Jean Ching-Yi Tien, Josh Vo, Abhijit Parolia, Lisha Wang, Mengyao Tan, Yuanyuan Qiao, Sudhanshu Shukla, Xiaoju Wang, Heng Zheng, Fengyun Su, Xuhong Cao, Arul Chinnaiyan. _University of Michigan, ann arbor, MI_.

Prostate cancer (Pca) remains the second leading cause of cancer-related deaths in men in the United States. Although advanced Pca usually responds to therapies that suppress androgen-axis signaling, resistance inevitably develops, leading to the emergence of castration-resistant prostate cancer (CRPC). Importantly, the clinical efficacy of novel therapies targeting androgen receptor (AR) signaling, such as abiraterone and enzalutamide, has confirmed that most CRPC cases demonstrate intact AR signaling. Since resistance to these therapies inevitably develops, approaches that improve the response duration and address the key pathways of resistance are warranted. Mechanisms underlying androgen deprivation therapy (ADT) resistance remain poorly defined, but may depend on epigenetic reprogramming. Our group previously showed that EZH2 is overexpressed and associates with Pca progression and poor prognosis. We substantiated this finding by screening a library of epigenetic inhibitors for their ability to render ADT-resistant CRPC cells sensitive to enzalutamide. Of these compounds, the EZH2 inhibitors facilitated a substantial increase in enzalutamide-mediated growth inhibition. Given these data, we hypothesized that EZH2-mediated epigenetic reprogramming might be crucial for the development and maintenance of ADT resistance, and targeting EZH2 might sensitize resistant CRPC cells to ADT. To test this hypothesis, we performed loss-of-function of EZH2 experiments on multiple CRPC cell lines, and determined that loss-of-function of EZH2 significantly sensitized Pca cells to ADT. Interestingly, by employing RNA-seq and ChIP-seq approaches to dissect the regulatory role of EZH2, we found that EZH2 inhibition significantly up-regulates AR signaling by changing the AR cistrome. Further experimentation confirmed that the loss-of-function of EZH2-mediated deregulation of AR signaling strengthens the AR-dependency of Pca, thus increasing the sensitivity of Pca to ADT. To co-target EZH2 and AR with greater efficiency, we employed antisense oligonucleotides (ASOs) directed against EZH2 and AR at the mRNA level. We determined that co-targeting EZH2 and AR by ASOs constitutes a novel, viable therapy for CRPC in vitro and in vivo. In conclusion, we have identified EZH2 as a critical epigenetic regulator for ADT resistance and have shown that co-targeting EZH2 and AR by ASOs to be a promising, potential strategy for preventing and treating CRPC.

#5835

Adipocyte-released agents induce resistance of breast cancer cells to lapatinib.

Aline Geneste,1 Minh Ngoc Duong,2 Aurore Cleret,1 Sabine Beaumel,1 Kamel Chettab,1 Emmanuel Delay,3 Philippe Valet,4 Lars Petter Jordheim,1 Charles Dumontet1. 1 _Cancer research center of Lyon, Lyon, France;_ 2 _Lausanne University Hospital Center (CHUV) and University of Lausanne, Epalinges, Switzerland;_ 3 _Centre Léon Bérard, Lyon, France;_ 4 _Université de Toulouse, Toulouse, France_.

Adipose tissue is often reduced to its role in fat storage and metabolic support for tumor cells. However, several studies have shown an adipocyte-mediated resistance effect to various anticancer therapies. However, the responsible agent(s), the mechanism(s) and the signaling pathway(s) involved in this resistance remain unclear. In the present study, we assessed the cytotoxic effect of lapatinib on several breast cancer cell lines in the presence or absence of adipocyte-conditioned medium. We performed different physical and chemical treatments on these conditioned media in order to separate and identify the different molecules and agents involved. In parallel, we investigated the changes that occurs in breast tumor cells following the contact with adipocyte-conditioned medium and the exposure to lapatinib both at transcriptional and protein level. In order to validate our hypothesis in vivo, we created a mouse model reproducing the contact between adipose tissue and tumors. Our results showed that tumor cells exposed to adipocyte-conditioned medium were less sensitive to lapatinib-mediated cytotoxic effects and cell cycle blockade than cells grown in standard culture medium. For example, we found that the IC50 was approximately seven-fold increased with lapatinib for tumor cells exposed to adipocyte-conditioned medium compared to the cells kept in control medium. This resistance was observed with the conditioned medium obtained both from human and murine adipocytes and confirmed on different breast tumor cell lines overexpressing HER2. Using a xenograft of normal human adipose tissue with implantation of tumor cells in contact to this adipose tissue, we also confirmed the protective effect against the antitumoral activity of lapatinib in vivo. The nature of the responsible agents of the resistance has been partly elucidated as well as the mechanisms. Indeed, it seems that the presence of soluble factors released from adipocytes such as lipolysis and metabolism products are required to limit the effect of lapatinib on tumor cells. The exposure of tumor cells to adipocyte-conditioned medium triggered changes on the lapatinib-mediated cell cycle blockade and on the expression of cell cycle regulation dependent proteins such as AKT, P27 and cyclins. In conclusion, compounds released from adipocytes reduced the lapatinib induced cytotoxicity on breast cancer cells overexpressing HER2 by interfering with the AKT signaling pathway. These results suggest possible strategies to counteract tumor cell resistance to lapatinib, by targeting adipocyte-released mediators.

#5836

Activation Induced Cytosine Deamination, AICDA, is induced after EGFR TKI exposure leading to secondary resistant mutations in lung adenocarcinoma.

Najwa El Kadi,1 April Davis,1 Alexander Cooke,1 Luo Wang,1 Hasan Korkaya,2 Gregory Kalemkerian,1 Khaled Hassan1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _Augusta University, Augusta, GA_.

Background: Epidermal growth factor receptor (EGFR) activation mutations occur in 10-50% of lung adenocarcinomas. This leads to constitutive activation of EGFR, which triggers multiple downstream survival and proliferation pathways. EGFR tyrosine kinase inhibitors (TKIs) are the mainstay of treatment for stage IV non-small cell lung cancer (NSCLC) patients with EGFR mutations. Acquired EGFR mutations are the main mechanism of on-target resistance to TKIs. T790M mutation that occurs after first line TKI treatment, is a cytosine to thymine (C>T) single nucleotide transition leading to a threonine to methionine amino acid change at position 790 (i.e. T790M). Interestingly, treatment with Osimertinib, that overcomes the T790M mutation, leads to other acquired resistant mutations, C797S, G796S/R and L792F/H. Our data suggest that resistant mutations are acquired events secondary to cytosine deamination through Activation Induced Cytosine Deamination enzyme (AICDA).

Results: Sub clones of the lung adenocarcinoma cell line PC9 with no evidence of T790M mutation by droplet digital PCR (ddPCR) at baseline, were treated with EGFR TKI. After serially increasing the treatment dose, T790M mutation was detected by ddPCR associated with a significant increase in AICDA expression. Knocking down AICDA by shRNA, decreases the development of T790M in PC9 cell lines after TKI exposure. Similarly, when the resistant T790M PC9 clones were treated with Osimertinib, the expression of AICDA was also induced. Using mass spectrometry, we established that cytosine at codon 790 is methylated; thus, deamination of 5-methylcytosine leads to thymine directly, explaining the T790M C>T mutation. In addition, using ChIP assay and pharmacological inhibition we confirm that upon TKI exposure, NFĸB binds AICDA promoter and induces its expression. In a mouse xenograft model, the induction of NFĸB and AICDA after EGFR TKI exposure is abrogated by concurrent use of an NFĸB inhibitor. Finally, patients treated with EFR TKI had an increased expression of AICDA upon progression.

Conclusion: In EGFR driven lung adenocarcinoma, NFĸB pathway is activated upon exposure to EGFR TKIs which induces AICDA expression. AICDA deaminates cytosine into other nucleotides leading to treatment resistance.

#5837

Acquired resistance to anticancer drug of high glucose-exposed HepG2.

Shuichi Kishimoto, Shoji Fukushima. _Faculty of Pharm. Sciences, Kobe Gakuin Univ., Kobe, Japan_.

Reports show that diabetic patients with cancer tend to show resistance to anticancer drug treatment and have poor prognosis compared to non-diabetic patients. We observed that HepG2-HG, which is a derivative of the hepatocellular carcinoma cell line HepG2 exposed continuously to high glucose, shows high resistance to etoposide. In this study, we aimed to understand the involvement of excretion transporter and apoptosis regulatory factor in anti-cancer drug resistance in HepG2-HG. HepG2 was cultured in minimum essential medium (MEM) (with 5.5 mM glucose), and HepG2-HG was cultured in MEM supplemented with up to 25 mM glucose. Sensitivity to anticancer drugs was evaluated by the SRB assay. mRNA expression was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) on Rotor-Gene Q using the KAPA SYBR FAST qPCR kit. Proteins extracted using the Ultra-RIPA kit were electrophoresed, transferred to a membrane by the iBlot 2 gel transfer device, reacted with antibodies, and evaluated for expression using enhanced chemiluminescence (ECL) prime reagent. IC50 values of etoposide in HepG2 and HepG2-HG were 0.59 µM and 17.5 µM, respectively. Comparison of the expression of the excretion transporter showed that the expression of p-glycoprotein (p-gp) was markedly enhanced in HepG2-HG compared to that in HepG2. Continuous contact of HepG2-HG with high glucose induced the expression of the glucose transporter GLUT1, which may provide a survival mechanism for cancer cells via escape from hypoxia-induced apoptosis. Furthermore, the expression of phospho-p70S6K was high in HepG2-HG, suggesting an increase in the activity of the mammalian target of rapamycin (mTOR). Therefore, we suggest that increased p-gp expression and mTOR activity in HepG2-HG provided resistance to etoposide by decreasing intracellular drug levels and suppressing the apoptotic mechanism. Estimation of the contribution of both mechanisms to etoposide resistance of HepG2-HG requires further examination.

#5838

Development of metabolic-targeting therapy for overcoming resistance to osimertinib in non-small cell lung cancer cells.

Masakuni Serizawa,1 Rina Umehara,1 Keiichi Ohshima,2 Kenichi Urakami,3 Takeshi Nagashima,4 Ken Yamaguchi,5 Kyoichi Kaira,6 Toshiaki Takahashi,7 Masatoshi Kusuhara8. 1 _Drug Discovery and Development Division, Shizuoka Cancer Center Research Institute, Nagaizumi-cho, Sunto-gun, Shizuoka, Japan;_ 2 _Medical Genetics Division, Shizuoka Cancer Center Research Institute, Nagaizumi-cho, Sunto-gun, Shizuoka, Japan;_ 3 _Cancer Diagnostics Division, Shizuoka Cancer Center Research Institute, Nagaizumi-cho, Sunto-gun, Shizuoka, Japan;_ 4 _SRL Inc., Shinjuku-ku, Tokyo, Japan;_ 5 _Shizuoka Cancer Center, Nagaizumi-cho, Sunto-gun, Shizuoka, Japan;_ 6 _Department of Oncology Clinical Development, Gunma University, School of medicine, Maebashi, Gunma, Japan;_ 7 _Division of Thoracic Oncology, Shizuoka Cancer Center, Nagaizumi-cho, Sunto-gun, Shizuoka, Japan;_ 8 _Region Resources Division, Shizuoka Cancer Center Research Institute, Nagaizumi-cho, Sunto-gun, Shizuoka, Japan_.

Background: The third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), osimertinib, is effective against patients with non-small-cell lung cancer (NSCLC) showing EGFR T790M-induced resistance to first generation EGFR-TKIs. Moreover, the potential of osimertinib as an effective initial therapy for treatment-naïve patients with NSCLC harboring EGFR-activating mutations has been reported. Unfortunately, cases of acquired osimertinib resistance exist, and the involvement of genetic alterations such as EGFR C797S mutation and MET amplification has been reported, although the resistance mechanisms are not fully understood. We have previously demonstrated that first-generation EGFR-TKI-resistant NSCLC cells possess altered metabolic pathways, which might be targeted for therapy to overcome this resistance. Therefore, we aimed to identify the metabolic profile relevant to osimertinib-resistance and develop a targeted therapy for the altered metabolic signature.

Methods: Osimertinib-resistant cell lines were generated by exposing HCC827 cells harboring the EGFR-activating mutation to increasing osimertinib concentrations (OSR1, OSR2). Moreover, we selected erlotinib-resistant cell lines that also showed osimertinib-resistance (EOSR1, EOSR2) from previously generated erlotinib-resistant cells obtained by prolonged exposure of HCC827 to erlotinib. Whole-exome sequencing, gene expression profiling, and metabolic profiling were performed to identify the genetic, gene expression, and metabolic signatures associated with osimertinib resistance, respectively. The growth-inhibitory effect of the inhibitors was evaluated by the WST assay.

Results: Four osimertinib-resistant cell lines showed resistance to whole-generation EGFR-TKIs, namely erlotinib, afatinib and osimertinib. EGFR T790M or C797S mutations were not detected, while MET amplification was commonly observed in these resistant cell lines. These cells also contained higher concentrations of fructose 1, 6-bisphosphate and lactic acid than HCC827, possibly because of a significant increase in hexokinase expression. Activation of the pentose phosphate pathway and subsequent nucleotide metabolism were also observed in the OSR cells, but not in the EOSR cells. A combination of a competitive glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), and osimertinib restored osimertinib sensitivity in the EOSR cells showing glycolysis dependence, but not in the OSR cells.

Conclusions: Four resistant cell lines showed the same genetic alteration, namely, MET gene amplification; however, the metabolic signature and response to the 2-DG-osimertinib combination differed between the EOSR and OSR cells. Stratification based on tumor metabolic signature may contribute to the selection of promising therapeutic strategies for overcoming osimertinib resistance.

#5839

Growth hormone receptor (GHR) expression confers resistance to ruxolitinib in endocrine-resistant breast cancer cells.

Anja N. Holtz, Douglas Yee, Heather Beckwith. _Univ. of Minnesota Masonic Cancer Ctr., Minneapolis, MN_.

More than 30% of patients with early-stage estrogen receptor-positive breast cancer (ER+) treated with adjuvant endocrine therapy will relapse within fifteen years and all patients with metastatic breast cancer expressing ER eventually acquire resistance to antiestrogen therapy. The insulin-like growth factor 1 (IGF-1) and its receptor (IGF-1R) are involved in the development of resistance to endocrine therapies such as tamoxifen. The IGF-1/growth hormone (GH) signaling axis has been implicated as a mitogenic pathway in the development and progression of breast carcinogenesis. Preclinical data demonstrate that blockade of the IGF-1R inhibits breast cancer growth, progression, and drug resistance. Unfortunately, IGF-1R targeted therapies have failed to show a benefit in prolonging either disease-free or overall survival in clinical trials. IGF-1R inhibition results in upregulation of GH due to disruption of a negative feedback pathway. We propose GH is able to stimulate mitogenic pathways independently of IGF-IR and the GHR is a potential therapeutic target in endocrine-resistant breast cancers. Tamoxifen-resistant (TamR) and long-term estrogen deprived (LTED) cells were derived from parental MCF-7L and T47D estrogen receptor-positive breast cancer cells. The endocrine-resistant cell lines express increased levels of growth hormone receptor (GHR) mRNA compared to the parental cell lines from which they were derived when measured by RT-PCR. TamR and LTED cell lines have reduced IGF-1R expression, but continue to phosphorylate insulin receptor substrate 1(IRS-1) downstream of IGF-1R. In these endocrine-resistant cells, GH treatment activates protein kinase B (Akt), phosphoinositide 3-kinase (PI3K), and mitogen activated protein kinase (MAPK) pathways in the absence of IGF-1. To determine if GHR signals through janus kinase-2 (JAK-2) in these cells, we treated MCF-7L and T47D TamR cells with 500 nM GH, which resulted in JAK-2 phosphorylation. To examine growth effects, we treated cells with the JAK-2 inhibitor ruxolitinib. Both MCF-7L and T47D endocrine-resistant cell lines treated with ruxolitinib have increased IC50 values compared to their parental counterparts. The MCF-7L parental cells had an IC50 value of 6.7 µM compared to 20 µM and 21.7 µM in TamR and LTED cells, respectively. The T47D parental cells had an IC50 value of 28.6 µM whereas the IC50 of T47D TamR cells was 45 µM. These data indicate endocrine-resistant breast cancer cell lines with elevated GHR expression have decreased sensitivity to JAK-2 inhibition. Based on these results, GHR and its downstream signaling should be considered as a target in the development of novel therapeutics to treat breast cancers that are resistant to traditional endocrine therapies.

#5840

Inhibition of cystathionine-β-synthase (CBS) sensitizes colon cancer cells to 5-FU via increasing apoptosis and inhibiting cellular bioenergetics.

Shanwen Chen, Pengyuan Wang, Yisheng Pan, Yucun Liu. _Peking University First Hospital, Beijing, China_.

Increased expression of cystathionine-β-synthase (CBS) has been validated in multiple kinds of tumor tissues, including colorectal cancer. Endogenous hydrogen sulfide (H2S), produced by CBS, was able to promote the proliferation and migration of colon cancer cells by inhibiting apoptosis and injuries caused by noxious environmental factors, including chemotherapeutic agents. Recently, overexpression of CBS has been reported to play a pivotal role in tumorigenesis of colon cancer cells, rendering the normal colon epithelial cell line acquiring the features of malignant cells. However, the potential role of CBS in the development of chemoresistance of colon cancer cells has not be illustrated. In the present study, overexpression of CBS in tumor tissues was further validated by realtime PCR and the effect of inhibition of both the activity and expression of CBS on the sensitivity to 5-fluorouracil (5-FU) was investigated in colon cancer cell lines. Inhibition of the activity of CBS by CBS inhibitor aminooxyacetate (AOAA) significantly increased the sensitivity of colon cancer cell lines to 5-FU, featured by decreased IC50 and colony forming efficiency. However, utilizing a slowly releasing H2S donor GYY4137, the present study suggested that exogenous hydrogen sulfide was able to increase the resistance to 5-FU in both colon cancer cell lines. What's intriguing was that, 5-FU, at clinically relevant concentrations, significantly inhibited the expression of CBS. Co-treatment of AOAA significantly increased the expression of Bax and decreased the expression of Bcl-2, indicating the involvement of increased apoptosis. AOAA also predisposed the colon cancer cells to the inhibition of cellular energetics induced by 5-FU, featured by the further reduced level of cellular ATP. These results suggested that inhibition of CBS promoted the sensitivity of colon cancer cells to 5-FU via increasing apoptosis and inhibiting cellular bioenergetics. Furthermore, inhibition of CBS expression by shRNA sensitizes the 5-FU resistant colon cancer cell line HCT-8/5-FU to treatment with 5-FU. The increased expression of CBS and the consequently increased level of endogenous H2S might be a potential therapeutic target for chemoresistance in colon cancer.

#5841

Ceramide synthase-6 binding to CD95/Fas confers resistance to chemotherapy in T-cell acute lymphoblastic leukemia cells.

Dattesh U. Verlekar, Hwangeui Cho, Sung-Jen Wei, Min H. Kang. _Texas Tech University Health Sciences Center, Lubbock, TX_.

Ceramide synthase (CERS) produces ceramides, key intermediators in the biosynthesis of complex sphingolipids and play an important role in cell growth, differentiation, apoptosis, and senescence. Six isoforms of ceramide synthases (CERS1-6) with varying substrate specificity generate ceramides of diverse chain lengths. In normal tissues, CERS2, synthesizing ceramides with C24 acyl chain (C24-Cer), is highly expressed in most of the tissues while CerS6 generates C16-Cer with low and tissue-specific distribution. Our data showed that CERS6 and C16-Cer levels were significantly higher in acute lymphoblastic leukemia (ALL) cells in comparison to peripheral blood mononuclear cells and T-Lymphocytes derived from healthy human volunteers. The purpose of this study was to investigate the role of CERS6 in chemo resistance in T-ALL models. Stable knockdown/overexpression of CERS6 was achieved by lentiviral vector transduction. Ceramide levels were measured by LC/MS. Cytotoxicity of conventional and investigational anti-leukemia agents was evaluated in T- ALL cells with altered CERS6 levels using DIMSCAN assay. CERS6 knockdown significantly decreased C16-Cer by 4-fold (p<0.01) while CERS6 overexpression increased C16-Cer by 2-fold (p<0.05). CERS6 knockdown in CCRF-CEM cells increased their sensitivity to a pan BCL-2 inhibitor, ABT-737 as well as a glucocorticoid, dexamethasone (DXM). The percent survival of CCRF-CEM cells with CERS6 knockdown treated with ABT-737 (100nM) or DXM (100nM) was 5% and 2% at 72h while cells transduced with non-targeting shRNA showed survival of 87% and 33% (p<0.001). In CCRF-CEM cells with CERS6 overexpression, the percent survival, when treated with ABT-737 (300nM) or DXM (300nM) for 72h, was significantly higher relative to the cells transduced with vector control: 39% vs 2% for ABT-737 and 33% vs 0.4% for DXM, p<0.01. Higher cleavage of PARP, Caspase 3 and Caspase 8 was seen in ABT-737 or DXM treated cells with CERS6 knockdown, which was reversed by the overexpression of CERS6. Sensitivity to ABT-737 in CERS6 knockdown cells was significantly reduced by a Caspase 8 inhibitor, suggesting that CERS6 alters ALL cell sensitivity to chemotherapy via extrinsic pathway of apoptosis. Pull down of CERS6 in CCRF-CEM cells identified CD95/Fas, a death receptor leading to activation of extrinsic apoptotic pathway, as a CERS6 binding partner. In CERS6 knockdown cells, a higher level of FADD was detected in Fas pull-down samples when treated with ABT-737 compared with cells transduced with non-targeting shRNA, indicating CERS6 interferes Fas/FADD assembly in inducing apoptosis. Taken together, CERS6 modulation affected the sensitivity of T-ALL cells to chemotherapy by interfering with Fas/FADD assembly in extrinsic apoptotic pathway. CERS6 may serve as a biomarker in determining the efficacy of anticancer drugs acting via extrinsic apoptotic pathway in T-ALL.

#5842

Oxidative stress and protein carbonylation towards multi-drug resistance in cancer.

Baikuntha P. Aryal,1 Jean-Pierre Gillet,2 V Ashutosh Rao1. 1 _FDA, Silver Spring, MD;_ 2 _University of Namur, Namur, Belgium_.

Reactive oxygen species (ROS) are constantly produced under normal as well as stress conditions and play important roles in cell signaling and homeostasis. An elevated level of ROS has been reported in most cancers which may play a critical role in both cancer progression or cancer suppression. One proposed therapeutic strategy is to alter the oxidative stress from ROS-induced tumor promoting event to a ROS-induced apoptotic signaling. Most ROS inducing therapeutic agents initially induce ROS to kill cancer cells by apoptosis; however, prolonged exposure of drugs may result in reduced ROS level and an adaptive microenvironment that makes cancer cells resistant to the same ROS-inducing therapeutic drugs. Chronic exposure of ROS may also upregulate the multidrug resistance protein expression in cancer cells. We hypothesize that differences in oxidative stress and the antioxidant response between drug sensitive and drug resistance cells may play a key role in drug resistance to redox-active drugs. We used two pairs of cell lines, a mitoxantrone sensitive and resistance MCF7 human breast adenocarcinoma (MCF7/MCF7MX) and H460 non-small lung cancer cell lines (H460/H460MX) for our experiments. We employed western blot and ELISA assays to determine the total and specific protein oxidation by carbonylation. Both MCF7MX and H460MX showed slightly lower level of total protein carbonylation compared to their mitoxantrone sensitive counterparts. In western blot analysis, we observed two specific proteins that were differentially carbonylated in MCF7MX compared to and MCF7. Both cytoskeletal and multifunctional proteins were identified by mass spectrometry and confirmed by two-color western blot analysis. Investigation of the potential roles of these specific protein carbonylation in drug resistance will be presented.

#5843

The efficacy of a histone deacetylase inhibitor in combination with a MEK inhibitor in lung cancer cells harboring RAS mutations.

Tadaaki Yamada,1 Joseph M. Amann,2 Azusa Tanimoto,3 Hirokazu Taniguchi,3 Takehito Shukuya,2 Seiji Yano,3 Koichi Takayama,1 David P. Carbone2. 1 _Kyoto Prefectural University of Medicine, Kyoto, Japan;_ 2 _Ohio State University Comprehensive Cancer Center, OH;_ 3 _Cancer Research Institute, Kanazawa University, Japan_.

Purpose: Non-small-cell lung cancer (NSCLC) can be identified by precise molecular subsets based on genomic alterations that drive tumorigenesis and include mutations in EGFR, KRAS, and various ALK fusions. However, despite effective treatment for EGFR and ALK, promising therapeutics have not been developed for patients with KRAS mutations. Therefore, novel therapeutic strategies for KRAS mutated cancer based on molecular mechanisms are needed to improve their prognosis. It has been reported that one way the RAS-ERK pathway contributes to tumorigenesis is by affecting stability and localization of FOXO3a protein, an important regulator of cell death and the cell cycle.

Experimental Design: We used NSCLC cells with RAS mutation to evaluate the effect of a MEK inhibitor in combination with a HDAC inhibitor through the expression and localization of FOXO proteins in vitro and in vivo. Protein expression was examined by Western blotting.

Results: Combined treatment with a MEK inhibitor and a HDAC inhibitor showed synergistic effects on cell viability of RAS mutated lung cancer cells through activation of FOXOs, with a subsequent increase in BIM and cell cycle inhibitors. Moreover, in a mouse xenograft model, the combination of belinostat and trametinib significantly decreases tumor formation through FOXOs by increasing BIM and increase in cell cycle inhibitors p21 and p27.

Conclusions: These findings demonstrate that FOXOs might be one of the critical pathways in RAS driven lung cancer cells, suggesting that the dual molecular targeted therapy for MEK and HDACs may be promising as novel therapeutic strategy in NSCLC with specific populations of RAS mutations.

#5844

Targeting DNA repair in Philadelphia positive B-ALL.

Danielle Johnson,1 Shwetha Tharkar,1 Simon Jones,2 Matt Jarpe,2 Mahesh Chandrasekharan,1 Srividya Bhaskara1. 1 _Univ. of Utah Huntsman Cancer Inst., Salt Lake City, UT;_ 2 _Regenacy Pharmaceuticals, Salt Lake City, UT_.

Introduction: The Philadelphia (Ph) chromosome was the first reported chromosomal rearrangement linked to a human malignancy. Ph-positive precursor B-cell acute lymphoblastic leukemic (Ph+Pre-B-ALL) expressing the BCR-ABL1 oncoprotein is a major subclass of ALL with poor prognosis and high relapse rates in adults. BCR-ABL1 drives an aberrantly hyperactive DNA repair that confers survival advantages to leukemic cells and serves as its 'Achilles' heel'. We used HDAC1,2 selective inhibitor in isolation or in combination with doxorubicin (a chemotherapy regimen component used for treating pre-B-ALL) to combat the aberrant DSB repair processes.

Experimental design: We used a combination of molecular and cell biology, proteomics and functional genomics-based approaches and primary patient-derived xenograft mouse models to understand the genome stability mechanisms targeted by HDAC1,2 inhibition to overcome oncogenic signals promoted by the BCR-ABL1 fusion protein.

Results: Mass-spectrometry analysis and DNA repair assays showed that HDAC1,2 inhibition and doxorubicin treatments impact common as well as distinct DNA repair and genome maintenance networks including decreased chromatin association of Mre11-Rad51-DNA ligase 1, the central hub of DNA repair. Translating this mechanistic knowledge, we then showed that the HDAC1,2 inhibitor either alone or when combined with a low dose doxorubicin caused cytotoxicity in primary patient Ph+ Pre-B-ALL cells, and decreased leukemia burden without adverse side-effects in vivo in refractory Ph+ Pre-B-ALL PDX models that we created. We recently found that HDAC1,2 targets H4K91ac mark in Ph+Pre-B-ALL cells. H4K91ac occurs within the body of nucleosome core and thus directly controls nucleosome or chromatin integrity. Increased H4K91ac following HDAC1,2 inhibition occurs around DSB sites genome-wide could cause a delay in the rate at which DSBs are repaired in Ph+Pre-B-ALL cells as a result of defective nucleosome structure around DSB sites.

Conclusion: Our comprehensive study using a multipronged approach showed that HDAC1,2 inhibition as a monotherapy or in combination with a low-dose doxorubicin can override DSB repair 'addiction' in Ph+ Pre-B-ALL. We are currently using the genome-wide ChIP-seq approach and an homing endonuclease DSB reporter system to examine how HDAC1,2 inhibition changes the chromatin landscape in Ph+Pre-B-ALL cells.

#5845

Roles for MLL2/ KMT2D or MLL3/ KMT2C in HER+ breast cancer stem cells.

Andrei Zlobin, Debra Wyatt, Mary Varsanik, Andrew Dingwall, Clodia Osipo.. _Loyola University Medical Center, Chicago, Maywood, IL_.

A major clinical challenge for the treatment of breast cancer (BC) is drug resistance. Cancer stem cells (CSCs) may contribute to drug resistance despite therapy. The HER2-positive subtype of BC contains a gene amplification for the ERBB2 proto-oncogene and these tumors are treated with anti-HER2 therapy. Anti-HER2 therapy, such as trastuzumab or lapatinib, increases Notch signaling and is required for HER2+ breast tumor resistance and tumor recurrence. Notch promotes survival of BC CSCs cells. MLL2/KMT2D (Lysine Methyltransferase 2D) and MLL3/KMT2C (Lysine Methyltransferase 2C) are histone methyltransferases that directly control gene enhancer activity through methylation of histone3 lysine4 within enhancer nucleosomes. These complexes collaborate with transcription factors, such as Notch, to drive gene expression by helping to shape the epigenetic landscape of a cell. Recently, it was shown that lapatinib or a PI3K inhibitor increases MLL2 activity and this increase in MLL2 contributes to resistance. Both MLL2 and MLL3 were found to be required for CSCs differentiation, while MLL2 was required for upregulation of c-Myc in HER2+ cells. Recent breast cancer xeno-engraftment studies found that clonal selection and expansion of primary and metastatic breast tumors was associated with amplification of MLL3 in 80% of breast cancer xenografts, suggesting that increased MLL3 expression may contribute to the survival of CSCs in breast tumors. High expression of MLL2 and/or MLL3 predict poor outcome for women with HER2+ breast cancer from cBio portal and Kaplan-Meier Plotter datasets. We measured the expression levels of MLL2 and MLL3 transcripts in both bulk HER2+ breast cancer cells that are trastuzumab sensitive, cells that have acquired resistance to trastuzumab, and cancer stem-enriched mammospheres. Results from PCR analysis showed that MLL2 and MLL3 transcripts were increased in mammospheres compared to bulk cells. Mammosphere forming efficiency of trastuzumab sensitive cells was decreased when MLL2 or MLL3 was knocked down using RNAi. Mammosphere forming efficiency of anti-HER2 therapy resistant (BT474 TR) cells was significantly decreased upon MLL3 knockdown, while HCC1954 mammospheres were inhibited by either MLL2 or MLL3 knockdown. We next measured transcript levels of Notch genes and other stem, mesenchymal, and luminal genes to determine whether MLL2 or MLL3 is necessary for the proper expression of cell fate genes. We found that Notch3 transcripts were decreased in both BT474 and HCC1954 cells upon MLL2 knockdown. SNAIL and Notch1 transcripts were also decreased, suggesting that MLL2 is required for the expression of genes involved in regulating cancer stem cell fates. These results suggest that MLL2/KMT2D and its paralog MLL3/KMT2C are increased in CSCs-derived from HER2+ breast cancer and may regulate CSC genes such Notch1 and Notch3 to promote resistance to anti-HER2 based therapy.

#5846

Drug resistance in chronic myeloid leukemia: Impact of methylation on gene expression in imatinib and nilotinib resistance.

Meike Kaehler,1 Inga Nagel,1 Henrike Bruckmueller,1 Ruwen Boehm,1 Ole Ammerpohl,2 Ingolf Cascorbi1. 1 _University of Kiel, Kiel, Germany;_ 2 _University of Ulm, Ulm, Germany_.

Background: Chronic myeloid leukemia (CML) is one of the most extensively studied hematopoietic disorders. Caused by reciprocal translocation t(9;22) and formation of the Philadelphia chromosome, it leads to constitutive expression of the BCR-ABL kinase and malignant transformation. Targeted therapy using tyrosine kinase inhibitors (TKI), such as imatinib or nilotinib, led to tremendous success in treatment. However, resistances are an uprising problem in patient cure. Besides BCR-ABL-dependent mechanisms (i.e. BCR-ABL amplification, mutations), cellular alterations of gene expression and its regulation are involved. Therefore, we performed an in vitro-study in K-562 CML cells to investigate changes of gene expression and epigenetics, as well as occurrence of mutations during the course of imatinib or nilotinib resistance.

Methods: We analyzed mRNA expression, DNA-methylation and mutation status of treatment-naïve and TKI-resistant sublines (0.5 µM, 2 µM imatinib; 100 nM nilotinib) of the in vitro-cell line model (K-562). mRNA expression was analyzed using Affymetrix Human Gene 2.0 ST arrays and DNA-methylation by Infinium MethylationEPIC BeadChips (Illumina); exome sequencing was performed at GATC, Konstanz, Germany. Target verification was performed using qRT-PCR, pyrosequencing or Sanger sequencing. Effects on TKI-susceptibility were analyzed using transfection experiments with analyses of cell viability (WST-1 assay) and apoptosis (Caspase Glo 9 assay).

Results: Cell resistant to low imatinib doses exhibited differentially expression of 485 mRNAs compared to treatment-naïve cells, whereas at high dose-resistance 166 mRNAs differed. 151 genes showed expression changes independent from the imatinib dose. In addition, we found 45 genes with expression changes during the course of imatinib resistance, i.e. the drug efflux transporter ABCG2, which was significantly upregulated at low and downregulated at high imatinib doses. A total of 254 genes was associated with nilotinib resistance, while 71 genes were differentially expressed in all resistant sublines. Regarding methylation and occurrence of mutations in imatinib resistance, we observed distinct variations pointing to genetic changes, especially at high-dose resistance, i.e. in the promoter region of microRNA-212, a proven regulator of ABCG2. In nilotinib resistance, we found 125 genes differentially methylated during development of resistance.

Conclusions: Our data indicate that development of resistance to tyrosine kinase inhibitors is based on complex expression profile changes that can in part be explained by epigenetic alteration e.g. in drug efflux transporters. Besides, the mechanisms underlying resistances to imatinib or nilotinib seem to be dissimilar. After verification in clinical studies, these in vitro findings could be relevant in attempts to overcome TKI-resistances.

#5847

A histone deacetylase inhibitor OBP-801 and eribulin synergistically inhibit the growth with apoptosis in triple-negative breast cancer cells.

Hisako Ono, Yoshihiro Sowa, Yosuke Iizumi, Mano Horinaka, Tetsuya Taguchi, Toshiyuki Sakai. _Kyoto Prefectural University of Medicine, Kyoto, Japan_.

Purpose: Triple negative breast cancer (TNBC) is the most aggressive subtype in breast cancer. Eribulin is one of the well-tolerated chemotherapeutic drugs for the treatment of metastatic breast cancer. We usually administer eribulin or eribulin with anti-HER2 antibodies. Eribulin was approved for metastatic breast cancer refractory to anthracyclins and taxanes through EMBRACE trial, and the subgroup analysis indicated the efficacy of eribulin was effective in patients with TNBC. However, the prognosis of patients with TNBC is still poor because of the various molecular characteristics. Therefore, there is an urgent need for more effective treatment for the management of TNBC.

Methods: We investigated the effect of eribulin in several TNBC cell lines. Eribulin inhibited the cell growth of TNBC cells in a dose-dependent manner. However, eribulin increased the expression of survivin, known to inhibit apoptosis. To counteract the up-regulation of survivin by eribulin, we used OBP-801, a novel histone deacetylase (HDAC) inhibitor, because HDAC inhibitors were reported to suppress the expression of survivin.

Results: Co-administration of OBP-801 with eribulin showed a synergistic inhibition of cell growth in TNBC cells. The mechanism of this combination is due to the enhancement of apoptosis. The apoptosis was partially dependent on caspase, and it was via the suppression of not only survivin as expected, but also Bcl-xL. OBP-801 could suppress the survivin expression induced by eribulin at both protein and mRNA level. Interestingly, the combination more potently suppressed a MAPK pathway than each agent.

Conclusion: We found that the combination of eribulin and OBP-801 synergistically inhibits the growth with apoptosis in TNBC cells, raising the possibility that the combination might be a novel promising strategy against TNBC patients.

#5848

Histone deacetylase inhibitor selectively targets CD123 positive chemoresistant AML cells.

Bowen Yan, Suming Huang, Yi Qiu. _Univ. of Florida, UF Health Cancer Center, Gainesville, FL_.

Acute Myeloid leukemia (AML) is the most common acute leukemia in adults. Chemoresistance remains a major burden for treatment of relapse patient. Studies suggest that AML relapse related to the survival of drug resistant leukemia stem cell (LSC) population. To better understand the mechanism of drug resistance and whether it is correlated to the expression of certain group of LSC surface markers, we collected various human AML cell lines and examined correlations of their IC50 of Ara-C and percentage of individual LSC surface markers in these cell lines. We found that only CD47 (R square = 0.6660, P value = 0.0476) and CD 123 (R square = 0.7421, P value = 0.0275) are positively correlated to chemoresistance. In order to rule out heterogeneous genetics background contribute to chemoresistance of cell lines, we generated a resistant cell line from a drug sensitive AML cell line, OCI-AML2, by gradually increased the Ara-C concentration in culture media. During the selection, the percentage of cells with CD47 and CD123 gradually increased. When the resistant cell line is established, the percent of CD123 positive cells are over 90 percent. RNA-seq analysis shows that genes related to cell cycle regulator and resistance of apoptosis changed significantly. However, signature genes that related to stemness, such as Oct4, Nanog, KLF4, Myc, FLT3, are no significant changed in resistant cells compare with the sensitive line, although Sox2 increased 50% and ALDH1A3 increased 3 folds. Therefore, chemoresistant cells may represent cells in a slow growing, de-differentiate stage, however, they may not bare all characteristics of LSCs. We further investigate the role of epigenetic factors in regulating the survival of chemoresistant leukemia cells. Our data shows epigenetic drugs that targeting histone deacetylases, bromodomain proteins and PRC1 complex are effective to induce apoptosis and cell death of chemoresistant cells, therefore they may be potential second line of chemotherapy drug for chemoresistant patients. Histone deacetylase inhibitor Romidepsin efficiently cleared chemoresistant AML blast in xenographed NSG mice. Furthermore, we found Romidepsin has synergetic effect when combine with Ara-C. Interestingly, Romedepsin preferentially target CD123 positive, slow growing cells while Ara-C mainly target fast growing, CD123 negative cells. Therefore, combine treatment with Romedepsin and Ara-C may prevent the development of chemoresistance in AML cells. In summary, we identify CD123 and CD47 cell surface markers may potentially serve as biomarker for chemresistance AML cells. Our study also sheds light on a new mechanism of drug resistance in leukemia, and provide a rationale to develop and test epigenetic-targeted therapies in leukemia, especially in drug resistant relapse patients.

#5849

Valproic acid reverses trastuzumab resistance in erbB2-positive breast cancer cells via downregulation of ErbB2/ErbB3 signaling through induction of ErbB family members-targeting miRNAs.

Tingting Lin,1 Qun Ren,1 Weimin Zuo,1 Rong Lin,1 Hu Zhao,1 Ping Wang,1 Jianming Tan,1 Hui Lyu,2 Bolin Liu,2 Shuiliang Wang1. 1 _Fujian Key Laboratory of Transplant Biology, Fuzhou General Hospital, Xiamen University, Fuzhou, China;_ 2 _Department of Pathology, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO_.

Amplification and/or overexpression of erbB2 occur in approximately 25% of invasive breast cancer and are significantly associated with a worse prognosis for breast cancer patients. Trastuzumab, a humanized monoclonal antibody against erbB2 receptor, has been successfully used in early-stage and metastatic breast cancer therapy of patients with erbB2-overexpressing tumors as monotherapy and in combination with other agents; however, both primary and acquired resistances to trastuzumab are common and currently represent a significant clinical problem. Thus, seeking of novel therapeutic strategies/agents to overcome trastuzumab resistance is vital to improve the survival of breast cancer patients whose tumors overexpress erbB2. In our current study, we explored whether valproic acid (VPA), a clinically used anticonvulsant drug with reported HDACi activity, reverses trastuzumab resistance in erbB2-positive breast cancer cells. We have found that VPA inhibits proliferation of both trastuzumab-sensitive SKBR3 and BT474 cell lines as well as their corresponding trastuzumab-resistant SKBR3-pool2 and BT474-HR20 cell lines in a dose- and time-dependent manner, which is accompanied by significant reduction of cyclin D1 as well as induction of P21. Meanwhile, VPA also induces caspase-dependent apoptosis in all aforementioned four cell lines. More interestingly, VPA not only significantly enhances the anti-proliferative effects of trastuzumab, but also potentiates trastuzumab-induced apoptosis in trastuzumab-sensitive and resistant cells. Our further mechanistic studies revealed that VPA treatment results in significant inactivation of PI3K/Akt signaling via simultaneous down-regulation of ErbB2 and ErbB3 in erbB2-positive breast cancer cancer cells, an effect which could be mainly arrtibuted to induction of ErbB family members-targeting microRNAs including miR-125a, miR-125b and miR-205 by VPA. Taken together, we presented here that VPA reverses trastuzumab resistance in erbB2-positive breast cance cells via downregulation of ErbB2/ErbB3 signaling in a miRNAs-inducing-dependent manner. Our data suggested that further investigation regarding the in vivo therapeutic effect of VAP against trastuzumab-resistant breast cancer should be of especial interest.

#5850

Nrf2-mediated oxidative stress response is altered during acquired resistance to the proteasome inhibitor, oprozomib, in multiple myeloma.

Snehal M. Gaikwad,1 Adriana Zingone,1 Aleksandra Michalowski,1 Susana Najera,1 Anaisa Quintanilla-Artega,1 Sayeh Gorjifard,1 John Simmons,1 Nick Watson,1 Ola Landgren,2 Jing Huang,1 Beverly Mock1. 1 _Centre for Cancer Research (CCR),National Cancer Institute (NCI), National Institute of Health (NIH), Bethesda, MD;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Multiple myeloma (MM) is a hematologic neoplasm characterized by malignant proliferation of plasma cells in the bone marrow. Proteasome inhibitors are widely used in treatment regimens for MM. Although initial responses to PI (e.g., bortezomib, carfilzomib) treatments have been promising, patients often develop resistance and become refractory to disease. Understanding molecular alterations in signaling cascades influenced by proteasome inhibitors and mechanisms underlying acquired resistance is needed. In this study, we have established a clinically relevant oproxomib-resistant subline (KMS28BMONYX) of the MM cell line KMS28BM. The KMS28BMONYX cell line is pan-resistant to PIs with a 10-fold increase in IC50 for oprozomib as compared to the parental line. To identify genes involved in modulating drug resistance, we analyzed gene expression profiles of both parental and resistant cell lines using the Affymetrix GeneChip Human Genome U133 Plus 2.0 array. Ingenuity Pathway Analysis of microarray data comparing the parental and resistant cells revealed an acute dependence on stress response proteins to maintain PI-resistance. Activation of nuclear factor-erythroid 2 (NF-E2)-related factor 2 (Nrf2; gene symbol NFE2L2) coupled with elevated levels of sequestosome 1/p62 (SQSTM1/p62) were prominent features of the KMS28BMONYX cell line. Altered levels of SQSTM1 correlated with resistance to oprozomib in several MM cell lines. Simultaneously, the KMS28BMONYX cell line showed increased expression of MYC and MCL1. Oprozomib treatment stabilized c-Myc expression in the KMS28BMONYX line. The Champion ChiP Transcription Factor Search Portal database DECODE predicted two c-Myc transcription factor binding sites in the SQSTM1 promoter. CHIP-seq data for MYC in MM1s cells also indicates strong binding in the promoter region of SQSTM1. Our data suggest that therapies targeting the SQSTM1/p62-Nrf2 pathway may help overcome proteasome inhibitor resistance in refractory MM patients.

#5851

Identifying novel genetic mechanism underlying ADT resistance in African American patients.

Arsheed A. Ganaie,1 Tabish Hussain,1 Marina Ferrari,1 Matteo Astone,1 Luke Hoeppner,1 Todd Schuster,1 Pari Murugan Murugan,2 Badrinath R. Konety,2 Mohammad Saleem1. 1 _Univ. of Minnesota Hormel Inst., Austin, MN;_ 2 _Univ. of Minnesota, Minneapolis, MN_.

Enzalutamide has recently been introduced in clinics to treat castration-resistant prostate cancer (CRPC) condition, however has failed to significantly improve the overall survival in patients. Similar to bicalutamide, Enzalutamide therapy is reported not to be significantly beneficial in blocking emergence of refractory CRPC in high-risk patients such as in African-American (AA) men who exhibit (i) aggressive prostate cancer (PCa), (ii) worse survival than Caucasians (CA), and (iii) poor response to ADT. Intrinsic genetic differences play an important role in race-related PCa disparities, and there is no concrete data available explaining Enzalutamide-therapy failure in AA PCa patients. In this study, we developed genetically distinct Enzalutamide-resistance cell models representing PCa in CA and AA men, and studied their characteristics in terms of proliferation, prostatosphere formation, in vivo metastasis in a transgenic zebra-fish model and responsiveness to Enzalutamide therapy in athymic mouse model. We show that Enzalutamide-therapy increases the enrichment of cancer stem (S/P) and epithelial-mesenchymal-transition (EMT) cell populations. We provide evidence that short-term and long-term (4-months) of Enzalutamide-therapy results in the induction of AR expression in resistant-Caucasian (LNCaP95-Enz) model, whereas no effect on AR-pathway is observed in resistant AA models (RC-77T/E and E006AA-hT). Because AKT pathway has been shown to drive CRPC in ADT-resistant cases, we tested efficacy of AKT-inhibitor therapy in Enzalutamide-resistant AA and CA models. Whereas Enzalutamide-resistant CA models were responsive to AKT-inhibitor therapy, AA models exhibited non-responsiveness. To understand the exact molecular mechanism of drug-resistance induced by Enzalutamide, we hypothesized that this phenomenon could be associated to the aberration of molecules (at genetic and epigenetic levels). We performed RNA-seq analysis to study global gene expression, mircoRNA and LncRNA expression profile in Enzalutamide-responsive and Enzalutamide-resistant AA PCa models. We show that non-AR mediated genetic and epigenetic mechanisms play significant role in drug-resistance in AA PCa. We established an AA-specific molecular signature of 2 genes; 3 micro-RNA's and 1 LncRNA. The data was validated in cell models and tumor specimens (primary and METS) of CA and AA patients. We identified BAMBi as a lead biomarker that differentiates responsive-PCa e from Enzalutamide-resistant PCa in AA men, and conducted functional studies to validate its significance as a therapeutic target. To summarize, our study provides detailed information about the changes in genetic and epigenetic landscape which occur during the PCa progression and form the basis of novel race-specific biomarkers of Enzalutamide-resistance and therapeutic targets in AA PCa patients.

#5852

FGF2 confers resistance to novel BET inhibitors in metastatic uveal melanoma.

Vivian Chua,1 Timothy Purwin,1 Connie Liao,1 Andrew Aplin2. 1 _Thomas Jefferson University, Philadelphia, PA;_ 2 _Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA_.

Uveal melanoma (UM) is the most common intraocular malignancy in adults and frequently metastasizes to the liver. Metastatic UM responds poorly to chemotherapy, immunotherapy and MAPK pathway inhibitors; hence, there is an urgent unmet need for new treatment strategies. UM has a low mutational burden and progression is associated with abberrant transcriptional programs. Inhibition of epigenetic modulators involved in transcriptional regulation such as the bromodomain and extraterminal domain (BET) proteins, may be feasible for the treatment of UM. Pre-clinically, JQ1, a first generation BET inhibitor, has been shown to suppress UM growth. Additionally, a multi-site clinical trial is ongoing testing a novel BET inhibitor, PLX51107 (Plexxikon Inc) in metastatic UM patients. Since growth factors in the tumor microenvironment potentially contribute to intrinsic resistance and poor responses to therapies, we determined effects of growth factors on the efficacy of BET inhibitors in metastatic UM cells. We show that FGF2 provides resistance of UM cells to the growth inhibitory effects of BET inhibitors, JQ1, PLX51107 and the third generation inhibitor, PLX72853. FGF2 rescued BET inhibitor-induced apoptosis and cell cycle arrest, which was associated with reversal of the decreased expression of cell cycle regulators (cyclin D1, phospho-RB) and increased expression of pro-apoptotic markers (cleaved PARP, Bim). Importantly, FGF receptor (FGFR) inhibitors reversed FGF2-induced BET inhibitor resistance. As UM predominantly metastasizes to the liver, we investigated FGF2 production by hepatic stellate cells. We show that hepatic stellate cells secrete FGF2 and stellate cell conditioned medium protects metastatic UM cells against the growth inhibitory effects of BET inhibitors. Interestingly, BET inhibitor treatment of UM cells elevated FGFR protein expression and this finding is also shown in UM cell xenograft tumors and in patient tumor samples following PLX51107 treatment. These results indicate that the FGF2/FGFR pathway is activated to promote resistance to BET inhibition via the secretion of FGF2 in the tumor stroma and adaptive upregulation of FGFR expression in UM. In conclusion, our studies suggest that co-targeting of FGFR signaling and BET proteins may be required to maximize the responses of metastatic UM to BET inhibitors.

#5853

**Targeting NAD** + **/PARP DNA repair pathway as a novel therapeutic approach to** SDHB **-mutated cluster I pheochromocytoma and paraganglioma.**

Ying Pang,1 Yanxin Lu,1 Veronika Caisova,1 Yang Liu,1 Petra Bullova,1 Thanh-Truc Huynh,1 Zdenek Frysak,2 Igor Hartmann,2 David Taïeb,3 Karel Pacak,1 Chunzhang Yang1. 1 _NIH, Bethesda, MD;_ 2 _Palacky University, Olomouc, Czech Republic;_ 3 _Aix-Marseille University, Marseille, France_.

Background: Cluster I pheochromocytomas and paragangliomas (PCPGs) tend to develop malignant transformation, tumor recurrence, and multiplicity. Transcriptomic profiling suggests that cluster I PCPGs and other related tumors exhibit distinctive changes in the tricarboxylic acid (TCA) cycle, the hypoxia signaling pathway, mitochondrial electron transport chain, and methylation status, suggesting that therapeutic regimen might be optimized by targeting these signature molecular pathways.

Experimental design: We investigated molecular signatures in clinical specimens from cluster I and II PCPGs, with a focus on the therapeutic resistance mechanisms. Further, we tested the applicability of a combination therapy including an FDA approved PARP inhibitor olaparib (Ola) and temozolomide (TMZ) in both in vitro cellular model and an in vivo allograft animal model.

Results: Our findings showed that cluster I PCPGs develop a distinctive dependency on mitochondrial complex I, evidenced by the upregulation of complex I components and enhanced NADH dehydrogenation. Mechanistically, alteration in mitochondrial function resulted in strengthened NAD\+ metabolism, which provides essential cofactor for PARP catalytic activity, prompting base excision repair and chemo resistance. Combining PARP inhibitor Ola with TMZ not only improved cytotoxicity but also reduced metastatic lesions, with prolonged overall survival in a mouse model with PCPG allograft.

Conclusions: Our results suggest that the NAD+/PARP pathway is a crucial targetable therapeutic resistance mechanism in SDHB-mutated PCPG. Combination therapy using TMZ and Ola could become an effective strategy against these and other advanced cluster I tumors.

Disclosure Statement: The authors have nothing to disclose. 

### Novel Targets and Therapeutics

#5854

BT5528, a Bicycle Toxin Conjugate targeting EphA2 has potent anti-tumor activity without bleeding or coagulation abnormalities in preclinical models.

Gavin Bennett,1 Philip Huxley,1 Amy Brown,1 Gemma Mudd,1 Peter U. Park,2 Nicholas Keen2. 1 _Bicycle Therapeutics Ltd, Cambridge, United Kingdom;_ 2 _Bicycle Therapeutics Ltd, Cambridge, MA_.

Ephrin receptor A2 (EphA2) is a member of the Ephrin receptor family of cell-cell junction proteins and is both highly overexpressed in several solid tumors and associated with poor prognosis in patients. EphA2 is considered a high-value target for cancer therapeutics, but development of an antibody drug conjugate targeting EphA2 (MEDI-547) was stopped in early clinical development after severe adverse events, including bleeding toxicity were seen (Annunziata et al, 2013). We have developed a series of Bicycle Toxin Conjugates comprising a constrained bicyclic peptide (Bicycle®) that binds with high affinity and specificity to EphA2, which is covalently linked to a toxin payload via a cleavable linker. The small size of BTCs offers a significant advantage over other targeted cytotoxic approaches such as antibody-drug conjugates due to rapid extravasation, renal clearance and improved tumor penetration. BT5528 was selected from a panel of >75 Bicycle Toxin Conjugates (BTCs) with variations in Bicycle binder, molecular spacer, cleavable linker and toxin payload, based on in vivo efficacy and tolerability. BT5528 is effective in EphA2-expressing xenograft models, with efficacy seen from 1mg/kg qw and complete tumor regression at 3mg/kg qw. Rapid, complete regression of tumors is seen across a range of EphA2-expressing cell lines, including HT1080 fibrosarcoma line, MDA-MB-231 triple negative breast cancer line and NCI-H1975 lung cancer line and NSCLC PDX models. In order to test the benefit of the enhanced tumor penetration of BTCs, NSCLC PDX tumors were grown to >1000mm3 before commencing treatment. Complete regression of tumor was seen following 3 weeks of dosing. The bleeding events reported in early clinical trials of MEDI-547 (EphA2 ADC) were consistent with effects on the coagulation system in preclinical models. Dose-limiting toxicology in non-human primate (NHP) was reportedly Disseminated Intravascular Coagulation (DIC) and changes were seen in APTT and D-Dimer, with associated elevations in liver enzymes. Toxicology studies of BT5528 in rat and NHP showed no laboratory or clinical evidence of DIC, with normal levels of platelets, D-Dimer, APTT, and other coagulation-related parameters. Bicycle Toxin Conjugates (BTC) targeting EphA2 show potent antitumor activity in a range of solid tumor xenograft models. The BTCs show profound efficacy, without the limiting toxicity observed with previous Antibody Drug Conjugate approaches. IND-enabling studies for BT5528 are currently underway.

#5855

Bicycle Drug Conjugates targeting EphA2 for the treatment of solid tumors: Discovery and selection of BT5528.

Gavin Bennett,1 Philip Huxley,1 Amy Brown,1 Gemma Mudd,1 Katerine van Rietschoten,1 Silvia Pavan,1 Liuhong Chen,1 Sophie M. Watcham,1 Peter U. Park,2 Nicholas Keen2. 1 _Bicycle Therapeutics Ltd, Cambridge, United Kingdom;_ 2 _Bicycle Therapeutics Ltd, Cambridge, MA_.

Ephrin receptor A2 (EphA2) is a member of the Ephrin receptor family of cell-cell junction proteins and is both highly overexpressed in several solid tumors and associated with poor prognosis in patients. Bicycles® are novel therapeutic agents: bicyclic peptides constrained via a chemical scaffold, which confer structural stability leading to high affinity and selectivity usually associated with antibodies. Bicycles can be elaborated to carry payloads to a specific target and their relatively small size (1.5-3 kDa) allows rapid tissue penetration and extravasation. Bicycle binders for EphA2 were identified using a proprietary phage display peptide technology consisting of highly diverse phage libraries of Bicycles. Initial screening identified Bicycle binders with high affinity for EphA2. EphA2 binding Bicycles were conjugated to cytotoxic payload via a cleavable linker to form a Bicycle Toxin Conjugate (BTC) and evaluated for EphA2 dependent cytotoxicity in vitro and in vivo. Efficacy was seen in EphA2-expressing xenograft models, but toxicity was seen at higher doses. These Bicycles were highly plasma protein bound, and relatively lipophilic. Visualization of Bicycle distribution by microPET showed localization to tumor, but with significant distribution to liver. Subsequent screening with a wider range of phage libraries and scaffolds identified Bicycle binders with alternate structures, lower PPB and lipophilicity. Crystal structures of these Bicycles bound to EphA2 enabled rational design to enhance potency and drug-like properties. Visualization of bio-distribution of these optimised Bicycles by microPET showed localization to tumor, with negligible localization to liver and renal clearance. A range of Bicycle Toxin Conjugates were produced by linking bicycle binders to cleavable linkers and toxin payloads. A series of BTCs were developed with DM1 payload, with linkers ranging from uncleavable to unhindered disulfide. Similarly, a series of BTCs were developed with MMAE payload, using uncleavable linker and a variety of cleavable peptidic linkers with different enzyme sensitivity. Binding affinity is maintained to EphA2 protein and cells expressing EphA2. BTC efficacy was evaluated in EphA2-expressing xenograft models, initially the HT-1080 fibrosarcoma model, but also MDA-MB-231 triple-negative breast cancer and NCI-H1975 lung cancer models. Efficacy was seen from 1mg/kg qw and complete regression of tumors from 2 weeks dosing at 2mg/kg qw. No efficacy was seen in xenograft models without EphA2 expression (eg MOLP-8).From >75 BTCs evaluated, BT5528 was chosen as a candidate molecule to progress to in-depth preclinical profiling. The development of Bicycle Toxin Conjugates against EphA2 allows fast and efficient targeting of solid tumors while sparing non-tumor tissues.

#5856

Identification and validation of Nrf2 inhibitors in esophageal squamous cell carcinoma.

Seth Weir,1 Xiaoxin Chen,2 Chorlada Paiboonrungruan2. 1 _University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 2 _North Carolina Central University, Chapel Hill, NC_.

Background: The Nrf2/Keap1 pathway is critical for human cells to respond to oxidative stress. In basal conditions the Keap1 homodimer is bound to Nrf2 in the cytoplasm. When stressed, Keap1 releases Nrf2 which translocates into the nucleus, binds the antioxidant response element, and activate antioxidative genes. Somatic mutations of Nrf2 or Keap1 gene can cause constitutive activation of this pathway, and thus results in chemoradioresistance and poor survival in patients with esophageal squamous cell carcinoma (ESCC). Our goal is to identify chemical Nrf2 inhibitors for targeted therapy of Nrf2high ESCC in the future.

Methods: Possible Nrf2 inhibitors were identified using high throughput screening of 1,280 compounds in the Prestwick library, and 35,000 compounds in the Asinex library with Nqo1-EGFP H1299 cells stimulated by an Nrf2 activator (CDDO). Nrf2 activity was monitored via Incucyte by measuring the total normalized green fluorescence divided by the total normalized red fluorescence (tagged nuclear cherry marker). The red channel assessed cell viability and cytotoxicity of compounds. Inhibitors were identified if they met a threshold of >50% inhibition. Lead compounds will be further validated in vitro with KYSE 450 (Nrf2low+Keap1 knockdown) and KYSE 70 (Nrf2high) by examining their effects on expression of nuclear Nrf2 and its downstream target gene (Nqo1). These compounds will also be validated in vivo using Sox2CreER;Nrf2Ki/Ki mice.

Results: We successfully established the in vitro assay with Nqo1-EGFP H1299 cells for high throughput screening of Nrf2 inhibitors. Eight lead compounds were identified from the Prestwick library and validated as

Nrf2 inhibitors via a dose-response experiment (IC50<1μM). Screening of the Asinex library and further validations are still ongoing.

Conclusion: We have identified Nrf2 inhibitors through high-throughput screening and will test them in vitro and finally in vivo.

#5857

Isorhapontigenin inhibits triple-negative breast cancer via activating NRF2-mediated pathway.

Yusha Zhu, Anthony Murphy, Thomas Kluz, Bo Li, Max Costa, Chuanshu Huang, Hong Sun. _NYU School of Medicine, New York, NY_.

Breast cancer is the most prevalent cancer among women worldwide, and is the second leading cause of cancer mortality among women. Triple-negative breast cancers (TNBCs; ER-/PR-/HER2-) are highly aggressive tumors with poor prognosis. Due to lack of these specific receptors, TNBCs cannot be treated with hormone therapies or anti-HER2 targeted therapies. Chemotherapy, combined with surgery and radiation therapy, is the primary strategy for treating TNBCs. Thus, discovery and evaluation of new alternative medications targeting triple-negative breast cancer is of tremendous importance for reducing breast cancer mortality. Isorhapontigenin (ISO), a new derivative of stilbene isolated from Chinese herb Gnetum Cleistostachyun, exhibits a strong inhibitory effect on human bladder cancers. However, its anticancer activity with respect to breast cancer remains unclear. In the present study, we investigated the potential anticancer effect of ISO on TNBCs. ISO treatment inhibited the proliferation and anchorage-independent growth of 4T1 cells, a murine triple-negative breast cancer cell line, in a dose-dependent manner. In addition, the results from wound healing and Transwell invasion assay indicated significantly reduced cell migration and invasion in ISO-treated cells. Moreover, we performed the whole transcriptome analysis using RNA-seq to elucidate the molecules and pathways that mediate ISO's anticancer activity. A total of 1972 differentially expressed genes were identified in ISO-treated cells. IPA analysis revealed that the top canonical pathway altered by ISO is NRF2-mediated pathway. ISO-induced NRF2 activation was further confirmed by nuclear translocation of NRF2 protein and upregulation of NRF2 downstream target genes (GSTA1, GSTM1, HMOX1, and NQO1). In summary, our study is the first to demonstrate that ISO inhibits breast cancer cell growth and invasion, in part, by activating NRF2-mediated signaling pathway. Our results provide a novel mechanism for ISO-induced anticancer effect, and strongly support ISO as a promising therapeutic agent for triple-negative breast cancers.

#5858

Development of a translation elongation inhibitor for the treatment of cancer.

Nathan Gomes,1 Barb Frederick,1 Daniel Gustafson,2 Bert Pronk,3 Tin Tin Su1. 1 _University of Colorado Boulder, Boulder, CO;_ 2 _Colorado State University, Fort Collins, CO;_ 3 _SuviCa Inc, CO_.

Translation is essential for cellular maintenance and growth, as well as survival in response to a variety of stresses. Translation factors are often overexpressed in cancers, and their experimental inhibition has been shown to retard cancer cell growth in a variety of pre-clinical models. Further, clinically relevant forms of cellular stress (e.g. ionizing radiation (IR), hypoxia) result in altered translational programs, and inhibition of translation following such stress results in impaired cellular recovery. Cumulatively these observations suggest that modulation of translation, alone or in combination with genotoxic stress, is of potential therapeutic relevance in oncology. Bouvardin (BVD) is a plant-derived protein synthesis inhibitor that we identified in a screen for enhancers of ionizing radiation (IR). We previously demonstrated that BVD functions as a translation elongation inhibitor by blocking dissociation of elongation factor 2 (eEF2) from the ribosome. In preclinical models of head and neck cancer (HNC) and glioblastoma two cancer indications for which IR is a standard of care, we demonstrated that BVD enhanced clonogenic death induced by IR. Further, BVD enhanced the growth inhibitory effects of IR in HNC tumor xenografts. We show here that a fully synthetic derivative of BVD, SVC112, exhibits strong therapeutic properties alone, and in combination with standards of care, in a variety of solid and hematological cancer models. As an example for solid tumors: in HNC cell lines SVC112 exhibits antiproliferative activity with an average IC50 of ≈150 nM, with no IC50 exceeding 500 nM. In clonogenic assays, physiologically relevant levels of SVC112 robustly synergistically enhance clonogenic death induced by HNC standards of care (e.g. IR and cisplatin). Finally, in cell line based HNC xenografts, SVC112 exhibits antitumorigenic activity as a single agent and enhances the growth inhibitory effects of IR-cisplatin treatment. As an example for hematological cancers: in acute myeloid leukemia (AML) cell lines SVC112 exhibits antiproliferative activity in sensitive cells with an average IC50 of ≈25 nM. Brief exposure to physiologically relevant levels of SVC112 induces rapid and robust apoptosis in sensitive AML cell lines concomitant with the loss of known drivers of AML growth (e.g. MCL-1). Finally, in cell line based AML xenograft models, SVC112 as a single agent induces robust and sustained antiproliferative activity. Cumulatively these data demonstrate that SVC112 mediated inhibition of translation elongation alone, and in combination with existing standards of care, is a potentially promising treatment option for a variety of solid and hematological cancers. This project has been funded in part with Federal funds from the NCI/NIH, Department of Health and Human Services, under Contract No. HHSN261201300021C

#5859

Enhanced therapy for MYC-driven medulloblastoma by targeting protein translation pathway.

Nagendra K. Chaturvedi, Don W. Coulter, Matthew J. Kling, Sutapa Ray, Timothy R. McGuire, Shantaram S. Joshi, J. Graham Sharp. _Univ. of Nebraska Medical Ctr., Omaha, NE_.

Medulloblastoma is the most frequent pediatric brain tumor of neuroectodermal cerebellar origin. High-risk MB patients with MYC overexpression and amplification usually show an extremely poor clinical outcome responding poorly to current therapies. Recent preclinical studies have revealed that the aberrant activation and interactions of PI3K/AKT/mTOR signaling is frequently associated with MYC-driven MB therapy-resistance. In addition, it is evident that both MYC and mTOR components of the PI3K/AKT/mTOR signaling axis can directly control and share a common translation pathway in increasing protein synthesis by regulating the expression of multiple components of the protein synthesis machinery, including ribosomal proteins and initiation factors of translation. Thus, inhibiting enhanced protein synthesis may represent a highly relevant strategy for the treatment of MYC-driven MB. Therefore, in this study, we investigated the single agents and combined anti-MB efficacies of a recently well-established BRD4 (bromodomain-containing protein 4)/MYC inhibitor JQ1 and PI3K-mTOR dual inhibitor BEZ235. Using four MB cell lines including three MYC amplified cell lines, the in vitro efficacy of this combined approach on cell growth/apoptosis and cell cycle along with associated molecular mechanism(s) were investigated. We used an MTT assay to measure survival and proliferation, flow-cytometric-based propidium-iodide staining to determine the effects of these inhibitors on cell-cycle, Annexin-V analyses to examine the cells undergoing apoptosis following treatments and western blot analyses to determine the expression levels of target molecules. Our results showed that both inhibitors as single agents, significantly decreased MB cell growth and induced apoptosis by targeting the key molecules of the associated pathways. Combined treatment of JQ1 and BEZ235 significantly decreased MB cell growth/survival in a dose-dependent fashion compared to single agent activity. Moreover, JQ1 and BEZ235 alone or combined significantly induced G1 cell-cycle arrest which coincided with decreased expression of cyclin-D1 and increased expression of p21 proteins in MYC-driven MB cells. Mechanistically, the co-treatment of JQ1 and BEZ235 significantly downregulated the expression levels of phosphorylated 4EBP1/p70-S6K (mTOR components) and BRD4/MYC proteins. Together, our findings demonstrate that the combination of JQ1 and BEZ235 showed significant synergistic efficacy against MYC-driven MB, by enhancing the inhibition of protein synthesis (translation) pathway. Initial evidence from this targeted approach, suggests that additional preclinical evaluation is warranted to determine likely clinical utility of targeted therapy for high-risk MB patients.

#5860

Novel, first-in-class dual inhibitors of lysine specific demethylase 1 (LSD1) and histone deacetylatse 1 (HDAC) for treatment of cancer.

Dhanalakshmi Sivanandhan,1 Sreekala Nair,1 Subramaniyam Tantry,2 Sridharan Rajagopal,1 Mahanandeesha Hallur,2 Kannan M,2 Srinatha KC,2 Damodara K,2 Dilipkumar M,1 Chandru G,2 Dimpy Ghosh,1 Pradeep N,1 Anuj Kumar Singh,1 Mohd Zainuddin,1 Rudresh G,1 Radha Sharma,2 Meghashree S,2 Durga P. Kumar,2 Purushottam Dewang,2 Sriram Rajagopal1. 1 _Jubilant Biosys Ltd., Bangalore, India;_ 2 _Jubilant Biosys Ltd., BENGALURU, India_.

Introduction: LSD1 is a flavin adenine dinucleotide (FAD)-dependent amine oxidase that removes methyl groups from mono- or dimethylated histone H3 lysine 4. Studies have shown that LSD1 contributes to acute myelogenous leukaemia pathogenesis and patients that are non responders to ATRA respond to LSD1-ATRA combination treatment in clinic. Recent studies have shown that there is cross-talk between two components of CoREST complex, LSD1 and HDAC1/2 and combined inhibition of these targets has been shown to be more efficacious in multiple cancers. Therefore, dual inhibitors targeting both LSD1 and HDAC could be useful in treating cancers. To test the hypothesis, we have developed a set of molecules that have dual activity on LSD1 and HDACs.

Methods: To assess in vitro LSD1potency, TR-FRET assay was used. For assessing HDAC activity fluorescence based HDAC activity assay was performed. Western blotting and RT-qPCR were used to assess biomarkers of LSD1 and HDAC1/2 inhibition. Alamar blue cytotoxicity assay was used to assess cell proliferation. Xenograft models were used to assess in vivo efficacy.

Results: Dual inhibitors show strong in vitro potency comparable to advanced clinical candidates, against LSD1. On HDAC1/2 these molecules showed comparable IC50 as marketed HDAC1/2 specific drugs. Interestingly, these dual inhibitors lead to stronger and complete inhibition of cell proliferation in broader types of cancer cell lines, including, acute myeloid leukemia, multiple myeloma, hepatocellular carinoma and small cell lung cancer when compared to LSD1 specific inhibitor GSK2879552. Target engagement studies confirm the concomitant increase in mRNA levels of GFI1b, CD11b, CD86 and H3K9 acetylation levels. In vitro activity also shows a clear translation in efficacy studies. JBI128, one of the lead molecules from this series was tested in multiple in vivo studies, including, non-small cell and small cell lung cancer, where it not only shows single agent activity, but can also be combined with other targeted agents or SoC safely. In addition, combination of JBI128 with immune checkpoint inhibitor anti-PDL1 antibody in the CT26 syngeneic model showed much stronger efficacy as compared to either of the single agents. This clearly suggests that these dual inhibitors in addition to being epigenetic modulators in the cancer cells also have a role in immune modulation thereby contributing to stronger efficacy. Further studies in terms of gene expression analysis are underway to understand the molecular mechanism behind the additional activity/efficacy of these dual inhibitors.

Conclusion: These LSD1-HDAC dual inhibitors have the potential to be developed as first-in-class therapies that could offer stronger clinical efficacy compared to LSD1 or HDAC inhibitor alone. Further mechanistic and combination studies with standard therapies are in progress.

#5861

RX-3117 promotes epigenetic effects in cancer cells through enhanced degradation of DNMT1.

Dzjemma Sarkisjan,1 Ferry Pronk,2 Rosan Kuin,3 Btissame el Hassouni,1 Young B. Lee,4 Deog J. Kim,4 Marc Dijk,3 Daan P. Geerke,3 G. J. Peters1. 1 _Department of Medical Oncology, VU University Medical Center, Amsterdam, Netherlands;_ 2 _Division of Applied Theoretical Chemistry, Amsterdam, Netherlands;_ 3 _Division of Computational Toxicology, VU University Amsterdam, Amsterdam, Netherlands;_ 4 _Rexahn Pharmaceuticals, Inc., Rockville, MD_.

RX-3117 is a novel, investigational, oral, small molecule nucleoside compound that shows promising antitumor activity in xenografts including patient-derived xenografts resistant to gemcitabine. RX-3117 is currently being evaluated in a Phase IIa multi-center, open-label clinical study in patients with relapsed or refractory pancreatic cancer and advanced bladder cancer. RX-3117 can downregulate DNMT1 and activate genes controlled by methylation, such as E-cadherin and p16. We aimed to elucidate the mechanism of RX-3117 mediated downregulation of DNMT1.

Expression of DNMT1 was evaluated by western blotting. Binding of DNMT1 to DNA was measured after DNA isolation, DNAse treatment and gel electrophoresis. DNMT1 trapping in the nucleus was also studied with ImageStream FACS analysis. DNMT1 ubiquitination was measured by immunoprecipitation and quantification of the ubiquitin tag K48. Translocation of DNMT1 was studied by confocal microscopy. Modeling studies were performed with Quantum Mechanics (QM) and Molecular Dynamics (MD).

DNMT1 acts on the DNA during the so-called base flipping necessary for methyl transfer. To determine whether RX-3117 treatment would lead to trapping of DNMT1, NSCLC A549 and PDAC SUIT-028 cells were treated for 24 hr with 1 µM RX-3117. Immunostaining of DNMT1-DNA and ImageStream FACS analysis demonstrated binding of DNMT1 to DNA. Treatment with RX-3117 also resulted in DNMT1 ubiquitination, as shown by an increase in the ubiquitin tag K48, reaching the highest level after 48 hr. Incubation of A549 and SUIT-028 cells with 80 nM of the specific proteasome inhibitor bortezomib prevented RX-3117 mediated degradation of DNMT1 (at 24 and 24 hr exposure to 1 µM RX-3117). This underlines the role of the proteasome in DNMT1 degradation. With confocal microscopy it was demonstrated that DNMT1 and the proteasome co-localized and translocated to the cytosol of A549 and SUIT-028 cells treated for 48 hours with 1 µM RX-3117. With molecular modelling using QM and MD, changes in nucleophilicity of C4-C5 double bound were observed indicating that DNMT1 would initiate methyl transfer but cannot proceed.

In summary we conclude that RX-3117 induces epigenetic changes in cancer cells by trapping DNMT1 in the nucleus followed by translocation of the protein to the proteasome for degradation.

#5862

3,5-bis(2,4-difluorobenzylidene)-4-piperidone, a novel compound potently inhibits HNSCC through a DCLK1 mediated mechanism.

David Standing, Jake New, Prasad Dandawate, Dharmalingam Subramaniam, Vusala Snyder, Jonathan Enders, Afreen Sayed, Shrikant Anant, Sufi M. Thomas. _University of Kansas Medical Center, Kansas City, KS_.

Background: Head and neck squamous cell carcinoma (HNSCC) affects 40,000 patients annually and is associated with <50% 5-year survival despite aggressive therapy. Cancer stem cells (CSC) have been reported to contribute to tumor aggressiveness and therapeutic resistance in several tumor types, including HNSCC. Very little is known about the molecular characterization of CSCs in HNSCC. High expression of Doublecortin-like Kinase 1 (DCLK1), a microtubule associated protein that has been previously reported to mark quiescent CSCs of the colon and pancreas, is correlated with poor prognostic outcomes according to TCGA databases. We hypothesized that DCLK1 expression confers stem cell properties and hence is a target for therapy in HNSCC.

Methods: HNSCC tumor micro array was stained with DCLK1 to evaluate DCLK1 expression in HNSCC compared to normal mucosal tissue. Additionally, HNSCC cells were screened for expression of DCLK1 to assess expression. HN5 and FaDu cells were treated with DiFiD (0-10µM) and analyzed for cell viability in 2-dimensional and 3-dimensional cultures. Expression for markers of apoptosis, stemness, and cell cycle regulation were determined by western blotting. Furthermore, Caspase 3/7 assay and Annexin V/Pi staining were performed to confirm induction of apoptosis. Thermal shift binding assays (CETSA) were performed to confirm DiFiD:DCLK1 interaction. Lastly, HN5 tumor xenograft toxicity studies were performed in Balb c/ nude FOXN1 mice to determine the antitumor efficacy of DiFiD in vivo.

Results: We identified expression of DCLK1 in HNSCC cell lines and patient samples. IHC demonstrated increased expression of DCLK1 in HNSCC compared to normal mucosal tissue. Furthermore, DCLK1 was upregulated in HNSCC cells lines. CETSA assay demonstrated increased thermal stability of DCLK1 following incubation with DiFiD, suggesting DiFiD binds to DCLK1. There was a dose and time dependent reduction in cell viability both in 2-dimensional and 3-dimensional culture models following treatment with DiFiD in both cell lines. DiFiD induced caspase mediated apoptosis following G2/M cell cycle arrest. DiFiD significantly inhibited tumor growth in vivo, as confirmed by reduction in tumor volume and weight. DiFiD also showed little off target toxicity, as mouse weights were not significantly altered following DiFiD treatment.

Conclusions: Taken together, these data demonstrate that DiFiD has potent anti-cancer activity and therefore may serve as a potential novel therapy for the treatment of HNSCC.

#5863

RGX-202, a first-in-class small-molecule inhibitor of the creatine transporter SLC6a8, is a robust suppressor of cancer growth and metastatic progression.

Isabel Kurth,1 Celia Andreu,1 Shugaku Takeda,1 Helen Tian,2 Foster Gonsalves,1 Katya Leites,1 Subhasree Sridhar,1 Jia Min Loo,3 Rob Busby,1 Sohail Tavazoie,4 Masoud Tavazoie1. 1 _Rgenix, Inc., New York, NY;_ 2 _University of Massachusetts, Amherst, MA;_ 3 _National University of Singapore, Singapore, Singapore;_ 4 _The Rockefeller University, New York, NY_.

Background: Colorectal cancer (CRC) is one of the leading causes of cancer deaths worldwide with more than 140,000 patients diagnosed and nearly 50,000 deaths annually in the U.S. alone. Roughly 60% of patients present with locally advanced or distant metastatic disease, with the liver being a primary site of metastatic colonization. Creatine metabolism has been implicated in colon cancer progression and metastatic colonization of the liver. Metastatic colon cancer cells upregulate and release creatine kinase-B (CKB) into the extracellular space, where it phosphorylates creatine to generate the high-energy metabolite phosphocreatine. Phosphocreatine is imported via the creatine transporter SLC6a8. Intracellular phosphocreatine can be converted to ATP to fuel the survival of metastatic cancer cells within the hypoxic hepatic microenvironment. Consistent with this finding, genetic depletion of SLC6a8 in colon and pancreatic cancer cell lines significantly reduced liver colonization in mouse xenograft models.

Results: We herein demonstrate that the novel small molecule RGX-202 is a robust inhibitor of creatine uptake in cancer cells, both in vitro and in vivo. Oral administration of RGX-202 induced apoptosis of colon cancer cells in vivo, and significantly suppressed colon cancer liver metastatic colonization and primary tumor growth, both in KRAS wild-type and KRAS mutant colon cancer cell lines as well as in human PDX mouse models. Using genetic studies, these effects were found to be dependent on tumoral expression of SLC6a8. In addition, combination treatment of the CT26 syngeneic colon cancer mouse model with 5-FU resulted in synergistic antitumor activity, with complete tumor regressions observed in more than 40% of treated mice. Similarly, combination treatment of the KPC syngeneic mouse model with gemcitabine significantly reduced the growth of primary pancreatic tumors. Definitive 28-day GLP toxicology and pharmacokinetics studies of RGX-202 are currently ongoing. Preliminary observations suggest good tolerability in several animal species with a favorable pharmacokinetic profile, including bioavailability.

Conclusion: These results strongly support clinical development of RGX-202 in patients with gastrointestinal cancers, including colorectal and pancreatic cancer, both as monotherapy and in combination with standard-of-care treatment.

#5864

Novel anti-BMI-1 therapy in non-small cell lung cancer.

Indira Krishnan,1 Giorgia Maroni,1 Sean Clohessy,1 Virginia Savova,2 Mahmoud Bassal,1 Rapolas Zilionis,2 Eva Csizmadia,1 Clara M. Kerwin,3 Sun Choi,3 Claire V. Meyerovitz,3 Nicole Pandell,1 Chee W. Fhu,4 Junyan Zhang,1 Daniela S. Basseres,5 Cristina M. Magli,6 Julian Goggi,7 Robert S. Welner,8 Allon M. Klein,2 Marla Weetall,9 Art Branstrom,9 Raphael Bueno,3 Azhar Ali,4 Daniel G. Tenen,4 Elena Levantini1. 1 _Beth Israel Deaconess Medical Center, Boston, MA;_ 2 _Harvard University, Boston, MA;_ 3 _Brigham and Women's Hospital, Boston, MA;_ 4 _Cancer Science Institute of Singapore, Singapore, Singapore;_ 5 _Universidade de São Paulo, São Paulo, Brazil;_ 6 _Consiglio Nazionale delle Ricerche ITB, Pisa, Italy;_ 7 _Singapore Bioimaging Consortium (A*STAR), Singapore, Singapore;_ 8 _University of Alabama at Birmingham, Birmingham, AL;_ 9 _PTC Therapeutics, South Plainfield, NJ_.

Lung cancer represents one of the most significant and lethal diseases worldwide. Advances in treatment protocols, the development of targeted agents, and our comprehensive understanding of the genetics and mechanisms driving this disease, have helped identify novel agents with therapeutic promise, yet mortality remains high. Thus, there is a real need to identify new therapeutic options that can target this disease to improve patient outcome. BMI-1 (B cell-specific Moloney murine leukemia virus integration site 1), a key component of the epigenetic Polycomb Repressive Complex 1, plays a substantial role in many solid tumors, including NSCLC. It represents a compelling therapeutic target for NSCLC cancer patients as the majority of NSCLCs (~75%) display positive BMI-1 protein expression. Several compounds that reduce levels of the oncogene BMI-1 protein have been discovered by PTC Therapeutics i.e., PTC596 and a related analog PTC-028. PTC596 completed Ph1 trials and is currently being further tested in Ph1b trials. By using xenografts and established murine models of NSCLC, we are investigating BMI-1 as a novel therapeutic target in lung cancer, the mechanisms through which BMI-1 confers tumorigenicity, and determine the efficacy of BMI-1 inhibition in in vivo models of lung cancer at the single cell level. One critical aspect of cancer development lies in the 3-dimensional network of interactions that occurs between cells within their microenvironment. Therefore, by means of droplet-based molecular barcoding techniques we are analyzing single cell transcripts and identifying the population clusters within the heterogeneous tumor cellular milieu, to determine which cellular populations exist within tumors and how they respond to anti-BMI-1 treatment. Our data show that mutant EGFR and K-Ras driven lung cancer transgenic mice, as well as xenograft mice, express high levels of BMI-1 protein. In addition, we show that tumor growth of both models is affected by PTC596 treatment, with a more rapid response than that of currently available therapeutics. Tumor growth was measured at different time-points by magnetic resonance imaging. Transcriptional deconvolution of single cell sequencing data has enabled us to identify tumor-associated pulmonary subpopulations displaying epithelial, immune, fibroblast, and endothelial features. Importantly, tumor associated epithelial cells display a positive BMI-1 signature, which we also identified in primary NSCLC samples, underscoring the beneficial effects of anti-BMI-1 therapy in NSCLC patients.

#5865

Gartanin, a 4-prenylated xanthone in the mangosteen fruit, inhibits the growth of prostate cancer cells associated with Skp2 degradation via neddylation inhibition and FBXW2 induction.

Victor Pham, Michelle Bui, Raymond Rendon, Ericka Agredano, Thanh Le, Liankun Song, Xiaolin Zi. _University of California, Irvine, Orange, CA_.

The S-phase kinase-associated protein 2 (Skp2) is overexpressed in prostate cancer and associated with tumor stage, disease recurrence, and worse patient survival. Studies also have demonstrated that Skp2 overexpression plays a critical role in development and progression of prostate cancer in several prostate cancer models, including PTEN knockout and TRAMP carcinogenesis and CWR22 xenograft models. Therefore, Skp2 is a promising target for developing new therapeutic and preventive approaches for prostate cancer. Here, we have found that gartanin, 4-prenylated xanthone from the Garcinia mangostana fruit (mangosteen), promoted Skp2 degradation in a proteasome-dependent manner in prostate cancer cells. In addition, gartanin induced the expression of FBXW2, a novel E3 ligase of SKP2 for targeted degradation, and downregulated the expression of NEDD8, leading to reduced neddylation levels of UBC12 and Cullin1. Molecular modelling analysis predicts that gartanin reasonably docks onto the regulatory subunit of NEDD8 activating enzyme E1 (NAE1) and next to the NEDD8 binding complex. More interestingly, gartanin selectively inhibits the growth of Skp2 overexpressing PC3 cells compared to parental PC cells (IC50 values for PC3/Skp2 vs. PC3 cells are approximately 4 μM vs. 14 μM). Together, our data indicate that gartanin is a novel natural agent for targeting Skp2 degradation via induction of FBXW2 expression and inhibition of neddylation. Gartanin deserves further investigation for its usefulness in prostate cancer prevention and treatment.

#5866

Discovery and profiling of a highly potent and selective ERK5 inhibitor: BAY-885.

Clara Lemos, Duy Nguyen, Lars Wortmann, Ulf Bömer, Simon Holton, Christian Lechner, Stefan Prechtl, Detlev Sülzle, Franziska Siegel, Dominik Mumberg, Marcus Bauser, Andrea Hägebarth. _Bayer AG, Berlin, Germany_.

ERK5 (Extracellular signal regulated kinase 5, also known as MAPK7, Gene ID: 5598) is a key integrator of cellular signal transduction and it has been shown to play a role in various cellular processes such as proliferation, differentiation, apoptosis and cell survival. Several studies have demonstrated that inhibition of ERK5 with siRNA or shRNA decreases proliferation and increases cell death in different tumor models, thereby highlighting the potential of ERK5 as a therapeutic target in cancer. By HTS and subsequent lead identification, we discovered a highly potent and selective ERK5 inhibitor, BAY-885, which was used to investigate the therapeutic potential of ERK5 in tumor cells displaying ERK5 genomic amplifications or with constitutive activation of the ERK5 pathway. BAY-885 inhibited ERK5 enzymatic activity with IC50 = 40 nM and was highly selective vs. 357 kinases (Eurofins panel). Inhibition by BAY-855 was confirmed in a cellular setting using a MEF2 reporter cell line (SN12C-MEF2-luc). The EGF-stimulated MEF2 transcriptional activity was strongly inhibited by BAY-885 (IC50 = 115 nM; IC90 = 691 nM). In contrast, the compound had no effect on a reporter cell line with constitutive luciferase expression (SN12C-CMV-luc, IC50 > 50 µM), thereby ruling out potential effects as a general inhibitor of transcription or translation. Importantly, despite its high potency, BAY-885 failed to inhibit the proliferation of cells with ERK5 genomic amplification (SN12C, SNU-449, MFM-223) or with constitutively active ERK5 signaling (BT-474, SK-BR-3). Altogether, our results demonstrate that inhibition of ERK5 kinase and transcriptional activity with a small molecule did not translate into antiproliferative activity in different relevant cell models, thus raising doubts as to the viability of ERK5 as a therapeutic target for anticancer drug development. The availability of potent and selective chemical probes, as the one described here, will contribute to further understanding the biology of ERK5 signaling in cancer.

#5867

Identification of novel inhibitors of the PAX3-FOXO1 oncogenic driver in rhabdomyosarcoma.

Robert G. Hawley,1 Girma M. Woldemichael,2 Berkley E. Gryder,1 Young Song,1 Sivasish Sindiri,1 Vineela Gangalapudi,1 Jun S. Wei,1 James B. McMahon,2 Jack F. Shern,1 Javed Khan1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _National Cancer Institute, Frederick, MD_.

Background: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood, representing 8% of all childhood tumors. The most aggressive subtype of RMS is associated with chromosomal translocations creating a fusion transcription factor, most commonly involving PAX3 and FOXO1 (fusion-positive (FP)-RMS). Our group has recently comprehensively characterized the genetic and epigenetic landscape of FP-RMS. We found that FP-RMS has a strikingly low somatic mutational burden and that the PAX3-FOXO1 fusion gene is the primary oncogenic driver. Specifically, we found that PAX3-FOXO1 reprograms the cis-regulatory epigenetic landscape by inducing de novo super enhancers in collaboration with the bromodomain and extra-terminal domain (BET) protein family member BRD4, and the master transcription factors MYOG, MYOD1, and MYCN. We therefore hypothesized that FP-RMS would be vulnerable to disruption of the PAX3-FOXO1 super enhancer oncogenic circuitry.

Method and Results: To test this, we developed a novel readout assay to simultaneously monitor PAX3-FOXO1 super enhancer and general transcriptional activity as well as cell viability, and screened against a 62,643 small-molecule library at a concentration of 10 µM. We found a total of 573 compounds with selective ALK super enhancer inhibition, which were selected for dose response assay with four 10-fold dilutions ranging from 20 μM to 2 nM. Further prioritization using a systematic computational model reduced this number to 64. Among the compounds with known activities, the multi-kinase PKC-412/midostaurin was previously identified in screens for PAX3-FOXO1 inhibitors. Other known compounds included topoisomerase I and topoisomerase II inhibitors, where both enzymes have recently been reported to play important roles in BRD4-facilitated DNA dynamics involving super enhancers and the transcriptional machinery. Among this list we identified 33 novel compounds of unknown mechanism of action. We therefore performed RNA-seq analysis to determine the transcriptional impact of the effect of these compounds. Pathway, gene set enrichment and Connectivity Map analyses of RNA-seq expression signatures implicate several of the novel compounds as inhibitors of epigenetic regulators, including inhibitors of BRD4, histone deacetylases, histone demethylases, histone methyltransferases, and chromodomain proteins.

Conclusions: Our systematic approach identified several small molecules that specifically disrupt the activity of the PAX3-FOXO1 fusion oncogene in RMS. Further characterization of the specificity and selectivity of the lead compounds is ongoing, and exact mechanism of action is currently being elucidated. Our ultimate goal is to develop a clinically effective precision therapeutic targeting the previously "undruggable" PAX3-FOXO1 oncogenic driver of aggressive FP-RMS.

#5868

Preclinical testing of 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide (ICA-1): A potent protein kinase C-ι inhibitor as a potential anticancer drug.

Christopher Apostolatos, Andre H. Apostolatos, Wishrawana S. Ratnayake, Marie Bourgeois, Mildred Acevedo-Duncan. _University of South Florida, Tampa, FL_.

Protein Kinase C iota (PKC-ἱ) is an oncogene overexpressed in many cancer cells including prostate, breast, ovarian, melanoma and glioma. Previous In- vitro studies have shown that ICA-1 a PKC-ἱ specific inhibitor, is effective against some cancer cell lines by decreasing cell growth and inducing apoptosis {Pillai et al. Int. J. Biochem. Cell Biol. 43 (2011) 784-794. To assess ICA-1 as a possible therapeutic, In- vivo studies using a murine model were performed. ICA-1 was tested for stability in blood serum and results demonstrated that ICA-1 was stable in human plasma at 25oC and 37oC over a course of two hours. Toxicity of ICA-1 was tested for both acute and sub-acute exposure. The acute exposure showed subject surviving after 48 hours of doses ranging from 5mg/kg to 5000 mg/kg. Sub-acute tests exposed the subjects to 14 days of treatment and was followed by serum and tissue collection. AST, ALK-P, GGT, Troponin, and CR-P serum levels were measured to assess organ function. Heart, liver, kidney, and brain tissues were analyzed for accumulation of ICA-1. Finally athymic nude mice were xenografted with DU-145 prostate cancer cells. After tumors reached approximately 1 cm2, they were either treated with ICA-1 or left as control until the tumor reached 2 cm2. Results showed tumors in treated mice took almost double the time to reach the experimental end point, showing a significant growth reduction. In conclusion, ICA-1 is stable, shows low toxicity, and is a potential therapeutic for some carcinoma tumors.

#5869

**TT-00420, a dual mechanism kinase inhibitor targeting both mitosis and tumor microenvironment, is highly active against TNBC both** in vitro **and** in vivo **.**

Peng Peng, Xiaoju Yang, Xiaoyan Qiang, Lin Li, Shumao Ni, Xiaofen Sun, Di Wu, Frank Wu. _Nanjing TransThera Biosciences Co. Ltd., Nanjing, Jiangsu, China_.

The hallmarks of cancer comprise dysregulated cell cycle and mitosis, active angiogenesis, tumor-promoting inflammation, and immune escape as well as other features. Therapeutic targeting one of the hallmarks has shown promising but short-lived efficacy in cancer therapy. Therefore, combination of two or more mechanisms holds great promise of next generation oncology drug development. Aurora kinase A and B are often over expressed in many malignancies and associated with poor prognosis. Several anti-angiogenesis agents have been approved alone or in combination with other oncology drugs. In addition to its role for tumor growth, recent studies illustrate that angiogenesis also plays critical roles in the complexity and dynamic regulation of tumor microenvironment (TME). With the rapid advancement of immune-oncology, several attractive targets involved in tumor-promoting inflammation and immune escape have emerged. We have focused our efforts on the discovery and development of small molecule kinase inhibitors with novel mechanisms to treat cancer. Kinase profiling showed that TT-00420, one of our leading compounds, inhibited Aurora A/B, RTKs involved in angiogenesis, and a few other targets directly involved in tumor-assocated inflammation and immune escape with low nano molar activities. In a screening of human breast cancer cell lines, TT-00420 was highly efficacious against triple negative breast cancer (TNBC) cells while leaving ER+ cells intact, which is completely complimentary to the activity of CDK4/6 inhibitors. This anti-TNBC activity was confirmed in a patient derived xenograft (PDX) TNBC model in vivo, in which TT-00420 was highly active both as first-line and second-line treatment. TT-00420 demonstrated good oral bioavailability and pharmacokinetic properties in mice, rats and dogs. 2-week repeated dose-range finding studies revealed mechanism-related but manageable toxicities of TT-00420 in the 3 species mentioned above. Its dual mechanism involving anti-mitosis and TME modulation will be discussed in this presentation. TT-00420 is now under active development for IND filings both in the US and China.

#5870

CPSI-1306: A novel macrophage migration inhibitory factor inhibitor against aggressive breast cancer.

Subhadip Das,1 Nabanita Chatterjee,1 Sanjay Mishra,1 Dinesh K. Ahirwar,1 Sanjay Varikuti,1 Kirti Kaul,1 Rajni K. Sukhla,1 Abhay R. Satoskar,2 Ramesh K. Ganju2. 1 _The Ohio State University Medical Center, Columbus, OH;_ 2 _The Ohio State University Medical Center & The Comprehensive Cancer Center, Columbus, OH_.

Breast cancer is the leading cause of mortality in women, accounting for 23% of all cancer deaths, and one in eight women will develop invasive breast cancer over the course of her life. Although there are therapies available, most tumors develop resistance. In addition, certain types of breast cancers, including high-grade metastatic and triple-negative breast cancer, have limited therapeutic options available. Thus, novel targeted therapeutic strategies are required for prevention of disease progression. Inflammation of breast tumors is a major confounding factor involved in tumor progression and metastasis. Therefore, targeting inflammatory microenvironment could be a major strategy to targets breast tumor progression. Macrophage migration inhibitory factor (MIF) induces severe proinflammatory responses through tautomerase and also functions as a chemokine that mediates the recruitment of inflammatory cells. It has been shown that overexpression of MIF helps in recruiting macrophages to the tumor microenvironment (TME). We are evaluating the clinical efficacy of CPSI-1306, a small-molecular inhibitor of MIF, using in vitro and in vivo assays. Previous reports show that CPSI-1306 specifically inhibits keto-enol tautomerase activity of MIF. In silico analysis of publicly available data showed that higher expression of MIF negatively correlates with breast cancer patient overall, distant metastasis and relapse-free survival. Next, we analyzed the effect of CPSI-1306 on breast cancer in vitro and found that CPSI-1306 significantly induces apoptosis and reduces the viability of metastatic breast cancer MDA-MB 468 and MDA-MB 231 cells in a dose- and time-dependent manner. Mechanistic studies showed that CPSI-1306 induced apoptosis by reducing mitochondrial membrane potential by increasing apoptogenic signals, including apoptosis induction factor (AIF) and Cytochrome-C. Further analysis revealed that CPSI-1306 inhibits activation of cell proliferation marker AKT in metastatic breast cancer cells. We further analyzed the clinical efficacy of CPSI-1306 in vivo, using preclinical MVT-1 mammary tumor orthotopic syngeneic mouse model, and observed that CPSI-1306 significantly reduces tumor growth and metastasis to the lungs. Histologic analysis revealed reduced number of Ki67-positive proliferative cells and CD31-positive blood vessels in CPSI-1306-treated tumors. Our studies revealed that CPSI-1306 could be used as a novel therapeutic agent against aggressive breast cancer.

#5871

Efficacy and selectivity of novel dually targeted kinase inhibitors for therapy of adult and pediatric high-grade glioma.

Trever R. Carter,1 Cavan P. Bailey,2 Yusha Y. Sun,3 Alexsandra B. Espejo,2 Christopher E. Whitehead,4 Judith Leopold,4 Joya Chandra2. 1 _University of Notre Dame, Notre Dame, IN;_ 2 _MD Anderson Cancer Center, Houston, TX;_ 3 _Princeton University, Princeton, NJ;_ 4 _University of MIchigan, Ann Arbor, MI_.

High grade gliomas (HGGs) in adults and children confer very poor prognosis, with median survival rates under two years post diagnosis. Genetic alterations in receptor tyrosine kinases (RTKs) are seen in both adult and pediatric glioblastoma. Amplification of epidermal growth factor receptor (EGFR), as well as mutations in the PI3K/AKT/mTOR pathway, are frequent and can be targeted by kinase inhibitor based strategies. However, clinical trials singly targeting either EGFR or PI3K have had marginal impact on HGG patient outcomes. Given data showing that compensatory kinase activation is responsible for refractory disease, we developed therapeutics that target more than one kinase. Employing a computational modeling approach, we exploited the known binding modes of structurally related ATP binding site inhibitors of EGFR and PI3K to design small molecules that simultaneously and selectively inhibit both kinases. A panel of novel inhibitors has been synthesized, screened for metabolic stability and characterized with respect to kinase inhibition. Six compounds with varying degrees of inhibition for EGFR and PI3K were evaluated in the NCI-60 COMPARE cell line panel, whereupon two molecules, MTX-216 and MTX-241, exhibited strong in vitro activity against a series of human HGG cell lines. We further evaluated cytotoxicity in five human glioblastoma (GBM) lines, some of which bear EGFRvIII, the truncation mutation frequently seen, and in three patient-derived pediatric diffuse intrinsic pontine glioma (DIPG) cells. Treatment with either MTX-216 or MTX-241 resulted in potent growth inhibition relative to targeting of EGFR alone or PI3K alone using gefitinib, lapatinib or alpelisib. Cytotoxic activity against DIPG patient-derived cell lines was observed at lower concentrations compared to adult GBM cell lines, suggesting that dosing could be de-escalated in children, thereby reducing risks of toxicities. Importantly, selectivity of these compounds was demonstrated against malignant cells versus normal human astrocytes. Furthermore, we have identified unique metabolism targeting features of these compounds in GBM and DIPG lines, indicating suppression of both glycolytic pathways and oxidative phosphorylation, which is not seen with clinically relevant EGFR or PI3K inhibitors. Given the documented metabolic dependencies of brain tumor cells on glycolytic pathways, future studies will evaluate whether biomarkers of sensitivity to the dual inhibitors can be identified. Taken together, our findings point toward proof that a single small molecule inhibitor with dual specificity for EGFR and PI3K represents a rational and promising treatment strategy for recurrent adult and pediatric HGG. Despite the knowledge that HGG is associated with deregulated EGFR and PI3K activity, no current treatment exists targeting both of these critical oncogenes with a single molecule approach.

#5872

Pediatric Preclinical Testing Consortium (PPTC) of eribulin in osteosarcoma (OS) patient-derived xenograft (PDX) models.

Wendong Zhang. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Introduction: Eribulin is a synthetic analogue of halichondrin B and inhibits cancer cell proliferation via blockade of microtubule function. It is FDA approved for patients with breast cancer with two prior chemotherapy regimens for the treatment of metastatic disease and for previously treated patients with inoperable or metastatic liposarcoma. In vivo testing of eribulin against osteosarcoma (OS) Pediatric Preclinical Testing Program (PPTP) PDX models, developed from primary tumors, demonstrated significant single-agent activity. However, a Children's Oncology Group (COG) phase 2 trial of eribulin in patients with relapsed OS did not demonstrate significant antitumor activity of single-agent eribulin. In the current study the efficacy of eribulin in PDX models generated from primary and relapsed tumors, as well as dose-response, were assessed.

Methods: Eribulin was evaluated in 6 PPTC models, 4 from primary OS specimens (OS46, OS51, OS55, OS56) and 2 from relapsed specimens (OS39R, OS60). Eribulin was administered via intraperitoneal (IP) injection at a dose of 1mg/kg on days 1 and 4, repeated every 21 days. Response to treatment was determined based on PPTP-established endpoints (1). In addition, 3 dose levels of eribulin were evaluated in 3 PPTP models developed from primary tumors (OS1, OS17, OS33) and 1 model developed from a relapsed tumor (OS60). In the dose-response studies, eribulin was administered IP at 1mg/kg, 0.5mg/kg and 0.25mg/kg on days 1 and 4, repeated every 21 days with follow-up through day 42.

Results: Eribulin at a dose level of 1mg/kg induced stable disease (SD) or partial response (PR) in 4/9 and 3/9 OS xenografts, respectively. SD/PR responses were observed in PDX models generated from primary tumors (5/7) as well as PDX models generated from relapsed tumors (2/2). Eribulin demonstrated maximal antitumor activity at the highest dose level, 1mg/kg (SD/PR in 3/4). At 0.5 mg/kg 1/4 models achieved SD/PR, and at 0.25mg/kg all 4 models showed progressive disease within the 6-week testing period. Minimum relative tumor volumes also showed a dose-response effect from 0.25 to 1.0 mg/kg.

Conclusions: Eribulin demonstrated antitumor activity against OS PDX models generated from primary and relapsed tumors at a dose level of 1mg/kg. Eribulin showed a clear dose-response effect in terms of its ability to induce SD/PR responses, highlighting the importance of dose selection. This dose-response effect suggests that an inability to achieve comparable serum drug concentrations in human clinical trials may explain the limited tumor responses observed in the COG phase 2 trial of eribulin. Pharmacokinetic studies from the COG phase 2 study are ongoing in an effort to understand the disparate tumor responses seen in the in vivo studies and the phase 2 clinical trial.

Reference: 1. Houghton PJ et al. Pediatr Blood Cancer 2007;49:928-40.

Supported by NCI Grants: CA199222, CA199221.

#5873

BAY-3827, a selective inhibitor of AMPK for the evaluation of the role of AMPK in Myc-dependent tumors.

Clara Lemos,1 Volker K. Schulze,1 Benjamin Bader,1 Clara D. Christ,1 Hans Briem,1 Oliver Politz,1 Florian Prinz,1 Simon Holton,1 Tobias Heinrich,1 Julien Lefranc,1 Philip Lienau,1 Arne Scholz,1 Franz von Nussbaum,2 Carl Friedrich Nising,1 Dominik Mumberg,1 Marcus Bauser,1 Andrea Hägebarth1. 1 _Bayer AG, Berlin, Germany;_ 2 _Bayer S.A.S., Lyon, France_.

The AMP-activated protein kinase (AMPK) is a sensor of the energy status in the cells, playing a key role in controlling their metabolism. For many years, AMPK was mainly perceived as a tumor suppressor in agreement with being a component of the LKB1 tumor suppressor cascade, which inhibits mTORC1. However, in the last few years, some studies suggested that AMPK might actually exert a pro-tumorigenic role in certain contexts. For instance, Liu and colleagues demonstrated that dysregulated MYC expression renders tumor cells sensitive to AMPK depletion (Liu et al, 2012. Nature). The authors showed that, due to their increased anabolism, MYC-dependent cells rely on AMPK to restore ATP levels and to prevent an energy crisis that results in apoptosis and cell death. Here we report the discovery of a new lead structure for the inhibition of AMPK by biochemical high throughput screening. The optimization of this lead structure towards potency and selectivity led to the probe compound BAY-3827 and the use of this tool compound to evaluate the therapeutic potential of AMPK inhibition in MYC-dependent tumors. To demonstrate a cellular effect of BAY-3827 an HRTF® assay (Homogeneous Time Resolved Fluorescence, cisbio) for phospho-Acetyl-CoA carboxylase (p-ACC, Ser79), a direct substrate of AMPK, was used. ACC phosphorylation was strongly inhibited by BAY-3827 in COLO 320DM and IMR-32 cells. However, despite its high potency, BAY-3827 failed to inhibit the proliferation of cells with dysregulated c-MYC or N-MYC. In conclusion, we have identified a potent and selective AMPK inhibitor. Despite demonstrated inhibition of AMPK kinase activity, BAY-3827 treatment did not translate into antiproliferative activity in MYC-dependent cells. While we could not confirm our initial hypothesis, one might speculate that inhibition of AMPK might be of therapeutic utility in other biological contexts. Therefore, the availability of potent and selective inhibitors, as described here, will contribute to further insight into the potential of AMPK inhibition as a therapeutic target in cancer.

#5874

Targeting DDX3 in medulloblastoma by RK-33.

Saritha Tantravedi,1 Farhad Vesuna,1 Allison Martin,1 Michael Lim,1 Charles G. Eberhart G. Eberhart,1 Paul J. van Diest,2 Venu Raman1. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _University Medical Center, Utrecht, Netherlands_.

Introduction: Medulloblastoma is the most common form of pediatric brain cancer. Current treatments of surgical resection, craniospinal irradiation, and chemotherapy increase survival but around 40% of patients experience disease recurrence. Classically, four molecular subgroups of medulloblastoma have been identified: Wnt; Sonic hedgehog (SHH); Group 3; and Group 4. More recent work has subtyped up to seven molecular subgroups. The RNA helicase DDX3 is involved in many biological activities such as transcription, mRNA translation, splicing, and nuclear export. About 8% of medulloblastoma cases involve mutations in DDX3, of which 11% involve the pediatric Wnt and SHH subtypes. In addition, coinciding DDX3 and CTNNB1 mutations associated with dysregulated Wnt /β-catenin signaling in Wnt-type medulloblastomas have also been reported.

Aims and Methods: The goal of this work was to study whether RK-33, a small molecule inhibitor of DDX3, would be effective against medulloblastoma as a single agent and in combination with radiotherapy. We studied DDX3 expression in medulloblastoma cell lines DAOY and UW228 and human tissue microarrays by immunohistochemistry, western blotting, and qRT-PCR. We targeted DDX3 by siRNA and by RK-33 and observed proliferation and viability changes by MTS assays. Flow cytometric analysis was used to determine effect of RK-33 on cell cycle and apoptosis. TOP/FOP promoter assay was utilized to study effect on TCF promoter activity. We used colony formation assays to study effect of combining radiation and RK-33 treatment on cells. DAOY tumors were generated in nude mice and treated with combination therapy to study effects in vivo.

Results: High DDX3 expression was observed in 55% of pediatric and 66% of adult medulloblastomas. DDX3 knockdown resulted in reduced cellular proliferation. Both the medulloblastoma cell lines were sensitive to RK-33, with IC50 values of 2.5 µM and 3.5 µM, respectively and resulted in decreased levels of DDX3 expression, with no observable toxicity. RK-33 resulted in a G1 phase cell cycle arrest before the onset of apoptosis. DDX3 knockdown resulted in a significant decrease of TCF activity and reduced transcription of Axin2, CCND1, MYC, and Survivin. A combination of RK-33 and radiation resulted in a significantly higher inhibitory effect than using single treatment of either RK-33 or radiation. We conclude that inhibiting DDX3 via the small molecule inhibitor RK-33 is effective in reducing medulloblastoma tumors and that the effect is synergistic with radiation treatment.

#5875

Development of LIFR inhibitor EC359 as a novel therapeutic for ovarian cancer.

Suryavathi Viswanadhapalli,1 Hareesh B Nair,2 Bindu Santhamma,2 Gangadhara R Sareddy,1 Yiliao Luo,1 Xinlei Pan,3 Edward R Kost,1 Ramachandran Murali,3 Rajeshwar Rao Tekmal,1 Klaus J Nickisch,2 Ratna K Vadlamudi1. 1 _UT Health San Antonio, San Antonio, TX;_ 2 _Evestra, Inc., San Antonio, TX;_ 3 _Cidars-Sinai Medical Center, Los Angeles, CA_.

Background: Ovarian cancer (OCa) is the deadliest of all gynecologic cancers. OCa patients initially respond to standard combinations of surgical and cytotoxic therapy; however, ~80% will develop recurrence and inevitably succumb to chemotherapy-resistant disease. OCa stem cells are implicated in the tumor initiation and therapy resistance. LIFR signaling plays a critical role in OCa progression and stemness. Further, high circulating LIF levels correlate with tumor recurrence and chemoresistance. The autocrine loop involving LIF, LIFR and STAT3 axis drives sustained fibroblast production of inflammatory mediators. This represents a significant problem and a critical need exists for development of novel therapies targeting the LIFR axis for treating OCa.

Methods: We have rationally designed and synthesized a small organic molecule (EC359) that emulates the LIF-LIFR binding site and functions as a LIFR inhibitor from a library of compounds. In silico docking studies were used to identify the putative interaction of the EC359 and LIF/LIFR complex. Binding of EC359 to LIFR was confirmed using surface plasmon resonance (SPR) and IP assays. Mechanistic studies were conducted using Western, RT-qPCR, and RNA-Seq analysis. Xenograft models were used for preclinical evaluation and toxicity. The efficacy of EC359 was tested using Patient-Derived eXplants (PDeX).

Results: Global analysis of online databases revealed negative correlation of OCa survival with LIFR expression. Molecular docking studies showed EC359 interacts at the LIF-LIFR binding interface. SPR studies confirmed interaction of EC359 to LIFR. Western analysis of eight cells that represent four subtypes of OCa confirmed higher expression of LIF and LIFR. EC359 reduced the growth of eight OCa cells with high potency (IC50 10-50 nM) and promoted apoptosis. EC359 treatment reduced stemness of OCa cells. EC359 activity is dependent on the level of expression of LIFR with little activity on cells that do not express LIFR. EC359 significantly reduced the viability of carboplatin- and taxol-resistant OCa cells. Mechanistic studies showed EC359 interacts with LIFR and block its interaction with LIF. EC359 treatment reduced the STAT3 phosphorylation, mTOR and downstream survival signaling cascades. RNA sequencing revealed unique pathways blocked by EC359. Treatment of xenograft tumors with EC359 significantly reduced the tumor volume compared to control. Further, using PDeX of OCa, we demonstrated that EC359 has potential to reduce the proliferation. Pharmacologically, EC359 exhibited high oral bioavailability and long half-life with a wide therapeutic window.

Conclusions: EC359 is a novel agent that targets LIF-LIFR axis and has activity against chemotherapy-resistant and primary OCa tumors. EC359 has the distinct pharmacologic advantages of oral bioavailability, in vivo stability, and is associated with minimal systemic side effects.

#5876

Exploratory testing of PM060184 compound in high-grade serous ovarian carcinoma cell lines.

Victoria Heredia-Soto,1 Andrés Redondo,1 Alejandro Gallego,1 María Miguel-Martín,1 Roberto Crespo,1 Alicia Hernández,1 David Hardisson,1 Jaime Feliu,1 Carlos Galmarini,2 Marta Mendiola1. 1 _La Paz Hospital, IdiPAZ, Madrid, Spain;_ 2 _Pharmamar, Madrid, Spain_.

Purpose of the Study: Marine sponges have developed mechanisms to protect themselves from a hostile marine microenvironment. One of these mechanisms is the use of biologically active metabolites, explored nowadays for their anticancer properties. PM060184 is one of these compounds, isolated from the Madagascan sponge Lithoplocamia lithistoides. This polyketide is currently under evaluation on clinical trials, and its antitumor activity was previously reported in the preclinical setting in a panel of cell lines and subcutaneous tumor xenografts of different origin. The purpose of the study is to explore the effect of PM060184 in a panel of ovarian high-grade serous (HGS) carcinoma cell lines with different sensitivity to cisplatin and paclitaxel.

Experimental Procedures: Cell growth inhibition was analyzed by exposure of increasing concentrations of PM060184 in 96 multiwell plates for 72h, by subsequent confluence evaluation and sulphorhodamine (SRB) staining. Cell cycle experiments were done by propidium iodide (PI) staining after treatment of cell lines at their IC50 value for 72h. Three independent experiments with 6 replicates per condition were performed for both approaches. Celigo Image Cytometer platform was employed for phase contrast and viable cells discrimination with a triple staining (Hoechst, PI and calcein AM), as well as for cell cycle analyses.

Results: We have focused on the effect of this drug on a panel of HGS ovarian carcinoma cell lines, previously characterized by their response to cisplatin and paclitaxel. We have observed antiproliferative activity in a concentration-dependent manner, with IC50 values at subnanomolar concentrations in all the cell lines tested. This effect was observed in platinum-sensitive and -resistant cell lines and all of them were also more sensitive to PM060184 than paclitaxel, another tubulin-binding agent usually used for the treatment of ovarian cancer. We have evaluated cell cycle and apoptosis at the IC50 values for each cell line. We have seen that this agent disrupts the cell cycle at different phases, as DNA synthesis and mitosis, depending on the cell line. Additionally, an increase in apoptotic cell death is detected as a sub G1 peak by flow cytometry in most of the cell lines tested.

Conclusions: PM060184 is a new tubulin-binding agent with a potent antitumor activity in a panel of HGS ovarian cancer cell lines, with different ranges of sensitivity to cisplatin and paclitaxel. Additionally, this effect is more potent than that exerted by other tubulin-binding compounds, such as paclitaxel. However, the underlying mechanism of PM060184 action on ovarian HGS cell lines will need to be further elucidated.

#5877

Antitumor activity of the mesoionic compound MI H 2.4 on breast cancer cell lines.

Luciana Amaral de Mascena Costa,1 Filipe Cássio Silva de Lima,1 Rodrigo da Silva Viana,1 Silvany de Sousa Araujo,2 Aurea Wischral,2 Helivaldo Diógenes da Silva Souza,3 Petrônio Filgueiras de Athayde-Filhoa,3 Leandro Araújo de Azevedo,4 Severino Alves Júnior,4 Manoel Adrião,2 J. Michael Mathis1. 1 _LSU School of Veterinary Medicine, Baton Rouge, LA;_ 2 _Universidade Federal Rural de Pernambuco, Recife, Brazil;_ 3 _Universidade Federal da Paraíba, João Pessoa, Brazil;_ 4 _Universidade Federal de Pernambuco, Recife, Brazil_.

Breast cancer is the most commonly diagnosed cancer in women under 60. Localized breast cancer is easily treated, resulting in high survival rates. However, treatment for advanced disease is inadequate, with a five-year survival rate of less than 24%. Thus, there is a great need for new therapies capable of increasing therapeutic efficacy. Synthetic mesoionic compounds, belonging to the 1,3-thiazolium-5-thiolate group, are recognized for their broad spectrum of biological activities including antibiotic, antiparasitic, antiviral, anticonvulsant, antidepressant, antioxidant, analgesic, anti-inflammatory, and more recently for their potential antitumor activity. These compounds have the ability to cross cell membranes; the characteristic of mesoionic structures having distinct regions of positive and negative charge associated with a poly-heterocyclic aromatic ring system, indicates the capability of strong interactions with biomolecules such as DNA and proteins. In this study, the cytotoxic effects of mesoionic compound MI H 2.4 alone and in combination with zinc was examined in breast cancer cell lines (4TI, BT-20, BT-549, MCF7, MDA-MB-231, MDA-MB-436, MM2MT, T-47D, and ZR-75-1) and normal breast cell lineages (HuMEC, MCF-10A, and MCF-12A) were evaluated. The effect of this agent on cell cycle was also investigated. Different concentrations of mesoionic compound MI H 2.4 (MI H 2.4 free) and in combination with zinc (MI H 2.4 Zinc) were added to the cultured cells and incubated for 24, 48, 72 and 96 h. Cell survival and cytotoxicity were evaluated using crystal violet and MTT assays. Cell cycle analysis was performed using MCF7 cells that were stained with propidium iodide and analyzed by flow cytometry. The cytotoxic effects of mesoionic compounds (MI H 2.4 free and MI H 2.4 Zinc) were highest at72 and 96 h. The MI H 2.4 free and MI H 2.4 Zinc showed a similar inhibitory effect on breast cancer cell growth in the μM range. In contrast, the normal breast cell lineages showed low cytotoxicity to treatment with the mesoionic compounds. Treatment of MCF7 cells cultured with MI H 2.4 free blocked cell cycle progression at the G2 phase of the cell cycle after 24 h of treatment. Mitochondrial function of MCF7 cells was determined using a Seahorse XF-24 Extracellular Flux Analyzer. Treatment with MI H 2.4 free and MI H 2.4 Zinc for 24 h resulted in a decreased basal and maximal mitochondrial respiration. In summary, mesoionic compound MI H 2.4 may offer a novel therapeutic strategy in the treatment of breast cancer, considering that it has significant antitumoral activity in breast cancer cell lines and low cytotoxicity in normal cells.

#5878

Effect of cannabinoid WIN 55,212-2 on prostate cancer cell proliferation, migration, invasion, and tumor growth.

Domenica Roberto, Laurence H. Klotz, Vasundara Venkateswaran. _Univ. of Toronto, Toronto, Ontario, Canada_.

Introduction and Objective: Prostate cancer (PCa) is the most commonly diagnosed cancer in men and the second leading cause of cancer related death in Canada. A large body of evidence supports a possible role for cannabinoids in certain aspects of human health and disease, acting as palliative agents and more importantly, as inhibitors of cancer cell proliferation, migration, invasion, and the angiogenesis of tumours. WIN 55,212-2 (WIN) is a highly potent synthetic cannabinoid, binding to the cannabinoid receptors 1 and 2. This study aims to determine the anti-cancer effect of WIN using preclinical models of prostate cancer.

Methodology: Human PCa cells (DU145, PC3) were treated with WIN at concentrations ranging from 1μM to 20μM and growth of cells (using the MTS assay) was assessed at various times following treatment. Wound-healing assays were conducted to investigate the migratory potential of cells following exposure to treatment, and trans-well invasion assays were performed to explore the influence of WIN on cell invasion. In vivo evaluation using PC3 xenografts was performed using swiss athymic mice. Treatment was administered thrice weekly and tumor volume assessed for a total of three weeks.

Results: Treatment with 10μM WIN resulted in a significant reduction in the proliferation of DU145 and PC3 cells after 24 h compared to control (p<0.05). Cell migration and invasion studies revealed a significant reduction in cell motility at 15μM WIN (p<0.05) and a significant reduction in cell invasion at a concentration as low as 1μM (p<0.05). Treatment with 5mg/kg WIN (ip) revealed significant differences in tumor volume compared to controls that received the vehicle (p<0.05). There were no significant alterations in body weight in both groups.

Conclusion: WIN 55,212-2 is a highly potent cannabinoid, having significant influences on cell proliferation, migration, and invasion, with reduction in the growth of tumors in prostate cancer xenografts. We are the first to demonstrate the use of WIN in PC3 xenografts and provide evidence for its use as a novel therapeutic option in patients with prostate cancer.

Funding/Conflicts of Interest: None

#5879

Effect of Minnelide, a prodrug, on cervical cancer growth.

Vivek Ramakrishnan,1 Bhuwan Giri,1 Urvashi Hooda,1 Peter Wilkinson,2 Christopher deHaydu,1 Janneth Oleas,1 Sabita Roy,1 Sundaram Ramakrishnan,1 Ashok Saluja1. 1 _University of Miami, Miami, FL;_ 2 _University of Minnesota, Minnesota, MN_.

Human Papilloma Virus (HPV) infection is associated with the development of Cervical cancer. Therapeutic resistance and recurrence remain the major obstacle to successful treatment of cervical cancer patients. Recently, a novel pro-drug Minnelide, derived from the diterpenoid triepoxide, triptolide, was found to be very effective against several human cancers in experimental animal models. The goal of the present study is to determine the effect of triptolide (in vitro) or Minnelide (in vivo) on HPV positive cervical cancer cells. Triptolide inhibited the proliferation of human cervical cancer cell lines, Ca Ski, SiHa, Me-180, and C-33a with an IC50 of 50 - 100 nM. Furthermore, triptolide inhibited the expression of viral oncoproteins E6 and E7 transcripts significantly. Consequently, p53, tumor suppressor protein levels were increased in the treated cells which led to increased apoptosis and reduced proliferation. Subsequently, we determined the effect of Minnelide on cervical cancer growth in athymic, nude mice. Tumor (SiHa and CaSki) bearing mice were treated with either 0.2 or 0.4 ug/kg doses of Minnelide. These studies showed that Minnelide inhibited tumor growth by about 70 % when compared to control group of mice and improved survival significantly. In a parallel study, combination treatment with carboplatin and Minnelide showed more than additive inhibition of tumor growth. In summary, Minnelide treatment reduced the transcript levels of HPV oncoproteins and inhibited cellular proliferation by upregulating p53. Our studies further demonstrate that Minnelide can be used either as a monotherapy or in combination with platinum drugs to inhibit the growth of cervical cancers.

#5880

Small-molecule modulators of PDE3/SLFN12 to kill cancer cells.

Timothy A. Lewis,1 Luc de Waal,1 Xiaoyun Wu,1 Manuel Ellerman,2 Charlotte Kopitz,2 Antje Wengner,2 Knut Eis,2 Martin Lange,2 Adrian Tersteegen,2 Philip Lienau,2 Heidi Greulich,1 Matthew Meyerson1. 1 _Broad Institute of MIT and Harvard, Cambridge, MA;_ 2 _Bayer AG, Berlin, Germany_.

We have previously reported1 our results with a small molecule called DNMDP which kills certain cancer cells by modulating the interaction of PDE3A and Schlafen 12 (SLFN12), a more recently discovered protein of unknown function. While DNMDP selectively inhibits PDE3, most PDE3 inhibitors have no cell killing effects and in fact rescue cancer cells from DNMDP-induced death. DNMDP is not suitable for advanced studies due to structural liabilities. Optimization of DNMDP to both increase activity and improve pharmacokinetic properties resulted in enantiomerically pure, low molecular weight, metabolically stable compounds which are active at low doses in animal models of cervical cancer and melanoma. While these compounds are selective PDE3A inhibitors and their biochemical activity mirrors their cellular activity, the activity of the compounds results not from PDE3 inhibition and increased cAMP levels, but from increased compound-induced binding of PDE3A to SLFN12, which most PDE3 inhibitors do not effect. Our results suggest that small molecule modulators of PDE3/SLFN12 binding may provide a novel treatment for the treatment of certain cancers.

1Nat. Chem. Bio. 2016, 12, 102-108.

#5881

Novel inhibitors of the epigenetic reader protein MBD2.

Hugh Giovinazzo, Zachary R. Reichert, Andries Bergman, Xiaohui Lin, Nicolas Wyhs, David Esopi, Ajay Vaghasia, Jianyong Liu, Yash Jain, Akshay Bhamidipati, Ruchama Steinberg, Traci Speed, Matthew Vaughn, Yonggang Zhang, Nate Brennen, Theodore Deweese, Srinivasan Yegnasubramanian, William G. Nelson. _Johns Hopkins University School of Medicine, Baltimore, MD_.

DNA hypermethylation can trigger silencing of tumor suppressor genes during cancer development and progression, partly through binding by methylated-DNA binding proteins (MBD), such as MBD2, that function as "epigenetic readers" and recruit co-repressor complexes to promote gene repression. Inhibiting MBD2-mediated repression represents an attractive cancer therapeutic strategy. Here, we used a cell-based screen to identify small molecules capable of reactivating hypermethylated promoter sequences. We used biochemical, molecular biologic, and pharmacologic approaches to characterize mechanism of action of identified MBD2 inhibitors. A subset of these compounds represent a new class of inhibitors capable of selectively antagonizing interactions between MBD2 and methylated DNA, leading to reactivation of the hypermethylated gene GSTP1 and the epigenetically silenced retinoic acid signaling pathway. Combinations of one of the newly identified MBD2 inhibitors, KCC-08, with the retinoic acid receptor agonist, isotretinoin, significantly reduced cancer cell growth/survival in vitro and in vivo. These novel MBD2 inhibitors are thus positioned for further pharmacologic lead development for use as probes to interrogate epigenetic gene silencing mechanisms and as cancer therapeutics.

#5882

Bench-to-bedside translation of ciclopirox prodrug for the treatment of non-muscle invasive and muscle-invasive bladder cancer.

Scott J. Weir,1 Partha Ranjarajan,1 Robyn Wood,1 Karl Schorno,2 Prabhu Ramamoorthy,1 Lian Rajweski,2 Kathy Heppert,2 Michael J. McKenna,3 William McCulloch,4 Greg A. Reed,1 Amanda Brinker,1 Michael J. Baltezor,2 Roy A. Jensen,1 John A. Taylor,1 Shrikant Anant1. 1 _Univ. of Kansas Medical Ctr., Kansas City, KS;_ 2 _Univ. of Kansas, Lawrence, KS;_ 3 _Navigator LSA, Wilmington, NC;_ 4 _Alba BioPharm Advisors Inc, Durham, NC_.

Ciclopirox (CPX) is contained in a number of FDA-approved topical antifungal drug products as the free acid and olamine salt. CPX possesses anticancer activity in a number of in vitro and in vivo preclinical models. Its clinical utility is limited as an oral anticancer agent, however. The oral bioavailability of CPX is quite low due to extensive first pass effect. The poor water solubility of CPX and its olamine salt prevent formulation as an injectable drug product. Thirdly, dose-limiting gastrointestinal toxicities were observed following four times daily oral dosing of CPX in patients with advanced hematologic malignancies. Ciclopirox Prodrug (CPX-POM), in contrast, has demonstrated excellent bioavailability via injectable routes of administration. Here we describe the preclinical characterization of CPX-POM, a novel anticancer agent being developed for the treatment of non-muscle invasive (NMIBC) and muscle invasive (MIBC) bladder cancer. Following IV, SQ and IP administration to mice, CPX-POM is rapidly and completely metabolized to CPX in blood via circulating phosphatases. CPX and its major, inactive glucuronide metabolite are extensively eliminated in urine. At well-tolerated doses, steady-state urine concentrations of CPX exceed in vitro IC50 values in mice by 15-30 fold. CPX inhibited cell proliferation, colony formation, and bladdosphere formation in vitro in T24 (NMIBC) and 253JBV (MIBC) human cell lines in both concentration- and time-dependent manners with IC50 values of 2-4 µM. CPX exposure increased the percentage of NMIBC and MIBC cells arrested at the S and G0/G1 phases, and induced cell death. CPX exposure significantly reduced expression of genes at the mRNA level involved in cancer stem cell signaling pathways including Notch, Wnt, and Hedgehog. CPX was shown to inhibit bladder cancer cell growth in vitro by inhibiting the Notch 1 signaling pathway. The validated N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) chemical carcinogen mouse model of bladder cancer was employed to establish in vivo preclinical proof of principle for CPX-POM. Over the once-daily IP dose range of 25-200 mg/kg, CPX-POM treatment resulted in significant decreases in bladder weight, a clear migration to lower stage tumors, dose-dependent reduction in Ki67 and PCNA staining, as well as a reduction in PCNA-expressing cells. All CPX-POM doses were well tolerated with no evidence of toxicity to the urinary tract based on blinded pathologic evaluation. There were also dose-dependent decreases in Notch 1, Presenilin 1, and Hey 1 in bladder cancer tissues obtained from CPX-POM treated animals. Tumor response was similar, in vivo, following once-daily and three-times weekly CPX-POM administration. CPX-POM has received FDA clearance to proceed to Phase I, and is currently being evaluated in a first-in-human trial in patients with advanced solid tumors.

#5883

Phosphodiesterase 7-A is a novel potential therapeutic target against ovarian cancer.

Nayara G. Tessarollo,1 Isabella S. Guimarães,1 Diandra Z. dos Santos,1 Taciane B. Henriques,1 Marcele LM Souza,1 Laura Maciel,2 João Carlos A. Almeida,2 Alan Branco,1 Ian V. Silva,1 Leticia BA Rangel1. 1 _Federal Univ. of Espirito Santo, Vitoria, Brazil;_ 2 _State University of North Fluminense Darcy Ribeiro, Brazil_.

Ovarian cancer (OC) is the leading cause of gynecologic cancer-related deaths due to late diagnosis and chemoresistance. RNA-seq data, conducted in collaboration with our group, compared the expression of transcription levels between normal Fallopian tube and high-grade serous ovarian carcinoma samples and revealed phosphodiesterase 7-A as a novel potential therapeutic target against the disease. Metabolic cell viability assays (MCV) were conducted following OC cells treatment with selective PDE7 inhibitor (BRL50481) in monotherapy or in association with paclitaxel (PTX). Mechanism of action of the drug was evaluated by cell cycle analysis, anexina V/PI assay, immunoblotting, real-time PCR and scanning/transmission electron microscopy. Data showed that the use of the BRL50481 in monotherapy reduced A2780 cells MCV by ~60% in a dose-dependent manner after 48h treatment, but nothing significant was observed in OVCAR3. On the other hand, the association of BRL50481 and PTX promoted inhibition of MCV in both cell lines analyzed. An increase in the potency of PTX was also observed, an aspect verified with the reduction of its IC50 in relation to monotherapy by 30% (p<0.01). Treatment chronology in cell survival was also verified. Thus, pretreatment of A2780 with BRL50481 200μM followed by its association with PTX promoted a reduction in cells MCV of ~70% (p<0.01) compared to treatment with PTX alone. With regard to OVCAR3, 400μM pretreatment of BRL50481 provided a VCM reduction of ~20% (p<0.001). Therefore, our results showed beneficial effect between the combination of PDE7 inhibitor and PTX, which allowed reduction of the PTX concentration used in A2780 and OVCAR3 by ~82.7 x 108 and 80.4 x 103uM, respectively. Moreover, the combination of BRL50481 and PTX promoted increased cell necrosis in OVCAR3. In addition, pretreatment of OVCAR3 with BRL50481 modulated the gene expression of the cytokines, as well as increased IL-6 secretion. The combination of BRL50481 and PTX further modulated negatively the PI3K/AKT/mTOR signaling pathway in both cell lines studied. Furthermore, the A2780-pretreated cells showed an increase in the expression of the pro-apoptotic Bax protein. Still, cell death may be related to the induction of autophagy seen in both by the increase of the expression of beclin. Moreover, BRL50481 promoted changes in OC cellular morphology, resembling the induction of cell death by BRL50481, as well as mitochondria and mitochondrial cristae modification. The association of the PDE7 inhibitor with PTX has been proved potentially beneficial in the fight against OC. The impressive additive effect between the drugs allows us to postulate a substantial decrease in PTX neurotoxicity caused in patients by the drug. Our current challenge is to synthesize less toxic BRL50481 analogues, which is only approved for experimental purposes, hence providing hope for better life quality to OC patients.

### Regulation of Gene Expression in Drug Resistance

#5884

4EBP1 **is a tumor suppressor gene unleashed by mTOR inhibition in head and neck squamous cell carcinoma.**

Zhiyong Wang,1 Daniel Martin,2 Lynn Vitale-Cross,2 Xiaodong Feng,1 Alfredo A. Molinolo,1 Michael M. Allevato,1 Victoria H. Wu,1 Mara Gilardi,1 Hao Xu,3 Qianming Chen,3 J.Silvio Gutkind1. 1 _UCSD, La Jolla, CA;_ 2 _NIDCR, Bethesda, MD;_ 3 _Sichuan University, Chengdu, China_.

Persistent aberrant activation of the PI3K/AKT/mTOR signaling pathway occurs in over 80% of head and neck squamous cell carcinomas (HNSCC). In prior studies we have shown that mTOR regulates proliferation, angiogenesis and apoptosis, thus playing an essential role in HNSCC initiation and progression. The frequent over-reliance on this signaling circuit for HNSCC growth may in turn represent a cancer vulnerability that can be exploited therapeutically. Indeed, we have shown that mTOR inhibitors (mTORi) promote tumor regression in multiple genetically defined and chemically induced carcinogenesis animal models, and encouraging results have been recently obtained using mTORi in HNSCC in the clinic. However, the complexity of genomic alterations in HNSCC may facilitate the emergence of drug resistance, especially in cases with patients who may receive multiple rounds of chemotherapy and/or radiation. Understanding the precise molecular processes by which mTOR acts in HNSCC may help predict intrinsic resistance mechanisms and select patients who may benefit the most from the administration of mTORi. Here, we focused on EIF4EBP1 (4EBP1), a direct target of mTOR that serves as key effector for protein synthesis. Remarkably, we found a negative correlation between 4EBP1 gene copy number loss and disease-free survival (p<0.001) and overall survival (p<0.01) in the HNSCC TCGA database (n=181). Furthermore, IHC analysis of 4EBP1 protein expression revealed that 16.3% of HNSCC cases analyzed (n=49) lack 4EBP1, with progressive protein loss in advanced cases. Aligned with a tumor-suppressive role, 4ebp1-knockout mice formed larger and more HNSCC lesions (p<0.001) in 4NQO oral carcinogenesis models. We engineered a mutant 4EBP1 that cannot be phosphorylated by mTOR (4EBP1-mut), resembling de-phospho-4EBP1, and generated HNSCC cells expressing this mutant in a tetracycline-inducible fashion. Protein-protein interactions and assembly of the translational complex were analyzed using 7-methyl GTP pull-down and co-IP assays. Expression of 4EBP1-mut disrupted the function of the translation initiation complex and was sufficient to prevent tumor growth in HNSCC xenograft models. Similarly, mTORi induced the accumulation of dephosphorylated endogenous wild-type 4EBP1 and resulted in HNSCC xenograft regression. Remarkably, CRISPR-CAS9-mediated knockout of 4EBP1 in HNSCC cells caused insensitivity to mTORi in vitro and in vivo. Overall, our findings suggest that 4EBP1 needs to be persistently repressed by mTOR phosphorylation in HNSCC and that mTORi may unleash 4EBP1's tumor-suppressive activity, thereby reducing the translation of growth-promoting and cancer cell stemness-related proteins. In turn, 4EBP1 gene and protein loss may underlie mTORi resistance, supporting that 4EBP1 expression and phosphorylation status may represent a mechanistic biomarker of mTORi sensitivity in HNSCC patients.

#5885

**Loss of** RAD51C **promoter hypermethylation confers PARP inhibitor resistance.**

Rachel M. Hurley,1 Ksenija Nesic,2 Cordelia McGehee,1 Olga Kondrashova,2 Maria I. Harrell,3 Paula A. Schneider,1 Xiaonan Hou,1 Cristina Correia,1 Karen S. Flatten,1 Giada V. Zapparoli,4 Alexander Dobrovic,4 Kevin K. Lin,5 Thomas C. Harding,5 Andrea E. Wahner Hendrickson,1 Elizabeth M. Swisher,3 Matthew Wakefield,2 S. John Weroha,1 Clare L. Scott,2 Scott H. Kaufmann1. 1 _Mayo Clinic, Rochester, MN;_ 2 _The Walter and Eliza Hall Institute, Melbourne, Australia;_ 3 _University of Washington, Seattle, WA;_ 4 _Olivia Newton-John Cancer Research Institute, Melbourne, Australia;_ 5 _Clovis Oncology, San Francisco, CA_.

Background: Acquired PARP inhibitor (PARPi) resistance in high-grade serous ovarian cancer (HGSOC) as a result of restored homologous recombination has been observed following secondary mutations that restore full-length protein in BRCA1, BRCA2, RAD51C, and RAD51D. Additionally, loss of BRCA1 methylation has also been shown to confer resistance. However, little is known about the role of RAD51C methylation in acquired PARPi resistance. In ARIEL2 Part 1, a phase 2 study of the PARPi rucaparib in ovarian carcinoma, four (2%) tumors demonstrated RAD51C methylation. The present study utilizes HGSOC patient derived xenografts (PDXs) and recurrent samples from ARIEL2 to assess the role of RAD51C methylation in the development of PARPi resistance.

Methods: To drive PARPi resistance, PDX039, an extremely PARPi-sensitive model lacking demonstrable mutations in DNA repair genes, was treated cyclically with niraparib (100 mg/kg) for 21 days, after which the tumor was allowed to regrow and re-established in new mice for the next treatment round. To evaluate the frequency of methylation change, RAD51C methylation was analyzed in 12 rucaparib-treated mice (300 or 450 mg/kg) harboring PDX183, a PARPi-sensitive model without mutations in DNA repair genes. Global changes in gene expression following development of PARPi resistance were assessed by RNA sequencing. RAD51C promoter methylation was evaluated by bisulfite sequencing. Subsequent functional analysis included qRT-PCR, IHC, and western blot. DNA damage response pathways are being evaluated by immunofluorescence ex vivo following niraparib, rucaparib, or IR.

Results: PDX039 grew through PARPi treatment by the third and fourth cycle of therapy. RAD51C was the only DNA repair gene to show significant change in RNAseq analysis (log2 fold-change=8.43; p=2e-192), corresponding with a loss of RAD51C methylation. Moreover, after just one round of PARPi treatment, RAD51C methylation was lost in 1 of 12 PARPi-treated PDX183 xenografts. RAD51C methylation loss ultimately resulted in restoration of expression, for which functional analysis is ongoing. Analysis of patient samples is currently underway.

Conclusions: In HGSOC PDX models, RAD51C methylation affords PARPi sensitivity in the absence of DNA repair gene mutations. Treatment pressure with PARPi can reverse RAD51C methylation and restore RAD51C expression. Isolated changes in methylation of the RAD51C locus are sufficient to restore HR and convey PARPi resistance.

#5886

Translational reprogramming of breast cancer and its role in tamoxifen resistance.

Sofia Bakogianni, Phillip Geter, Amanda Ernlund, Rezina Arju, Abhilash Gadi, Robert J. Schneider. _NYU School of Medicine, New York, NY_.

Breast cancer is the most commonly diagnosed malignancy among women in the U.S. and the second leading cause of cancer-related deaths in women. The estrogen receptor-positive (ER+) breast cancer subtype comprises the majority (70-80%) of breast cancer cases and the majority of late recurrences. Anti-estrogen therapy with tamoxifen remains a cornerstone of adjuvant therapy for ER+ breast cancers in the premenopausal setting, but resistance is inevitable for a third of patients, leading to metastatic disease, which is responsible for the majority of breast cancer deaths. Our work demonstrates that tamoxifen resistance in ER+ breast cancer involves selective mRNA translation mediated by hyperactivation of mTOR and MNK1, two kinases that play major roles in mRNA translation. The hyperactivation of mTOR and MNK1 converges on eukaryotic translation initiation factor 4E (eIF4E) to increase its availability and mRNA selectivity. Using tamoxifen-sensitive and -resistant ER+ cells, we conducted genome-wide transcriptomic and translatomic analyses +/- silencing of eIF4E. We identified key genes responsible for tamoxifen resistance, whose mRNAs are selectively translationally upregulated with increased levels of eIF4E. RUNX2 is a key mRNA that is selectively translationally upregulated, which we show provides resistance by opposing estrogenic signaling and possibly phenotypically reprogramming the cell. Silencing of RUNX2 strongly resensitizes the cells to tamoxifen. We also show that RUNX2 impairs cellular proliferation and cell survival of previously drug-resistant cells in the presence of tamoxifen. Silencing of key translationally upregulated mRNAs, such as RUNX2, can partially restore endocrine therapy sensitivity. Using a tamoxifen-resistant ER+ PDX model, and patient-derived sensitive and resistant tumor tissues (no recurrence or recurrence within 5 years), we validated the hyperactivation of mTOR and MNK1, the overexpression of eIF4E, and selective mRNA translation in tamoxifen resistance. We hypothesize that selection for increased expression of RUNX2 and other selectively translated mRNAs promotes a more "basal-like," estrogen-independent phenotype. We aim to determine how tamoxifen resistance translationally reprograms breast cancer cells to a different breast cancer phenotype (e.g., cancer stem cell-like, basal-like, luminal with disrupted ER signaling).

#5887

A microRNA signature panel predicts differential sensitivity of liver cancer cells to chemotherapeutic drugs.

Volodymyr Tryndyak,1 Iryna Kindrat,1 Brigit McDannell,2 Frederick A. Beland,1 Igor P. Pogribny1. 1 _FDA National Center for Toxicological Research, Jefferson, AR;_ 2 _Smith College, Northampton, MA_.

Hepatocellular carcinoma (HCC) is one of the deadliest human cancers, with a steadily rising incidence in the United States and worldwide. Most HCC patients are diagnosed at intermediate and advanced stages of the disease when treatment options are limited to systematic therapy; however, the individual patient response to treatment varies significantly. In the present study, we investigated an ability of the base-line mircoRNA (miRNA) expression profile in human liver cancer cells to predict the response to chemotherapeutic drugs currently used in the clinical management of HCC. Next-generation sequencing analysis of the base-line miRNA expression in naïve SK-Hep-1, Hep3B, HepG2, Huh7, and PLC/PRF/5 cells showed different miRNA expression profiles, among which 70 miRNAs were in common among all cell lines. A detailed analysis of the common miRNAs revealed that SK-Hep-1 and HepG2 cells exhibited the major differences in their base-line miRNA expression. The cancer cells were then treated with sorafenib, doxorubicin hydrochloride, 5-fluorouracil, and cisplatin, and cell survival was determined by cell viability assays. The sensitivity of liver cancer cells to these chemotherapeutic drugs varied among the cell lines, with SK-Hep-1 cells being the most resistant to sorafenib and the most sensitive to 5-fluorouracil. In contrast, HepG2 cells were the most sensitive to sorafenib, doxorubicin hydrochloride, and cisplatin treatment and the most resistant to 5-fluorouracil. Further analysis of miRNA expression in these two cell lines identified 107 distinct differentially expressed miRNAs (cut-off > 5-fold) involved in the regulation of critical cancer-related pathways. Among these differentially expressed miRNAs, the basal expression pattern of several miRNAs corresponded to the miRNA expression patterns associated with an acquired cancer-cell-resistant phenotype to sorafenib. Specifically, miR-10a and miR-181 were up-regulated and miR-27b, miR-34a, miR-122, miR-200a, and miR-200b were down-regulated in naïve SK-Hep-1 cells. Likewise, the low basal expression of miR-30a, miR-30b, miR-27a, miR-125b, miR-135b, miR-149, and miR-218 and over-expression of miR-143 and miR-200a in naïve HepG2 were similar to that in cells with an acquired 5-fluorouracil cancer-resistant phenotype. These findings were confirmed in functional transfection experiments that showed ectopic modulation of miRNA levels in SK-Hep-1 and HepG2 modified their drug sensitivity to sorafenib and 5-fluorouracil. These results suggest that analysis of the expression of these miRNAs in HCC at the onset of chemotherapy may help to determine the optimal treatment options, prevent the development of multidrug resistance, and improve overall clinical management of HCC.

#5888

Disruption of FOXO3a-miRNAs mediated feedback inhibition of IGF2-IRS1 signaling contributes to Herceptin resistance in HER2 positive breast cancer cells.

Zhimin He. _Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou, China_.

Resistance to HER2-target therapy remains a clinical challenge for HER2+ breast cancer subtype. The molecular mechanisms for Herceptin resistance remain elusive. Here, we demonstrated that the IGF2 and IRS1 protein levels were increased in Herceptin resistant breast cancer cells and that the IGF2/IGF1R/IRS1/Akt/mTOR signaling was involved in Herceptin resistance. In Herceptin sensitive HER2+ breast cancer cells, IRS1 protein level resulted from mTOR2 mediated FOXO3a status determined the feedback regulation of IGF2. Upon high dose rhIGF2 treatment, the mTOR1 translationally upregulated PPP3CB protein level that recovered the function of FOXO3a inactivated by mTOR2. FOXO3a positively transcriptionally regulated miR-128-3p and miR-30-5p expression that targeted IRS1 mRNA to control the feedback regulation of IGF2 signaling. In agreement, in Herceptin resistant cells, the decrease of miR-128-3p and miR-30-5p resulted from constitutively inactivation of FOXO3a by mTOR2, led to increase of IRS1 protein level, even in response to high endogenous IGF2 protein level in culture medium. High IGF2 protein level was resulted from decrease of miR-193a-5p that was also regulated by FOXO3a. Unexpectedly, in Herceptin resistant cells, Src/p-STAT5 signaling cooperating with HDAC1 transcriptionally repressed PPP3CB expression resulting into FOXO3a inactivation. Moreover, we confirmed that IRS1 knockdown reversed the Herceptin resistance in vivo using a xenograft mouse model. Serums form patients with poor response to Herceptin, had higher IGF2 protein level. Tissues from patients with poor response had higher IRS1, p-Akts473, p-FOXO3a and p-Src levels, but had lower PPP3CB, miR-128-3p, miR-30-5p and miR-193a-5p levels. In conclusions, disruption of PPP3CB-FOXO3a-miRNAs mediated feedback inhibition of IGF2-IRS1 signaling plays pivotal role in Herceptin resistance of HER2+ breast cancer. Related molecules in this signaling may represent potential biomarkers and therapeutic targets for HER2+ breast cancer treatment.

Keywords: Breast cancer, Herceptin resistance, IGF2, IRS1, FOXO3a

This study was supported by grants from the National Natural Science Foundation of China: No.81672616, No.81402196 and No.81272450, supported by grants from Guangdong Natural Science Funds for Distinguished Young Scholars No.2016A030306003 (G Zheng).

#5889

**TWIST1 is a key mediator of HGF-MET-driven resistance to targeted therapies in** EGFR **mutant and MET-driven lung cancer.**

Zachary A. Yochum,1 Suman Chatterjee,1 Eric H. Huang,1 Deena M. Maurer,1 Myriam A. Attar,1 Sanja Dacic,2 Laura P. Stabile,3 Timothy F. Burns1. 1 _University of Pittsburgh, Hillman Cancer Center, Pittsburgh, PA;_ 2 _University of Pittsburgh Medical Center, Pittsburgh, PA;_ 3 _University of Pittsburgh, Pittsburgh, PA_.

The c-Met (MET) receptor and its ligand, hepatocyte growth factor (HGF), have been shown to mediate epithelial-mesenchymal transition (EMT), proliferation, invasion, motility, and angiogenesis. The HGF/MET pathway is frequently altered in non-small cell lung cancer (NSCLC) and has emerged as a targetable oncogenic driver, as patients with MET amplification and/or mutations have demonstrated marked responses to the MET tyrosine kinase inhibitor (TKI), crizotinib. However, long-term efficacy of MET TKIs is limited as acquired resistance is inevitable and almost half of patients with MET alterations fail to respond to MET TKIs. HGF overexpression has been identified as a mechanism of resistance to both MET and EGFR TKIs in MET altered and EGFR mutant NSCLC. Furthermore, MET amplification has been implicated in EGFR TKI resistance. However, the mechanism(s) by which the HGF-MET pathway causes resistance are poorly understood. We have previously shown that the EMT-transcription factor, TWIST1, is required for MET-driven NSCLC. Here, we investigated the requirement of TWIST1 in HGF-mediated resistance to MET and EGFR TKIs and the role of TWIST1 in de novo and acquired resistance to MET and EGFR TKIs. We found that HGF treatment induced EMT in NSCLC cell lines and increased TWIST1 protein expression through a post-translational mechanism. We demonstrated that targeting TWIST1 pharmacologically with the TWIST1 inhibitor, harmine, overcame HGF-mediated resistance to both MET and EGFR TKIs in MET and EGFR-driven NSCLC. This suggests that TWIST1 is specifically required for HGF-mediated resistance to targeted therapies. We also found that TWIST1 is overexpressed in a subset of MET and EGFR altered cell lines and TWIST1 overexpression was sufficient to cause resistance to MET and EGFR TKIs. In MET-driven and EGFR mutant cell lines that express TWIST1 and are resistant to targeted therapies, we demonstrated that harmine treatment resensitized resistant cells to MET and EGFR TKIs, respectively. To investigate the role of TWIST1 overexpression in Hgf-driven lung cancer, we utilized a CCSP-Hgf (CH) mouse model that constitutively overexpresses Hgf in the lung and develops crizotinib-sensitive tumors following treatment with the tobacco carcinogen, nicotine-derived nitrosamine ketone (NNK). We demonstrated that the Twist1 overexpressing CTH (CCSP-rtTA/Twist1-tetO-luc/CCSP-Hgf) mice developed significantly larger tumors in response to NNK as compared to CH and CCSP-rtTA/Twist1-tetO-luc (CT) mice. In summary, we established that HGF-regulated TWIST1 expression and that TWIST1 expression is required for resistance to MET and EGFR TKIs in the presence and absence of HGF overexpression. These studies suggest that targeting TWIST1 may be an effective therapeutic strategy to overcome HGF-MET-driven resistance in EGFR mutant NSCLC as well as MET TKI resistance in MET-driven NSCLC.

#5890

Aurora-A potentiates chemoresistance via regulating the microRNA biogenesis pathway.

Li Jyuan Lin. _Institute of Bioinformatics and Biosignal Transduction, National Cheng-Kung University, Tainan, Taiwan_.

Chemotherapy has a good success rate in colorectal cancer; however, recurrence of colorectal cancer is still frequent due to the development of drug resistance. MicroRNA (miRNA) is a type of non-coding RNA with a size of around 19-22 nucleotides and plays an important role in regulating many biological functions, the onset of diseases, as well as drug response. It was reported that Dicer, one of the key enzymes of the miRNA biogenesis pathway, may be involved in the chemoresistance through regulating the expression of miRNAs. Aurora-A, a cell cycle-regulated kinase, plays a vital role in cancer development and chemoresistance. According to the TCGA database, the expression levels of Dicer and Aurora-A are both increased in colorectal cancer. Here, we demonstrated that overexpressed Aurora-A can regulate the chemodrug response by influencing the miRNA biogenesis pathway via Dicer in colorectal cancer. Our results showed that the protein level, but not the mRNA expression, of Dicer was decreased upon 5-FU or oxaliplatin treatment in drug sensitive cells; whereas, the expression level of Dicer was increased in oxaliplatin resistant cell lines. Knockdown expression of Dicer in oxaliplatin resistant cell lines could reverse the resistance of oxaliplatin. The expression levels of several potential miRNAs changed upon chemodrugs treatment. Interestingly, we found that overexpressed Aurora-A could increase the expression of Dicer and potentiate drug resistance in colorectal cancer cells. In contrast, knockdown expression of Aurora-A decreased Dicer protein expression and led to enhanced drug sensitivity. Taken together, our results suggested that Aurora-A and Dicer can collaborate to regulate drug response through influencing the miRNA biogenesis pathway in colorectal cancer. The further mechanism of Dicer and Aurora-A in chemoresistance is currently under our investigation.

#5891

ABCG2 expression contributes to CC-115 resistance.

Jenny Beebe. _Indiana University School of Medicine, Indianapolis, IN_.

Introduction: A major obstacle in the treatment of cancers is innate or acquired resistance. Multi-drug resistance proteins such as MDR1, ABCG2, MRP1, and LRP have been shown to be involved in resistance and are predictors of clinical outcomes. Expression of ABCG2, an ATP-binding cassette efflux transporter, may be responsible for observed variability of the efficacy of CC-115, a dual mTOR/DNA-PK inhibitor, currently in clinical trials. Here we evaluate the role of ABCG2 in CC-115 resistance.

Methods: Western blots, methylene blue and MTT assays, and FACS were used to determine ABCG2 expression, cell survival/viability with and without inhibitors, and accumulation of CC-115 respectively.

Results: MCF7/AdVp3000 (M3K) cells have been shown to have a significant increase in ABCG2 as compared to parental MCF7 cells. M3K cells had a 50-fold increase in IC50 to CC-115 compared to MCF7. Use of ABCG2 inhibitors FTC and C8 (1uM), decreased the IC50 of CC-115 in M3K cells by 10-fold. Accumulation studies using FACS showed that M3K cells had significantly lower accumulation of CC-115 as compared to MCF7 and addition of FTC and C8 (1uM) significantly enhanced accumulation of CC-115 to the same extent as seen in MCF7 cells. Next, HEK293/ABCG2 stable clone with overexpression of ectopic ABCG2 and its control HEK293/vec cells were used to determine ABCG2's direct impact on CC-115 resistance. HEK293/ABCG2 cells had a 10-fold increase in resistance to CC-115 with an IC50 of 2.05 uM as compared to 0.23 uM in HEK293/vec cells. Addition of FTC and C8 (1uM) decreased the IC50 by 10-fold in HEK293/ABCG2 cells. Accumulation of CC-115 in HEK293/ABCG2 cells was also significantly lower than that in HEK293/vec cells and addition of FTC and C8 (1uM) significantly increased its accumulation only in HEK293/ABCG2 cells.

Discussion: The results from this study suggest that ABCG2 contributes to and possibly is responsible for CC-115 resistance and that inhibiting ABCG2 could lead to reversal of CC-115 resistance, increased response rate, highlights the importance of personalized therapies, and has the potential to increase patient survival.

#5892

miR-217 sensitizes chronic myelogenous leukemia cells to tyrosine kinase inhibitors by targeting pro-oncogenic anterior gradient 2.

Bin Pan,1 Xiangmin Wang,1 Kailin Xu,2 Takayuki Ikezoe1. 1 _Fukushima Medical University, Fukushima, Japan;_ 2 _The Affiliated Hospital of Xuzhou Medical University, Xuzhou, China_.

BCR-ABL1 independent mechanisms had been indicated to mediate drug resistance to tyrosine kinase inhibitors (TKIs) in patients with chronic myelogenous leukemia (CML). In this study, we observed increased level of pro-oncogenic anterior gradient 2 (AGR2) in TKIs-resistant CML cells. Silencing of AGR2 in dasatinib-resistant K562 (K562DR) cells led to restored sensitivity to dasatinib both in vitro and in vivo. Exposure to dasatinib induced up-regulation of AGR2 in K562 cells, which indicated a probable treatment related drug resistance. We further investigated the potential interaction between microRNA (miRNA) and AGR2 in K562DR cells, and found that down-regulation of miR-217 was associated with over-expression of AGR2 in K562DR cells. Luciferase reporter assay identified that miR-217 negatively regulated expression of AGR2 through binding 3'-untranslated region (3'-UTR) of AGR2. Forced expression of miR-217 led to decreased expression of AGR2 as well as compromised TKIs-resistant potential of K562DR cells. Similarly, over-expression of miR-217 re-sensitized K562DR cells to dasatinib-treatment in a murine xenograft transplantation model. Taken together, TKIs-treatment induced drug-resistance is correlated with decrease of miR-217 and up-regulation of AGR2. miR-217/AGR2 interaction might be a potential therapeutic target in treating CML patients with TKIs-resistant.

#5893

Expression and role of autophagy-associated p62 (SQSTM1) in multidrug-resistant ovarian cancer.

Jinglu Wang,1 Cassandra Garbutt,2 Francis J Hornicek,1 Zhenfeng Duan1. 1 _UCLA David Geffen School of Medicine, Los Angeles, CA;_ 2 _Massachusetts General Hospital, Boston, MA_.

Multidrug resistance is the major cause of treatment failure in ovarian cancer. p62 (SQSTM1) is a multifunctional protein involved in multiple cellular processes including proliferation, drug sensitivity and autophagy-associated cancer cell growth. p62 is a critical indicator of autophagic flux, which is inversely associated with autophagy activity. However, the role of p62 remains controversial in drug resistance in human ovarian cancer. In this study, we examined p62 expression by immunohistochemistry in a unique ovarian cancer tissue microarray (TMA), which was constructed with paired primary, metastatic, and recurrent tumor tissues from 26 individual patients. Results showed that both the metastatic and recurrent tumor tissues expressed less p62 than the patient-matched primary tumor. A significant inverse correlation has been found between p62 expression and both the disease free survival and overall survival. In addition, multidrug resistant cancer cell lines expressed lower levels of p62 as compared with their parental drug sensitive cell lines. Importantly, cell viabilities determined by MTT assay after exposure to different concentrations of paclitaxel showed inhibition of autophagy or accumulation of p62 enhances paclitaxel sensitivity in ovarian cancer drug resistant cells. Furthermore, the wound healing assay exhibited that inhibition of autophagy significantly decreased multidrug resistant ovarian cancer cell migration in vitro. Collectively, these data highlight that autophagy pathway may be a promising therapeutic target to prevent metastasis, recurrence and to reverse drug resistance in ovarian cancer.

#5894

Snail reduces the antitumor efficacy of mTOR kinase inhibitors by transcriptional repression of 4E-BP1.

Qing Ye,1 Jun Wang,1 Yanan Cao,1 Yubin Guo,1 Xiuping Huang,1 Wenting Mi,1 Side Liu,2 Chi Wang,1 Hsin-Sheng Yang,1 Binhua Zhou,1 Bernard M. Evers,1 Qing-Bai She1. 1 _Univ. of Kentucky, Lexington, KY;_ 2 _Southern Medical University, Guangzhou, China_.

The mTOR inhibitors, including the second generation of mTOR kinase inhibitors, for cancer treatment have been evaluated in clinical trials, but their overall activity as monotherapy is limited. The cap-dependent translation repressor, 4E-BP1, is an important downstream effector of the mTOR kinase in controlling cell proliferation and tumor growth. Loss of 4E-BP1 expression has been linked to cancer progression and resistance to mTOR inhibitors. However, the molecular mechanism underlying 4E-BP1 downregulation in tumors remains unclear. Here, we identify Snail as a strong transcriptional repressor of 4E-BP1. We find that 4E-BP1 expression inversely correlates with Snail level in cancer cell lines and clinical specimens. Snail binds to three E-boxes present in the human 4E-BP1 promoter to block the transcription of the 4E-BP1 gene. Ectopic expression of Snail in cancer cell lines lacking Snail profoundly represses 4E-BP1 expression, promotes the formation of the eIF4F translation initiation complex for active translation in polysomes, and reduces the antiproliferative effect of mTOR kinase inhibitors. Conversely, genetic disruption of Snail or pharmacologic inhibition of Snail activity using the Snail co-repressor HDAC inhibitors restores 4E-BP1 expression and sensitizes cancer cells to mTOR kinase inhibitors by enhancing 4E-BP1-mediated translation-repressive effect on cell proliferation and tumor growth. Together, our findings reveal a novel oncogenic function of Snail in mTOR/4E-BP1-mediated translational control of tumorigenesis, and provide a rationale for targeting Snail to improve mTOR-targeted therapies.

#5895

Targeting RhoA-regulated gene transcription in drug-resistant melanoma.

Sean A. Misek, Kathleen A. Gallo, Richard R. Neubig. _Michigan State Univ., East Lansing, MI_.

Much of the recent focus of melanoma targeted therapy has been on the ERK pathway, which is aberrantly activated in approximately 90% of melanoma tumors. Over half of these express BRAFV600E. Current targeted therapies such as Vemurafenib (BRAFV600E inhibitor), or a combination therapy of BRAF + MEK inhibitors show profound initial effects in a majority of BRAFV600E expressing tumors. However, these responses are often short-lived and resistance typically develops within months. The goal of this work is to identify pharmacologically targetable resistance mechanisms so that effective combination therapies can be developed.

Preliminary bioinformatics analysis suggests that the RhoA subfamily of Rho GTPases is activated in BRAFi-resistant cancer cell lines and BRAFi/MEKi-resistant human melanoma tumors. Thus, we hypothesize that RhoA promotes BRAFi resistance, so simultaneously targeting RhoA and BRAF may reverse drug resistance. Using cell line models of acquired BRAFi-resistance we demonstrated that the RhoA pathway is activated in a subset of resistant cell lines. The cell lines with increased RhoA activation have increased sensitivity to multiple ROCK inhibitors.

In addition to regulating the cytoskeleton, RhoA can also regulate gene transcription through activation of multiple transcriptional co-activators, including MRTF and YAP. MRTF and YAP regulated gene transcription through their role as transcriptional co-activators and their interaction with chromatin remodeling complexes. MRTF and YAP are both activated in BRAFi-resistant cell lines. Indirectly targeting MRTF with CCG-222740 or YAP with the YES1 inhibitor Dasatinib effectively kills BRAFi-resistant melanoma cells.

Sox10 was previously identified as a gene which promotes BRAFi resistance. Expression of Sox10 is downregulated, and Sox9 is upregulated, 100-1000-fold exclusively in the resistant cell lines which have RhoA activation. Further, a Sox10 gene signature is inversely correlated with a RhoA/C signature in the TCGA dataset and melanoma scRNA-seq data. Sox10 loss is a major drug resistance melanoma in melanoma. These data suggest that targeting RhoA-regulated gene transcription may be an effective mechanism to target Sox9High/Sox10Low cells, and ultimately prevent or reverse drug resistance.

Taken together these data suggest that MRTF and YAP promote Vemurafenib resistance in Sox9High/Sox10Low melanoma cells.

#5896

Downregulation of calcium-sensing receptor enhances MET-mediated prostate cancer chemosensitivity.

Luying Huang,1 Fuhao Wang,2 Haibo Tong,2 Atsushi Mizokami,3 Evan T. Keller,4 Yi Lu,2 Jian Zhang2. 1 _Guangxi Medical University, China;_ 2 _Southern University of Science and Technology, China;_ 3 _Kanazawa University, Japan;_ 4 _University of Michigan, Ann Arbor, MI_.

Chemoresistance, in various types of cancer including prostate cancer (PCa), is one of the major challenges in cancer therapy. Others and our lab have reported that the epithelial-to-mesenchymal transition (EMT) plays critical roles in development of chemoresistance and metastasis. The calcium-sensing receptor (CaSR) plays important roles in maintenance of calcium homeostasis and altered CaSR expression, and functional changes are involved in certain diseases, including tumorigenesis and its development. However, the mechanism of CaSR in PCa chemoresistance remains unclear. In current study, the CaSR expression level was detected in parental PC3 and DU145 cells and their chemoresistant PC3-TxR and DU145-TxR cells. We found the CaSR was highly expressed in chemoresistant PC3-TxR and DU145-TxR cells. Then by using siRNA technique, CaSR expression was transiently knocked down in chemoresistant cells. The tumor cell migration, invasion, and colony formation were evaluated by wound healing, transwell, and colony formation assays, respectively. The drug sensitivity was evaluated using MTS assay. We observed that the abilities of tumor cell migration and invasion were significantly diminished in CaSR knockdown PC3-TxR and DU145-TxR cells. The ability of colony formation was also reduced. Interestingly, the sensitivity to paclitaxel was reactivated in CaSR knockdown PC3-TxR and DU145-TxR cells. We finally observed that E-cadherin expression was increased in CaSR knockdown chemoresistant cells, and Snail, Vimentin and β-catenin were decreased; these results are consistent with EMT phenotypic alterations. Taken together, we conclude that downregulation of CaSR enhances MET-mediated prostate cancer chemosensitivity.

Supported by NSFC Key Project 81130046; NSFC projects 81773146,81272415; JCYJ20170412152943794, JCYJ20170412154619484, JCYJ20170307105128101, JCYJ2017030711041760; Guangxi Key Projects 2013GXNSFEA053004.

#5897

Role of polymerase II associated factor 1, PAF1, in docetaxel resistant prostate cancer cells.

Sakthivel Muniyan, Rama Krishna Nimmakayala, Saswati Karmakar, Satyanarayana Rachagani, Jawed A. Siddiqui, Parthasarathy Seshacharyulu, Ming-Fong Lin, Kaustubh Datta, Moorthy P. Ponnusamy, Surinder K. Batra. _University of Nebraska Medical Center, Omaha, NE_.

Background: Docetaxel remains the first-line therapeutic intervention for metastatic and castration-resistant (CR) prostate cancer (PCa). However, the therapeutic efficacy is limited due to unresponsiveness, toxicity, and drug-resistance. The availability of additional therapies increases the PCa patient survival modestly, but the development of cross-resistance limit the therapeutic efficacy. Hence, there is a need to understand the mechanisms of resistance and identify a novel target for the better management of CR PCa patients. Methods: Docetaxel resistant 22Rv1, LNCaP C-81, and PC3 PCa cells were established, and its resistance phenotype was determined by cell growth inhibition (MTT assay), apoptosis markers (western blot), tumor sphere assay, drug efflux property (SP analysis by FACS), auto fluorescence (FACS). qRT-PCR, western blot, and confocal microscopic analysis were also performed to confirm the drug-resistant marker phenotype. Additional experiments were performed to determine the underlying molecular mechanisms of docetaxel resistance. Results: The docetaxel resistant PCa cells grew well at the higher concentration of docetaxel (120, 15 and 50 nM of docetaxel, respectively) and did not result in apoptosis as measured by cPARP and caspase-3 cleavage. Docetaxel resistant PCa cells confer cross-resistance to second-generation chemotherapeutic agent cabazitaxel and show altered cell proliferation and invasion. Blocking by the ABCB1 specific inhibitor enhances docetaxel-induced cell death on par with parental cells. Side population analysis by flow cytometry confirms the acquired drug efflux property. Side population, autofluorescence and tumor sphere analyses confirmed the drug-resistance and stem-like cell phenotype. qRT-PCR, western blot and confocal microscopy show the abundant expression levels of the drug transporters, ABCB1 and ABCG2. Further, the docetaxel resistant PCa cells show higher stem cell network proteins such as PAF1, POU5F1, NANOG and SOX9 expression levels compared to age-matched control cells. Conversely, Tet-inducible PAF1 knockdown reduces embryonic stem cell network proteins and reverses docetaxel-resistance phenotype. Conclusions: Collectively, our study suggests that the stem cell factors such as PAF1 play a major role in docetaxel resistance and aggressiveness to PCa cells. Understanding the associated mechanisms and targeting these factors could lead a better management approach for CRPCa patients.

#5898

A systems approach for determining the mechanism of resistance to tumor treating fields in glioblastoma.

Dongjiang Chen, Son Le, Nagheme Thomas, David D. Tran. _University of Florida, Gainesville, FL_.

Glioblastoma (GBM) is the most common and deadliest malignant brain cancer in adults despite aggressive chemoradiotherapy. Recently, Tumor Treating Fields (TTFields) was approved in combination with adjuvant temozolomide chemotherapy for newly diagnosed GBM. The addition of TTFields resulted in a significant improvement in overall survival. TTFields are low-intensity alternating electric fields that are thought to disturb mitotic macromolecules' assembly, leading to disrupted chromosomal segregation and cell death. However, the majority of GBM are not sensitive to TTFields and most of TTFields responders eventually develop resistance. However the mechanism of resistance remains largely unexplored. Understanding how cancer cells gain the ability to circumvent the biophysical forces of TTFields and their downstream effects will provide new opportunities to improve therapeutic efficacy of this novel anti-cancer treatment modality. To accomplish these objectives, we have developed several human GBM cell lines that demonstrated relative resistance to the cytotoxic effects of TTFields compared to the parental cells. Importantly TTFields-induced chromosomal instability such as the formation of micronuclei was unchanged in resistant cells compared to their sensitive counterparts, suggesting that resistance to TTFields may be conferred by a selective loss of deleterious downstream reactions to the biophysical damages. To systematically identify the mechanism of resistance, we have recently developed two innovative computational platforms, GeneRep and nSCORE and experimentally validated them in several published datasets. The tandem platforms complemented each other and allowed us to 1) interrogate large-scale gene expression profiles of pairs of TTFields resistant cells vs. sensitive counterparts to extract candidate resistance-specific gene regulatory networks, and 2) functionally rank specific core factors responsible for resistance, respectively. Once identified, biological confirmation of these factors using cell lines, mouse models and samples of patient's derived TTFields sensitive and resistant GBM. Our long-term goal is to develop targeted therapy to prevent tumor's resistance to TTFields therapy.

#5899

Genome-wide CRISPR-Cas9 screening identifies genetic vulnerabilities and potential therapeutic targets in castration resistant prostate cancer.

Jakob Haldrup, Linnéa Schmidt, Jakob S. Pedersen, Karina D. Sørensen. _Department of Molecular Medicine (MOMA), Aarhus University Hospital, Aarhus N, Denmark_.

Background: Treatment options for castration-resistant prostate cancer (CRPC) are limited and only a few agents (e.g. enzalutamide, docetaxel) are routinely used in the clinic. Unfortunately, CRPC tumors will invariable develop resistance to these agents and only a subset of patients will respond. Thus, novel predictive biomarkers are urgently needed to ensure that an expensive and potentially harmful agent is given only to patients who will benefit from it. Furthermore, to enable new treatment strategies, a better understanding of drug resistance mechanisms is required.

Methods: To identify novel drug resistance genes and mechanisms of therapy resistance, we performed genome-wide CRISPR-Cas9 knockout screens in LNCaP (hormone naïve) and in the isogenic C4 (castration resistant) PC cell line, respectively. Using 77,441 unique sgRNAs (Brunello library), a total of 19,114 protein-coding genes were tested for their potential functional role in enzalutamide or docetaxel resistance. Both IC50 and IC90 values were used for selection. MAGeCK was used to identify enriched and depleted sgRNAs (treatment vs. DMSO).

Results: Among the highest ranked hits for the C4 dropout screens, several potential genetic vulnerabilities and mechanisms of resistance were identified, as knockout of specific genes sensitized C4 cells to enzalutamide. None of these hits were observed in LNCaP, which suggest CRPC-specific resistance mechanisms. For the positive screens, increased enzalutamide resistance was observed after knockout of genes encoding e.g. phosphokinases and specific solute carriers (SLCs) for C4 and LNCaP, respectively. Lastly, our results suggest that knockout of specific zinc finger nucleases (ZFNs), posttranslational modification enzymes or microtubule components may modulate docetaxel resistance in C4 cells.

Conclusion: Drug resistance is a major clinical problem. Here, we identified genetic vulnerabilities that may be translated into predictive biomarkers, combination therapies and/or novel drug development strategies for CRPC.

#5900

Inhibition of CYP3A5 down-regulates cancer drug resistance genes and induces synthetic lethality in prostate cancer cells.

Priyatham Gorjala,1 Ranjana Mitra,1 Oscar Goodman2. 1 _Roseman Univ. of Health Sciences, Las Vegas, NV;_ 2 _Roseman Univ. of Health Sciences and Comprehensive Cancer Centers of Nevada, Las Vegas, NV_.

Background and objective: Androgen deprivation therapy (ADT) is among the most effective of all cancer therapies for prostate cancer; however, resistance to therapy leading to castrate resistant prostate cancer (CRPC) is inevitable in most patients. Recently we demonstrated that CYP3A5 expressed in prostate cancer cells facilitates the nuclear translocation of AR, directly impacting the transcriptional regulation of AR-dependent genes promoting prostate cancer cell growth. Our preliminary data show that CYP3A5 is a part of the HSP90-AR complex, known to regulate AR nuclear localization and activation. Additionally, we observed that CYP3A5 inhibition also blocks growth of prostate cancer cells lacking AR ligand binding site (22RV1). This observation suggested presence of an alternative mechanism of regulation of prostate cell growth by CYP3A5, independent of its AR activation. We hypothesize that CYP3A5 inhibition can also sensitize prostate cancer cells to therapy by downregulating expression of drug resistance genes independent of its AR regulation. Our aim is to decipher the role of CYP3A5 in therapeutic resistance, by screening for CYP3A5 regulated cancer drug resistance genes and to further understand the underlying mechanism of this regulation.

Method: We used cancer drug resistance PCR array after CYP3A5 siRNA knockdown to identify CYP3A5 regulated genes involved in therapeutic resistance. Further we evaluated the role of CYP3A5 in inducing synthetic lethality in combination with DNA damage repair inhibiting drugs by imagining the γ-H2AX foci.

Results: CYP3A5 siRNA knockdown downregulates 11 genes involved in cancer drug resistance: TOP2A, BRCA1/2, CCNE1, CDK2/4, DHFR, MVP, MYC, RARB and HPRT1 with a p≤0.005 value. Of the list of downregulated genes, the three genes TOP2A, BRCA2 and BRCA1 have the most fold change and their loss is known to impair homologous recombination (HR), leading to synthetic lethality. HR defects in CRPC, sensitizes these tumors to PARP and topoisomerase inhibitors. We tested the effect of CYP3A5 inhibition in inducing HR defects by analyzing γ-H2AX foci counts in combination with topoisomerase inhibitor etoposide. Increased number of γ-H2AX foci suggest that CYP3A5 siRNA may induce synthetic lethality in prostate cancer cells when used in combination with DNA damage repair inhibiting drugs by impairing HR and downregulating TOP2A and BRCA1/2 expression.

Conclusions:

CYP3A5 inhibition downregulates DNA damage repair genes (TOP2A and BRCA1/2) and cell cycle progression genes (CCNE1, CDK 2/4) and impairs HR. CYP3A5 inhibition can sensitize CRPC to PARP and topoisomerase inhibitors as it induces synthetic lethality by downregulating TOP2A and BRCA1/2 expression. CYP3A5 inhibition can be potentially exploited therapeutically in combination with PARP inhibitors as it functionally impairs HR and causes synthetic lethality.

#5901

The role of POU3F2 in the aggressive behavior of neuroendocrine prostate cancer.

Naoko Kobayashi, Dai Hong, Chun Wang, Joyce Yamashiro, Johnny Guan, Robert E. Reiter. _UCLA, Los Angeles, CA_.

Neuroendocrine and Small Cell Prostate Cancer (NEPC) is a lethal disease emerging from Castrate Resistant Prostate Cancer (CRPC) after treatment with next-generation anti-androgen therapies such as abiraterone and enzalutamide. The mechanisms by which treatment resistant prostate epithelial cancer cells trans-differentiate to poorly differentiated small cell cancer are not well understood. We and others found that Nervous System-Specific Octamer-Binding Transcription Factor (POU3F2) expression is induced in NEPC. POU3F2 is a reprogramming transcription factor that controls cellular differentiation and can induce stem-like cells from differentiated glioblastoma. POU3F2 also plays a critical role in melanoma progression and in small cell carcinoma of the lung. NEPC shares similarities with small cell carcinoma from other organs. We hypothesized that POU3F2 may be a key transcription factor that is required for transdifferentiation and the survival of NEPC cells. We further hypothesize that the underlying mechanism of therapy resistance in NEPC is that NEPC consists of stem-like cells that were reprogrammed from differentiated prostate cancer cells under the selective pressure of androgen receptor (AR) pathway-targeted therapy. We elucidated the functional role of POU3F2 in the aggressive cancer behavior of NEPC —including stem-cell properties, tumorigenesis, invasion, and chemo-resistance. We also characterized the mechanisms that regulate POU3F2 expression in therapy-resistant NEPC. The study provides a rational for targeted therapy for treatment of NEPC. It could also be used to design a strategy to halt the emergence of NEPC from CRPC.

#5902

RNA sequencing analysis of taxane-resistant prostate cancer cells reveals potential candidate genes for therapeutic targeting.

Christina K. Cajigas-Du Ross, Leanne Woods-Burnham, Joshua Ramirez, Xin Chen, Charles Wang, Carlos A. Casiano. _Loma Linda Univ. School of Medicine, Loma Linda, CA_.

There is no cure for metastatic prostate cancer (PCa), the most commonly diagnosed cancer and third leading cause of cancer mortality among U.S. men. Patients with advanced PCa often develop resistance to conventional therapies resulting in metastatic castration-resistant PCa (mCRPC). Docetaxel (DTX) chemotherapy is the standard-of-care for mCRPC, followed by secondary chemotherapy with cabazitaxel (CBZ), yet chemoresistance and death inevitably occurs. While pharmacological agents targeting single survival pathways have shown promise at the bench side, clinical trials have failed. This is mostly due to the presence of redundant pathways utilized by PCa tumor cells to maintain chemoresistance. Therefore, we are interested in further defining molecular pathways associated with DTX-resistance in order to identify novel therapeutic targets to overcome this resistance. We hypothesized that RNA sequencing (RNAseq) analysis comparing the transcriptomes of chemosensitive and chemoresistant mCRPC cells will reveal potential targets for combinatorial therapies. RNAseq analysis of DTX-sensitive and -resistant PC3 and DU145 cells revealed a number of differentially regulated genes that were either unique to each cell line or common to both. We found that dipeptidyl peptidase 4 (DPP4), fatty acid binding protein 5 (FABP5), nestin (NES), and tetraspanin 8 (TSPAN8) were among the top most robustly and significantly upregulated genes in both DTX-resistant PC3 and DU145 cell lines. In-house qPCR studies validated the RNAseq results for these genes, and Western blotting analysis showed increased expression of these proteins in DTX-resistant cells, which correlated with increased mRNA expression. Interestingly, NES, TSPAN8, DPP4 are genes associated with development of the cancer stem-cell (CSC) phenotype. Other genes associated with CSCs were also upregulated in the DTX-resistant cells, suggesting that in vitro selection of mCRPC cells for DTX resistance may enrich for CSCs. This novel finding is consistent with recent studies implicating CSCs in PCa chemoresistance. Further analysis and validation of our RNAseq data will increase our understanding of DTX-resistance mechanisms in PCa cells, including the yet undefined functional role of CSC-related genes. These studies also reveal potential therapeutic targets to circumvent DTX resistance in PCa.

#5903

The molecular mechanism for producing BQ323636.1 in Tamoxifen resistance breast cancer cells.

Ka Chun Mok, Ho Tsoi, Ui-Soon Khoo, Ellen Man. _Univ. of Hong Kong, Hong Kong, Hong Kong_.

Breast cancer is one of the prevalent causes of cancer in women. Two thirds of breast cancer patients are ER-positive and can be benefited from tamoxifen treatment. However, 50% of the patients will eventually develop the resistance. Identifying molecular targets associated with tamoxifen resistance would help in designing better therapeutic strategies. Previous study from our group identify a novel factor BQ323636.1 (BQ), a spliced variant of NCOR2, could confer tamoxifen resistance. From our in vivo study, nuclear enrichment of BQ in primary breast tumor was found to be associated with tamoxifen resistance. However, the molecular mechanism governed the nuclear import of BQ and was not clear. From bioinformatics prediction, we identified a functional nuclear localization signal (NLS) on BQ. We therefore performed a protein screening to search for the import receptor which could interact with BQ. KPNA1 was found to interact with BQ. To further validate such an interaction, we fused the NLS to GPF reporter protein (GFP-NLS) and performed a co-immunoprecipitation. The result showed that the GFP-NLS was able to interact with KPNA1. Knockdown of KPNA1 by RNAi could prevent the nuclear import of BQ. This confirmed the functional role of KPNA1 in the nuclear import of BQ. In addition, knockdown of KPNA1 in BQ overexpressing breast cancer cells could reverse the tamoxifen resistance. The results highlighted the functional significance of KPNA1 in the tamoxifen resistance. Subsequently, we investigated the functional role of BQ in the nucleus. We found BQ could deplete the gene repressive function of NCOR2. We found that NCOR2 could interact with HSF4 and should repress the expression of HIF1α. In the presence of BQ, the repressive function of NCOR2 on HIF1α was compromised, leading to up-regulation of HIF1α. To further validate the effect of HIF1α in the tamoxifen resistance, HIF1α inhibitor was employed and found that the treatment of the inhibitor could reduce the resistance in BQ overexpressing breast cancer cells. Therefore, our study uncovered the role of KPNA1 in the development of tamoxifen resistance through which KPNA1 could promote the nuclear import of BQ. Subsequently, BQ could lead to the up-regulation of HIF1α which also play significant role in the development of tamoxifen resistance.

#5904

Post-transcriptional regulation of androgen receptor by DDX3 in prostate cancer progression.

Jordan E. Vellky, William Ricke. _University of Wisconsin-Madison, Madison, WI_.

Purpose

Prostate cancer (CaP) driven by androgen receptor (AR) can be targeted therapeutically by androgen deprivation therapy (ADT); however, in 10-20% of cases, ADT fails, allowing disease recurrence. Mechanisms for recurrence include dysregulation of AR at the gene, RNA, and protein level. Recently, AR negative cell growth has been implicated as a mechanism of resistance. This study aims to identify mechanisms of drug resistance in castration-resistant prostate cancer (CRPC) that can be exploited therapeutically to reduce disease recurrence.

Experimental Procedures

Using the BCaP model, a novel series of cell lines derived from a single human prostate epithelial cell, we can study the progression of CaP from benign to metastasis. Additionally, using the LNCaP-C4 series we can study progression to CRPC. To determine the RNA binding capacity of DDX3, we utilized 1) RIP-qPRC for identification of mRNA targets and 2) RNAscope for visual colocalization. Changes in RNA and protein expression were determined using qPCR, Western blot, and IF.

Results

DDX3 is an ATP-dependent RNA helicase that can aid translation of target mRNAs, or in stress conditions, form ribonucleoprotein granules that prevent translation. While DDX3 is implicated in several cancers, its role as a translational regulator in CaP remains unstudied. In BCaP and LNCaP-C4, DDX3 protein expression increases through progression, concurrent with localization to cytoplasmic puncta. RIP-qPCR identified AR as an mRNA target of DDX3 in the metastatic and CRPC cell lines. Because of the dual role of DDX3 in translational control, AR expression in these models was investigated; while AR mRNA expression increased through progression, AR protein expression decreased. Treatments in vitro with RK33, a commercially available inhibitor of DDX3, restored the protein expression of AR and downstream AR signaling (PSA, Nkx3.1). These data suggest DDX3 acts as a translational repressor of AR in metastasis and CRPC. To confirm that increased DDX3 expression is sufficient to reduce AR protein expression, we overexpressed DDX3 using two models: induced cellular stress and a DDX3 expression vector. Here, we saw DDX3 localized to puncta, and subsequent reduction in AR and PSA protein. Because DDX3 is sufficient to reduce AR protein expression in metastases and CRPC, co-treatment of DDX3 inhibitors with anti-androgen therapy may prevent AR negative cell growth underlying recurrence. in vitro co-treatments with RK33 and bicalutamide, an AR antagonist, show decreased proliferation compared to either treatment alone, suggesting increased efficacy of bicalutamide with DDX3 inhibition.

Conclusions

DDX3 as a repressor of AR translation could have clinical implications as a mechanism of resistance to ADT. Based on preliminary data, DDX3 could be contributing to the regulation of AR post-transcriptionally, and targeting DDX3 could reduce resistance and disease recurrence.

#5905

Sp1 acetylation associates with stemness characteristics in temozolomide-resistant glioblastoma.

Jian-Ying Chuang,1 Che-Chia Hsu,1 Kwang-Yu Chang,2 Wen-Chang Chang1. 1 _Taipei Medical University, Taipei, Taiwan;_ 2 _National Health Research Institutes, Tainan, Taiwan_.

The prognosis of glioblastoma (GBM) is usually poor even following treatment with the first-line chemotherapeutic agent temozolomide (TMZ). One most known resistant mechanism is the presence of DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). However, compared with MGMT-mediated innate TMZ resistance, the development of acquired resistance is considered more complex with multi-factorial involvement such as the presence of cancer stem cells (CSCs). In this study, we treated the MGMT-negative GBM cells with TMZ to investigate the acquired resistance, and found that both histone deacetylases (HDACs) and Sp1 are key factors protecting GBM against TMZ. These results include the following: (1) Stemness markers were highly increased in TMZ-resistant GBMs; (2) The activity of HDACs affected the stem-like characteristics and cell survival of GBM stem cells (GSCs); (3) An HDAC1/2/6-selective inhibitor MPT0B291 increased TMZ-sensitivity and induced senescence in TMZ-resistant cells; (4) MPT0B291 suppressed anti-senescence genes (hTERT and BMI1) expression via inhibition Sp1 transactivation; (5) Both HDACs and Sp1 were elevated and interacted with each other in GSCs and resistant GBM cells; (6) TMZ treatment induced Sp1 deacetylation, but MPT0B291 attenuated that. In summary, we verified that HDACs increases Sp1 activation via protein deacetylation and causes Sp1-downstream target upregulation, which may enrich stemness properties and protect GBM against chemotherapeutic drugs.

#5906

EGFR and SOX2 crosstalk determines EGFR-TKI.

An-Chun Lee,1 Chia-Cherng Yu,2 Yuan-Hung Wang,3 Yu-Ting Chou1. 1 _National Tsing Hua University, HsinChu, Taiwan;_ 2 _National Taiwan University Hospital, Taipei, Taiwan;_ 3 _Taipei Medical University-Shuang Ho Hospital, New Taipei City, Taiwan_.

Activating mutations in the epidermal growth factor receptor (EGFR), while initiating lung tumorigenesis, render tumors susceptible to EGFR-tyrosine kinase inhibitors (TKIs). SOX2 functions as a cell fate determination factor in lung progenitor cells. However, how SOX2 expression affects TKI treatment and tumor invasion in lung cancer is obscure. Here we report that EGFR mutations crosstalk with SOX2 to maintain cell proliferation and barrier properties in lung cancer cells, some of which generate heterogeneity by switching off SOX2 expression, thus promoting TKI resistance and invasiveness. Downregulation of SOX2 expression by shRNA against SOX2 or TGF-beta treatment induced epithelial-to-mesenchymal transition (EMT) and endowed cell resistant to TKIs. TKI treatment selected EGFR-mutated lung cancer cells harboring low SOX2 expression with the EMT signature. TKI naïve EGFR-mutated lung cancer cells harboring low SOX2 expression exhibits intrinsic TKI resistance. Conversely, enhancing endogenous SOX2 expression reverses EMT and increases sensitivity to TKIs. Immunohistochemistry analysis revealed that low SOX2 expression predicts a poor survival in patients harboring EGFR mutations. Taken together, our findings show how cancer plasticity elicited by SOX2 expression and TKI selection generates distinct oncogenic properties and TKI sensitivity, providing critical insights into tumor progression in EGFR-mutated lung cancer.

#5907

Fulvestrant resistance in estrogen receptor positive breast cancer models is driven by heterogeneous ER independent transcriptional programs.

Kamila K. Kaminska, Nina Akrap, Johan Staaf, Åke Borg, Ana Bosch, Gabriella Honeth. _Lund University, Lund, Sweden_.

Endocrine therapy is the mainstay of treatment for metastatic estrogen receptor (ER) positive breast cancer. The selective ER degrader (SERD) fulvestrant is increasingly relevant due to its high therapeutic efficacy and often used in combination with other targeted treatments in the metastatic setting. However, most metastatic patients inevitably progress during therapy and develop resistance, constituting a major clinical problem. Our aim is to identify mechanisms conferring resistance to fulvestrant and evaluate potential therapeutic interventions. We have generated several fulvestrant-resistant (FR) in vitro models through chronic exposure of established breast cancer cell lines (i.e. MCF7, Cama1, T47D and HCC1428) to increasing concentrations of fulvestrant. The models have been characterized by assessing gene expression alterations using microarrays, protein expression by western blots, cell cycle profiling by flow cytometry and drug sensitivity by proliferation assays. These established FR models demonstrate a marked decrease in sensitivity to fulvestrant and ability to progress through the cell cycle in the presence of fulvestrant despite downregulation of ERα and decrease in ER signaling. Gene Ontology analysis revealed involvement of mitotic cell cycle, cell proliferation and DNA replication and repair genes in development of resistance. Investigation of cell cycle regulation showed cell line specific alterations in the FR models. Striking molecular alterations included upregulation of cyclin D3, as well as cyclin E2 and its binding partner CDK2 in Cama1 FR cells, and upregulation of CDK6 in MCF7 FR cells. These changes indicate importance of cyclin D-CDK4/6 and cyclin E-CDK2 nodes in supporting cell cycle progression of cells that have developed resistance to fulvestrant. Subsequently, we evaluated response of the FR cells to pharmacological inhibition of CDK4/6 by palbociclib and CDK2 by dinaciclib. MCF7 parental and FR cells were equally responsive to palbociclib treatment, while Cama1 FR cells displayed less sensitivity to palbociclib than their parental counterpart. In contrast, dinaciclib induced comparable growth inhibition in Cama1 parental and FR cells, while demonstrating lower efficacy in MCF7 FR model comparing to parental cells. Over all, our findings show heterogeneous mechanisms of endocrine resistance and demonstrate that our in vitro models can represent diverse mechanisms of fulvestrant resistance. This heterogeneity likely occurs also in breast cancer patients whose tumors develop resistance to endocrine therapy. The FR models display ER-independent growth, for which the underlying mechanisms include alterations in cell cycle regulation. Importantly, some of the changes could predispose FR cells to respond better to specific therapeutic interventions, such as treatment with CDK4/6 or CDK2 inhibitors.

#5908

Resistance mechanisms to TP53-MDM2 inhibition identified by in vivo piggyBac transposon mutagenesis screen.

Emilie A. Chapeau. _Novartis Pharma AG, Basel, Switzerland_.

Inhibitors of MDM2-TP53 interaction are predicted to be effective in tumors where the TP53 gene is wild-type by preventing TP53 protein degradation. One such setting is represented by the frequent CDKN2A deletion in human cancer that, through inactivation of p14ARF, activates MDM2 protein, which in turn degrades TP53 tumor suppressor. Here we utilized piggyBac transposon insertional mutagenesis to anticipate resistance mechanisms that occur under intermittent high dose treatment with the MDM2-TP53 inhibitor HDM201. Constitutive piggyBac mutagenesis in Arf-/- mice provided a collection of spontaneous tumors with characterized insertional genetic landscapes. Tumors were allografted in large cohorts of mice to assess HDM201 pharmacological effects. 16 out of 21 allograft models were sensitive to HDM201 but ultimately relapsed under treatment. When tumors with acquired resistance to HDM201 were compared to untreated tumors, 87 genes were found differentially and significantly targeted by piggyBac transposon. Resistant tumors displayed a complex clonality pattern suggesting emergence of several resistant subclones. Among the most frequent alterations conferring resistance, we observed somatic and insertional loss-of-function mutations in Trp53 in 54% of tumors, and transposon-mediated gain-of-function alterations in Bcl-xL, Mdm4, and two TP53-family members, resulting in expression of the TP53 dominant negative truncations ΔNTrp63 and ΔNTrp73. Enhanced BCL-xL and MDM4 proteins expression was confirmed in resistant tumors, as well as in HDM201 resistant patient-derived tumor xenografts. Interestingly, concomitant inhibition of MDM2 and BCL-xL demonstrated significant synergy in p53 wild-type cell lines in vitro.

Moreover, we performed an additional piggyBac screen with daily HDM201 treatment, at lower dose. Surprisingly, we identified Bcl-XL expression as a unique mechanism of resistance in intermittent high dose scheduling. These data suggested that different apoptotic inductions between the two HDM201 dose regimens (intermittent high dose/daily lower dose) likely play a role in the way the resistance to HDM201 takes place.

Collectively, our study has identified several potential mechanisms by which TP53 wild-type tumors may escape MDM2 targeted therapy, and provide evidence that Bcl-XL inhibition may be beneficial for relapsing patients that were treated with HDM201 intermittent scheduling.

### Therapeutic Approaches Based on Gene Delivery and Vector System

#5909

**Inhibition of Cd39 and Cd73 by 3** rd **-generation antisense oligonucleotides to improve immunity against tumors.**

Tamara Hilmenyuk,1 Sandra Kallert,2 Richard Klar,1 Lisa Hinterwimmer,1 Monika Schell,1 Marina Van Ark,2 Alfred Zippelius,2 Frank Jaschinski1. 1 _Secarna Pharmaceuticals GmbH & Co. KG, Planegg/Martinsried, Germany; _2 _University Hospital and University of Basel, Basel, Switzerland_.

During the last decades it became obvious that the immune system can be utilized to evoke effective antitumor responses. However, cancer cells develop mechanisms to circumvent this. The two ectonucleotidases CD39 and CD73 are promising drug targets, as they act in concert to convert extracellular immune-stimulating ATP to immunosuppressive adenosine. CD39 and CD73 are expressed on different immune cells as well as on a range of cancer cells and the latter are recognized to co-opt both ectonucelotidases for circumventing antitumor immune responses. In order to enhance immunity against tumors it would be favorable to increase extracellular ATP- and to simultaneously reduce adenosine concentrations in the tumor microenvironment. We designed antisense oligonucleotides (ASOs) with specificity for human and mouse CD39 and CD73. Knockdown efficacy of ASOs on mRNA and protein level was investigated in cancer cell lines and primary human T cells. CD39 and CD73 activity was evaluated by measuring levels of ATP, AMP and adenosine in cell supernatants. As functional readout we analyzed T cell proliferation and viability in presence of extracellular ATP and adenosine. The impact of ASO-mediated target knockdown in vivo on antitumor immune responses was analyzed in a syngeneic mouse tumor model. CD39- and CD73-specific ASOs suppressed expression of CD39 and CD73 mRNA and protein in different cancer cell lines and in primary human T cells without the need of any transfection reagent in vitro. Furthermore, degradation of extracellular ATP or AMP was significantly blocked by CD39- or CD73-specific ASOs while formation of adenosine was suppressed. Supplementation of cell culture medium with ATP impaired proliferation and viability of CD39- expressing CD8+ T cells. Strikingly, CD39-knockdown by ASO reversed the inhibitory effects of ATP on cell proliferation and viability. Extracellular AMP strongly inhibited proliferation and viability of CD73-expressing CD4+ T cells. Notably, AMP-mediated inhibition of proliferation was considerably less pronounced in cells treated with CD73-specific ASO. Systemic treatment of mice with CD39- and CD73-specific ASOs resulted in a knockdown of CD39 and CD73 mRNA expression in spleen and liver. Moreover, CD39-ASO treatment of tumor-bearing mice significantly reduced CD39-protein expression on tumor-associated macrophages, as well as intratumoral CD8+ T cells and Tregs. Remarkably, frequency of intratumoral Tregs was potently reduced by ASO-mediated CD39 knockdown. Taken together, targeting of CD39 and CD73 by ASOs represents a very promising state-of-the art therapeutic approach to improve immunity against tumors.

#5910

Oncolytic gene editing therapy for the treatment of pediatric rhabdomyosarcoma.

Michael P. Phelps, Heechang Yang, Shivani Patel, Eleanor Chen. _University of Washington, Seattle, WA_.

The inability to directly target many tumor oncogenes remains a major challenge for current cancer therapy. Small molecule inhibitors targeting these potent cancer genes have had limited success, therefore a new therapeutic approach is urgently needed. Recent advances in high efficiency gene editing technology have shown remarkable promise for the ability to destroy tumor oncogenes by targeting the genomic DNA (gDNA) of cancer cells. Unlike traditional small molecule inhibitors, cancer gene editing therapy is not sensitive to modifications in protein structure and can target any potential cancer gene. Gene editing therapy can also be rapidly adapted to the unique genetics of individual patients, providing the flexibility needed to target essential genes in rare cancers or individuals with relapsed tumors, for which there is no treatment options. Despite promising developments in this technology, major challenges remain in establishing an effective gene therapy delivery system, as well as improving cancer specific gene editing selectivity. To help overcome these challenges, we have developed high efficiency CRISPR/Cas9 gene editing approaches targeting the activated RAS oncogene in embryonal rhabdomyosarcoma (ERMS) and the PAX3/FOXO1 fusion gene in alveolar RMS (ARMS). Rhabdomyosarcoma is an aggressive pediatric sarcoma characterized by myogenic differentiation arrest. We demonstrate that targeting the exons of these driver oncogenes can effectively regress human RMS tumor xenografts in mice, using a tamoxifen- inducible gene editing approach. We also used a tumor-specific gene editing strategy to target the activating point mutation found in the NRAS and HRAS oncogenes as well as the breakpoint in the PAX3/FOXO1 fusion gene. We employed CRISPR systems from both Streptococcus pyogenes and Staphylococcus aureus to expand the number of genetic mutations that can be targeted in cancer cells. Finally, to test the therapeutic viability of cancer gene editing technology we engineered myxoma replicating oncolytic viruses with the CRISPR/Cas9 gene editing components, targeting both NRAS and PAX3/FOXO1 oncogenes. Myxoma virus is a non-integrating rabbit DNA poxvirus capable of selectively replicating in cancer cells with reduced anti-viral response. Our approach seeks to synergize the tumor selectivity of myxoma oncolytic viral replication with CRISPR/Cas9 technology, to create a novel cancer gene editing therapy that can be customized to the unique genetic mutations found in each cancer patient. While we have examined this technology on RMS tumors, the approach could also be widely applied to a number of different cancer types, creating one of the first customizable cancer therapies.

#5911

A bicistronic adenoviral vector carrying cytosine deaminase and GM-CSF genes significantly improves antitumor immunity and overall survival in colon cancer.

Hakan Akbulut,1 Arzu Coleri,2 Gunce Sahin,3 Yucheng Tang,4 Albert Deisseroth,5 Fikri Icli1. 1 _Ankara Univ. School of Medicine, Ankara, Turkey;_ 2 _Ankara Univ. Faculty of Science, Ankara, Turkey;_ 3 _Izzet Baysal Univ. Faculty of Science, Bolu, Turkey;_ 4 _Sidney Kimmel Cancer Center, Ankara, CA;_ 5 _U.S. Food and Drug Administration, Ankara, MD_.

Background: Combination of cytotoxic treatments with immunotherapeutic agents seem more efficacious than using either strategy alone. Oncolytic viral vectors carrying immunostimulatory genes like GM-CSF have emerged as treatment options in various tumors. Previously, we have shown that combination of suicide gene therapy either with GM-CSF or vector-induced dendritic cells has significant anti-tumor efficacy. In the current study, we aimed to combine the cytotoxic effect of the suicide gene cytosine deaminase and the immunostimulatory effect of granulocyte macrophage colony stimulating factor in a single vector construct. Methods: We have constructed a bicistronic adenoviral vector carrying cytosine deaminase (CD) and granulocyte macrophage-colony stimulating factor (GM-CSF) genes combined with an IRES element and driven by CMV promoter (Ad-CMV-CDiresGMCSF). We tested the in vitro efficacy of the vector in various tumor cell lines and in a mouse model of colon cancer. Results: Our bicistronic vector Ad-CMV-CDiresGMCSF showed significant in-vitro cytotoxic effects on tumor cell lines similar to the vector carrying CD gene alone when 5-FC added. Likewise, the GM-CSF producing efficacy of the bicistronic vector was comparable to the vector carrying GM-CSF gene alone. In the syngeneic colon cancer model, the bicistronic vector carrying CD and GM-CSF genes yielded significant tumor shrinkage and overall survival when compared to the control suicide vector carrying CD gene alone. Accordingly, anti-tumor immune response parameters including CTL assay and immune cell infiltration of tumor tissue were significantly improved in the Ad-CMV-CDiresGMCSF vector treated group (p<0.01). Conclusions: The Ad-CMV-CDiresGMCSF vector construct suggests a potential for the treatment of established tumors by inducing significant tumor killing and tumor-specific immune response.

#5912

Sentinel lymph node targeted therapy by oncolytic sendai virus suppresses micrometastasis of head and neck squamous cell carcinoma in an orthotopic nude mouse model.

Yuya Tanaka,1 Koji Araki,1 Shingo Tanaka,1 Yoshihiro Miyagawa,1 Hiroshi Suzuki,1 Daisuke Kamide,1 Masayuki Tomifuji,1 Kosuke Uno,1 Eiko Harada,1 Taku Yamashita,2 Yasuji Ueda,3 Makoto Inoue,3 Akihiro Shiotani1. 1 _National Defense Medical College, Tokorozawa, Japan;_ 2 _Kitasato University School of Medicine, Sagamihara, Japan;_ 3 _ID Pharma Co., Ltd., Chiyoda-ku, Japan_.

Background: Lymph node metastasis is one of the most important prognostic factors in head and neck squamous cell carcinoma (HNSCC). In clinical N0 cases, however, it is difficult to decide whether we should select elective neck dissection or watchful waiting even at present. We recently reported the successful oncolytic virotherapy against head and neck cancer using recombinant Sendai virus vector (rSeV). We herein focus on sentinel lymph node (SLN)-targeted therapy by oncolytic Sendai virus as a novel less invasive therapy. The objectives of this study are to investigate rSeV migration into SLN, and to elucidate anti-tumor effect on SLN micrometastasis of HNSCC in an orthotopic nude mouse model.

Experimental design: We established SLN micrometastasis of HNSCC in an orthotopic nude mouse model. We inoculated HSC-3-M3, human highly metastatic tongue squamous carcinoma cells, into the left edge of the tongue, and two weeks after tumor inoculation we intratumorally injected a type of rSeV-GFP that selectively shows urokinase type plasminogen activator (uPA)-specific cell killing activity via cell-cell fusion, which is named "BioKnife". We investigated rSeV migration into SLN by using immunostaining, RT-PCR, and in vivo imaging system. We also examined anti-tumor effect and its mechanisms through serial section examination of SLN by using anti-SeV antibody and the ApopTag peroxidase in situ apoptosis detection kit.

Results: rSeV migration into SLN was clearly visible by fluorescent signals of rSeV in SLN using immunostaining and in vivo imaging system, and clarified by significant increases of relative mRNA expressions of GFP compared to control group in both tongue and SLN using RT-PCR (p < 0.05 respectively). Serial section examination of SLN showed that BioKnife selectively infected cancer cells and exhibited significant anti-tumor effect against SLN micrometastases compared to control groups (p < 0.05). We also detected apoptosis in SLN micrometastasis infected by BioKnife.

Conclusions: This study demonstrated that BioKnife can migrate into metastatic SLN after intratumoral injection into primary tumor, and effectively suppresses SLN micrometastases in an orthotopic nude mouse model, suggesting that SLN targeted therapy of oncolytic rSeV has a great potential to provide a novel and promising alternative to elective neck dissection for clinical N0 patients with HNSCC.

#5913

ECO nanoparticle delivery of siRNA targeting DNA repair to enhance the radiation response of glioblastoma.

Jennifer A. Lee,1 Nadia Ayat,2 Zhanhu Sun,2 Tatsuya Kawai,1 Zheng-Rong Lu,2 Kevin Camphausen1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Case Western Reserve University, Cleveland, OH_.

Background: Glioblastoma (GBM) is among the most lethal of human cancers with a median survival of ~15 months. Novel treatment strategies targeting DNA damage response proteins such as ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNApk) are being investigated for the radiosensitization of GBM. Previously, (1-aminoethyl)iminobis[N-(oleicylcysteinyl-1-amino-ethyl)-propionamide (ECO) nanoparticles (NPs) were shown to effectively deliver siRNA targeting tumor growth and metastasis. Here we investigate the ability of these NPs to deliver siRNA targeting ATM and DNApk to enhance the radiation response of glioma and glioma stem cell populations.

Methods: Normal astrocytes, glioma (U251), and glioma stem cells (NSC11, GBMJ1) were used. Protein analysis revealed the degree of silencing using ECO NPs with siRNA targeting ATM or DNApk. The effects of each siRNA alone (40nM) and in combination (20nM each) on radiation response was evaluated using clonogenic cell survival and γH2AX foci assays. A cranial guide screw model was used to assess the effectiveness of NPs in vivo. U251 tumor cells expressing luciferase were orthotopically implanted in nude mice and NPs targeting luciferase or DNApk were injected intratumorally. The efficacy of siRNA delivery was assessed via bioluminescence signaling and immunohistochemical staining.

Results: ECO NPs effectively silenced both target genes in all tumor cells. The effect of this silencing on double strand break (DSB) repair varied with each cell line. γH2AX studies showed significantly impaired DSB repair in U251 cells with DNApk and ATM/DNApk combination treatment and in GBMJ1 stem cells with combination treatment. Treatment of NSC11 stem cells resulted in trends towards impaired DSB repair however the results were not statistically significant. Importantly, each treatment had no significant effect on DSB repair in normal astrocytes. Evaluation of radiation response in the long term using clonogenic assays resulted in dose enhancement factors for ATM, DNApkcs, and combination treatments of 1.24, 1.43, and 1.34 for U251, 1.34, 1.35, and 1.48 for NSC11, and 1.35, 1.36, and 1.51 for GBMJ1. In vivo studies showed evidence that ECO NPs were able to deliver siRNA targeting luciferase and reduce the bioluminescence signal output in vivo.

Conclusions: The use of ECO NPs in normal astrocytes resulted in no significant effect on DSB repair demonstrating a potential therapeutic advantage to ECO NP/siRNA therapies. In contrast, NPs effectively delivered siRNA targeting ATM and DNApk to glioma and glioma stem cells showing the ability to target and treat a heterogeneous GBM cell population. The subsequent enhancement of radiation response in vitro coupled with in vivo observations of bioluminescence silencing provide the basis for future investigation into the use of ECO/siRNA complexes to enhance the radiation response of GBM in vivo.

#5914

The porcine model for onocolytic adenovirus-based therapy.

Malavika Chandrashekar, Lisa Koodie, Michele Dunning, George Ruth, Richard Bianco, Masato Yamamoto, Julia Davydova. _University of Minnesota, Minneapolis, MN_.

Oncolytic adenoviruses (Ad) are promising tools in the development of cancer therapeutics. A majority of Ad-based therapies utilize serotypes of species C, with Adenovirus type 5 (Ad5) being the most commonly employed. Previously, clinical trials have demonstrated the low efficiency of Ad5 vectors, mainly due to absence of the Ad5 primary receptor (Coxsackie Adenovirus Receptor, CAR) in cancer cells. Engineering Ad vectors utilizing the species B (Ad3, Ad35, Ad11) receptors have greatly improved the oncolytic potential of Ad-based therapies. However, the lack of a viable animal model has impeded clinical translation of these tropism-modified vectors. Mouse models are insufficient because Ad does not replicate in murine tissue. Non-human apes are not feasible due to availability and cost. Cotton rats and Syrian hamsters, although permissive of Ad5 replication, are not suitable for Ad3-, Ad35-, and Ad11-retargeted vectors due to the lack of species B primary receptors (CD46 and desmoglein 2) in rodent systems. In this study, we explored pigs as a model to study performance of the group B oncolytic adenoviruses by employing the fiber-modified Ad5/Ad3 chimeric vector. As a control, the Ad5 fiber-unmodified virus was used. First, we demonstrated the ability of swine cell lines to support replication of both Ad5 and Ad5/Ad3. Second, we analyzed binding, gene transfer, cytolytic, and replication ability of Ad5 and Ad5/Ad3 in various non-human cell lines (swine, hamster, murine, rat, bovine, canine). Our data confirmed that while hamster cell lines (HP1 and HapT1) were able to support binding and replication of Ad5-based vectors, they failed to support that with the Ad3-retargeted vectors. Additionally, we showed that among all tested cell lines, only porcine cells (PK15 and PTK75) were supportive of both binding and replication of the Ad3-retargeted virus. Of note, Ad5/Ad3 outperformed Ad5 in its cytolytic effect in porcine cell lines. These in vitro results prompted evaluation of the vectors in vivo. Immunocompetent Yorkshire pigs were systemically injected with a single dose of Ad5- and Ad5/3- expressing luciferase (Luc) from the Ad E3 region. Quantitative PCR analyses of the primary organs collected 7 days post-infection revealed Ad5 and Ad5/3 viral DNA in the lungs and spleen. Replication-dependent Luc expression was also observed in these tissue samples suggesting active viral replication. The quantity of viral DNA in other tissue such as the kidneys, liver, and pancreas was negligible. The results of these in vitro and in vivo studies indicate that pigs are a promising model to assess unmodified and tropism-modified adenoviral vectors.

#5915

**Overexpression of tumor suppressor** p53 **gene by oncolytic adenovirus boosts immune responses in human pancreatic ductal adenocarcinoma cells.**

Hiroyuki Araki,1 Hiroshi Tazawa,1 Takeshi Ieda,1 Takuro Fushimi,1 Shinji Kuroda,1 Ryuichi Yoshida,1 Hiroyuki Kishimoto,1 Masahiko Nishizaki,1 Yasuo Urata,2 Shunsuke Kagawa,1 Toshiyoshi Fujiwara1. 1 _Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan;_ 2 _Oncolys BioPharma, Inc., Okayama, Japan_.

Background: Pancreatic ductal adenocarcinoma (PDAC) is the worst prognosis disease with an overall 5-year survival rate of less than 5%. Although immune checkpoint therapy has been emerged as a novel antitumor therapy, PDAC is less sensitive to immunotherapy due to small number of infiltrating T cells. Recently, oncolytic virotherapy has been shown to promote intratumoral T cell infiltration as an immunogenic antitumor therapy. In the current study, we explored whether overexpression of the wild-type p53 tumor suppressor gene could synergize with oncolysis for induction of immunogenic cell death in human PDAC cells.

Methods: OBP-702 is a tumor-specific replication-competent oncolytic adenovirus, in which the hTERT promoter drives the expression of the viral E1A and E1B genes for tumor-specific viral replication and the Egr-1 promoter induces wild-type p53 expression. We used 4 PDAC cell lines (MIA PaCa-2, Capan-1, BxPC-3, Panc-1). Molecular mechanisms of OBP-702-mediated cell death was investigated. Migration and invasion properties were assessed using transwell chamber assay. In addition, virus-induced immunogenic cell death was assessed by analyzing the level of extracellular ATP and high-mobility group box protein B1 (HMGB1). In vivo antitumor effect of OBP-702 was assessed using an orthotopic BxPC-3 xenograft tumor model.

Results: At 72 hours after virus infection, OBP-702 induced profound antitumor effect in all human PDAC cells. OBP-702 induced autophagy- and apoptosis-related cell deaths. In migration and invasion assays, MIA Paca-2 and Capan-1 cells were low-invasive type, and Panc-1 and BxPC-3 cells were high-invasive type. OBP-702 inhibited migration and invasion properties of high-invasive type PDAC cells At 24 and 48 hours after virus infection, the secretion of ATP was significantly higher in OBP-702-infected PDAC cells compared to p53-null virus-infected cells. Moreover, OBP-702 significantly induced the higher amount of extracellular HMGB1 in all PDAC cells. Finally, OBP-702 significantly suppressed tumor growth in an orthotopic BxPC-3 xenograft tumor model compared to p53-null virus.

Conclusions: Our data suggest that oncolytic adenovirus-mediated p53 overexpression induces more profound immunogenic cell death to boost the immune responses in PDAC. This additional immunological function of p53 is quite attractive and warrants clinical trials combined with immune checkpoint inhibitors.

#5916

Engineering an oncolytic adenovirus targeted to the CXCR4 chemokine receptor for breast cancer therapy.

Samia M. O'Bryan, J. Michael Mathis. _Louisiana State University, Baton Rouge, LA_.

Breast cancer is the most commonly diagnosed cancer in women under 60. Localized breast cancer is easily treated, resulting in high survival rates. However, treatments for advanced disease are inadequate, resulting in poor five-year survival rates at less 24%. Thus, there is a great need for new therapies capable of increasing treatment efficacy. Oncolytic virotherapy using the human adenovirus is a novel therapeutic approach designed to specifically target cancer cells while sparing normal tissues. As a well-characterized vector, adenovirus is easily manipulated and results in high gene transfer efficiency. Combined with systemic stability, low pathogenicity and the ability to infect a broad range of dividing and non-dividing cells, this vector is uniquely suited for oncolytic virotherapy. Used as an oncolytic therapy, replication of the vector within cancer cells causes the lysis of the cells and subsequent spread of progeny virions within the surrounding tumor stroma. One challenge to targeting cancer cells with adenovirus has been the low expression of the endogenous adenovirus receptor, the Coxsackie and adenovirus receptor (CAR), prompting the search for new targets. This study engineered an adenovirus vector to target CXCR4, a seven-membrane spanning G-protein-coupled receptor, whose role is implicated in a wide variety of tumors, including breast cancer. Altered expression of CXCR4 drives cancer cell migration and invasion, which has been associated with metastasis. Previously, we developed a bispecific adaptor molecule targeting CXCR4 to retarget a replication-deficient adenovirus to breast cancer cells overexpressing CXCR4. In the current study, we have engineered a replication-competent oncolytic adenovirus targeting CXCR4, by replacing the adenovirus fiber gene with a modified fiber containing the SDF-1 ligand of CXCR4. The modified fiber was constructed using the T4 fibritin protein fused to the tail of the adenovirus fiber and attached to the CXCL12 ligand via a spacer protein. Also, the red fluorescent protein (RFP) sequence was fused to the capsid protein IX (pIX) gene for visual tracking. The virus was then rescued and amplified in HEK-293 cells for characterization and downstream applications. We confirmed the presence of a modified fiber and the specificity of CXCR4 binding. Subsequently, the vector was tested in a panel of breast cancer cells for infection and cell killing efficiency. Together these studies addressed the hypothesis that constructing a retargeted oncolytic adenovirus using the CXCL12 ligand provide selective infection and killing of breast cancer cells overexpressing CXCR4. These results provide a strong rationale for further developing the retargeted vector for oncolytic virotherapy in patients.

#5917

A novel lipid nanoparticle (NBF-006) encapsulating glutathione S-transferase P (GSTP) siRNA for the treatment of KRAS-driven non-small cell lung cancer.

Zhihong O'Brien*, Li Wang*, Bharat Majeti*, Jeanpierre Clamme, Robiel Baclig, James Chu, Steven Fong, Jens Harborth, Jose Ibarra, Haiqing Yin, Jing Yu, Clayton Zhang, Roger Adami, Sonya Zabludoff, Wenbin Ying. _NITTO BIOPHARMA, INC, San Diego, CA_.

Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancers and KRAS mutation occurs in 25-30% of NSCLC. Therapeutics directly targeting KRAS mutant proteins are currently not available. We developed a novel siRNA therapeutic against Glutathione S-Transferase P (GSTP), a gene target that is highly expressed in KRAS mutant NSCLC and is also involved in modulating RAS signaling pathway proteins. GSTP has been known as a Phase II detoxification enzyme and a modulator of MAP kinase-related cell-signaling pathways. Therefore, developing a GSTP inhibiting siRNA may be an effective therapeutic approach to treat KRAS mutant NSCLC. The lead drug product candidate (NBF-006) is a proprietary siRNA-based lipid nanoparticle (LNP) with a size of less than 70 nm, comprising GSTP siRNA (NDT-05-1040) and a novel ionizable, non-immunogenic, biodegradable lipid developed to target tumors in the lung. Our studies evaluated biodistribution and in vivo antitumor activities of NBF-006 in KRAS mutant NSCLC xenograft models. NBF-006 delivered siRNA molecules favorably to the lungs and tumor as compared to other tissues, with 70-80% of tumor cells showing siRNA uptake. Significant tumor growth inhibition (P <0.05) was observed in xenograft models derived from A549 (KRASMUT/TP53WT) and H23 (KRASMUT/TP53MUT) NSCLC cells, with notable tumor regression in A549 tumors and highly significant tumor regression in the H23 tumors following once-a-week administration. In the bioluminescent orthotopic lung tumor model, the average luminescent signal response in the NBF-006 treatment group was markedly lower than the vehicle control group (14.3 vs. 69.6 million photon/second, P <0.05). Additionally, in a surgically implanted orthotopic lung tumor model, the survival rate of the NBF-006 treatment group was significantly prolonged (P <0.005) at a 4 mg/kg dose compared to the vehicle control group. Finally, NBF-006 treatment was well tolerated in the preclinical efficacy models. In summary, we conclude that NBF-006 is selectively delivered to lung tumor tissue and its considerable uptake by tumor cells results in significant tumor growth inhibition and overall survival benefit as demonstrated in our preclinical models of KRAS mutant NSCLC.

*These authors contributed equally.

#5918

A novel glutathione S-transferase P (GSTP) siRNA (NDT-05-1040) for the treatment of KRAS-driven non-small cell lung cancer.

Cima Cina, Jens Harborth, Zhihong O'Brien, Nish Beltran-Raygoza, Jung-kang Jin, Jessica Xu, Sang Jun Lee, Kwok Tsang, Jiping Yao, Roger Adami, Sonya Zabludoff, Wenbin Ying. _NITTO BIOPHARMA, INC, San Diego, CA_.

Lung cancer is the most common form of cancer worldwide and the leading cause of cancer-related deaths. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers, and KRAS mutation occurs in 25-30% of NSCLC. However, to date, direct therapeutic targeting of KRAS has not been achieved. Our approach to developing a KRAS mutant NSCLC therapeutic agent was to identify a selective novel target involved in modulating signaling proteins in the RAS pathway without impacting healthy cells. Numerous studies have shown that glutathione S-transferase P (GSTP) is strongly upregulated in many cancer types, specifically lung, colon, and pancreas. Besides being a well-known Phase II detoxification enzyme, GSTP is also known to influence cell signaling through interaction with MAP kinases such as JNK and c-jun. We therefore hypothesized that GSTP knockdown (KD) could be an effective therapeutic approach to treat KRAS mutant tumors. To test this hypothesis, we designed a series of novel GSTP siRNAs and identified our lead candidate, a highly potent and selective GSTP siRNA (NDT-05-1040) to specifically target and downregulate GSTP protein. A panel of KRAS mutant NSCLC cell lines was utilized to determine the GSTP KD potency and antiproliferative activity of NDT-05-1040. Western blot analysis and co-immunoprecipitation (co-IP) were conducted to identify the proteins that were regulated by GSTP in the proliferation and apoptosis pathways. These studies demonstrated that NDT-05-1040 is a very potent and selective GSTP siRNA. Furthermore, transfection of NDT-05-1040 led to significant growth inhibition and induced apoptosis in NSCLC cells without affecting normal cell viability. Western blot analysis showed that NDT-05-1040 effectively decreased the phosphorylation of CRAF, ERK, MEK, in the MAP kinase pathway. Interestingly, with NDT-05-1040 treatment, the phosphorylation of Akt and mTOR in the PI3K pathway was also decreased. Moreover, the activity of p-JNK, PUMA, caspase-3/7, and p53 in the KRAS mutant cells was upregulated by NDT-05-1040. Co-IP experiments demonstrated that GSTP formed a complex with both p-CRAF and JNK. In summary, GSTP KD by NDT-05-1040 targeted the RAS/MEK/ERK, Akt, mTOR and apoptosis pathways. This selective multipronged attack on the growth and survival pathways of cancer cells makes it a compelling target and could potentially serve as a novel therapeutic agent for KRAS mutant NSCLC.

#5919

Component-specific qPCR assays for characterization and identity testing of multigenome ZVex®, a dendritic cell-targeting lentiviral vector platform.

Anshika Bajaj,1 Tsai-Yu Lin,1 Lisa Y. Ngo,1 Michele Murphy,1 Brenna Kelley-Clarke,2 Wayne R. Gombotz,1 Jan H. ter Meulen,1 Peter Berglund1. 1 _Immune Design, Seattle, WA;_ 2 _Silverback Therapeutics, Seattle, WA_.

Background: ZVex is a novel, integration-deficient, dendritic cell-targeting lentiviral vector platform currently being evaluated in clinical trials in sarcoma patients. The vector is capable of delivering multiple full-length genes simultaneously. Here, we describe ZVex preparations encoding the human cancer testis antigens MAGE-A1, 3, 4, 10 and Interleukin 12. Because lentiviral vectors package two RNA molecules each, this ZVex approach results in the generation of 15 possible permutations of homozygous or heterozygous vector genotypes. The homologous nature of the MAGEA family members (50-80% sequence identity) poses significant challenges to their specific detection and quantification in a MAGEA gene pool. Here, we describe highly specific qPCR assays for detection of the 4 MAGEA genes and IL12 for characterization and identity testing of multigenome ZVex preparations.

Materials and Methods: Genes were codon-modified to enable specific detection of vector-expressed genes over cellular sequences. The entire IL12B and short, non-overlapping, divergent target regions in each of the 4 MAGEA genes were carefully selected as templates for primer/probe design. Primer-blast was used to predict absence of off-target detection of cross-reacting genomic sequences. To further enhance the specificity of the primers/probes to each of their target MAGEA, potential off-target amplicons that can be generated in the other 3 MAGEA genes due to base pair mismatch were further sequence-modified to reduce sequence identities. ZVex vectors were generated by transfecting 4 or 5 different plasmids encoding the codon modified full-length MAGEA and/or IL12 genes in a producer cell line along with essential vector packaging plasmids. Single/multiplex qPCR assays were performed to analyze vector RNA/reverse-transcribed DNA.

Results: The primer/probe combinations were highly specific for each of the 4 MAGEA genes and IL12 as indicated by melt curve analysis and producing a single qPCR product at the expected size. The primer/probes specifically detected from 10 to 107 copies of their target MAGEA with >=93% efficiency and specifically detected reverse-transcribed vector DNA in a cell-based vector transduction assay. The assays demonstrated relative gene specific titers that correlate with transfected plasmid ratios. MAGEA/IL12 specific qPCR assays benchmark well against previously established "gold standard" qPCR assays used for quantifying total ZVex vector genome titers.

Conclusions: Component-specific qPCR assays were developed for the specific assessment of constituent genes in multigenome ZVex preparations. These assays demonstrate inter/intragene specificity, are amenable to multiplexing, and can be used for quality control, titration, characterization, and identity testing of ZVex lots in a cGMP setting.

#5920

Antitumor efficacy of a dual stromal and tumor targeted oncolytic measles virus in breast and colon cancer models.

Yuqi Jing, Valery Chavez, Natasha Karishma Khatwani, Jaime Merchan. _Univ. of Miami Sylvester Comp. Cancer Ctr., Miami, FL_.

Efficient systemic delivery of oncolytic viruses remains an unresolved issue and an obstacle to the success of oncolytic virotherapy. One of the main barriers that lead to decreased oncolytic viral delivery to tumor cells is the tumor stromal component. A potential strategy to circumvent this problem is to use a retargeted oncolytic virus that is able to target, and replicate in tumor stromal cells. The aim of this study is to evaluate the consequences of tumor stromal/vascular targeting by oncolytic measles virus via urokinase receptor (uPAR) as they relate to improved viral delivery and antitumor efficacy.

We rescued and characterized a novel dual targeting MV-un-muPA, which binds human cancer cells via CD46 and murine tissues via murine uPAR. This novel virus variant was generated as a tool to investigate the ability of a virus to target tumor stromal cells, especially tumor microvasculature (via murine uPAR) and deliver the infection to tumor cells (via CD46) in vivo. In vitro, MV-un-muPA infected and induced cytotoxicity to human or murine cancer cells in a species specific manner. Efficient MV-un-muPA replication was demonstrated in multiple human and murine cancer cells as well as murine fibroblasts in vitro. MV-un-muPA mediated viral transfer was demonstrated between murine endothelial or murine fibroblast to (RFP expressing) human cancer cells. To further validate the consequences of tumor stromal/vascular targeting by oncolytic measles virus via uPAR as they relate to improved viral delivery, the effects of MV-un-muPA vs a non-targeted MV virus (MV-GFP) were assessed in human breast (MDA-MD231) and colon (HT-29) cancer xenograft models. In these models, tumor cells express human CD46 and the host stroma expresses murine uPAR. Tumor bearing mice were treated with MV-GFP (targeting tumor cells via CD46), dual targeting virus MV-un-muPA (target tumor stroma via murine uPAR, and tumor cells via CD46) by intravenous administration (3 doses). Systemic administration of MV-un-muPA resulted in a significant delay in tumor progression as well as improved survival compared to mice treated with vehicle control or MV-GFP. Tumor based studies showed increased levels of viral RNA, tumor apoptosis, resulted in a significantly reduced stroma and the reduction of cancer associated fibroblasts and endothelial cells, in tumors treated with the dual targeted virus, compared to MV-GFP.

In conclusion, our results demonstrate for the first time that dual targeting of tumor stroma and tumor cells by MV-un-m-uPA enhance in vivo viral delivery and oncolytic virus antitumor efficacy in two in vivo models of cancer. This serves as clear proof that vascular/stromal targeting is an effective strategy to enhance viral delivery and antitumor effects and support future studies to further uncover the potential of stromal targeted oncolytic viruses.

#5921

Systemic treatment with mesothelin-targeted oncolytic adenovirus shows efficacy patient-derived xenograft of pancreatic cancer.

Mizuho Sato-Dahlman, Yoshiaki Miura, Praveensingh Hajeri, Hideki Yoshida, Kari Jacobsen, Chikako Yanagiba, Masato Yamamoto. _Univ. of Minnesota, Minneapolis, MN_.

Pancreatic cancer is an aggressive malignant disease. Despite extensive efforts, systemic therapies have provided only limited efficacy for patients with this disease. Oncolytic Adenovirus (OAd) is a promising therapeutics, and it is also known for its efficient in vivo gene delivery. However, when adenovirus vectors are injected intravenously into mice, most of the virus goes to the liver and can lead to liver toxicity at high dosage. One of the reason for liver tropism is that hepatocytes express high levels of the primary adenovirus receptor, and non-parenchymal liver cells, such as Kupffer cell and epithelial cell, also capture the viral particle. As a consequence of large sequestration of adenovirus by liver, the tumor transduction rate is low and the in vivo efficacy is limited. Therefore, the improvement of cancer selective transduction and vector distribution to avoid liver sequestration would overcome the obstacles for systemic delivery required for efficient systemic treatment of spread and/or metastatic lesions of pancreatic cancer with OAd.

To improve the tumor transduction, we have generated the pancreatic cancer-targeted OAd by high-throughput screening of Ad-fiber library in mesothelin (MSLN) expressing cells. The pancreatic cancer-targeted OAd binds to MSLN protein, which is overexpressed on the surface of pancreatic cancer. MSLN-targeted OAd showed selective and powerful anti-tumor effect against Panc-1 xenograft tumor model in both intratumoral (i.t.) and intravenous (i.v.) injection. Importantly, when we assessed viral distribution after i.v. injection, the liver sequestration of MSLN-targeted OAd was lower than untargeted OAd (Ad5 WT virus) at 48 hrs after injection. By day 7, the viral copy number of MSLN-targeted OAd in the tumor was significantly higher than Ad5 WT virus. These results suggest that systemic injection of the tumor targeted-OAd showed significantly lower liver sequestration and better tumor accumulation. Additionally, we performed multiple time point injection of MSLN-targeted OAd against regrown tumors. Four out of six tumors were controlled with repeated injection. Next, antitumor effect of MSLN-targeted OAd was assessed in patient-derived xenograft (PDX) model. After intravenous administration, only the MSLN-targeted OAd showed significant antitumor effect compared to the untreated group (p<0.05), while the growth of Ad5 WT virus injected group was same as untreated group.

In this study, systemic injection of MSLN-targeted OAd showed remarkable anti-tumor effect in both systemic and intratumoral injections at low dose. Our results indicated that tumor targeted-OAd can embody efficient systemic treatment for pancreatic cancer which are mostly found with spread or metastatic lesions.

#5922

"Gammora": Selective killing of cancer cells bearing CD24 by viral integrase.

Shiran Shapira,1 Eynat Finkelshtein,2 Abraham Loyter,2 Nadir Arber1. 1 _Tel Aviv Sourasky Medical Ctr., Tel Aviv, Israel;_ 2 _KAplan Medical Center, Hadassah-Hebrew University Medical School Jerusalem, Rehovot, Israel_.

The HIV integrase is important in the integration of viral DNA to the host genome resulting in double-stranded breaks (DSB). Only 1-2 copies are integrated, due to inhibition of HIV-1 Rev protein, making the cells invisible to the immune system. CD24 is mostly expressed on cancer and not normal cells. We hypothesize that specific targeting of the viral particles using mAb to CD24 together with stimulating the enzyme activity and increasing the frequency of integration events, would increase the occurrence of DSB, that will lead to genomic instability, and ultimately to apoptosis of the target cells.

Aim

To develop a therapeutic platform, for selective eradication of cancer cells, which consists of targeted lentivirus particles and IN-derived peptides.

Methods

Integrase derived peptides (designated as INR2 and INS) were synthesized. Humanized anti-CD24 antibody (scFv) was engineered and fused to the lentivirus envelope. Stability, solubility and toxicity of the peptides were analyzed by HPLC-MS and MTD, respectively, as well as their activity and permeability into cells. Cell death was measured qualitatively by fluorescent microscopy and quantified by the enzymatic MTT assay. Colorectal, pancreatic and lung cancer cells were used for testing the potency of the lentiviral-based system. The efficacy of the suggested system was assessed in vivo, in nude mice bearing xenografts of lung cancer. The mice were treated systemically twice weekly.

Results

INS and INR2 stimulate viral integrase in vitro. A single IV administration of INR2 and INS was well tolerated without adverse effects. These lentivirus particles contain nonfunctional DNA with flanked LTRs allowing their integration into the target cells DNA and formation of DSB due to the action of the integrase whose activity was stimulated by the IN-derived peptides. Massive cell death (40-70%) in a dose-

dependent manner was achieved. Raltegravir, an antiretroviral FDA-approved drug, was able to inhibit cell death (2-3 times). In vivo, the INR2 and INS combination led to a significant reduction (50%) in tumor volume. Live imaging (IVIS device) of the tumors and selected organs confirmed that the viruses indeed reach the tumor after systemic injection and that the anti-CD24 enhanced the selectivity of infection.

Conclusions

The use of IN-derived peptides together with the CD24-targeted lenti-therapy approach suggests a novel strategy that specifically promotes killing and removal of a wide range of cancer cells.

#5923

CD133-targeted oncolytic adenovirus exhibits anti-tumor effect in colorectal cancers, and its combination with irradiation inhibits liver metastasis.

Jing Li Huang, Mizuho Sato-Dahlman, Kari Jacobsen, Masato Yamamoto. _Univ. of Minnesota, Minneapolis, MN_.

Colorectal cancer (CRC) is the third most common cancer in the world, and about 50% of patients relapse after treatment. Cancer stem cells (CSCs) have contribution to recurrence, metastasis and chemotherapy resistant of CRC. CD133 (Prominin-1), a member of the transmembrane glycoprotein family, is a marker of CSCs in several cancers including CRC: its expression correlates with recurrence, metastases and chemotherapy resistance, as well as poor prognosis in CRC. It is therefore reasonable to develop a CSC-directed CRC therapeutic strategy by employing CD133as a target molecule. Recently, we have established a method for isolating transductionally-targeted infectivity-selective adenovirus by high-throughput screening. Using this adenovirus library screening system, we isolated the CD133-specific Oncolytic Adenovirus (OAd) and tested the oncolytic activity of CD133-targeted OAd (CD133-OAd) in both in vitro and in vivo, with and without irradiation which induces CD133 expression. The infectivity-selective OAd (ISOAd) with CD133-targeting motif (TYML motif) selectively infected CD133+ CRC cell lines and lysed them efficiently. In the context of modulation of stemness, CD133-OAd inhibited colony formation in vitro. In tumor formation assay in nude mice, treatment with CD133-OAd prior to tumor inoculation inhibited the establishment of tumor of CD133+ CRC cell lines. Intra-tumor (i.t.) administration of CD133-OAd into established subcutaneous tumor exhibited significantly stronger antitumor effect compared to OAd without targeting.

We have reported that the irradiation increases CD133 expression in CRC cells, and the replication of CD133-OAd increased significantly after irradiation. In athymic nude mice, treatment of irradiated cells with CD133-OAd abolished tumor-forming capacity, compared to cells without irradiated and cells treated with radiation alone (5% vs 94% and 5% vs 73% respectively, p<0.0001). When the antitumor effect of CD133-OAd was analyzed on subcutaneously established tumors, treatment with radiation and CD133-OAd significantly reduced tumor growth compared to no treatment and treatment with radiation only (both p<0.0001).

In addition to the effect on the main tumor, CD133-OAd suppressed liver metastasis of CRC. The mice injected with human CRC cells pretreated with CD133-OAd combined with irradiation had significantly lower incidence of liver metastasis compared with the untreated control group or the groups received irradiation or CD133-OAd treatment alone.

Our CD133-targeted ISOAd is effective for cytotoxic killing, reduces tumor formation, and mitigates tumor growth in radiation resistant CRC cells. This targeted OAd therapy may be applicable to address therapeutic resistance and prevent the establishment of recurrent colorectal cancer.

#5924

Priming tumor cells by immunogenic modulation to enhance IL-27 immune therapy efficacy.

Marxa L. Figueiredo, Manoel Figueiredo Neto. _Purdue University, West lafayette, IN_.

Immunogenic modulation is an emerging strategy for treating bone-metastatic prostate tumors, which present an altered balance between bone resorption and bone formation, with enhanced tumor growth and severe pain and fractures. There remains a critical lack of therapies that can significantly reduce tumor burden while simultaneously restoring the bone. Emerging immune therapies include Interleukin-27 (IL-27), a multifunctional cytokine that drives antitumor activity through STAT1/T-bet/IFNγ pathway immune-activation and anti-angiogenic mechanisms. IL-27 also is a pro-osteogenic factor, as it inhibits osteoclast formation and increases the pool of mature functional osteoblasts. Our goal for this work was to examine whether IL-27 immunetherapy efficacy still could be improved by the addition of 'priming' strategies such as chemotherapy-induced immunogenic modulation in order to more efficiently promote tumor cell elimination by the immune system. Immunogenic modulation (IMO) develops following tumor cell exposure to chemotherapy, with alterations in tumor cell phenotype that prime cells for elimination by the immune system. Our results suggest that chemotherapy can reduce tumor cell viability, however, aggressive prostate tumor cells still are relatively resistant even at high doses. We have identified a potential cytokine combination that can sensitize aggressive prostate cancer cells (PC3) to low or sublethal doses of recently FDA-approved chemotherapy agents. Gene expression analyses using quantitative real time PCR arrays suggest that single chemotherapuetic agents promote a fairly low modulation and magnitude of apoptotic transcripts. Immune therapy alone (IL27 plus OP1 or 27OP1) upregulated several apoptosis genes, however, the combination of 27OP1 synergized with either cabozantinib or cabazitaxel, especially in augmenting expression of proapoptotic genes BNIP2, and CARD8 and immune modulatory gene CD70. Preliminary pathway analyses with the gene expression data (Pathgen) generated hypotheses of possible mechanisms involving gene regulation by AP1 or SMAD related pathways. Functional assays using luciferase reporter assays showed that AP1 activity was significantly upregulated by 27OP1, Cabozantinib, Cabazitaxel+27OP1, and Cabozantinib+27OP1. SMAD1/5/8 activity was significantly upregulated by OP1, 27OP1, Cabozantinib, Cabazitaxel+27OP1, and Cabozantinib+27OP1. These results suggest that 27OP1 synergizes with cabozantinib or cabazitaxel at least through AP1 and SMAD-regulatory mechanisms. Future studies will examine the effectiveness of these combinations in vivo, and preclinical studies are planned to examine whether low-dose chemotherapy can augment 27OP1 immunotherapy efficacy. Ultimately, our goal is to identify the optimal therapeutic niche for immunotherapy in combination with current immunomodulatory chemotherapies.

#5925

A novel oncolytic adenovirus for radioiodine therapy and imaging.

Lisa Koodie,1 Eriko Iguchi Kawakami,2 Kari Jacobsen,1 Zuzan Cayci,1 Andrew Taylor,1 Edward W. Greeno,1 Robert J. Schumacher,1 John C. Morris,2 Martin E. Fernandez-Zapico,2 Julia Davydova1. 1 _Univ. of Minnesota, Minneapolis, MN;_ 2 _Mayo Clinic, Rochester, MN_.

Recently, the use of oncolytic viruses encoding the sodium-iodide symporter (NIS) has become an attractive approach to achieve radionuclide imaging of cancer. However, the potential of virus-induced NIS expression to facilitate therapy with radioactive 131I has not been fully explored. We designed a tumor-selective, infectivity-improved oncolytic adenovirus (Ad5/3-Cox2-NIS-ADP) modified to express high quantities of NIS. This virus induced radioisotope uptake in prostate and lung cancer cells and facilitated SPECT/CT imaging, but more importantly, supported the effective therapy with 131I. In our original vector, enhanced oncolysis was mediated by overexpression of adenoviral death protein (ADP). Although this structure was operative in detection and therapeutic regimens, we were concerned that the cytolytic effect of ADP may affect NIS membrane localization, and diminish its effectiveness as a theranostic tool. We therefore designed an identical Ad5/3-Cox2-NIS-ADP-deleted virus {ADP(-)} and assessed the impact of ADP on NIS expression and radioisotope uptake. Western blot and immunocytochemical analyses of pancreatic cancer cells infected with ADP(-) demonstrated higher NIS levels when compared to the ADP(+) counterpart. This correlated with an improved 125I uptake in vitro. SPECT/CT imaging studies assessing 99mTcO4- accumulation in mice to visualize pancreatic adenocarcinoma (PDAC) using patient-derived xenografts (PDX) showed that a single dose of ADP(-) accumulated far greater levels of 99mTcO4- when compared to ADP(+). Remarkably, ADP(-) produced stronger signal that was maintained up until day 32, and this outlasted ADP(+) and the control vector, replication-deficient AdCMV-NIS, currently employed in a clinical trial. Notably, within the PDAC, ADP(-) showed a distinct NIS cell membrane distribution pattern as it co-localized with cell membrane bound-cytokeratin-4. Unlike ADP(-), ADP(+) produced a punctate NIS staining pattern, with little to no cell membrane localization. These results support our hypothesis that ADP-cytolytic effect reduces NIS membrane localization, subsequently affecting radionuclide uptake. To evaluate the therapeutic potential of NIS-based radiotherapy, we treated mice bearing PDAC tumors with virotherapy alone or in combination with 131I. NIS-expressing OAds in combination with 131I significantly reduced tumor growth when compared to viro- or radiotherapy alone. The therapeutic effect in mice treated with ADP(-) combined with 131I outperformed that with ADP(+) or AdCMV-NIS vectors. Quantitative analyses of 131I uptake in tumor tissues with a gamma counter showed a clear trend where ADP(-) retained higher 131I uptake than ADP(+). These findings support the clinical applicability of the ADP-deleted OAd as the more sensitive tool for NIS-based cancer diagnosis and therapy. We are currently investigating the biodistributon and toxicity of our vectors in a pig model.

#5927

Assessing the efficacy of a novel gene therapy approach for treating ovarian cancer: Combination RRV-mediated prodrug activated suicide gene therapy.

James Grosso, Sara Collins, Aki Inagaki, Brian Slomovitz, Noriyuki Kasahara. _University of Miami, Miami, FL_.

Introduction/Aim: Retroviral replicating vectors (RRVs) afford a unique strategy for gene therapy targeting cancer cells. By "activating" RRVs with prodrug activator suicide genes, these RRVs become strong gene delivery vehicles that replicate selectively in cancer cells, and stably persist through proviral genomic integration, leading to inducible cell death upon prodrug administration. This strategy has shown highly promising clinical results in early phase trials (published), and is currently being evaluated in an international Phase III clinical trial for patients with recurrent high-grade glioma. Recently, we have also applied RRV-mediated gene therapy to preclinical models of ovarian cancer. The aim of this current study was to evaluate feasibility and efficacy of combination suicide gene therapy using two RRVs encoated ('pseudotyped') with different envelope proteins and delivering different prodrug activators for the treatment of ovarian cancer.

Methods: Viral spread was monitored in established and primary patient-derived ovarian cancer cell lines (SKOV3, OCI-P5X, OCI-C5X) over the course of ≥27 days using amphotropic murine leukemia virus (AMLV) envelope- and gibbon ape leukemia virus (GALV) envelope-pseudotyped RRVs delivering either fluorescent protein reporter genes (GFP, mStrawberry) or prodrug activator genes (yeast cytosine deaminase, E. coli nitroreductase), either individually or in combination, at various multiplicities of infection (MOI). Flow cytometry for GFP, mStrawberry, and Gag viral protein was used to determine transduction levels and transgene expression levels of RRVs at serial time points. Each therapeutic RRV was tested by MTS assay for its ability to induce cytotoxicity upon exposure to different concentrations prodrug. To determine viral integration and vector stability, PCR of genomic DNA extracted from infected cells was performed using RRV specific primers, and qRT-PCR was performed to determine vector copy number per cell.

Results: All ovarian cancer cell lines tested could be >80% transduced by AMLV- and GALV-pseudotyped RRVs, both individually and in combination, within 6-24 days even after initial inoculation at MOI 0.001, with replication kinetics varying between different cell lines. Both RRVs could stably integrate into the target cell genome without evidence of transgene deletion. MTS assays demonstrated that prodrug activated cytotoxicity led to efficient killing of RRV-transduced ovarian cancer cells in vitro, with synergistic cytotoxicity observed at lower prodrug concentrations after combined treatment.

Conclusion: RRV-mediated prodrug activator gene therapy may be an effective approach for efficiently and selectively inducing cytotoxicity in ovarian cancer cells. These data support further evaluation of this strategy through translational studies in vivo.

#5928

Suicide gene therapy of ovarian cancer.

Obeid M. Malekshah, Siddik Sarkar, Arash Hatefi. _Rutgers University, Piscataway, NJ_.

Purpose: The objective of this study was to develop a gene directed enzyme prodrug therapy (GDEPT) method that can effectively kill both differentiating and cancer stem-like cells (CSCs) in ovarian cancer.

Methods: Ovarian cancer cell lines including A2780, A2780-Cis, SKOV-3, OVCAR-3 and OVASC-1 (malignant cells from ascitic fluid of an ovarian cancer patient) were examined to identify the most drug resistant one. Various chemotherapeutic drugs which are used for suicide gene therapy were tested to identify one that is the most effective against ovarian cancer cells. These include 6-Methylpurine, 5-Fluorouracil, and SN-38. Cisplatin was used as a standard of care control. The anticancer activities of the drugs were studied by a cell toxicity assay and clonogenic assay. Flow cytometry was performed to study surface stem markers, such as CD133, CXCR4, HER2. ALDH1 activity in ovarian cancer cells was studied by ALDH assay kit. The ability of the most effective enzyme/prodrug system to kill drug resistant ovarian cancer cells was studied by a cell viability assay.

Results: The results of in vitro dose-response experiments on various ovarian cancer cells showed that SN-38 was the most efficient drug with IC50 in a range of 10 -100 nM, in comparison to 10-500 μM range of all other selected active drugs. OVASC-1 and OVCAR-3 cell lines showed a higher resistance to drug therapy as compared with other cell lines. It was observed that these two cell lines have a higher number of drug resistant cancer cells in their populations. It was also observed that CSCs are strongly associated with resistance to chemotherapy. ALDH assay, clonogenic assay and sphere formation assay showed a direct correlation between the therapy resistance and the percentage of CSCs. OVASC-1 cells in comparison to the other cell lines were found to be a suitable model for therapy resistant studies. After treatment by 100 nM SN-38, no visible colonies of Ascites were observed. Adipose-derived stem cells (ADSCs) were genetically modified to express Carboxylesterase-2 (CE2) and thus could convert CPT-11 to its active form (SN-38). Using this enzyme/prodrug system an effective eradication of highly resistant OVASC-1 ovarian cancer cells was demonstrated.

Conclusions: Our findings suggest that Carboxylesterase/CPT-11 enzyme/prodrug system is the most effective GDEPT method against highly drug resistant ovarian cancer cells.

#5929

microRNA control of an oHSV vector allows for robust oncolysis and selective control of viral replication in normal tissues.

Edward M. Kennedy, Terry Farkaly, Caitlin Goshert, Allison Colthart, Prajna Behera, Peter Grzesik, Kyle Grant, Michael Paglia, Paola Grandi, Christophe Quéva, Mitchell Finer, Lorena Lerner. _Oncorus, Cambridge, MA_.

Effective treatment of advanced stage cancer with oncolytic viruses requires potent cancer cell-specific oncolytic activity. We modified oncolytic HSV (oHSV) to utilize a novel conditional-lethal strategy that retains all viral genes to maintain potency of the oncolytic virus, yet block replication in normal cells. In addition, we armed the virus with extracellular matrix decreasing enzyme to promote viral spread and immunomodulatory payloads to increase recruitment and effector function of immune cells. To enhance the selectivity of viral replication to tumor cells, Oncorus engineered oHSVs containing microRNA (miRNA)-binding cassettes (miR-T) in essential genes required for viral replication. The first Oncorus clinical candidate, ONCR-1, under development for the treatment recurrent glioblastoma multiform (GBM), has been attenuated for replication in neurons, the most trophic cell type for HSV. Neuronal attenuation was achieved by inserting binding sites for a highly specific neuronal miRNA, miR-124, into the 3′UTR of the essential ICP4 gene. We demonstrate that these engineered miR-124 response elements can selectively inhibit the expression of ICP4 and VP5 as well as the expression of ONCR-1 payloads. This attenuation strategy eliminates viral replication in miR-124 expressing cells without compromising oncolytic activity in cancer cells, thereby providing a therapeutic window. We extended the design oHSV attenuation by profiling of miRNAs expression in panels of solid tumors and non-diseased tissue counterparts in other potentially high-risk tissues, prioritized to indications amenable to local or systemic administration. We leveraged our comprehensive normal and malignant tissue dataset to identify numerous highly expressed unique non-cancerous tissue-specific candidate miRNAs. Insertion of miR-T cassettes into HSV-1 genes collaborated to stall the early steps in the viral replication cycle and further increased the depth and breadth of oHSV attenuation in non-disease tissue relative to the single miR-T cassette in ICP4. We found that miR-122, a highly expressed miRNA in normal hepatocytes, is low or absent in many carcinoma cells. Insertion of miR-122 cassette into the 3′UTR of an immediate early gene, ICP27, inhibited completely viral replication in miR-122 expressing cells. oHSV with dual miR-124 and miR-122 was potently attenuated cells expressing one or both miRs while displaying a robust oncolytic activity in malignant cells. We have shown that insertion of miR-T cassettes in essential viral genes effectively attenuates oHSV replication in normal human cells, and most importantly, robust oncolysis was retained in cancer cells. This work extends the miR-T paradigm to multiple viral genes with diverse miRNA targets, thus expanding the protection of normal tissues and facilitating the application of oHSV to the treatment of multiple malignancies.

#5930

Oral siRNA delivery for colorectal liver metastases cancer therapy.

Sung-Hun Kang,1 Vishnu Revuri,1 Sungpil Cho,1 In-Kyu Park,2 Kwang Jae Cho,3 Woo Kyun Bae,4 Yong-Kyu Lee1. 1 _Korea National University of Transportation, Chungju, Republic of Korea;_ 2 _Chonnam National University Medical School, Gwangju, Republic of Korea;_ 3 _The Catholic University of Korea, Seoul, Republic of Korea;_ 4 _Chonnam National University Hwasun Hospital, Hwasun, Republic of Korea_.

Liver metastasis (CLM) is the lethal secondary hepatic cancer with 5 - 20 months' average survival rate and mostly originated from colorectal cancer (CRC). Surgery and systemic chemotherapy are the currently best options to treat patients. However, over 60% of recurrence rate and severe side effects such as sinusoidal obstruction syndrome and nodular regenerative hyperplasia from the systemic chemotherapy significantly impact on patient's recovery from the treatment. To overcome these side effects and also achieve the synergistic therapeutic effect, we developed a dual padlock system (AR-GT) for oral siRNA delivery to regulate PI3K/Akt signaling pathway, which is highly associated with the progression of CRC to CLM. AR-GT is an about 130 nm nanoparticle consisted of a gold nanoparticle core anchoring siAkt RNA (AR) and a single outer layer of glycol chitosan-taurocholic acid (GT) conjugate. GT layer plays several roles in oral siRNA delivery such as the protection of siRNA in the gastrointestinal (GI) environment, facilitation of ileac uptake of AR-GT in the small intestine and transportation of AR-GT through enterohepatic circulation into the liver. In vitro characterization of AR-GT demonstrated the protection of siRNA under simulated GI pHs for 5 days, facilitation of the trans-epithelial transport in the intestinal Caco2 and ASBT positive cells, and reduction of Akt protein by successful release of siAKT from the AR-GT. Following oral delivery of AR-GT to CLM mouse model, we observed high biodistribution of AR-GT in the ileum, liver, and kidney through intestinal uptake, enterohepatic transportation, and renal excretion. Moreover, CLM mouse after oral delivery of AR-GT (100 μg/kg) manifested about 58 % reduction of the numbers of tumor nodules in the liver and near 100% survival benefit compared to control mouse fed with either saline or AR-Chitosan nanoparticles. Further analysis of liver samples obtained from CLM mouse after oral delivery of AR-GT indicated about 38% reduction of pAkt expression and the enhancement of apoptotic protein expressions such as 2 times or 1.5 times increase of caspase 9 and bax. Theses observation suggests survival benefit and tumor nodule reduction in CLM mouse root on successful AR-GT mediated oral siAkt RNA delivery. we conclude AR-GT oral siRNA delivery system is beneficial in the CLM treatment and further suggest its application to target other cancers. 
